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Lonely Receptors: RXR – Jensen, Chambon, and Evans

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 7.2

 

Nuclear receptors provoke RNA production in response to steroid hormones

Albert Lasker Basic Medical Research Award

Pierre Chambon, Ronald Evans and Elwood Jensen

For the discovery of the superfamily of nuclear hormone receptors and elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways.

Hormones control a vast array of biological processes, including embryonic development, growth rate, and body weight. Scientists had known since the early 1900s that tiny hormone doses dramatically alter physiology, but they had no idea that these signaling molecules did so by prodding genes. The 1950s, when Jensen began his work, was the great era of enzymology. Conventional wisdom held that estradiol—the female sex hormone that instigates growth of immature reproductive tissue such as the uterus—entered the cell and underwent a series of chemical reactions that produced a particular compound as a byproduct. This compound—NADPH—is essential for many enzymes’ operations but its small quantities normally limit their productivity. A spike in NADPH concentrations would stimulate growth or other activities by unleashing the enzymes, the reasoning went.

In 1956, Jensen (at the University of Chicago) decided to scrutinize what happened to estradiol within its target tissues, but he had a problem: The hormone is physiologically active in minute quantities, so he needed an extremely sensitive way to track it. He devised an apparatus that tagged it with tritium—a radioactive form of hydrogen—at an efficiency level that had not previously been achieved. This innovation allowed him to detect a trillionth of a gram of estradiol.

When he injected this radioactive substance into immature rats, he noticed that most tissues—skeletal muscle, kidneys and liver, for example—started expelling it within 15 minutes. In contrast, tissues known to respond to the hormone—those of the reproductive tract—held onto it tightly. Furthermore, the hormone showed up in the nuclei of cells, where genes reside. Something there was apparently grabbing the estradiol.

Jensen subsequently showed that his radioactive hormone remained chemically unchanged once inside the cell. Estrogen did not act by being metabolized and producing NADPH, but presumably by performing some job in the nucleus. Subsequent work by Jensen and Jack Gorski established that estradiol converts a protein in the cytoplasm, its receptor, into a form that can migrate to the nucleus, embrace DNA, and turn on specific genes.

From 1962 to 1980, molecular endocrinologists built on Jensen’s work to discover the receptors for the other major steroid hormones—testosterone, progesterone, glucocorticoids, aldosterone, and the steroid-like vitamin D. In addition to Jensen and Gorski, many scientists—notably Bert O’Malley, Jan-Ake Gustafsson, Keith Yamamoto, and the late Gordon Tompkins—made crucial observations during the early days of steroid receptor research.

Clinical Applications of Estrogen-Receptor Detection

Clinicians knew that removing the ovaries or adrenal glands of women with breast cancer would stop tumor growth in one out of three patients, but the molecular basis for this phenomenon was mysterious. Jensen showed that breast cancers with low estrogen-receptor content do not respond to surgical treatment. Receptor status could therefore indicate who would benefit from the procedure and who should skip an unnecessary operation. In the mid-1970s, Jensen and his colleague Craig Jordan found that women with cancers that contain large amounts of estrogen receptor are also likely to benefit from tamoxifen, an anti-estrogen compound that mimics the effect of removing the ovaries or adrenal glands. The other patients—those with small numbers of receptors—could immediately move on to chemotherapy that might combat their disease rather than waiting months to find out that the tumors were growing despite tamoxifen treatment. By 1980, Jensen’s test had become a standard part of care for breast cancer patients.

In the meantime, Jensen set about generating antibodies that bound the receptor—a tool that provided a more reliable way to measure receptor quantities in excised breast tumor specimens. His work has transformed the treatment of breast cancer patients and saves or prolongs more than 100,000 lives annually.

Long-Lost Relatives

By the early 1980s, interest in molecular endocrinology had shifted toward the rapidly developing area of gene control. Chambon and Evans had long wondered how genes turn on and off, and recognized nuclear hormone signaling as the best system for studying regulated gene transcription. They wanted to know exactly how nuclear receptors provoke RNA production in response to steroid hormones. To manipulate and analyze the receptors, they would need to isolate the genes for them.

By late 1985 and early 1986, Evans (at the Salk Institute in La Jolla) and Chambon (at the Institute of Genetics and Molecular and Cellular Biology in Strasbourg, France) had pieced together the glucocorticoid and estrogen receptor genes, respectively. They noticed that the sequences resembled that of v-erbA, a miscreant viral protein that fosters uncontrolled cell growth. This observation raised the possibility that v-erbA and its well-behaved cellular counterpart, c-erbA, would also bind DNA and control gene activity in response to some chemical activator, or ligand. In 1986, Evans and Björn Vennström simultaneously reported that c-erbA was a thyroid hormone receptor that was related to the steroid hormone receptors, thus uniting the fields of thyroid and steroid biology.

Chambon and Evans set to work deconstructing the glucocorticoid and estrogen receptors. By creating mutations at different spots and probing which activities the resulting proteins lost, they dissected the receptor into three domains: one bound hormone, one bound DNA, and one activated target genes. The structure of each domain strongly resembled the analogous one in the other receptor.

Chambon and Evans wanted to match other members of the growing receptor gene family with their chemical triggers. Because the DNA- and ligand-binding regions functioned independently, it was possible to hook the DNA-binding domain of, say, the glucocorticoid receptor to the ligand-binding domain of another receptor whose ligand was unknown. The ligand for that receptor would then activate a glucocorticoid-responsive test gene.

Evans would use this method to identify ligands for several novel members of the nuclear receptor family, and both he and Chambon exploited it to discover a physiologically crucial receptor. In the late 1970s, scientists had suggested that the physiologically active derivative of vitamin A, retinoic acid, could exert its effects by binding to a nuclear receptor. This nutrient is essential from fertilization through adulthood, and researchers were eager to understand its activities on a molecular level. During embryonic development, deficiency of retinoic acid impairs formation of most organs, and the compound can hinder cancer cell proliferation. So Chambon set out to find a receptor that responded to retinoic acid. He isolated a member of the nuclear receptor gene family whose production increased in breast cancer cells that slowed their growth upon exposure to the chemical. Simultaneously, Evans identified the same protein. He tested whether more than a dozen compounds activated an unknown receptor and one passed: retinoic acid.

Remarkably, in 1986, the two scientists had independently—and unbeknownst to each other—identified the same retinoic acid receptor, a molecule of tremendous significance. The discovery of this molecule provided an entry point for detailing vitamin A biology.

Rx for Lonely Receptors: RXR

The list of presumptive nuclear receptors was growing quickly as scientists realized that the common DNA sequences provided a handle with which to grab these molecules from the genome. Because their chemical activators weren’t known, they were called “orphan” receptors, and researchers were keen on “adopting” them to ligands. Some of these ligands, they reasoned, would represent previously unknown classes of gene activators. The test system that Chambon and Evans used to match up retinoic acid with its receptor, in which they stitched an unknown ligand-binding domain to a DNA-binding domain for a receptor with known target sequences, could be harnessed to accomplish this task.

Evans had identified some potential nuclear receptors from fruit flies. He decided to pursue a human orphan receptor that closely resembled one of these receptor genes, reasoning that a protein that functioned in both flies and mammals was likely to perform an important job.

This receptor responded to retinoic acid in intact cells but did not bind it in the test tube, so Evans called it the Retinoid X Receptor (RXR), thinking that its ligand was some retinoic acid derivative. In cells, enzymes convert retinoic acid to metabolites and it seemed possible that one of these compounds was RXR’s ligand. In 1992, Evans’s group and one at Hoffmann-La Roche discovered that 9-cis-retinoic acid, a stereoisomer of retinoic acid, could activate RXR, identifying the first new receptor ligand in 25 years. This finding launched the orphan receptor field because it provided strong evidence that the strategy could unearth previously unknown ligands.

In the meantime, Chambon had found that the purified retinoic acid receptor, in contrast to the estrogen receptor, did not bind efficiently to its target DNA. Other nuclear receptors, too, needed help grasping genes. In the test tube, the retinoic acid, thyroid hormone, and vitamin D3 receptors could attach well to their target DNA only when supplemented with cellular material, which presumably contained some crucial substance. Chambon and Michael Rosenfeld independently purified a single protein that performed this feat, and it turned out to be none other than RXR. This ability of RXR to pair with other receptors—forming so-called heterodimers—would turn out to be key for switching on many orphan receptors. These heterodimeric couplings yield large numbers of distinct gene-controlling entities.

Chambon revealed the power of mixing and matching in these molecular duos through his thorough and extensive genetic manipulations in mice. He has shown that vitamin A exerts its wide-ranging effects on organ development in the embryo through the action of eight different forms of the retinoic acid receptor and six different forms of RXR, interacting with each other in a multitude of combinations.

Clinical Applications of the Superfamily Work

The concept of RXR as a promiscuous heterodimeric partner for certain nuclear receptors led to the unexpected identification of a number of clinically relevant receptors. These proteins include the peroxisome proliferator-activated receptor (PPAR), which stimulates fat-cell maturation and sits at the center of Type 2 diabetes and a number of lipid-related disorders; the liver X receptors (LXRs) and bile acid receptor (FXR), which help manage cholesterol homeostasis; and the steroid and xenobiotic receptor (PXR), which turns on enzymes that dispose of chemicals that need to be detoxified, such as drugs.

Because the nuclear receptors wield such physiological power, they have provided excellent targets for disease treatment. The anti-diabetes compounds glitazones, for example, work by stimulating PPAR, and the clinically used lipid-lowering medications called fibrates work by binding a closely related receptor, PPAR. Retinoic acid therapy has dramatically altered the prognosis of people with acute promyelocytic leukemia by triggering specialization of the immature white blood cells that accumulate in these individuals. The three-dimensional structure of nuclear receptors with and without their ligands, which Chambon and his colleagues first solved, promises to accelerate drug discovery in the whole field.

Nuclear hormone receptors have touched on human health in other ways as well. Genetic perturbations in the genes for these proteins cause a variety of illnesses. For example, certain forms of rickets arise from mutations in the vitamin D receptor and several disorders of male sexual differentiation stem from defects in the androgen receptor.

The discoveries of Jensen, Chambon, and Evans revealed an unimagined superfamily of proteins. At the start of this work almost 50 years ago, no one would have anticipated that steroids, thyroid hormone, retinoids, vitamin D, fatty acids, bile acids, and many lipid-based drugs transmit their signal through similar pathways. Four dozen human nuclear receptors are now known, and scientists are working out the roles of these proteins in normal and aberrant physiology. These discoveries have revolutionized the fields of endocrinology and metabolism, and pointed toward new tactics for drug discovery.

by Evelyn Strauss, Ph.D.

 

The 2004 Lasker Award for Basic Medical Research will be presented to Elwood Jensen, Ph.D., the Charles B. Huggins Distinguished Service Professor Emeritus in the Ben May Institute for Cancer Research at the University of Chicago, one of three scientists whose discoveries “revolutionized the fields of endocrinology and metabolism,” according to the award citation. Jensen’s work had a rapid, direct and lasting impact on treatment and prevention of breast cancer.

The Lasker Awards are the nation’s most distinguished honor for outstanding contributions to basic and clinical medical research. Often called “America’s Nobels,” the Lasker Award has been awarded to 68 scientists who subsequently went on to receive the Nobel Prize, including 15 in the last 10 years.

Jensen will share the basic medical research award with two colleagues, Pierre Chambon, of the Institute of Genetics and Molecular and Cellular Biology (Strasbourg, France), and Ronald M. Evans of the Salk Institute for Biological Studies (La Jolla, California) and the Howard Hughes Medical Institute.

They were selected for their discovery of the “superfamily of nuclear hormone receptors and the elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways.” The implications of this research for understanding human disease and accelerating drug discovery “have been profound and hold much promise for the future,” notes the announcement from the Lasker Foundation.

Jensen is being honored for his pioneering research on how steroid hormones, such as estrogen, exert their influence. His discoveries explained how these hormones work, which has led to the development of drugs that can enhance or inhibit the process.

Hormones control a vast array of biological processes, including embryonic development, growth rate and body weight. Before Jensen, however, the way which hormones cause these effects was “a complete mystery,” recalled Gene DeSombre, Ph.D., professor emeritus at the University of Chicago, who worked with Jensen in the Ben May Institute as a post-doctoral fellow and then as a colleague.

In the 1950s, biochemists thought a hormone entered a cell, where a series of oxidation and reductions reactions with the estrogen provided needed energy for the growth stimulation and other specific actions shown by estrogens.

From the late 1950s to the 1970s Jensen entirely overturned that notion. Working with estrogen, he proved that hormones do not undergo chemical change. Instead, they bind to a receptor protein within the cell. This hormone-receptor complex then travels to the cell nucleus, where it regulates gene expression.

At the time, this idea was heresy. “That really got him into some hot water,” recalled DeSombre. “Jensen struggled quite a lot,” echoes Shutsung Liao, Ph.D., another Ben May colleague, who subsequently found a similar system for testosterone action. But for Jensen, just getting into hot water was a struggle. When he first presented preliminary data at a 1958 meeting in Vienna, only five people attended, three of whom were the other speakers. More than 1,000 attended a simultaneous symposium on the metabolic processing of estrogen.

In the next 20 years, Jensen convinced his colleagues by publishing a series of major and highly original discoveries in four related areas of hormone research:

  • Jensen discovered the estrogen receptor, the first receptor found for any hormone. In 1958, using a radioactive marker, he showed that only the tissues that respond to estrogen, such as those of the female reproductive tract, were able to concentrate injected estrogen from the blood. This specific uptake suggested that these cells must contain binding proteins, which he called “estrogen receptors.”
  • In 1967, Jensen and Jack Gorski of the University of Wisconsin showed that these putative receptors were macromolecules that could be extracted from these tissues. With this method, Jensen showed that when estrogen bound to this receptor, the compound then migrated to the nucleus where it bound avidly and activated specific genes, stimulating new RNA synthesis.
  • By 1968, Jensen had devised a reliable test for the presence of estrogen receptors in breast cancer cells. It had been known for decades that about one-third of premenopausal women who had advanced breast cancer would respond to estrogen blockade brought about by removing their ovaries, the source of estrogen, but there was no way to predict which women would respond. In 1971, Jensen showed that women with receptor-rich breast cancers often have remissions following removal of the sources of estrogen, but cancers that contain few or no estrogen receptors do not respond to estrogen-blocking therapy.
  • By 1977, Jensen and Geoffrey Greene, Ph.D., also in the University of Chicago’s Ben May Institute, had developed monoclonal antibodies directed against estrogen receptors, which enabled then to quickly and accurately detect and count estrogen receptors in breast and other tumors. By 1980, this test had become a standard part of care for breast cancer patients

This work “transformed the treatment of breast cancer patients,” notes the Lasker Foundation, “and saves or prolongs more than a 100,000 lives annually.”

”Jensen’s revolutionary discovery of estrogen receptors is beyond doubt one of the major achievements in biochemical endocrinology of our time,” said DeSombre. “His work is hallmarked by great technical ingenuity and conceptual novelty. His promulgation of simple yet profound ideas concerning the role of receptors in estrogen action have been of the greatest importance for research on the basic and clinical physiology not only of estrogens but also of all other categories of steroid hormones.”

By the early 1970s, Jensen was searching for chemical, rather than surgical, ways to shield estrogen-dependent tumors from circulating hormones. He and colleague Craig Jordan (then at the Worcester Foundation for Experimental Biology in Massachusetts) subsequently found that women with cancers that contain large amounts of estrogen receptor are also likely to benefit from tamoxifen, a compound that blocks some of the effects of estrogen. Patients with few or no receptors could immediately move on to chemotherapy rather than waiting months to find out that the tumors were growing despite tamoxifen treatment.

Following Jensen’s lead, researchers soon found that the receptors for the other major steroid hormones, such as testosterone, progesterone, and cortisone, worked essentially the same way.

In 1986, Pierre Chambon and Ronald Evans separately but simultaneously discovered that the steroid hormone receptors were merely the tip of the iceberg of what would turn out to be a large family of structurally related nuclear receptors, now known to consist of 48 members. Evans and Chambon unearthed a number of these receptors, which revealed new regulatory systems that control the body’s response to essential nutrients (such as Vitamin A), fat-soluble signaling molecules (such as fatty acids and bile acids), and drugs (such as the glitazones used to treat Type 2 diabetes and retinoic acid for certain forms of acute leukemia).

These three individuals “created the field of nuclear hormone receptor research, which now occupies a large area of biological and medical investigation,” said Dr. Joseph L. Goldstein, chairman of the international jury of researchers that selects recipients of the Lasker Awards, and recipient of the Lasker Award for Basic Medical Research and the Nobel Prize in Medicine in 1985.

They revealed the “unexpected and unifying mechanism by which many signaling molecules regulate a plethora of key physiological pathways that operate from embryonic development through adulthood. They discovered a family of proteins that allows chemicals as diverse as steroid hormones, Vitamin A, and thyroid hormone to perform in the body.”

Jensen, known for concluding his lectures in verse, neatly summed up what his extraordinary series of discoveries might mean to a woman who has been diagnosed with breast cancer:

“A lady with growth neoplastic
Thought surgical ablation too drastic.
She preferred that her ill
Could be cured with a pill,
Which today is no longer fantastic.”

JBC THEMATIC MINIREVIEW SERIES 2011

Nuclear Receptors in Biology and Diseases

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases

Sohaib Khan and Jerry B Lingrel

Although a connection between breast cancer and the ovary was made by Sir George Beatson in 1896 and estrogen was purified in 1920, it remained puzzling as to how the hormone exerted its biological effects. In the late 1950s, when Elwood Jensen delved into this problem by asking, essentially, “What does tissue do with this hormone?” little did he know that his quest would lead to the establishment of the nuclear receptor field. The late 1950s was the era of intermediary metabolism and enzymology, when steroid hormones were considered likely substrates in the formation of metabolites that functioned as cofactors in an essential metabolic pathway. The biological responses to estrogens and other steroids were thought to be mediated by enzymes. Against this background and prevailing dogma, Jensen and colleagues defined the biochemical mechanisms by which steroid hormones exert their effects. While working at the University of Chicago’s Ben May Institute for Cancer Research, they synthesized tritium-labeled estradiol and concurrently developed a new method to measure its uptake in biological material. These tools enabled them to determine the biochemical fate of physiological amounts of hormone. They discovered that the reproductive tissues of the immature rat contain characteristic hormone-binding components with which estradiol reacts to induce uterine growth without itself being chemically changed. From the close correlation between the inhibition of binding and inhibition of growth response, Jensen established that the binding substances were receptors. Thus, we saw the birth of the first member of the nuclear receptor family (known as the estrogen receptor). These findings stimulated the search for other physiological receptors, and the pioneering works by Pierre Chambon, Ronald Evans, Jan-Åke Gustafsson, Bert W. O’Malley, and Keith Yamamoto led to the discoveries of the glucocorticoid receptor (GR),2 progesterone receptor, retinoic acid receptor, and orphan receptors. In a rather short span of time, the nuclear receptor family has grown into a 49-member-strong “superfamily.” This is a family whose members, functioning as sequence-specific transcription factors, have defined the many intricacies of the mechanism of transcription. These ligand-dependent transcription factors generally possess similar “domain organizations,” of which the DNA-binding domain and the ligand-binding domain are critical in amplifying the hormonal signals via the receptor target genes. The nuclear receptor family is divided into four groups: (i) Group 1 is composed of steroid hormone receptors that control target gene transcription by binding as homodimers to response element (RE) palindromes; (ii) in Group 2, the nuclear receptors heterodimerize with retinoid X receptor and generally bind to direct repeat REs; (iii) Group 3 consists of those orphan receptors that function as homodimers and bind to direct repeat REs; and (iv) orphan receptors in Group 4 function as monomers and bind to single REs.

Since the early demonstration by Jack Gorski and Jensen that the estrogen receptor (ER) activates transcription, the nuclear receptor field has come a long way. In addition to the first cloning of the polymerase II transcription factors (GR and ER cDNAs), of note is the discovery of steroid receptor coactivators (SRCs), a truly major piece of the transcriptional jigsaw puzzle, described by the laboratories of O’Malley and Myles Brown. The induction of coactivators and corepressors in the transcriptional machinery has expanded tremendously our understanding of this complex process. We now know that ligand binding to the respective receptors triggers a fascinating chain of events, including the translocation of the receptors to the nucleus, ligand-induced changes in the receptor conformations, receptor dimerization, interaction with the target gene promoter elements, recruitment of coactivators (or corepressors), chromatin remodeling, and subsequent interaction with the polymerase II complex to initiate transcription.

By virtue of their abilities to regulate a myriad of human developmental and physiological functions (reproduction, development, metabolism), nuclear receptors have been implicated in a wide range of diseases, such as cancer, diabetes, obesity, etc. Not surprisingly, drug companies are spending billions of dollars to develop medicines for cancer and metabolic disorders that involve nuclear receptors. More than 50 years after the discovery of the ER, the scientific community owes Jensen and other founding members of the nuclear receptor family much gratitude, for they have taken us through a remarkable expedition filled with eureka moments to understand how hormones and other ligands function!

This thematic minireview series will cover a range of topics in the nuclear receptor field. The minireviews include the current studies of identifying subtypes of the GR. Different receptors arise from alternative mRNA splicing and from the use of different promoter start sites and post-translational modifications, such as phosphorylation. The series covers the physiological roles of the different GRs. The field of orphan nuclear receptors and the search for possible ligands also are reviewed. One minireview concentrates largely on the following nuclear receptors: peroxisome proliferator-activated receptor (PPAR) α, PPARγ, Rev-erbα, and retinoic acid receptor-related orphan receptor α. ERα was the first identified and has been studied the most, whereas ERβ has not been studied in the same detail. ERβ is very important, and one of the minireviews provides a summary of the new biological functions that are being ascribed to it. Also, the development of small molecule inhibitors for the ER will be considered. An important aspect of nuclear receptor function is how these receptors function in transcription. The role of transcriptional coactivators in nuclear receptor gene regulation will be reviewed as well as how signal amplification and interaction are involved in transcription regulation by steroids. The SRC/p160 family of coregulators includes SRC-1, SRC-2, and SRC-3, and the latter has been shown to act as an oncogene, particularly in breast cancer. Molecular analysis of its role in breast cancer progression and metastasis will be the focus of one of the minireviews. In addition, interactions of nuclear receptors with the genome will be reviewed, as will the role of the homeodomain protein HoxB13 in specifying the cellular response to androgens. Mining nuclear receptor cistromes and how nuclear receptors reset metabolism also will be considered. The association of nuclear receptors (e.g. PPARδ) with physiological functions, such as circadian rhythm and muscle functions, will also be addressed. Finally, the role of nuclear receptors in disease using the retinoid X receptor α/β knock-out and transgenic mouse model skin syndromes and asthma will be reviewed. These are diverse and important topics that are critical in understanding the regulation of nuclear receptors and the biological roles they play in normal function and disease.

The Nuclear Receptor Superfamily: A Rosetta Stone for Physiology

Ronald M. Evans
Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037
Molecular Endocrinology 19(6):1429–143   http://dx.doi.org:/10.1210/me.2005-0046

In the December 1985 issue of Nature, we described the cloning of the first nuclear receptor cDNA encoding the human glucocorticoid receptor (GR) (1). In the 20 yr since that event, our field has witnessed a quantum leap by the subsequent discovery and functional elaboration of the nuclear receptor superfamily (2)—a family whose history is linked to the evolution of the entire animal kingdom and whose actions, by decoding the genome, span the vast diversity of biological functions from development to physiology, pathology, and treatment. A messenger is an envoy or courier charged with transmitting a communication or message. In one sense, the cloning of that first messenger (the GR) represented the completion of a prediction that began with Elwood Jensen’s characterization of the first steroid receptor protein (3) and continued with the pioneering work of others in the steroid receptor field (including Gorski, O’Malley, Gustafsson, and Yamamoto). Yet, like the discovery of the Rosetta stone in 1799, the revelation of the GR sequence heralded a completely unpredictable demarcation in the field, helping to solve mysteries unearthed nearly 100 yr ago as well as opening a portal to the future. The beginnings of the adventure lie in disciplines such as medicine and nutrition, which gave rise to the emergent field of endocrinology in the first half of the last century. The purification of chemical messengers ultimately known as hormones from organs and vitamins from foods spurred the study of these compounds and their physiologic effects on the body. At about the same time, the field of molecular biology was emerging from the disciplines of chemistry, physics, and their application to biological problems such as the structure of DNA and the molecular events surrounding its replication and transcription. It would not be until the late 1960s and 1970s that endocrinology and molecular biology would begin to intersect as the link between receptors and transcriptional control were being laid down. During this time, the work of Jensen (4) and Gorski (5) identified a high-affinity estrogen receptor (ER) that suggested an action in the nucleus. Gordon Tomkins and his associates (J. Baxter, G. Ringold, E. B. Thompson, H. Samuels, H. Bourne, and others) were one of the most creative forces studying glucocorticoid action (6). Concurrent work by O’Malley, Gustafsson, and Yamamoto provided further, important evidence supporting a link between steroid receptor action and transcription (see accompanying perspective articles in this issue of Molecular Endocrinology). But whereas the steroid hormone field continued to evolve in this direction, it is of interest to note that the mechanism of action of thyroid hormone and retinoids remained clouded and controversial until the eventual cloning of their receptors in the late 1980s. Likewise, no one had foreseen the possibility that other lipophilic molecules (like oxysterols, bile acids, and fatty acids) would also function through a similar mechanism, or that other steroid receptor-like proteins existed that would play an important role in transcriptional regulation of so many diverse pathways. Thus, the GR isolation in 1985 led to the concept of a hidden superfamily of receptors that in a very real way provided the needed molecular code to unravel the puzzle of physiologic homeostasis.

Unconventional Gene-Ology

The study of RNA tumor viruses was ascendant, and the concept that they evolved by pirating key signaling pathways greatly influenced my future studies. With this training, I went on to work with Jim Darnell at the Rockefeller University on adenovirus transcription, a model brought to the lab by Lennart Philipson. At the time, adenovirus was one of the best tools to study programmed gene expression in an animal cell. My sole focus was to localize the elusive major late promoter, which provided my first Nature paper (7). Ed Ziff, a newly hired assistant professor from Cambridge, brought innovative unpublished DNA and RNA sequencing techniques that, after much technical angst, allowed us to sequence the major late promoter and derive the structure of the first eukaryotic polymerase II promoter (8). This thrilling result convinced me that the problem of gene control could be solved at the molecular level. Our next goal, which I shared with Michael Harpold in the Darnell lab, was to translate the concepts developed around adenovirus into cellular systems. My model was to analyze the glucocorticoid and thyroid hormone regulation of the GH gene. Under the strict federal guidelines for newly approved recombinant DNA research, we cloned the GH cDNA in 1977 and the first genomic clones in 1978 (9) after I moved on to The Salk Institute. However, to fully address the hormone signaling problem, I realized that it would be necessary to clone the GR and thyroid hormone receptors (TRs), which began in earnest in 1981. Up until that time, the purification and cloning of any polymerase II transcription factor had eluded researchers because of their low abundance. Four years later, the GR would be the first transcription factor for a defined response element to be cloned, sequenced, and functionally identified.

A Rock and A Hard Place

A key question was whether the GR protein encoded by the receptor was sufficient, when expressed in a heterologous cell, to convey the hormonal message. Before the publication, a new postdoc, Vincent Giguere, began tinkering with the isolated GR, trying to address this question. The rate of development of any field is limited by the existing techniques and depends on the development of new ones. Vincent devised a revolutionary technique—the cotransfection assay that required two plasmids to be taken up in the same cell, the expression vector to be transcribed, the encoded protein to be functional and an inducible promoter linked to a chloramphenicol acetyltransferase reporter in the nucleus ready to flicker on (10, 12). With so many variables and unknowns, I was stunned and expressionless when it worked the very first time. Cotransfection was an easy, fast, and quantitative technique. It would become (and still remains) the dominant assay to characterize receptor function. It would also become the mainstay for drug discovery in the pharmaceutical industry. The development of this technique proved a great advantage because existing technology involved creating stable cell lines, a tedious process prone to integration artifacts that ultimately could not match the explosive pace of the field. Indeed, within 4 months Stan and Vincent had fully characterized 27 insertional mutants delineating the DBD, LBD, and two activation domains (12). The route to understanding the signaling mechanism now had a solid structural foundation. A serendipitous gift to my retroviral origins was the homology of the GR sequence to the v-erbA oncogene product of the avian erythroblastosis virus genome (13). With this discovery, erbA advanced to a candidate nuclear transcription factor potentially involved in a signal transduction pathway. Thus, while Stan concentrated on the GR, Cary began to delve into the erbA discovery. Within months of the GR publication, the human c-erbA gene was in hand (14). Unbeknownst to us, Bjorn Vennstrom, one of the first to characterize the avian erythroblastosis virus genome, had also isolated c-erbA and was searching for a function. Based on the low homology of the LBD region to the GR and ER, both groups deduced that the imaginary erbA ligand would be nonsteroidal.

The work of our two groups (15, 16), published in December of 1986, broadened the principles of the signal transduction pathway by demonstrating that thyroid and steroid hormone receptor signaling had a common evolutionary origin and provided an entree to understand how mutations within a receptor could activate it to an oncogene. Although we did not know it at the time, this work would also lead us to the concept of the corepressor. In the meantime, my student, Catherine Thompson, zeroed in on an erb-A-related gene and soon identified a second TR expressed at high levels in the central nervous system (17). Thus came into existence the and forms of the TR. Jeff Arriza, the third graduate student in the lab, purified a genomic fragment that had weakly hybridized to the GR resulting in the isolation of the human mineralocorticoid receptor (MR) (18). MR proved to have an at least 10-fold higher affinity for glucocorticoids than the GR itself and was further distinguished by its ability to bind and be activated by aldosterone. This enabled the development of GR- and MR-selective drugs such as the recent MR antagonist eplerenone. Thus, in a 2-yr time span our lab had progressed on three distinct, albeit related, receptor systems, and in doing so molecular biology and endocrinology were irrevocably linked. The field of molecular endocrinology (and coincidentally the eponymous journal) was born.

Ligands From Stone

I have often been asked how we could handle so many divergent systems. Indeed, from a medical perspective, these systems seem widely unrelated. Studies of ER, progesterone receptor, and androgen receptor (AR) fall under reproductive physiology, vitamin D under bone and mineral metabolism, with vitamin A part of nutritional science. Medical fields are naturally idiosyncratic because of the specialized knowledge required to conduct experiments. With my training as a molecular biologist, physiology was the complex output of genes and thus control of gene expression was the overriding problem. This conceptual approach had a great unifying effect because all receptors transduce their signaling through the gene. As an “outsider,” my goal was to exploit multiple receptor systems to seek general principles. This philosophical approach afforded us a freedom to redefine the signaling problem from the nucleus outward and thus even poorly characterized, even unknown, physiologic systems fell into the crosshairs of our molecular gun.

Vincent, while screening a testes Fig. 1. Models of Nuclear Receptor Structure Top, Original hand-shaped wire model (circa 1992) of the nuclear receptor DBD. Bottom, Schematic representation of the GR DBD. Conserved residues in zinc fingers, P-box and D-box are indicated isolated what would become the vitamin A or retinoic acid receptor (RAR) (19). Initially, Vincent thought he had isolated the AR, although this later proved not to be the case. By that stage, the lab had perfected a new technique—the domain swap—by which the GR DBD could be introduced into any receptor and confers on the chimeric protein the ability to activate a mouse mammary tumor virus reporter. This clever technique, independently developed in the Chambon lab, would prove to be essential. Effectively, the domain swap would enable us to screen for ligands without any knowledge of their physiologic function. Activation of a target gene was all that was needed! By creating this GR chimera, Vincent was able to screen the new receptor against a ligand cocktail including androgens, steroids, thyroid hormone, cholesterol, and the vitamin A metabolite retinoic acid. From the first assay, it was clear that he had isolated a high-affinity selective RAR that had no response to any other test ligand. Thus, without knowing any true direct target gene for retinoic acid, we were nonetheless able to isolate and characterize its receptor. Remarkably, Martin Petkovich in the Chambon lab isolated the same gene. Once again, this is an example where a new technique offered an entirely new approach to a problem. Both papers were published in the December 1987 issue of Nature (19, 20). With the combination of steroids, thyroid hormones, and vitamin A, the three elemental components of the nuclear receptor superfamily were in hand. By the time the RAR papers were published, Vincent with Na Yang, had already isolated two estrogen-related receptors termed ERR1 and 2 that would represent the first true orphan receptors in the evolving superfamily (21). A third receptor (ERR3) would be isolated 10 yr later (22). The three ERRs are distinguished by their ability to activate through ER response elements, but required no ligand. However, of potential major medical relevance, estrogen antagonists such as 4-hydroxy-tamoxifen silences ERR constitutive activity (23). The superfamily was growing exponentially, transforming into a new field, driven by a new breed of exceptional students and fellows attracted by the mechanics of transcription and its emerging link to physiology. For example, the RAR and TR offered an unprecedented look at understanding the action of vitamin A as a morphogen and the role of thyroxin in setting the basal metabolic rate of the body. We were a relatively small group, and our decision to work on multiple different receptor systems created a unique environment. Because there was so little overlap between projects, postdocs and students readily discussed all results, exchanged reagents and freely collaborated, resulting in a tremendous acceleration of progress. The high level of camaraderie was powered by the joie de vivre of the exciting discoveries and the ability of our family of students and postdocs to each adopt their own receptors. We all felt we were in a golden age and even more was to come.

In 1989, Jan Sap in Vennstrom’s group and Klaus Damm in our group demonstrated that the TR becomes oncogenic by mutation in the LBD (24, 25). Although we expected ligand-independent activation, it was clearly a constituitive repressor becoming the first example of a dominant-negative oncogene. The concept of the dominant-negative oncogene had been proposed one year earlier by Ira Herskowitz (26). This discovery changed our thinking on hormone action, and repression soon would be shown to be a common feature of receptor antagonists. David Mangelsdorf, who had arrived in the lab the year before was captivated by the glow of weakly hybridizing DNA bands and, in 1989, had amassed his own collection of orphan receptors, among which was the future retinoid X receptor (RXR) (27). In search for biological activity, a candidate ligand was found in lipid extracts from outdated human blood. However, the key test came from demonstrating that addition of all-trans retinoic acid to cultured cells would lead to its rapid metabolism coupled with the release of an inducing activity for RXR, which we termed retinoid X. David and his benchmate, Rich Heyman, began working on the chemistry of this inducer along with Gregor Eichele and Christine Thaller, then at Baylor College of Medicine (Houston, TX). This work led to the identification of 9-cis retinoic acid by our lab and a group at Hoffman LaRoche (Nutley, NJ) (28, 29). Like the retinal molecule in rhodopsin, 9-cis-retinoic acid represents the exploitation of retinoid isomerization by nature to control a key signaling pathway. More importantly, in the 39 yr since the discovery of aldosterone in 1953, this revelation would reawaken and reinvent the single most defining but dormant tool of endocrinology—ligand discovery. Indeed, the discovery that new receptors could lead to new ligands opened up an entirely new avenue of research. Like the puzzle of the structure of the benzene ring, which was solved in 1890 when Fredrick Kekule dreamed of a snake biting its own tail, the physiologic head of the “endocrine snake” and the molecular biology tail had come full circle. The era of reverse endocrinology was now upon us.

Response Elements: Deciphering The Scripts

One problem in addressing the downstream effects of our newly discovered receptors was that their response elements and target genes were by definition unknown. Kaz Umesono delved into this mystery and would produce a paradigm shift that would both solve the problem and further unify the field. With the view that the DBD functioned as a molecular receptor for its cognate hormone response element, meticulous mutational studies revealed two key DBD sequences, termed the P-box and D-box, that controlled the process (30).

The D-box was shown to direct dimerization, a feature previously thought to be unique to the LBD. One perplexing point was that the P-boxes of the nonsteroidal receptors were conserved, leading to the improbable prediction that many different receptors would recognize the same target sequence. By manual compilation and comparison of all known response elements, Kaz proposed a core hexamer— AGGTCA—as the prototypic common target sequence. By requiring the half-site to be an obligate hexamer an underlying pattern—the direct repeat—emerged. In the direct repeat paradigm, we proposed that half-site spacing, not sequence difference, was the key ingredient to distinguishing the response elements. The metric was referred to as the 3-4-5 rule (31). According to the rule, direct repeats of AGGTCA spaced by three nucleotides, would be a vitamin D response element (DR-3), the same repeat spaced by four nucleotides a thyroid hormone response element (DR-4), and the same repeat spaced by five nucleotides a vitamin A response element (DR-5). Eventually, all steps from 0–5 on the DR ladder would be filled (Fig. 2). The validity of this paradigm was ensured by a crystal structure solved in collaboration with Paul Sigler’s group at Yale (32). Indeed, of the remaining 40 nonsteroidal receptors, all but three can be demonstrated to have a preferred binding site within some component of the direct repeat ladder. Exceptions include SHP and DAX, which lack DBDs, and farnesoid X receptor (FXR) that binds to the ecdysone response element as a palindrome with zero spacing. Kaz’s insight, by drawing commonality from diversity, came to solve a problem that impacted on virtually every receptor. Remarkably, each new receptor in the superfamily could immediately be assigned a place on the ladder. The ladder also provided a simple means to conduct a ligand screening assay in absence of any knowledge of an endogenous target gene. Kaz’s ladder was a turbo charge for the field. The next major advance in the field was the discovery of the RXR heterodimer. Although we knew that retinoid and thyroid receptors required a nuclear competence factor for DNA binding, its identity was unknown. We tested RXR, but our initial experiments were flawed. Of the first four papers describing the discovery, that from Chambon’s lab was most elegant because they simply purified an activity to homogeneity to find RXR (33)! Rosenfeld was first to publish, and Ozato, Pfahl and Kliewer all concurred (34–37). Tony Oro and Pang Yao in our lab soon published that the ecdysone receptor functions as a heterodimer with ultraspiracle, the insect homolog of RXR (38, 39), revealing that the ancient origins of the heterodimer which arose before the divergence of vertebrates and invertebrates.

Reverse Endocrinology: Decoding Physiology

The orphan receptors would transform our view of endocrine physiology with unexpected links to toxicology, nutrition, cholesterol, and triglyceride metabolism as well as to a myriad of diseases including atherosclerosis, diabetes, and cancer. The three RXR isoforms formed the core with 14 heterodimer partners including the vitamin D receptor (VDR), TR/, and RAR//. The initial adopters of orphan receptors included Giguere, Mangelsdorf, Weinberger, Bruce Blumberg, Steve Kliewer, and Barry Forman. Barry unlocked the first secret to for peroxisome proliferator-activated receptor (PPAR) by identifying prostaglandin J2 (PGJ2) as a high-affinity ligand (40). The second step, in collaboration with Peter Tontonoz in Bruce Spiegleman’s lab, revealed that PGJ2 was adipogenic in cell lines and perhaps more importantly that the synthetic antidiabetic drug Troglitazone was a potent PPAR agonist (41). Similar work was conducted and published by Kliewer, who had now moved to Glaxo (42). By acquiring a ligand, a physiologic response, and a drug, PPAR was suddenly transported to the center of a physiologic cyclone that would spin into its own specialty field. Since 1995, more than 1000 papers (see PubMed) have been published on PPAR and its natural and synthetic ligands. This early work illuminated the molecular strategy of reverse endocrinology and the emerging importance of the orphan receptors in human disease and drug discovery. Cary returned to the lab for a sabbatical and, with Barry, demonstrated that FXR was responsive to farnesoids and other molecules in the mevalonate pathway. The findings by Mangelsdorf that liver X receptors (LXRs) bound oxysterols (43) and by Kliewer, Mangelsdorf, and Forman that FXR is a bile acid receptor (44–46) provided a whole new conceptual approach to cholesterol and triglyceride homeostasis. The steroid and xenobiotic receptors (SXR)/pregnane X receptor (PXR) (47–49) and the constituitive androstane receptor (CAR) (50) respond to xenobiotics to activate genes for P450 Fig. 2. Examples of Receptor Heterodimer Combinations that Fill the Direct Repeat (DR) Response Element Ladder from DR1 to DR5 Evans enzymes, conjugation and transport systems that detoxify drugs, foreign chemicals, and endogenous steroids. RXR and its associated heterodimeric partners quickly established a new branch of physiology, shedding its dependence on endocrine glands and allowing the body to decode signals from environmental toxins, dietary nutrients, and common metabolites of intermediary metabolism.

Continued…

ROCK OF AGES

The human body is, after all a living machine, a complex device that consumes and uses energy to sustain itself, defend against predators, and ultimately reproduce. One might reasonably ask, “If the superfamily acts through a common molecular template, can the family as a whole be viewed as a functional entity?” In other words, is there yet some overarching principle that we have yet to grasp. . . and might this imaginary principle lie at the heart of systems physiology? Simply stated, what led to the evolution of integrated physiology as the functional output of the superfamily? One obvious speculation is survival. To survive, all organisms must be able to acquire, absorb, distribute, store, and use energy. The receptors are exquisitely evolved to manage fuel—everything from dietary and endogenous fats (PPARs), cholesterol (LXR, FXR), sugar mobilization (GR), salt (MR), and calcium (VDR) balance and maintenance of basal metabolic rate (TR). Because only a fraction of the material we voluntarily place in our bodies is nutritional, the xenobiotic receptors (PXR, CAR) are specialized to defend against the innumerable toxins in our environment. Survival also means reproduction, which is controlled by the gonadal steroid receptors (progesterone receptor, ER, AR). However, fertility is dependent on nutritional status, indicating the presumptive communication between these two branches of the family. The third key component managed by the nuclear receptor family is inflammation. During viral, bacterial, or fungal infection, the inflammatory response defends the body while suppressing appetite, conserving fuel, and encouraging sleep (associated with cytokine release). However, if needed, even an ill body is capable of defending itself by releasing adrenal steroids, mobilizing massive amounts of fuel, and transiently suppressing inflammation. In fact, clinically, (with the exception of hormone replacement) glucocorticoids are only used as antiinflammatory agents. Other receptors including the RARs, LXRs, PPAR and , and vitamin D receptor protect against inflammation. Thus, nature evolved within the structure of the receptor the combined ability to manage energy and inflammation, indicating the important duality between these two systems. In aggregate, this commonality between distinct physiologic branches suggests that the superfamily might be approached as an intact functional dynamic entity.

Historically, endocrinologists and geneticists rarely saw eye to eye. As I have indicated in this perspective article, the disciplines have now become united in a new subject—transcriptional physiology. With this in mind, we might expect the existence of larger organizational principles that establish how the various evolutionary branches of the superfamily integrate to form whole body physiology. The existence of molecular rules governing the function and evolution of a megagenetic entity like the nuclear receptor superfamily, if correct, may be useful in understanding complex human disease and provide a conceptual basis to create more effective pharmacology. With so much accomplished in the last 20 yr (see Fig. 3), there are glimpses of clarity—enough to see the enormity and wonder of the problem and enough to know there is still a long and challenging voyage ahead. But who knows, the next breakthrough may only be a stone’s throw away.

http://press.endocrine.org/doi/pdf/10.1210/me.2005-0046

 

Pierre Chambon MD

Recipient of the Canada Gairdner International Award, 2010
“For the elucidation of fundamental mechanisms of transcription in animal cells and to the discovery of the nuclear receptor superfamily.”

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch-Graffenstaden, France

Dr. Pierre Chambon is Honorary Professor at the College de France (Paris), and Emeritus Professor at the Faculté de Médecine of the Strasbourg University. He was the Founder and former Director of the IGBMC, and also the Founder and former Director of the Institut Clinique de la Souris (ICS/MCI), in Strasbourg.

Dr. Pierre Chambon is a world expert in the fields of gene structure, and transcriptional control of gene expression. During the last 25 years his studies on the structure and function of nuclear receptors has changed our concept of signal transduction and endocrinology. By cloning the estrogen and progesterone receptors, and discovering the retinoic acid receptor family, he markedly contributed to the discovery of the superfamily of nuclear receptors and to the elucidation of their universal mechanism of action that links transcription, physiology and pathology. Through extensive site-directed mutagenesis and genetic studies in the mouse, Pierre Chambon has unveiled the paramount importance of nuclear receptor signaling in embryonic development and homeostasis at the adult stage. The discoveries of Pierre Chambon have revolutionized the fields of development, endocrinology and metabolism, and their disorders, pointing to new tactics for drug discovery, and finding important applications in biotechnology and modern medicine.

These scientific achievements are logically inscribed in an uninterrupted series of discoveries made by Pierre Chambon over the last 45 years in the field of transcriptional control of gene expression in higher eukaryotes: discovery of PolyADPribose (1963), discovery of multiple RNA polymerases differently sensitive to a-amanitin (1969), contribution to elucidation of chromatin structure: the Nucleosome (1974), discovery of animal split genes (1977), discovery of enhancer elements (1981), discovery of multiple promoter elements and their cognate factors (1980-1993).

Pierre Chambon has received numerous international awards, including the 2004 Lasker Basic Medical Research Award for the discovery of the superfamily of nuclear hormone receptors and the elucidation of a unifying mechanism that regulates embryonic development and diverse metabolic pathways. He is a member of the French Académie des Sciences, and also a Foreign Member of the National Academy of Sciences (USA) and of the Royal Swedish Academy of Sciences. Pierre Chambon serves on a number of editorial boards, including Cell, and Molecular Cell. Pierre Chambon is author of more than 900 publications. He has been ranked fourth among most prominent life scientists for the 1983-2002 period.

An Interview with Pierre Chambon
2004 Albert Lasker Basic Medical Research Award
http://www.laskerfoundation.org/media/v_chambon.htm

Pierre Chambon, MD

​Honorary Professor at the Collège-de-France and Professor of Molecular Biology and Genetics, Institute for Advanced Study, University of Strasbourg; Group Leader, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch-Graffenstaden, Strasburg, France

A pioneer in the fields of gene structure and transcriptional control of gene expression, Dr. Chambon has fundamentally changed our understanding of signal transduction, which has led to revolutionary new tactics for drug discovery. His work elucidated how molecules that promote gene transcription are organized and regulated in eukaryotic organisms and, independently of Dr. Ronald Evans, he discovered in 1987 the retinoid receptor families, which led to the discovery and characterization of the superfamily of nuclear hormone receptors, including steroid and retinoid receptors.

Dr. Chambon’s previous research led to the discovery of PolyADPribose, multiple RNA polymerases differentially sensitive to α-amaniti, and has markedly contributed to the elucidation of the nucleosome and chromatin structure, as well as to the discovery of animal split genes, DNA sequences called enhancer elements, and multiple promoter elements and their cognate factors. These discoveries have greatly enhanced understanding of embryonic development and cell differentiation. To further studies of various nuclear receptors, Dr. Chambon has developed a method that allows in the mouse the generation of somatic mutations of any gene, at any time, and in any specific cell type, a tool valuable in generating mouse models of cancer.

In 1994, Dr. Chambon took on the role of founding a major research institute in France. As the first director of IGBMC, he built the institute to encompass hundreds of top researchers and multiple research programs funded by public agencies and private industry. In 2002, he founded and was the first director of the Institut Clinique de la Souris in Strasbourg. In these positions, he has succeeded in supporting and influencing a generation of scientists.

Career Highlights

​2010  Canada Gairdner International Award

2004  Albert Lasker Basic Medical Research Award

2003  Alfred P. Sloan, Jr., Prize, General Motors Cancer Foundation

1999  Louisa Gross Horwitz Prize, Columbia University

1998  Robert A. Welch Award in Chemistry

1991  Prix Louis-Jeantet de médecine, Fondation Louis-Jeantet

1990  Sir Hans Krebs Medal, Federation of European Biochemical Societies

1988  King Faisal International Prize for Science, King Faisal Foundation

1987  Harvey Prize, Technicon-Israel Institute of Technology

more…

 

Minireviews In This Series:

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases

Sohaib Khan and Jerry B Lingrel

Steroid Receptor Coactivator (SRC) Family: Masters of Systems Biology

Brian York and Bert W. O’Malley

Estrogen Signaling via Estrogen Receptor β

Chunyan Zhao, Karin Dahlman-Wright, and Jan-Åke Gustafsson

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

David J. Shapiro, Chengjian Mao, and Milu T Cherian

Cellular Processing of the Glucocorticoid Receptor Gene and Protein: New Mechanisms for Generating Tissue Specific Actions of Glucocorticoids

Robert H Oakley and John A Cidlowski

Endogenous Ligands for Nuclear Receptors: Digging Deeper

Michael Schupp and Mitchell A. Lazar

 

 

 

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Summary of Signaling and Signaling Pathways


Summary of Signaling and Signaling Pathways

Author and Curator: Larry H Bernstein, MD, FCAP

In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it.  Furthermore, he observes –  Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

acetylCoA

acetylCoA

In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view.  The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics.  The inherent difficulty lies in two places:

  1. Interactions between differently weighted determinants
  2. A large part of the genome is concerned with regulatory function, not expression of the code

The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell.  Completion of the tutorial would provide

  1. a basic understanding signal transduction and
  2. the role of phosphorylation in signal transduction.
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

In addition – detailed knowledge of –

  1. the role of Tyrosine kinases and
  2. G protein-coupled receptors in cell signaling.
serine

serine

threonine

threonine

protein kinase

protein kinase

We are constantly receiving and interpreting signals from our environment, which can come

  • in the form of light, heat, odors, touch or sound.

The cells of our bodies are also

  • constantly receiving signals from other cells.

These signals are important to

  • keep cells alive and functioning as well as
  • to stimulate important events such as
  • cell division and differentiation.

Signals are most often chemicals that can be found

  • in the extracellular fluid around cells.

These chemicals can come

  • from distant locations in the body (endocrine signaling by hormones), from
  • nearby cells (paracrine signaling) or can even
  • be secreted by the same cell (autocrine signaling).

Notch-mediated juxtacrine signal between adjacent cells. 220px-Notchccr

Signaling molecules may trigger any number of cellular responses, including

  • changing the metabolism of the cell receiving the signal or
  • result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.

controlling the output of ribosomes.

To which I would now add..

  • result in either an inhibitory or a stimulatory effect

The three stages of cell signaling are:

Cell signaling can be divided into 3 stages:

Reception: A cell detects a signaling molecule from the outside of the cell.

Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.

Response: Finally, the signal triggers a specific cellular response.

signal transduction

signal transduction

http://www.hartnell.edu/tutorials/biology/images/signaltransduction_simple.jpg

The initiation is depicted as follows:

Signal Transduction – ligand binds to surface receptor

Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.

  • by changing shape or
  • by joining with another protein
  • once a specific ligand binds to it.

Examples of membrane receptors include

  • G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

  • Receptor Tyrosine Kinases.
intracellular signaling

intracellular signaling

http://www.hartnell.edu/tutorials/biology/images/membrane_receptor_tk.jpg

Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).

Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation.  That does not have to be the case in the normal homeostatic case.

This point is the differentiating case between what JEDS Roselino has referred as

  1. a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
  2. a one-to-one transcription of the genetic code.

The rate of transcription can be controlled, or it can be blocked.  This is in large part in response to the metabolites in the immediate interstitium.

This might only be

  • a change in the rate of a transcription or a suppression of expression through RNA.
  • Or through a conformational change in an enzyme
 Swinging domains in HECT E3 enzymes

Swinging domains in HECT E3 enzymes

Since signaling systems need to be

  • responsive to small concentrations of chemical signals and act quickly,
  • cells often use a multi-step pathway that transmits the signal quickly,
  • while amplifying the signal to numerous molecules at each step.

Signal transduction pathways are shown (simplified):

Signal Transduction

Signal Transduction

Signal transduction occurs when an

  1. extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
  2. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
  3. Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
  4. The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Guanosine monophosphate structure

Guanosine monophosphate structure

In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.

This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.

Integrin

Integrin

Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.

steroid hormone receptor

steroid hormone receptor

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

signal transduction pathways

signal transduction pathways

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.

The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.

RTKs

RTKs

The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.

Allosteric_Regulation.svg

Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.

Signal-Transduction-Pathway

Signal-Transduction-Pathway

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are

  • members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.

They act as molecular switches usually

  • tethered to membranes by isoprenyl groups linked to their carboxyl ends.

Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate

  • small G proteins that activate guanine nucleotide exchange factors such as SOS1.

Once activated, these exchange factors can activate more small G proteins, thus

  • amplifying the receptor’s initial signal.

The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.

Integrin

 

Integrin

Integrin

Integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).

Integrins are produced by a wide variety of cells; they play a role in

  • cell attachment to other cells and the extracellular matrix and
  • in the transduction of signals from extracellular matrix components such as fibronectin and collagen.

Ligand binding to the extracellular domain of integrins

  • changes the protein’s conformation,
  • clustering it at the cell membrane to
  • initiate signal transduction.

Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.

As shown in the picture, cooperative integrin-RTK signaling determines the

  1. timing of cellular survival,
  2. apoptosis,
  3. proliferation, and
  4. differentiation.
integrin-mediated signal transduction

integrin-mediated signal transduction

Integrin signaling

Integrin signaling

ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation

  • to open a channel in the cell membrane
  • through which ions relaying signals can pass.

An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels

  1. induces action potentials, such as those that travel along nerves,
  2. by depolarizing the membrane of post-synaptic cells,
  3. resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR

RyR and Ca+ release from SR

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;

  • it acts as a second messenger
  • initiating signal transduction cascades and
  • altering the physiology of the responding cell.

This results in amplification of the synapse response between synaptic cells

  • by remodelling the dendritic spines involved in the synapse.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,

cAMP

cAMP

Inositol_1,4,5-trisphosphate.svg

Inositol_1,4,5-trisphosphate.svg

  • the latter controlling the release of intracellular calcium stores into the cytoplasm.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,

  • calcium ions bind to the EF hand domains of calmodulin,
  • allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor

calcium movement and RyR2 receptor

PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.

Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_via_metabolomics_and_systems_biology_driven_approaches

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Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are

  • α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,

  • DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.

DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.

On the other hand,

  • zinc is the most abundant trace metal in the human brain.

There are many scientific studies linking zinc, especially

  • excess amounts of free zinc, to cellular death.

Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between

  • omega-3 fatty acids, zinc transporter levels and
  • free zinc availability at cellular levels.

Many other studies have also suggested a possible

  • omega-3 and zinc effect on neurodegeneration and cellular death.

Therefore, in this review, we will examine

  • the effect of omega-3 fatty acids on zinc transporters and
  • the importance of free zinc for human neuronal cells.

Moreover, we will evaluate the collective understanding of

  • mechanism(s) for the interaction of these elements in neuronal research and their
  • significance for the diagnosis and treatment of neurodegeneration.

Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to

  • reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in

Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as

  • cellular differentiation,
  • photoreceptor membrane biogenesis and
  • active synaptogenesis.

Despite the significance of these

two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both

  • omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
  • opposing physiological effects. In the human body,
  • high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
  • thereby increase inflammatory processes [10].

the reverse process can be seen with increased omega-3 fatty acids in the body.

Many other factors, such as

  1. thromboxane A2 (TXA2),
  2. leukotriene
  3. B4 (LTB4),
  4. IL-1,
  5. IL-6,
  6. tumor necrosis factor (TNF) and
  7. C-reactive protein,

which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.

Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in

  • immune responses,
  • gene expression and
  • intercellular communications.

omega-3 fatty acids are known to be vital in

  • the prevention of fatal ventricular arrhythmias, and
  • are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels

.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.

The main

In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that

  • only high fish consumption, but
  • not dietary omega-3 acid intake,
  • had a protective effect on cognitive decline

In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown

a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.

A  trial of subjects with mild memory complaints demonstrated

  • an improvement with 900 mg of DHA.

We review key findings on

  • the effect of the omega-3 fatty acid DHA on zinc transporters and the
  • importance of free zinc to human neuronal cells.

DHA is the most abundant fatty acid in neural membranes, imparting appropriate

  • fluidity and other properties,

and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated

  • in membrane phospholipids at synapses and
  • in retinal photoreceptors and
  • also in the testis and sperm.

In adult rats’ brain, DHA comprises approximately

  • 17% of the total fatty acid weight, and
  • in the retina it is as high as 33%.

DHA is believed to have played a major role in the evolution of the modern human –

  • in particular the well-developed brain.

Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.

Analysis of human cadaver brains have shown that

  • people with AD have less DHA in their frontal lobe
  • and hippocampus compared with unaffected individuals

Furthermore, studies in mice have increased support for the

  • protective role of omega-3 fatty acids.

Mice administrated with a dietary intake of DHA showed

  • an increase in DHA levels in the hippocampus.

Errors in memory were decreased in these mice and they demonstrated

  • reduced peroxide and free radical levels,
  • suggesting a role in antioxidant defense.

Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary

  • DHA supplementation had a protective effect against reduction in
  • drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
  • decreased caspase activity.

 

Zinc

Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to

  • growth,
  • development,
  • differentiation,
  • immune response,
  • receptor activity,
  • DNA synthesis,
  • gene expression,
  • neuro-transmission,
  • enzymatic catalysis,
  • hormonal storage and release,
  • tissue repair,
  • memory,
  • the visual process

and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects,  as

  • a component of over 300 different enzymes
  • an integral component of a metallothioneins
  • a gene regulatory protein.

Approximately 3% of all proteins contain

  • zinc binding motifs .

The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;

  1. catalytic,
  2. coactive and

Indeed, it is the only metal found in all six different subclasses

of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.

Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated

  • through glutamate receptors, and
  • it inhibits excitatory and inhibitory receptors.

Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and

  • its release is dependent on neural activity.

Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.

The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to

  • form insoluble amyloid in the presence of
  • high physiological levels of zinc.

Insoluble amyloid is thought to

  • aggregate to form plaques,

which is a main pathological feature of AD. Further studies have shown that

  • chelation of zinc ions can deform and disaggregate plaques.

In AD, the most prominent injuries are found in

  • hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
  • somatostatin-containing neurons in the forebrain.

All of these neurons are known to favor

  • rapid and direct entry of zinc in high concentration
  • leaving neurons frequently exposed to high dosages of zinc.

This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of

  • inhibiting Ca2+ and Na+ voltage gated channels
  • and up-regulating the cellular levels of reactive oxygen species (ROS).

High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.

If the human diet is deficient in zinc, the body

  • efficiently conserves zinc at the tissue level by compensating other cellular mechanisms

to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and

  • redistribution of available zinc to more zinc dependent cells or organs.

A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the

  • assessment of the zinc status of individuals and populations.

In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting

  • as a target for zinc ion binding and thus
  • assisting in the trafficking of zinc ions through the cell,
  • which may be similar to that of zinc transporters

Zinc Transporters

Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur

  • via intermembrane proteins and zinc transporter (ZnT) proteins

These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).

Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).

The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where

  • it is responsible for the transport of zinc into the synaptic vesicles of
  • glutamatergic neurons in the hippocampus and neocortex,

Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/.   http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg

Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].

omega-3 fatty acids in the mammalian body are

  1. α-linolenic acid (ALA),
  2. docosahexenoic acid (DHA) and
  3. eicosapentaenoic acid (EPA).

In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between

  • increased fish consumption and reduced risk of AD
  • and eight out of ten studies have reported a link between higher blood omega-3 levels

DHA and Zinc Homeostasis

Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in

  • increased zinc levels in the hippocampus and
  • elevated expression of the putative zinc transporter, ZnT3, in the rat brain.

Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,

  • a significant reduction in plaque load
  • and the presence of insoluble amyloid were observed.

In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant

  • reduction in free zinc availability in the hippocampus
  • and cerebral cortex.

Collectively, the findings from this study are very interesting and indicate a clear connection between

  • zinc availability and amyloid plaque formation,

thus indicating a possible link to AD.

DHA supplementation has also been reported to limit the following:

  1. amyloid presence,
  2. synaptic marker loss,
  3. hyper-phosphorylation of Tau,
  4. oxidative damage and
  5. cognitive deficits in transgenic mouse model of AD.

In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.

In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.

When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible

  • neuroprotective mechanism of DHA,

which we believe is exerted through

  • a reduction in cellular zinc levels (through altering zinc transporter expression levels)
  • that in turn inhibits apoptosis.

DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and

  • free zinc levels in the cytosol were significantly low compared to the control

This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had

  • low levels of active caspase-3 and
  • high Bcl-2 levels compared to the control treatment.

These findings are consistent with previous published data and further strengthen the possible

  • correlation between zinc, DHA and neurodegeneration.

On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of

  • ZnT3 in memory and AD pathology.

For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of

  • ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
  • required for hippocampus-dependent memory

Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.

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