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

Address all correspondence and requests for reprints to: Ronald M. Evans, Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037.

ROMANCING THE STONE

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

Unlike many others working in the field of nuclear receptors that had their start as endocrinologists, my interest developed from my early work in molecular biology and transcription. In 1970, I entered graduate school at UCLA, focusing on RNA tumor viruses with Marcel Baluda, a protégé of Renato Dulbecco. 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

The isolation would not be easy as the GR was buried deep within the cell. Although several regulatory factors including the ER, GR, progesterone receptor, and pregnancy-specific-ß1-glycoprotein (SP1) had been partially purified, there was no available amino acid sequence to either guide the cloning or help confirm the identity of the products. A new strategy would have to be employed. To make a long story short (for the longer version, see Ref. 10), our approach relied on antibodies independently prepared by our collaborators John Cidlowski and Brad Thompson for both enrichment of mRNA and the screening of expression cDNA libraries.

In the absence of functional assays, the eventual identification of the full-length human GR by Stan Hollenberg and Cary Weinberger was dependent on the reaction of an in vitro translation product with the antibody and more importantly, by the demonstration that this in vitro product bound glucocorticoids (11). Two forms of the human GR were isolated (termed {alpha} and ß), which differed only at their extreme carboxy termini. Because the ß isoforms did not bind hormone, we concluded that the ligand binding domain (LBD) was located in this region (1).

We proposed that a cysteine-rich amino acid stretch in the middle of the protein encoded the DNA binding domain (DBD) and even speculated on a zinc finger motif, which to our delight turned out to be correct (Fig. 1Go). The receptor mapped to human chromosome 5, and low stringency hybridization suggested other related sequences in the genome.



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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.

 
A key question was whether the 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. Indeed, Cary firmly believed the nonsteroidal ligand was thyroid hormone. No doubt this was influenced by our ongoing commitment to thyroid hormone regulation of the GH gene, although few in the lab took Cary’s belief seriously.

Over a drink at an EMBO meeting, Cary confessed his idea to Bjorn, who promptly confirmed this suspicion. 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 {alpha} and ß 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.

This transcription centric approach would bear fruit in the coming year as Vincent, while screening a testes library, 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). Remarkably, a single amino acid change within the P-box could interconvert glucocorticoid and ER specificity. 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. 2Go).



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Fig. 2. Examples of Receptor Heterodimer Combinations that Fill the Direct Repeat (DR) Response Element Ladder from DR1 to DR5

 
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, 35, 36, 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{alpha}/ß, and RAR{alpha}/ß/{gamma}.

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) {gamma} 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{gamma} 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{gamma} 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{gamma} 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, 45, 46) provided a whole new conceptual approach to cholesterol and triglyceride homeostasis. The steroid and xenobiotic receptors (SXR)/pregnane X receptor (PXR) (47, 48, 49) and the constituitive androstane receptor (CAR) (50) respond to xenobiotics to activate genes for P450 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.

FROM TABLET TO PILL

Neither wealth nor royalty wields power over the frailties of the human body. But nuclear receptors do. This makes them excellent targets for disease treatment. Our work impacted therapy in several aspects.

First, the cloning of the human GR started off the collection of the human superfamily genes. This is important because human receptors often respond differently to drugs than other mammalian receptors. The most dramatic examples of species variation are the xenobiotic receptors, but even small differences can be important in drug development. Over the intervening years, our group and others have added to and completed the human nuclear receptor set.

Secondly, the cotransfection assay provided an innovative and quantitative means to screen chemical libraries for new and specific classes of both agonists and antagonists. This was a great equalizing factor for poorly characterized receptors.

Third, the orphan receptors, particularly RXR and its partners, offered unprecedented new targets. RXR compounds such as 9-cis-retinoic acid (Panretin) and a synthetic analog (Targretin) are now approved by the Food and Drug Administration as compounds for cancer. The antidiabetic thiazolidinedione compounds (e.g. rosiglitazone and pioglitazone) work by activating PPAR{gamma} and are the only approved drugs that act as insulin sensitizers. Many more potent forms are under development. The t(15:17) translocated form of the RAR produces acute promyelocytic leukemia, providing the first direct link between a receptor mutation and cancer (although this is now confirmed for many receptors including ER, AR, and PPAR{gamma}). Retinoic acid treatment was the first and perhaps the most dramatic example of differentiation therapy with a natural compound. It acts by triggering the maturation of immature leukemic white blood cells. Acute promyelocytic leukemia, which only 10 yr ago was almost uniformly fatal, now enjoys a greater than 90% cure rate! LXR and FXR represent promising new drug targets for cholesterol and triglyceride control, although these have not yet advanced into clinical trials.

Perhaps one of the most powerfully quiet impacts that we have participated in is that of the xenobiotic receptor PXR. One of the most vexing problems in drug development is the inappropriate stimulation of PXR, which is responsible for approximately 60% of drug-drug interactions. Since its identification, the cloned human receptor [also known as SXR (47)] has progressively been incorporated by the pharmaceutical industry as a counter screen to develop drugs with little or no activity on PXR (51, 52). This one simple screen alone should help to produce a future generation of markedly safer drugs.

GENETIC TECTONICS

Twenty-seven years ago, my infatuation and driving commitment to nuclear receptors was based on the question of how genes can be controlled at both the cellular and organismal level. Shortly after the GR cloning, nuclear receptors quickly became one of the dominant models for studying transcriptional control. We naturally assumed that the receptors would somehow link to the initiation machinery via direct physical interaction. This was wrong. A number of key events intervened to change our thinking of the hormonal switch. In 1989, we and Vennstrom’s lab (24, 25) demonstrated that the oncogenic form of the TR was a transcriptional repressor. This demonstration of active repression by a nonliganded transcription factor was the first link in piecing together the now standard two-state model for receptor activation. The attempt to understand this mechanism led to the isolation of silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor corepressor (N-CoR), which helped to initiate the cofactor era (53, 54). Brown and O’Malley isolated the first p160 proteins [ERAP160, SRC-1 (55, 56)], and in the following year we and Rosenfeld (57, 58) described cAMP response element binding protein-binding protein (CBP) /P300 as potent mediators of nuclear receptor activation. Thus, in the span of 18 months, corepressors and coactivators were on the map. Allis’ discovery of histone acetyltransferases directly implicated the enzymatic activity of described CBP in transcriptional activation (59). After Stu Schreibers’ discovery of histone deacetylase-1, we (and four other labs) (60, 61, 62, 63, 64, 65) showed that transcriptional repressors such as the SMRT and N-CoR act by recruiting this enzyme to chromatin.

The concept that ligand acts by dismissing histone deacetylases and activate by recruiting histone acetyltransferases in a ligand-dependent fashion transformed our view of the two-step signaling process. Suddenly, it appeared that modification of the histone tail was the actual physical substrate of hormone action. This concept that divergent receptors employ a common molecular operating system was simply not anticipated. For this reason, our focus on transcription instead of physiology provided the needed forward theme to decipher a unified signaling hypothesis.

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{gamma} and {delta}, 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 mega-genetic 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. 3Go), 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.



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Fig. 3. Time Line of Progress in the Nuclear Receptor Field over the Past Two Decades

The general trend of the field is shown above the time line, with specific discoveries from our lab indicated below. See Refs. 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89–91.*, Virtual Physiology—refers to generation of new traits (e.g. marathon mouse) to study disease; Digital Medicine—employs computational infrastructure to integrate large bodies of knowledge for predictive and preventative medicine. **, Barish, G. D., M. Downes, W. A. Alaynick, C. B. Ocampo, R. T. Yu, A. L. Bookout, D. J. Mangelsdorf, and R. M. Evans, manuscript in preparation; Xang X., M. Downes, R. T. Yu, A. L. Bookout, M. Straume, D. J. Mangelsdorf, and R. M. Evans, manuscript in preparation; and Fu, M., T. Sun, A. L. Bookout, M. Downes, R. T. Yu, R. M. Evans, and D. J. Mangelsdorf, manuscript in preparation. VLDL, Very low-density lipoprotein receptor; NR, nuclear receptor; RA, retinoic acid; SMRTER, Drosophila nuclear receptor coregulator; TLX, vertebrate tailless-like nuclear receptor; PML, human acute promyelocytic leukemia; LTP, long-term potentiation; LTD, long-term depression; NMR, nuclear magnetic resonance.

 



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Fig. 4.
 
ACKNOWLEDGMENTS

I am indebted to a remarkable group of teachers and institutions, especially Jim Darnell and the Rockefeller University, and for the last 27 yr my students, post-docs, and technicians at the Salk Institute for their loyalty, dedication, and enthusiasm for science. I am grateful to my many colleagues and collaborators who have been generous in sharing their insight and expertise. In preparing this article, special thanks go to Ruth Yu for comments, persistence, and behind-the-scene work on figures and text. I also thank Vincent Giguere, David Mangelsdorf, and Emily Kagan for commentary and correction, my assistants Lita Ong and Elaine Stevens for their innumerable contributions and friendship, and my wife Ellen and daughter Lena for unwaversing support.

FOOTNOTES

The Rosetta Stone theme was inspired by Joe Goldstein’s commentary at the 2004 Lasker Awards ceremony.

RME is an Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies. This work was supported in part by National Institutes of Health Grants DK57978, HD27183, Program Project CA54418, and the Hillblom Foundation.

Abbreviations: AR, Androgen receptor; CAR, constituitive androstane receptor; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; DR, vitamin D response element; ER, estrogen receptor; ERR, estrogen-related receptor; FXR, farnesoid X receptor; GR, glucocorticoid receptor; LBD, ligand binding domain; LXR, liver X receptor; MR, mineralocorticoid receptor; N-CoR, nuclear receptor corepressor; PGJ2, prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; SMRT, silencing mediator of retinoid and thyroid hormone receptors; TR, thyroid hormone receptor; VDR, vitamin D receptor.

Received for publication January 18, 2005. Accepted for publication March 28, 2005.

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