Our appreciation of the physiological functions of estrogens and the mechanisms through which estrogens bring about these functions has changed during the past decade. Just as transgenic mice were produced in which estrogen receptors had been inactivated and we thought that we were about to understand the role of estrogen receptors in physiology and pathology, it was found that there was not one but two distinct and functional estrogen receptors, now called ER alpha and ER beta. Transgenic mice in which each of the receptors or both the receptors are inactive have revealed a much broader role for estrogens in the body than was previously thought. This decade also saw the description of a male patient who had no functional ER alpha and whose continued bone growth clearly revealed an important function of estrogen in men. The importance of estrogen in both males and females was also demonstrated in the laboratory in transgenic mice in which the aromatase gene was inactivated. Finally, crystal structures of the estrogen receptors with agonists and antagonists have revealed much about how ligand binding influences receptor conformation and how this conformation influences interaction of the receptor with coactivators or corepressors and hence determines cellular response to ligands.

There was a time when the classification of sex hormones was simple. Androgens were male and estrogens female. What remains true today is that in young adults androgen levels are higher in males and estrogen levels higher in females. More recently we have learned that estrogens are necessary in males for regulation of male sexual behavior, maintenance of the skeleton and the cardiovascular system, and for normal function of the testis and prostate. The importance of androgen in females was never in doubt, it is after all the precursor of estrogen as the substrate for aromatase, the enzyme that produces estrogen. In addition, the tissue distribution of androgen receptors suggests that androgens themselves are important in the ovary, uterus, breast, and brain. New information promises to clarify some of the complex issues of the physiological roles of estrogen and the contribution of estrogen to the development of neoplastic diseases in humans. The discovery of the second estrogen receptor, the creation of mutant mice defective in both estrogen receptors and in the aromatase gene, the solution of the structures of the ligand-binding domains of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), the finding of novel routes through which estrogen receptors can modulate transcription, and the identification of a man with a bi-allelic disruptive mutation of the ERalpha gene are but some of the milestones. This review focuses on the mechanistic aspects of signal transduction mediated by ERs and on the physiological consequences of deficiency of estrogen or estrogen receptor in the available mouse models.

The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.

The physiological e ects of estrogens are mediated by two intracellular transcription factors, the estrogen receptors (ERs), that regulate transcription of target genes through binding to speci®c DNA target sequences. Here we describe alterations in cellular responses to di erent ER agonists and to the anti-estrogenic compound tamoxifen resulting from co-expression of the two ERs in transient co-transfection experiments. Our results demonstrate that ERb can act as a negative or positive dominant regulator of ER activity. This is manifested through reduced transcriptional activity at low concentrations of estradiol (E 2 ); increased antagonistic e ects of tamoxifen on E 2 stimulated activity; and enhanced agonistic action of the phytoestrogenic compound genistein. Furthermore, using chimeric proteins lacking the N-terminal activation function 1 (AF-1), we show that the di erential responses of ERa and ERb to di erent agonists and antagonists are primarily dictated by inherent di erences in the C-terminal ligand-binding domains of the receptors, whereas the magnitude of transcriptional activity is in¯uenced by ERa AF-1, but not ERb AF-1. The ERa AF-1 activity appears to be modulated upon co-expression of both ERs. The alterations in transcriptional activity resulting from co-expression of ERa and ERb are probably due to the formation of a/b heterodimeric complexes. This study demonstrates that co-localization and subsequent heterodimerization of ERa and ERb may result in receptor activity distinct from that of ER homodimers.

The physiological activities of estrogens are thought to be mediated by specific nuclear receptors, ERα and ERβ. However, certain tissues, such as the bone, that are highly responsive to estrogens only express a low level of these receptors. Starting from this apparent contradiction, we have evaluated the potentials of two related receptors ERRα and ERRβ to intervene in estrogen signaling. ERα, ERRα and ERRβ bind to and activate transcription through both the classical estrogen response element (ERE) and the SF-1 response element (SFRE). In contrast, ERβ DNA-binding and transcriptional activity is restricted to the ERE. Accordingly, the osteopontin gene promoter is stimulated through SFRE sequences, by ERRα as well as by ERα, but not by ERβ. Analysis of the cross-talk within the ER/ERR subgroup of nuclear receptors thus revealed common targets but also functional differences between the two ERs.

Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor ␣ (ER␣) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ER␤ is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ER␤ gene (ER␤ ؊/؊ ) revealed decreased numbers of alveoli in adult female ER␤ ؊/؊ mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Plateletderived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ER␤ ؊/؊ mice, and direct transcriptional regulation of these genes by ER␤ was demonstrated. This suggests that estrogens act via ER␤ in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ER␤ as a previously unknown regulator of postnatal lung development and homeostasis.The vital function of the lung is to provide a gas-exchange surface to meet the organism's needs for oxygen uptake and carbon dioxide elimination. Several parameters in lung biology and pathology, both during development and in the adult, are sexually dimorphic. A role for estrogen in these dimorphisms was suggested in 1980 when Mendelson et al. (21) showed an estrogen-binding component in human fetal lung tissue. Lung maturation during fetal development is more rapid in female fetuses than in male fetuses, and the onset of surfactant synthesis occurs later in the male fetus. This difference appears to be mediated mainly by inhibitory effects of androgens, but stimulatory effects of estrogens have also been demonstrated (2). Postnatal sex differences in the rodent lung have been described by Massaro et al. (20). Adult females have a larger number of alveoli, smaller in size, than males, probably to allow for elevated oxygen consumption during pregnancy and lactation. This difference develops as animals reach sexual maturity and seems to be mediated mainly by estrogens (19). In the human population, women are more prone than men to developing chronic obstructive pulmonary disease (29) and incur a higher risk of developing lung cancer (13, 41), indicating that women are more susceptible to the deleterious effects of tobacco smoking. The reasons for these sex differences are unknown, but estrogens are likely to play a major role, since in animal models, there are estrogen-dependent gender differences in susceptibility towards tobacco-associated lung carcinogens (23), and furthermore, epidemiological studies suggest that hormone replacement therapy with estrogen is associated with a higher risk of lung cancer in postmenopausal women (1,39).Although previous data suggest that estrogen...

Human estrogen receptors alpha (ERalpha) and beta (ERbeta) are ligand-inducible transcription factors that are highly homologous in their central DNA-binding and carboxyl-terminal ligand-binding domains. In contrast, there is very little conservation between ERalpha and ERbeta in the amino-terminal domain. Using different human cell lines, we show that wild-type ERbeta transcriptional activity is lower or similar to that of ERalpha, depending on the cell type. Deletion of the amino-terminal domain in both ER subtypes resulted in no or a lower decrease of transcriptional activity of ERbeta compared with ERalpha, suggesting that the ERbeta amino-terminal domain contains a weaker transcriptional activation function-1. Using ERalpha and ERbeta deletion mutants, we showed that the amino-terminal transcriptional activity of ERbeta maps to amino acids 1-31. Interestingly, this domain contains a six amino-acid motif (amino acids 5-10 in human ERbeta) that is part of the ERalpha-activation function-1 region (amino acids 49-54 in human ERalpha) and highly conserved among all mammalian ERalpha amino-terminal domains. Despite this similarity between the two ER subtypes, no autonomous and ligand-independent activity of the ERbeta-amino-terminal domain was observed in yeast and mammalian cells in contrast to ERalpha. This study provides a molecular basis for the difference in transcriptional activity between ERalpha and ERbeta and establishes that ERbeta contains a structurally and functionally restricted amino-terminal transcriptional activity.

The biological effects of estrogens are mediated by the estrogen receptors ER␣ and ER␤. These receptors regulate gene expression through binding to DNA enhancer elements and subsequently recruiting factors such as coactivators that modulate their transcriptional activity. Here we show that ARNT (aryl hydrocarbon receptor nuclear translocator), the obligatory heterodimerization partner for the aryl hydrocarbon receptor and hypoxia inducible factor 1␣, functions as a potent coactivator of ER␣-and ER␤-dependent transcription. The coactivating effect of ARNT depends on physical interaction with the ERs and involves the C-terminal domain of ARNT and not the structurally conserved basic helixloop-helix and PAS (Per-ARNT-Sim) motifs. Moreover, we show that ARNT͞ER interaction requires the E2-activated ligand binding domain of ER␣ or ER␤. These observations, together with the previous role of ARNT as an obligatory partner protein for conditionally regulated basic helix-loop-helix-PAS proteins like the aryl hydrocarbon receptor or hypoxia inducible factor 1␣, expand the cellular functions of ARNT to include regulation of ER␣ and ER␤ transcriptional activity. ARNT was furthermore recruited to a natural ER target gene promoter in a estrogen-dependent manner, supporting a physiological role for ARNT as an ER coactivator.cross-talk ͉ chromatin immunoprecipitation E strogens regulate important physiological processes, such as development and function of the male and female reproductive system and maintenance of bone mass in women, and represent a protective factor against cardiovascular disease (1). The physiological effects of estrogens are mediated by estrogen receptors ␣ (ER␣) and ␤ (ER␤), which belong to the nuclear receptor superfamily (1, 2). Nuclear receptors are liganddependent transcription factors characterized by a conserved structural arrangement composed of a centrally located DNA binding domain (DBD) containing two highly conserved Zn finger motifs. This domain is flanked in the N terminus by a variable A͞B region, which contains an activation function (AF-1). The ligand binding domain (LBD), located C-terminally of the DBD, contains a second AF (AF-2) and is also responsible for ligand binding, receptor dimerization, and cofactor interaction (3).The ERs are, in the absence of ligand, present in the nucleus in a nonactivated form. The latent ERs interact with corepressors such as N-Cor, SHP, or SMRT, which inhibit constitutive transcriptional activity (1). Binding of agonists induces release of repressor proteins and allows interaction with coactivators of the p160 class like SRC-1 or transcription intermediary factor 2 (TIF-2). These proteins have been shown to increase access to chromatin through acetylation of histones, mediate contact with general transcription factors, and enhance receptor AF-1͞AF-2 synergy (4, 5).Interestingly, p160 coactivators share considerable sequence homology to basic helix-loop-helix (bHLH)-PAS (Per-ARNTSim) transcription factors. This family also includes factors such as the aryl h...