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Blocking miRNAs in Triple Negative Breast Cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Why Blocking microRNAs in Triple Negative Breast Cancer Is so Difficult

http://www.genengnews.com/gen-news-highlights/why-blocking-micrornas-in-triple-negative-breast-cancer-is-so-difficult/81252048/

New research uncovers why attempts at blocking microRNAs for triple negative breast cancer often fail. [National Breast Cancer Foundation]

http://www.genengnews.com/Media/images/GENHighlight/thumb_Dec2_2015_NationalBreastCancerFoundation_MicroRNAs4198253108.jpg

 

While a triple negative score is hardly ever a good thing, for breast cancer it is especially troubling. Triple-negative breast cancer (TNBC) refers to a disease scenario where the cancer cells do not express the genes for estrogen, progesterone, or HER2/neu receptors simultaneously—making this cancer particularly aggressive and difficult to treat as most chemotherapies target one of these receptors.

Over the past several years, researchers have discovered that various microRNAs (miRNAs) underlie the expression of certain genes that can enable cancer cells to proliferate faster. However, the ability to block these miRNAs in TNBC has been met with failure. Yet now, scientists from Thomas Jefferson University in Philadelphia believe they have discovered the reason conventional methods to block these miRNAs has been thus far unsuccessful.

“Triple-negative breast cancer is one of the most aggressive forms of breast cancer, and there’s been a lot of excitement in blocking the microRNAs that appear to make this type of cancer grow faster and resist conventional treatment,” explained senior author Eric Wickstrom, Ph.D., professor in the department of biochemistry and molecular biology at TJU. “However blocking microRNAs hasn’t met with great success and this paper offers one explanation for why that might be the case.”

The findings from this study were published online today in PLOS ONE through an article entitled “Non-specific blocking of miR-17-5p guide strand in triple negative breast cancer cells by amplifying passenger strand activity.” Insight from this study may enable new and more effective design of blockers against previously intractable miRNAs.

“Triple negative breast cancer strikes younger women, tragically killing them in as little as two years,” noted lead author Yuan-Yuan Jin, a doctoral candidate in the department of biochemistry and molecular biology at TJU. “Only chemotherapy and radiation are approved therapies for triple negative breast cancer. We want to treat a genetic target that will keep patients alive with a good quality of life.”

The investigators targeted the miRNA molecule miR-17, which has been shown previously to cause a surge in TNBC growth by alternating genes that would normally signal a diseased or early cancerous cell to die—specifically, the tumor suppressor genes PDCD4 and PTEN.

Although, when the TJU researchers tried to reduce the levels of miR-17 in TNBC cells, rather than increase the levels of the tumor suppressor genes, as they had anticipated, they saw an even larger decrease in these genes than unmodified controls.

The TJU team was acting under the current assumptions that miRNA, which are double stranded, only silence genes using one of their two strands, which is complementary to parts of the messenger RNA coding sequence. The matching, or so-called passenger strand, was thought to be discarded and degraded by the cell.

Using a method to silence RNAs, which involves flooding the cell with modified RNA sequences that mimic the passenger strand and bind to the single-stranded microRNA before it reaches its target, the TJU team saw more silencing of the PDCD4 and PTEN genes.  After some bioinformatic and folding energy calculations, the authors realized that both strands of miR-17 were active in downregulating the tumor suppressor genes.

“Rather than blocking miR-17, we were inadvertently boosting its levels, and, therefore, boosting the cell’s cancerous potential,” noted Jin.

The results of the current study should help to open a pathway to designing specific blockers of one microRNA strand without imitating the opposite strand. Dr. Wickstrom added that “we are now testing new miR-17 blocker designs made possible by these results.”

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Clinical Laboratory Challenges

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

CLINICAL LABORATORY NEWS   

The Lab and CJD: Safe Handling of Infectious Prion Proteins

Body fluids from individuals with possible Creutzfeldt-Jakob disease (CJD) present distinctive safety challenges for clinical laboratories. Sporadic, iatrogenic, and familial CJD (known collectively as classic CJD), along with variant CJD, kuru, Gerstmann-Sträussler-Scheinker, and fatal familial insomnia, are prion diseases, also known as transmissible spongiform encephalopathies. Prion diseases affect the central nervous system, and from the onset of symptoms follow a typically rapid progressive neurological decline. While prion diseases are rare, it is not uncommon for the most prevalent form—sporadic CJD—to be included in the differential diagnosis of individuals presenting with rapid cognitive decline. Thus, laboratories may deal with a significant number of possible CJD cases, and should have protocols in place to process specimens, even if a confirmatory diagnosis of CJD is made in only a fraction of these cases.

The Lab’s Role in Diagnosis

Laboratory protocols for handling specimens from individuals with possible, probable, and definitive cases of CJD are important to ensure timely and appropriate patient management. When the differential includes CJD, an attempt should be made to rule-in or out other causes of rapid neurological decline. Laboratories should be prepared to process blood and cerebrospinal fluid (CSF) specimens in such cases for routine analyses.

Definitive diagnosis requires identification of prion aggregates in brain tissue, which can be achieved by immunohistochemistry, a Western blot for proteinase K-resistant prions, and/or by the presence of prion fibrils. Thus, confirmatory diagnosis is typically achieved at autopsy. A probable diagnosis of CJD is supported by elevated concentration of 14-3-3 protein in CSF (a non-specific marker of neurodegeneration), EEG, and MRI findings. Thus, the laboratory may be required to process and send CSF samples to a prion surveillance center for 14-3-3 testing, as well as blood samples for sequencing of the PRNP gene (in inherited cases).

Processing Biofluids

Laboratories should follow standard protective measures when working with biofluids potentially containing abnormally folded prions, such as donning standard personal protective equipment (PPE); avoiding or minimizing the use of sharps; using single-use disposable items; and processing specimens to minimize formation of aerosols and droplets. An additional safety consideration is the use of single-use disposal PPE; otherwise, re-usable items must be either cleaned using prion-specific decontamination methods, or destroyed.

Blood. In experimental models, infectivity has been detected in the blood; however, there have been no cases of secondary transmission of classical CJD via blood product transfusions in humans. As such, blood has been classified, on epidemiological evidence by the World Health Organization (WHO), as containing “no detectible infectivity,” which means it can be processed by routine methods. Similarly, except for CSF, all other body fluids contain no infectivity and can be processed following standard procedures.

In contrast to classic CJD, there have been four cases of suspected secondary transmission of variant CJD via transfused blood products in the United Kingdom. Variant CJD, the prion disease associated with mad cow disease, is unique in its distribution of prion aggregates outside of the central nervous system, including the lymph nodes, spleen, and tonsils. For regions where variant CJD is a concern, laboratories should consult their regulatory agencies for further guidance.

CSF. Relative to highly infectious tissues of the brain, spinal cord, and eye, infectivity has been identified less often in CSF and is considered to have “low infectivity,” along with kidney, liver, and lung tissue. Since CSF can contain infectious material, WHO has recommended that analyses not be performed on automated equipment due to challenges associated with decontamination. Laboratories should perform a risk assessment of their CSF processes, and, if deemed necessary, consider using manual methods as an alternative to automated systems.

Decontamination

The infectious agent in prion disease is unlike any other infectious pathogen encountered in the laboratory; it is formed of misfolded and aggregated prion proteins. This aggregated proteinacious material forms the infectious unit, which is incredibly resilient to degradation. Moreover, in vitro studies have demonstrated that disrupting large aggregates into smaller aggregates increases cytotoxicity. Thus, if the aim is to abolish infectivity, all aggregates must be destroyed. Disinfectant procedures used for viral, bacterial, and fungal pathogens such as alcohol, boiling, formalin, dry heat (<300°C), autoclaving at 121°C for 15 minutes, and ionizing, ultraviolet, or microwave radiation, are either ineffective or variably effective against aggregated prions.

The only means to ensure no risk of residual infectious prions is to use disposable materials. This is not always practical, as, for instance, a biosafety cabinet cannot be discarded if there is a CSF spill in the hood. Fortunately, there are several protocols considered sufficient for decontamination. For surfaces and heat-sensitive instruments, such as a biosafety cabinet, WHO recommends flooding the surface with 2N NaOH or undiluted NaClO, letting stand for 1 hour, mopping up, and rinsing with water. If the surface cannot tolerate NaOH or NaClO, thorough cleaning will remove most infectivity by dilution. Laboratories may derive some additional benefit by using one of the partially effective methods discussed previously. Non-disposable heat-resistant items preferably should be immersed in 1N NaOH, heated in a gravity displacement autoclave at 121°C for 30 min, cleaned and rinsed in water, then sterilized by routine methods. WHO has outlined several alternate decontamination methods. Using disposable cover sheets is one simple solution to avoid contaminating work surfaces and associated lengthy decontamination procedures.

With standard PPE—augmented by a few additional safety measures and prion-specific decontamination procedures—laboratories can safely manage biofluid testing in cases of prion disease.

 

The Microscopic World Inside Us  

Emerging Research Points to Microbiome’s Role in Health and Disease

Thousands of species of microbes—bacteria, viruses, fungi, and protozoa—inhabit every internal and external surface of the human body. Collectively, these microbes, known as the microbiome, outnumber the body’s human cells by about 10 to 1 and include more than 1,000 species of microorganisms and several million genes residing in the skin, respiratory system, urogenital, and gastrointestinal tracts. The microbiome’s complicated relationship with its human host is increasingly considered so crucial to health that researchers sometimes call it “the forgotten organ.”

Disturbances to the microbiome can arise from nutritional deficiencies, antibiotic use, and antiseptic modern life. Imbalances in the microbiome’s diverse microbial communities, which interact constantly with cells in the human body, may contribute to chronic health conditions, including diabetes, asthma and allergies, obesity and the metabolic syndrome, digestive disorders including irritable bowel syndrome (IBS), and autoimmune disorders like multiple sclerosis and rheumatoid arthritis, research shows.

While study of the microbiome is a growing research enterprise that has attracted enthusiastic media attention and venture capital, its findings are largely preliminary. But some laboratorians are already developing a greater appreciation for the microbiome’s contributions to human biochemistry and are considering a future in which they expect to measure changes in the microbiome to monitor disease and inform clinical practice.

Pivot Toward the Microbiome

Following the National Institutes of Health (NIH) Human Genome Project, many scientists noted the considerable genetic signal from microbes in the body and the existence of technology to analyze these microorganisms. That realization led NIH to establish the Human Microbiome Project in 2007, said Lita Proctor, PhD, its program director. In the project’s first phase, researchers studied healthy adults to produce a reference set of microbiomes and a resource of metagenomic sequences of bacteria in the airways, skin, oral cavities, and the gastrointestinal and vaginal tracts, plus a catalog of microbial genome sequences of reference strains. Researchers also evaluated specific diseases associated with disturbances in the microbiome, including gastrointestinal diseases such as Crohn’s disease, ulcerative colitis, IBS, and obesity, as well as urogenital conditions, those that involve the reproductive system, and skin diseases like eczema, psoriasis, and acne.

Phase 1 studies determined the composition of many parts of the microbiome, but did not define how that composition affects health or specific disease. The project’s second phase aims to “answer the question of what microbes actually do,” explained Proctor. Researchers are now examining properties of the microbiome including gene expression, protein, and human and microbial metabolite profiles in studies of pregnant women at risk for preterm birth, the gut hormones of patients at risk for IBS, and nasal microbiomes of patients at risk for type 2 diabetes.

Promising Lines of Research

Cystic fibrosis and microbiology investigator Michael Surette, PhD, sees promising microbiome research not just in terms of evidence of its effects on specific diseases, but also in what drives changes in the microbiome. Surette is Canada research chair in interdisciplinary microbiome research in the Farncombe Family Digestive Health Research Institute at McMaster University
in Hamilton, Ontario.

One type of study on factors driving microbiome change examines how alterations in composition and imbalances in individual patients relate to improving or worsening disease. “IBS, cystic fibrosis, and chronic obstructive pulmonary disease all have periods of instability or exacerbation,” he noted. Surette hopes that one day, tests will provide clinicians the ability to monitor changes in microbial composition over time and even predict when a patient’s condition is about to deteriorate. Monitoring perturbations to the gut microbiome might also help minimize collateral damage to the microbiome during aggressive antibiotic therapy for hospitalized patients, he added.

Monitoring changes to the microbiome also might be helpful for “culture negative” patients, who now may receive multiple, unsuccessful courses of different antibiotics that drive antibiotic resistance. Frustration with standard clinical biology diagnosis of lung infections in cystic fibrosis patients first sparked Surette’s investigations into the microbiome. He hopes that future tests involving the microbiome might also help asthma patients with neutrophilia, community-acquired pneumonia patients who harbor complex microbial lung communities lacking obvious pathogens, and hospitalized patients with pneumonia or sepsis. He envisions microbiome testing that would look for short-term changes indicating whether or not a drug is effective.

Companion Diagnostics

Daniel Peterson, MD, PhD, an assistant professor of pathology at Johns Hopkins University School of Medicine in Baltimore, believes the future of clinical testing involving the microbiome lies in companion diagnostics for novel treatments, and points to companies that are already developing and marketing tests that will require such assays.

Examples of microbiome-focused enterprises abound, including Genetic Analysis, based in Oslo, Norway, with its high-throughput test that uses 54 probes targeted to specific bacteria to measure intestinal gut flora imbalances in inflammatory bowel disease and irritable bowel syndrome patients. Paris, France-based Enterome is developing both novel drugs and companion diagnostics for microbiome-related diseases such as IBS and some metabolic diseases. Second Genome, based in South San Francisco, has developed an experimental drug, SGM-1019, that the company says blocks damaging activity of the microbiome in the intestine. Cambridge, Massachusetts-based Seres Therapeutics has received Food and Drug Administration orphan drug designation for SER-109, an oral therapeutic intended to correct microbial imbalances to prevent recurrent Clostridium difficile infection in adults.

One promising clinical use of the microbiome is fecal transplantation, which both prospective and retrospective studies have shown to be effective in patients with C. difficile infections who do not respond to front-line therapies, said James Versalovic, MD, PhD, director of Texas Children’s Hospital Microbiome Center and professor of pathology at Baylor College of Medicine in Houston. “Fecal transplants and other microbiome replacement strategies can radically change the composition of the microbiome in hours to days,” he explained.

But NIH’s Proctor discourages too much enthusiasm about fecal transplant. “Natural products like stool can have [side] effects,” she pointed out. “The [microbiome research] field needs to mature and we need to verify outcomes before anything becomes routine.”

Hurdles for Lab Testing

While he is hopeful that labs someday will use the microbiome to produce clinically useful information, Surette pointed to several problems that must be solved beforehand. First, molecular methods commonly used right now should be more quantitative and accurate. Additionally, research on the microbiome encompasses a wide variety of protocols, some of which are better at extracting particular types of bacteria and therefore can give biased views of communities living in the body. Also, tests may need to distinguish between dead and live microbes. Another hurdle is that labs using varied bioinfomatic methods may produce different results from the same sample, a problem that Surette sees as ripe for a solution from clinical laboratorians, who have expertise in standardizing robust protocols and in automating tests.

One way laboratorians can prepare for future, routine microbiome testing is to expand their notion of clinical chemistry to include both microbial and human biochemistry. “The line between microbiome science and clinical science is blurring,” said Versalovic. “When developing future assays to detect biochemical changes in disease states, we must consider the contributions of microbial metabolites and proteins and how to tailor tests to detect them.” In the future, clinical labs may test for uniquely microbial metabolites in various disease states, he predicted.

 

Automated Review of Mass Spectrometry Results  

Can We Achieve Autoverification?

Author: Katherine Alexander and Andrea R. Terrell, PhD  // Date: NOV.1.2015  // Source:Clinical Laboratory News

https://www.aacc.org/publications/cln/articles/2015/november/automated-review-of-mass-spectrometry-results-can-we-achieve-autoverification

 

Paralleling the upswing in prescription drug misuse, clinical laboratories are receiving more requests for mass spectrometry (MS) testing as physicians rely on its specificity to monitor patient compliance with prescription regimens. However, as volume has increased, reimbursement has declined, forcing toxicology laboratories both to increase capacity and lower their operational costs—without sacrificing quality or turnaround time. Now, new solutions are available enabling laboratories to bring automation to MS testing and helping them with the growing demand for toxicology and other testing.

What is the typical MS workflow?

A typical workflow includes a long list of manual steps. By the time a sample is loaded onto the mass spectrometer, it has been collected, logged into the lab information management system (LIMS), and prepared for analysis using a variety of wet chemistry techniques.

Most commercial clinical laboratories receive enough samples for MS analysis to batch analyze those samples. A batch consists of a calibrator(s), quality control (QC) samples, and patient/donor samples. Historically, the method would be selected (i.e. “analysis of opiates”), sample identification information would be entered manually into the MS software, and the instrument would begin analyzing each sample. Upon successful completion of the batch, the MS operator would view all of the analytical data, ensure the QC results were acceptable, and review each patient/donor specimen, looking at characteristics such as peak shape, ion ratios, retention time, and calculated concentration.

The operator would then post acceptable results into the LIMS manually or through an interface, and unacceptable results would be rescheduled or dealt with according to lab-specific protocols. In our laboratory we perform a final certification step for quality assurance by reviewing all information about the batch again, prior to releasing results for final reporting through the LIMS.

What problems are associated with this workflow?

The workflow described above results in too many highly trained chemists performing manual data entry and reviewing perfectly acceptable analytical results. Lab managers would prefer that MS operators and certifying scientists focus on troubleshooting problem samples rather than reviewing mounds of good data. Not only is the current process inefficient, it is mundane work prone to user errors. This risks fatigue, disengagement, and complacency by our highly skilled scientists.

Importantly, manual processes also take time. In most clinical lab environments, turnaround time is critical for patient care and industry competitiveness. Lab directors and managers are looking for solutions to automate mundane, error-prone tasks to save time and costs, reduce staff burnout, and maintain high levels of quality.

How can software automate data transfer from MS systems to LIMS?

Automation is not a new concept in the clinical lab. Labs have automated processes in shipping and receiving, sample preparation, liquid handling, and data delivery to the end user. As more labs implement MS, companies have begun to develop special software to automate data analysis and review workflows.

In July 2011, AIT Labs incorporated ASCENT into our workflow, eliminating the initial manual peak review step. ASCENT is an algorithm-based peak picking and data review system designed specifically for chromatographic data. The software employs robust statistical and modeling approaches to the raw instrument data to present the true signal, which often can be obscured by noise or matrix components.

The system also uses an exponentially modified Gaussian (EMG) equation to apply a best-fit model to integrated peaks through what is often a noisy signal. In our experience, applying the EMG results in cleaner data from what might appear to be poor chromatography ultimately allows us to reduce the number of samples we might otherwise rerun.

How do you validate the quality of results?

We’ve developed a robust validation protocol to ensure that results are, at minimum, equivalent to results from our manual review. We begin by building the assay in ASCENT, entering assay-specific information from our internal standard operating procedure (SOP). Once the assay is configured, validation proceeds with parallel batch processing to compare results between software-reviewed data and staff-reviewed data. For new implementations we run eight to nine batches of 30–40 samples each; when we are modifying or upgrading an existing implementation we run a smaller number of batches. The parallel batches should contain multiple positive and negative results for all analytes in the method, preferably spanning the analytical measurement range of the assay.

The next step is to compare the results and calculate the percent difference between the data review methods. We require that two-thirds of the automated results fall within 20% of the manually reviewed result. In addition to validating patient sample correlation, we also test numerous quality assurance rules that should initiate a flag for further review.

What are the biggest challenges during implementation and continual improvement initiatives?

On the technological side, our largest hurdle was loading the sequence files into ASCENT. We had created an in-house mechanism for our chemists to upload the 96-well plate map for their batch into the MS software. We had some difficulty transferring this information to ASCENT, but once we resolved this issue, the technical workflow proceeded fairly smoothly.

The greater challenge was changing our employees’ mindset from one of fear that automation would displace them, to a realization that learning this new technology would actually make them more valuable. Automating a non-mechanical process can be a difficult concept for hands-on scientists, so managers must be patient and help their employees understand that this kind of technology leverages the best attributes of software and people to create a powerful partnership.

We recommend that labs considering automated data analysis engage staff in the validation and implementation to spread the workload and the knowledge. As is true with most technology, it is best not to rely on just one or two super users. We also found it critical to add supervisor level controls on data file manipulation, such as removing a sample that wasn’t run from the sequence table. This can prevent inadvertent deletion of a file, requiring reinjection of the entire batch!

 

Understanding Fibroblast Growth Factor 23

Author: Damien Gruson, PhD  // Date: OCT.1.2015  // Source: Clinical Laboratory News

https://www.aacc.org/publications/cln/articles/2015/october/understanding-fibroblast-growth-factor-23

What is the relationship of FGF-23 to heart failure?

A Heart failure (HF) is an increasingly common syndrome associated with high morbidity, elevated hospital readmission rates, and high mortality. Improving diagnosis, prognosis, and treatment of HF requires a better understanding of its different sub-phenotypes. As researchers gained a comprehensive understanding of neurohormonal activation—one of the hallmarks of HF—they discovered several biomarkers, including natriuretic peptides, which now are playing an important role in sub-phenotyping HF and in driving more personalized management of this chronic condition.

Like the natriuretic peptides, fibroblast growth factor 23 (FGF-23) could become important in risk-stratifying and managing HF patients. Produced by osteocytes, FGF-23 is a key regulator of phosphorus homeostasis. It binds to renal and parathyroid FGF-Klotho receptor heterodimers, resulting in phosphate excretion, decreased 1-α-hydroxylation of 25-hydroxyvitamin D, and decreased parathyroid hormone (PTH) secretion. The relationship to PTH is important because impaired homeostasis of cations and decreased glomerular filtration rate might contribute to the rise of FGF-23. The amino-terminal portion of FGF-23 (amino acids 1-24) serves as a signal peptide allowing secretion into the blood, and the carboxyl-terminal portion (aa 180-251) participates in its biological action.

How might FGF-23 improve HF risk assessment?

Studies have shown that FGF-23 is related to the risk of cardiovascular diseases and mortality. It was first demonstrated that FGF-23 levels were independently associated with left ventricular mass index and hypertrophy as well as mortality in patients with chronic kidney disease (CKD). FGF-23 also has been associated with left ventricular dysfunction and atrial fibrillation in coronary artery disease subjects, even in the absence of impaired renal function.

FGF-23 and FGF receptors are both expressed in the myocardium. It is possible that FGF-23 has direct effects on the heart and participates in the physiopathology of cardiovascular diseases and HF. Experiments have shown that for in vitro cultured rat cardiomyocytes, FGF-23 stimulates pathological hypertrophy by activating the calcineurin-NFAT pathway—and in wild-type mice—the intra-myocardial or intravenous injection of FGF-23 resulted in left ventricular hypertrophy. As such, FGF-23 appears to be a potential stimulus of myocardial hypertrophy, and increased levels may contribute to the worsening of heart failure and long-term cardiovascular death.

Researchers have documented that HF patients have elevated FGF-23 circulating levels. They have also found a significant correlation between plasma levels of FGF-23 and B-type natriuretic peptide, a biomarker related to ventricular stretch and cardiac hypertrophy, in patients with left ventricular hypertrophy. As such, measuring FGF-23 levels might be a useful tool to predict long-term adverse cardiovascular events in HF patients.

Interestingly, researchers have documented a significant relationship between FGF-23 and PTH in both CKD and HF patients. As PTH stimulates FGF-23 expression, it could be that in HF patients, increased PTH levels increase the bone expression of FGF-23, which enhances its effects on the heart.

 

The Past, Present, and Future of Western Blotting in the Clinical Laboratory

Author: Curtis Balmer, PhD  // Date: OCT.1.2015  // Source: Clinical Laboratory News

https://www.aacc.org/publications/cln/articles/2015/october/the-past-present-and-future-of-western-blotting-in-the-clinical-laboratory

Much of the discussion about Western blotting centers around its performance as a biological research tool. This isn’t surprising. Since its introduction in the late 1970s, the Western blot has been adopted by biology labs of virtually every stripe, and become one of the most widely used techniques in the research armamentarium. However, Western blotting has also been employed in clinical laboratories to aid in the diagnosis of various diseases and disorders—an equally important and valuable application. Yet there has been relatively little discussion of its use in this context, or of how advances in Western blotting might affect its future clinical use.

Highlighting the clinical value of Western blotting, Stanley Naides, MD, medical director of Immunology at Quest Diagnostics observed that, “Western blotting has been a very powerful tool in the laboratory and for clinical diagnosis. It’s one of many various methods that the laboratorian brings to aid the clinician in the diagnosis of disease, and the selection and monitoring of therapy.” Indeed, Western blotting has been used at one time or the other to aid in the diagnosis of infectious diseases including hepatitis C (HCV), HIV, Lyme disease, and syphilis, as well as autoimmune disorders such as paraneoplastic disease and myositis conditions.

However, Naides was quick to point out that the choice of assays to use clinically is based on their demonstrated sensitivity and performance, and that the search for something better is never-ending. “We’re constantly looking for methods that improve detection of our target [protein],” Naides said. “There have been a number of instances where we’ve moved away from Western blotting because another method proves to be more sensitive.” But this search can also lead back to Western blotting. “We’ve gone away from other methods because there’s been a Western blot that’s been developed that’s more sensitive and specific. There’s that constant movement between methods as new tests are developed.”

In recent years, this quest has been leading clinical laboratories away from Western blotting toward more sensitive and specific diagnostic assays, at least for some diseases. Using confirmatory diagnosis of HCV infection as an example, Sai Patibandla, PhD, director of the immunoassay group at Siemens Healthcare Diagnostics, explained that movement away from Western blotting for confirmatory diagnosis of HCV infection began with a technical modification called Recombinant Immunoblotting Assay (RIBA). RIBA streamlines the conventional Western blot protocol by spotting recombinant antigen onto strips which are used to screen patient samples for antibodies against HCV. This approach eliminates the need to separate proteins and transfer them onto a membrane.

The RIBA HCV assay was initially manufactured by Chiron Corporation (acquired by Novartics Vaccines and Diagnostics in 2006). It received Food and Drug Administration (FDA) approval in 1999, and was marketed as Chiron RIBA HCV 3.0 Strip Immunoblot Assay. Patibandla explained that, at the time, the Chiron assay “…was the only FDA-approved confirmatory testing for HCV.” In 2013 the assay was discontinued and withdrawn from the market due to reports that it was producing false-positive results.

Since then, clinical laboratories have continued to move away from Western blot-based assays for confirmation of HCV in favor of the more sensitive technique of nucleic acid testing (NAT). “The migration is toward NAT for confirmation of HCV [diagnosis]. We don’t use immunoblots anymore. We don’t even have a blot now to confirm HCV,” Patibandla said.

Confirming HIV infection has followed a similar path. Indeed, in 2014 the Centers for Disease Control and Prevention issued updated recommendations for HIV testing that, in part, replaced Western blotting with NAT. This change was in response to the recognition that the HIV-1 Western blot assay was producing false-negative or indeterminable results early in the course of HIV infection.

At this juncture it is difficult to predict if this trend away from Western blotting in clinical laboratories will continue. One thing that is certain, however, is that clinicians and laboratorians are infinitely pragmatic, and will eagerly replace current techniques with ones shown to be more sensitive, specific, and effective. This raises the question of whether any of the many efforts currently underway to improve Western blotting will produce an assay that exceeds the sensitivity of currently employed techniques such as NAT.

Some of the most exciting and groundbreaking work in this area is being done by Amy Herr, PhD, a professor of bioengineering at University of California, Berkeley. Herr’s group has taken on some of the most challenging limitations of Western blotting, and is developing techniques that could revolutionize the assay. For example, the Western blot is semi-quantitative at best. This weakness dramatically limits the types of answers it can provide about changes in protein concentrations under various conditions.

To make Western blotting more quantitative, Herr’s group is, among other things, identifying losses of protein sample mass during the assay protocol. About this, Herr explains that the conventional Western blot is an “open system” that involves lots of handling of assay materials, buffers, and reagents that makes it difficult to account for protein losses. Or, as Kevin Lowitz, a senior product manager at Thermo Fisher Scientific, described it, “Western blot is a [simple] technique, but a really laborious one, and there are just so many steps and so many opportunities to mess it up.”

Herr’s approach is to reduce the open aspects of Western blot. “We’ve been developing these more closed systems that allow us at each stage of the assay to account for [protein mass] losses. We can’t do this exactly for every target of interest, but it gives us a really good handle [on protein mass losses],” she said. One of the major mechanisms Herr’s lab is using to accomplish this is to secure proteins to the blot matrix with covalent bonding rather than with the much weaker hydrophobic interactions that typically keep the proteins in place on the membrane.

Herr’s group also has been developing microfluidic platforms that allow Western blotting to be done on single cells, “In our system we’re doing thousands of independent Westerns on single cells in four hours. And, hopefully, we’ll cut that down to one hour over the next couple years.”

Other exciting modifications that stand to dramatically increase the sensitivity, quantitation, and through-put of Western blotting also are being developed and explored. For example, the use of capillary electrophoresis—in which proteins are conveyed through a small electrolyte-filled tube and separated according to size and charge before being dropped onto a blotting membrane—dramatically reduces the amount of protein required for Western blot analysis, and thereby allows Westerns to be run on proteins from rare cells or for which quantities of sample are extremely limited.

Jillian Silva, PhD, an associate specialist at the University of California, San Francisco Helen Diller Family Comprehensive Cancer Center, explained that advances in detection are also extending the capabilities of Western blotting. “With the advent of fluorescence detection we have a way to quantitate Westerns, and it is now more quantitative than it’s ever been,” said Silva.

Whether or not these advances produce an assay that is adopted by clinical laboratories remains to be seen. The emphasis on Western blotting as a research rather than a clinical tool may bias advances in favor of the needs and priorities of researchers rather than clinicians, and as Patibandla pointed out, “In the research world Western blotting has a certain purpose. [Researchers] are always coming up with new things, and are trying to nail down new proteins, so you cannot take Western blotting away.” In contrast, she suggested that for now, clinical uses of Western blotting remain “limited.”

 

Adapting Next Generation Technologies to Clinical Molecular Oncology Service

Author: Ronald Carter, PhD, DVM  // Date: OCT.1.2015  // Source: Clinical Laboratory News

https://www.aacc.org/publications/cln/articles/2015/october/adapting-next-generation-technologies-to-clinical-molecular-oncology-service

Next generation technologies (NGT) deliver huge improvements in cost efficiency, accuracy, robustness, and in the amount of information they provide. Microarrays, high-throughput sequencing platforms, digital droplet PCR, and other technologies all offer unique combinations of desirable performance.

As stronger evidence of genetic testing’s clinical utility influences patterns of patient care, demand for NGT testing is increasing. This presents several challenges to clinical laboratories, including increased urgency, clinical importance, and breadth of application in molecular oncology, as well as more integration of genetic tests into synoptic reporting. Laboratories need to add NGT-based protocols while still providing old tests, and the pace of change is increasing.What follows is one viewpoint on the major challenges in adopting NGTs into diagnostic molecular oncology service.

Choosing a Platform

Instrument selection is a critical decision that has to align with intended test applications, sequencing chemistries, and analytical software. Although multiple platforms are available, a mainstream standard has not emerged. Depending on their goals, laboratories might set up NGTs for improved accuracy of mutation detection, massively higher sequencing capacity per test, massively more targets combined in one test (multiplexing), greater range in sequencing read length, much lower cost per base pair assessed, and economy of specimen volume.

When high-throughput instruments first made their appearance, laboratories paid more attention to the accuracy of base-reading: Less accurate sequencing meant more data cleaning and resequencing (1). Now, new instrument designs have narrowed the differences, and test chemistry can have a comparatively large impact on analytical accuracy (Figure 1). The robustness of technical performance can also vary significantly depending upon specimen type. For example, LifeTechnologies’ sequencing platforms appear to be comparatively more tolerant of low DNA quality and concentration, which is an important consideration for fixed and processed tissues.

https://www.aacc.org/~/media/images/cln/articles/2015/october/carter_fig1_cln_oct15_ed.jpg

Figure 1 Comparison of Sequencing Chemistries

Sequence pile-ups of the same target sequence (2 large genes), all performed on the same analytical instrument. Results from 4 different chemistries, as designed and supplied by reagent manufacturers prior to optimization in the laboratory. Red lines represent limits of exons. Height of blue columns proportional to depth of coverage. In this case, the intent of the test design was to provide high depth of coverage so that reflex Sanger sequencing would not be necessary. Courtesy B. Sadikovic, U. of Western Ontario.

 

In addition, batching, robotics, workload volume patterns, maintenance contracts, software licenses, and platform lifetime affect the cost per analyte and per specimen considerably. Royalties and reagent contracts also factor into the cost of operating NGT: In some applications, fees for intellectual property can represent more than 50% of the bench cost of performing a given test, and increase substantially without warning.

Laboratories must also deal with the problem of obsolescence. Investing in a new platform brings the angst of knowing that better machines and chemistries are just around the corner. Laboratories are buying bigger pieces of equipment with shorter service lives. Before NGTs, major instruments could confidently be expected to remain current for at least 6 to 8 years. Now, a major instrument is obsolete much sooner, often within 2 to 3 years. This means that keeping it in service might cost more than investing in a new platform. Lease-purchase arrangements help mitigate year-to-year fluctuations in capital equipment costs, and maximize the value of old equipment at resale.

One Size Still Does Not Fit All

Laboratories face numerous technical considerations to optimize sequencing protocols, but the test has to be matched to the performance criteria needed for the clinical indication (2). For example, measuring response to treatment depends first upon the diagnostic recognition of mutation(s) in the tumor clone; the marker(s) then have to be quantifiable and indicative of tumor volume throughout the course of disease (Table 1).

As a result, diagnostic tests need to cover many different potential mutations, yet accurately identify any clinically relevant mutations actually present. On the other hand, tests for residual disease need to provide standardized, sensitive, and accurate quantification of a selected marker mutation against the normal background. A diagnostic panel might need 1% to 3% sensitivity across many different mutations. But quantifying early response to induction—and later assessment of minimal residual disease—needs a test that is reliably accurate to the 10-4 or 10-5 range for a specific analyte.

Covering all types of mutations in one diagnostic test is not yet possible. For example, subtyping of acute myeloid leukemia is both old school (karyotype, fluorescent in situ hybridization, and/or PCR-based or array-based testing for fusion rearrangements, deletions, and segmental gains) and new school (NGT-based panel testing for molecular mutations).

Chemistries that cover both structural variants and copy number variants are not yet in general use, but the advantages of NGTs compared to traditional methods are becoming clearer, such as in colorectal cancer (3). Researchers are also using cell-free DNA (cfDNA) to quantify residual disease and detect resistance mutations (4). Once a clinically significant clone is identified, enrichment techniques help enable extremely sensitive quantification of residual disease (5).

Validation and Quality Assurance

Beyond choosing a platform, two distinct challenges arise in bringing NGTs into the lab. The first is assembling the resources for validation and quality assurance. The second is keeping tests up-to-date as new analytes are needed. Even if a given test chemistry has the flexibility to add analytes without revalidating the entire panel, keeping up with clinical advances is a constant priority.

Due to their throughput and multiplexing capacities, NGT platforms typically require considerable upfront investment to adopt, and training staff to perform testing takes even more time. Proper validation is harder to document: Assembling positive controls, documenting test performance criteria, developing quality assurance protocols, and conducting proficiency testing are all demanding. Labs meet these challenges in different ways. Laboratory-developed tests (LDTs) allow self-determined choice in design, innovation, and control of the test protocol, but can be very expensive to set up.

Food and Drug Administration (FDA)-approved methods are attractive but not always an option. More FDA-approved methods will be marketed, but FDA approval itself brings other trade-offs. There is a cost premium compared to LDTs, and the test methodologies are locked down and not modifiable. This is particularly frustrating for NGTs, which have the specific attraction of extensive multiplexing capacity and accommodating new analytes.

IT and the Evolution of Molecular Oncology Reporting Standards

The options for information technology (IT) pipelines for NGTs are improving rapidly. At the same time, recent studies still show significant inconsistencies and lack of reproducibility when it comes to interpreting variants in array comparative genomic hybridization, panel testing, tumor expression profiling, and tumor genome sequencing. It can be difficult to duplicate published performances in clinical studies because of a lack of sufficient information about the protocol (chemistry) and software. Building bioinformatics capacity is a key requirement, yet skilled people are in short supply and the qualifications needed to work as a bioinformatician in a clinical service are not yet clearly defined.

Tumor biology brings another level of complexity. Bioinformatic analysis must distinguish tumor-specific­ variants from genomic variants. Sequencing of paired normal tissue is often performed as a control, but virtual normal controls may have intriguing advantages (6). One of the biggest challenges is to reproducibly interpret the clinical significance of interactions between different mutations, even with commonly known, well-defined mutations (7). For multiple analyte panels, such as predictive testing for breast cancer, only the performance of the whole panel in a population of patients can be compared; individual patients may be scored into different risk categories by different tests, all for the same test indication.

In large scale sequencing of tumor genomes, which types of mutations are most informative in detecting, quantifying, and predicting the behavior of the tumor over time? The amount and complexity of mutation varies considerably across different tumor types, and while some mutations are more common, stable, and clinically informative than others, the utility of a given tumor marker varies in different clinical situations. And, for a given tumor, treatment effect and metastasis leads to retesting for changes in drug sensitivities.

These complexities mean that IT must be designed into the process from the beginning. Like robotics, IT represents a major ancillary decision. One approach many labs choose is licensed technologies with shared databases that are updated in real time. These are attractive, despite their cost and licensing fees. New tests that incorporate proprietary IT with NGT platforms link the genetic signatures of tumors to clinically significant considerations like tumor classification, recommended methodologies for monitoring response, predicted drug sensitivities, eligible clinical trials, and prognostic classifications. In-house development of such solutions will be difficult, so licensing platforms from commercial partners is more likely to be the norm.

The Commercial Value of Health Records and Test Data

The future of cancer management likely rests on large-scale databases that link hereditary and somatic tumor testing with clinical outcomes. Multiple centers have such large studies underway, and data extraction and analysis is providing increasingly refined interpretations of clinical significance.

Extracting health outcomes to correlate with molecular test results is commercially valuable, as the pharmaceutical, insurance, and healthcare sectors focus on companion diagnostics, precision medicine, and evidence-based health technology assessment. Laboratories that can develop tests based on large-scale integration of test results to clinical utility will have an advantage.

NGTs do offer opportunities for net reductions in the cost of healthcare. But the lag between availability of a test and peer-evaluated demon­stration of clinical utility can be considerable. Technical developments arise faster than evidence of clinical utility. For example, immuno­histochemistry, estrogen receptor/progesterone receptor status, HER2/neu, and histology are still the major pathological criteria for prognostic evaluation of breast cancer at diagnosis, even though multiple analyte tumor profiling has been described for more than 15 years. Healthcare systems need a more concerted assessment of clinical utility if they are to take advantage of the promises of NGTs in cancer care.

Disruptive Advances

Without a doubt, “disruptive” is an appropriate buzzword in molecular oncology, and new technical advances are about to change how, where, and for whom testing is performed.

• Predictive Testing

Besides cost per analyte, one of the drivers for taking up new technologies is that they enable multiplexing many more analytes with less biopsy material. Single-analyte sequential testing for epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase, and other targets on small biopsies is not sustainable when many more analytes are needed, and even now, a significant proportion of test requests cannot be completed due to lack of suitable biopsy material. Large panels incorporating all the mutations needed to cover multiple tumor types are replacing individual tests in companion diagnostics.

• Cell-Free Tumor DNA

Challenges of cfDNA include standardizing the collection and processing methodologies, timing sampling to minimize the effect of therapeutic toxicity on analytical accuracy, and identifying the most informative sample (DNA, RNA, or protein). But for more and more tumor types, it will be possible to differentiate benign versus malignant lesions, perform molecular subtyping, predict response, monitor treatment, or screen for early detection—all without a surgical biopsy.

cfDNA technologies can also be integrated into core laboratory instrumentation. For example, blood-based EGFR analysis for lung cancer is being developed on the Roche cobas 4800 platform, which will be a significant change from the current standard of testing based upon single tests of DNA extracted from formalin-fixed, paraffin-embedded sections selected by a pathologist (8).

• Whole Genome and Whole Exome Sequencing

Whole genome and whole exome tumor sequencing approaches provide a wealth of biologically important information, and will replace individual or multiple gene test panels as the technical cost of sequencing declines and interpretive accuracy improves (9). Laboratories can apply informatics selectively or broadly to extract much more information at relatively little increase in cost, and the interpretation of individual analytes will be improved by the context of the whole sequence.

• Minimal Residual Disease Testing

Massive resequencing and enrichment techniques can be used to detect minimal residual disease, and will provide an alternative to flow cytometry as costs decline. The challenge is to develop robust analytical platforms that can reliably produce results in a high proportion of patients with a given tumor type, despite using post-treatment specimens with therapy-induced degradation, and a very low proportion of target (tumor) sequence to benign background sequence.

The tumor markers should remain informative for the burden of disease despite clonal evolution over the course of multiple samples taken during progression of the clinical course and treatment. Quantification needs to be accurate and sensitive down to the 10-5 range, and cost competitive with flow cytometry.

• Point-of-Care Test Methodologies

Small, rapid, cheap, and single use point-of-care (POC) sequencing devices are coming. Some can multiplex with analytical times as short as 20 minutes. Accurate and timely testing will be possible in places like pharmacies, oncology clinics, patient service centers, and outreach programs. Whether physicians will trust and act on POC results alone, or will require confirmation by traditional laboratory-based testing, remains to be seen. However, in the simplest type of application, such as a patient known to have a particular mutation, the advantages of POC-based testing to quantify residual tumor burden are clear.

Conclusion

Molecular oncology is moving rapidly from an esoteric niche of diagnostics to a mainstream, required component of integrated clinical laboratory services. While NGTs are markedly reducing the cost per analyte and per specimen, and will certainly broaden the scope and volume of testing performed, the resources required to choose, install, and validate these new technologies are daunting for smaller labs. More rapid obsolescence and increased regulatory scrutiny for LDTs also present significant challenges. Aligning test capacity with approved clinical indications will require careful and constant attention to ensure competitiveness.

References

1. Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012; doi:10.1155/2012/251364.

2. Brownstein CA, Beggs AH, Homer N, et al. An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge. Genome Biol 2014;15:R53.

3. Haley L, Tseng LH, Zheng G, et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal ­cancers. [Epub ahead of print] Modern Pathol July 31, 2015 as doi:10.1038/modpathol.2015.86.

4. Butler TM, Johnson-Camacho K, Peto M, et al. Exome sequencing of cell-free DNA from metastatic cancer patients identifies clinically actionable mutations distinct from primary ­disease. PLoS One 2015;10:e0136407.

5. Castellanos-Rizaldos E, Milbury CA, Guha M, et al. COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing. Methods Mol Biol 2014;1102:623–39.

6. Hiltemann S, Jenster G, Trapman J, et al. Discriminating somatic and germline mutations in tumor DNA samples without matching normals. Genome Res 2015;25:1382–90.

7. Lammers PE, Lovly CM, Horn L. A patient with metastatic lung adenocarcinoma harboring concurrent EGFR L858R, EGFR germline T790M, and PIK3CA mutations: The challenge of interpreting results of comprehensive mutational testing in lung cancer. J Natl Compr Canc Netw 2015;12:6–11.

8. Weber B, Meldgaard P, Hager H, et al. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays. BMC Cancer 2014;14:294.

9. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013;339:1546–58.

10. Heitzer E, Auer M, Gasch C, et al. Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. Cancer Res 2013;73:2965–75.

11. Healy B. BRCA genes — Bookmaking, fortunetelling, and medical care. N Engl J Med 1997;336:1448–9.

 

 

 

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Cancer Companion Diagnostics

Curator: Larry H. Bernstein, MD, FCAP

 

Companion Diagnostics for Cancer: Will NGS Play a Role?

Patricia Fitzpatrick Dimond, Ph.D.

http://www.genengnews.com/insight-and-intelligence/companion-diagnostics-for-cancer/77900554/

Companion diagnostics (CDx), in vitro diagnostic devices or imaging tools that provide information essential to the safe and effective use of a corresponding therapeutic product, have become indispensable tools for oncologists.  As a result, analysts expect the global CDx market to reach $8.73 billion by 2019, up from from $3.14 billion in 2014.

Use of CDx during a clinical trial to guide therapy can improve treatment responses and patient outcomes by identifying and predicting patient subpopulations most likely to respond to a given treatment.

These tests not only indicate the presence of a molecular target, but can also reveal the off-target effects of a therapeutic, predicting toxicities and adverse effects associated with a drug.

For pharma manufacturers, using CDx during drug development improves the success rate of drugs being tested in clinical trials. In a study estimating the risk of clinical trial failure during non-small cell lung cancer drug development in the period between 1998 and 2012 investigators analyzed trial data from 676 clinical trials with 199 unique drug compounds.

The data showed that Phase III trial failure proved the biggest obstacle to drug approval, with an overall success rate of only 28%. But in biomarker-guided trials, the success rate reached 62%. The investigators concluded from their data analysis that the use of a CDx assay during Phase III drug development substantially improves a drug’s chances of clinical success.

The Regulatory Perspective

According to Patricia Keegen, M.D., supervisory medical officer in the FDA’s Division of Oncology Products II, the agency requires a companion diagnostic test if a new drug works on a specific genetic or biological target that is present in some, but not all, patients with a certain cancer or disease. The test identifies individuals who would benefit from the treatment, and may identify patients who would not benefit but could also be harmed by use of a certain drug for treatment of their disease. The agency classifies companion diagnosis as Class III devices, a class of devices requiring the most stringent approval for medical devices by the FDA, a Premarket Approval Application (PMA).

On August 6, 2014, the FDA finalized its long-awaited “Guidance for Industry and FDA Staff: In Vitro Companion Diagnostic Devices,” originally issued in July 2011. The final guidance stipulates that FDA generally will not approve any therapeutic product that requires an IVD companion diagnostic device for its safe and effective use before the IVD companion diagnostic device is approved or cleared for that indication.

Close collaboration between drug developers and diagnostics companies has been a key driver in recent simultaneous pharmaceutical-CDx FDA approvals, and partnerships between in vitro diagnostics (IVD) companies have proliferated as a result.  Major test developers include Roche Diagnostics, Abbott Laboratories, Agilent Technologies, QIAGEN), Thermo Fisher Scientific, and Myriad Genetics.

But an NGS-based test has yet to make it to market as a CDx for cancer.  All approved tests include PCR–based tests, immunohistochemistry, and in situ hybridization technology.  And despite the very recent decision by the FDA to grant marketing authorization for Illumina’s MiSeqDx instrument platform for screening and diagnosis of cystic fibrosis, “There still seems to be a number of challenges that must be overcome before we see NGS for targeted cancer drugs,” commented Jan Trøst Jørgensen, a consultant to DAKO, commenting on presentations at the European Symposium of Biopathology in June 2013.

Illumina received premarket clearance from the FDA for its MiSeqDx system, two cystic fibrosis assays, and a library prep kit that enables laboratories to develop their own diagnostic test. The designation marked the first time a next-generation sequencing system received FDA premarket clearance. The FDA reviewed the Illumina MiSeqDx instrument platform through its de novo classification process, a regulatory pathway for some novel low-to-moderate risk medical devices that are not substantially equivalent to an already legally marketed device.

Dr. Jørgensen further noted that “We are slowly moving away from the ‘one biomarker: one drug’ scenario, which has characterized the first decades of targeted cancer drug development, toward a more integrated approach with multiple biomarkers and drugs. This ‘new paradigm’ will likely pave the way for the introduction of multiplexing strategies in the clinic using gene expression arrays and next-generation sequencing.”

The future of CDxs therefore may be heading in the same direction as cancer therapy, aimed at staying ahead of the tumor drug resistance curve, and acknowledging the reality of the shifting genomic landscape of individual tumors. In some cases, NGS will be applied to diseases for which a non-sequencing CDx has already been approved.

Illumina believes that NGS presents an ideal solution to transforming the tumor profiling paradigm from a series of single gene tests to a multi-analyte approach to delivering precision oncology. Mya Thomae, Illumina’s vice president, regulatory affairs, said in a statement that Illumina has formed partnerships with several drug companies to develop a universal next-generation sequencing-based oncology test system. The collaborations with AstraZeneca, Janssen, Sanofi, and Merck-Serono, announced in 2014 and 2015 respectively, seek to  “redefine companion diagnostics for oncology  focused on developing a system for use in targeted therapy clinical trials with a goal of developing and commercializing a multigene panel for therapeutic selection.”

On January 16, 2014 Illumina and Amgen announced that they would collaborate on the development of a next-generation sequencing-based companion diagnostic for colorectal cancer antibody Vectibix (panitumumab). Illumina will develop the companion test on its MiSeqDx instrument.

In 2012, the agency approved Qiagen’s Therascreen KRAS RGQ PCR Kit to identify best responders to Erbitux (cetuximab), another antibody drug in the same class as Vectibix. The label for Vectibix, an EGFR-inhibiting monoclonal antibody, restricts the use of the drug for those metastatic colorectal cancer patients who harbor KRAS mutations or whose KRAS status is unknown.

The U.S. FDA, Illumina said, hasn’t yet approved a companion diagnostic that gauges KRAS mutation status specifically in those considering treatment with Vectibix.  Illumina plans to gain regulatory approval in the U.S. and in Europe for an NGS-based companion test that can identify patients’ RAS mutation status. Illumina and Amgen will validate the test platform and Illumina will commercialize the test.

Treatment Options

Foundation Medicine says its approach to cancer genomic characterization will help physicians reveal the alterations driving the growth of a patient’s cancer and identify targeted treatment options that may not have been otherwise considered.

FoundationOne, the first clinical product from Foundation Medicine, interrogates the entire coding sequence of 315 cancer-related genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers.  Based on current scientific and clinical literature, these genes are known to be somatically altered in solid cancers.

These genes, the company says, are sequenced at great depth to identify the relevant, actionable somatic alterations, including single base pair change, insertions, deletions, copy number alterations, and selected fusions. The resultant fully informative genomic profile complements traditional cancer treatment decision tools and often expands treatment options by matching each patient with targeted therapies and clinical trials relevant to the molecular changes in their tumors.

As Foundation Medicine’ s NGS analyses are increasingly applied, recent clinical reports describe instances in which comprehensive genomic profiling with the FoundationOne NGS-based assay result in diagnostic reclassification that can lead to targeted drug therapy with a resulting dramatic clinical response. In several reported instances, NGS found, among the spectrum of aberrations that occur in tumors, changes unlikely to have been discovered by other means, and clearly outside the range of a conventional CDx that matches one drug to a specific genetic change.

TRK Fusion Cancer

In July 2015, the University of Colorado Cancer Center and Loxo Oncology published a research brief in the online edition of Cancer Discovery describing the first patient with a tropomyosin receptor kinase (TRK) fusion cancer enrolled in a LOXO-101 Phase I trial. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors.

While the authors say TRK fusions occur rarely, they occur in a diverse spectrum of tumor histologies. The research brief described a patient with advanced soft tissue sarcoma widely metastatic to the lungs. The patient’s physician submitted a tumor specimen to Foundation Medicine for comprehensive genomic profiling with FoundationOne Heme, where her cancer was demonstrated to harbor a TRK gene fusion.

Following multiple unsuccessful courses of treatment, the patient was enrolled in the Phase I trial of LOXO-101 in March 2015. After four months of treatment, CT scans demonstrated almost complete tumor disappearance of the largest tumors.

The FDA’s Elizabeth Mansfield, Ph.D., director, personalized medicine staff, Office of In Vitro Diagnostics and Radiological Health, said in a recent article,  “FDA Perspective on Companion Diagnostics: An Evolving Paradigm” that “even as it seems that many questions about co-development have been resolved, the rapid accumulation of new knowledge about tumor biology and the rapid evolution of diagnostic technology are challenging FDA to continually redefine its thinking on companion diagnostics.” It seems almost inevitable that a consolidation of diagnostic testing should take place, to enable a single test or a few tests to garner all the necessary information for therapeutic decision making.”

Whether this means CDx testing will begin to incorporate NGS sequencing remains to be seen.

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Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

The Changing Face of Obesity

Science tells us obesity is a chronic disease. Why does the outmoded and injurious notion that it is a problem of willpower persist?

By Joseph Proietto | November 1, 2015   http://www.the-scientist.com//?articles.view/articleNo/44288/title/The-Changing-Face-of-Obesity/

In Dante Alighieri’s Divine Comedy the narrator meets a man named Ciacco who had been sent to Hell for the “Damning sin of Gluttony.” According to Catholic theology, in order to end up in Hell one must willfully commit a serious sin. So Dante believed that fat people chose to be fat. This antiquated view of the cause of obesity is still widespread, even among medical professionals. The consequences of this misconception are significant, because it forms the basis for the discrimination suffered by the obese; for the wasting of scarce resources in attempts to change lifestyle habits by public education; and for the limited availability of subsidized obesity treatments.

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While obesity is often labeled a lifestyle disease, poor lifestyle choices alone account for only a 6 to 8 kg weight gain. The body has a powerful negative feedback system to prevent excessive weight gain. The strongest inhibitor of hunger, the hormone leptin, is made by fat cells. A period of increased energy intake will result in fat deposition, which will increase leptin production. Leptin suppresses hunger and increases energy expenditure. This slows down weight gain. To become obese, it may be necessary to harbor a genetic difference that makes the individual resistant to the action of leptin.

Evidence from twin and adoption studies suggests that obesity has a genetic basis, and over the past two decades a number of genes associated with obesity have been described. The most common genetic defect in European populations leading to severe obesity is due to mutations in the gene coding for the melanocortin 4 receptor (MCR4). Still, this defect can explain severe obesity in only approximately 6 percent to 7 percent of cases (J Clin Invest, 106:271-79, 2000). Other genes have been discovered that can cause milder increases in weight; for example, variants of just one gene (FTO) can explain up to 3 kg of weight variation between individuals (Science, 316:889-94, 2007).

Genes do not directly cause weight gain. Rather, genes influence the desire for food and the feeling of satiety. In an environment with either poor access to food or access to only low-calorie food, obesity may not develop even in persons with a genetic predisposition. When there is an abundance of food and a sedentary lifestyle, however, an obesity-prone person will experience greater hunger and reduced satiety, increasing caloric intake and weight gain.

Since the 1980s, there has been a rapid rise in the prevalence of obesity worldwide, a trend that likely results from a variety of complex causes. There is increasing evidence, for example, that the development of obesity on individual or familial levels may be influenced by environmental experiences that occur in early life. For example, if a mother is malnourished during early pregnancy, this results in epigenetic changes to genes involved in the set points for hunger and satiety in the developing child. These changes may then become fixed, resulting in a tendency towards obesity in the offspring.

The biological basis of obesity is further highlighted by the vigorous defense of weight following weight loss. There are at least 10 circulating hormones that modulate hunger. Of these, only one has been confirmed as a hunger-inducing hormone (ghrelin), and it is made and released by the stomach. In contrast, nine hormones suppress hunger, including CCK, PYY, GLP-1, oxyntomodulin, and uroguanylin from the small bowel; leptin from fat cells; and insulin, amylin, and pancreatic polypeptide from the pancreas.

 

After weight loss, regardless of the diet employed, there are changes in circulating hormones involved in the regulation of body weight. Ghrelin levels tend to increase and levels of multiple appetite-suppressing hormones decrease. There is also a subjective increase in appetite. Researchers have shown that even after three years, these hormonal changes persist (NEJM, 365:1597-604, 2011; Lancet Diabetes and Endocrinology, 2:954-62, 2014). This explains why there is a high rate of weight regain after diet-induced weight loss.

Given that the physiological responses to weight loss predispose people to regain that weight, obesity must be considered a chronic disease. Data show that those who successfully maintain their weight after weight loss do so by remaining vigilant and constantly applying techniques to oppose weight regain. These techniques may involve strict diet and exercise practices and/or pharmacotherapy.

It is imperative for society to move away from a view that obesity is simply a lifestyle issue and to accept that it is a chronic disease. Such a change would not only relieve the stigma of obesity but would also empower politicians, scientists and clinicians to tackle the problem more effectively.

Joseph Proietto was the inaugural Sir Edward Dunlop Medical Research Foundation Professor of Medicine in the Department of Medicine, Austin Health at the University of Melbourne in Australia. He is a researcher and clinician investigating and treating obesity and type 2 diabetes.

 

 

A Weighty Anomaly

Why do some obese people actually experience health benefits?

By Jyoti Madhusoodanan | November 1, 2015     http://www.the-scientist.com//?articles.view/articleNo/44304/title/A-Weighty-Anomaly/

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THE ENDOCRINE THEORY: Some researchers have posited that fat cells may secrete molecules that affect glucose homeostasis in muscle or liver tissue.COURTESY OF MITCHELL LAZAR

In the early 19th century, Belgian mathematician Adolphe Quetelet was obsessed with a shape: the bell curve. While helping with a population census, Quetelet proposed that the spread of human traits such as height and weight followed this trend, also known as a Gaussian or normal distribution. On a quest to define a “normal man,” he showed that human height and weight data fell along his beloved bell curves, and in 1823 devised the “Quetelet Index”—more familiar to us today as the BMI, or body mass index, a ratio of weight to height.

Nearly two centuries later, clinicians, researchers, and fitness instructors continue to rely on this metric to pigeonhole people into categories: underweight, healthy, overweight, or obese. But Quetelet never intended the metric to serve as a way to define obesity. And now, a growing body of evidence suggests these categories fail to accurately reflect the health risks—or benefits—of being overweight.

Although there is considerable debate surrounding the prevalence of metabolically healthy obesity, when obesity is defined in terms of BMI (a BMI of 30 or higher), estimates suggest that about 10 percent of adults in the U.S. are obese yet metabolically healthy, while as many as 80 percent of those with a normal BMI may be metabolically unhealthy, with signs of insulin resistance and poor circulating lipid levels, even if they suffer no obvious ill effects. “If all we know about a person is that they have a certain body weight at a certain height, that’s not enough information to know their health risks from obesity,” says health-science researcher Paul McAuley of Winston-Salem State University. “We need better indicators of metabolic health.”

The dangers of being overweight, such as a higher risk of heart disease, type 2 diabetes, and other complications, are well known. But some obese individuals—dubbed the “fat fit”—appear to fare better on many measures of health when they’re heavier. Studies have found lower mortality rates, better response to hemodialysis in chronic kidney disease, and lower incidence of dementia in such people. Mortality, it’s been found, correlates with obesity in a U-shaped curve (J Sports Sci, 29:773-82, 2011). So does extra heft help or hurt?

To answer that question, researchers are trying to elucidate the metabolic reasons for this obesity paradox.

In a recent study, Harvard University epidemiologist Goodarz Danaei and his colleagues analyzed data from nine studies involving a total of more than 58,000 participants to tease apart how obesity and other well-known metabolic risk factors influence the risk of coronary heart disease. Controlling these other risk factors, such as hypertension or high cholesterol, with medication is simpler than curbing obesity itself, Danaei explains. “If you control a person’s obesity you get rid of some health risks, but if you control hypertension or diabetes, that also reduces health risks, and you can do the latter much more easily right now.”

Danaei’s team assessed BMI and metabolic markers such as systolic blood pressure, total serum cholesterol, and fasting blood glucose. The three metabolic markers only explained half of the increased risk of heart disease across all study participants. In obese individuals, the other half appeared to be mediated by fat itself, perhaps via inflammatory markers or other indirect mechanisms (Epidemiology, 26:153-62, 2015). While Danaei’s study was aimed at understanding how obesity hurts health, the results also uncovered unknown mechanisms by which excess adipose tissue might exert its effects. This particular study revealed obesity’s negative effects, but might these unknown mechanisms hold clues that explain the obesity paradox?

Other researchers have suggested additional possibilities—for example, that inflammatory markers such as TNF-α help combat conditions such as chronic kidney disease, or that obesity makes a body more capable of making changes to, and tolerating changes in, blood flow depending on systemic needs (Am J Clin Nutr, 81:543-54, 2005).

According to endocrinologist Mitchell Lazar at the University of Pennsylvania, the key to explaining the obesity paradox may be two nonexclusive ways fat tissue is hypothesized to function. One mechanism, termed the endocrine theory, suggests that fat cells secrete, or don’t secrete enough of, certain molecules that influence glucose homeostasis in other tissues, such as muscle or liver. The first such hormone to be discovered was leptin; later studies reported several other adipocyte-secreted factors, including adiponectin, resistin, and various cytokines.

The other hypothesis, dubbed the spillover theory, suggests that storing lipids in fat cells has some pluses. Adipose tissue might sequester fat-soluble endotoxins, and produce lipoproteins that can bind to and clear harmful lipids from circulation. When fat cells fill up, however, these endotoxins are stashed in the liver, pancreas, or other organs—and that’s when trouble begins. In “fat fit” people, problems typically linked to obesity such as high cholesterol or diabetes may be avoided simply because their adipocytes mop up more endotoxins.

“In this model, one could imagine that if you could store even more fat in fat cells, you could be even more obese, but you might be protected from problems [associated with] obesity because you’re protecting the other tissues from filling up with lipids that cause problems,” says Lazar. “This may be the most popular current model to explain the fat fit.”

Although obesity greatly increases the risk of type 2 diabetes—up to 93-fold in postmenopausal women, for example—not all obese people suffer from the condition. Similarly, a certain subtype of individuals with “normal” BMIs are at greater risk of developing insulin resistance and type 2 diabetes than others with BMIs in the same range. Precisely what distinguishes these two cohorts is still unclear. “Just as important as explaining why some obese people don’t get diabetes is to explain why other subgroups—normal-weight people or those with lipodystrophy—sometimes get it,” Lazar says. “If there are multiple subtypes of obesity and diabetes, can we figure out genetic aspects or biomarkers that cause one of these phenotypes and not the other?”

To Lazar, McAuley, and other researchers, it’s increasingly evident that BMI may not be that metric. Finding better ways to assess a healthy weight, however, has proven challenging. Researchers have tested measures, such as the body shape index (ABSI) or the waist-hip ratio, which attempt to gauge visceral fat—considered to be more metabolically harmful than fat in other body locations. However, these metrics have yet to be implemented widely in clinics, and few are as simple to understand as the BMI (Science, 341:856-58, 2013).

Independent of metrics, however, the health message regarding weight is still unanimous: exercise and healthy dietary choices benefit everyone. “At a certain point, despite all the so-called fit-fat people, the demographics say that there’s a huge risk of diabetes and heart disease at very high BMI,” notes Lazar. “We can’t assume we’ll be one of the lucky ones who will have a BMI in the obese category but will still be protected from heart disease.”

Correction (November 2): The original version of this article misattributed the pull quote above. The attribution for this quote has been corrected, and The Scientist regrets the error.

 

 

THE HEALTH RISK OF OBESITY—BETTER METRICS IMPERATIVE

 Science 23 Aug 2013;  341(6148): 856858     DOI: http://dx.doi.org:/10.1126/science.1241244
Obesity paradoxes.
In this review, we examine the original obesity paradox phenomenon (i.e. in cardiovascular disease populations, obese patients survive better), as well as three other related paradoxes (pre-obesity, “fat but fit” theory, and “healthy” obesity). An obesity paradox has been reported in a range of cardiovascular and non-cardiovascular conditions. Pre-obesity (defined as a body mass index of 25.0-29.9 kg · m⁻²) presents another paradox. Whereas “overweight” implies increased risk, it is in fact associated with decreased mortality risk compared with normal weight. Another paradox concerns the observation than when fitness is taken into account, the mortality risk associated with obesity is offset. The final paradox under consideration is the presence of a sizeable subset of obese individuals who are otherwise healthy. Consequently, a large segment of the overweight and obese population is not at increased risk for premature death. It appears therefore that low cardiorespiratory fitness and inactivity are a greater health threat than obesity, suggesting that more emphasis should be placed on increasing leisure time physical activity and cardiorespiratory fitness as the main strategy for reducing mortality risk in the broad population of overweight and obese adults.
Obesity, insulin resistance, and cardiovascular disease.
Recent Prog Horm Res. 2004;59:207-23.
The ability of insulin to stimulate glucose disposal varies more than six-fold in apparently healthy individuals. The one third of the population that is most insulin resistant is at greatly increased risk to develop cardiovascular disease (CVD), type 2 diabetes, hypertension, stroke, nonalcoholic fatty liver disease, polycystic ovary disease, and certain forms of cancer. Between 25-35% of the variability in insulin action is related to being overweight. The importance of the adverse effects of excess adiposity is apparent in light of the evidence that more than half of the adult population in the United States is classified as being overweight/obese, as defined by a body mass index greater than 25.0 kg/m(2). The current epidemic of overweight/obesity is most-likely related to a combination of increased caloric intake and decreased energy expenditure. In either instance, the fact that CVD risk is increased as individuals gain weight emphasizes the gravity of the health care dilemma posed by the explosive increase in the prevalence of overweight/obesity in the population at large. Given the enormity of the problem, it is necessary to differentiate between the CVD risk related to obesity per se, as distinct from the fact that the prevalence of insulin resistance and compensatory hyperinsulinemia are increased in overweight/obese individuals. Although the majority of individuals in the general population that can be considered insulin resistant are also overweight/obese, not all overweight/obese persons are insulin resistant. Furthermore, the cluster of abnormalities associated with insulin resistance – namely, glucose intolerance, hyperinsulinemia, dyslipidemia, and elevated plasma C-reactive protein concentrations — is limited to the subset of overweight/obese individuals that are also insulin resistant. Of greater clinical relevance is the fact that significant improvement in these metabolic abnormalities following weight loss is seen only in the subset of overweight/obese individuals that are also insulin resistant. In view of the large number of overweight/obese subjects at potential risk to be insulin resistant/hyperinsulinemic (and at increased CVD risk), and the difficulty in achieving weight loss, it seems essential to identify those overweight/obese individuals who are also insulin resistant and will benefit the most from weight loss, then target this population for the most-intensive efforts to bring about weight loss.
Long-Term Persistence of Hormonal Adaptations to Weight Loss

Priya Sumithran, Luke A. Prendergast, Elizabeth Delbridge, Katrina Purcell, Arthur Shulkes, Adamandia Kriketos, and Joseph Proietto

N Engl J Med 2011; 365:1597-1604   October 27, 2011http://dx.doi.org:/10.1056/NEJMoa1105816

After weight loss, changes in the circulating levels of several peripheral hormones involved in the homeostatic regulation of body weight occur. Whether these changes are transient or persist over time may be important for an understanding of the reasons behind the high rate of weight regain after diet-induced weight loss.

Weight loss (mean [±SE], 13.5±0.5 kg) led to significant reductions in levels of leptin, peptide YY, cholecystokinin, insulin (P<0.001 for all comparisons), and amylin (P=0.002) and to increases in levels of ghrelin (P<0.001), gastric inhibitory polypeptide (P=0.004), and pancreatic polypeptide (P=0.008). There was also a significant increase in subjective appetite (P<0.001). One year after the initial weight loss, there were still significant differences from baseline in the mean levels of leptin (P<0.001), peptide YY (P<0.001), cholecystokinin (P=0.04), insulin (P=0.01), ghrelin (P<0.001), gastric inhibitory polypeptide (P<0.001), and pancreatic polypeptide (P=0.002), as well as hunger (P<0.001).

What’s new in endocrinology and diabetes mellitus

Large genome wide association studies have demonstrated that variants in the FTO gene have the strongest association with obesity risk in the general population, but the mechanism of the association has been unclear. However, a nonocoding causal variant in FTO has now been identified that changes the function of adipocytes from energy utilization (beige fat) to energy storage (white fat) with a fivefold decrease in mitochondrial thermogenesis [17]. When the effect of the variant was blocked in genetically engineered mice, thermogenesis increased and weight gain did not occur, despite eating a high-fat diet. Blocking the gene’s effect in human adipocytes also increased energy utilization. This observation has important implications for potential new anti-obesity drugs. (See “Pathogenesis of obesity”, section on ‘FTO variants’.)

Liraglutide for the treatment of obesity (July 2015)

Along with diet, exercise, and behavior modification, drug therapy may be a helpful component of treatment for select patients who are overweight or obese. Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist, used for the treatment of type 2 diabetes, and can promote weight loss in patients with diabetes, as well as those without diabetes.

In a randomized trial in nondiabetic patients who had a body mass index (BMI) of ≥30 kg/m2 or ≥27 kg/m2 with dyslipidemia and/or hypertension, liraglutide 3 mg once daily, compared with placebo, resulted in greater mean weight loss (-8.0 versus -2.6 kg with placebo) [18]. In addition, cardiometabolic risk factors, glycated hemoglobin (A1C), and quality of life improved modestly. Gastrointestinal side effects transiently affected at least 40 percent of the liraglutide group and were the most common reason for withdrawal (6.4 percent). Liraglutide is an option for select overweight or obese patients, although gastrointestinal side effects (nausea, vomiting) and the need for a daily injection may limit the use of this drug. (See “Obesity in adults: Drug therapy”, section on ‘Liraglutide’.)

In a trial designed specifically to evaluate the effect of liraglutide on weight loss in overweight or obese patients with type 2 diabetes (mean weight 106 kg), liraglutide, compared with placebo, resulted in greater mean weight loss (-6.4 kg and -5.0 kg for liraglutide 3 mg and 1.8 mg, respectively, versus -2.2 kg for placebo) [19]. Treatment with liraglutide was associated with better glycemic control, a reduction in the use of oral hypoglycemic agents, and a reduction in systolic blood pressure. Although liraglutide is not considered as initial therapy for the majority of patients with type 2 diabetes, it is an option for select overweight or obese patients with type 2 diabetes who fail initial therapy with lifestyle intervention and metformin.  (See “Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes mellitus”, section on ‘Weight loss’.)

The Skinny on Fat Cells

Bruce Spiegelman has spent his career at the forefront of adipocyte differentiation and metabolism.

By Anna Azvolinsky | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44312/title/The-Skinny-on-Fat-Cells/

Bruce Spiegelman
Stanley J. Korsmeyer Professor of Cell Biology
and Medicine
Harvard Medical School
Director, Center for Energy Metabolism
and Chronic
Disease, Dana-Farber Cancer Institute, Boston

It’s hard to know whether you have the right stuff to be a scientist, but I had a passion for the research,” says Bruce Spiegelman, professor of cell biology at Harvard Medical School and the Dana-Farber Cancer Institute. After receiving his PhD in biochemistry from Princeton University in 1978, Spiegelman sent an application to do postdoctoral research to just one lab. “I wasn’t thinking I should apply to five different labs. I just marched forward more or less in a straight line,” he says. Spiegelman did know that he had no financial backup and depended on research fellowships throughout the early phase of his science career. “I thought it was fantastic, and still think so, that a PhD in science is supported by the government. I certainly appreciated that, because many of my friends in the humanities had to support themselves by cobbling together fellowships and teaching every semester, whereas we didn’t face similar challenges in the sciences.”

Since his graduate student days, Spiegelman has realized his potential, pioneering the study of adipose tissue biology and metabolism. He was introduced to the field in Howard Green’s laboratory, then at MIT, where Spiegelman began his one and only postdoc in 1978. Green had recently developed a system for culturing adipose cells and asked Spiegelman if he wanted to study fat cell differentiation. “I knew nothing about adipose tissue, but I was really interested in any model of how one cell switches to another. Whether skin or fat didn’t matter too much to me, because I was not coming at this from the perspective of physiology but from the perspective of how do these switches work at a molecular level?”

Spiegelman has stuck with studying the biology and differentiation of fat cells for more than 30 years. While looking for the master transcriptional regulator of fat development—which his laboratory found in 1994—Spiegelman’s group also discovered one of the first examples of a nuclear oncogene that functions as a transcription factor, and, more recently, the team found that brown fat and white fat come from completely different origins and that brown and beige fat are distinct cell types. Spiegelman was also the first to provide evidence for the connection between inflammation, insulin resistance, and fat tissue.

Here, Spiegelman talks about his strong affinity for the East Coast, his laboratory’s search for molecules that can crank up brown fat production and activity, and the culture of his laboratory’s weekly meeting.

Spiegelman Sets Out

First publication. Spiegelman grew up in Massapequa, New York, a town on Long Island. “Birds, insects, fish, and animals were fascinating to me. As a kid, I imagined I would be a wildlife ranger,” he says. Spiegelman and his brother were the first in their family to attend college; Spiegelman entered the College of William and Mary in 1970 thinking he would major in psychology. But before taking his first psychology course, he had to take a biology course, really loved it, and switched his major. For his senior thesis, he chose one of the few labs that did biochemistry-related research. He studied cultures of the filamentous fungus Aspergillus ornatus in which he induced the upregulation of a metabolic enzyme. Spiegelman applied a calculus transformation that related the age of the culture to the age of individual cells, something that had not been previously done. The work earned him his first first-author publication in 1975. “It was not a great breakthrough, but I think it showed that I was maybe applying myself more than the typical undergraduate.”

Full steam ahead. “My interest in laboratory research was intense. Even though it was not particularly inspired work, the first-author publication in a college where not many of the professors published a lot gave me a lot of confidence. It was probably out of proportion to the quality of the actual work.” That confidence and Spiegelman’s interest in the chemistry of living things led him to pursue a PhD in biochemistry at Princeton University. “Very early on, I felt that I couldn’t understand biology if it didn’t go to the molecular level. To me, just describing how an animal lived without understanding how it worked was very unsatisfying. I think it was one of the best decisions that I made in my life, to do a PhD in biochemistry,” he says, “because if you really want to understand living systems, you are very limited in how you can understand them without having a strong background in biochemistry because these are, essentially, chemical systems.”

Embracing molecular biology. Spiegelman initially joined Arthur Pardee’s laboratory, but switched when Pardee left Princeton for Harvard University in 1975. Because he was already collaborating with Marc Kirschner, a cell biologist and biochemist who studies the regulation of the cell cycle and how the cytoskeleton works, it was an easy transition to transfer to the new laboratory. In Kirschner’s group, Spiegelman became the cell biologist among many protein biochemists working on microtubule assembly in vitro. Rather than understanding how the proteins fit together to form the filamentous structures, Spiegelman wanted to understand what controlled their assembly inside cells. Working in mammalian cells, Spiegelman published three consecutive Cell papers on how microtubule assembly occurs in vivo. The firstpaper, from 1977, demonstrated that a nucleotide functions to stabilize the tubulin molecule rather than to regulate tubulin assembly in vivo.

Spiegelman Simmers

A new tool. For his next move, Spiegelman wanted to marry his background in biochemistry and molecular biology with a good cellular model system. He became interested in differentiation at the end of his PhD, while studying how the cytoskeleton is reorganized during neural differentiation, and settled on Green’s MIT laboratory for his postdoc. Green had developed a way to study both skin and fat cell differentiation. Again, Spiegelman was the odd man out, working on the molecular biology of fat cell differentiation while most of the graduate students and postdocs focused on the cellular biology of skin cell differentiation. While there, Spiegelman learned how to clone cDNA—a new method that some researchers thought was just another new fad, he says. “I thought it was pretty obvious that this was a tool that would be a game changer. I could see how I could clone some of the cDNAs and genes that were regulated in the fat cell lineage and then try to understand the regulation of these genes.”

Setting the stage. Spiegelman demonstrated that cAMP regulates the synthesis of certain enzymes in fat cells during differentiation. But while this was the most influential paper from his postdoc, says Spiegelman, it was his demonstration of cloning mRNAs from adipocytes, published in 1983, that set the stage for cloning fat-selective genes. The work, mostly done when Spiegelman was already a new faculty member at the Dana-Farber Cancer Institute, stemmed from his learning molecular cloning in Phillip Sharp’s lab at MIT and Bryan Roberts’s lab at Harvard. “This was the raw material from which we eventually cloned PPARγ and showed it to be the master regulator of fat [cell] development.”

Roots. Spiegelman became an assistant professor at the Harvard Medical School in 1982, when he was not yet 30. Although he had entertained the idea of moving to the West Coast with his wife, whom he had met at Princeton where she obtained a PhD in French literature, Spiegelman says he is really an East Coaster at heart. “My wife and I came to love Boston and were very comfortable there. Our families were both in New York, which was close, but not too close, and we really enjoyed the culture and pace of Boston; it was more ‘us.’ We really liked to visit California but didn’t particularly want to move there. We’re both real Northeastern people.”

Relating to Sisyphus. The transition from doing a postdoc to setting up his own laboratory was “very exciting and terribly stressful,” says Spiegelman. “When I think back, I always tried to be professional with my laboratory, but I was so stressed at suddenly being on my own with no management training.” The people resources he had encountered in his graduate and postdoctoral training labs were also not there yet, and he says his first publication as a principal investigator was like pushing a rock up a hill. But eventually, Spiegelman’s lab built a reputation and reached a critical mass of talented people who advanced the science. Again in 1983, Spiegelman produced a publication showing that morphological manipulation can affect gene expression and adipose differentiation.

End goal. Spiegelman’s goal was to find a master molecule that  orchestrates the conversion of adipocyte precursor cells into bona fide fat cells. Piece by piece, his lab identified the enhancers, promoters, and other regulatory elements involved in adipocyte differentiation. In 1994, graduate student Peter Tontonoz finallyfound that the PPARγ gene, inserted via a retroviral vector into fibroblasts, could induce the cells to become adipose cells. “It took 10 years,” Spiegelman says. Along the way, the laboratory found that c-fos, the product of a famous nuclear oncogene, bound to the promoters of fat-specific genes and worked as a transcription factor. “It was not really known how nuclear oncogenes worked. This was one of the first papers showing that these oncogenes bound to gene promoters and were transcription factors.”

A wider scope. In 1993, graduate student Gökhan Hotamisligil found that tumor necrosis factor-alpha(TNF-α), is induced in the fat tissue of rodent models of obesity and diabetes. The paper sparked the formation of the field of immunometabolism and resulted in the expansion of Spiegelman’s lab into the physiology arena, partly thanks to the guidance of C. Ronald Kahn and Jeff Flier, who both study metabolism and diabetes. But the work initially encountered pushback, says Spiegelman, partly because it was the merging of two fields.

Spiegelman Scales Up

Fat color palette. Brown fat tissue, abundant in infants but scarce in adults, is a metabolically active form of fat that is chock full of mitochondria and is found in pockets in the body distinct from white fat tissue.Pere Puigserver, then a postdoc in Spiegelman’s lab, found that the coactivator PCG-1, binding to PPARγ and other nuclear receptors, could stimulate mitochondrial biogenesis. The PCG-1 gene is turned on by stimuli such as exercise or a cold environment. Later, postdoc Patrick Seale, Spiegelman, and their colleagues showed brown fat cells derive from the same lineage that gives rise to skeletal muscle. “This was a big surprise, maybe the biggest surprise we ever uncovered in the lab,” says Spiegelman.

A paler shade of brown. More recently, in 2012, Spiegelman’s laboratory showed that within adult white adipose tissue, there are pockets of a yet another type of fat tissue that he called beige fat. “I think the evidence is very good from rodents that if you activate brown and beige fat, you get metabolic benefit both in obesity and diabetes. So the question now is: Can that be done in humans in a way that’s beneficial and not toxic?”  The lab is now looking to identify molecules that can either ramp up the activity of brown and beige fat or increase the production of both cell types as possible therapeutics for metabolic disorders or even cancer-associated cachexia. “Anyone who says that either approach will work better is being foolish. We just don’t know enough to go after just one or the other.”

On the irisin controversy. After reporting in 2012 that a muscle-related hormone called irisin could switch white fat to metabolically active brown fat, Spiegelman became embroiled in a media-covered debate about whether the molecule really exists; he was also the victim of a potential fraud plot. Most recently, Spiegelman provided thorough evidence that irisin does in fact exist. On the controversy, he says it’s a fine line between defending his scientific integrity and not adding more fuel to the fire or engaging with his harassers. “We have a long track record of doing credible and reproducible science and it was not that complicated to address the paper that claimed irisin was ‘a myth.’ That study used very outmoded scientific approaches.”

Raw talent. Many of Spiegelman’s trainees have gone on to become very successful scientists, including Tontonoz, Hotamisligil, Evan Rosen, and Randy Johnson. “It’s a quantum change in the experience of doing science when you get people who have their own visions. I would have thought that interacting with smart people would mainly help me get my scientific vision accomplished. And that was partly true, but also it changed my vision. When you have people challenging you on a day-to-day basis, you learn from them through the questions they ask and the way they challenge you in a constructive way. They made me a much better scientist.”

Rigorous mentorship.  “I feel very passionately that a major part of my job is to prepare the next generation of scientists. Everyone who comes through my lab will tell you that I take that very seriously. We make sure my students give a lot of talks and get critical assessments of their presentations to our lab group. I am very hands-on both scientifically and in developing the way students project their vision. I had a very good mentor, Marc Kirschner, and I’d like to think that I learned how to be a mentor from him. I want to make sure that when people walk out of my lab they are prepared to run independent research programs.”

Greatest Hits

  • Identified the master regulator of adipogenesis, the nuclear receptor PPARγ
  • Was the first to show that a nuclear oncogene, c-fos, codes for a transcription factor that binds to the promoters of genes
  • Demonstrated that adipose tissue synthesizes tumor necrosis factor-alpha (TNF-α), providing the first direct link between obesity, inflammation, insulin resistance, and fat tissue.
  • Showed that brown fat cells are not developmentally related to white fat
  • Identified beige fat as a distinct cell type, different from either white or brown fat

 

Fanning the Flames

Obesity triggers a fatty acid synthesis pathway, which in turn helps drive T cell differentiation and inflammation.

By Kate Yandell | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44306/title/Fanning-the-Flames/

EDITOR’S CHOICE IN IMMUNOLOGY

The paper
Y. Endo et al., “Obesity drives Th17 cell differentiation by inducing the lipid metabolic kinase, ACC1,” Cell Reports, 12:1042-55, 2015.

Cell Rep. 2015 Aug 11;12(6):1042-55.   http://dx.doi.org:/10.1016/j.celrep.2015.07.014. Epub 2015 Jul 30.
Obesity Drives Th17 Cell Differentiation by Inducing the Lipid Metabolic Kinase, ACC1.
  • A high-fat diet augments Th17 cell development and the expression of Acaca
  • ACC1 controls Th17 cell development in vitro and Th17 cell pathogenicity in vivo
  • ACC1 modulates RORγt function in developing Th17 cells
  • Obesity in humans induces ACACA and IL-17A expression in CD4 T cells

Chronic inflammation due to obesity contributes to the development of metabolic diseases, autoimmune diseases, and cancer. Reciprocal interactions between metabolic systems and immune cells have pivotal roles in the pathogenesis of obesity-associated diseases, although the mechanisms regulating obesity-associated inflammatory diseases are still unclear. In the present study, we performed transcriptional profiling of memory phenotype CD4 T cells in high-fat-fed mice and identified acetyl-CoA carboxylase 1 (ACC1, the gene product of Acaca) as an essential regulator of Th17 cell differentiation in vitro and of the pathogenicity of Th17 cells in vivo. ACC1 modulates the DNA binding of RORγt to target genes in differentiating Th17 cells. In addition, we found a strong correlation between IL-17A-producing CD45RO(+)CD4 T cells and the expression of ACACA in obese subjects. Thus, ACC1 confers the appropriate function of RORγt through fatty acid synthesis and regulates the obesity-related pathology of Th17 cells.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2035221719/2050630604/fx1.jpg

 

 

http://www.the-scientist.com/November2015/NovMediLit_310px.jpg

FEEDING INFLAMMATION: When mice eat a diet high in fat, their CD4 T cells show increased expression of the fatty acid biosynthesis gene Acaca, which encodes the enzyme ACC1 (1). Products of the ACC1 fatty acid synthesis pathway encourage the transcription factor RORγt to bind near the gene encoding the cytokine IL-17A (2). There, RORγt recruits an enzyme called p300 to modify the genome epigenetically and turn on IL-17A. The memory T cells then differentiate into inflammatory T helper 17 cells.
See full infographic: PDF
© STEVE GRAEPEL

Obesity often comes with a side of chronic inflammation, causing inflammatory chemicals and immune cells to flood adipose tissue, the hypothalamus, the liver, and other areas of the body. Inflammation is a big part of what makes obesity such an unhealthy condition, contributing to Type 2 diabetes, heart disease, cancers, autoimmune disorders, and possibly even neurodegenerative diseases.

To better understand the relationship between obesity and inflammation, Toshinori Nakayama, Yusuke Endo, and their colleagues at Chiba University in Japan started with what often leads to obesity: a high-fat diet. They fed mice rich meals for a couple of months and looked at how gene expression in the animals’ T cells compared to gene expression in the T cells of mice fed a normal diet. Most notably, they found increased expression ofAcaca, a gene that codes for a fatty acid synthesis enzyme called acetyl coA carboxylase 1 (ACC1). They went on to show that the resulting increase in fatty acid levels pushed CD4 T cells to differentiate into inflammatory T helper 17 (Th17) cells.

Th17 cells help fight off invading fungi and some bacteria. But these immune cells can also spin out of control in autoimmune diseases such as multiple sclerosis. Nakayama’s team showed that either blocking ACC1 activity with a drug called TOFA or deleting a key portion of Acaca in mouse CD4 T cells reduced the generation of pathologic Th17 cells. Overexpressing Acaca increased Th17-cell generation.

The researchers also demonstrated that mice fed a high-fat diet had elevated susceptibility to a multiple sclerosis–like disease, and that TOFA reduced the symptoms.

“This is a very intriguing finding, suggesting not only that obesity can directly induce Th17 differentiation but also indicating that pharmacologic targeting of fatty acid synthesis may help to interfere with obesity-associated inflammation,” Tim Sparwasser of the Twincore Center for Experimental and Clinical Infection Research in Hannover, Germany, says in an email. Sparwasser and his colleagues had previously shown that ACC1 is required for the differentiation of Th17 cells in mice and humans.

Nakayama explains that CD4 T cells must undergo profound metabolic changes as they mature and differentiate. “The intracellular metabolites, including fatty acids, are essential for cell proliferation and cell growth,” he says in an email. When fatty acid levels in T cells increase, the cells are activated and begin to proliferate.

“It’s a nice illustration of how, really, immune response is so highly connected to the metabolic state of the cell,” says Gökhan S. Hotamisligil of Harvard University’s T.H. Chan School of Public Health who was not involved in the study. “The immune system launches its responses commensurate with the sources of nutrients and energy from the environment,” he adds in an email.

There are still missing pieces in the path from high-fat diet to increased Acaca expression to ACC1’s influence on T-cell differentiation. It also remains to be seen how this plays out in obese humans, although Nakayama and colleagues did show that inhibiting ACC1 reduced pathologic Th17 generation in human immune cell cultures, and that the T cells of obese humans contain elevated levels of ACC1 and show signs of increased differentiation into Th17 cells.

 

The prevalence of obesity has been increasing worldwide, and obesity is now a major public health problem in most developed countries (Gregor and Hotamisligil, 2011, Ng et al., 2014). Obesity-induced inflammation contributes to the development of various chronic diseases, such as autoimmune diseases, metabolic diseases, and cancer (Kanneganti and Dixit, 2012, Kim et al., 2014,Osborn and Olefsky, 2012, Winer et al., 2009a). A number of studies have pointed out the importance of reciprocal interactions between metabolic systems and immune cells in the pathogenesis of obesity-associated diseases (Kaminski and Randall, 2010, Kanneganti and Dixit, 2012, Kim et al., 2014, Mauer et al., 2014, Stienstra et al., 2012, Winer et al., 2011).

Elucidating the molecular mechanisms by which naive CD4 T cells differentiate into effector T cells is crucial for understanding helper T (Th) cell-mediated immune pathogenicity. After antigen stimulation, naive CD4 T cells differentiate into at least four distinct Th cell subsets: Th1, Th2, Th17, and inducible regulatory T (iTreg) cells (O’Shea and Paul, 2010, Reiner, 2007). Several specific master transcription factors that regulate Th1/Th2/Th17/iTreg cell differentiation have been identified, including T-bet for Th1 (Szabo et al., 2000), GATA3 (Yamashita et al., 2004, Zheng and Flavell, 1997) for Th2, retinoic-acid-receptor-related orphan receptor γt (RORγt) for Th17 (Ivanov et al., 2006), and forkhead box protein 3 (Foxp3) for iTreg (Sakaguchi et al., 2008). The appropriate expression and function of these transcription factors is essential for proper immune regulation by each Th cell subset.

Among these Th cell subsets, Th17 cells contribute to the host defense against fungi and extracellular bacteria (Milner et al., 2008). However, the pathogenicity of IL-17-producing T cells has been recognized in various autoimmune diseases, including multiple sclerosis, psoriasis, inflammatory bowel diseases, and steroid-resistant asthma (Bettelli et al., 2006, Coccia et al., 2012, Ivanov et al., 2006,Leonardi et al., 2012, McGeachy and Cua, 2008, Nylander and Hafler, 2012,Stockinger et al., 2007, Sundrud et al., 2009).

An HFD Promotes Th17 Cell Differentiation and Affects the Expression of Fatty Acid Enzymes in Memory CD4 T Cells In Vivo

Inhibition of ACC1 Function Results in Decreased Th17 Cell Differentiation and Ameliorates the Development of Autoimmune Disease

ACC1 Controls the Differentiation of Th17 Cells Both In Vitro and In Vivo

ACC1 Controls the Function, but Not Expression, of RORγt in Differentiating Th17 Cells

Extrinsic Fatty Acid Supplementation Restored Acaca−/− Th17 Cell Differentiation through the Functional Improvement of RORγt

Obese Subjects Show Upregulation of ACACA and Increased Th17 Cells in CD45RO+ Memory CD4 T Cells

We herein identified a critical role that ACC1 plays in Th17 cell differentiation and the pathogenicity of Th17 cells through the control of the RORγt function under obese circumstances. High-fat-induced obesity augments Th17 cell differentiation and the expression of enzymes involved in fatty acid metabolism, including ACC1. Pharmacological inhibition or genetic deletion of ACC1 resulted in impaired Th17 cell differentiation in both mice and humans. In contrast, overexpression of Acaca induced Th17 cells in vivo, leaving the expression ofIfng and Il4 largely unchanged. ACC1 modulated the binding of RORγt to theIl17a gene and the subsequent p300 recruitment in differentiating Th17 cells. Memory CD4 T cells from peripheral blood mononuclear cells (PBMCs) of obese subjects showed increased IL-17A production and ACACA expression. Furthermore, a strong correlation was detected between the proportion of IL-17A-producing cells and the expression level of ACACA in memory CD4 T cells in obese subjects. Thus, our findings provide evidence of a mechanism wherein obesity can exacerbate IL-17-mediated pathology via the induction of ACC1.

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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Vitamin D debates

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Vitamin D: Time for Rational Decision-Making

JoAnn E. Manson, MD, DrPH

Hello. This is Dr JoAnn Manson, professor of medicine at Harvard Medical School and Brigham and Women’s Hospital. I would like to talk with you about the vitamin D dilemma. The question of whether to screen routinely for vitamin D deficiency or to recommend high-dose vitamin D supplementation for our patients continues to be one of the most perplexing and vexing issues in clinical practice, and many clinicians are seeking guidance on these issues.

There appears to be a growing disconnect between the observational studies and the randomized clinical trials of vitamin D. For example, the observational studies are showing a fairly consistent relationship between low blood levels of vitamin D and an increased risk for heart disease, cancer, diabetes, and many other chronic diseases. Yet, the randomized clinical trials of vitamin D supplementation to date have been generally disappointing. This includes several randomized trials published over the past few months, including a meta-analysis[1] of randomized trials of vitamin D supplementation showing minimal, if any, benefit in terms of lowering blood pressure; a trial[2] of high-dose vitamin D supplementation showing no clear benefit for muscle strength, bone mineral density, or even the risk for falls; and, most recently, a randomized trial[3] of vitamin D supplementation with and without calcium showing no clear benefit in reducing the risk for colorectal adenomas. The latter trial was very recently published in the New England Journal of Medicine.

The Institute of Medicine (IOM)[4] and the US Preventive Services Task Force[5] do not endorse routine universal screening for vitamin D deficiency. They also recommend more moderate intakes [of vitamin D]. For example, the IOM recommends 600-800 IU a day for adults and also recommends avoiding daily intakes above 4000 IU, which has been set as the tolerable upper intake level.

However, it is important to keep in mind that these are public health population guidelines for a generally healthy population, and they by no means preclude individual decision-making by the clinician in the context of a patient who may have health conditions or risk factors that would indicate a benefit from targeted screening for vitamin D deficiency or higher-dose supplementation. For example, some patients may have higher vitamin D requirements. This may include patients with bone health problems (osteoporosis, osteomalacia) or poor diets, those who spend minimal time outdoors, those with malabsorption syndromes, or those who take medications that may interfere with vitamin D metabolism (glucocorticoids, anticonvulsant medications, and antituberculosis drugs). Therefore, overall, there is a role for individualized decision-making, in terms of screening for vitamin D deficiency in patients who have bone health problems or special risk factors, and even treating with higher doses of vitamin D, which may go above 4000 IU a day in patients who have higher requirements.

In the next several years, large-scale, randomized trials of vitamin D supplementation, including high-dose vitamin D supplementation, will be completed—and these results will be published. They will help to inform clinical decision-making, so stay tuned for those results.

Thank you so much for your attention. This is JoAnn Manson.

References

  1. Beveridge LA, Struthers AD, Khan F, et al. D-PRESSURE Collaboration. Effect of vitamin D supplementation on blood pressure: a systematic review and meta-analysis incorporating individual patient data. JAMA Intern Med. 2015;175:745-754. Abstract
  2. Hansen KE, Johnson RE, Chambers KR, et al. Treatment of vitamin D insufficiency in postmenopausal women: a randomized clinical trial. JAMA Intern Med. 2015;175:1612-1621. Abstract
  3. Baron JA, Barry EL, Mott LA, et al. A trial of calcium and vitamin D for the prevention of colorectal adenomas. N Engl J Med. 2015;373:1519-1530. Abstract
  4. Institute of Medicine. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: National Academies Press; 2011. http://iom.nationalacademies.org/Reports/2010/Dietary-Reference-Intakes-for-Calcium-and-Vitamin-D.aspx Accessed October 28, 2015.
  5. US Preventive Services Task Force. Final Recommendation Statement: Vitamin D Deficiency: Screening, 2014.http://www.uspreventiveservicestaskforce.org/Page/Document/RecommendationStatementFinal/vitamin-d-deficiency-screening Accessed October 28, 2015.

 

Isn’t there much more to this than the debates entail?

The vitamin D hormone and its nuclear receptor: molecular actions and disease states.
 J Endocrinol. 1997 Sep;154 Suppl:S57-73.      http://dx.doi.org:/10.1677/joe.0.154S057

Vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure that the blood levels of these ions are sufficient for the normal mineralization of type I collagen matrix in the skeleton. In contrast to classic vitamin D-deficiency rickets, a number of vitamin D-resistant rachitic syndromes are caused by acquired and hereditary defects in the metabolic activation of the vitamin to its hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or in the subsequent functions of the hormone in target cells. The actions of 1,25(OH)2D3 are mediated by the nuclear vitamin D receptor (VDR), a phosphoprotein which binds the hormone with-high affinity and regulates the expression of genes via zinc finger-mediated DNA binding and protein-protein interactions. In hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), natural mutations in human VDR that confer patients with tissue insensitivity to 1,25(OH)2D3 are particularly instructive in revealing VDR structure function relationships. These mutations fall into three categories: (i) DNA binding/nuclear localization, (ii) hormone binding and (iii) heterodimerization with retinoid X receptors (RXRs). That all three classes of VDR mutations generate the HVDRR phenotype is consistent with a basic model of the active receptor as a DNA-bound, 1,25(OH)2D3-liganded heterodimer of VDR and RXR. Vitamin D responsive elements (VDREs) consisting of direct hexanucleotide repeats with a spacer of three nucleotides have been identified in the promoter regions of positively controlled genes expressed in bone, such as osteocalcin, osteopontin, beta 3-integrin and vitamin D 24-OHase. The 1,25(OH)2D3 ligand promotes VDR-RXR heterodimerization and specific, high affinity VDRE binding, whereas the ligand for RXR, 9-cis retinoic acid (9-cis RA), is capable of suppressing 1,25(OH)2D3-stimulated transcription by diverting RXR to form homodimers. However, initial 1,25(OH)2D3 liganding of a VDR monomer renders it competent not only to recruit RXR into a heterodimer but also to conformationally silence the ability of its RXR partner to bind 9-cis RA and dissociate the heterodimer. Additional probing of protein-protein interactions has revealed that VDR also binds to basal transcription factor IIB (TFIIB) and, in the presence of 1,25(OH)2D3, an RXR-VDR-TFIIB ternary complex can be created in solution. Moreover, for transcriptional activation by 1,25(OH)2D3, both VDR and RXR require an intact short amphipathic alpha-helix, known as AF-2, positioned at their extreme C-termini. Because the AF-2 domains participate neither in VDR-RXR heterodimerization nor in TFIIB association, it is hypothesized that they contact, in a ligand-dependent fashion, transcriptional coactivators such as those of the steroid receptor coactivator family, constituting yet a third protein-protein interaction for VDR. Therefore, in VDR-mediated transcriptional activation, 1,25(OH)2D3 binding to VDR alters the conformation of the ligand binding domain such that it: (i) engages in strong heterodimerization with RXR to facilitate VDRE binding, (ii) influences the RXR ligand binding domain such that it is resistant to the binding of 9-cis RA but active in recruiting coactivator to its AF-2 and (iii) presents the AF-2 region in VDR for coactivator association. The above events, including bridging by coactivators to the TATA binding protein and associated factors, may position VDR such that it is able to attract TFIIB and the balance of the RNA polymerase II transcription machinery, culminating in repeated transcriptional initiation of VDRE-containing, vitamin D target genes. Such a model would explain the action of 1,25(OH)2D3 to elicit bone remodeling by stimulating osteoblast and osteoclast precursor gene expression, while concomitantly triggering the termination of its hormonal signal by inducing the 24-OHase catabolizing enzyme.

 

Classic nutritional rickets is caused by the simultaneous deprivation of sunlight exposure and dietary vitamin D. As depicted in Fig. 1, the pathways comprising the metabolic activation of the vitamin to its hormonal form and subsequent functions in target tissues present a number of additional steps where defects elicit vitamin D-resistant rachitic syndromes. Two of these disorders involve the inadequate bioactivation of 25-hydroxy¬ vitamin D3 (25(OH)D3) to 1,25-dihydroxyvitamin D3 (l,25(OH)2D3) by the kidney as catalyzed by the 1-OHase enzyme (Fig. 1).

Figure 1 Bioactivation of vitamin D3 and actions of the 1,25(OH)2D3 hormonal metabolite on intestine, bone and kidney, along with related rachitic syndromes. The production of 1,25(OH)2D3 is depicted in the lowet portion and its functions on mineral ttansport in target cells ate pictured in the upper portion. Defects eliciting rachitic syndromes ate boxed, with the televant mutated gene and chromosomal location denoted where appropriate

Acquired chronic renal failure results in renal rickets and secondary hyperparathyroidism (renal osteodystrophy) when the compromising of renal mass reduces 1-OHase activity (Haussier oc McCain 1977). The etiology of pseudo-vitamin D-deficiency rickets (PDDR) apparently involves a hereditary defect in the gene coding for the 1-OHase enzyme (Labuda et al. 1992). Interestingly, the PDDR locus is resolvable from that of the vitamin D receptor (VDR) but maps very close to it on chromosome 12 in the 12ql3—14 region (Labuda et al. 1992). Recently, a cDNA was cloned for the rat 1-OHase (St-Arnaud et al. 1996) and it is expected that the human renal 1-OHase gene will soon be cloned and its chromosomal location determined. The likelihood that both the gene encoding the enzyme that generates the l,25(OH)2D3 hormone and the cognate hormone receptor gene lie in close proximity on chromosome 12 invites speculation about the evolution of the vitamin D ligand receptor system. The traditional actions of vitamin D, via its l,25(OH)2D3 hormonal metabolite, are to effect calcium and phosphate homeostasis to ensure the deposition of bone mineral on type I collagen matrix (summarized in Fig. 1).

Figure 1 Bioactivation of vitamin D3 and actions of the 1,25(OH)2D3 hormonal metabolite on intestine, bone and kidney, along with related rachitic syndromes. The production of 1,25(OH)2D3 is depicted in the lowet portion and its functions on mineral ttansport in target cells ate pictured in the upper portion. Defects eliciting rachitic syndromes ate boxed, with the televant mutated gene and chromosomal location denoted where appropriate

 

l,25(OH)2D3 stimulates intestinal calcium and phosphate absorption, bone calcium and phosphate résorption, and renal calcium and phosphate reabsorption, all resulting in a sufficient CaP04 ion product to precipitate hydroxyapatite. Failure to achieve normal bone mineral accretion by these mechanisms leads to rachitic syndromes. Recently, a breakthrough has occurred in our understand¬ ing of what was originally known as hypophosphatemic vitamin D-resistant rickets, a familial disorder of renal phosphate wasting more appropriately referred to as dominant X-linked hypophosphatemic (HYP) rickets (Fig. 1). The gene defect responsible for HYP rickets has been fine mapped in the Xp22T region, harboring a gene identified as PEX, or phosphate regulating gene with homologies to endopeptidases located on the X-chromosome (Francis et al. 1995). One hypothesis is that PEX codes for an endopeptidase that apparently correctly processes a peptide precursor to yield a novel, as yet unidentified, phosphate retaining hormone. The normal function of this hormone may be to oppose the action of parathyroid hormone (PTH) and stimulate phosphate reabsorption by the renal tubule by inducing the Na -phosphate cotransporter. However, the existence of tumor-induced osteomalacia, an acquired disorder that closely resembles the phosphate wasting of HYP rickets and is characterized by low circulating l,25(OH)2D3 (Parker et al. 1981), combined with renal cross-transplantation (Nesbitt et al. 1992) and parabiosis (Meyer et al. 1989) studies in normal and hyp mice, indicates strongly that the HYP phenotype is caused by excessive amounts of a phosphaturic hormone in the circulation. This humoral peptide is distinct from PTH and has been named phosphatonin (Cai et al. 199A, Econs & Drezner 1994). Thus, instead of PEX mutations result¬ ing in insufficient generation of a novel phosphate retaining peptide, they may instead elicit the appearance of abnormally high circulating levels of phosphatonin, with the normal role of the PEX gene product postulated to be the proteolytic inactivation of this phosphaturic principle. Most germane to the vitamin D endocrine system is the fact that serum l,25(OH)2D3 levels are inappropriately low for the prevailing phosphate concentrations in HYP rickets and patients can be cured with a therapeutic combination of phosphate and l,25(OH)2D3 (Harrel et al. 1985). Because it is well known that hypophosphatemia stimulates l,25(OH)2D3 production (Hughes et al. 1975), the PEX/phosphatonin system might constitute yet another regulatory loop in maintaining normal phosphate homeostasis. One could hypothesize that under hypo- phosphatemic conditions, when l,25(OH)2D3 levels are elevated, the sterol hormone not only increases intestinal phosphate absorption (Fig. 1) and suppresses PTH synthesis (DeMay et al. 1992) to conserve phosphate, but also induces the PEX gene product (Rowe et al. 1996) to cleave phosphatonin and further promote renal phosphate reclamation. l,25(OH)2D3 is primarily recognized as a calcémic hormone, perhaps due to the abundance of dietary phosphate, or because calcium homeostasis is more vitamin D-dependent than the regulation of extracellular phos¬ phate. Regardless of the mechanism, traditional vitamin D-deficiency and clinically significant defects in the vitamin D receptor lead invariably to hypocalcemia and secondary hyperparathyroidism, with phosphate being somewhat less affected. As illustrated in Fig. 1, target tissue insensitivity to l,25(OH)2D3 is known as hereditary hypocalcémie vitamin D-resistant rickets (HVDRR) and is caused by defects in the gene on chromosome 12 coding for the VDR. A review of the etiology of HVDRR and the natural mutations in the VDR that confer tissue insensitivity and clinical resistance to l,25(OH)2D3 is particularly instructive in illuminating the physiologic relevance of the l,25(OH)-,D3-VDR hormone-receptor complex as well as structure/function relationships in the receptor itself.

Natural mutations in the nuclear vitamin D receptor Clinically significant hereditary hypocalcémie vitamin D-resistant rickets is an autosomal recessive disorder resulting in a phenotype characterized by severe bowing of the lower extremities, short stature and, often, alopecia (Rut et al. 199A). The serum chemistry in HVDRR includes frank hypocalcemia, secondary hyperpara¬ thyroidism, elevated alkaline phosphatase, variable hypophosphatemia and markedly increased l,25(OH)2D3. The symptoms of HVDRR, with the exception of alopecia, mimic classic vitamin D-deficiency rickets, suggesting that VDR not only mediates the bone mineral homeostatic actions of vitamin D but may also participate in the differentiation of hair follicles in utero. Recently, VDR knockout mice have been created (Yoshizawa et al. 1996), revealing apparently normal hétérozygotes but severely affected homozygotes (VDR-/-), 90% ofwhich die within 8—10 weeks. Surviving mice lose their hair and possess low bone mass, hypocalcemia, hypophosphatemia and 10-fold elevated l,25(OH)2D3 coincident with extremely low 24,25(OH)2D3. All of these parameters in the VDR knockout mouse mimic the phenotype of patients with HVDRR, confirming that VDR normally mediates all of the bone mineral regulating functions of vitamin D. Interestingly, although natural point mutations in other receptors related to VDR, such as thyroid hormone receptor ß (TRß) (Collingwood et al. 1994), are charac¬ terized by dominant negative receptors that generate the thyroid hormone resistant phenotype in the heterozygotic context, no natural, dominant negative mutations have yet been identified in HVDRR patients (Whitfield et al. 1996). Thus, all HVDRR cases studied to date are homozygous for the particular VDR mutation.

Figure 2 Natural mutations in the human vitamin D receptor leading to 1,25(OH)2D¡ hormone resistance. See text for details and citations. N37, K91 and E92 are not sites of VDR natural mutations, but are so designated because they ate heterodimerization contacts that lie within the DNA binding domain (Hsieh et al. 1995, Rastinejad et al. 1995). The eight cysteine residues (C) that tetrahedrally coordinate two zinc atoms in the finger sttucture are also denoted.

Figure 2 illustrates a number of point mutations in VDR that have been detected in HVDRR patients (reviewed in Rut et al. 199A, Haussler et al. 1995). Three of these genetic alterations result in nonsense mutations that introduce stop codons in VDR (K73stop, Q152sfo|> and Y295stop), creating truncated VDRs that lack both hormone- and DNA-binding (heterodimerization) capacities and are associated with unstable mRNAs. More revealing are the series of missense mutations (Fig. 2) that can be classified according to three of the basic molecular functions of VDR: (i) DNA binding/nuclear localization by the N-terminal zinc finger region, (ii) l,25(OH)2D3 hormone binding by the C-terminal domain and (iii) heterodimerization with retinoid X receptors (RXRs) through subregions of the C-terminal domain. As depicted schematically in Fig. 2 and discussed in detail later, VDR is a ligand-dependent transcription factor that controls gene expression by heterodimerizing with RXR and associating specifically with vitamin D responsive elements (VDREs) in target genes. Since VDR is a member of the steroid, retinoid, thyroid hormone receptor superfamily, and belongs to the VDR/retinoic acid receptor (RAR)/TR subfamily of RXR heterodimerizing species (Haussler et al. 1991), it is reasonable to draw from data on RAR and TR for comparison with VDR.

The greatest number of VDR natural mutations char¬ acterized to date are localized to the DNA binding, zinc finger region (Fig. 2). The first two discovered, G33D and R73Q (Hughes et al. 1988), reside at the ‘tips’ of the fingers and affect charge—charge interactions between VDR and the phosphate backbone of DNA. When viewed in toto, the zinc finger region mutations in HVDRR (Fig. 2) have the following two general prop¬ erties: (i) they occur in residues conserved across the entire nuclear receptor superfamily and (ii) most lie within -helices on the C-terminal side of the first and second fingers which are intimately involved in DNA base recognition and phosphate backbone contacts respectively (Rastinejad et al. 1995). These observations suggest that many of the clinically significant mutations in VDR which are still compatible with life may not greatly perturb the fundamental structure of the DNA binding domain of the receptor, but instead compromise its ability to recog¬ nize DNA with specificity and high affinity. Whether HVDRR cases with mutations in zinc finger region residues unique to VDR will be uncovered depends upon the properties of such alterations, which could range from innocuous to lethal.

Mutations located within the hormone binding domain of VDR also elicit the HVDRR phenotype (Fig. 2), including R274L (Kristjansson et al. 1993) and H305Q (Malloy et al 1995). Transcriptional activation by R274L and H305G VDR is attenuated as a result of inefficient l,25(OH)2D3 binding, ranging from severe in the case of R274L to a modest increase in Kd for H305Q. In both instances, transcriptional activation is restored when the dose of l,25(OH)2D3 is raised to pharmacologie levels (10 m) in transfection experiments (Kristjansson et al. 1993, Malloy et al. 1995). Our laboratory has recently characterized two novel VDR hormone binding domain mutations in HVDRR patients, I314S and R391C, that significantly affect the heterodimerization of VDR with RXR (Whitfield et al. 1996). Both of these C-terminal replacements (Fig. 2), however, do display some degree of what may be a hormone binding deficit, a phenomenon not observable in typical in vitro ligand binding kinetic assays at 4 °C. Thus, only at 37 °C in intact cells do R391C and I314S exhibit apparent slight and significant impairment of l,25(OH)2D3 high affinity retention respectively (Whitfield et al. 1996). Further, the two mutations in question are situated in or adjacent to heptad repeats (Fig. 2), hypothetical coiled-coil-like structures that were originally proposed to participate in the heterodimerization of VDR, RAR, and TR with RXR (Forman & Samuels 1990, Nakajima et al. 1994). Consist¬ ent with this concept, both R391C and I314S VDRs do not bind RXR with normal affinity when assayed in vitro, with the greatest impairment of heterodimerization occur¬ ring with R391C (affinity reduced by one order of magnitude) (Whitfield et al. 1996). Additional evidence supporting blunted RXR heterodimerization by these two mutant VDRs is provided by transfection experiments in restored to that of normal fibroblasts when fibroblasts from patients harboring either the R391C or the I314S mutation are cotransfected with exogenous RXR. Yet this apparent RXR rescue of the mutated VDRs requires approximately 10-fold elevated l,25(OH)2D3 doses com¬ pared with the response to hormone in normal fibroblasts (Whitfield et al. 1996). This latter observation reveals that the hormone binding and heterodimerization functions of VDR are not entirely separable, an aspect which is also apparent from fundamental biochemical analysis of the hormone dependency of VDR-RXR heterodimer binding to VDREs as discussed in detail below.

Understanding the molecular properties of natural VDR mutations in HVDRR allows us to comprehend why the patients respond differentially to therapy with massive doses of l,25(OH)2D3, or suitable analogs. For example, cases with zinc finger region aberrations are unresponsive to the hormone because DNA binding is precluded by the absence of structural complemen¬ tarity between VDR and the VDRE, regardless of the l,25(OH)2D3 liganding or heterodimerization of the receptor in solution. Conversely, patients harboring mutations in the hormone binding/heterodimerization domain can be responsive to pharmacologie doses of l,25(OH)2D3 or analogs, even though the hormone already is increased in the circulation because of the hypocalcemia caused by tissue insensitivity. For example, patient I314S was essentially cured by excess vitamin D metabolite, indicating that compensating for the hormone binding deficit was able to override the milder heterodimerization defect and allow sufficient VDRE binding by the VDR-RXR heterodimer. Conversely, patient R391C responded only modestly to treatment with excess l,25(OH)2D3 analog, presumably because the fundamental heterodimerization defect could not be overcome and therefore normal VDRE binding could not be achieved (Whitfield et al. 1996).

The final insights gained from the natural VDR mutations summarized in Fig. 2 are structural in nature. We have discussed previously that the zinc finger mutations are confined to absolutely conserved residues. In the crystal structure of the DNA binding domain heterodimers of RXRa and TRß (Rastinejad et al. 1995), the lysine and arginine residues corresponding to K45 and R50 in human VDR (hVDR) make direct base contacts with DNA, while the arginines corresponding to R73 and R80 in hVDR make direct DNA phosphate backbone contacts. That mutations in these four residues are clinically important in the etiology of HVDRR argues for structural congruity between the VDR finger region and that of TR. Rastinejad et al. (1995) have extended this assumption to include a modeling of RXR-TR vs RXRVDR bound to DNA which accommodates the fact that TR binds as a heterodimer to a direct hexanucleotide repeat spaced by four nucleotides (DR+4), while VDR binds as a heterodimer to a similar set of half elements spaced by three nucleotides (DR+3). In addition to verifying the common protein-DNA interfaces, their modeling predicts that hVDR residues N37 in the first finger and K91/E92 C-terminal of the second finger (see Fig. 2) engage in heterodimeric contacts with residues in the second zinc finger ofRXR to form effectively a stable, DNA-supported heterodimer. Indeed, recent site-directed mutational studies (Hsieh et al. 1995) indicate that the alteration ofK91 and E92 in hVDR in fact grossly reduces transactivation while moderately attenuating hetero¬ dimerization and DNA binding, thus confirming the importance of K91 and E92. An additional surprising finding was that the K91/E92 double mutant manifested dominant negative characteristics (Hsieh et al. 1995), distinguishing it from the natural HVDRR replacements discussed above. Apparently, the K91/E92 mutant VDR is able to bind DNA sufficiently through its native zinc finger and strong heterodimerization function in the ligand binding domain such that it can block binding by wild type receptor, but is rendered inactive in stimulating transcription because of a presumed conformational per¬ turbation initiated by unstable or improper alignment of the heterodimer on the VDRE.

Based upon recently reported X-ray crystal structures of the ligand binding domains of ligand-occupied hRARy (Renaud et al. 1995), agonist-occupied rat TRa, (Wagner et al. 1995) and unoccupied, but dimeric hRXRa (Bourguet et al. 1995), it is also possible to incorporate the HVDRR mutations in the hormone binding domain (Fig. 2) into a hypothetical structural context. Figure 3 constitutes a schematic compilation of the existing crystallographic data and compares them with natural and artificially generated mutations in hVDR. At the top of Fig. 3, the residue numbers for VDR in the ligand binding domain appear in relation to the older heptad repeat nomenclature (heptads 1—9, dotted boxes). At least some of these heptads, particularly heptads 4 and 9, are thought to facilitate heterodimerization (Nakajima et al. 1994). The El region is a highly conserved area that supports heterodimerization (Whitfield et al. 1995è). The helices depicted schematically in Fig. 3 (open boxes) are those determined for hRARy; this general pattern of -helices and ß-strands (solid boxes) appears to be well conserved across the TR, RAR and RXR members of the subfamily crystallized thus far (Bourguet et al. 1995, Renaud et al. 1995, Wagner et al. 1995). Although the heterodimerization domains have yet to be elucidated by structural analysis, the homodimerization domain of RXR is comprised of helices 7, 9 and 10 (Fig. 3 and Bourguet et al. 1995). Flanking the dimerization region are clusters of ligand binding contacts, shown for RAR and TR in Fig. 3, which paint a picture of hormone binding involving helices 3, 5, 11 and 12 plus portions of helices 6 and 7 along with their intervening loop, as well as the loop between ß-strands 1 and 2.

Figure 3 Hormone binding (R274L and H305Q) and heterodimerization (I314S and R391C) natural mutations in VDR that confer the HVDRR phenotype are positioned in the context of retinoid and thyroid hormone receptor subfamily ligand binding domain structures. See text for details and citations.

As summarized in Fig. 3 and discussed by Whitfield et al. (1995a, 1996), a number of artificially generated mutants in hVDR support the con¬ cept that the dimerization and honnone binding regions in VDR are well aligned with those in RXR, RAR and TR. Of even greater interest and relevance to the present monograph, the four clinically important hVDR mutants under consideration correspond to pertinent locations in the known structures of the retinoid and thyroid hormone receptor ligand binding domains. We postulate that this general structural organization represents that of the VDR ligand binding domain. As shown in Fig. 3, the pure hormone binding mutant hVDRs, namely R274L and H305Q, are located precisely within ligand clusters in helix 5 and in the loop between helix 6 and 7 respectively. I314S, which endows hVDR with combined defects in hormone retention and heterodimerization, lies within helix 7 at a presumed interface of ligand binding and dimerization activities of the receptor (Fig. 3). Finally, R391C is positioned well within the helix 10 dimerization surface, but not far removed from C-terminal ligand binding contacts that are likely influenced by replacement of this amino acid in hVDR. Thus, at least within the context of the assumed structural organization of VDR derived from that of other subfamily members, the I314S and R391C mutations are situated precisely where they would be predicted to lie, given the biological properties of the mutant receptors and the phenotype of the patients. These results not only have profound implications con¬ cerning the putative structure of VDR in relation to its closest relatives, but prove unequivocally that the calcémic actions of l,25(OH)2D3 are mediated by the vitamin D receptor, existing as a l,25(OH)2D3-liganded heterodimer with RXR that is bound to DNA.

Physiology and cellular actions of l,25(OH)2D3

In order to delineate the physiologic roles for the vitamin D hormone, it is appropriate first to place the VDR mediator into the context of vitamin D metabolism and cellular actions. Figure 4 summarizes the integration of vitamin D metabolism and cellular actions introduced in Fig. 1, with physiologic regulatory events now super¬ imposed on the metabolic pathway and the inclusion of an expanded list of physiologic actions for the 1,25( )2 4 hormone. The conversion of vitamin D3 to 25(OH)D3 by the liver is a constitutive metabolic step, followed by the 1-hydroxylation of25(OH)D3 to l,25(OH)2D3, a reaction under exquisite control (Haussler & McCain 1977). When blood calcium is low, activation of this latter step occurs, either as a result of the hypocalcémie state per se, or in response to elevated PTH, each of which serves indepen¬ dently to enhance renal 1-OHase activity. Low phosphate is also capable of separately upregulating the 1-OHase enzyme. To limit activation, the hormonal product, l,25(OH)2D3, effects an ultra-short feedback loop to suppress its own biosynthesis in the kidney and also represses PTH synthesis to remove the peptide hormone stimulus of the 1-OHase via a longer feedback loop (Fig. 4). However, the dominant negative feedback controls of 1-OHase activity appear to result from the concerted actions of l,25(OH)2D3 to stimulate bone mineral résorption and to promote intestinal calcium and phosphate absorption, which together elicit an increase in blood calcium and phosphate levels, each of which down-regulates the 1-OHase.

Figure 4 Vitamin D metabolism and cellulat actions, mediated by the VDR-RXR heterodimer binding to a VDRE

The process by which l,25(OH)2D3 causes bone remodeling is complex, involving stimulation of osteoclast differentiation and osteoblastic production of osteopontin, both of which activate résorption in part through the recognition of bone matrix osteopontin by osteoclast surface avß3-integrin. The résorption effect is supported by l,25(OH)2D3-elicited suppression of bone formation via the induction of osteocalcin and the repression of type I collagen. This latter insight that the normal function of osteocalcin is to curtail bone matrix formation arises from the creation of osteocalcin knockout mice (Ducy et al. 1996). In addition to stimulating the transcription of bone-related genes such as osteopontin and osteocalcin, the l,25(OH)2D3 hormone also induces its own eatab¬ olism in kidney as well as other target tissues like bone by enhancing the expression of the vitamin D-24-OHase enzyme. 24-Hydroxylation of l,25(OH)2D3 is the first step in deactivating the hormone, which is eventually metabolized by side chain cleavage to calcitróle acid (Haussler 1986). Thus, the synthesis of l,25(OH),D3 is not only governed by feedback mechanisms that sense l,25(OH)2D3, calcium, PTH and phosphate concentrations, but the hormone induces the termination of its own signal in target tissues, qualifying l,25(OH)2D3 as a bonafide hormone by any definition.

Figure 4 Vitamin D metabolism and cellulat actions, mediated by the VDR-RXR heterodimer binding to a VDRE

As introduced in the section on HVDRR, mediation of the cellular functions of l,25(OH)2D3 requires that VDR bind the hormonal ligand specifically and with high affinity (Fig. 4). Upon such binding, VDR becomes hyperphosphorylated (Jurutka et al. 1993, Haussler et al. 1994) and recruits RXR into a hetero¬ dimeric complex that binds strongly to DNA (Fig. 4). The l,25(OH)2D3-hganded RXR-VDR heterocomplex selectively recognizes VDREs in the promoter regions of positively controlled genes such as osteocalcin (MacDonald et al. 1991), osteopontin (Noda et al. 1990), vitamin D-24-OHase (Ohyama et al. 199A) and ß3-integrin (Cao et al. 1993). Negative VDREs (Haussler et al. 1995) exist in the 5′-regions of the genes for type I collagen (Pavlin et al. 199A), bone sialoprotein (Li & Sodek 1993), PTH (DeMay et al. 1992) and PTH-related peptide (Falzon 1996, Kremer et al. 1996). The mechanisms whereby VDR accomplishes positive and negative control of DNA transcription after VDRE association are not well under¬ stood, although substantial progress has been made in comprehending the stimulation of transcription as detailed in later sections of this article. Moreover, as summarized in Fig. 5, a number of VDREs have been definitively characterized. The prototypical VDBJS is found in the osteocalcin gene, consisting of an imperfect direct repeat of hexanucleotide estrogen responsive element (ERE)-like, half-sites with a spacer of three nucleotides (DR+3). Classic EREs possess a central GT core at positions 3 and 4 of the hexanucleotide, but this feature is only partially conserved in the six natural positive VDREs listed in Fig. 5. There is, however, absolute conservation of the A in position 6 of the 5′ half-element and of the G at position 2 of the 3′ half-element. A preliminary working consensus for the positive VDRE can be derived from these natural VDREs (see boxed sequence in Fig. 5). This generaliz¬ ation is supported, in part, by PCR experiments that were designed to select, from random oligonucleotides, the highest affinity DNA ligand for the RXR-VDR heterodimer (Nishikawa et al. 1994, Colnot et al. 1995).

Figure 5 Natural vitamin D responsive elements (DR+3s) in genes positively tegulated by l,25(OH)2D3. The consensus VDREs are based on either sequence comparisons (boxed) or a selection of random sequences (at bottom).

The random selection process yields an identical VDRE 5′ half-element of GGGTCA (Fig. 5, bottom), which is also a preferred RXR target when RXR homodimers bind to DNA (Yang et al. 1995). This observation is in concert with the conclusion (Jin & Pike 1996) that, with respect to association ofRXR-VDR with VDREs, RXR lies on the 5′ half-element whereas VDR is situated on the 3′ half-element. Examination of both consensus sequences suggests that the G at position 3 of the spacer is important in VDR binding, a deduction consistent with the finding (MacDonald et al. 1991) that this base is partially protected by RXR-VDR in methylation interference assays. How¬ ever, interesting differences arise when one compares the most frequently encountered 3′ half-element bases in natural VDREs, namely the GGGGCA composite which actually occurs in human osteocalcin, with the GGTTCA random consensus selection for the 3′ half-element (Fig. 5). Clearly, GGTTCA represents a potent VDR binding site, a supposition that is bolstered by the fact that osteopontin, which possesses a perfect DR+3 of GGTTCA, is the highest affinity VDRE we have tested (data not shown). Intriguingly, Ts at positions 3 and 4 in the 3′ VDR half-site occur infrequently in the balance of natural VDREs (Fig. 5). The paucity of Ts in the 3′ half-element could be related to a need for varying potency of VDREs in regulated genes, or may even provide for a repertoire of different VDR conformations that could be induced by contact with distinct 3′ half-site core sequences. This postulated range of VDR conforma¬ tions might endow the receptor with the ability to recruit a variety of different coactivators and corepressors, or even to favor the binding of one vitamin D metabolite ligand over another. Irrespective of the above considerations, it is evident that the primary VDRE is a DR+3 recognition site in DNA that directs the VDR to the promoter region of l,25(OH)2D3 regulated genes, ultimately altering the functions of target cells as a result of transcriptional control of gene expression.

Significance of lipophilic ligands in the association of RXR-VDR with DNA

Dimeric complexes are a feature commonly employed in the regulation of eukaryotic transcriptional systems. This process of protein dimerization often will generate novel heterodimeric complexes which display highly cooperative binding to DNA as well as an altered target sequence specificity (Glass 1994). Among the classical steroid hormone receptors, dimerization results in the formation of symmetrical homodimeric protein complexes on palindromic DNA half sites. Dimerization has been shown to be mediated in part by residues within the DNA binding domain of the receptor (Luisi et al. 1991) and is enhanced by residues within the ligand binding domain (Falwell et al. 1990). The other subfamily of nuclear hormone receptors, including VDR, TR and RAR, apparently binds with highest affinity to direct repeat elements either as homodimers or, more commonly, as heterodimers with RXR (Kliewer et al. 1992). In both subgroups of nuclear receptors, protein-protein interactions serve to align the DNA binding domains so that they are optimally positioned to bind to their specific DNA target sequences (Kurokawa et al. 1993, Perlmann et al. 1993, Rastinejad et al. 1995). The ligand binding region of these receptors is multifunctional, in that this domain not only binds the cognate ligand, but also it possesses a dimerization surface as well as the ligand-dependent transactivation function, AF-2 (Gronemeyer 1991, Chambón 1994). The dimerization surface consists of packed helices which are stabilized by hydrophobic heptad repeats interspersed throughout the structure. Ligand apparently can influence different functional components, including the dimerization interface, and the activating AF-2 domain (Renaud et al. 1995, Wagner et al. 1995). Therefore, a likely role for ligand is to regulate the association and dissociation of dimeric protein complexes and hence regulate specific binding to DNA target sequences.

In this regard the following three questions remain regarding l,25(OH)2D3-mediated control of positively regulated genes: (i) does VDR bind as a homodimer (Freedman et al. 1994, Nishikawa et al. 1994) as well as a heterodimer to DR+3 VDREs? (ii) What is the effect of the l,25(OH)2D3 ligand on VDR or VDR-RXR binding to VDREs? (iii) What role does 9-cis retinoic acid, the RXR ligand, play m RXR-VDR binding to VDREs and enhanced transcription of l,25(OH)2D3-responsive genes? It is generally accepted that TR forms homodimers as well as heterodimers with RXR on thyroid hormone responsive elements (TREs), although recent data suggest that the TR homodimer, when unoccupied by thyroid hormone, operates as a repressor of transcription (Chin & Yen 1996, Schulman et al. 1996). Thyroid hormone is proposed to dissociate TR homodimers to facilitate TRRXR heterodimerization on the TRE and stimulate transcription. In contrast, RAR does not appear to be capable of forming homodimers on DR+5 retinoic acid responsive elements (RAREs) (Perlmann et al. 1996), instead cooperating exclusively with RXR in RARE association and vitamin A metabolite-responsive transcrip¬ tion. When present in excess in gel mobility shift DNA binding assays in vitro, both TR and RAR display RXR heterodimeric association with their respective hormone responsive elements (HREs) in the absence of added lipophilic ligand. These in vitro studies are consistent with immunocytochemical data indicating that, unlike classic steroid honnone receptors that reside in the cytoplasm complexed with Hsp-90 and other proteins in their unoccupied state, unliganded TR, RAR and VDR (Clemens et al. 1988) exist in the nucleus in general association with DNA. These findings have led to the dogma that ligand is not required for TR, RAR and VDR to associate with target HREs. Indeed, we have observed that addition of 260 ng baculovirus-expressed hVDR to a gel shift reaction generates weak homodimeric VDR as well as strong VDR-RXR-heterodimeric binding to a rat osteocalcin VDRE probe, both of which are independent of the presence of l,25(OH)2D3 (Nakajima et al. 1994). However, in vivo footprinting experiments (Blanco et al. 1996, Chen et al. 1996) have led to the conclusion that, at least in the case of RAR-RXR heterodimers, RAR ligands are required for RARE binding. We, therefore, sought to devise an in vitro gel shift assay that would more accurately reflect the in vivo situation, primarily consisting of the use of physiologic salt (0-15 m KCl) concentrations and limited amounts of partially purified, baculovirusexpressed VDR and RXRs (Thompson et al. 1997). Utilizing this assay, we have addressed the three questions regarding VDR/RXR listed above, namely heterodimer versus homodimer, the potential role of l,25(OH)2D3 and the effect of 9-cis retinoic acid (9-cis RA).

When 20 ng VDR (~ 10 nM) or 20 ng VDR plus 20 ng RXR are incubated with either the rat osteocalcin or mouse osteopontin VDREs (see Fig. 5), no DNA-bound homodimeric VDR species is apparent, but a VDRE complexed VDR-RXR heterodimer occurs that is strik¬ ingly dependent upon the presence of the l,25(OH)2D3 ligand (Thompson et al. 1997). Thus, at receptor levels approaching that in a typical target cell, a VDR liganddependent heterodimer with RXR is the preferred VDRE binding species. Only when VDR or VDR plus RXR levels are raised to 100 ng of each receptor with the mouse osteopontin VDRE (Thompson et al. 1997), or 260 ng with the weaker rat osteocalcin VDRE (Nakajima et al. 1994), can faint homodimers of VDR bound to the probe be visualized. In addition, at these greater amounts ofreceptors, neither the VDR homodimer nor the VDRRXR heterocomplexes are modulated significantly by inclusion of l,25(OH)2D3 in the incubation (Thompson et al. 1997). We, therefore, conclude that higher receptor levels in vitro generate artifactual VDR homodimers as well as attenuate the normal physiological ligand dependence of VDR-RXR binding to the VDRE. To explain seemingly ligand-independent VDR-RXR association with the VDRE, we postulate the existence of a subpopulation of VDR that is unstably activated in the absence of l,25(OH)2D3 (Schulman et al. 1996) and therefore capable of heterodimerization to generate a positive gel mobility shift under conditions of vast receptor excess. In contrast, our physiologically relevant gel shift assay at <10nM receptor levels and 0-15 m KCl reflects the presumed in vivo events of ligand triggered heterodimerization (Blanco et al. 1996, Chen et al. 1996), and extends earlier in vitro data showing that l,25(OH)2D3 enhances VDRRXR complex formation (Sone et al. 1991, MacDonald et al. 1993, Ohyama et al. 1994).

Next, we tested the effect of 9-cis RA in this gel shift assay. A spectrum of data exists on the role of 9-cis RA in l,25(OH)2D3-stimulated transcription, including demon¬ stration of synergistic action with l,25(OH)2D3 (Carlberg et al. 1993, Schrader et al. 1994, Kato et al. 1995, Sasaki et al. 1995), negligible action (Ferrara et al. 1994), or an inhibitory effect (MacDonald et al. 1993, Jin & Pike 1994, Lemon & Freedman 1996). These marked differences likely result from varying transfection and ligand addition protocols, as well as cell and species specificity. Employing the physiological gel shift procedure with biochemically defined components, we obtained clear evidence that 9-cis RA is a potent inhibitor of l,25(OH)2D3-enhanced, VDR-RXR binding to VDREs such as osteocalcin, with dramatic attenuation by the retinoid occurring at concentrations as low as 10 m (Thompson et al. 1997). Previous gel shift data had also hinted at 9-cis RA inhibition (MacDonald et al. 1993, Cheskis & Freedman 1994), even though higher concentrations of 9-cis RA were utilized in these earlier studies. One somewhat puzzling finding, however, was that the suppressive effect of 9-cis RA seemed more pronounced in vitro than in transfected cells, where retinoid inhibition of l,25(OH)2D3-stimulated transcription is significant, but 50% or less in magnitude (MacDonald et al. 1993). This suggested that multiple pathways may exist for the assembly of the RXR-VDR heterocomplex in vivo. To probe for distinct routes of assembly, we varied the order of addition ofVDR, RXR, l,25(OH)2D3 and 9-cis RA in the gel shift assay for VDRE binding (Thompson et al. 1997). The results showed that 9-cis RA is a potent inhibitor of VDR-RXR heterodimerization on the VDRE in all situations except when VDR alone is preincubated with l,25(OH)2D3 followed by addition of RXR (Thompson et al. 1997). To explain these data, we have developed the model depicted in Fig. 6, which hypothesizes two alternative allosteric pathways for the interaction ofVDR-RXR with the VDRE.

Figure 6 Model of two different allosteric pathways for VDR-RXR-1,25(OH)2D3 binding to DNA.

In pathway A (Fig. 6), l,25(OH)2D3 occupies monomeric VDR, altering the conformation of the ligand binding domain such that it recruits RXR for heterodimeric binding to DNA and subsequent VDRE recognition. Importantly, we pos¬ tulate that previously occupied VDR conformationally influences RXR in the resulting heterodimer such that it is incapable of being liganded by 9-cis RA (pathway A, Fig. 6). This action to abolish RXR ligand responsiveness both silences the ability of 9-cis RA spuriously to trigger vitamin D hormone signal transduction, and prevents 9-cis RA from dissociating the RXR-VDR complex in order to divert RXR for retinoid signal transduction. On the other hand, as illustrated in pathway (Fig. 6), we propose that RXR exists in a different, 9-cis RA-receptive, allosteric state in most other circumstances, such as when present as a monomer, in an apoheterodimer with VDR, or even when the apoheterodimer of RXR and VDR is subsequently liganded with l,25(OH)2D3. This latter species of RXR-VDR-l,25(OH)2D3 (pathway B) is hypothesized to be fully competent in VDRE recognition, but the 9-cis RA binding function of the RXR partner has not been conformationally repressed, rendering this form sensitive to dissociation by 9-cis RA, which would then favor the formation of retinoid-occupied RXR homo¬ dimers. Therefore, unless VDR monomers are first occu¬ pied by l,25(OH)2D3 (pathway A), 9-cis RA can operate to divert or dissociate RXR and direct it to form RXR homodimers (pathway B). It is tempting to speculate that the l,25(OH),D3-liganded heterodimer in pathway A is more potent in transcriptional stimulation than the analogous species in pathway B, perhaps because the AF-2 function of the RXR partner is allosterically activated only in the former instance. The l,25(OH)2D3-occupied VDR-RXR in pathway has the advantage of flexible regulation because it is effectively a two-ligand switch. It likely occurs in vivo because, as stated above, the fact that 9-cis RA blunting significant but incomplete suggests that at least two populations of RXR-VDR heterodimers exist. Finally, when our model (Fig. 6) is compared with those for RXR-RAR and RXR-TR (Forman et al. 1995), it is evident that VDR is closer in mechanism of action to the TR, where 9-cis RA inhibits TR signal transduction by diversion of BJÍR (Lehmann et al. 1993). Also analogous is the fact that thyroid hormone occupation of the TR partner abolishes 9-cis RA binding to the RXR counter¬ part (Forman et al. 1995). Finally, the action of RXRPJ\R heterodimers seems to be fundamentally different from that of RXR-VDR in that RAR liganding by a retinoid facilitates RXR occupation by its retinoid ligand, resulting in cooperative stimulation of gene transcription by the repertoire of vitamin A metabolites.

VDR protein-protein interactions that effect gene transcription

Although we now have at least a rudimentary understand¬ ing of ligand-induced VDR binding to a VDRE, the next logical question is how does VDR regulate the machinery for gene transcription? In the basal state ofDNA transcrip¬ tion, the TATA-box binding protein (TBP) and its associated factors (TAFs) are bound to the TATA box at approximately position — 20 in the 5′ region of controlled genes, but the frequency of transcriptional initiations is very low because the RNA polymerase II-basal transcription factor IIB (TFIIB) enzyme complex is not stably associated with TBP-TAFs. The recruitment of the TFIIB-RNA polymerase II complex appears to be the rate limiting step in preinitiation complex formation, and is stimulated dramatically when a transacting factor or factors bind to upstream enhancers. In a process involving DNA looping, transactivators are thought to attract TFIIB and also interact with TAFs, forming a stable preinitiation complex that executes repeated rounds of productive transcription. Recent data indicate that the activation function in the hormone binding domain of the estrogen receptor, AF-2, associates specifically with a TAF known as TAFn30 (Jacq et al. 1994) and that the estrogen receptor (ER) binds to TFIIB in vitro (lng et al. 1992). In collaboration with Ozato and associates and Tsai and O’Malley, we have observed that hVDR also specifically associates with hTFIIB (Blanco et al. 1995). In this work, Blanco et al. (1995) showed that VDR binds to a TFIIB-glutathione S transferase fusion protein linked to glutathione-laden beads. Additionally, it was observed that both TRa and RARa interact with hTFIIB (Blanco et al. 1995), but that RXR does so only very weakly (P W Jurutka, L S Remus and M R Haussler, unpublished results). This last result suggests that, while the ligand binding partners in the VDR/TR/RAR subfamily provide a hard-wired connection to the assembly and en¬ hancement of the transcription machinery, the RXR partner is not primarily engaged in TFIIB contact.

Independent data obtained by MacDonald et al. (1995) using the powerful yeast two-hybrid system to detect protein-protein interactions also revealed that hVDR binds efficiently to TFIIB. Moreover, MacDonald et al. (1995) further exploited the yeast two-hybrid system to prove that, while hVDR and RXR interact, no homodimeric association occurs for hVDR alone, providing further evidence against the existence of physiologically significant VDR homodimers. Utilizing fusion protein technology, they also showed that VDR interacts directly with RXR to form a heterodimer in solution in the absence of DNA and, further, that this process was enhanced 8-fold by the presence of l,25(OH)2D3 hor¬ mone (MacDonald et al. 1995). Because hVDR-TFIIB association is not dependent upon the l,25(OH)2D, ligand (Blanco et al. 1995, MacDonald et al. 1995), the role of l,25(OH)2D3 can now be further resolved to an early participation in conforming VDR such that it attracts RXR followed by the targeting of the resulting RXR-VDR heterodimer to VDREs (see Fig. 6).

Figure 6 Model of two different allosteric pathways for VDR-RXR-1,25(OH)2D3 binding to DNA.

Interestingly, the presence of BJCR further facilitates VDR-TFIIB association, especially in the presence of l,25(OH)2D3 (PW Jurutka, LS Remus and MR Haussler, unpublished results). In fact, because of its capacity to enhance VDR-RXR heterodimerization, the l,25(OH)2D3 ligand is capable ofgenerating high levels of an RXR-VDR-TFIIB ternary complex in solution, sig¬ nificantly in excess ofthat occurring with either RXR and TFIIB or even with VDR and TFIIB (P W Jurutka, L S Remus and M R Haussler, unpublished results). These data not only reaffirm the interaction ofVDR with TFIIB, but also they imply that the l,25(OH)2D3-liganded VDR-RXR complex is the most efficient binder of TFIIB. This latter effect may be the result of positive conformational influences of RXR on liganded VDR, since VDR is the primary attachment moiety for TFIIB.

Because VDR-TFIIB interactions have been detected either in vitro or in the yeast system where certain mammalian cell restrictions may be relaxed, it was import¬ ant to confirm the relevance ofVDR-TFIIB association in mammalian cells. Blanco et al. (1995) have reported functional studies which, for the first time, show the interaction ofTFIIB with a member ofthe steroid receptor superfamily in ligand-dependent activation oftranscription in intact cells. In pluripotent PI9 mouse embryonal carcinoma cells, transfection of hVDR or hTFIIB alone produced no better than a 2-fold induction of VDREluciferase reporter expression by l,25(OH)2D3. However, when transfected together, hVDR and hTFIIB mediated a synergistic transcriptional response of approximately 30-fold when l,25(OH)2D3 was added, an effect which was absolutely dependent on the presence of the VDRE in the luciferase construct. It should be noted that the VDR-TFIIB positive cooperation appears to be cellspecific because similar experiments in contact-inhibited NIH/3T3 Swiss mouse embryo cells resulted in squelching of transcription by TFIIB. Therefore, in more differentiated cells, perhaps including osteoblasts or fibro¬ blasts, accessory coactivators may be present to modulate TFIIB or bridge between VDR and TFIIB.

In summary, VDR and TFIIB are hypothesized to exist in a multi-subunit transcription complex which also con¬ tains TAFs and/or coactivators that may be promoter- or tissue-specific. Further characterization of this complex will require the discovery of cell type and promoterspecific components via transfection and biochemical interaction studies. Ultimately, an in vitro transcription system must be devised which utilizes defined components to replicate faithfully l,25(OH)2D3-stimulated gene expression.

One subdomain of VDR that likely interacts with coactivators and/or basal transcription factors is the extreme C-terminus. We have previously shown that 403 hVDR, a truncated receptor that lacks the C-terminal 25 amino acids, binds l,25(OH),D3 ligand with reasonable affinity and heterodimerizes normally with RXR, but is devoid of transcriptional activity (Nakajima et al. 1994). These data suggest that VDR contains a transcriptional activation domain near its C-terminus.

Figure 7 The extreme C-terminal amino acid sequence compared across the nuclear receptor superfamily: VDR appears to share the ligand-dependent transcription activation function (AF-2). AR, androgen receptor; CR, glucocorticoid receptor; PR, progesterone receptor.

Indeed, as illustrated in Fig. 7, the region of VDR from residues 416 to 422 possesses a high degree of similarity to the analogous sequences in the entire nuclear receptor superfamily. One hallmark of this conserved sequence is the glutamic acid residue at position 420 of hVDR (Fig. 7) included in a consensus of (where cp=a hydrophobic amino acid) for this domain (Renaud et al. 1995, Wagner et al. 1995). Allowing for conservative replace¬ ments, it seems virtually certain that hVDR forms an amphipathic helix (corresponding to helix 12 in the other receptors) surrounding glutamic acid-420 that is analogous to the ligand-dependent activation function (AF-2) char¬ acterized for TR (Barettino et al. 199A), RAR (Renaud et al. 1995), RXR (Leng et al. 1995) and ER (Danielian et al. 1992). Although this AF-2 domain is capable of autonomously activating transcription (Leng et al. 1995), that such activity is modest may be because of the fact that the AF-2 region is proposed to operate in a liganddependent fashion, involving a structural rearrangement to reposition the AF-2 for both intramolecular and intermolecular protein—protein interactions. Specifically, based upon the crystal structure of unoccupied RXR (Bourguet et al. 1995) and liganded RAR (Renaud et al. 1995) and TR (Wagner et al. 1995), helix 12/AF-2 appears to protrude outward from the more globular ensemble of helices 1—11 in the absence ofligand, such that it is unable to interact efficiently with coactivator/transcription factor. Upon liganding, a conformational signal is then transmit¬ ted to helix 12 that causes it to fold back on helix 11 and attach to the face of the globular ligand binding domain. The pivoting of helix 12 seemingly accomplishes two feats that mediate ligand-activated transcription by the receptor: (i) closing of a ‘door’ on the channel through which the lipophilic ligand enters the internal binding pocket of the receptor, and (ii) locking helix 12 into a stable confor¬ mation that facilitates its interaction with coactivator/ transcription factor. Ligand binding contacts on or near helix 12 (see Fig. 3) probably are significant in maintaining this active positioning of helix 12, essentially trapping ligand in the binding pocket to effect more sustained transactivation events.

Figure 7 The extreme C-terminal amino acid sequence compared across the nuclear receptor superfamily: VDR appears to share the ligand-dependent transcription activation function (AF-2). AR, androgen receptor; CR, glucocorticoid receptor; PR, progesterone receptor.

In order to evaluate the relevance of the above proposed mechanism for VDR action, we (Jurutka et al. 1997) have altered E-420 and L-417 (see Fig. 7) individually to alanine residues, which preserves the putative -helical character of this region. The altered VDRs bind ligand near-normally, with only a mild increase (about 3-fold) in the Kd for the E420A receptor. Both E420A and L417A hVDRs also heterodimerize efficiently with RXR and associate with VDREs similarly to wild-type hVDR, yet their capacity for l,25(OH)2D3-stimulated transcription is abolished, even at high doses ofligand (Jurutka et al. 1997). These point mutations, therefore, identify a C-terminal AF-2 in VDR which corresponds to similar activation domains in other nuclear receptor superfamily members. Because VDR interacts with TFIIB, one of the first questions we asked was whether the VDR AF-2 consti¬ tutes a contact site for this basal transcription factor. Although some very preliminary evidence existed for an association between TFIIB and the C-terminus of hVDR (MacDonald et al. 1995), we observed that neither the E420A nor the L417A mutant VDRs are impaired in their interaction with TFIIB as probed with glutathione-S transferase—TFIIB fusion protein binding technology (Jurutka et al. 1997). Thus, the domain(s) of VDR that interfaces with TFIIB apparently lies elsewhere in the receptor, possibly in the N-terminal portion of the ligand-binding region (Blanco et al. 1995), in the hinge (MacDonald et al. 1995), or in the vicinity of the DNA-binding zinc fingers.

The present experiments with VDR are in concert with recent insight into the function of AF-2 in other nuclear receptors, which is to recruit coactivators of the type of steroid receptor coactivator-1 (SRC-1) (Oñate et al. 1995). A number of candidate coactivators have been isolated in addition to SRC-1 (Halachmi et al. 199A, Baniahmad et al. 1995, CavaiUes et at. 1995, Lee et al. 1995, Hong et al. 1996) and, in several cases, interaction with nuclear receptors requires intact AF-2 core regions (Baniahmad et al. 1995, CavaiUes et al. 1995). Moreover, AF-2 mutations act as dominant negative receptors, for example in the case of hRARy (Renaud et al. 1995). Indeed, we have observed that VDR AF-2 mutants E420A and L417A exhibit dominant negative properties with respect to transcriptional activation (Jurutka et al. 1997). Such AF-2 altered receptors are inactive transcriptionally, but can bind l,25(OH)2D3 ligand and heterodimerize normally on VDREs, the consequence being competition with wild-type VDR-RXR heterodimers for VDRE binding. These data argue that the AF-2 of the primary VDR partner in an RXR-VDR heterodimer is absolutely required for the mediation of l,25(OH)2D3-activated transcription, not only for its intrinsic activation potential, but also because of its presumed role in stabilizing the retention of l,25(OH)2D3 ligand in the VDR binding pocket.

Figure 6 Model of two different allosteric pathways for VDR-RXR-1,25(OH)2D3 binding to DNA.

What part, if any, is played by the AF-2 domain (Fig. 7) of the RXR ‘silent’ partner in the RXR-VDRl,25(OH)2D3 signal transduction pathway? To investigate this phenomenon, AF-2 truncated mutants of RXRa or RXRß were created and tested for their ability to function as dominant negative modulators of l,25(OH)2D3- stimulated transcription (Blanco et al. 1996). Because previous data with RXR-RAR control of gene expression seemed to indicate that the RXR AF-2 was dispensable (Durand et al. 1994), we were surprised to find that AF-2 truncated RXRs were potent dominant negative effectors of l,25(OH)2D3 action in transfected cells (Blanco et al. 1996). We, therefore, conclude that although the RXR ‘silent’ partner in VDR signaling apparently is not occupied by retinoid ligand (see Fig. 6), its AF-2 does play an active role in transcriptional stimulation. A similar conclusion has also been reached recently by two other groups studying RXR-RAR action (Chen et al. 1996, Schulman et al. 1996), with the use of RAR-specific ligands precluding ligand binding by the RXR partner. However, Schulman et al. (1996) have introduced a caveat to the above theory as they point out that AF-2-truncated RXRs in heterodimers become strong, constitutive binders of corepressors like the silencing mediators of retinoid and thyroid hormone receptors (SMRTs). Thus, an alternative explanation to an active coactivator-binding role for RXR AF-2 in heterodimers is that it plays a more passive role in excluding corepressors. In this latter scenario, truncation or point mutation (Schulman et al. 1996) of RXR AF-2 generates spurious corepressor binding rather than compromising coactivator contact. Only additional research into coactivator and corepressor associations of VDR-RXR heterodimers will resolve this issue.

General mechanism for vitamin D hormone action on transcription

In order to provide a working hypothesis for l,25(OH)2D3 action at the molecular level, we have developed the model illustrated in Fig. 8. It is based primarily on data from our laboratory and others studying 1,25(OH)2D3 and VDR, and it relies on the assumed similarities between VDR action and that of TR and RAR. VDR is proposed to exist in target cell nuclei, perhaps very weakly associ¬ ated with DNA, in a monomeric, inactive conformation with the C-terminal AF-2 domain extended away from the hormone binding cavity. Upon liganding with l,25(OH)2D3, VDR assumes an active conformation, with the AF-2 pivoted into correct position for both ligand retention and coactivator contact. In addition, the hormone facilitates interaction of VDR and RXR through a stabilized heterodimerization interface. In turn, 1,25(OH)2D3-occupied VDR may itselffunction as a kind of allosteric regulator of RXR, perhaps by conveying a confonnational signal through the juxtapositioned dimer¬ ization domains to induce the AF-2 ofRXR into an active conformation for coactivator binding. As discussed above (see Fig. 6), the joining of preliganded VDR and unliganded RXR apparently renders the RXR partner unresponsive to binding and either activation or dissocia¬ tion by 9-cis RA. Alternatively, if 9-cis RA encounters RXR monomer first (Fig. 8), or binds to RXR that is complexed with VDR in an apoheterodimer (Fig. 6), the retinoid is able to divert the RXR to generate homo¬ dimers and effectively blunt l,25(OH)2D3-driven transcription In the primary activation pathway pictured in Fig. 8, the RXR-VDR-l,25(OH)2D3 complex recognizes and targets the genes to be controlled through high affinity association with the VDRE in a gene promoter region. Coactivators that are presumed to bind to VDR and RXR AF-2 s are then postulated to link with TAFs/TBP, thereby looping out DNA 5′ of the TATA box. This series of events positions VDR such that it can independently recruit TFIIB to the promoter complex, a process that initiates the assembly of the RNA polymerase II holoenzyme into the preinitiation complex. Precedents exist for transcription factors independently attracting TFIIB, such as hepatocyte nuclear factor-4 (Malik & Karathanasis 1996), as well as for a sequential, two-step pathway for activator-stimulated transcriptional initiation (Struhl 1996, Stargell & Struhl 1996). Using the latter model as an analogy, the VDR activator would contact both TBP/ TAFs (via – coactivator bridges) and TFIIB in order to initiate RNA polymerase II holoenzyme assembly. The order of attachment of these two ‘arms’ of activation has not been determined but, at least, in the case of acidic activators, recruitment to the TATA element precedes interaction with components of the initiation complex (Stargell & Struhl 1996). It is of interest that the mechan¬ ism of l,25(OH)2D3 action depicted in Fig. 8 is not only essential for induction of bone remodeling and other vitamin D functions, but is also self-limiting via 24-OHase induction. In addition, these actions of l,25(OH),D, would be blunted under conditions within a cell where 9-cis RA concentrations dominate over those of l,25(OH)2D3.

Figure 8 Model for transcriptional activation by 1,25(OH)2D3 on the promoter of a target gene

The above described molecular mechanism whereby the vitamin D hormone controls gene expression requires further experimental evaluation. To advance our under¬ standing of the structure/function relationships in VDR, a physical characterization of the structure of VDR via X-ray crystallography will be required. Furthermore, in order to comprehend the genomic action of vitamin D in calcium homeostatic and other target cells, it will be necessary to elucidate the detailed involvement of various RXR isoforms, specific TAFs and novel coactivators/ corepressors that might influence the regulation of differ¬ ent vitamin D-controlled promoters. This information in its entirety should assist in determining the potential role for VDR and l,25(OH)2D3 in the pathophysiology of osteoporosis and other endocrine-related bone diseases.

References

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Barettino D, Ruiz MdMV & Stunnenberg HG 1994 Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBOJournal 13 3039-3049.

Blanco JCG, Wang I-M, Tsai SY, Tsai MJ, O’Malley BW, Jurutka PW, Haussler MR & Ozato 1995 Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. Proceedings of the National Academy of Sciences of the USA 92 1535-1539.

Blanco JCG, Dey A, Leid M, Minucci S, Park B-K, Jurutka PW, Haussler MR & Ozato 1996 Inhibition of ligand induced promoter occupancy in vivo by a dominant negative RXR. Genes to Cells 1 209-221.

Bourguet W, Ruff M, Chambón , Gronemeyer & Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-a. Nature 375 377-382.

Cai Q, Hodgson SF, Kao PC, Lennon VA, Klee GG, Zinmeister AR & Kumar R 1994 Inhibition of renal phosphate transport by a tumor product in a patient with oncogeneic osteomalacia. New England Journal of Medicine 330 1645-1649.

Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J & Teitelbaum SL 1993 Cloning of the promoter for the avian integrin ß3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3. Journal of Biological Chemistry 268 27371-27380.

Carlberg C, Bendik I, Wyss A, Meier E, Sturzenbecker LJ, Grippo JF & Hunziker W 1993 Two nuclear signalling pathways for vitamin D. Nature 361 657-660.

more…

 

The structure of the nuclear hormone receptors.
  • 1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, 77555-0645, USA.

Steroids. 1999 May;64(5):310-9.   http://www.ncbi.nlm.nih.gov/pubmed/10406480

The functions of the group of proteins known as nuclear receptors will be understood fully only when their working three-dimensional structures are known. These ligand-activated transcription factors belong to the steroid-thyroid-retinoid receptor superfamily, which include the receptors for steroids, thyroid hormone, vitamins A- and D-derived hormones, and certain fatty acids. The majority of family members are homologous proteins for which no ligand has been identified (the orphan receptors). Molecular cloning and structure/function analyses have revealed that the members of the superfamily have a common functional domain structure. This includes a variable N-terminal domain, often important for transactivation of transcription; a well conserved DNA-binding domain, crucial for recognition of specific DNA sequences and protein:protein interactions; and at the C-terminal end, a ligand-binding domain, important for hormone binding, protein: protein interactions, and additional transactivation activity. Although the structure of some independently expressed single domains of a few of these receptors have been solved, no holoreceptor structure or structure of any two domains together is yet available. Thus, the three-dimensional structure of the DNA-binding domains of the glucocorticoid, estrogen, retinoic acid-beta, and retinoid X receptors, and of the ligand-binding domains of the thyroid, retinoic acid-gamma, retinoid X, estrogen, progesterone, and peroxisome proliferator activated-gamma receptors have been solved. The secondary structure of the glucocorticoid receptor N-terminal domain, in particular the taul transcription activation region, has also been studied. The structural studies available not only provide a beginning stereochemical knowledge of these receptors, but also a basis for understanding some of the topological details of the interaction of the receptor complexes with coactivators, corepressors, and other components of the transcriptional machinery. In this review, we summarize and discuss the current information on structures of the steroid-thyroid-retinoid receptors.

 

 Cellular retinoid-binding proteins.
Ong DE1.  Author information
Arch Dermatol. 1987 Dec;123(12):1693-1695a.

A number of specific carrier proteins for members of the vitamin A family have been discovered. Two of these proteins bind all-trans-retinol and are found within cells important in vitamin A metabolism or function. These two proteins have considerable sequence homology and have been named cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II (CRBP [II]). A third intracellular protein, cellular retinoic acid-binding protein (CRABP) also is structurally similar but binds only retinoic acid. Although retinol appears to be bound quite similarly by the two retinol-binding proteins, subtle differences are apparent that appear to be related to the different functions of the two proteins. That, coupled with the specific cellular locations of the two proteins, suggests their roles. Cellular retinol-binding protein appears to have several roles, including (1) delivering retinol to specific binding sites within the nucleus and (2) participating in the transepithelial movement of retinol across certain blood-organ barriers. In contrast, CRBP (II) appears to be involved in the intestinal absorption of vitamin A and, in particular, may direct retinol to a specific esterifying enzyme, resulting in the production of fatty acyl esters of retinol that are incorporated into chylomicrons for release to the lymph. Like CRBP, CRABP can deliver its ligand retinoic acid to specific binding sites within the nucleus, sites different from those for retinol. The nuclear binding of retinol and retinoic acid may be part of the mechanism by which vitamin A directs the state of differentiation of epithelial tissue.

 

Interaction of the Retinol/Cellular Retinol-binding Protein Complex with Isolated Nuclei and Nuclear Components
GENE LIAU, DAVID E. ONG, and FRANK CHYTIL
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
http://jcb.rupress.org/content/91/1/63.full.pdf

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding protein (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei . CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [ 3H]retinol-CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei . Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin . The binding of retinol to these sites may affect gene expression.

Early histological studies have clearly shown that when animals become vitamin A deficient various epithelial tissues of these animals lose the ability to maintain proper differentiation (1) . However, providing retinol (vitamin A alcohol) to the animal permits tissue repair, with improperly differentiated cells rapidly replaced by normal cells (2) . This indicates that vitamin A has an essential role in cellular differentiation . The action of retinol appears to be mediated by a specific intracellular protein called cellular retinol-binding protein (CRBP). CRBP binds retinol with great avidity and specificity and has been detected in a number oftissues (3, 4) . Recently, CRBP was purified and partially characterized (5, 6) . It is distinct from the well-known serum retinol transport protein called retinol-binding protein (5, 7) . That CRBP plays an important role in the action of vitamin A is suggested by the following observations: It is found complexed with retinol in vivo (4, 8). It binds cis-isomers of retinol with a specificity that parallels the in vivo activity of these isomers (9), Finally, if retinol is first complexed with CRBP, the retinol can bind to the nucleus in a specific and saturable manner (10) . In this study we compare the interaction of the CRBP-retinol complex with isolated nuclei to its interaction with isolated chromatin and follow the fate of both the protein and the ligand . The nuclear binding sites for retinol were localized using autoradiography .

……

The experiments described here were designed to gain insight concerning the still unknown molecular mechanisms by which retinol exerts its effects on the differentiation of epithelia. Alterations in genomic expression appear to be induced in animals fed a retinol-deficient diet, as shown by changes in nuclear RNA synthesis observed in vivo (27-30) as well as in vitro (13) . A working hypothesis has been used that retinol, being a small molecule, might exert its action in a way similar to the accepted model for the mode of action of steroid hormones in differentiation . This model involves binding of the steroid hormone inside the target cell to a specific binding protein called a receptor . The resulting cytoplasmic ligand receptor complex, after undergoing a not fully understood conformational change, translocates to the nucleus . The receptor protein can then be detected in nuclear extracts by its ability to bind specifically the steroid hormone. The receptor steroid complex has been shown to interact with chromatin. Such interaction is believed to lead to an altered expression of the genome, which is the basis for the steroid hormone-induced differentiation (31) .

The steroid hormone model has been used profitably to investigate the mode of retinol action . Indeed, a specific binding protein for retinol, CRBP, was discovered to be present in many tissues (3) . Moreover, after purifying this protein to homogeneity, it was demonstrated that CRBP is able to deliver retinol to the nucleus in a specific manner (10) .

However, we report here a unique feature which appears to be distinct from the steroid hormone model. Using retinol CRBP complex in which the radioactive label is on the protein, we find that CRBP delivers retinol in a specific manner to the nucleus; the retinol associates with chromatin, but the protein itself does not remain bound. This conclusion is based on the observation that the radioactively-labeled protein is still able to deliver retinol inside the nucleus, but it cannot be recovered with the nucleus, in contrast to steroid hormone receptors.

The interaction of the specifically delivered retinol appears to be primarily with chromatin. The outer nuclear envelope is apparently not significantly involved in the interaction as Triton X-100-treated nuclei retain 70% of the retinol binding sites found in intact nuclei. It is still possible that the isolated chromatin and the Triton-treated nuclei contain some of the nuclear matrix and that it is actually the matrix which contains the specific binding sites for retinol. However, preliminary evidence indicates that the specific binding sites remain with a soluble chromatin preparation prepared by mild nuclease digest of nuclei rather than with the nuclear matrix. That the CRBP is necessary for delivering retinol to the nucleus is clearly documented by autoradiography. Free retinol, not bound to CRBP, binds nonspecifically to the nuclei, and to chromatin, and autoradiography shows indiscriminate localization of retinol in the lipid-rich nuclear membrane areas.

The data presented here invite the proposal that the retinolCRBP complex enters the nucleus in some manner which is apparently not dependent on the nuclear membrane. The complex then recognizes a limited number (generally an order of magnitude greater than for steroid hormones) of specific sites on the chromatin where the transfer of retinol from CRBP to these sites takes place. The sites were not detectable and may not be accessible if the retinol is free from CRBP. After the transfer CRBP does not remain associated with the specific sites . The functional significance of the specific interaction between retinol and chromatin remains to be demonstrated .

 

Inhibition of vitamin D receptor-retinoid X receptor-vitamin D response element complex formation by nuclear extracts of vitamin D-resistant New World primate cells.
Most New World primate (NWP) genera evolved to require high circulating levels of steroid hormones and vitamin D. We hypothesized that an intracellular vitamin D binding protein (IDBP), present in both nuclear and cytoplasmic fractions of NWP cells, or another protein(s) may cause or contribute to the steroid hormone-resistant state in NWP by disruption of the receptor dimerization process and/or by interference of receptor complex binding to the consensus response elements present in the enhancer regions of steroid-responsive genes. We employed electromobility shift assay (EMSA) to screen for the presence of proteins capable of binding to the vitamin D response element (VDRE). Nuclear and post-nuclear extracts were prepared from two B-lymphoblastoid cell lines known to be representative of the vitamin D-resistant and wild type phenotypes, respectively. The extracts were compared for their ability to retard the migration of radiolabeled double stranded oligomers representative of the VDREs of the human osteocalcin and the mouse osteopontin gene promoters. A specific, retarded band containing VDR-RXR was identified when wild type cell but not when vitamin D-resistant cell nuclear extract was used in the binding reaction with either probe. In addition, vitamin D-resistant cell nuclear extract contained a protein(s) which was bound specifically to the VDRE and was capable of completely inhibiting VDR-RXR-VDRE complex formation; these effects were not demonstrated with nuclear extract from the wild type cell line or with the post-nuclear extract of the vitamin D-resistant cell line. We conclude that a VDRE-binding protein(s), distinct from IDBP and present in nuclear extract of cells from a prototypical vitamin D-resistant NWP, is capable of inhibiting normal VDR-RXR heterodimer binding to the VDRE.
Reversing Bacteria-Induced Vitamin D Receptor Dysfunction to Treat Chronic Disease: Why Vitamin D Supplementation Can Be Immunosuppressive, Potentially Leading to Pathogen Increase
by J.C. Waterhouse, PhD
Recent attempts to increase vitamin D supplementation to prevent and treat chronic disease have arisen primarily out of observations of low vitamin D levels (25-D) being associated with a variety of diseases. However, new research indicates that these low vitamin D levels are often the result rather than the cause of the disease process, just as in the autoimmune disease, sarcoidosis. Trevor Marshall, PhD, recently summarized this alternative perspective on vitamin D, in a session he co-chaired at the 6th International Congress on Autoimmunity. He and his colleagues presented in silico* and clinical data from the last eight years, indicating that intraphagocytic bacteria are able to block the vitamin D receptor (VDR), and this leads to abnormally low measured vitamin D levels. A second consequence of the bacteria-induced VDR blockage is inhibition of innate immunity. By blocking the VDR, bacteria are able to cause persistent infection and inflammation and thus cause many chronic diseases. Short-term symptom reduction observed from vitamin D supplementation appears to be due to immune suppression by precursor forms of vitamin D that add to the bacterial blockage of the VDR. In silico data also indicates that high levels of vitamin D metabolites suppress antimicrobial peptide production by binding to other nuclear receptors (e.g., thyroid-alpha-1, glucocorticoid). Increasingly, epidemiological, geographical and clinical data are lending support to this model of disease. Studies using more advanced cell culture and molecular techniques are confirming the presence of previously undetected bacteria, including biofilm and cell wall deficient bacteria, as well as “persisters.” A greater understanding of how bacteria resist standard antibiotic approaches is also being gained. A protocol has been developed that is successfully restoring VDR and innate immune function with a VDR agonist and eliminating pathogens with low-dose, pulsed combinations of antibiotics. Immunopathological reactions (a.k.a., Jarisch-Herxheimer reactions) occur due to increased pro-inflammatory cytokines resulting from bacterial killing. The result is an exacerbation of symptoms with each dose of antibiotic, but improvement occurs over the long-term. Remission is being achieved in numerous chronic conditions, including many autoimmune diseases and fibromyalgia, as well as many diseases of aging. Although vitamin D ingestion is avoided as part of this protocol, the evidence indicates that the net result of the protocol is improved vitamin D receptor activation.

Introduction
Vitamin D is a topic of increasing interest and has been implicated in many physiological processes beyond its initially recognized role in calcium absorption and metabolism.1 Vitamin D is found in supplements and a few foods (e.g., fish, liver, egg yolk, fortified products). The majority of vitamin D is produced in the skin when exposed to UV radiation from sunlight. But some have begun advocating consumption of levels of vitamin D above the RDA, and some advocate very high levels, ranging from 1,000 to 5,000 IU or more daily.2 Vitamin D is a secosteroid, with a close resemblance in structure to immunosuppressive steroids. Levels of the various vitamin D metabolites are the result of complex feedback mechanisms involving multiple enzymes and receptors, indicating that it is regulated more like a steroid than a nutrient.1

Short-term symptom reduction has sometimes been observed through increases in sun exposure 3,4 or vitamin D supplementation.5 However, this appears to be due to the anti-inflammatory effect arising from immune suppression, analogous to the effect of a steroid, such as prednisone. If one were to assume that the inflammation is purely pathological, this might be considered beneficial, but evidence that has been accumulating over many decades indicates that inflammation in most chronic diseases is occurring in response to undetected chronic bacterial infection (see below). Since immune suppression can promote the increase of pathogens, the effect of vitamin D supplementation is not likely to be harmless in this situation, but appears to have long-term effects associated with increased levels of bacterial pathogens. The role of this microbiota in producing the inflammation and oxidative stress observed in so many diseases will be discussed near the end of this article.6-8

Vitamin D from food or sun is first converted to 25-D (25-hydroxyvitamin-D) and then converted in a second step to the active 1,25D form (1,25-dihydroxyvitamin-D) that is able to activate the vitamin D receptor (VDR). The type of vitamin D usually measured in the blood is the precursor form, 25-D, rather than 1,25-D, the form that activates the receptor. Activation of the vitamin D receptor is extremely important, as it has numerous effects, including effects on the immune system1 and cancer.9,10 However, recent research indicates that increasing vitamin D via supplementation or sun exposure is not the way to achieve more VDR activation in chronic disease, due to blockage of the VDR by bacterial products.6 This insight has been put to use in a new model of chronic disease and a new protocol.6,8,11-14

A New Perspective on Vitamin D and a New Treatment Approach

Trevor Marshall, PhD, (Murdoch University, Australia) has developed a model of chronic autoimmune and inflammatory diseases in which intraphagocytic bacteria cause disease by producing a substance that binds to and blocks the VDR.1 One such substance has been already identified providing proof of principle.1

  • The VDR is important for adequate innate immune function, including the production of numerous antimicrobial peptides.15

These include

  1. cathelicidin and
  2. beta-defensin,

two of the body’s own arsenal of internally produced antibiotics.

Thus, VDR blockage would seem to be an excellent bacterial strategy, as it would lead to poor innate immune system function and further growth of bacteria and other pathogens. A functioning VDR also appears to be important in controlling cell growth and metastasis, so as to help prevent and control cancerous growths.9,10

A protocol based on this model of disease has been achieving a high rate of improvement/remissions in a wide array of conditions.6,11-14,16-18 It involves the use of

  • a VDR agonist, olmesartan, which is able to activate the VDR effectively and safely.

In addition, low dosages of combinations of select pulsed antibiotics are used to eliminate the bacteria, which also helps restore VDR functioning. The protocol also involves avoidance of vitamin D supplementation. When faced with VDR dysfunction, the evidence indicates that

  • attempting to increase 25-D only adds to the dysregulation of the vitamin D metabolites without being able to adequately overcome the bacteria-induced VDR blockage.6,8

Too much vitamin D can be harmful in two ways, according to Marshall’s work.1,6

  1. In silico data from highly sophisticated molecular modeling shows that high vitamin D levels can block the VDR and thus block innate immune function.18 In addition,
  2. high levels of various vitamin D metabolites can affect thyroid-alpha-1, glucorticoid, and androgen receptors and disrupt hormonal control and further affect innate immune function.1

Thus, any short-term symptom reduction from high levels of vitamin D that may occur is probably occurring at the cost of long-term pathogen increase. This has been supported by observations of patient’s responses over time. In the short-term, even for ten years or more in some cases, the person may feel better with high vitamin D intake. But in the long-term, the chronic infection progresses, because the high 25-D is only adding to the bacterial blockage of the VDR and the suppression of bacterial killing.18

Symptoms increase when the immune system is better able to kill the pathogens, due to the high levels of inflammatory cytokine levels that occur. This is called the immunopathological reaction or Jarisch-Herxheimer reaction.6,11 The symptoms range from pain and fatigue to cognitive impairment and depression, but include numerous other symptoms characteristic of the underlying inflammatory condition.6,11 By suppressing the immune response, vitamin D supplementation may suppress these symptoms in the short-term and may even result in a sort of dependence on vitamin D supplementation or sun exposure to keep the symptoms at bay.

The long-term efficacy of the protocol (sometimes called the Marshall Protocol or MP) in activating the VDR is also supported by improved or stabilized bone density, which is typical in patients on the protocol, if the RDA of calcium is consumed. The protocol replaces vitamin D supplementation with use of the VDR agonist olmesartan (120 to 160 mg in divided doses) and reduces the level of bacteria blocking the VDR with antibiotics and, in this way, is apparently effective in activating the VDR.6,12

Marshall proposes that vitamin D receptor blockage results in the low levels of 25-D that have been observed in numerous diseases. The precursor, 25-D form is the form that is most frequently measured. The VDR blockage typically leads to dysregulation of metabolite levels, and one effect is down-regulation of the conversion of vitamin D to 25-D.1 Thus, according to this perspective, low 25-D levels are the result, not a cause, of the disease process. It follows that a low serum 25-D is not indicative of a true vitamin D deficiency in this situation. Both laboratory19 and clinical findings20 have supported the existence of an apparently similar type of down-regulation of conversion to 25-D.

At the same time that low 25-D is observed, high 1,25-D levels are also usually observed. In fact, elevated 1,25-D has been shown to be a good indicator of inflammatory and autoimmune disease.13,16 When interpreting the results, however, it should be remembered that samples must be frozen until analyzed for accurate 1,25-D results. And occasionally, in cases of quite advanced disease or elderly patients, 1,25-D will be low as well, yet still be consistent with VDR blockage and inflammatory disease.21

Marshall’s protocol was first used to treat sarcoidosis. It is well established that a dysregulation of vitamin D levels, often with very high 1,25-D and low 25-D, occurs in this condition.22 Marshall’s and other’s work has confirmed that this dysregulation also occurs in a wide range of other diseases.12,13,23,24 This pattern of high 1,25-D and low 25-D also exists in VDR knockout mice.25 These mice are genetically engineered to lack a VDR, a situation analogous to a bacteria-blocked VDR.

The very complex relationships among genes, metabolites, enzymes, and receptors that Marshall recently summarized1,6 show that vitamin D is not a mere nutrient. In fact, the active form is a secosteroid transcriptional factor. It is part of a highly regulated and complex system influencing many aspects of metabolism and immune function. There are several feedback and feedforward pathways that influence the levels of various vitamin D forms that Marshall reviewed in depth.1

Marshall was recently invited to co-chair a session on vitamin D at the 6th Annual International Autoimmunity Conference, and he gave one of the keynote presentations of the session.6 Several other presentations were given that support the protocol and model. For example, Perez presented data on treatment response in 20 autoimmune conditions that support Marshall’s model.11 The autoimmune diseases successfully treated in this open-label trial include rheumatoid arthritis, systemic lupus erythematosis, diabetes type 1 and 2, psoriasis, Hashimoto’s thyroiditis, Sjogren’s syndrome, scleroderma, uveitis, myasthenia gravis, and ankylosing spondylitis. Chronic fatigue syndrome and fibromyalgia were shown to respond to the protocol in another presentation.17 And another study indicated that dysregulation of nuclear receptors in the endometrium by vitamin D, along with chronic bacterial infection, can help explain the higher prevalence of some autoimmune diseases in women.26

Epidemiological and Short-Term Clinical and Experimental Data
The in silico and clinical data discussed above provide strong evidence for Marshall’s model, and some might argue it is more reliable than epidemiological and short-term evidence. It is widely recognized that there are many limitations inherent in epidemiological and short-term experimental data due to difficulties in obtaining relevant and accurate results. Confounding factors and the inability to assess the effects of long-term immune suppression from high levels of vitamin D make the results less reliable.13,21 Experiments using animal models have the problem of genetic differences and different disease causation methods.1,13Studies of supplementation are often not randomized and thus are subject to unknown confounding factors that may affect the choice to take vitamin D supplements.13 Furthermore, sun exposure is hard to quantify and is often left out of the analyses. Any of the above can lead to invalid conclusions.

Despite this, a number of recent studies that may be relevant will be discussed here to show that there is much independent support for Marshall’s model among these types of studies. In addition, some lesser-known aspects of some of the studies used to support a high vitamin D intake will be reviewed, which cast doubt on some of their conclusions.

Cancer and All-Cause Mortality
In the case of cancer prevention, a recent randomized controlled trial of calcium and vitamin D by Lappe et al.27 is used to support vitamin D supplementation. However, it has a number of serious limitations. One problem is the assumption that removing the data from the first year is justified. If one looks at Figure 1, in the article by Lappe et al,27 in which the data from the first year was included, there is very little difference between calcium and vitamin D vs. calcium alone throughout the study period. No group of patients was given vitamin D alone. Also, there is not yet long-term data on incidence, since the study lasted only four years. Any reduced incidence may reflect delay in diagnosis. In addition, long-term survival may not ultimately improve. In fact, patients taking vitamin D might even die sooner (see below). In addition to the above critique, a number of published comments have also taken issue with this trial, pointing to other problems and limitations.9,28

Another recent study29 reported finding barely significant lower cancer rates in premenopausal women (95% confidence interval, 0.42-1.0) who consumed more vitamin D. However, they found a marginally significant higher rate of moderately differentiated tumors in postmenopausal women who had higher vitamin D intake. And since postmenopausal women make up a much higher proportion of breast cancer cases, this is particularly concerning. This is just one example of the rather inconclusive, mixed data on vitamin D supplementation that becomes apparent when the vitamin D studies are looked at as a whole (see Discussion section in ref. 29). Even the benefit for premenopausal women is questionable. Bertone-Johnson et al.30 pointed out a quite plausible rationale for the existence of a bias toward low estrogen in those who choose to take vitamin D supplements.

A number of limitations found in the other studies are used as a basis for supporting vitamin D supplementation. For instance, the data is rarely long-term enough and rarely covers all the effects possible. Although there may be an appearance of benefit in the short-term or for subsets of the populations studied, a large, long-term prospective study showed no effect of 25-D on the overall cancer mortality rate in the long-term.31 Freedman et al.31 even showed a suggestion of a negative effect of higher vitamin D levels. There was a non-significant increase in overall mortality in the two groups with 25-D at higher levels (80 to <100 nmol/L: Risk Ratio = 1.21, 95% CI =0.83 to 1.78; =100 nmol/L: Risk Ratio = 1.35; 95% CI = 0.78 to 2.31, where 100 nmol/L corresponds to about 40 ng/ml).

This is in accord with a study in prostate cancer32 (also see discussion in ref. 21) and one in pancreatic cancer33 that found higher cancer rates when 25-D was high. Cancer rates increased among patients with a 25-D level above approximately 32 ng/ml. Evidence regarding solar radiation and geographical/latitudinal analyses are also used as evidence, yet solar radiation has many other effects besides raising 25-D.34,35 Many other relevant factors, such as pathogen distributions, climate effects on pathogen spread36,37 and host susceptibility,38 diet, and pollution levels also vary with geographical location.

It was recently pointed out in the Bulletin of the World Health Organization that high 25-D has been found to be associated with greater cancer risk in some studies.39 Studies mentioned, included one that found that there was a higher rate of many internal cancers in patients who have a type of skin cancer that is considered to be the best indicator of long-term sun exposure.40 Another study discussed failed to find a geographical pattern that would support a protective effect of increased 25-D.41 On the whole, in these epidemiological studies, the data is mixed and inconsistent, which is to be expected when there are so many unknown confounding factors affecting 25-D levels and disease incidence that may bias the results.13 In addition, a recent large prospective study presented evidence suggesting that circulating 25-D concentrations may be associated with increased risk of aggressive prostate cancer.42 For all types of prostate cancer, the data failed to support the hypothesis that higher vitamin D decreases prostate cancer risk.42

Studies looking at overall mortality benefits of vitamin D are sometimes misleading at first glance. In the large meta-analysis done recently on the effect of vitamin D and calcium on mortality rates,43 the abstract attributes reduced mortality to vitamin D, yet the only statistically significant results were for calcium together with vitamin D. Another serious problem is that most of the studies analyzed in the meta-analysis were only a few years in duration, so long-term effects on mortality and morbidity could not be accurately assessed.

Bone Density, Parathyroid Hormone
Another area that should be re-evaluated is the negative association between parathyroid hormone and 25-D levels. This association is often used to assert that high levels of 25-D (e.g., 40 –50 ng/ml or more) are optimal. Aloia et al.44 has pointed out that the studies that conclude these high levels of vitamin D are needed fail to require adequate calcium intake, and that is why such high levels are suggested. It should also be considered whether both low 25-D and high PTH are due to the disease process rather than the low 25-D causing the elevated PTH. In addition, only a small percentage of patients with low 25-D have elevated PTH. The low 25-D may be indicating a systemic chronic bacterial infection, and the abnormally high PTH levels in a small percentage of patients may merely be pointing to those cases in which bacteria have infected the parathyroid gland to a greater degree.

In a study comparing vitamin D supplementation with calcium supplementation,45“the effect of calcium on bone loss was blunted in subjects with the highest levels of serum 25OH vitamin D [25-D].” This last finding is supportive of Marshall’s in silico work indicating that high 25-D actually blocks the VDR.6,18 The largest meta-analysis so far clearly showed benefit from calcium supplementation; however, benefit for vitamin D was much less clear.46 No significant benefit for fracture risk was found when comparing vitamin D and calcium to calcium alone, though some differences were found between vitamin D levels.

Another factor that needs to be considered is whether immune suppression is the cause of bone density improvement when high vitamin D levels are used. Immunosuppressive drugs that lower TNF-alpha using antibodies can improve bone density by reducing inflammation.47 High levels of vitamin D supplementation can also lower TNF-alpha48 and suppress the immune response. Thus, it is possible that an increase in bone density from vitamin D supplementation could be the result of immune suppression via TNF reduction, rather than correction of a vitamin D deficiency. TNF-lowering drugs such as infliximab (Remicade) increase risk of cancer and tuberculosis. Thus, the desirability of improving bone density through immune suppression is questionable. This immunosuppressive effect of vitamin D may even explain what seems to be a beneficial effect on falls and muscle strength of elevating vitamin D through supplementation.21 This may be only a symptom reduction in the short-term and may be harmful in the long-term due to the immune suppression.

Autoimmune Disease
In the area of autoimmune disease, the data is equally mixed, and sometimes the larger, more recent studies fail to show any effect of vitamin D levels. For example, a recent large study failed to find an association between serum 25-D levels and the incidence of systemic lupus erythematosis and rheumatoid arthritis.49 Research has found that the average age at which patients acquired rheumatoid arthritis is 12 years earlier in Mexico than in Canada and pointed to the possible role of infectious agents in causing the disease.50 And clearly this study does not support the idea that sun exposure is beneficial for rheumatoid arthritis, since Mexico gets far more sun than Canada.

Although some studies in type 2 diabetes have indicated vitamin D supplementation may be preventive,51 these studies were not randomized and thus are subject to many known and unknown confounding factors affecting a parent’s decision to give a child supplemental vitamin D.13 And even if it were clearly established that vitamin D supplementation reduced the incidence of diabetes in infants and small children, that would not mean that it would help in established disease or older patients, nor would it necessarily mean it is the optimal way to achieve diabetes prevention and long-term health. The positive response of both type 1 and type 2 diabetes patients to the Marshall Protocol11 indicates research on the role of bacteria in diabetes should be a priority.

Influenza and Colds
It has been proposed that vitamin D levels’ decline in winter best accounts for the seasonality of colds and influenza52 and that this potentially supports the need for increased supplementation.52,53 However, new evidence indicates that changes in the viral coat properties can account for the seasonal outbreaks at higher latitudes.36,37 Effects on the airways in dry, cold climates also appear to increase susceptibility to viral and bacterial infections in winter and could contribute to higher winter prevalence of respiratory infections in cold climates.38

Another important point is that the patients being followed on the Marshall Protocol include a number of individuals who report that during the worst period of their chronic illness, they had few or no colds or flu-like illnesses, sometimes for many years at a time. And sometimes this low rate of colds was apparent even years before their illness. This has also been reported in Parkinson’s disease, with the decrease in viral respiratory infections also occurring several years before the disease was diagnosed.54 Thus, even if future research were to establish that vitamin D supplementation reduced colds and influenza, this is by no means an adequate argument for its use. The above observations in chronically ill patients indicate that observing a reduction in respiratory viral infections is not always a sign of good overall health.

Indications of Long-Term Negative Effects of Vitamin D Supplementation
Brannon et al.55 pointed out in a recent report from a roundtable discussion of vitamin D data needs that many studies so far have not yet adequately investigated potential negative consequences such as soft tissue calcification. Vitamin D has been implicated in arterial calcification in the past56 as well as other negative effects.13 The report by the roundtable of vitamin D experts expressed concern that many studies may be shortsighted with regard to adverse outcomes.55

A disturbing new study showed a highly significant correlation (p=0.007) between increased vitamin D intake from food and supplements and the volume of brain lesions shown by MRI in elderly adults.57 In the multivariable regression model, vitamin D intake retained its significant correlation with brain lesion volume even after the effects of calcium were statistically removed. However, calcium did not retain a significant independent correlation with the lesions when the study controlled for vitamin D. Thus, the analysis points to vitamin D supplementation as the key factor in higher lesion volume in this study. These types of brain lesions have been linked to adverse effects in many studies, e.g., stroke,58 psychiatric disorders,59,60 brain atrophy,61 and earlier death.62 Interestingly, the levels of vitamin D intake were not particularly high by some standards, with the highest intake estimated at 1015 mg daily (mean of 341 mg), about half coming from supplements and the rest from food.

The correlation between vitamin D intake and brain lesions seems to lend further support to Marshall’s work. In another study, the finding that over a three-year period, a small percentage of patients were found to have a slight regression of their brain lesions,63 leaves room for hope that the lesions are potentially reversible. Reversibility would be in accord with the improvement of depression and cognitive deficits and other neurological symptoms reported in patients on the Marshall Protocol.6,64

Elusive Bacterial Pathogens Are Detected with Improved Methods
Over many decades, researchers have reported evidence that hard-to-detect bacterial infections are the cause of many diseases,65,66 including autoimmune disease,65-68 cardiovascular disease,69-71 and even cancer.72-77 Some have noted the recent trend toward finding more infectious causes of disease and suggested this is likely to increase in the coming years.6,71,77-80

Recently, Barry Marshall received the Nobel Prize for discovering that the bacteria Helicobacter pylori causes ulcers. And it is now known that H. pylori is a causal factor in stomach cancer.77

New techniques using 16s ribosomal RNA shotgun sequencing,81,82 as well as more advanced culturing and observational techniques65,66,80,83-85 are suggesting that, up until now, most microbiologists have failed to detect a large percentage of potential disease-causing agents. “Persister” cells have been identified that escape antibiotic treatment.86 Cell wall deficient organisms have long been studied,65-66and just recently, advances have been made in understanding their structure and in culturing techniques.80 Research is also indicating that a bacterial biofilm-like microbiota of multiple species even exists within human cells.6,8

Bacteria that grow on a surface in a multi-species community, protected by both a biofilm and the combined effect of their individual resistance strategies, have been a growing area of research.79 Bacterial biofilms have been found to cause the non-healing ulcers in diabetics and may be successfully treated using novel approaches, thus reducing the need for limb amputation.88

Other examples of studies detecting unexpected bacterial pathogens include work linking pathogens in amniotic fluid to pre-term birth89 and research showing numerous previously undetected species in the biofilms that coat prosthetic hip joints.82 Many species of bacteria have been in wounds that were previously undetected using older techniques.81 Macfarlane et al.90 used a combination of more advanced techniques to study bacteria in biofilm communities in patients with Barrett’s esophagus, a pre-cancerous condition. Their methods revealed significant differences between patients and controls in the types and numbers of bacterial species, differences that were previously undetected using older techniques.

Increasingly, inflammation is observed in chronic diseases ranging from depression to cardiovascular disease and cancer.87 The above trends, when combined with observations of bacteria in numerous diseases6,13,65,66,71,91 and the success of the anti-bacterial protocol developed by Marshall6,8,11,13 suggest an extensive role for previously unidentified chronic bacterial infections.

Research is also supporting the ineffectiveness of most standard antibiotic protocols against these bacteria70 and suggesting why other approaches may work better. For instance, some antibiotics target cell walls, and this actually promotes the production of cell wall deficient forms of bacteria that resist many antibiotics.80 Furthermore, many antibiotics are known to inhibit phagocytosis and other aspects of the immune response when taken at high, constant dosages.92

The ability of bacteriostatic antibiotics such as clindamycin to be effective at low doses has been documented.93,94 The survival of “persister” cells mean that pulsed antibiotics are likely to be more effective.86 And fascinating investigations of biofilm communities have revealed many ways in which bacteria can resist antibiotics when used in traditional ways.95 The existence of communities of many bacterial species means that combinations of antibiotics are probably needed to be effective against all the species present. Thus, there is increasing support for the use of pulsed, low dosages of combinations of bacteriostatic antibiotics as used in the anti-bacterial protocol discussed here.

What is particularly encouraging is that the effectiveness of Marshall’s protocol in many systemic chronic disease indicates that these elusive pathogens do respond to select currently available bacteriostatic antibiotics when innate immune function is restored through restoring vitamin D receptor function.6,11 Not only do the bacterial infections appear to resolve, the evidence so far suggests that the improved immune response leads to reduced viral, fungal, and protozoal infections as well.

Conclusions
In silico and clinical data indicate that it is likely that associations between low vitamin D levels and chronic diseases are not evidence of deficiency, but result from a bacteria-induced blockage of the vitamin D receptor, leading to down-regulation of 25-D levels.1,6 According to this model of chronic disease, the short-term benefits sometimes perceived with high vitamin D levels are not due to correction of a vitamin D deficiency but due to suppression of bacterial killing and the immunopathological reaction that accompanies it. Data on reversal of a range of inflammatory and autoimmune diseases through an anti-bacterial protocol that includes vitamin D avoidance and a VDR agonist support this view.6,11

As discussed in detail above, it appears that increasing vitamin D supplementation is not the answer to these chronic diseases and is likely to be counter-productive. Other researchers have also raised concerns regarding vitamin D supplementation’s potential adverse effects. Potential dangers include increased aortic calcification55,56 and brain lesions shown by MRI57 (also see above). In addition, some studies have even found evidence of increased danger from cancer in association with higher levels of vitamin D.32,33,39,40,42

Many have been attracted to the area of vitamin D research, recognizing interesting patterns and responses to supplementation that at first seemed to indicate widespread deficiency and, at the very least, indicate that vitamin D plays a powerful role in physiological processes. Great strides have been made in the last 30 years by scientists with a range of perspectives, and this has led to great excitement and a laudable commitment to use that knowledge to help patients.

However, new genomic and molecular research and the positive response to a new anti-bacterial protocol that involves the avoidance of vitamin D indicate the need for a reappraisal of the data gathered so far. It appears that attempting to raise 25-D through vitamin D supplementation or sun exposure is not the right approach to many, if not most, common chronic diseases. Instead, as discussed above, the evidence supports the effectiveness of a new protocol in restoring vitamin D receptor function, which appears to be a crucial factor in recovery.

One of the most commendable attributes of a truly objective scientist is the willingness to be open to changing long-held positions in the light of new evidence. It will be interesting to see how many have this all-too-rare quality, as research and discussion of vitamin D and the VDR continues. It is to be hoped that the tremendous healing potential likely to be available from eliminating the pathogens that cause chronic disease will inspire an especially high level of open-minded discussion and cooperation.

Caution: The immunopathological reactions from killing the high levels of bacteria that have accumulated in chronically ill patients can be severe and even life-threatening, and thus the Marshall Protocol must be done very carefully and slowly, according to the guidelines.7,96 For the sake of safety, antibiotics must be started at quite low dosages, starting with only one antibiotic. Health care providers are responsible for the use of this information. Neither Autoimmunity Research, Inc., nor the author assume responsibility for the use or misuse of this protocol.

Note: Neither the author, Prof. Marshall, nor the non-profit Autoimmunity Research, Inc. have any financial connection with any product or lab mentioned with regard to the Marshall Protocol. The information needed to implement the Marshall Protocol is available free of charge fromwww.AutoimmunityResearch.org.

Vitamin D3 and Its Nuclear Receptor Increase the Expression and Activity of the Human Proton-Coupled Folate Transporter

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D3 (vitamin D3) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D3 ex vivo. The PCFTpromoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-α (RXRα) in the presence of vitamin D3. In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region −1694/−1680. DNA binding assays showed direct and specific binding of the VDR:RXRα heterodimer to the PCFT(−1694/−1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(−1694/−1680) motif mediates a transcriptional response to vitamin D3. In functional support of this regulatory mechanism, treatment with vitamin D3 significantly increased the uptake of [3H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D3 and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D3 may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

Folates are water-soluble B vitamins that act as one-carbon donors required for purine biosynthesis and for cellular methylation reactions. They are essential for de novo synthesis of nucleic acids, and thus for production and maintenance of new cells, particularly in rapidly dividing tissues such as bone marrow and intestinal epithelium (Kamen, 1997). Adequate dietary folate availability is especially important during periods of rapid cell division, such as during pregnancy and infancy. Folate deficiency has been associated with reduced erythropoiesis, which can lead to megaloblastic anemia in both children and adults (Ifergan and Assaraf, 2008). Deficiency of folate availability in pregnant women has been linked to neural tube defects, such as spina bifida, in children (Pitkin, 2007). This has prompted the application of folate supplementation schemes either as pills or via fortification of grain products with folates (Eichholzer et al., 2006). Folates have also been proposed to act as protective agents against colorectal neoplasia, although contradictory results have also been reported (Sanderson et al., 2007).

The availability of diet-derived folates is primarily determined by the rate of their uptake into the epithelial cells of the intestine, mediated by the proton-coupled folate transporter (PCFT, gene symbol SLC46A1), localized at the apical brush-border membranes of enterocytes (Subramanian et al., 2008a). PCFT is an electrogenic transporter that functions optimally at a low pH (Qiu et al., 2006;Umapathy et al., 2007). Despite being abundantly expressed in enterocytes, the second folate transporter, termed reduced folate carrier (RFC, gene symbolSLC19A1), has recently been shown not to play an important role in intestinal folate absorption (Zhao et al., 2004; Wang et al., 2005).

The human PCFT gene resides on chromosome 17, contains 5 exons, and is expressed as two prominent mRNA isoforms of 2.1 and 2.7 kilobase pairs (Qiu et al., 2006). Mutations in the PCFT gene have been associated with hereditary folate malabsorption, a rare autosomal recessive disorder (Qiu et al., 2006; Zhao et al., 2007). The PCFT protein is predicted to have a structure harboring 12 transmembrane domains (Qiu et al., 2007; Subramanian et al., 2008a). Although the transport function of PCFT has been studied extensively, relatively little is known about the regulation of PCFT gene expression. PCFT promoter activity has been shown possibly to be epigenetically regulated by its methylation status in human tumor cell lines (Gonen et al., 2008). Furthermore, both the PCFT mRNA expression levels and PCFT promoter activity positively correlate with the level of differentiation of colon-derived Caco-2 cells (Subramanian et al., 2008b).

In addition to its well known roles in regulating calcium homeostasis and bone mineralization, 1,25-dihydroxyvitamin D3 (vitamin D3), the biologically active metabolite of vitamin D, executes many other important functions, particularly in the intestine. For example, vitamin D3 promotes the integrity of mucosal tight junctions (Kong et al., 2008). Many effects of vitamin D3 are mediated via its action as a ligand for the vitamin D receptor (VDR; gene symbol NR1I1), a member of the nuclear receptor family of transcription factors (Dusso et al., 2005). VDR typically regulates gene expression by directly interacting with so-called direct repeat-3 (DR-3; a direct repeat of AGGTCA-like hexamers separated by three nucleotides) motifs within the target promoters, as a heterodimer with another nuclear receptor, retinoid X receptor-α (RXRα; gene symbol NR2B1) (Haussler et al., 1997). Genetic variants of VDR have been associated with inflammatory bowel disease (Simmons et al., 2000; Naderi et al., 2008). Similarly to folates, both VDR and its ligand vitamin D3 have been proposed to be protective against intestinal neoplasia (Ali and Vaidya, 2007). Dietary folate intake has been suggested to regulate gene expression of the components of the vitamin D system, possibly via epigenetic control through the function of folates as methyl donors (Cross et al., 2006). Several intestinally expressed transporter genes, such as those encoding the multidrug resistance protein 1 and multidrug resistance-associated protein 2, have recently been shown to be induced by vitamin D3 (Fan et al., 2009). We investigated whether vitamin D3 regulates the expression of the PCFT gene, encoding a transporter crucial for intestinal folate absorption. The human well polarized enterocyte-derived Caco-2 cells exhibit many of the characteristics associated with mature enterocytes and were used here to investigate the effects of vitamin D3 on PCFT gene expression and folate transport activity.

……..

Vitamin D3 regulates the expression of its target genes primarily by acting as an agonistic ligand for its DNA-binding nuclear receptor VDR, although nongenomic actions by vitamin D3 have also been described previously (Christakos et al., 2003;Dusso et al., 2005). VDR, an important regulator of differentiation and proliferation of enterocytes, typically activates gene expression by heterodimerizing with its nuclear receptor partner RXRα. VDR:RXRα heterodimers then directly bind to DR-3-like elements on the target genes. It should be noted that other modes of VDR-mediated regulation, either via direct interaction with other DNA-binding factors or through nongenomic actions, have also been reported (Dusso et al., 2005).

Here we demonstrate that VDR is a ligand-dependent transactivator of the humanPCFT gene, coding for a vital transporter for intestinal absorption of dietary folates. PCFT mRNA is also abundantly expressed in the liver (Qiu et al., 2006). However, VDR is expressed at very low levels in primary human hepatocytes or hepatocyte-derived cell lines (Gascon-Barre et al., 2003; data not shown), suggesting that VDR-mediated regulation of the PCFT gene may not occur in hepatocytes.

Endogenous PCFT mRNA levels were induced by vitamin D3 in a dose-dependent manner in Caco-2 cells (Fig. 1A). This increase was not further enhanced by cotreatment of cells with the RXRα ligand 9-cis retinoic acid (data not shown), consistent with a previous report that VDR:RXRα heterodimers, at least in some promoter contexts, may not respond to RXRα ligands (Forman et al., 1995). Alternatively, saturating levels of RXRα ligands may already be endogenously present in cells in these experimental conditions. In transient transfection assays, the PCFT promoter fragment −2231/+96 exhibited significant response to exogenous expression of VDR alone in the presence of its ligand (Fig. 2), most probably supported by endogenously expressed RXRα in Caco-2 cells.

Supporting the importance of the VDR:RXRα heterodimer formation for PCFTpromoter regulation, the luciferase values were further significantly elevated upon exogenous expression of RXRα. Exogenous expression of VDR in the absence of vitamin D3 did not notably influence the activity of the PCFT(−2231/+96) promoter, indicating ligand-dependence of VDR action. In deletional transfection analysis, the strongest induction in response to VDR and RXRα in the presence of their ligands was achieved with the PCFT(−2231/+96) promoter fragment (Fig. 3A). Induction of the shortest deletion variant tested [PCFT(−843/+96)luc] was approximately 50% of that achieved for the PCFT(−2231/+96), indicating that this more proximal region is likely to contain further DNA elements mediating a response to vitamin D3. However, in our current study, we focused on the distal region between the nucleotides −2231 and −1674 upstream of the transcriptional start site of the human PCFT gene, which confers maximal response to vitamin D3. In our computational analysis, we identified a putative VDRE within the PCFTpromoter region between nucleotides −1694 and −1680. We have not so far been successful in identifying further binding sites for the VDR:RXRα heterodimer in the more proximal region of the PCFT promoter. It may be that, in addition to direct DNA-binding to the PCFT(−1694/−1680) element identified here, VDR may also affect PCFT promoter activity indirectly, via interactions with other DNA-binding factors. For example, it has been proposed that the p27Kip1 gene is regulated by VDR via response elements for unrelated DNA-binding transcription factors Sp1 and NF-Y (Huang et al., 2004).

Both endogenously expressed and recombinant VDR and RXRα bound to thePCFT(−1694/−1680) element specifically and as obligate heterodimers (Fig. 4). The interaction between VDR and this region of the PCFT promoter within living cells treated with VDR and RXRα ligands was confirmed by chromatin immunoprecipitation tests (Fig. 5). Heterologous promoter assays proved that thePCFT(−1694/−1680) element can function as an independent VDR response element. The significant decrease in VDR:RXRα-mediated induction upon mutagenesis of the PCFT(−1694/−1680) element confirmed that it is an important functional mediator of the effect (Fig. 6, A and B).

Although we observed vitamin D3-mediated increase of rat Pcft mRNA expression ex vivo (Fig. 1C), the rat Pcft promoter (chromosome 10; GenBank accession number NW_047336) exhibits no significant overall homology with the humanPCFT promoter over the proximal 3000-bp regions. This suggests that despite the divergence of the promoter sequences between human and rodent PCFT/Pcftgenes, the functional response to vitamin D3 is conserved.

The activation of PCFT gene transcription by VDR also translates into an increase in PCFT protein function. Vitamin D3 treatment of Caco-2 cells led to significantly increased uptake of folate across the apical membrane, in a dose-dependent manner (Fig. 7). In keeping with the fact that PCFT strongly prefers an acidic milieu for its transport function (Qiu et al., 2006; Nakai et al., 2007; Unal et al., 2009), we only observed vitamin D3-stimulated transport activity at pH5.5, but not at neutral pH. These data strongly suggest that vitamin D3-mediated transcriptional activation of PCFT gene expression leads to an increase of PCFT transport function. Consistent with our model, mRNA expression of the other known folate carrier expressed in Caco-2 cells, RFC, which functions efficiently at neutral pH (Ganapathy et al., 2004; Wang et al., 2004), was not affected by vitamin D3treatment (Fig. 1B). It has been reported that vitamin D3-induced gene expression increases as Caco-2 cells differentiate (Cui et al., 2009). Thus, our current findings on VDR-mediated regulation of PCFT expression provide a possible molecular mechanism for a prior observation that folate uptake into Caco-2 cells is enhanced upon confluence-associated differentiation (Subramanian et al., 2008b).

Our results suggest that intestinal folate absorption may be enhanced by an increase in dietary vitamin D3 intake. Food products are often supplemented with folates, because of their proposed beneficial health effects. Based on our current study, supplementation of vitamin D3 may enhance the intestinal absorption of folates. PCFT also transports the antifolate drug methotrexate (MTX) (Inoue et al., 2008; Yuasa et al., 2009) widely used in the treatment of autoimmune diseases and cancer. MTX interferes with folate metabolism by competitively inhibiting the enzyme dihydrofolate reductase. Our results may further suggest a potential mechanism to increase intestinal absorption of MTX via simultaneous treatment with vitamin D3, thereby affecting the bioavailability of MTX. Patients suffering from inflammatory bowel disease are frequently on long-term treatment with calcium and vitamin D3 as a prophylaxis against osteopenia and osteoporosis (Lichtenstein et al., 2006). This patient group is frequently treated with folates (in the case of folate deficiency) or MTX (as a second-line immunosuppressant) (Rizzello et al., 2002). MTX therapy per se requires prophylactic administration of folates, and these patients often receive additional calcium/vitamin D3. Our current results may warrant a closer investigation into potential drug-drug interactions between pharmacologically administered vitamin D3, MTX, and folates. Taking into account the previous report that folates regulate the expression of genes involved in vitamin D3 metabolism, it may be that folate and vitamin D3 homeostasis are closely interlinked through such mutual regulatory interactions.

 

Innate immune response and Th1 inflammation
http://mpkb.org/home/pathogenesis/innate_immunity

The innate immune response is the body’s first line of defense against and non-specific way for responding to bacterial pathogens.1 Located in the nucleus of a variety of cells, the Vitamin D nuclear receptor (VDR) plays a crucial, often under-appreciated, role in the innate immune response.

When functioning properly, the VDR transcribes between hundreds2 and thousands of genes3including those for the proteins known as the antimicrobial peptides. Antimicrobial peptides are “the body’s natural antibiotics,” crucial for both prevention and clearance of infection.4The VDR also expresses the TLR2 receptor, which is expressed on the surface of certain cells and recognizes foreign substances.

The body controls activity of the VDR through regulation of the vitamin D metabolites. 25-hydroxyvitamin D (25-D) antagonizes or inactivates the Receptor while 1,25-dihydroxyvitamin D (1,25-D) agonizes or activates the Receptor.

Greater than 36 types of tissue have been identified as having a Vitamin D Receptor.5

Another component of the innate immune response is the release of inflammatory cytokines. The result is what medicine calls inflammation, which generally leads to an increase in symptoms.

Before the Human Microbiome Project, scientists couldn’t link bacteria to inflammatory diseases. But with the advent of DNA sequencing technology, scientists have detected many of the bacteria capable of generating an inflammatory response. All diseases of unknown etiology are inflammatory diseases.

Nuclear receptors and ligands

Nuclear receptors are a class of proteins found within the interior of cells that are responsible for sensing the presence of hormones and certain other molecules. A unique property of nuclear receptors which differentiate them from other classes of receptors is their ability to directly interact with and control the expression of genomic DNA. Some of the molecules (or ligands) which bind the nuclear receptor activate (agonize) it and some inactivate (antagonize) it.

It is commonly accepted that most ligands, approximately 95% to 98%, inactivate the nuclear receptors. Since the nuclear receptors play a significant role in the immune response, this factor alone may explain why so many drugs and substances found in food and drink are immunosuppressive.

Because the expression of a large number of genes is regulated by nuclear receptors, ligands that activate these receptors can have profound effects on the organism. Many of these regulated genes are associated with various diseases which explains why the molecular targets of approximately 13% of FDA approved drugs are nuclear receptors.6

Different cell types have different nuclear receptors. One of the nuclear receptors seen in immune cells is the Vitamin D Receptor (VDR). The VDR has two endogenous or “native” ligands, which are also the two main forms of vitamin D in the human body: 25-hydroxyvitamin D (25-D) and 1,25-dihydroxyvitamin D (1,25-D). Non-native or exogenous ligands can also inactivate or activate a nuclear receptor, depending on its molecular structure.

Ligands compete to dock at nuclear receptors. When is a given kind of ligand such as 25-D as opposed to 1,25-D more likely to bind to the VDR? It depends. 1,25-D tends to be much less common than 25-D – by a factor of 1,000 or more – so it binds to the receptor much more infrequently. A greater concentration of a given molecule can displace competing molecules off the nuclear receptor. Affinity occurs in logarithmic fashion, which is to say that it operates on the basis of a sliding scale. In short, an increase in 1,25-D and a decrease in 25-D can tilt the odds in favor of 1,25-D, and vise versa.

Affinity as well as the question of whether a ligand inactivates or activates a nuclear receptor can all be validated using in silicomodeling. Although less precise, it is also possible to measure these properties in vitro.

Activated by 1,25-D and inactivated by 25-D, the Vitamin D nuclear receptor (VDR) transcribes a number of genes crucial to the function of the innate immune response.

Role of Vitamin D Receptor in innate immunity

Vitamin D/VDR have multiple critical functions in regulating the response to intestinal homeostasis, tight junctions, pathogen invasion, commensal bacterial colonization, antimicrobe peptide secretion, and mucosal defense…. The involvement of Vitamin D/VDR in anti-inflammation and anti-infection represents a newly identified and highly significant activity for VDR.

Jun Sun 7

When activated by 1,25-D, the Vitamin D Receptor (also called the calcitriol receptor) transcribes thousands of genes.8 It is commonly known that the VDR functions in regulating calcium metabolism.9 It is becoming increasingly clear, however, that the clinically accepted role of the Vitamin D metabolites, that of regulating calcium homeostasis, is just a small subset of the functions actually performed by these hormones. 

Transcription of antimicrobial peptides

One of the VDR’s key functions is the transcription of antimicrobial peptides.10 11 See below.  

Other antimicrobial activity of the VDR

Additionally, when the VDR is activated, TLR2 is expressed.12 TLR2 is a receptor, which is expressed on the surface of certain cells and recognizes native or foreign substances, and passes on appropriate signals to the cell and/or the nervous system.

When activated TLR2 allows the immune system to recognize gram-positive bacteria, including Staphylococcus aureus13 14Chlamydia pneumoniae15 and Mycoplasma pneumoniae.16 TLR2 also protects from intracellular infections such as Mycobacteria tuberculosis.17  

Antimicrobial peptides

The antimicrobial peptides (AMPs), of which there are hundreds, are families of proteins, which have been called “the body’s natural antibiotics,” crucial for both prevention and clearance of infection. AMPs are broad-spectrum, responding to pathogens in a non-specific manner.18

For example, consider cathelicidin, a protein transcribed the VDR, which not unlike a Swiss Army knife, has many different functions. Because it can be differentially spliced, the cathelicidin protein itself can respond to a range of very different microbial challenges. In humans, the cathelicidin antimicrobial peptide gene encodes an inactive precursor protein (hCAP18) that is processed to release a 37amino-acid peptide (LL-37) from the C-terminus. LL-37 is susceptible to proteolitic processing by a variety of enzymes, generating many different cathelicidin-derived peptides, each of which has specific targets. For example, LL-37 is generated in response toStaphylococcus aureus, yet LL-37 represents 20% of the cathelicidin-derived peptides, with the smaller peptides being much more abundant and able to target even more diverse microbial forms.19

AMPs have been documented to kill bacteria and disrupt their function through the following modes of action:

  • interfering with metabolism
  • targeting cytoplasmic components
  • disrupting membranes
  • act as chemokines and/or induce chemokine production, which directs traffic of bacteria

Also, AMPs aid in recovery from infection by:

  • promoting wound healing
  • inhibiting inflammation

In many cases, the exact mechanism by which antimicrobial peptides kill bacteria is unknown. In contrast to many conventional antibiotics including those used by the Marshall Protocol, AMPs appear to be bacteriocidal (a killer of bacteria) instead of bacteriostatic (an inhibitor of bacterial growth).

Two of the more significant families of AMPs are cathelicidin and the beta-defensins. Of these two families, cathelicidin is the most common.

The full extent by which microbes interfere with AMP expression is the subject of a rapidly growing body of research.20 21 22

Antimicrobial peptides target fungi and viruses

The antimicrobial peptides play a role in mitigating the virulence of the virome and other non-bacterial infectious agents. In addition to its antibacterial activity, alpha-defensin human neutrophil peptide-1 inhibits HIV and influenza virus entry into target cells.23 It diminishes HIV replication and can inactivate cytomegalovirus, herpes simplex virus, vesicular stomatitis virus and adenovirus.24 In addition to killing both gram positive and gram-negative bacteria, human beta-defensins HBD-1, HDB-2, and HBD-3 have also been shown to kill the opportunistic yeast species Candida albicans.25 Cathelicidin also possesses antiviral and antifungal activity.26 27

In other words, there is a reason why this group of proteins are named antimicrobial peptides rather than antibacterial peptides.

Unexpected antimicrobial peptides

There are now several examples of substances believed to cause disease, which have since been proven to be part of host defense.

  • amyloid beta (amyloid-β) – In a seminal 2010 study, a team of Harvard researchers showed that amyloid beta – the hallmark of Alzheimer’s disease – can act as an antimicrobial peptide, having antimicrobial activity against eight common microorganisms, including Streptococcus, Staphylococcus aureus, and Listeria.28 This led study author Rudolph E. Tanzi, PhD to conclude that amyloid beta is “the brain’s protector.” However, a 2010 study suggests that toxic levels of amyloid beta “dramatically suppresses VDR expression.” This suggests that overexpression of amyloid beta serves the interests of at least some microbes.29Read more.
  • certain human prion proteins   

Evolutionarily conserved

The TLR2/1 and cathelicidin-vitamin D pathway has long played a “powerful force” in protecting the body against infection. This is evidenced by the fact that the Alu short interspersed element (SINE), which transcribes the vitamin D receptor binding element (VDRE), has been evolutionarily conserved for 55-60 million years, but not prior.30 The differences in this pathway between humans/primates and other mammals call into question animal models that try to emulate the vitamin D system and indeed the immune system.

Inflammation

Another component of the innate immune response is inflammation, the universal initial response of the organism to any injurious agent.31 Inflammation is a systemic physiological process fundamental for survival.32 The identification of bacteria and other pathogens triggers the release of inflammatory cytokines. These cytokines include interferon-gamma, tumor necrosis factor-alpha (TNF-alpha), and Nuclear Factor-kappa B (NF-kappaB). Cytokines are regulatory proteins, such as the interleukins and lymphokines, that are released by cells of the immune system and act as intercellular mediators in the generation of an immune response. The result is what medicine calls inflammation, which generally leads to an increase in symptoms.

Th1/Th17 inflammation

One key type of inflammation is the Th1/Th17 (T-helper) inflammatory response. In the interests of concision, the Th1/Th17, on this site and others, the Th1/Th17 response is referred to as the Th1 response. This reaction occurs in response to intracellular pathogens, which according to the Marshall Pathogenesis, play a driving force in chronic disease.

All Th1 diseases are marked by an inflammatory response

Before the Human Microbiome Project, scientists couldn’t consistently link bacteria to inflammatory diseases. But with the advent of DNA sequencing technology, scientists have detected many of the bacteria capable of generating an inflammatory response. All diseases of unknown etiology are inflammatory diseases.

An inflammatory immune response—one of the body’s primary means to protect against infection—defines multiple established infectious causes of chronic diseases, including some cancers. Inflammation also drives many chronic conditions that are still classified as (noninfectious) autoimmune or immune-mediated (e.g., systemic lupus erythematosus, rheumatoid arthritis, Crohn’s disease). Both [the innate and adaptive immune systems] play critical roles in the pathogenesis of these inflammatory syndromes. Therefore, inflammation is a clear potential link between infectious agents and chronic diseases.

Siobhán M. O’Connor et al. 33

Th2 inflammation

According to the Marshall Pathogenesis, generally speaking, any activity of the Th2 cytokines in chronic disease is a result of the primary Th1-inducing pathogens.

Many palliative therapies interfere with inflammation

While inflammation is associated with disease, inflammation often serves an invaluable role as the immune system fights off chronic pathogens. Numerous medications artificially suppress inflammation including anti-TNF drugs, interferon, corticosteroids, antifungals, and anti-pyreutics. While interfering with the inflammatory response typically reduces immunopathology and makes a patient feel less symptomatic in the near term, doing so allows the bacteria which cause chronic disease to proliferate.

The release of cytokines appears to be essential for recovery after an infection. One study found that the cytokine TNF-alpha – which is blocked by anti-TNF drugs – is necessary for the proper expression of acquired specific resistance following infection withMycobacterium tuberculosis.34 35 36 Another effect of the use of TNF blockers is to break or reduce the formation of granuloma, one of the body’s mechanisms to control bacterial pathogens.37

Commensal microbes

The host innate immune defense system is highly active in healthy tissue.38 Commensal bacteria can activate innate immune responses.39 40

Keywords:
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15 Cao F, Castrillo A, Tontonoz P, Re F, Byrne GI Chlamydia pneumoniae–induced macrophage foam cell formation is mediated by Toll-like receptor 2. Infect Immun. 2007;75:753-9.
16 Chu HW, Jeyaseelan S, Rino JG, Voelker DR, Wexler RB, Campbell K, Harbeck RJ, Martin RJ TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression. J Immunol. 2005;174:5713-9.
17 Carlos D, Frantz FG, Souza-Júnior DA, Jamur MC, Oliver C, Ramos SG, Quesniaux VF, Ryffel B, Silva CL, Bozza MT, Faccioli LHTLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection. Microbes Infect.2009;11:770-8.
20 Chakraborty K, Ghosh S, Koley H, Mukhopadhyay AK, Ramamurthy T, Saha DR, Mukhopadhyay D, Roychowdhury S, Hamabata T, Takeda Y, Das S Bacterial exotoxins downregulate cathelicidin (hCAP-18/LL-37) and human beta-defensin 1 (HBD-1) expression in the intestinal epithelial cells. Cell Microbiol. 2008;10:2520-37.

 

Role of Dihydroxyvitamin D3 and Its Nuclear Receptor in Novel Directed Therapies for Cancer

S. Ondková, D. Macejová and J. Brtko
Gen. Physiol. Biophys. (2006), 25, 339—353   http://www.gpb.sav.sk/2006_04_339.pdf

Dihydroxyvitamin D3 is known to affect broad spectrum of various biochemical and molecular biological reactions in organisms. Research on the role and function of nuclear vitamin D receptors (VDR) playing a role as dihydroxyvitamin D3 inducible transcription factor belongs to dynamically developing branches of molecular endocrinology. In higher organisms, full functionality of VDR in the form of heterodimer with nuclear 9-cis retinoic acid receptor is essential for biological effects of dihydroxyvitamin D3. This article summarizes selected effects of biologically active vitamin D3 acting through their cognate nuclear receptors, and also its potential use in therapy and prevention of various types of cancer.

……

Vitamin D family consists of 9,10-secosteroids which differ in their side-chain structures. They are classified into five forms: D2, ergocalciferol; D3, cholecalciferol; D4, 22,23-dihydroergocalciferol; D5, sitosterol (24-ethylcholecalciferol) and D6, stigmasterol (Napoli et al. 1979). The main forms are vitamin D2 (ergocalciferol: plant origin) and vitamin D3 (cholecalciferol: animal origin). Both 25-hydroxyvitamin D2 and 1α,25-dihydroxyvitamin D2 have been evaluated for their biological functions. Vitamin D itself is a prohormone that is metabolically converted to the biologically active metabolite, 1,25-dihydroxyvitamin D3 in kidney. This vitamin D3, currently considered a steroid hormone, activates its cognate nuclear receptor (vitamin D receptor or VDR) which alter transcription rates of the target genes responsible for its biological responses. In general, vitamin D is essential for mineral homeostasis, for absorption and utilization of both calcium and phosphate and it aids in the mobilization of bone calcium and maintenance of serum calcium concentrations. Through these function, it plays an important role in ensuring proper functioning of muscles, nerves, blood clotting, cell growth and energy utilization. It has been proposed that vitamin D is also important for insulin and prolactin secretion, immune and stress responses, melanin synthesis and for differentiation of skin and blood cells (Lips 2006). Vitamin D metabolites also play a role in the prevention of auto-immune diseases and cancer (Pinette et al. 2003; Dusso et al. 2005). The steroid hormone 1α,25-dihydroxyvitamin D3 (calcitriol) exerts biological responses by interaction with both the well-characterized nuclear receptor (VDRnuc) responsible for activation gene transcription and not fully characterized membrane-associated protein/receptor (VDRmem) involved in generating a variety of rapid, non-genotropic responses (Evans 1988; Norman et al. 2002).

Vitamin D metabolism

Vitamin D, the “sunshine” vitamin, is synthesized under the influence of ultraviolet light in the skin. Many mammals have provitamin D (7-dehydrocholesterol) which is converted to provitamin D3 in their skin. When human skin is exposed to sunlight, the UV-B photons (wavelengths 290–315 nm) interact with 7-dehydrocholesterol causing photolysis and cleavage of the B-ring of the steroid structure, which upon thermoisomerization yields a secosteroid. Thus, provitamin D3 which is inherently unstable rapidly converts by a temperature-dependent process to vitamin D3 (MacLaughlin et al. 1982; Holick 1994). Vitamin D3 enters the blood circulation and binds to vitamin D binding protein (DBP) (Haddad et al. 1993) which carries vitamin D3 to liver and kidney for bioactivation (Wikvall 2001). In the first activation step, vitamin D3 is hydroxylated by the enzyme 25-hydroxylase to 25- hydroxyvitamin D3 mainly in the liver. This metabolite is present in the circulation at the concentration of more than 0.05 µmol/l (20 ng/ml). In the second step, the biologically active hormone 1α,25-dihydroxyvitamin D3 is generated by hydroxylation of 25-hydroxyvitamin D3 at 1α-position in kidney. The enzyme 1α-hydroxylase has been shown to be also present in keratinocytes and prostate epithelial cells, suggesting that those organs may also be able to generate 1α,25-dihydroxyvitamin D3 from 25-dihydroxyvitamin D3 (Schwartz et al. 1998). The activity of 1α-hydroxylase in the kidney serves as the major control point in production of the active hormone. The active metabolite 1α,25-dihydroxyvitamin D3 is present in human plasma at the concentration ranging from 0.05 to 0.15 nmol/l (20–60 pg/ml) (Hartwell et al. 1987; Gross et al. 1996). In general, 90 to 100% of the most human being vitamin D requirement comes from exposure to sunlight (Holick 2003) and the rest of the vitamin D3 content is obtained from diet (Malloy and Feldman 1999). The catabolism of vitamin D occurs by further hydroxylation of 25-dihydroxyvitamin D3 by 24-hydroxylase to yield 24,25-dihydroxyvitamin D3. The 24-hydroxylase is ubiquitous enzyme and is expressed in all the cells expressing VDR. This enzyme is regulated by parathyroid hormone and 1α,25-dihydroxyvitamin D3. The major significance of 24-hydroxylation is inactivation of vitamin D (Nishimura et al. 1994; Brenza and DeLuca 2000). The combinations of 1,25-dihydroxyvitamin D3 with inhibitors of 24-hydroxylase such as ketoconazole or liarozole may enhance its antitumour effects in prostate cancer therapy.

Vitamin D3 receptor

More than 2000 synthetic analogues of the biological active form of vitamin D, 1α,25-dihydroxyvitamin D3, are presently known. Basically, all of them interfere with the molecular switch of nuclear 1α,25-dihydroxyvitamin D3 signalling, which is the complex of the VDR, the retinoid X receptor (RXR), and a 1α,25-dihydroxyvitamin D3 response element (VDRE) (Carlberg 2003).

VDR is the only nuclear protein that binds the biologically most active vitamin D metabolite, 1α,25-dihydroxyvitamin D3, with high affinity (Kd = 0.1 nmol/l). This classifies the VDR into the classical endocrine receptor subgroup of the nuclear receptor superfamily, which also contains the nuclear receptors for hormones as retinoic acid, thyroid hormone, estradiol, progesterone, testosterone, cortisol, and aldosterol (Carlberg 1995). Similarly, like other biologically active ligand for nuclear hormone receptors, 1,25-dihydroxyvitamin D3 can modulate expression of selected ion transport protein genes (Van Baal et al. 1996; Hudecova et al. 2004).

The VDR was first isolated after trancfection of COS-1 cells with cloned sequences of complementary DNA that was isolated from human intestine (Baker et al. 1988). VDR has been found in more than 30 tissues including intestine, colon, breast, lung, ovary, bone, kidney, parathyroid gland, pancreatic β-cells, monocytes, keratinocytes, and many cancer cells, suggesting that the vitamin D endocrine system may also be involved in regulating the immune systems, cellular growth, differentiation and apoptosis (Jones et al. 1998). The active form of vitamin D binds to intracellular receptors that then function as transcription factors to modulate gene expression. Like the receptors for other steroid hormones and thyroid hormones, the VDR has specific hormone-binding and DNA-binding domains. It contains two zinc finger structures forming a characteristic DNA-binding domain (DBD) of 66 amino acids and a carboxy-terminal ligand-binding domain (LBD) of approximately 300 amino acids, which is formed by 12 α-helices. Ligand binding causes a conformational change within the LBD, in which helix 12, the most carboxy-terminal α-helix, closes the ligand-binding pocket via a “mouse-trap like” intramolecular folding (Moras and Gronemeyer 1998). Moreover, the LBD is involved in a variety of interactions with nuclear proteins, such as other nuclear receptors, corepressor and coactivator proteins. These ligand-triggered protein-protein interactions are the central molecular event of nuclear 1α,25-dihydroxyvitamin D3 signalling.

….

Role of vitamin D3 in cancer

Some of biologically active ligands for nuclear receptors exert tumour-suppressive activity, and they have therapeutical exploitation due to their antiproliferative and apoptosis-inducing effects (Brtko and Thalhamer 2003).

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During the last decade, evidence for vitamin D3 effects has been accumulating not only for prostate cancer (Feldman et al. 1995; Ma et al. 2004) but also for colon cancer (Cross et al. 1997; Bischof et al. 1998). 1α-hydroxylase was found to be Vitamin D and Cancer Treatment 347 expressed and active in colorectal cancer (Bareis et al. 2001; Cross et al. 2001; Tangpricha et al. 2001; Ogunkolade et al. 2002) and ovarian cancer (Miettinen et al. 2004). In both colon and also lung tumours, CYP24A1 mRNA was significantly up-regulated, while VDR mRNA was generally down-regulated when compared to respective normal tissues. When the level of VDR in 12 malignant colonic tumours was compared with that of adjacent normal tissue, in 9 cases out of 12, expression of VDR in tumours was decreased. However, in that study, the expression of CYP24A1 was not assessed. It has also been shown that, at least in human colon cancer cell lines, the level of VDR correlates with the degree of cell differentiation (Shabahang et al. 1993; Anderson et al. 2006).

Recently, it has been suggested that actually 20–30% of colorectal cancer incidence might be due to insufficient exposure to sunlight. This fact was strengthen by correlation between reduced colorectal cancer incidence and sunlight exposure, low skin pigmentation, nutritional vitamin D intake and high serum levels of 25- hydroxyvitamin D3 (Grant and Garland 2003). In the colon at least, CYP27B1 and VDR expression was described to be actually elevated during early tumour progression and that described dual positivity was found in many, but not all the tumour cells. In human colon tumours, CYP24 mRNA is quite highly expressed and the studies also demonstrated that with the exception of differentiated Caco-2 cells, CYP24 activity is constitutively present or can be induced by 1α,25- dihydroxyvitamin D3. During tumour progression in the colon, not only VDR but CYP27B1 and CYP24 expression were found to be increased in tumour tissues (Bareis et al. 2001; Bises et al. 2004).

Androgens, retinoids, glucocorticoids, estrogens and agonists of peroxisome proliferator-activated receptor directly or indirectly have reasonable impact on vitamin D signalling pathways, and vice versa. It was proposed that sex hormones might reduce colorectal cancer risk (McMichael and Potter 1980). The studies suggested that current and long-term use of estrogens is associated with a substantial decrease in risk of fatal colon cancer. The mechanism, however, by which estrogens could inhibit colonic tumour growth, remains an enigma. There are at least two distinct estrogen receptors in the human body: ERα and ERβ. In the normal human colon, ERβ is widely regarded to be the predominant subtype (CampbellThompson et al. 2001). In a recently terminated pilot study together with Strang Cancer Prevention Centre at Rockefeller University (NY, USA), tissues from postmenopausal women receiving 17β-estradiol for expression of CYP27B1 by real time RT-PCR were examined. CYP27B1 was found to be elevated significantly in all subjects after receiving 17β-estradiol for 4 weeks.

Amplification of chromosomal region 20q12q13 containing the CYP24A1 gene has been reported in ovarian cancer, as well (Tanner et al. 2000). Although inhibition of ovarian cancer cell growth by 1α,25-dihydroxyvitamin D3 has been reported (Saunders et al. 1992, 1995), a clinical trial testing the efficacy of 1α,25- dihydroxyvitamin D3 combined with isotretinoin in treating 22 epithelial ovarian cancer patients for 74 weeks has not produced positive results (Rustin et al. 1996).

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DNA Replication

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Decades Old DNA Replication Models Called into Question

http://www.genengnews.com/gen-news-highlights/decades-old-dna-replication-models-called-into-question/81251929/

 

Decades Old DNA Replication Models Called into Question

http://www.genengnews.com/media/images/GENHighlight/102252_web8123122217.jpg

A series of electron micrographs show the barrel-shaped helicase, which is the enzyme that separates the two DNA strands, along with other components of the replisome, including polymerase-epsilon (green).[Brookhaven National Laboratory]

  • It may be time to update biology texts to reflect newly published data from a collaborative team of scientists at Rockefeller University, Stony Brook University, and the U.S. Department of Energy’s Brookhaven National Laboratory. Using cutting-edge electron microscopy (EM) techniques, the investigators gathered the first ever images of the fully assembled replisome, providing new insight into the molecular mechanisms of replication.

    “Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

    “Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

http://www.genengnews.com/Media/images/GENHighlight/102254_web2322915422.jpg

Previously (left), the replisome’s two polymerases (green) were assumed to be below the helicase (tan), the enzyme that splits the DNA strands. The new images reveal one polymerase is located at the front of the helicase, causing one strand to loop backward as it is copied (right). [Brookhaven National Laboratory]

The researcher’s findings focused on the replisome found in eukaryotic organisms, a category that includes a broad swath of living things, including humans and other multicellular organisms. Over the past several decades, there has been an array of data describing the individual components comprising the complex nature of replisome. Yet, until now no pictures existed to show just how everything fit together.

“This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell,” explained co-senior author Huilin Li, Ph.D., biologist with joint appointments at Brookhaven Lab and Stony Brook University. “These new images show the fully assembled and fully activated ‘helicase’ protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two ‘polymerase’ enzymes that duplicate each strand to copy the genome.”

The image and implications from this study were described in a paper entitled “The architecture of a eukaryotic replisome,” published recently through Nature Structural & Molecular Biology.

Traditional models of DNA replication show the helicase enzyme moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands. However, the images that the researchers collected of intact replisomes revealed that only one of the polymerases is located at the back of the helicase. The other is on the front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

“DNA replication is one of the most fundamental processes of life, so it is every biochemist’s dream to see what a replisome looks like,” stated lead author Jingchuan Sun, EM biologist in Dr. Li’s laboratory. “Our lab has expertise and a decade of experience using electron microscopy to study DNA replication, which has prepared us well to tackle the highly mobile therefore very challenging replisome structure. Working together with the O’Donnell lab, which has done beautiful, functional studies on the yeast replisome, our two groups brought perfectly complementary expertise to this project.”

The positioning of one polymerase at the front of the helicase suggests that it may have an unforeseen function—the possibilities of which the collaborative group of scientists is continuing to study. Whatever the function the offset polymerase ends up having, Drs. Li and O’Donnell hope that it will not only provide them better insight into the replication machinery but that they may uncover useful information that can be exploited for disease intervention.

“Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here,” the scientists concluded.

 

RELATED CONTENT

 

Fifth Histone Found to Recruit Proteins for DNA Repair   

http://www.genengnews.com/gen-news-highlights/fifth-histone-found-to-recruit-proteins-for-dna-repair/81251895/

Scientists at the University of Copenhagen say they have located a previously unknown function for histones, which allows for an improved understanding of how cells protect and repair DNA damages. This new discovery may be of great importance to the treatment of diseases caused by cellular changes such as cancer and immune deficiency syndrome.

The study (“Histone H1 couples initiation and amplification of ubiquitin signaling after DNA damage”) is published in Nature.

“I believe that there’s a lot of work ahead. It’s like opening a door onto a previously undiscovered territory filled with lots of exciting knowledge. The histones are incredibly important to many of the cells’ processes as well as their overall wellbeing,” said Niels Mailand, Ph.D., from the Novo Nordisk Foundation Center for Protein Research at the Faculty of Health and Medical Science.

Histones enable the tight packaging of DNA strands within cells. The strands are two meters in length and the cells usually about 100,000 times smaller. Generally speaking, there are five types of histones. Four of them are core histones and they are placed like beads on the DNA strands, which are curled up like a ball of wool within the cells. The role of the histones is already well described in research, and in addition to enabling the packaging of the DNA strands they also play a central part in practically every process related to the DNA-code, including repairing possibly damaged DNA.

The four core histones have tails and, among other things, they signal damage to the DNA and thus attract the proteins that help repair the damage. Between the histone “yarn balls” we find the fifth histone, Histone H1, but up until now its function has not been thoroughly examined.

Using a mass spectrometer, Dr. Mailand and his team have discovered that, surprisingly, the H1 histone also helps summon repair proteins.

“In international research, the primary focus has been on the core histones and their functionality, whereas little attention has been paid to the H1 histone, simply because we weren’t aware that it too influenced the repair process. Having discovered this function in the H1 constitutes an important piece of the puzzle of how cells protect their DNA, and it opens a door onto hitherto unknown and highly interesting territory,” noted Dr. Mailand.

He expects the discovery to lead to increased research into Histone H1 worldwide, which will lead to increased knowledge of cells’ abilities to repair possible damage to their DNA and thus increase our knowledge of the basis for diseases caused by cellular changes. It will also generate more knowledge about the treatment of these diseases.

“By mapping the function of the H1 histone, we will also learn more about the repair of DNA damages on a molecular level. In order to provide the most efficient treatment, we need to know how the cells prevent and repair these damages,” point out Dr. Mailand.

 

Cover All the Bases for Oligonucleotide Analysis

Stephen Luke

Synthetic oligonucleotides have emerged as promising therapeutic agents for the treatment of a variety of diseases, including viral infections and cancer. Researchers are looking at several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and aptamers, for therapeutic applications.

However, various impurities – product-related, in the starting materials, and arising from incomplete capping of coupling reactions – must be identified and removed and postsynthesis processing must be monitored. Thus, a key challenge in the development and manufacture of oligonucleotide therapeutics is to establish analytical methods that are capable of separating and identifying impurities.

Exploring Better Options for Oligonucleotide LC Separations

 

 

Table 1. Options for oligonucleotide LC separations

Ion-pair, reversed-phase separation of the trityl-on oligos and is relatively simple to perform. This method separates the full-length target oligo, which still has the dMT group attached, from the deprotected failure sequences. The analytical information obtained is limited, so this is generally considered a purification method.

An alternate method, ion-exchange separations of the trityl-off, deprotected oligos uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column, making the technique unsuitable for use with LC/MS.

Finally, ion-pair, reversed-phase separation of the trityl-off, deprotected oligos makes use of organic solvents and mobile phase additives such as TEAA (triethylammonium acetate) or TEA-HFIP (triethylamine and hexafluoroisopropanol) to ion-pair with the negatively charged phosphodiester backbone of the oligonucleotide. High-performance columns deliver excellent resolution. What’s more, methods with volatile mobile phase constituents such as TEA-HFIP are suitable for use with LC/MS, providing useful information to help characterize oligonucleotide structures and sequences.

In Table 1 we summarize some of the options for oligonucleotide analysis by liquid chromatography.

Designed for ion-pair, reversed-phase separation of the trityl-off, deprotected oligos using either TEAA or TEA-HFIP mobile phases –Agilent AdvanceBio Oligonucleotide columns meet these challenges.

more….   http://www.genengnews.com/gen-articles/cover-all-the-bases-for-oligonucleotide-analysis/5594/

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