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Lesson 8 Cell Signaling and Motility: Lesson and Supplemental Information on Cell Junctions and ECM: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Please click on the following link for the PowerPoint Presentation for Lecture 8 on Cell Junctions and the Extracellular Matrix: (this is same lesson from 2018 so don’t worry that file says 2018)
(for this lesson pay attention to the part that shows how Receptor Tyrosine Kinase activation (RTK) can lead to signaling to an integrin and also how the thrombin receptor leads to cellular signals both to GPCR (G-protein coupled receptors like the thrombin receptor, the ADP receptor; but also the signaling cascades that lead to integrin activation of integrins leading to adhesion to insoluble fibrin mesh of the newly formed clot and subsequent adhesion of platelets, forming the platelet plug during thrombosis.)
The retina is responsible for capturing images from the visual field. Retinitis pigmentosa, which refers to a group of inherited diseases that cause retinal degeneration, causes a gradual decline in vision because retinal photoreceptor cells (rods and cones) die. Images on the left are courtesy of the National Eye Institute, NIH; image on the right is courtesy of Robert Fariss, Ph.D., and Ann Milam, Ph.D., National Eye Institute, NIH.
Metabolomics, the comprehensive evaluation of the products of cellular processes, can provide new findings and insight in a vast array of diseases and dysfunctions. Though promising, metabolomics lacks the standing of genomics or proteomics. It is, in a manner of speaking, the new kid on the “omics” block.
Even though metabolomics is still an emerging discipline, at least some quarters are giving it a warm welcome. For example, metabolomics is being advanced by the Common Fund, an initiate of the National Institutes of Health (NIH). The Common Fund has established six national metabolomics cores. In addition, individual agencies within NIH, such as the National Institute of Environmental Health Sciences (NIEHS), are releasing solicitations focused on growing more detailed metabolomics programs.
Whether metabolomic studies are undertaken with or without public support, they share certain characteristics and challenges. Untargeted or broad-spectrum studies are used for hypotheses generation, whereas targeted studies probe specific compounds or pathways. Reproducibility is a major challenge in the field; many studies cannot be reproduced in larger cohorts. Carefully defined guidance and standard operating procedures for sample collection and processing are needed.
While these challenges are being addressed, researchers are patiently amassing metabolomic insights in several areas, such as retinal diseases, neurodegenerative diseases, and autoimmune diseases. In addition, metabolomic sleuths are availing themselves of a growing selection of investigative tools.
A Metabolomic Eye on Retinal Degeneration
The retina has one of the highest metabolic activities of any tissue in the body and is composed of multiple cell types. This fact suggests that metabolomics might be helpful in understanding retinal degeneration. At least, that’s what occurred to Ellen Weiss, Ph.D., a professor of cell biology and physiology at the University of North Carolina School of Medicine at Chapel Hill. To explore this possibility, Dr. Weiss began collaborating with Susan Sumner, Ph.D., director of systems and translational sciences at RTI International.
Retinal degeneration is often studied through the use of genetic-mouse models that mimic the disease in humans. In the model used by Dr. Weiss, cells with a disease-causing mutation are the major light-sensing cells that degenerate during the disease. Individuals with the same or a similar genetic mutation will initially lose dim-light vision then, ultimately, bright-light vision and color vision.
Wild-type and mutant phenotypes, as well as dark- and light-raised animals, were compared, since retinal degeneration is exacerbated by light in this genetic model. Retinas were collected as early as day 18, prior to symptomatic disease, and analyzed. Although data analysis is ongoing, distinct differences have emerged between the phenotypes as well as between dark- and light-raised animals.
“There is a clear increase in oxidative stress in both light-raised groups but to a larger extent in the mutant phenotype,” reports Dr. Weiss. “There are global changes in metabolites that suggest mitochondrial dysfunction, and dramatic changes in lipid profiles. Now we need to understand how these metabolites are involved in this eye disease and the relevance of these perturbations.”
For example, the glial cells in the retina that upregulate a number of proteins in response to stress to attempt to save the retina are as likely as the light-receptive neurons to undergo metabolic changes.
“One of the challenges in metabolomics studies is assigning the signals that represent the metabolites or compounds in the samples,” notes Dr. Sumner. “Signals may be ‘unknown unknowns,’ compounds that have never been identified before, or ‘known unknowns,’ compounds that are known but that have not yet been assigned in the biological matrix.”
Internal and external libraries, such as the Human Metabolome Dictionary, are used to match signals. Whether or not a match exists, fragmentation patterns are used to characterize the metabolite, and when possible a standard is obtained to confirm identity. To assist with this process, the NIH Common Fund supports Metabolite Standard Synthesis Cores (MSSCs). RTI International holds an MSSC contract in addition to being a NIH-designated metabolomics core.
Mitochondrial Dysfunction in Alzheimer’s Disease
Alzheimer’s disease (AD) is difficult to diagnose early due to its asymptomatic phase; accurate diagnosis occurs only in postmortem brain tissue. To evaluate familial AD, a rare inherited form of the disease, the laboratory of Eugenia Trushina, Ph.D., associate professor of neurology and associate professor of pharmacology at the Mayo Clinic, uses mouse models to study the disease’s early molecular mechanisms.
Synaptic loss underlies cognitive dysfunction. The length of neurons dictates that mitochondria move within the cell to provide energy at the site of the synapses. An initial finding was that very early on mitochondrial trafficking was affected reducing energy supply to synapses and distant parts of the cell.
During energy production, the major mitochondrial metabolite is ATP, but the organelle also produces many other metabolites, molecules that are implicated in many pathways. One can assume that changes in energy utilization, production, and delivery are associated with some disturbance.
“Our goal,” explains Dr. Trushina, “was to get a proof of concept that we could detect in the blood of AD patients early changes of mitochondria dysfunction or other changes that could be informative of the disease over time.”
A Mayo Clinic aging study involves a cohort of patients, from healthy to those with mild cognitive impairment (MCI) through AD. Patients undergo an annual battery of tests including cognitive function along with blood and cerebrospinal fluid sampling. Metabolic signatures in plasma and cerebrospinal fluid of normal versus various disease stages were compared, and affected mitochondrial and lipid pathways identified in MCI patients that progressed to AD.
“Last year we published on a new compound that goes through the blood/brain barrier, gets into mitochondria, and very specifically, partially inhibits mitochondrial complex I activity, making the cell resistant to oxidative damage,” details Dr. Trushina. “The compound was able to either prevent or slow the disease in the animal familial models.
“Treatment not only reduced levels of amyloid plaques and phosphorylated tau, it also restored mitochondrial transport in neurons. Now we have additional compounds undergoing investigation for safety in humans, and target selectivity and engagement.”
“Mitochondria play a huge role in every aspect of our lives,” Dr. Trushina continues. “The discovery seems counterintuitive, but if mitochondria function is at the heart of AD, it may provide insight into the major sporadic form of the disease.”
Distinguishing Types of Asthma
In children, asthma generally manifests as allergy-induced asthma, or allergic asthma. And allergic asthma has commonalities with allergic dermatitis/eczema, food allergies, and allergic rhinitis. In adults, asthma is more heterogeneous, and distinct and varied subpopulations emerge. Some have nonallergic asthma; some have adult-onset asthma; and some have obesity-, occupational-, or exercise-induced asthma.
Adult asthmatics may have markers of TH2 high verus TH2 low asthma (T helper 2 cell cytokines) and they may respond to various triggers—environmental antigens, occupational antigens, irritants such as perfumes and chlorine, and seasonal allergens. Exercise, too, can trigger asthma.
One measure that can phenotype asthmatics is nitric oxide, an exhaled breath biomarker. Nitric oxide is a smooth muscle relaxant, vasodilator, and bronchodilator that can have anti-inflammatory properties. There is a wide range of values in asthmatics, and a number of values are needed to understand the trend in a particular patient. L-arginine is the amino acid that produces nitric oxide when converted to L-citrulline, a nonessential amino acid.
According to Nicholas Kenyon, M.D., a pulmonary and critical care specialist who is co-director of the University of California, Davis Asthma Network (UCAN), some metabolomic studies suggest that there is a state of L-arginine depletion during asthma attacks or in severe asthma suggesting a lack of substrate to produce nitric oxide. Dr. Kenyon is conducting clinical work on L-arginine supplementation in a double-blind cross-over intervention trial of L-arginine versus placebo. The 50-subject study in severe asthmatics should be concluded in early 2017.
Many new biologic therapies are coming to market to treat asthma; it will be challenging to determine which advanced therapy to provide to which patient. Therapeutics mostly target severe asthma populations and are for patients with evidence of higher numbers of eosinophils in the blood and lung, which include anti-IL-5 and (soon) anti-IL-13, among others.
Tools Development
Waters is developing metabolomics applications that use multivariate statistical methods to highlight compounds of interest. Typically these applications combine separation procedures, accomplished by means of liquid chromatography or gas chromatography (LC or GC), with detection methods that rely on mass spectrometry (MS). To support the identification, quantification, and analysis of LC-MS data, the company provides bioinformatics software. For example, Progenesis QI software can interrogate publicly available databases and process information about isotopic patterns, retention times, and collision cross-sections.
Mass spectrometry (MS) is the gold standard in metabolomics and lipidomics. But there is a limit to what accurate mass and resolution can achieve. For example, neither isobaric nor isomeric species are resolvable solely by MS. New orthogonal analytical tools will allow more confident identifications.
To improve metabolomics separations before MS detection, a post-ionization separation tool, like ion mobility, which is currently used to support traditional UPLC-MS and MS imaging metabolomics protocols, becomes useful. The collision-cross section (CCS), which measures the shape of molecules, can be derived, and it can be used as an additional identification coordinate.
Other new chromatographic tools are under development, such as microflow devices and UltraPerformance Convergence Chromatography (UPC2), which uses liquid CO2 as its mobile phase, to enable new ways of separating chiral metabolites. Both UPC2 and microflow technologies have decreased solvent consumption and waste disposal while maintaining UPLC-quality performance in terms of chromatographic resolution, robustness, and reproducibility.
Informatics tools are also improving. In the latest versions of Waters’ Progenesis software, typical metabolomics identification problems are resolved by allowing interrogation of publicly available databases and scoring according to accurate mass, isotopic pattern, retention time, CCS, and either theoretical or experimental fragments.
MS imaging techniques, such as MALDI and DESI, provide spatial information about the metabolite composition in tissues. These approaches can be used to support and confirm traditional analyses without sample extraction, and they allow image generation without the use of antibodies, similar to immunohistochemistry.
“Ion-mobility tools will soon be implemented for routine use, and the use of extended CCS databases will help with metabolite identification,” comments Giuseppe Astarita Ph.D., principal scientist, Waters. “More applications of ambient ionization MS will emerge, and they will allow direct-sampling analyses at atmospheric pressure with little or no sample preparation, generating real-time molecular fingerprints that can be used to discriminate among phenotypes.”
Microflow Technology
Microflow technology offers sensitivity and robustness. For example, at the Proteomics and Metabolomics Facility, Colorado State University, peptide analysis was typically performed using nanoflow chromatography; however, nanoflow chromatography is slow and technically challenging. Moving to microflow offered significant improvements in robustness and ease-of-use and resulted in improved chromatography without sacrificing sensitivity.
Conversely, small molecule applications were typically performed with analytical-scale chromatography. While this flow regime is extremely robust and fast, it can sometimes be limited in sensitivity. Moving to microflow offered significant improvements in sensitivity, 5- to 10-fold depending on the compound, without sacrificing robustness.
But broad-scale microflow adoption is hampered by a lack of available column chemistries and legacy HPLC or UPLC infrastructure that is not conducive to low-flow operation.
“We utilize microflow technology on all of our tandem quadrupole instruments for targeted quantitative assays,” says Jessica Prenni, Ph.D., director, Proteomics and Metabolomics Facility, Colorado State University. “All of our peptide quantitation is exclusively performed with microflow technology, and many of our small molecule assays. Application examples include endocannabinoids, bile acids and plant phytohormone panels.”
Compound annotation and comparability and transparency in data processing and reporting is a challenge in metabolomics research. Multiple groups are actively working on developing new tools and strategies; common best practices need to be adopted.
The continued growth of open-source spectral databases and new tools for spectral prediction from compound databases will dramatically impact the ability for metabolomics to result in novel discoveries. The move to a systems-level understanding through the combination of various omics data also will have a huge influence and be enabled by the continued development of open-source and user-friendly pathway-analysis tools.
Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging
Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.
In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.
In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.
At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.
Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.
Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.
Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.
Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.
“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”
In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.
Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.
“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”
Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.
“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”
This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.
Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).
Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response. …..
Researchers Uncover How ‘Silent’ Genetic Changes Drive Cancer
“Traditionally, it has been hard to use standard methods to quantify the amount of tRNA in the cell,” says Tavazoie. The lead authors of the article, Hani Goodarzi, formerly a postdoc in the lab and now a new assistant professor at UCSF, and research assistant Hoang Nguyen, devised and applied a new method that utilizes state-of-the-art genomic sequencing technology to measure the amount of tRNAs in different cell types.
The team chose to compare breast tissue from healthy individuals with tumor samples taken from breast cancer patients–including both primary tumors that had not spread from the breast to other body sites, and highly aggressive, metastatic tumors.
They found that the levels of two specific tRNAs were significantly higher in metastatic cells and metastatic tumors than in primary tumors that did not metastasize or healthy samples. “There are four different ways to encode for the protein building block arginine,” explains Tavazoie. “Yet only one of those–the tRNA that recognizes the codon CGG–was associated with increased metastasis.”
The tRNA that recognizes the codon GAA and encodes for a building block known as glutamic acid was also elevated in metastatic samples.
The team hypothesized that the elevated levels of these tRNAs may in fact drive metastasis. Working in mouse models of primary, non-metastatic tumors, the researchers increased the production of the tRNAs, and found that these cells became much more invasive and metastatic.
They also did the inverse experiment, with the anticipated results: reducing the levels of these tRNAs in metastatic cells decreased the incidence of metastases in the animals.
How do two tRNAs drive metastasis? The researchers teamed up with members of the Rockefeller University proteomics facility to see how protein expression changes in cells with elevated levels of these two tRNAs.
“We found global increases in many dozens of genes,” says Tavazoie, “so we analyzed their sequences and found that the majority of them had significantly increased numbers of these two specific codons.”
According to the researchers, two genes stood out among the list. Known as EXOSC2 and GRIPAP1, these genes were strongly and directly induced by elevated levels of the specific glutamic acid tRNA.
“When we mutated the GAA codons to GAG– a “silent” mutation because they both spell out the protein building block glutamic acid–we found that increasing the amount of tRNA no longer increased protein levels,” explains Tavazoie. These proteins were found to drive breast cancer metastasis.
The work challenges previous assumptions about how tRNAs function and suggests that tRNAs can modulate gene expression, according to the researchers. Tavazoie points out that “it is remarkable that within a single cell type, synonymous changes in genetic sequence can dramatically affect the levels of specific proteins, their transcripts, and the way a cell behaves.”
Testing Blood Metabolites Could Help Tailor Cancer Treatment
Scientists have found that measuring how cancer treatment affects the levels of metabolites – the building blocks of fats and proteins – can be used to assess whether the drug is hitting its intended target.
This new way of monitoring cancer therapy could speed up the development of new targeted drugs – which exploit specific genetic weaknesses in cancer cells – and help in tailoring treatment for patients.
Scientists at The Institute of Cancer Research, London, measured the levels of 180 blood markers in 41 patients with advanced cancers in a phase I clinical trial conducted with The Royal Marsden NHS Foundation Trust.
They found that investigating the mix of metabolic markers could accurately assess how cancers were responding to the targeted drug pictilisib.
Their study was funded by the Wellcome Trust, Cancer Research UK and the pharmaceutical company Roche, and is published in the journal Molecular Cancer Therapeutics.
Pictilisib is designed to specifically target a molecular pathway in cancer cells, called PI3 kinase, which has key a role in cell metabolism and is defective in a range of cancer types.
As cancers with PI3K defects grow, they can cause a decrease in the levels of metabolites in the bloodstream.
The new study is the first to show that blood metabolites are testable indicators of whether or not a new cancer treatment is hitting the correct target, both in preclinical mouse models and also in a trial of patients.
Using a sensitive technique called mass spectrometry, scientists at The Institute of Cancer Research (ICR) initially analysed the metabolite levels in the blood of mice with cancers that had defects in the PI3K pathway.
They found that the blood levels of 26 different metabolites, which were low prior to therapy, had risen considerably following treatment with pictilisib. Their findings indicated that the drug was hitting its target, and reversing the effects of the cancer on mouse metabolites.
Similarly, in humans the ICR researchers found that almost all of the metabolites – 22 out of the initial 26 – once again rose in response to pictilisib treatment, as seen in the mice.
Blood levels of the metabolites began to increase after a single dose of pictilisib, and were seen to drop again when treatment was stopped, suggesting that the effect was directly related to the drug treatment.
Metabolites vary naturally depending on the time of day or how much food a patient has eaten. But the researchers were able to provide the first strong evidence that despite this variation metabolites can be used to test if a drug is working, and could help guide decisions about treatment.
New Metabolic Pathway Reveals Aspirin-Like Compound’s Anti-Cancer Properties
Researchers at the Gladstone Institutes say they have found a new pathway by which salicylic acid, a key compound in the nonsteroidal anti-inflammatory drugs aspirin and diflunisal, stops inflammation and cancer.
In a study (“Salicylate, Diflunisal and Their Metabolites Inhibit CBP/p300 and Exhibit Anticancer Activity”) published in eLife, the investigators discovered that both salicylic acid and diflunisal suppress two key proteins that help control gene expression throughout the body. These sister proteins, p300 and CREB-binding protein (CBP), are epigenetic regulators that control the levels of proteins that cause inflammation or are involved in cell growth.
By inhibiting p300 and CBP, salicylic acid and diflunisal block the activation of these proteins and prevent cellular damage caused by inflammation. This study provides the first concrete demonstration that both p300 and CBP can be targeted by drugs and may have important clinical implications, according to Eric Verdin, M.D., associate director of the Gladstone Institute of Virology and Immunology .
“Salicylic acid is one of the oldest drugs on the planet, dating back to the Egyptians and the Greeks, but we’re still discovering new things about it,” he said. “Uncovering this pathway of inflammation that salicylic acid acts upon opens up a host of new clinical possibilities for these drugs.”
Earlier research conducted in the laboratory of co-author Stephen D. Nimer, M.D., director of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, and a collaborator of Verdin’s, established a link between p300 and the leukemia-promoting protein AML1-ETO. In the current study, scientists at Gladstone and Sylvester worked together to test whether suppressing p300 with diflunisal would suppress leukemia growth in mice. As predicted, diflunisal stopped cancer progression and shrunk the tumors in the mouse model of leukemia. ……
Novel Protein Agent Targets Cancer and Host of Other Diseases
Researchers at Georgia State University have designed a new protein compound that can effectively target the cell surface receptor integrin v3, mutations in which have been linked to a number of diseases. Initial results using this new molecule show its potential as a therapeutic treatment for an array of illnesses, including cancer.
The novel protein molecule targets integrin v3 at a novel site that has not been targeted by other scientists. The researchers found that the molecule induces apoptosis, or programmed cell death, of cells that express integrin v3. This integrin has been a focus for drug development because abnormal expression of v3 is linked to the development and progression of various diseases.
“This integrin pair, v3, is not expressed in high levels in normal tissue,” explained senior study author Zhi-Ren Liu, Ph.D., professor in the department of biology at Georgia State. “In most cases, it’s associated with a number of different pathological conditions. Therefore, it constitutes a very good target for multiple disease treatment.”
“Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, which binds to integrin αvβ3 outside the classical ligand-binding site,” the authors wrote. “We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3.”
The findings from this study were published recently in Nature Communications in an article entitled “Rational Design of a Protein That Binds Integrin αvβ3 Outside the Ligand Binding Site.” …..
“We took a unique angle,” Dr. Lui noted. “We designed a protein that binds to a different site. Once the protein binds to the site, it directly triggers cell death. When we’re able to kill pathological cells, then we’re able to kill the disease.”
The investigators performed extensive cell and molecular testing that confirmed ProAgio interacts and binds well with integrin v3. Interestingly, they found that ProAgio induces apoptosis by recruiting caspase 8—an enzyme that plays an essential role in programmed cell death—to the cytoplasmic area of integrin v3. ProAgio was much more effective in inducing cell death than other agents tested.
Noncoding RNAs Not So Noncoding
Bits of the transcriptome once believed to function as RNA molecules are in fact translated into small proteins.
In 2002, a group of plant researchers studying legumes at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, discovered that a 679-nucleotide RNA believed to function in a noncoding capacity was in fact a protein-coding messenger RNA (mRNA).1 It had been classified as a long (or large) noncoding RNA (lncRNA) by virtue of being more than 200 nucleotides in length. The RNA, transcribed from a gene called early nodulin 40 (ENOD40), contained short open reading frames (ORFs)—putative protein-coding sequences bookended by start and stop codons—but the ORFs were so short that they had previously been overlooked. When the Cologne collaborators examined the RNA more closely, however, they found that two of the ORFs did indeed encode tiny peptides: one of 12 and one of 24 amino acids. Sampling the legumes confirmed that these micropeptides were made in the plant, where they interacted with a sucrose-synthesizing enzyme.
Five years later, another ORF-containing mRNA that had been posing as a lncRNA was discovered inDrosophila.2,3 After performing a screen of fly embryos to find lncRNAs, Yuji Kageyama, then of the National Institute for Basic Biology in Okazaki, Japan, suppressed each transcript’s expression. “Only one showed a clear phenotype,” says Kageyama, now at Kobe University. Because embryos missing this particular RNA lacked certain cuticle features, giving them the appearance of smooth rice grains, the researchers named the RNA “polished rice” (pri).
Turning his attention to how the RNA functioned, Kageyama thought he should first rule out the possibility that it encoded proteins. But he couldn’t. “We actually found it was a protein-coding gene,” he says. “It was an accident—we are RNA people!” The pri gene turned out to encode four tiny peptides—three of 11 amino acids and one of 32—that Kageyama and colleagues showed are important for activating a key developmental transcription factor.4
Since then, a handful of other lncRNAs have switched to the mRNA ranks after being found to harbor micropeptide-encoding short ORFs (sORFs)—those less than 300 nucleotides in length. And given the vast number of documented lncRNAs—most of which have no known function—the chance of finding others that contain micropeptide codes seems high.
Overlooked ORFs
From the late 1990s into the 21st century, as species after species had their genomes sequenced and deposited in databases, the search for novel genes and their associated mRNAs duly followed. With millions or even billions of nucleotides to sift through, researchers devised computational shortcuts to hunt for canonical gene and mRNA features, such as promoter regions, exon/intron splice sites, and, of course, ORFs.
ORFs can exist in practically any stretch of RNA sequence by chance, but many do not encode actual proteins. Because the chance that an ORF encodes a protein increases with its length, most ORF-finding algorithms had a size cut-off of 300 nucleotides—translating to 100 amino acids. This allowed researchers to “filter out garbage—that is, meaningless ORFs that exist randomly in RNAs,” says Eric Olsonof the University of Texas Southwestern Medical Center in Dallas.
Of course, by excluding all ORFs less than 300 nucleotides in length, such algorithms inevitably missed those encoding genuine small peptides. “I’m sure that the people who came up with [the cut-off] understood that this rule would have to miss anything that was shorter than 100 amino acids,” saysNicholas Ingolia of the University of California, Berkeley. “As people applied this rule more and more, they sort of lost track of that caveat.” Essentially, sORFs were thrown out with the computational trash and forgotten.
Aside from statistical practicality and human oversight, there were also technical reasons that contributed to sORFs and their encoded micropeptides being missed. Because of their small size, sORFs in model organisms such as mice, flies, and fish are less likely to be hit in random mutagenesis screens than larger ORFs, meaning their functions are less likely to be revealed. Also, many important proteins are identified based on their conservation across species, says Andrea Pauli of the Research Institute of Molecular Pathology in Vienna, but “the shorter [the ORF], the harder it gets to find and align this region to other genomes and to know that this is actually conserved.”
As for the proteins themselves, the standard practice of using electrophoresis to separate peptides by size often meant micropeptides would be lost, notes Doug Anderson, a postdoc in Olson’s lab. “A lot of times we run the smaller things off the bottom of our gels,” he says. Standard protein mass spectrometry was also problematic for identifying small peptides, says Gerben Menschaert of Ghent University in Belgium, because “there is a washout step in the protocol so that only larger proteins are retained.”
But as researchers take a deeper dive into the function of the thousands of lncRNAs believed to exist in genomes, they continue to uncover surprise micropeptides. In February 2014, for example, Pauli, then a postdoc in Alex Schier’s lab at Harvard University, discovered a hidden code in a zebrafish lncRNA. She had been hunting for lncRNAs involved in zebrafish development because “we hadn’t really anticipated that there would be any coding regions out there that had not been discovered—at least not something that is essential,” she says. But one lncRNA she identified actually encoded a 58-amino-acid micropeptide, which she called Toddler, that functioned as a signaling protein necessary for cell movements that shape the early embryo.5
Then, last year, Anderson and his colleagues reported another. Since joining Olson’s lab in 2010, Anderson had been searching for lncRNAs expressed in the heart and skeletal muscles of mouse embryos. He discovered a number of candidates, but one stood out for its high level of sequence conservation—suggesting to Anderson that it might have an important function. He was right, the RNA was important, but for a reason that neither Anderson nor Olson had considered: it was in fact an mRNA encoding a 46-amino-acid-long micropeptide.6
“When we zeroed in on the conserved region [of the gene], Doug found that it began with an ATG [start] codon and it terminated with a stop codon,” Olson says. “That’s when he looked at whether it might encode a peptide and found that indeed it did.” The researchers dubbed the peptide myoregulin, and found that it functioned as a critical calcium pump regulator for muscle relaxation.
With more and more overlooked peptides now being revealed, the big question is how many are left to be discovered. “Were there going to be dozens of [micropeptides]? Were there going to be hundreds, like there are hundreds of microRNAs?” says Ingolia. “We just didn’t know.”
Little things mean a lot. To any biologist, this time-worn maxim is old news. But it’s worth revisiting. As several articles in this issue of The Scientist illustrate, how researchers define and examine the “little things” does mean a lot.
Consider this month’s cover story, “Noncoding RNAs Not So Noncoding,” by TS correspondent Ruth Williams. Combing the human genome for open reading frames (ORFs), sequences bracketed by start and stop codons, yielded a protein-coding count somewhere in the neighborhood of 24,000. That left a lot of the genome relegated to the category of junk—or, later, to the tens of thousands of mostly mysterious long noncoding RNAs (lncRNAs). But because they had only been looking for ORFs that were 300 nucleotides or longer (i.e., coding for proteins at least 100 amino acids long), genome probers missed so-called short ORFs (sORFs), which encode small peptides. “Their diminutive size may have caused these peptides to be overlooked, their sORFs to be buried in statistical noise, and their RNAs to be miscategorized, but it does not prevent them from serving important, often essential functions, as the micropeptides characterized to date demonstrate,” writes Williams.
How little things work definitely informs another field of life science research: synthetic biology. As the functions of genes and gene networks are sussed out, bioengineers are using the information to design small, synthetic gene circuits that enable them to better understand natural networks. In “Synthetic Biology Comes into Its Own,” Richard Muscat summarizes the strides made by synthetic biologists over the last 15 years and offers an optimistic view of how such networks may be put to use in the future. And to prove him right, just as we go to press, a collaborative group led by one of syn bio’s founding fathers, MIT’s James Collins, has devised a paper-based test for Zika virus exposure that relies on a freeze-dried synthetic gene circuit that changes color upon detection of RNAs in the viral genome. The results are ready in a matter of hours, not the days or weeks current testing takes, and the test can distinguish Zika from dengue virus. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” commented one researcher about the test.
Moving around little things is the name of the game when it comes to delivering a package of drugs to a specific target or to operating on minuscule individual cells. Mini-scale delivery of biocompatible drug payloads often needs some kind of boost to overcome fluid forces or size restrictions that interfere with fine-scale manipulation. To that end, ingenious solutions that motorize delivery by harnessing osmotic changes, magnets, ultrasound, and even bacterial flagella are reviewed in “Making Micromotors Biocompatible.”
Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany).
This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.
Integrins are heterodimeric receptors that are important for cell-cell and cell-extracellular matrix (ECM) interactions and are composed of one α and one β-subunit [1, 2]. These cell adhesion molecules act as transmembrane linkers between their extracellular ligands and the cytoskeleton, and modulate various signaling pathways essential in the biological functions of most cells. Integrins play a crucial role in processes such as cell migration, differentiation, and survival during embryogenesis, angiogenesis, wound healing, immune and non-immune defense mechanisms, hemostasis and oncogenic transformation [1]. The fact that many integrins are also linked with pathological conditions has converted them into very promising therapeutic targets [3]. In particular, integrins αvβ3, αvβ5 and α5β1 are involved in angiogenesis and metastasis of solid tumors, being excellent candidates for cancer therapy [4–7].
There are a number of different integrin subtypes which recognize and bind to the tripeptide sequence RGD (arginine, glycine, aspartic acid), which represents the most prominent recognition motif involved in cell adhesion. For example, the pro-angiogenic αvβ3 integrin binds various RGD-containing proteins, including fibronectin (Fn), fibrinogen (Fg), vitronectin (Vn) and osteopontin [8]. It is therefore not surprising that this integrin has been targeted for cancer therapy and that RGD-containing peptides and peptidomimetics have been designed and synthesized aiming to selectively inhibit this receptor [9, 10].
One classical strategy used in drug design is based on the knowledge about the structure of the receptor-binding pocket, preferably in complex with the natural ligand. However, this strategy, the so-called “rational structure-based design”, could not be applied in the field of integrin ligands since the first structures of integrin’s extracellular head groups were not described until 2001 for αvβ3 [11] (one year later, in 2002 the structure of this integrin in complex with Cilengitide was also reported [12]) and 2004 for αIIbβ3 [13]. Therefore, initial efforts in this field focused on a “ligand-oriented design”, which concentrated on optimizing RGD peptides by means of different chemical approaches in order to establish structure-activity relationships and identify suitable ligands.
We focused our interest in finding ligands for αvβ3 and based our approach on three chemical strategies pioneered in our group: 1) Reduction of the conformational space by cyclization; 2) Spatial screening of cyclic peptides; and 3)N-Methyl scan.
The combination of these strategies lead to the discovery of the cyclic peptidec(RGDf(NMe)V) in 1995. This peptide showed subnanomolar antagonistic activity for the αvβ3 receptor, nanomolar affinities for the closely related integrins αvβ5 and α5β1, and high selectivity towards the platelet receptor αIIbβ3. The peptide was patented together with Merck in 1997 (patent application submitted in 15.9.1995, opened in 20.3.1997) [14] and first presented with Merck’s agreement at the European Peptide Symposium in Edinburgh (September 1996) [15]. The synthesis and activity of this molecule was finally published in 1999 [16]. This peptide is now developed by Merck-Serono, (Darmstadt, Germany) under the name “Cilengitide” and has recently entered Phase III clinical trials for treating glioblastoma [17]. …..
The discovery 30 years ago of the RGD motif in Fn was a major breakthrough in science. This tripeptide sequence was also identified in other ECM proteins and was soon described as the most prominent recognition motif involved in cell adhesion. Extensive research in this direction allowed the description of a number of bidirectional proteins, the integrins, which were able to recognize and bind to the RGD sequence. Integrins are key players in the biological function of most cells and therefore the inhibition of RGD-mediated integrin-ECM interactions became an attractive target for the scientific community.
However, the lack of selectivity of linear RGD peptides represented a major pitfall which precluded any clinical application of RGD-based inhibitors. The control of the molecule’s conformation by cyclization and further spatial screening overcame these limitations, showing that it is possible to obtain privileged bioactive structures, which enhance the biological activity of linear peptides and significantly improve their receptor selectivity. Steric control imposed in RGD peptides together with their biological evaluation and extensive structural studies yielded the cyclic peptide c(RGDfV), the first small selective anti-angiogenic molecule described. N-Methylation of this cyclic peptide yielded the much potentc(RGDf(NMe)V), nowadays known as Cilengitide.
The fact that brain tumors, which are highly angiogenic, are more susceptible to the treatment with integrin antagonists, and the positive synergy observed for Cilengitide in combination with radio-chemotherapy in preclinical studies, encouraged subsequent clinical trials. Cilengitide is currently in phase III for GBM patients and in phase II for other types of cancers, with to date a promising therapeutic outcome. In addition, the absence of significant toxicity and excellent tolerance of this drug allows its combination with classical therapies such as RT or cytotoxic agents. The controlled phase III study CENTRIC was launched in 2008, with primary outcome measures due on September 2012. The results of this and other clinical studies are expected with great hope and interest.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Integrins are heterodimeric cell surface receptors found in nearly all metazoan cell types, composed of non-covalently linked α and β subunits. In mammals, eighteen α-subunits and eight β-subunits have been identified to date 1. From this pool, 24 distinct heterodimer combinations have been observed in vivo that confer cell-to-cell and cell-to-ligand specificity relevant to the host cell and the environment in which it functions 2. Integrin-mediated interactions with the extracellular matrix (ECM) are required for the attachment, cytoskeletal organization, mechanosensing, migration, proliferation, differentiation and survival of cells in the context of a multitude of biological processes including fertilization, implantation and embryonic development, immune response, bone resorption and platelet aggregation. Integrins also function in pathological processes such as inflammation, wound healing, angiogenesis, and tumor metastasis. In addition, integrin binding has been identified as a means of viral entry into cells 3. ….
Combination of cilengitide and radiation therapy and temozolomide. The addition of cilengitide to radiotherapy and temozolomide based treatment regimens has shown promising preliminary results in ongoing Phase II trials in both newly diagnosed and progressive glioblastoma multiforme 139–140. In addition to the Phase II objectives sought, these trials are significant in that they represent progress that has made in determining tumor drug uptake and in identifying a subset of patients that may benefit from treatment. In a Phase II trial enrolling 52 patients with newly diagnosed glioblastoma multiforme receiving 500 mg cilengitide twice weekly during radiotherapy and in combination with temozolomide for 6 monthly cycles following radiotherapy, 69% achieved 6 months progression free survival compared to 54 % of patients receiving radiotherapy followed by temozolomide alone. The one-year overall survival was 67 and 62 % of patients for the cilengitide combination group and the radiotherapy and temozolomide group, respectively. Non-hematological grade 3-4 toxcities were limited, and included symptoms of fatigue, asthenia, anorexia, elevated liver function tests, deep vein thrombosis and pulmonary embolism in across a total of 5.7% of the patients. Grade 3-4 hematological malignancies were more common and included lymphopenia (53.8%), thrombocytopenia (13.4%) and neutropenia (9.6%). This trial is significant in the fact that is has provided the first evidence correlating a molecular biomarker with response to treatment. Decreased methylguanine methyltransferase (MGMT) expression was associated with favorable outcome. Patients harboring increased MGMT promoter methylation appeared to benefit more from combined treatment with cilengitide than did patients lacking promoter methylation. The significance of the MGMT promoter methylation in predicting response is likely due to inclusion of temozolomide in the treatment combination.
A similar Phase II study evaluating safety and differences in overall survival among newly diagnosed glioblastoma multiforme patients receiving radiation therapy combined with temozolomide and varying doses of cilengitide is nearing completion. Preliminary reports specify that initial safety run-in studies in 18 patients receiving doses 500, 1000 and 2000 mg cilengitide found no dose limiting toxicities. Subsequently 94 patients were randomized to receive standard therapy plus 500 or 2000 mg cilengitide. Median survival time in both cohorts was 18.9 months. At 12 months the overall survival was 79.5 % (89/112 patients).
In the last two decades great progress has been made in the discovery and development of integrin targeted therapeutics. Years of intense research into integrin function has provided an understanding of the potential applications for the treatment of disease. Advances in structural characterization of integrin-ligand interactions has proved beneficial in the design and development of potent, selective inhibitors for a number of integrins involved in platelet aggregation, inflammatory responses, angiongenesis, neovascularization and tumor growth.
The αIIbβ3 integrin antagonists were the first inhibitors to make their way into clinical use and have proven to be effective and safe drugs, contributing to the reduction of mortality and morbidity associated with acute coronary syndromes. Interestingly, the prolonged administration of small molecules targeting this integrin for long-term prevention of thrombosis related complications have not been successful, for reasons that are not yet fully understood. This suggests that modulating the intensity, duration and temporal aspects of integrin function may be more effective than simply shutting off integrin signaling in some instances. Further research into the dynamics of platelet activation and thrombosis formation may elucidate the mechanisms by which integrin activation is modulated.
The introduction of α4 targeted therapies held great promise for the treatment of inflammatory diseases. The development of Natalizumab greatly improved the quality of life for multiple sclerosis patients and those suffering with Crohn’s Disease compared to previous treatments, but the role in asthma related inflammation could not be validated. Unfortunately for MS and Crohn’s patients, immune surveillance in the central nervous system was also compromised as a direct effect α4β7 antagonism, with potentially lethal effects. Thus Natalizumab and related α4β7 targeting drugs are now limited to patients refractory to standard therapies. The design and development of α4β1 antagonists for the treatment of Crohn’s Disease may offer benefit with decreased risks. The involvement of these integrins in fetal development also raises concerns for widespread clinical use.
Integrin antagonists that target angiogenesis are progressing through clinical trials. Cilengitide has shown promising results for the treatment of glioblastomas and recurrent gliomas, cancers with notoriously low survival and cure rates. The greatest challenge facing the development of anti-angiogenic integrin targeted therapies is the overall lack of biomarkers by which to measure treatment efficacy.
Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach
Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by α5β1 is poorly understood. Here we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of RGD and synergy sites in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish α5 subunit with the corresponding regions of human α5 we show that blades 1-4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller (D3-A3 loop) contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human α5β1 supports an important function for D3-A3 loop residues Trp-157 and Ala-158 in the binding of antagonists. These results will aid the development of reagents that block α5β1 functions in vivo.
Structural Basis of Integrin Regulation and Signaling
Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cellpathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 Å couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
The immune system relies heavily on integrins for (a) adhesion during leukocyte trafficking from the bloodstream, migration within tissues, immune synapse formation, and phagocytosis; and (b) signaling during costimulation and cell polarization. Integrins are so named because they integrate the extracellular and intracellular environments by binding to ligands outside the cell and cytoskeletal components and signaling molecules inside the cell. Integrins are noncovalently associated heterodimeric cell surface adhesion molecules. In vertebrates, 18 α subunits and 8 β subunits form 24 known αβ pairs (Figure 1). This diversity in subunit composition contributes to diversity in ligand recognition, binding to cytoskeletal components and coupling to downstream signaling pathways. Immune cells express at least 10 members of the integrin family belonging to the β2, β7, and β1 subfamilies (Table 1). The β2 and β7 integrins are exclusively expressed on leukocytes, whereas the β1 integrins are expressed on a wide variety of cells throughout the body. Distribution and ligand-binding properties of the integrins on leukocytes are summarized in Table 1. For reviews, see References 1 and 2. Mutations that block expression of the β2 integrin subfamily lead to leukocyte adhesion deficiency, a disease associated with severe immunodeficiency (3).
As adhesion molecules, integrins are unique in that their adhesiveness can be dynamically regulated through a process termed inside-out signaling or priming. Thus, stimuli received by cell surface receptors for chemokines, cytokines, and foreign antigens initiate intracellular signals that impinge on integrin cytoplasmic domains and alter adhesiveness for extracellular ligands. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction (outside-in signaling). These dynamic properties of integrins are central to their proper function in the immune system. Indeed, mutations or small molecules that stabilize either the inactive state or the active adhesive state—and thereby block the adhesive dynamics of leukocyte integrins—inhibit leukocyte migration and normal immune responses.
Integrins, Cadherins, Signaling and the Cytoskeleton
Curator: Larry H. Bernstein, MD, FCAP
We have reviewed the cytoskeleton, cytoskeleton pores and ionic translocation under lipids. We shall now look at this again, with specific attention to proteins, transporters and signaling.
Integrins and extracellular matrix in mechanotransduction
Lindsay Ramage
Queen’s Medical Research Institute, University of Edinburgh,
Edinburgh, UK
Cell Health and Cytoskeleton 2012; 4: 1–9
Integrins are a family of cell surface receptors which
mediate cell–matrix and cell–cell adhesions.
Among other functions they provide an important
mechanical link between the cells external and intracellular environments while
the adhesions that they form also have critical roles in cellular signal-transduction.
Cell–matrix contacts occur at zones in the cell surface where
adhesion receptors cluster and when activated
the receptors bind to ligands in the extracellular matrix.
The extracellular matrix surrounds the cells of tissues and forms the
structural support of tissue which is particularly important in connective tissues.
Cells attach to the extracellular matrix through
specific cell-surface receptors and molecules
including integrins and transmembrane proteoglycans.
Integrins work alongside other proteins such as
cadherins,
immunoglobulin superfamily
cell adhesion molecules,
selectins, and
syndecans
to mediate
cell–cell and
cell–matrix interactions and communication.
Activation of adhesion receptors triggers the formation of matrix contacts in which
bound matrix components,
adhesion receptors,
and associated intracellular cytoskeletal and signaling molecules
form large functional, localized multiprotein complexes.
Cell–matrix contacts are important in a variety of different cell and
tissue properties including
embryonic development,
inflammatory responses,
wound healing,
and adult tissue homeostasis.
This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.
Integrins are a family of αβ heterodimeric receptors which act as
cell adhesion molecules
connecting the ECM to the actin cytoskeleton.
The actin cytoskeleton is involved in the regulation of
cell motility,
cell polarity,
cell growth, and
cell survival.
The integrin family consists of around 25 members which are composed of differing
combinations of α and β subunits.
The combination of αβ subunits determines
binding specificity and
signaling properties.
In mammals around 19 α and eight β subunits have been characterized.
Both α and β integrin subunits contain two separate tails, which
penetrate the plasma membrane and possess small cytoplasmic domains which facilitate
the signaling functions of the receptor.
There is some evidence that the β subunit is the principal
site for
binding of cytoskeletal and signaling molecules,
whereas the α subunit has a regulatory role. The integrin
tails
link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,
such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.
The ECM is a complex mixture of matrix molecules, including -glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
and nonmatrix proteins, – including growth factors.
These can be categorized as insoluble molecules within the ECM, soluble molecules, and/or matrix-associated biochemicals, such as systemic hormones or growth factors and cytokines that act locally.
The integrin receptor formed from the binding of α and β subunits is shaped like a globular head supported by two rod-like legs (Figure 1). Most of the contact between the two subunits occurs in the head region, with the intracellular tails of the subunits forming the legs of the receptor.6 Integrin recognition of ligands is not constitutive but is regulated by alteration of integrin affinity for ligand binding. For integrin binding to ligands to occur the integrin must be primed and activated, both of which involve conformational changes to the receptor.
The integrins are composed of well-defined domains used for protein–protein interactions. The α-I domains of α integrin subunits comprise the ligand binding sites. X-ray crystallography has identified an α-I domain within the β subunit and a β propeller domain within the α subunit which complex to form the ligand-binding head of the integrin.
The use of activating and conformation-specific antibodies also suggests that the β chain is extended in the active integrin. It has since been identified that the hybrid domain in the β chain is critical for integrin activation, and a swing-out movement of this leg activates integrins.
Figure Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.
Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell.15 This bidirectional signaling requires
dynamic,
spatially, and
temporally regulated formation and
disassembly of multiprotein complexes that
form around the short cytoplasmic tails of integrins.
Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain
not only adhesion to the ECM
but are involved in complex signaling pathways
which include establishing
cell polarity,
directed cell migration, and
maintaining cell growth and survival.
Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules
to assemble the focal adhesion complex
which is capable of responding to environmental stimuli efficiently.
Mapping of the integrin
adhesome binding and signaling interactions
identified a network of 156 components linked together which can be modified by 690 interactions.
The binding of the adaptor protein talin to the β subunit cytoplasmic tail is known to have a key role in integrin activation. This is thought to occur through the disruption of
inhibitory interactions between α and β subunit cytoplasmic tails.
Talin also binds
to actin and to cytoskeletal and signaling proteins.
This allows talin to directly link activated integrins
to signaling events and the cytoskeleton.
Genetic programming occurs with the binding of integrins to the ECM
Signal transduction pathway activation arising from integrin-
ECM binding results in changes in gene expression of cells
and leads to alterations in cell and tissue function. Various
different effects can arise depending on the
cell type,
matrix composition, and
integrins activated.
One way in which integrin expression is important in genetic programming is in the fate and differentiation of stem cells.
Osteoblast differentiation occurs through ECM interactions
with specific integrins
to initiate intracellular signaling pathways leading to osteoblast-specific gene expression
disruption of interactions between integrins and collagen;
fibronectin blocks osteoblast differentiation and
Disruption of α2 integrin prevents osteoblast differentiation, and activation of the transcription factor
It has been suggested that integrin-type I collagen interaction is necessary for the phosphorylation and activation of osteoblast-specific transcription factors present in committed osteoprogenitor cells.
A variety of growth factors and cytokines have been shown to be important in the regulation of integrin expression and function in chondrocytes. Mechanotransduction in chondrocytes occurs through several different receptors and ion channels including integrins. During osteoarthritis the expression of integrins by chondrocytes is altered, resulting in different cellular transduction pathways which contribute to tissue pathology.
In normal adult cartilage, chondrocytes express α1β1, α10β1 (collagen receptors), α5β1, and αvβ5 (fibronectin) receptors. During mechanical loading/stimulation of chondrocytes there is an influx of ions across the cell membrane resulting from activation of mechanosensitive ion channels which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides. Using these strategies it was identified that α5β1 integrin is a major mechanoreceptor in articular chondrocyte responses to mechanical loading/stimulation.
Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation in contrast to the hyperpolarization response of normal chondrocytes. The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage both involve recognition of the mechanical stimulus by integrin receptors resulting in the activation of integrin signaling pathways leading to the generation of a cytokine loop. Normal and osteoarthritic chondrocytes show differences at multiple stages of the mechanotransduction cascade (Figure 3). Early events are similar; α5β1 integrin and stretch activated ion channels are activated and result in rapid tyrosine phosphorylation events. The actin cytoskeleton is required for the integrin-dependent Mechanotransduction leading to changes in membrane potential in normal but not osteoarthritic chondrocytes.
Cell–matrix interactions are essential for maintaining the integrity of tissues. An intact matrix is essential for cell survival and proliferation and to allow efficient mechanotransduction and tissue homeostasis. Cell–matrix interactions have been extensively studied in many tissues and this knowledge is being used to develop strategies to treat pathology. This is particularly important in tissues subject to abnormal mechanical loading, such as musculoskeletal tissues. Integrin-ECM interactions are being used to enhance tissue repair mechanisms in these tissues through differentiation of progenitor cells for in vitro and in vivo use. Knowledge of how signaling cascades are differentially regulated in response to physiological and pathological external stimuli (including ECM availability and mechanical loading/stimulation) will enable future strategies to be developed to prevent and treat the progression of pathology associated with integrin-ECM interactions.
Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels
Matthews, DR. Overby, R Mannix and DE. Ingber
1Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital, and 2Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA J Cell Sci 2006; 119: 508-518. http://dx.doi.org:/10.1242/jcs.02760
To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead isplacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response;
(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;
(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and
(4) a large-scale repositioning response with prolonged (>1 minute) stress.
Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by
inhibiting tension through interference with Rho signaling,
similar to the case of the immediate and early responses, but it was also prevented by
blocking mechanosensitive ion channels or
by inhibiting Src tyrosine kinases.
All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.
Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins
J Atherton, I Farabella, I-Mei Yu, SS Rosenfeld, A Houdusse, M Topf, CA Moores
1Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, United Kingdom; 2Structural Motility, Institut Curie, Centre National de la Recherche Scientifique, Paris, France; 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
eLife 2014;3:e03680. http://dx.doi.org:/10.7554/eLife.03680
Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including
mitosis,
motility, and
intracellular transport
Their involvement in a range of pathological processes also highlights their significance as therapeutic targets and the importance of understanding the molecular basis of their function They are defined by their motor domains that contain both the microtubule (MT) and ATP binding sites. Three ATP binding motifs—the P-loop, switch I, switch II–are highly conserved among kinesins, myosin motors, and small GTPases. They share a conserved mode of MT binding such that MT binding, ATP binding, and hydrolysis are functionally coupled for efficient MT-based work.
The interior of a cell is a hive of activity, filled with proteins and other items moving from one location to another. A network of filaments called microtubules forms tracks along which so-called motor proteins carry these items. Kinesins are one group of motor proteins, and a typical kinesin protein has one end (called the ‘motor domain’) that can attach itself to the microtubules.
The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together, flexible regions of the neck allow the two motor domains to move past one another, which enable the kinesin to essentially walk along a microtubule in a stepwise manner.
Atherton et al. use a technique called cryo-electron microscopy to study—in more detail than previously seen—the structure of the motor domains of two types of kinesin called kinesin-1 and kinesin-3. Images were taken at different stages of the cycle used by the motor domains to extract the energy from ATP molecules. Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.
When a motor domain binds to the microtubule, its shape changes, first stimulating release of the breakdown products of ATP from the previous cycle. This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding are relatively small but produce larger changes in the flexible neck region that enable individual motor domains within a kinesin pair to co-ordinate their movement and move in a consistent direction. This mechanism involves tight coupling between track binding and fuel usage and makes kinesins highly efficient motors.
A number of kinesins drive long distance transport of cellular cargo with dimerisation allowing them to take multiple 8 nm ATP-driven steps toward MT plus ends. Their processivity depends on communication between the two motor domains, which is achieved via the neck linker that connects each motor domain to the dimer-forming coiled-coil
Kinesins are a superfamily of microtubule-based
ATP-powered motors, important for multiple, essential cellular functions.
How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy.
All our reconstructions have, as their asymmetric unit, a triangle-shaped motor domain bound to an αβ-tubulin dimer within the MT lattice (Figure 1). The structural comparisons below are made with respect to the MT surface, which, at the resolution of our structures (∼7 Å, Table 1), is the same (CCC > 0.98 for all). As is well established across the superfamily, the major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4, which lies at the tubulin intradimer interface (Figure 1C, Kikkawa et al., 2001).
However, multiple conformational changes are seen throughout the rest of each domain in response to bound nucleotide (Figure 1D). Below, we describe the conformational changes in functionally important regions of each motor domain starting with the nucleotide-binding site, from which all other conformational changes emanate.
The nucleotide-binding site (Figure 2) has three major elements: (1) the P-loop (brown) is visible in all our reconstructions;
(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and
(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4,
the conformation and flexibility of which is determined by MT binding and motor nucleotide state.
Movement and extension of helix-α6 controls neck linker docking
the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that its conformation alters in response to different nucleotide states. In addition, because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and because helix-α4 is held against the MT during the ATPase cycle,
conformational changes in helix-α6 control movement of the neck linker.
Mechanical amplification and force generation involves conformational changes across the motor domain
A key conformational change in the motor domain following Mg-ATP binding is peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation (Figure 3—figure supplement 2); this is required to accommodate rotation of helix-α6 and consequent neck linker docking (Figure 3B–E).
Peeling of the central β-sheet has previously been proposed to arise from tilting of the entire motor domain relative to static MT contacts, pivoting around helix-α4 (the so-called ‘seesaw’ model; Sindelar, 2011). Specifically, this model predicts that the major difference in the motor before and after Mg-ATP binding would be the orientation of the motor domain with respect to helix-α4.
Kinesin mechanochemistry and the extent of mechanistic conservation within the motor superfamily are open questions, critical to explain how MT binding, and ATP binding and hydrolysis drive motor activity. Our structural characterisation of two transport motors now allows us to propose a model that describes the roles of mechanochemical elements that together drive conserved MT-based motor function.
Model of conserved MT-bound kinesin mechanochemistry. Loop11/N-terminus of helix-α4 is flexible in ADP-bound kinesin in solution, the neck linker is also flexible while loop9 chelates ADP. MT binding is sensed by loop11/helix-α4 N-terminus, biasing them towards more ordered conformations.
We propose that this favours crosstalk between loop11 and loop9, stimulating ADP release. In the NN conformation, both loop11 and loop9 are well ordered and primed to favour ATP binding, while helix-α6—which is required for mechanical amplification–is closely associated with the MT on the other side of the motor domain. ATP binding draws loop11 and loop9 closer together; causing
(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,
(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and
(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.
In both motors, microtubule binding promotes
ordered conformations of conserved loops that
stimulate ADP release,
enhance microtubule affinity and
prime the catalytic site for ATP binding.
ATP binding causes only small shifts of these nucleotide-coordinating loops but induces
large conformational changes elsewhere that
allow force generation and
neck linker docking towards the microtubule plus end.
Family-specific differences across the kinesin–microtubule interface account for the
distinctive properties of each motor.
Our data thus provide evidence for a
conserved ATP-driven
mechanism for kinesins and
reveal the critical mechanistic contribution of the microtubule interface.
Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function
I Timmerman, PL Hordijk, JD van Buul
Cell Health and Cytoskeleton 2010; 2: 23–31
Endothelial cell–cell junctions are strictly regulated in order to
control the barrier function of endothelium.
Vascular endothelial (VE)-cadherin is one of the proteins that is crucial in this process. It has been reported that
phosphorylation events control the function of VE-cadherin.
This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.
The vascular endothelium is the inner lining of blood vessels and
forms a physical barrier between the vessel lumen and surrounding tissue;
controlling the extravasation of fluids,
plasma proteins and leukocytes.
Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,
transient hyperpermeability
in response to stimulation with inflammatory mediators,
which takes place primarily in postcapillary venules.
However, when severe, inflammation may result in dysfunction of the endothelial barrier in various parts of the vascular tree, including large veins, arterioles and capillaries. Dysregulated permeability is observed in various pathological conditions, such as tumor-induced angiogenesis, cerebrovascular accident and atherosclerosis.
Two fundamentally different pathways regulate endothelial permeability,
the transcellular and paracellular pathways.
Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes
vesicular transport systems, fenestrae, and biochemical transporters.
The paracellular route is controlled by
the coordinated opening and closing of endothelial junctions and
thereby regulates traffic across the intercellular spaces between endothelial cells.
Endothelial cells are connected by
tight, gap and
adherens junctions,
of which the latter, and particularly the adherens junction component,
vascular endothelial (VE)-cadherin,
are of central importance for the initiation and stabilization of cell–cell contacts.
Although multiple adhesion molecules are localized at endothelial junctions, blocking the adhesive function of VE-cadherin using antibodies is sufficient to disrupt endothelial junctions and to increase endothelial monolayer permeability both in vitro and in vivo. Like other cadherins, VE-cadherin mediates adhesion via homophilic, calcium-dependent interactions.
This cell–cell adhesion
is strengthened by binding of cytoplasmic proteins, the catenins,
to the C-terminus of VE-cadherin.
VE-cadherin can directly bind β-catenin and plakoglobin, which
both associate with the actin binding protein α-catenin.
Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that
α-catenin cannot bind to both β-catenin and actin simultaneously.
Data using purified proteins show that
monomeric α-catenin binds strongly to cadherin-bound β-catenin;
in contrast to the dimer which has a higher affinity for actin filaments,
indicating that α-catenin might function as a molecular switch regulating cadherin-mediated cell–cell adhesion and actin assembly.
Thus, interactions between the cadherin complex and the actin cytoskeleton are more complex than previously thought. Recently, Takeichi and colleagues reported that
the actin binding protein EPLIN (epithelial protein lost in neoplasm)
can associate with α-catenin and thereby
link the E-cadherin–catenin complex to the actin cytoskeleton.
Although this study was performed in epithelial cells,
an EPLIN-like molecule might serve as
a bridge between the cadherin–catenin complex and
the actin cytoskeleton in endothelial cells.
Next to β-catenin and plakoglobin, p120-catenin also binds directly to the intracellular tail of VE-cadherin.
Numerous lines of evidence indicate that
p120-catenin promotes VE-cadherin surface expression and stability at the plasma membrane.
Different models are proposed that describe how p120-catenin regulates cadherin membrane dynamics, including the hypothesis
that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
with the endocytic membrane trafficking machinery.
In addition, p120-catenin might regulate VE-cadherin internalization through interactions with small GTPases. Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to
decrease RhoA activity,
elevate active Rac1 and Cdc42, and thereby is thought
to regulate actin cytoskeleton organization and membrane trafficking.
The intact cadherin-catenin complex is required for proper functioning of the adherens junction. Mutant forms of VE-cadherin which
lack either the β-catenin, plakoglobin or p120 binding regions reduce the strength of cell–cell adhesion.
Moreover, our own results showed that
interfering with the interaction between α-catenin and β-catenin,
using a cell-permeable peptide which encodes the binding site in α-catenin for β-catenin,
resulted in an increased permeability of the endothelial monolayer.
Several mechanisms may be involved in the regulation of the organization and function of the cadherin–catenin complex, including endocytosis of the complex, VE-cadherin cleavage and actin cytoskeleton reorganization. The remainder of this review primarily focuses on the
role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.
Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation
It is a widely accepted concept that tyrosine phosphorylation of components of the VE–cadherin-catenin complex
Correlates with the weakening of cell–cell adhesion.
One of the first reports that supported this idea showed that the level of phosphorylation of VE-cadherin was
high in loosely confluent endothelial cells, but
low in tightly confluent monolayers,
when intercellular junctions are stabilized.
In addition, several conditions that induce tyrosine phosphorylation
of adherens junction components, like
v-Src transformation
and inhibition of phosphatase activity by pervanadate,
have been shown to shift cell–cell adhesion from a strong to a weak state. More physiologically relevant;
permeability-increasing agents such as
histamine,
tumor necrosis factor-α (TNF-α),
thrombin,
platelet-activating factor (PAF) and
vascular endothelial growth factor (VEGF)
increase tyrosine phosphorylation of various components of the cadherin–catenin complex.
A general idea has emerged that
tyrosine phosphorylation of the VE-cadherin complex
leads to the uncoupling of VE-cadherin from the actin cytoskeleton
through dissociation of catenins from the cadherin.
However, tyrosine phosphorylation of VE-cadherin is required for efficient transmigration of leukocytes.
This suggests that VE-cadherin-mediated cell–cell contacts
are not just pushed open by the migrating leukocytes, but play
a more active role in the transmigration process.
A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.
Notes: A) Permeability-inducing agents such as thrombin, histamine and VEGF, induce tyrosine phosphorylation (pY) of VE-cadherin and the associated catenins. Although the specific consequences of catenin tyrosine phosphorylation in endothelial cells are still unknown, VE-cadherin tyrosine phosphorylation results in opening of the cell–cell junctions (indicated by arrows) and enhanced vascular permeability. How tyrosine phosphorylation affects VE-cadherin adhesiveness is not yet well understood; disrupted binding of catenins, which link the cadherin to the actin cytoskeleton, may be involved. VEGF induces phosphorylation of VE-cadherin at specific residues, Y658 and Y731, which have been reported to regulate p120-catenin and β-catenin binding, respectively. Moreover, VEGF stimulation results in serine phosphorylation (pSer) of VE-cadherin, specifically at residue S665, which leads to its endocytosis. B) Adhesion of leukocytes to endothelial cells via ICAM-1 increases endothelial permeability by inducing phosphorylation of VE-cadherin on tyrosine residues. Essential mediators, such as the kinases Pyk2 and Src, and signaling routes involving reactive oxygen species (ROS) and Rho, have been shown to act downstream of ICAM-1. Different tyrosine residues within the cytoplasmic domain of VE-cadherin are involved in the extravasation of neutrophils and lymphocytes, including Y658 and Y731. (β: β-catenin, α: α-catenin, γ: γ-catenin/plakoglobin).
N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs
BT Jamal, MN Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009; 1: 67–80
N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how
N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.
Using the hypoglycosylated E-cadherin variant, V13, we show that
V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.
This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that
increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.
On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they
mediated the interaction of AJs with the actin cytoskeleton.
N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin.
These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through
the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.
Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle
Matteo Vatta, and Georgine Faulkner,
1 Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX 2 Department of Reproductive and Developmental Sciences, University of Trieste, Trieste, Italy
3 Muscular Molecular Biology Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy
The heart is a force-generating organ that responds to
self-generated electrical stimuli from specialized cardiomyocytes.
This function is modulated
by sympathetic and parasympathetic activity.
In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes
depend on their highly evolved and specialized cytoskeletal apparatus.
Defects in components of the cytoskeleton, in the long term,
affect the ability of the cell to compensate at both functional and structural levels.
In addition to the structural remodeling,
the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.
The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review
the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels
and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.
Myocardial dysfunction in the end-stage failing heart is very often associated with increasing
susceptibility to ventricular tachycardia (VT) and ventricular fibrillation (VF),
both of which are common causes of sudden cardiac death (SCD).
Among the various forms of HF,
myocardial remodeling due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM)
is characterized by alterations in baseline ECG,
which includes the
prolongation of the QT interval,
as well as QT dispersion,
ST-segment elevation, and
T-wave abnormalities,
especially during exercise. In particular, subjects with
severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure. LBBB and RBBB have both been repeatedly associated with AV block in heart failure.
The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest. In LVAD-treated subjects,
QRS- and both QT- and QTc duration decreased,
suggesting that QRS- and QT-duration are significantly influenced by mechanical load and
that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.
Despite the increasing use of LVAD supporting either continuous or pulsatile blood flow in patients with severe HF, the benefit of this treatment in dealing with the risk of arrhythmias is still controversial.
Large epidemiological studies, such as the REMATCH study, demonstrated that the
employment of LVAD significantly improved survival rate and the quality of life, in comparison to optimal medical management.
An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,
HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.
In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that
the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,
while no effect was observed on the development of polymorphic ventricular tachycardia (PVT)/ventricular fibrillation (VF).
The sarcomere
The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,
it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,
which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.
Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also
maintain the shape and flexibility of the different sub-cellular compartments, including the
plasma membrane,
the double lipid layer, which defines the boundaries of the cell and where
ion channels are mainly localized.
The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole. Titin connects
the Z-line to the M-line of the sarcomeric structure
(Figure 1).
In addition to the strategic
localization and mechanical spring function,
titin is a length-dependent sensor during
stretch and promotes actin-myosin interaction
Titin is stabilized by the cross-linking protein
telethonin (T-Cap), which localizes at the Z-line and is also part of titin sensor machinery (Figure 1).
The complex protein interactions in the sarcomere entwine telethonin to other
Z-line components through the family of the telethonin-binding proteins of the Z-disc, FATZ, also known as calsarcin and myozenin.
FATZ binds to
calcineurin,
γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2,
the major component of the Z-line and a pivotal
F-actin cross-linker (Figure 1).contractile unit of striated muscles, and
the sarcolemma,
the plasma membrane surrendering the muscle fibers in skeletal muscle and the muscle cell of the cardiomyocyte,
determines the mechanical plasticity of the cell, enabling it to complete and re-initiate each contraction-relaxation cycle.
At the level of the sarcomere,
actin (thin) and myosin (thick) filaments generate the contractile force,
while other components such as titin, the largest protein known to date, are responsible for
the passive force during diastole and for the restoring force during systole, and (titin).
the Z-line to the M-line of the sarcomeric structure
(Figure 1).
In addition to the strategic
localization and mechanical spring function,
it acts as a length-dependent sensor during stretch and
promotes actin-myosin interaction.
Stabilized by the cross-linking protein telethonin (T-Cap),
titin localizes at the Z-line and is
part of titin sensor machinery
Another cross-linker of α-actinin-2 in the complex Z-line scaffold is
the Z-band alternatively spliced PDZ motif protein (ZASP),
which has an important role in maintaining Z-disc stability
in skeletal and cardiac muscle (Figure 1).
ZASP contains a PDZ motif at its N-terminus,
which interacts with C-terminus of α-actinin-2,
and a conserved sequence called the ZASP like motif (ZM)
found in the alternatively spliced exons 4 and 6.
It has also been reported
to bind to the FATZ (calsarcin) family of Z-disc proteins (Figure 1).
The complex protein interactions in the sarcomere entwine telethonin to other Z-line components through the family of the telethonin-binding proteins of the
Z-disc,
FATZ, also known as calsarcin and
myozenin
FATZ binds to calcineurin,
γ-filamin as well as the
spectrin-like repeats (R3–R4) of α-actinin-2, the major component of the Z-line and a pivotal F-actin cross-linker (Figure 1).
sarcomere structure
Figure 1. Sarcomere structure
The diagram illustrates the sarcomeric structure. The Z-line determines the boundaries of the contractile unit, while Titin connects the Z-line to the M-line and acts as a functional spring during contraction/relaxation cycles.
Sarcomeric Proteins and Ion Channels
In addition to systolic dysfunction characteristic of dilated cardiomyopathy (DCM) and diastolic dysfunction featuring hypertrophic cardiomyopathy (HCM), the clinical phenotype of patients with severe cardiomyopathy is very often associated with a high incidence of cardiac arrhythmias. Therefore, besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially
affect ion channel anchoring, and trafficking, as well as
functional regulation by second messenger pathways,
causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.
Intense investigation of
the sarcomeric actin network,
the Z-line structure, and
chaperone molecules docking in the plasma membrane,
has shed new light on the molecular basis of
cytoskeletal interactions in regulating ion channels.
In 1991, Cantiello et al., demonstrated that
although the epithelial sodium channel and F-actin are in close proximity,
they do not co-localize.
Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that
the integrity of the filamentous actin (F-actin) network was essential
for the maintenance of normal Na+ channel function.
Later, the group of Dr. Jonathan Makielski demonstrated that
actin disruption induced a dramatic reduction in Na+ peak current and
slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation.
These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.
F-actin is intertwined in a multi-protein complex that includes
the composite Z-line structure.
Further, there is a direct binding between
the major protein of the Z-line, α-actinin-2 and
the voltage-gated K+ channel 1.5 (Kv1.5), (Figure 2).
The latter is expressed in human cardiomyocytes and localizes to
the intercalated disk of the cardiomyocyte
in association with connexin and N-cadherin.
Maruoka et al. treated HEK293 cells stably expressing Kv1.5 with cytochalasin D, which led to
a massive increase in ionic and gating IK+ currents.
This was prevented by pre-incubation with phalloidin, an F-actin stabilizing agent. In addition, the Z-line protein telethonin binds to the cytoplasmic domain of minK, the beta subunit of the potassium channel KCNQ1 (Figure 2).
Molecular interactions between the cytoskeleton and ion channels
Figure 2. Molecular interactions between the cytoskeleton and ion channels
The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties. The cardiac sodium channel Nav1.5 associates with the DGC, while potassium channels such as Kv1.5, associate with the Z-line.
Ion Channel Subunits and Trafficking
Correct localization is essential for ion channel function and this is dependent upon the ability of auxiliary proteins to
shuttle ion channels from the cytoplasm to their final destination such as
the plasma membrane or other sub-cellular compartments.
In this regard, Kvβ-subunits are
cytoplasmic components known to assemble with the α-subunits of voltage-dependent K+ (Kv) channels
at their N-terminus to form stable Kvα/β hetero-oligomeric channels.
When Kvβ is co-expressed with Kv1.4 or Kv1.5, it enhances Kv1.x channel trafficking to the cell membrane without changing the overall protein channel content. The regulatory Kvβ subunits, which are also expressed in cardiomyocytes, directly decrease K+ current by
accelerating Kv1.x channel inactivation.
Therefore, altered expression or mutations in Kvβ subunits could cause abnormal ion channel transport to the cell surface, thereby increasing the risk of cardiac arrhythmias.
Ion Channel Protein Motifs and Trafficking
Cell membrane trafficking in the Kv1.x family may occur in a Kvβ subunit-independent manner through specific motifs in their C-terminus. Mutagenesis of the final asparagine (N) in the Kv1.2 motif restores the leucine (L) of the Kv1.4 motif
re-establishing high expression levels at the plasma membrane in a Kvβ-independent manner
Cytoskeletal Proteins and Ion Channel Trafficking
Until recently, primary arrhythmias such as LQTS have been almost exclusively regarded as ion channelopathies. Other mutations have been identified with regard to channelopathies. However, the conviction that primary mutations in ion channels were solely responsible for
the electrical defects associated with arrhythmias
has been shaken by the identification of mutations in the
ANK2 gene encoding the cytoskeletal protein ankyrin-B
that is associated with LQTS in animal models and humans.
Ankyrin-B acts as a chaperone protein, which shuttles the cardiac sodium channel from the cytoplasm to the membrane. Immunohistochemical analysis has localized ankyrin-B to the Zlines/T-tubules on the plasma membrane in the myocardium. Mutations in ankyrin-B associated with LQTS
alter sodium channel trafficking due to loss of ankyrin-B localization at the Z-line/transverse (T)-tubules.
Reduced levels of ankyrin-B at cardiac Z-lines/T-tubules were associated with the deficiency of ankyrin-B-associated proteins such as Na/K-ATPase, Na/Ca exchanger (NCX) and inositol-1, 4, 5-trisphosphate receptors (InsP3R).
Dystrophin component of the Dystrophin Glycoprotien Complex (DGC)
Synchronized contraction is essential for cardiomyocytes, which are connected to each other via the extracellular matrix (ECM) through the DGC. The N-terminus domain of dystrophin
binds F-actin, and connects it to the sarcomere, while
the cysteine-rich (CR) C-terminus domain ensures its connection to the sarcolemma (Figure 2).
The central portion of dystrophin, the rod domain, is composed of
rigid spectrin-like repeats and four hinge portions (H1–H4) that determine the flexibility of the protein.
Dystrophin possesses another F-actin binding domain in the Rod domain region, between the basic repeats 11- 17 (DysN-R17).
Dystrophin, originally identified as the gene responsible for Duchenne and Becker muscular dystrophies (DMD/BMD), and later for the X-linked form of dilated cardiomyopathy (XLCM), exerts a major function in physical force transmission in striated muscle. In addition to its structural significance, dystrophin and other DGC proteins such as syntrophins are required for the
correct localization,
clustering and
regulation of ion channel function.
Syntrophins have been implicated in ion channel regulation. Syntrophins contain two pleckstrin homology (PH) domains, a PDZ domain, and a syntrophin-unique (SU) C-terminal region. The interaction between syntrophins and dystrophin occurs at the PH domain distal to the syntrophin N-terminus and through the highly conserved SU domain. Conversely, the PH domain proximal to the N-terminal portion of the protein and the PDZ domain interact with other membrane components such as
phosphatidyl inositol-4, 5-bisphosphate,
neuronal NOS (nNOS),
aquaporin-4,
stress-activated protein kinase-3, and
5,
thereby linking all these molecules to the dystrophin complex (Figure 2).
Among the five known isoforms of syntrophin, the 59 KDa α1-syntrophin isoform is the most highly represented in human heart, whereas in skeletal muscle it is only present on the
sarcolemma of fast type II fibers.
In addition, the skeletal muscle γ2-syntrophin was found at high levels only at the
postsynaptic membrane of the neuromuscular junctions.
In addition to syntrophin, other scaffolding proteins such as caveolin-3 (CAV3), which is present in the caveolae, flask-shaped plasma membrane microdomains, are involved
in signal transduction and vesicle trafficking in myocytes,
modulating cardiac remodeling during heart failure.
CAV3 and α1-syntrophin, localizes at the T-tubule and are part of the DGC. In addition, α1-syntrophin binds Nav1.5, while
caveolin-3 binds the Na+/Ca2+ exchanger, Nav1.5 and the L-type Ca2+ channel as well as nNOS and the DGC (Figure 2).
Although ankyrin-B is the only protein found mutated in patients with primary arrhythmias, other proteins such as caveolin-3 and the syntrophins if mutated may alter ion channel function.
Conclusions
It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular, the recent findings of structural and functional links between
the cytoskeleton and ion channels
could expand the therapeutic interventions in
arrhythmia management in structurally abnormal myocardium, where aberrant binding
between cytoskeletal proteins can directly or indirectly alter ion channel function.
Executive Summary
Arrhythmogenesis and myocardial structure
Rhythm alterations can develop as a secondary consequence of myocardial structural abnormalities or as a result of a primary defect in the cardiac electric machinery.
Until recently, no molecular mechanism has been able to fully explain the occurrence of arrhythmogenesis in heart failure, however genetic defects that are found almost exclusively in ion channel genes account for the majority of primary arrhythmias such as long QT syndromes and Brugada syndrome. The contractile apparatus is linked to ion channels
The sarcomere, which represents the contractile unit of the myocardium not only generates the mechanical force necessary to exert the pump function, but also provides localization and anchorage to ion channels.
Alpha-actinin-2, and telethonin, two members of the Z-line scaffolding protein complex in the striated muscle associate with the potassium voltage-gated channel alpha subunit Kv1.5 and the beta subunit KCNE1 respectively.
Mutations in KCNE1 have previously been associated with the development of arrhythmias in LQTS subjects.
Mutations in both alpha-actinin-2, and telethonin were identified in individuals with cardiomyopathy. The primary defect is structural leading to ventricular dysfunction, but the secondary consequence is arrhythmia.
Ion channel trafficking and sub-cellular compartments
Ion channel trafficking from the endoplasmic reticulum (ER) to the Golgi complex is an important check-point for regulating the functional channel molecules on the plasma membrane. Several molecules acting as chaperones bind to and shuttle the channel proteins to their final localization on the cell surface
Ion channel subunits such as Kvβ enhance Kv1.x ion channel presentation on the sarcolemma. The α subunits of the Kv1.x potassium channels can be shuttled in a Kvβ-independent manner through specific sequence motif at Kv1.x protein level.
In addition, cytoskeletal proteins such as ankyrin-G bind Nav1.5 and are involved in the sodium channel trafficking. Another member of the ankyrin family, ankyrin-B was found mutated in patients with LQTS but the pathological mechanism of ankyrin-B mutations is still obscure, although the sodium current intensity is dramatically reduced.
The sarcolemma and ion channels
The sarcolemma contains a wide range of ion channels, which are responsible for the electrical propagating force in the myocardium.
The DGC is a protein complex, which forms a scaffold for cytoskeletal components and ion channels.
Dystrophin is the major component of the DGC and mutations in dystrophin and DGC cause muscular dystrophies and X-linked cardiomyopathies (XLCM) in humans. Cardiomyopathies are associated with arrhythmias
Caveolin-3 and syntrophins associate with Nav1.5, and are part of the DGC. Syntrophins can directly modulate Nav1.5 channel function.
Conclusions
The role of the cytoskeleton in ion channel function has been hypothesized in the past, but only recently the mechanism underlying the development of arrhythmias in structurally impaired myocardium has become clearer.
The recently acknowledged role of the cytoskeleton in ion channel function suggests that genes encoding cytoskeletal proteins should be regarded as potential candidates for variants involved in the susceptibility to arrhythmias, as well as the primary target of genetic mutations in patients with arrhythmogenic syndromes such as LQTS and Brugada syndrome.
Studies of genotype-phenotype correlation and and patient risk stratification for mutations in cytoskeletal proteins will help to tailor the therapy and management of patients with arrhythmias.
“A research team from Stanford University’s School of Medicine is now one step closer to uncovering a cancer treatment that could be applicable across the board in killing every kind of cancer tumor” (1). It appeared that their antibody-drug against the CD47 protein, enabled the shrinking of all tumor cells. After completing their animal studies the researchers now move into a human phase clinical trials. CD47 has been previously studied and evaluated for its role in multiple cells, some of this data however, is somewhat controversy. So where do we stand?
CD47
CD47 (originally named integrin-associated protein (IAP)) is a cell surface protein of the immunoglobulin (Ig) superfamily, which is heavily glycosylated and expressed by virtually all cells in the body and overexpressed in many types of cancer including breast, ovarian, colon, prostate and others (3). CD47 was first recognized as a 50 kDa protein associated and copurified with the Alpha-v-Beta-3 integrin in placenta and neutrophil granulocytes and later shown to have the capacity to regulate integrin function and the responsiveness of leukocytes to RGD-containing extracellular matrix proteins. CD47 has also been shown to be identical to the OA-3/OVTL3 antigen highly expressed on most ovarian carcinomas (4,5).
CD47 consists of an extracellular IgV domain, a five times transmembrane-spanning domain, and a short alternatively spliced cytoplasmic tail. In both humans and mice, the cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids, showing different tissue expression patterns (3).
CD47 interactions (3, 6):
Thrombospondin-1 (TSP-1) – a secreted glycoprotein that plays a role in vascular development and angiogenesis. Binding of TSP-1 to CD47 influences several fundamental cellular functions including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation.
Signal-regulatory protein-alpha (SIRPα) – an inhibitory transmembrane receptor present on myeloid cells. The CD47/SIRPα interaction leads to bidirectional signaling, resulting in different cell-to-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T-cell activation.
Integrins – several membrane integrins, most commonly integrin avb3. These interactions result in CD47/integrin complexes that effect a range of cell functions including adhesion, spreading and migration
These interactions with multiple proteins and cells types create several important functions, which include:
Cell proliferation – cell proliferation is heavily dependent on cell type as both activation and loss of CD47 can result in enhanced proliferation. For example, activation of CD47 with TSP-1 in wild-type cells inhibits proliferation and reduces expression of stem cell transcription factors. In cancer cells however, activation of CD47 with TSP-1 increases proliferation of human U87 and U373 astrocytoma. it is likely that CD47 promotes proliferation via the PI3K/Akt pathway in cancerous cells but not normal cells (7). Loss of CD47 allows sustained proliferation of primary murine endothelial cells and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters (8).
Apoptosis – Ligation of CD47 by anti-CD47mAbs was found to induce apoptosis in a number of different cell types (3). For example: Of the two SIRP-family members known to bind the CD47 IgV domain (SIRPα and SIRPγ), SIRPα as a soluble Fc-fusion protein does not induce CD47-dependent apoptosis, hile SIRPα or SIRPγ bound onto the surface of beads induces apoptosis through CD47 in Jurkat T cells and the myelomonocytic cell line U937.
Migration – CD47 role on cell migration was first demonstrated in neutrophils, these effects were shown to be dependent on avb3 integrins, which interact with and are activated by CD47 at the plasma membrane. In cancer, Blocking CD47 function has been shown to inhibit migration and metastasis in a variety of tumor models. Blockade of CD47 by neutralizing antibodies reduced migration and chemotaxis in response to collagen IV in melanoma, prostate cancer and ovarian cancer-derived cells (9).
Angiogenesis – The mechanism of the anti-angiogenic activity of CD47 is not fully understood, but introduction of CD47 antibodies and TSP-1 have been shown to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells (10). More so, CD47 signaling influences the SDF-1 chemokine pathway, which plays a role in angiogenesis (11). (12)
Inflammatory response – Interactions between endothelial cell CD47 and leukocyte SIRPγ regulate T cell transendothelial migration (TEM) at sites of inflammation. CD47 also functions as a marker of self on murine red blood cells which allows RBC to avoid phagocytosis. Tumor cells can also evade macrophage phagocytosis through the expression of CD47 (2, 13).
It appears that CD47 ligation induce different responses, depending on cell type and partner for ligation.
Therapeutic and clinical aspect of CD47 in human cancer:
CD47 is overexpressed in many types of human cancers and its known function as a “don’t eat me” signal, suggests the potential for targeting the CD47-SIRPα pathway as a common therapy for human malignancies (2,13). Upregulation of CD47 expression in human cancers also appears to influence tumor growth and dissemination. First, increased expression of CD47 in several hematologic malignancies was found to be associated with a worse clinical prognosis, and in ALL to predict refractoriness to standard chemotherapies (13, 14-16). Second, CD47 was demonstrated to regulate tumor metastasis and dissemination in both MM and NHL (13, 17).
Efforts have been made to develop therapies inhibiting the CD47-SIRPα pathway, principally through blocking monoclonal antibodies directed against CD47, but also possibly with a recombinant SIRPα protein that can also bind and block CD47.
Chao MP et al. 2012 Combination strategies targeting CD47 in cancer
While monotherapies targeting CD47 were efficacious in several pre-clinical tumor models, combination strategies involving inhibition of the CD47-SIRPα pathway offer even greater therapeutic potential. Specifically, antibodies targeting CD47-SIRPα can be included in combination therapies with other therapeutic antibodies, macrophage-enhancing agents, chemo-radiation therapy, or as an adjuvant therapy to inhibit metastasis (13).
For example, anti-SIRPα antibody was found to potentiate antibody-dependent cellular cytotoxicity (ADCC) mediated by the anti-Her2/Neu antibody trastuzumab against breast cancer cells (18). CD47–SIRPα interactions and SIRPα signaling negatively regulate trastuzumab-mediated ADCC in vitro and antibody-dependent elimination of tumor cells in vivo
More so, chemo-radiation therapy-mediated upregulation of cell surface calreticulin may potentially augment the activity of anti-CD47 antibody. However, this approach may also lead to increased toxicity as cell surface calreticulin is expressed on non-cancerous cells undergoing apoptosis, a principle effect of chemo-radiation therapy (19).
Highlights:
Phagocytic cells, macrophages, regulate tumor growth through phagocytic clearance
CD47 binds SIRPα on phagocytes which delivers an inhibitory signal for phagocytosis
A blocking anti-CD47 antibody enabled phagocytic clearance of many human cancers
Phagocytosis depends on a balance of anti-(CD47) and pro-(calreticulin) signals
Anti-CD47 antibody synergized with an FcR-engaging antibody, such as rituximab
Summary
Evasion of immune recognition is a major mechanism by which cancers establish and propagate disease. Recent data has demonstrated that the innate immune system plays a key role in modulating tumor phagocytosis through the CD47-SIRPα pathway. Careful development of reagents that can block the CD47/SIRPα interaction may indeed be useful to treat many forms of cancer without having too much of a negative side effect in terms of inducing clearance of host cells. Therapeutic approaches inhibiting this pathway have demonstrated significant efficacy, leading to the reduction and elimination of multiple tumor types.
Dr. Weissman says: “We are now hopeful that the first human clinical trials of anti-CD47 antibody will take place at Stanford in mid-2014, if all goes well. Clinical trials may also be done in the United Kingdom”. These clinical trials must be designed so that the data they generate will produce a valid scientific result!!!
2. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J, Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP, Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM, Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M, Raveh T, Tseng D, Jaiswal S, Enger PØ, Steinberg GK, Li G, So SK, Majeti R, Harsh GR, van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6662-6667. http://www.pnas.org/content/early/2012/03/20/1121623109
3. Oldenborg PL. CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease. ISRN Hematology Volume 2013 (2013), Article ID 614619, 19 pages. http://www.hindawi.com/isrn/hematology/2013/614619/
4. G. Campbell, P. S. Freemont, W. Foulkes, and J. Trowsdale, “An ovarian tumor marker with homology to vaccinia virus contains an IgV- like region and multiple transmembrane domains,”Cancer Research, vol. 52, no. 19, pp. 5416–5420, 1992. http://cancerres.aacrjournals.org/content/52/19/5416.long
5. L. G. Poels, D. Peters, Y. van Megen et al., “Monoclonal antibody against human ovarian tumor-associated antigens,” Journal of the National Cancer Institute, vol. 76, no. 5, pp. 781–791, 1986. http://www.ncbi.nlm.nih.gov/pubmed/3517452
7. Sick E, Boukhari A, Deramaudt T, Rondé P, Bucher B, André P, Gies JP, Takeda K (February 2011). “Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway”. Glia59 (2): 308–319. http://www.ncbi.nlm.nih.gov/pubmed/21125662
9. Shahan TA, Fawzi A, Bellon G, Monboisse JC, Kefalides NA. “Regulation of tumor cell chemotaxis by type IV collagen is mediated by a Ca(2+)-dependent mechanism requiring CD47 and the integrin alpha(V)beta(3)”. J. Biol. Chem 2000.275 (7): 4796–4802. http://www.jbc.org/content/275/7/4796
10. Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD. “CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1”. J. Biol. Chem 2006. 281 (36): 26069–26080. http://www.jbc.org/content/281/36/26069
11. Smadja DM, d’Audigier C, Bièche I, Evrard S, Mauge L, Dias JV, Labreuche J, Laurendeau I, Marsac B, Dizier B, Wagner-Ballon O, Boisson-Vidal C, Morandi V, Duong-Van-Huyen JP, Bruneval P, Dignat-George F, Emmerich J, Gaussem P. “Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties”. Arterioscler. Thromb. Vasc. Biol 2011. 31 (3): 551–559. http://atvb.ahajournals.org/content/31/3/551
14. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr, van Rooijen N, Weissman IL. Cd47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286–299. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726837/
15. Chao MP, Alizadeh AA, Tang C, Jan M, Weissman-Tsukamoto R, Zhao F, Park CY, Weissman IL, Majeti R. Therapeutic antibody targeting of cd47 eliminates human acute lymphoblastic leukemia.Cancer Res. 2011;71 (4):1374–1384. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041855/
16. Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Jan M, Cha AC, Chan CK, Tan BT, Park CY, et al. Anti-cd47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-hodgkin lymphoma. Cell. 2010;142(5):699–713. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943345/
17. Chao MP, Tang C, Pachynski RK, Chin R, Majeti R, Weissman IL. Extranodal dissemination of non-hodgkin lymphoma requires cd47 and is inhibited by anti-cd47 antibody therapy. Blood.2011;118(18):4890–4901. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208297/
18. Zhao XW, van Beek EM, Schornagel K, Van der Maaden H, Van Houdt M, Otten MA, Finetti P, Van Egmond M, Matozaki T, Kraal G, Birnbaum D, et al. Cd47-signal regulatory protein-alpha (sirpalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci U S A.2011;108(45):18342–18347. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215076/
19. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, Castedo M, Mignot G, Panaretakis T, Casares N, Metivier D, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13(1):54–61. http://www.ncbi.nlm.nih.gov/pubmed/17187072
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