Feeds:
Posts
Comments

Posts Tagged ‘progesterone receptor’


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

 

 

 

Read Full Post »


Cilia and the Oviduct

Author: Aashir Awan, PhD

In a previous article, there was a discussion on the role of primary cilia in ovarian cancers with specific context to the hedgehog signal transduction system.  The article helped to highlight not only the role that this organelle plays in ovarian cancer tumorigenesis but also hints at perhaps a mechanistic explanation at the molecular level (Egeberg et al., 2012).  In this review, we focus on primary cilia and some of the signal transduction pathways it helps to coordinate within the oviduct.  Motile cilia are probably better known in their roles  aiding in the movement of the oocyte.  But, in the last few years, research has been undertaken to study the sensory role of the cilium in the female reproductive system.  As such, Drs. Christensen and Stefan Teilmann (University of Copenhagen) undertook a few studies to show the importance of three different signal transduction systems that are being coordinated by the cilium in this particular tissue.

Fig2Their first paper demonstrated that progesterone receptor was localized to the cilia on the epithelial layer of cells surrounding the oviduct and specifically to the lower half of the ciliary length which can be seen in the immunofluorescence analysis of the progesterone receptor profile in the left hand-side figure (Teilmann et al., 2006).  Furthermore, the expression of this receptor is markedly increased upon exposure to gonadotrophin hormones indicating that there is a feedback loop that is sensitive to hormonal regulation.  Previously, it had been shown that progesterone regulates the activity of the outer dynein arms of the cilium through specific effector molecules (Fliegauf et al., 2005).  Thus, the progesterone released upon ovulation would be thought to directly affect the ciliated epiethlium in order to help facilitate the movement of the oocyte through the oviduct thereby highlighting the important role of the cilium (and the signal transduction pathway) to the overall physiology of the female reproductive system.  This work has recently been reproduced by Dr. Larrson’s group in Sweden (Bylander et al., 2013).

Fig1

The Christensen group continued further studies by localizing the angipoeiten receptors, Tie-1 and Tie-2, to the primary cilia of the ovarian surface epithelial as well as the oviduct as seen in the figure on right showing an immunoflourescent micrograph of the infundibulum (Teilmann and Christensen 2005).  Since the expression of their agonist, Ang1, increases during ovulation (Hazzard et al., 1999), both these receptors are thought to play a role in vascularization of the tissue surrounding the developing follicles.  Also, using this  reasoning, the paper argues that the Ang/Tie signaling axis plays an important and general role by serving as an anti-apoptotic system to maintain a dedifferentiated phenotype of both endothelial cells.

Fig3

Finally, Dr. Christensen’s group also demonstrated a unique localization of polycystins 1 and 2 to the primary cilia of ovarian granulose cells (Teilmann et al., 2005).  These calcium cation channels have been shown to sense the flow of urine in the kidney in monitoring general homeostasis and whose mutations have been shown to cause polycystic kidney disesase (Pazour et al.,2002; Yoder et al., 2002).  As with the progesterone receptor, there is a marked effect on polycystins concentration upon gonadrotrophin stimulation as clearly seen on the left-hand side figure (the arrow show ciliary localization of the polycystin 2 receptor; also, note the dramatic increase in polycystin 2 immunofluorescence in the infundibulum).  Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4) was found to be localized to the motile cilia in specific subpopulation of epithelial cells within the ampulla and isthmus.  Thus, the localization of these receptors  indicates that the primary cilia would again be involved in a sensory role perhaps by affecting the differentiation and maturation of the emerging oocyte and in relaying physiological information upon ovulatation to the epithelial cells of the surrounding oviduct.

One can imagine that these are probably only a partial list of the important receptor molecules localized thus far to the  cilia that exist within the female reproductive system.  Since more and more receptor molecules are being found within the relatively small confines of this organelle, one can hypothesize that perhaps the signal transduction mechanism between different receptor molecules is ocurring within the cilium itself perhaps even independent of what may be occurring in the cell body. Since reproductive and fertility issues remain a problem in the medical field, it behooves us to continue research into the overall contributions of  this organelle within the female reproductive system.

REFERENCES

Bylander ALind KGoksör MBillig HLarsson DJ. 2013 The classical progesterone receptor mediates the rapid reduction of fallopian tube ciliary beat frequency by progesterone. Reprod Biol Endocrinol. 11:33.

Egeberg DLLethan MManguso RSchneider LAwan AJørgensen TSByskov AGPedersen LBChristensen ST. 2012 Primary cilia and aberrant cell signaling in epithelial ovarian cancer. Cilia. 1:15.

Fliegauf MOlbrich HHorvath JWildhaber JHZariwala MAKennedy MKnowles MROmran H. 2005 Mislocalization of DNAH5 and DNAH9 in respiratory cells from patients with primary ciliary dyskinesia. Am J Respir Crit Care Med. 171:1343-1349.

Hazzard TMMolskness TAChaffin CLStouffer RL. 1999 Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval. Mol Hum Reprod. 5:1115-1121.

Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB.20002 Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol. 12:R378-R380.

Teilmann SC, Christensen ST. 2005 Localization of the angiopoietin receptors Tie-1 and Tie-2 on the primary cilia in the femalereproductive organs. Cell Biol Int.29:340-346.

Teilmann SCByskov AGPedersen PAWheatley DNPazour GJChristensen ST. 2005 Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs. Mol Reprod Dev. 71:444-452.

Teilmann SCClement CAThorup JByskov AGChristensen ST. 2006 Expression and localization of the progesterone receptor in mouse and human reproductive organs. J Endocrinol. 191:525-535.

Yoder BKHou XGuay-Woodford LM. 2002 The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol. 13:2508-2516.

 

Read Full Post »