Perspective: Female Steroid Hormone Action

The steroid hormones estrogen (E) and progesterone (P) play a central role in the regulation of all aspects of female reproductive activity leading to the establishment and maintenance of pregnancy. Together, they act at the level of the hypothalamus, pituitary, ovary, and uterus to coordinate cyclic neuroendocrine gonadotropin production, ovulatory activity, and uterine development in preparation for implantation of fertilized embryos. Both hormones are also essential for postnatal mammary gland development regulating postpubertal mammary ductal morphogenesis in the case of E and pregnancy-associated lateral ductal branching and lobular alveolar differentiation in the case of P. The diverse physiological activities of E and P, however, are not restricted to the female reproductive system. Estrogen is essential for male fertility, and both hormones have been implicated in the cardiovascular, immune, and central nervous systems and in bone function. In particular, estrogen has been shown to play an important role in protection against osteoporosis in postmenopausal women (1), in the prevention of coronary heart disease (2) and in the maintenance of cognitive function (3). In addition to positive effects in both reproductive and nonreproductive organs, estrogen plays an important role in the development of uterine cancers, and both hormones have been implicated in the development of breast cancer (4–6). The critical role of estrogen in development of breast cancer is evidenced by the significant protective response observed in women after treatment with the antiestrogen, 4-hydroxy-tamoxifen (4-OHT) resulting in a 25% decrease in mortality and 45% decrease in incidence (6, 7). The conflict between positive and negative activities of these hormones has fueled a search for selective receptor modulators (SRMS) for use in hormone replacement therapy that possess the capability of harnessing the tissue selective beneficial effects of the steroids while lacking adverse activities in breast and uterus. The effects of E and P are mediated through interaction with specific intracellular receptors that are members of the nuclear receptor superfamily of transcription factors (8). Binding of the steroids to their cognate receptors induces conformational changes in receptor structure leading to receptor dimerization, posttranslational modification, and binding to specific enhancer DNA elements in the promoters of specific genes and recruitment of coregulator proteins that interact with general transcriptional machinery to elaborate hormone-triggered changes in promoter activity. In general, agonist ligands of receptors promote binding of coactivator proteins that promote transcription initiation while binding of antagonists promote interaction with corepressor proteins that facilitate transcription repression (9). Recent advances in our understanding of the molecular mechanisms of action of estrogen and progesterone receptors, together with molecular genetic approaches to examine the physiological consequences of receptor and coregulator protein ablation, have provided important insights into how physiological diversity of female steroid hormone action is achieved. Emerging from these studies is the general principle that the modular nature of receptors allows ligand, tissue and promoter specific interaction with select subsets of coregulators capable of elaborating distinct transcriptional and hence physiological responses to steroid signal. E and P responsive tissues are initially determined by the tissue distribution of receptor proteins whose restricted spatiotemporal expression identifies tissues targeted for hormonal response. However, tissues that express receptors for estrogen and progesterone exhibit physiologically diverse responses to the same steroidal ligand. Functional diversity arises from the existence of two structurally related but nonidentical receptors for each hormone and by the ability of a single receptor subtype to elicit diverse transcriptional responses to a specific ligand. Molecular dissection of the structural and functional relationships of steroid receptors and of the mechanisms by which they interact with ligand, DNA, and the transcriptional apparatus has provided valuable information on the molecular pathways by which steroid receptors can generate functionally diverse transcriptional responses to their cognate steroid ligand. Steroid receptors including those for estrogen and progesterone have a modular protein structure consisting of distinct functional domains capable of binding steroidal ligand, dimerization of liganded receptors, interaction with hormone-responsive DNA elements, and interaction with coregulator proteins required for bridging receptors to the transcriptional apparatus (8, 9). Binding of estrogen and progestin agonists to their receptors induces conformational changes in receptor structure that promote interaction of coactivator proteins with distinct activation domains (AFs) located within both the amino and carboxy terminal regions of the receptor. Such coactivators promote chromatin remodeling and bridging with general transcription factors resulting in the formation of productive transcription initiation complexes at the receptor responsive promoter. In contrast, binding of receptor antagonist compounds induces receptor conformational changes that render AFs nonpermissive to coactivator binding and instead promote interaction with corepressor proteins that inhibit transcriptional activity of the receptor. The ability of steroid receptors to interact with a variety of coactivator and corepressor proteins, together with the differing spatiotemporal expression of coregulators, illustrate a key role of coregulators in mediating different tissue specific responses to steroidal ligand. Finally, receptors for estrogen and progesterone can be activated in the absence of steroidal ligand by phosphorylation pathways that modulate their interactions with coregulator proteins. Receptors for estrogen are expressed as two structurally related subtypes, ERα and ERβ, that are encoded by two distinct genes (10, 11). Both proteins share a high degree of amino acid conservation in their DNA binding domains (97%) and exhibit a significant but lesser degree of homology in their ligand binding domains (58%). Two functionally distinct transactivation domains have been identified in both proteins; the first (AF1) located in the poorly conserved amino terminal domain and a second (AF2) located in the ligand binding domain. AF1 and AF2 can contribute independently and synergistically to receptor transcriptional activity in response to agonist ligands and to ligand-independent phosphorylation pathways of receptor activation and their relative activities vary depending on cellular and promoter context (12–14). ERα and ERβ exhibit significant functional differences when examined under similar conditions in cell-based transactivation assays. Ligand binding profiles show both similar and distinct affinities of each receptor for different estrogen agonist and antagonist ligands (15). Transcriptional responses of each receptor to ligands with which they interact with equal affinity (including 17β-estradiol) also vary significantly due in part to sequence divergence in their AF-1 domains (16) and to a differential preference of individual subtypes for specific coactivator proteins (17). Receptor sequence divergence, however, accounts only in part for the cell- and promoter-based variations in transcriptional responses to a specific ligand. The transcription regulatory activity of either receptor in response to ligand is highly dependent on the cellular and promoter environment (12, 18). The identification of a complex group of coregulator proteins that are recruited in a cell- and promoter-specific manner to the ligand occupied estrogen receptors reflects one of the most important recent advances in our understanding of the cellular mechanisms leading to tissue diversity in transcriptional responses to estrogen (19). Superimposed upon this transcriptional diversity is the ability of different estrogen receptor agonist and antagonist ligands to induce distinct conformational changes in receptor structure, thereby generating a spectrum of transcriptional responses with altered cell and promoter dependency through ligand specific modulation of the conformational context of AF domains (20–23). In addition to providing a mechanistic explanation for ability some estrogen receptor ligands (SERMS) to elicit select tissue-specific agonist activities of estrogen, the physiological implications of these findings are that ligand-specific manipulation of coregulator interaction can be used to achieve tissue and promoter specificity in transcriptional responses to receptor. In contrast to estrogen, receptors for progesterone are expressed as two distinct isoforms, PR-A and PR-B that arise from a single gene (24, 25). The expression of both isoforms is conserved in rodent and humans and overlaps spatiotemporally in female reproductive tissues. However, the ratios of the individual isoforms vary in reproductive tissues as a consequence of developmental (26) and hormonal status (27) and during carcinogenesis (28, 29). The PR-A and PR-B differ in that the PR-B protein contains an additional sequence of amino acids at its amino terminus that is not contained in PR-A. This PR-B-specific domain encodes a third transactivation function (AF3) that is absent from PR-A (30, 31). Recent evidence has demonstrated that the presence of AF3 allows binding of a subset of coactivators to PR-B that are not efficiently recruited by progestin-bound PR-A (32). Thus, when expressed individually in cultured cells, PR-A and PR-B display different transactivation properties that are specific to both cell type and target gene promoter context (33–36). Agonist-bound PR-B functions as a strong activator of transcription of several PR dependent promoters and in a variety of cell types in which PR-A is inactive. Further, when both isoforms are coexpressed in cultured cells, in cell and promoter contexts in which agonist bound PR-A is inactive, the PR-A can repress the activity of PR-B. This repressor capability of PR-A also extends to other steroid receptors including ERα (31, 37). Finally, the PR-A and PR-B proteins also respond differently to P antagonists (reviewed in Ref. 38). While antagonist bound PR-A is inactive, antagonist bound PR-B can be converted to a strongly active transcription factor by modulating intracellular phosphorylation pathways (39–41). Although the sequence of the ligand binding domain of the PR-A and PR-B is identical, the ability of different ligands to induce different conformational changes in PR, together with the synergistic activity of the amino and carboxy terminal activation domains (42), predicts that PR-A or PR-B selective transcriptional regulation can be achieved by manipulating ligand interactions with the carboxy terminal. The use of genetically altered mouse mutants in which expression of individual progesterone or estrogen receptor genes has been specifically ablated has allowed direct examination of the essential roles of these receptors in mediating physiological responses to E and P. In addition to defining the individual and collective contributions of receptor subtypes to the overall repertoire of hormone action, these models facilitate examination of the contribution of specific receptor subtypes to the activities of tissue-selective receptor modulators. They have also proved a valuable means of identifying alternative pathways of steroid action that are independent of receptor activity as well as addressing the physiological significance of ligand-independent pathways of receptor activation. Finally, the receptor null mutant models serve as powerful tools to dissect the molecular genetic pathways that are regulated by these steroid receptors invivo. Selective ablation of ERα and ERβ in mice has provided definitive evidence that these receptor subtypes mediate distinct physiological responses to estrogen both within the reproductive tract and in nonreproductive tissues (11, 43–45). In general, the different roles of these subtypes are a reflection of a mostly segregated spatiotemporal distribution of each receptor. ERα is the dominant subtype expressed throughout the female reproductive tract and its ablation results in infertility due to defects in sexual behavioral expression, neuroendocrine gonadotropin regulation, ovulation, uterine function, and postpubertal mammary gland morphogenesis. The ERα-subtype also plays an essential role in male fertility and mediates many of the nonreproductive activities of estrogen including regulation of bone resorption-remodeling in females, postnatal endochondral bone growth in both sexes, cardiovascular endothelial regeneration, adipogenesis, and sexual behavior (11). In contrast to ERα, ablation of ERβ results in less severe phenotypic consequences with regard to estrogen signaling. ERβ is expressed in both the male and female reproductive tracts in a pattern largely distinct from that of ERα and its ablation results in a subfertile phenotype restricted to impaired female ovarian function (44). Its expression and activity in this tissue are complimentary to but distinct from those of ERα. Expression of ERβ has also been detected in several nonreproductive estrogen responsive tissues including bone-forming osteoblasts, epiphyseal chondrocytes, and the cardiovascular and central nervous systems. The protein was recently shown to play an essential role in regulation of cortical neuronal survival (46) and appears to contribute together with ERα to protection against cardiovascular injury (47) While the tissue-selective contributions of ERα and ERβ to estrogen and SERM signaling are still under active investigation, the distinct roles identified to date highlight the importance of these receptor subtypes in mediating tissue selective physiological responses to estrogen. The availability of ERα and ERβ knockout models has also allowed physiological testing and validation of the existence of alternative signaling pathways that are independent of either ligand or receptor. Thus, while uterotrophic responses stimulated by estrogen require functional ERα, the ability of some catechol and zenoestrogens to elicit such responses is independent of ERα (48, 49). In contrast, ERα is an essential mediator of proliferative responses that are stimulated in this tissue by epidermal growth factor acting in the absence of estrogen (50). This latter observation has provided an important physiological validation of the ligand-independent activation of estrogen receptors previously observed in cell based transactivation assays. The differences in transcriptional activities and coregulator interactions between the PR-A and PR-B observed in vitro predicted that these proteins also may mediate different physiological responses to progesterone. In addition, the selective ability of PR-A to inhibit transcriptional responses induced by both PR-B and the estrogen receptors suggested that PR-A has the capacity to diminish overall progesterone responsiveness in certain tissues as well as contribute to the antiestrogenic activities of progesterone previously observed in the uterus. Null mutation of the PR gene encoding both isoforms has provided evidence of an essential role of PRs in a variety of female reproductive and nonreproductive activities (51). Female mice lacking both PRs exhibit impaired sexual behavior, neuroendocrine gonadotropin regulation, anovulation, uterine dysfunction, and impaired ductal branching morphogenesis and lobuloalveolar differentiation of the mammary gland. PRs also play an essential role in regulation thymic involution during pregnancy and in the cardiovascular system through regulation of endothelial cell proliferation (52, 53). Receptors for progesterone have also been identified in the central nervous system and bone where progesterone has been implicated in both cognitive function and bone maintenance. However, the essential role of PRs in these regions has not yet been confirmed. Recent studies have begun to address the individual contributions of the PR-A and PR-B proteins to the physiological actions of progesterone using mouse mutants in which expression of the PR-A (PRAKO) or PR-B (PRBKO) isoform has been selectively ablated. Analysis of the phenotypic consequences of these mutations on female reproductive function has provided physiological proof of principle that the distinct transcriptional responses to PR-A and PR-B observed in cell-based transactivation assays are indeed reflected in an ability of the individual isoforms to elicit distinct physiological responses to progesterone. In PRAKO mice (54), the PR-B isoform functions in a tissue-specific manner to mediate a subset of the reproductive functions of PRs. Ablation of PR-A does not affect responses of the mammary gland or thymus to P but results in severe abnormalities in ovarian and uterine function. Surprisingly, the absence of PR-A in PRAKO uteri revealed an unexpected P dependent proliferative activity of PR-B in the epithelium and demonstrated that PR-A is essential to diminish both progesterone (acting via PRB) and estrogen-mediated proliferative responses in this tissue. The observation that PR-A is essential to inhibit estrogen induced proliferation in the uterus is consistent with previous observations that agonist bound PR-A is capable of inhibiting estrogen-dependent transcriptional activation in cell-based transactivation assays (37). Notably, this inhibitory activity of PRA was tissue specific and did not extend to the mammary gland where both PR-A and PR-B act as proliferative mediators of P. Consistent with the distinct tissue- and promoter-specific activities of PR-A and PR-B observed in tissue culture studies, the tissue-selective activities of PR-B observed in PRAKO mice were associated with an ability of this isoform to regulate a subset of progesterone responsive target genes rather than to differences in its spatiotemporal expression relative to the PR-A isoform (54). In contrast to the reproductive defects observed in PRAKO mice, more recent studies using PRBKO mice have shown that ablation of PR-B does not affect either ovarian, uterine, or thymic responses to progesterone but results in reduced mammary ductal morphogenesis (Jericevic, B., and O. M. Conneely, unpublished observations). Thus, PR-A is both necessary and sufficient to elicit these P-dependent reproductive responses while the PR-B isoform is required to elicit normal proliferative responses of the mammary gland to P. From a mechanistic standpoint, the differences in physiological activities observed between the PR-A and PR-B isoforms provides an important illustration of the key role played by the amino terminal AF domains in distinguishing tissue specific responses to steroidal ligand. The results demonstrate that the inclusion or deletion of the N-terminal AF3 domain in PR is sufficient to alter tissue specific physiological responses to P. The characterization and mode of action of coregulator proteins that mediate the transcriptional activity of steroid receptors have been intensely examined in recent years. Agonist ligand or ligand-independent activation of receptors is associated with recruitment of a complex group of coactivators including nucleosome-disrupting histone acetyltransferases (SRC family members, PCAF and P300), mediator proteins that bridge receptor complexes with the general transcription factor complexes (e.g. DRIP/TRAP220/ARC, and mediator), an RNA helicase, p68 and components of the ubiquitin proteosome degradation system including the ubiquitin ligases, E6AP and RPF1(9, 14, 55, 56). A growing number of corepressors associated with antagonist ligand repression of transcriptional activation have also been identified including the histone deacetylases, N-COR and SMRT (57, 58) and the estrogen receptor interacting proteins RIP140 (59) and REA (60). Considering the complex array of coregulator proteins that can interact with both estrogen and progesterone receptors, tissue selective expression of distinct subsets of coregulator proteins would be expected to strongly influence receptor dependent biological responses in specific tissues. Results from recent studies on the comparative spatiotemporal expression of coregulators and steroid receptors in mammalian tissues, together with the generation of knockout mouse models carrying null mutations of several coregulator proteins, have provided a key proof of concept of the essential role of coregulator proteins in mediating tissue selective physiological responses to steroidal ligand. It is becoming apparent that the spatiotemporal expression of some coregulator proteins in steroid responsive tissues is both developmentally and hormonally controlled. For example, the expression of coactivator SRC-1 is dissociated from estrogen receptor-expressing cells during postpubertal mammary gland morphogenesis but becomes colocalized with ER-positive cells during pregnancy (61, 62). A growing number of recent reports have also associated aberrant expression of coregulator proteins with the development of breast cancer. Significantly, these reports reveal a developing pattern of increased coactivator levels associated with tumorigenesis while the expression of corepressors is significantly decreased. For example, levels of CBP, TRAP220, and the SRC family members, SRC-2/TIF2 and AIB-1/SRC-3 are all elevated breast tumors (63, 64). However, most these is which is in of breast levels of the are in relative to and with the development of in a mouse of breast cancer evidence of the essential role of specific coregulators in of tissue-specific responses to estrogen and progesterone has recently been provided by genetic ablation of a coregulator proteins in Analysis of the reproductive of mice carrying a null mutation of SRC-1 and that these coactivators regulate mostly distinct physiological activities that are due to a segregated spatiotemporal expression pattern of the two proteins. However, with regard to their role in and P-dependent reproductive deletion of either coactivator results in a hormone in mammary gland developmental responses to E and P, essential roles for both proteins in this tissue. In contrast, only SRC-1 is expressed in the uterus and its expression is essential to elicit growth and responses of this tissue to E and P, uterine function is by deletion of of the essential role of steroid receptor corepressors in mediating the transcriptional activity of estrogen receptors has also recently been provided by gene Analysis of the transcriptional responses of mouse carrying a null mutation of to the estrogen receptor demonstrated that this protein was essential to mediate the inhibitory activity of the Ablation of in a of to a receptor agonist Finally, ablation of the in mice ovulatory and an ovarian phenotype that previously observed in mice, uterine implantation was However, while the phenotype of RIP140 null mice a tissue-specific contribution of the protein to reproductive function, a direct between the phenotype and the corepressor activity of RIP140 to be the have in our understanding of the molecular pathways by which steroid receptors elicit diverse physiological responses to hormonal It is that tissue and promoter in hormone action is determined not only by the tissue-selective expression of distinct receptor subtypes but also of a complex group of receptor interacting coregulator proteins whose function is essential in the diverse repertoire of transcriptional responses to The central role of coregulators in mediating physiological responses to estrogen and progesterone has only recently begun to be The availability of and models to facilitate examination of the physiological roles of individual coregulators, together with the use of differential gene array to tissue-specific of the hormonal facilitate dissection of the molecular genetic pathways by specific From the physiological to is becoming apparent that coregulator function may contribute to a variety of related including steroid reproductive dysfunction, and to alter coregulator recruitment to receptors by manipulation of receptor using ligands together with a understanding of the tissue-specific molecular pathways by specific coregulators facilitate the development of tissue specific ligands for hormonal