A Life-Long Search for the Molecular Pathways of Steroid Hormone Action

Bert W. O’Malley

Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Bert O’Malley, Professor and Chairman, Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: berto{at}bcm.tmc.edu.

ABSTRACT

The O’Malley laboratory first showed that estrogen and progesterone act in the nucleus to stimulate synthesis of specific mRNAs (ovalbumin and avidin), coding for their respective inducible proteins. The overall molecular pathway of steroid-receptor-DNA-mRNA-protein-function was then established and provided a coherent foundation for future studies of the impact of estrogen and progesterone receptors on endocrine tissue development, adult function, and in pathologies such as cancer. The lab group went on to: biochemically demonstrate ligand-induced conformational activation of progesterone and estrogen receptors, discover the concept of ligand-independent activation of steroid receptors, discover key steroid receptor coactivator intermediary coactivators for receptor function, and define the role of coactivators/corepressors in selective receptor modulator drug action and in cell homeostasis. This body of work advanced our molecular understanding of the critical role of steroid hormones in normal and abnormal physiology and also generated a base of scientific knowledge that served to further modern hormonal therapy and disease management.

THE BEGINNING

MY INTEREST in steroid hormones began in earnest when I entered the National Institutes of Health (NIH) as a clinical associate in the Endocrine Branch of the National Cancer Institute in 1965, then under the direction of Mort Lipsett. My plan was to do steroid chemistry for a year and then to move my efforts toward examining the actions of these hormones. I remember the discussions at Branch coffee breaks about the radiolabeling of estradiol and the tissue-binding experiments performed by Jensen in his pioneering studies. At that time, there was considerable argument among Jensen and certain other scientists as to whether the estradiol binding protein he discovered was a true receptor, but I found the whole concept to be fascinating and almost certainly correct as presented by Elwood. It was at this point that I became interested in studying aspects of the molecular pathway of steroid hormone action. My first and memorable mentor for my work on the chicken oviduct was Stan Korenman, but he then left the NIH at the end of that first year. I stayed at NIH and continued to develop the chick oviduct as a model for hormone action. Why the oviduct? The oviduct was chosen because of the work of Roy Hertz in the early 1940s. He was attempting to devise a new bioassay for estrogenic substances and published weight changes in the chicken oviduct in response to estrogens. Knowing the protein composition of egg white and guessing that the proteins were made in the oviduct, Stan and I employed the chicken oviduct for the early studies on induction of specific radiolabeled protein synthesis. I remember my around-the-clock injections of chickens during that first year. Indeed, we showed that estrogen and progesterone induced de novo synthesis of the specific proteins, ovalbumin and avidin, respectively (1, 2).

DEMONSTRATING THAT NUCLEAR RECEPTOR (NR) LIGANDS REGULATE NUCLEAR RNAs

These protein experiments provided the critical background information to encourage me to attempt to define the overall pathway and mechanism of action of estrogen and progesterone. The sum total of my subsequent life’s work has been focused on this goal. Although it was suspected by some that steroid hormones could induce new protein synthesis, there was considerable confusion as to the molecular mechanism in the late 1960s and early 1970s. Some biochemists were still wed to the theory that steroids activated enzymes directly, and many prominent physiologists believed that steroid hormones exerted their primary effects at the cell membrane; the latter is a thought that only recently has been reborn and now appears to be bearing some fruit. At the time that I entered the field in the 1960s, studies by other labs showed that steroids caused an increase in incorporation of radiolabeled precursor nucleotides into trichloroacetic acid-precipitable RNA. A widespread interpretation that steroids increased RNA synthesis only by enhancement of radiolabeled nucleotide uptake into cellular pools was set to rest by the sum of our 1968 study showing that new species of RNA, measured by RNA-DNA hybridization and RNA composition analyses, were induced in chick oviduct target tissue by steroid hormones, and that their induction was coincident with the synthesis of new specific proteins (3). With these analyses, carried out by my first postdoctoral fellow, Bill McGuire, we predicted that steroid hormones acted on DNA to turn on the synthesis of genes. This result strongly focused my thoughts on the nucleus and gene expression, where they remained for the next 30 yr. I should mention that we did not forget about the receptor itself during this time period. In 1970, Merry Sherman and I published the most complete physical chemical characterization of a steroid receptor [oviduct PR (progesterone receptor)] to that point in time, and suggested that this information would soon allow us to trace the receptor directly to nuclear gene targets (4).

THE MECHANISM OF STEROID HORMONE ACTION: LIGANDS INDUCE SPECIFIC mRNAs

At this stage, I was almost certain that steroid hormones (and their receptors) acted at the level of DNA to turn on (and off) genes. Nevertheless, even among those who favored the nucleus as the primary target of steroids, transcriptional regulation was not the universally accepted theory. In fact, major labs promoted both posttranscriptional regulation (5) and translational regulation (6) to explain the increase in protein synthesis. Thus, it was imperative that we prove that the specific mRNAs that coded for our oviduct proteins were induced by steroid hormones. Unfortunately, the methodology required to prove this was only in its infancy. I moved to Vanderbilt University in 1969, and for the next decade of my scientific career, I was very fortunate to have the opportunity to enter into a critical and invaluable collaboration with Tony Means. In 1969, our primary goal was to demonstrate unequivocally that steroid hormones can induce actual specific mRNAs (i.e. ovalbumin and avidin mRNAs) that would then code for their respective specific proteins. I also wanted to carry out these experiments in a living animal because of prior criticisms of cell culture artifacts. We were fortunate to be the first to publish that estrogen induced ovalbumin mRNA levels in the chick oviduct (7, 8, 9) (Fig. 1Go). We also next published that progesterone induced avidin mRNA, and the concept of nuclear action of steroid receptors was born. In the subsequent year, the Schimke lab substantiated our estrogen induction of ovalbumin mRNA in the same tissue (10). The overall pathway for steroid hormone action now could be predicted, i.e. steroid hormones induce synthesis of proteins by first inducing levels of their specific mRNAs. We proposed that the hormonal induction of mRNAs for avidin and ovalbumin was due to new mRNA synthesis because there was no mRNA produced before hormone administration to the chick oviduct. Therefore, the possibility, suggested by others, of increasing mRNA (i.e. ovalbumin) posttranscriptionally to levels equivalent to almost 50% of the cellular mRNA by inhibiting the degradation of the mRNAs could be calculated as mathematically infeasible. Also, translational regulation by the hormones could be ruled against because there was no mRNA detectable in tissue and no mRNA on oviduct polysomes in the absence of hormone stimulation. Most importantly, the test of time proved these results and our interpretation of transcriptional regulation to be entirely correct.



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Fig. 1. Illustration of our 1972 experiment (see Refs. 8 and 9 ) showing the effect of DES (diethylstilbestrol) on the rate of ovalbumin protein synthesis (dashed lines) and levels of ovalbumin mRNA (bars) during 48 h after administration of the hormone to immature chicks. The coincidence of the curves implies cause and effect and was interpreted as a direct transcriptional response to a steroid hormone.

 
Our elucidation of the overall action pathway for steroid hormones by the demonstrations of estrogen induction of ovalbumin mRNA and progesterone induction of avidin mRNA in 1972 was exciting indeed. I realize that some of the readers of this perspective were likely in grade school at the time and cannot fathom any possibilities for regulation other than transcriptional. Nevertheless, it was a period of intense excitement in our lab group, and we could sense the whole climate changing in our field. In my opinion, it was our lab’s single most important contribution to the field of steroid hormone action. I say that because many have suggested that this series of publications changed the field of endocrinology, fostering the emergence of the new fields of hormone action and molecular endocrinology. Hormone action and transcriptional regulation were now inextricably linked in all meetings, beginning with the then heavily oversubscribed Gordon Conferences on Hormone Action. There was a logarithmic explosion of publications on steroid hormone action and steroid receptors (see the "About NURSA" section in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/Nuclear Receptor Signaling Atlas (NURSA) web site: www.NURSA.org), which then extended to studies encompassing all other hormones. To add to the excitement, Jensen had applied his estrogen receptor (ER) antibody to analysis of breast tumors to predict the patients who would benefit from hormonal therapy. The tree of molecular endocrinology had some transcriptional roots, as can be noted in the early works of Tomkins, Williams-Ashman, Mueller, Tata, Kenny, Baxter, Thompson, Gorski, Liao, Wilson, and many others. However, in the mid-to-late 1970s, this hormone action tree sprouted many more branches as an influx of a large number of additional outstanding molecular scientists [i.e. Gustafsson (1974), Yamamoto (1975), Chambon (1981), Evans (1982), Rosenfeld (1982), Roeder (1993), etc.] expanded the hormone/receptor/action field. They were drawn by an interest in transcriptional regulation, and they elevated further the quality of our field’s experimental science and our scientific meetings. The NR field is one of the most innovative, progressive, and productive fields of all of the medical science subspecialties.

I next moved my lab group to Baylor College of Medicine in 1973 and continued to generate additional substantiating data for our gene regulation model. During the next decade, we carried out studies to purify an mRNA (ovalbumin), to clone the first cDNAs for ovalbumin and avidin, to determine the exact number of copies of the mRNA in cells before and after hormone stimulation, to study the structure of the ovalbumin gene, and to show that the hormone-receptor complex acted on target cell chromatin to effect transcription. The pace and level of competition was impressive among the labs in the field. Yamamoto published glucocorticoid induction of radiolabeled mRNA synthesis of a viral gene (mouse mammary tumor virus) in 1975 (11), substantiating hormone action to be at the level of transcription; since then, the model has been confirmed repeatedly by other labs. Hormone response elements were next identified by Gustafsson and Yamamoto. A few years later, we and others were able to accomplish stimulation of gene transcription by a purified PR in a chromatin cell-free transcription system; this system allowed us to directly demonstrate that the receptor acted via the promoter region to somehow stabilize general transcription factor complexes on the TATA element (12). Although we saw some ability for purified receptor to interact with certain key components of the basic transcriptional machinery such as transcription factor IIB, we repeatedly observed that there was some powerful magical protein component in nuclear extracts that greatly enhanced the action of the receptor—and that our purified receptor did not act efficiently in the presence of purified TATA transcriptional components unless we added back small aliquots of a nuclear extract. It was not for another 5 yr that we were able to isolate and clone one of these elusive magical components, the protein coactivator SRC (steroid receptor coactivator)-1 (13). More excitement was to come before then, however.

NRs CAN BE ACTIVATED BY SECOND MESSENGER PATHWAYS IN THE ABSENCE OF LIGAND

By 1990, I thought we knew almost everything about the basic aspects of ligand binding and activation of steroid receptors. We certainly were in for a surprise. In the course of doing some control experiments, a postdoctoral fellow (Larry Denner) in the lab, along with my close collaborators, Bill Schrader and Nancy Weigel, made the observation that the avian PR could be activated by phosphorylation pathways in the absence of ligand. Because the results were initially difficult to believe, we repeated the experiments many times, but the conclusion was inescapable—-a ligand-independent pathway existed for activating certain receptors. The paper describing this work presented a concept not previously predicted over the preceding 25 yr of research in the receptor field—and had future consequences to the biology and pathology of NRs. As one can imagine, there was considerable skepticism both among reviewers and my colleagues when we presented these data. We first showed that cellular phosphorylation pathways could activate a receptor in the absence of ligand (14). Next, we demonstrated that dopamine, not by binding to PR but by acting at its own D1 membrane receptor, could activate PR both in cultured cells, and in living animals (15, 16). This novel steroid-independent pathway for activation of a steroid receptor is now known to occur via signaling cascades from membrane regulatory molecules such as cAMP, dopamine, growth factors, cytokines, and possibly other cellular regulators acting at the membrane (16). Ligand-independent activation represents a prime means by which membrane receptors and NRs communicate at the level of the genome, and a mechanism by which the cellular environment can modulate NR function and transcription. These ligand-independent pathways for receptor function are particularly important in the mechanism of action of neurotransmitters such as dopamine in central nervous system neurobehavior (16) and of growth factors, as demonstrated clearly by the Korach lab (17). Moreover, applications are substantiated now in pathologies such as inflammation and in breast/prostate cancers where the ER/androgen receptors have been shown to be activated by phosphorylation cascades induced by tumor growth factors in the absence of ligand. Importantly, because approximately 50% of the orphan receptors of the NR family are thought not to have primary endogenous physiological ligands, our discovery of ligand-independent activation uncovered the mechanism by which this large group of orphan receptors now are considered to be activated and/or regulated (18). In this latter case, we solved a conundrum before it arose (19).

After Bill Schrader and Nancy Weigel purified the PR and demonstrated the first isoform structures of a steroid receptor (PRa; PRb) (20), Donald McDonnell showed that the PRa and PRb forms of PR have differing and opposite functional activities. Our early PRa and PRb work was the harbinger of NR isoforms as a later developing concept in our field, one that proved important for retinoic acid receptor, thyroid hormone receptor (TR), and retinoid X receptor. Unfortunately, our efforts to clone the first receptor clearly failed, being led astray by a false immunogenic protein that contaminated our antibody pool. After the clonings of a partial cDNA for rat glucocorticoid receptor (GR) by Gustafsson (21) and Yamamoto, the first full-length clone of human GR by Evans (22) and Rosenfeld, and ER by Jensen (23) and Chambon (24) in this issue, Orla Conneely succeeded in cloning PR. We next went on to clone the vitamin D receptor and confirmed its role in human vitamin D-resistant rickets (25). The cloning of receptors led to another increase in the publication rate in our field and made important reagents available. The Evans lab was first to identify an orphan receptor (the ER relateds), but soon many more were discovered. Although we cloned five of the original orphans, I chose to maintain my focus on classical steroid receptors and mechanisms. However, in 1992, we did publish the prediction that most of the orphan receptors would turn out to be activated (or inactivated) either by metabolic ligands or by phosphorylation signaling pathways (19). This prediction turned out to be correct.

LIGANDS DIRECTLY ACTIVATE RECEPTORS FOR TRANSCRIPTIONAL RESPONSES AND THE C-TERMINAL REGION OF RECEPTORS CONTAINS THE KEY TO LIGAND ACTIVATION

Although we accepted that liganded receptor was the mediator of the transcriptional response, the precise mechanistic details of its function were unclear. Next, we needed to directly couple the events of receptor binding to DNA with receptor-mediated transcription and prove that the ligand mediated this series of reactions in a sequential process. Using a cell free transcription system and purified PR in 1990, Milan Bagchi demonstrated directly that progesterone specifically set off a chain of distinguishable receptor events leading to heat shock protein dissociation, conversion from an inactive to an active receptor form, binding to a PR element, and finally, induction of transcription of the target gene; the RU486 antagonist promoted all events except conversion to an active receptor form in our transcription system (26). (Only much later, in 1998, did we use GR protein produced by Gustafsson to carry out an experiment to show that steroid receptors bind to their inverted repeat hormone response elements in DNA as homodimers (27); Chambon showed identical results for ER that same month.) After Bagchi’s experiment described above, we now understood that the activation of receptor by ligand was direct, but we didn’t understand the conformational step of receptor activation by agonist and inactivation by antagonist in structural terms. In a further collaboration with Ming and Sophia Tsai in 1992, in what I believe was one of our more important but lesser known biochemical studies, my postdoctoral fellow, George Allen, used molecular mapping with proteolytic enzymes and monoclonal antibodies produced by Dean Edwards to predict that the structural allosteric mechanism of action by agonist/antagonist ligands occurred at the C-terminal tail (now also known as helix 12) of NRs (28) (Fig. 2Go). These results led us to postulate that the C-terminal tail of the receptor flipped over like a lid and compacted and covered the ligand binding site when the ligand binding domain (LBD) was occupied by hormone; the ligand-induced surface predicted was later shown to be the coregulator interaction region of the molecule. We were excited, however, when the liganded crystals of two receptors were published in 1995. Our structural model in our 1992 paper (Fig. 2Go) (28) was proven to be surprisingly accurate in terms of that found in the first important crystal structures of two agonist-bound NR LBDs published simultaneously by Baxter (TR) and Chambon (retinoic acid receptor).



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Fig. 2. A model diagram of receptor structure-function relationships induced by agonist and antagonist ligands and published in our 1992 paper (see Ref. 28 ). It shows the receptor in oval shape and illustrates the results of our molecular mapping studies of receptors using proteolytic enzymes and site-specific antibodies. When the receptor LBD is unoccupied by ligand and inactive, the C-terminal tail is extended; when agonist binds, the tail flips over and covers the ligand binding site, providing an activation surface for interaction with coactivators (not discovered at that time); when the LBD is occupied by antagonist, the tail does not position similarly and assumes more the inactive/unoccupied configuration (preventing coactivator from binding to the activation surface). In this model, the primary regulatory structural alterations of receptors induced by ligand binding occur via C-terminal tail positioning. Subsequent later detailed crystallizations and x-ray analyses of NRs confirmed the general accuracy of this model. hsp, Heat shock protein.

 
EVIDENCE FOR THE EXISTENCE OF CELLULAR COREPRESSORS FOR NRs

My main thoughts, however, still remained on the missing molecular link between the receptor and the general transcription factor machinery at the TATA box. I remember being encouraged by the experiments of Mark Ptashne, who was studying regulatory activator molecules in yeast that did not bind to DNA, but rather, acted via squelchable protein-protein interactions (29). We performed an ER-PR squelching experiment that was published in 1989 and predicted that ER and PR interact with some common transcription factor(s) and that this may be a general mechanism for regulation by the steroid receptors (30). The Chambon lab published a more detailed confirmation of this squelching hypothesis the next year. However, we were not happy with this approach because squelching experiments provided no real information on the specificity or the precise targets of the squelching reaction. I felt that we needed to back up and rethink this problem in a manner that identified specific molecules. As a first step toward this goal, Donald McDonnell showed in yeast that incorporated mammalian ER and PRs were able to interact with a specific yeast repressor protein (i.e. SSN6) that repressed the transcriptional activity of an activation [activation function (AF) 1] domain of the steroid receptors when tamoxifen was bound. On the other hand, the SSN6 corepressor dissociated when estradiol was bound, leading to receptor activation (31).

We then turned our attention to AF2-mediated repression mechanisms (32, 33). In a series of experiments using TR, my postdoctoral fellow, Aria Baniahmad, first demonstrated a silencing activity associated with a human activation-mutant receptor; the silencing activity was contained in the C terminus (33, 34). He next went on to uncover the existence of a corepressor protein for the AF2 of a NR in mammalian cells. I presented this complete data and our corepressor theory at the winter Keystone Nuclear Receptor meeting in 1994 (submitted for publication in September 1994 and published in January 1995) (33). To the best of my knowledge, our studies using mutational analyses of NRs (TR, ER, PR) were the first to claim the existence of a new class of unknown soluble NR corepressors in animal cells that functioned by protein-protein interactions and not by competitive interactions with DNA binding sites. Consequently, I guess Aria’s paper (33) represents the biochemical discovery of coregulators for steroid receptors, and in it, I believe we were first to predict a ligand-induced coactivator exchange with corepressor at the C terminus of a NR to achieve ligand regulation of gene expression. Later that year, Rosenfeld and Evans separately cloned the first cDNAs for corepressors. Now, more than ever, we were determined to find this elusive coactivator molecule for NRs. We were not alone in our quest, however.

THE FIRST SELECTIVE NR COACTIVATOR IS CLONED

The search for coactivators was ongoing in more than one lab, beginning with the early description of receptor-associated protein fragments by David Moore in yeast assays and cell-free work on a 160-kDa protein band that bound to activated ER by Myles Brown and a 140-kDa band studied by Parker. A detailed description of everyone’s activities is beyond the limitations of this perspective, but it is safe to say that at that time the cloning of an authentic molecule containing demonstrable coactivation activity was an elusive goal to all. As a field, we had no specific guidelines as to what coactivators should do when added back to animal cells. In collaboration with Ming and Sophia Tsai, my postdoctoral fellow, Sergio Oñate, identified the first selective NR coactivator cDNA clone with clear biological activity, SRC-1 (13). In his publication, we established the criteria that subsequently were used to categorize the extensive number of coactivators discovered later by our lab and many others. The SRC-1 coactivator (and its two related family members) turned out to be a key to understanding the transcriptional activity of NRs. As we expected, SRC-1 was found to be a major booster of the power of transcription in the presence of the liganded receptor, but it also directed histone acetylase activity to the gene. McDonnell’s lab cloned hRPF1, and we next cloned a second coactivator, E6-AP; both were found to be dual function proteins containing an activation domain and a second domain with proteolytic/ubiquitinylation activity (35). Other members of the SRC-1 family were cloned by other labs, and more and more coactivators began to appear, each with the same interesting structure of a coactivation domain linked to some enzyme function.

Soon after cloning SRC-1, we made an important pharmacologic prediction of principle for coactivators, when Carolyn Smith and I published that the intracellular coactivator/corepressor ratio can determine the cellular activity of a mixed antagonist/agonist ligand-like tamoxifen (36). This experiment explained in large part the puzzling mechanism of the tissue-selective actions of selective ER modulators (SERMs) and contributed to the expansion of selective receptor modulator development in the pharmaceutical field. Later, we substantiated this explanatory model in a controlled cell-free chromatin transcription system with purified receptor and purified SRC coactivators (37).

Surprisingly, as we were cloning additional coactivators, other labs found more, and the number expanded logarithmically. There are now approximately 150 identified coactivator proteins that function with NRs, and most function in a hormone-dependent manner (see the "About NURSA" section in NIDDK/NURSA web site: www.NURSA.org). Many of these coactivators are enzymes that are recruited to NR-occupied promoters to carry out the multiple steps inherent to gene activation and repression; some coactivators are even noncoding RNA molecules (38). The difficulty in proving suspected coactivators to be authentic was a continuing problem. Consequently, after cloning SRC-1, we felt it critical to quickly go on to achieve in vivo evidence to prove the biological activity of coactivators [SRC-1 and SRC-3/amplified in breast cancer 1 (AIB1)] in living animals via a series of gene knockout experiments performed by a postdoctoral fellow, Jianming Xu. Our newly generated SRC-1 KO mice were shown to have partial resistance to multiple steroid hormones (39). Next, SRC-3 KO mice (40) were constructed and proved to have a separate and distinct phenotype from the SRC-1 KO, thereby demonstrating the impressive tissue-specific selectivity even among multiple closely related members of this SRC-1 family of coactivators. With publication of these initial KO mouse studies, coactivators were accepted generally to be biologically important in the NR field.

UNSUSPECTED ROLES OF COACTIVATORS IN TRANSCRIPTION AND CELLULAR SIGNALING

To say the least, we were quite naive at that time as to the extensive biology of the coactivators. Subsequent experimental observations showed that steroid receptors not only mediate initiation/reinitiation of transcription but also can alter the nature of the gene product by regulating the downstream alternative splicing of pre-mRNAs in a receptor-specific manner (41). Our initial experiments were based on principles defined earlier by the Spiegelman lab for PGC-1 (42). We described multiple receptor coactivators that can regulate the alternative RNA splicing process and defined the specific peptide domains that provide the coactivation and the alternative splicing activities (43). Furthermore, after Zafar Nawaz and I showed that certain dual function coactivators have ubiquitin ligase (i.e. E2/E3) activity that ubiquitinylates proteins within the receptor-coactivator complex, a postdoctoral fellow, David Lonard, demonstrated that degradation of the receptor-coactivator complex by the 26S proteasome was obligatory for efficient continued receptor-mediated transcription (44). This work led to our realization that the receptor-coactivator complex has a built-in enzymatic self-destruct code, and that if the gene was to remain on, new molecules would need to be continually supplied to the site. There are now well over a dozen dual function coactivators known that contain completely distinct enzyme activities—and all are brought to the target gene by the receptors bound to the hormone response elements located within promoter regions.

In recent years, our laboratory continued to expand the biology of coactivators by searching for novel pleiotropic signaling activities of these molecules in other pathways such as inflammation, growth, and stress (45). Our work showed that coactivators act as homeostats to sense environmental signals and to coordinate the signals emanating from membrane receptors with NRs and gene transcription, a concept of importance to both normal and pathological signaling by hormones. The coactivators do this by receiving phosphorylation signals from the environment via membrane receptors. More importantly, a coactivator phosphorylation code exists whereby signals from kinase pathways selectively phosphorylate serine/threonine residues in the coactivator molecule, and depending upon the combination of sites phosphorylated, the coactivator is preferentially directed to bind and activate distinct sets of downstream transcription factors (46) (Fig. 3Go). This code represents an elegant mechanism by which membrane-initiated signaling pathways can direct limiting quantities of a coactivator toward their own relevant downstream transcriptional activators bound to the target promoter DNA. It is the product of the cellular concentration of coactivator and the phosphorylation state of coactivator that drives the potency and the selectivity of coactivators for target DNA-bound activator proteins (46). In addition to the concentration of coactivators (or corepressors) in the cell, the site-specific phosphorylations on the coactivator provide a cell-specific input to a coactivator’s activity, and thus to a ligand’s activity.



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Fig. 3. Signaling through the cell from the environment to the genome, can occur by differential phosphorylation of coactivators, which in turn differentially activate distinct sets of nuclear DNA-binding transcription factors. The figure illustrates our current hypothesis for the activations of the SRC-3/AIB1 coactivator. In the figure, estrogen, TNF{alpha}, or other stimuli activate distinct kinase pathways, and differentially phosphorylate SRC-3. The differential phosphorylation selectively directs SRC-3 to form distinct coactivator complexes with different downstream transcription factors, thereby creating differential gene activations (see Ref. 46 ). NF-{kappa}B, Nuclear factor {kappa}B; CBP, cAMP response element binding protein-binding protein; CoA, coenzyme A; CoA-X and CoA-Y, any unknown coactivator.

 
ROLES OF COACTIVATORS IN PHARMACOLOGY AND PATHOLOGY

Our understanding of the complex protein interactions involved in NR functions has been unraveled now to a point where our current understanding of mechanisms has provided a major impact in medicine. The early studies of Baxter and Tomkins on GR mutants verified their connection to genetic disease predisposition. The work of Jensen showed the utility of ER analyses in therapeutic decisions for breast cancer, and Craig Jordan’s important observations typified tamoxifen as the primary prototype SERM for breast cancer therapy. Fueled by both basic and clinical research, interest in selective receptor modulators (SRMs) of NRs because pharmacologic drugs has been explosive. After the clinical studies on tamoxifen, second generation SERMs have been developed by a number of pharmaceutical firms and new SRMs for androgens, glucocorticoids, and progesterone are in early phase development in biotechnology/pharmaceutical companies. SRMs also have been synthesized recently for the orphan receptors that are ligand regulatable. The new information on the molecular mechanisms of action of coactivators also has been rapidly applied to abnormal physiology. Coactivators for steroid receptors now have been reported to be overexpressed in many endocrine tumors, after the initial report directed to AIB1/SRC-3 (47). We found that SRC-3 can cooperate with oncogenes in transformation of normal cells in culture (47), and that our SRC-3 KO mice were resistant to carcinogenic/oncogenic induction of mammary cancers (48). In a recent important study published by Myles Brown (49), overexpression of AIB1/SRC-3 in transgenic mice was found to result in spontaneous appearance of breast cancers in older animals. The totality of available experimental evidence proves this coactivator to be an authentic oncogene. In fact, recent analyses of clinical human breast tumor samples indicate that AIB1/SRC-3 is overexpressed along with HER2/neu in a subset of human breast cancers (50). This cooperative stimulation of SRC-3 expression and phosphorylation leads to extremely aggressive growth of breast cancers and is instrumental in predicting those women who will manifest early resistance to tamoxifen therapy (50). These results highlight the pathologic importance of our discoveries of the deleterious synergism between coactivators (SRC-3) and ligand-independent pathway activators such as HER2/neu, which cause phosphorylation and maximal activation of SRC-3 in the endocrine cancer cells. Finally, coactivators (and corepressors) are important determinants of normal response to steroid hormones and provide explanations for variance in tissue-hormone kinetics and sensitivity, in growth selectivity, and even in phenotypic diversity among individuals.

In conclusion, I want to emphasize very clearly that my work over the past 35 yr has been as leader of a series of small teams of extremely talented faculty collaborators, postdoctoral fellows, and students. No modern scientist works without important contributions from others, and the contributions of my trainees and collaborators cannot be overstated. I am deeply appreciative of my outstanding scientific colleagues and my administrative assistants. In no way do I envision myself as other than the spiritual leader of a talented scientific choir, whose members also became close and lifelong friends. Although the vast majority of our hypotheses over the years were correct, they were wrong in certain instances. I guess the latter can be expected when one attempts to push the edge of current knowledge. Importantly, in the field of science one presents his/her conclusions, and the scientific body keeps working toward an accurate verification of the secrets of nature, continually modifying and retooling hypotheses and models until they reach near certainty. To keep things in perspective, I should also make clear my great admiration for the large and expert group of scientists that work in the field of NRs and who have made the numerous and immense contributions that have emanated from our field. Their intelligence, good humor, and friendship have been a source of both critical feedback and great pleasure to me for the past 35 yr. Most important of all, I am grateful for the devotion of my wife, Sally, and my four children (Sally Jr., Bert Jr., Becky, and Erin) and for their understanding of the considerable demands of my career in science. All in all, it is these above individuals who have made possible any small successes that I have achieved in my career.


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FOOTNOTES

This work was supported by National Institutes of Health Grants HD-07857 and HD-08188.

Abbreviations: AF, Activation function; AIB1, amplified in breast cancer 1; ER, estrogen receptor; GR, glucocorticoid receptor; LBD, ligand binding domain; NR, nuclear receptor; PR, progesterone receptor; SERM, selective ER modulator; SRC, steroid receptor coactivator; SRM, selective receptor modulator; TR, thyroid hormone receptor.

Received for publication November 30, 2004. Accepted for publication December 24, 2004.

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