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.

During the past decade there has been a substantial advance in our understanding of estrogen signaling both from a clinical as well as a preclinical perspective. Estrogen signaling is a balance between two opposing forces in the form of two distinct receptors (ERα and ERβ) and their splice variants. The prospect that these two pathways can be selectively stimulated or inhibited with subtype-selective drugs constitutes new and promising therapeutic opportunities in clinical areas as diverse as hormone replacement, autoimmune diseases, prostate and breast cancer, and depression. Molecular biological, biochemical, and structural studies have generated information which is invaluable for the development of more selective and effective ER ligands. We have also become aware that ERs do not function by themselves but require a number of coregulatory proteins whose cell-specific expression explains some of the distinct cellular actions of estrogen. Estrogen is an important morphogen, and many of its proliferative effects on the epithelial compartment of glands are mediated by growth factors secreted from the stromal compartment. Thus understanding the cross-talk between growth factor and estrogen signaling is essential for understanding both normal and malignant growth. In this review we focus on several of the interesting recent discoveries concerning estrogen receptors, on estrogen as a morphogen, and on the molecular mechanisms of anti-estrogen signaling.

Estrogens inf luence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ER␣ and ER␤. We previously generated and studied knockout mice lacking estrogen receptor ␣ and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor ␤ Estrogens are critical to the functioning and maintenance of a diverse array of tissues and physiological systems in mammals. The actions of estrogen on such classical targets as the reproductive tract, gonads, mammary tissue, and hypothalamic͞pituitary axis have been well characterized. A role in nonreproductive tissues, such as maintenance of bone mineral density and cardiovascular health in women, also has been described (1, 2). The physiological responses to estrogen are known to be mediated within specific tissues by at least two estrogen receptors (ERs), ER␣ and ER␤ (3-5). The ERs are a class I member of the nuclear hormone receptor family and act as ligand-activated nuclear transcription factors (6). Studies of the receptors' tissue distribution and expression pattern indicate that ER␣ has a broad expression pattern, whereas ER␤ has a more focused pattern with high levels in the ovary, prostate, epididymis, lung, and hypothalamus (7,8). However, the exact physiological responses attributable to each receptor are unknown. We previously described the pleiotropic effects of disruption of the ER␣ gene in ER␣ knockout mice (␣ERKO), including absence of breast development in females and infertility caused by reproductive tract and gonadal and behavioral abnormalities in both sexes (9-13). Here, we describe the generation of mice homozygous for a disruption of the ER␤ gene; initial characterizations indicate that the ER␤ Ϫ͞Ϫ mice exhibit phenotypes that are distinct from those of the ␣ERKO mice.

Many of the effects of estrogens on the uterus are mediated by ERalpha, the predominant ER in the mature organ. Because of the poor reproductive capacity of ERbeta knockout (BERKO) female mice (small litter size, multiple-resorbed fetuses), the role of uterine ERbeta was explored. In the immature uterus, ERalpha and ERbeta are expressed at comparable levels in the epithelium and stroma, and 17beta-estradiol (E(2)) treatment decreases ERbeta in the stroma. The immature uterus of untreated BERKO mice exhibits elevated levels of progesterone receptor (PR) and the proliferation-associated protein, Ki-67. It also exhibits exaggerated responsiveness to E(2), as indicated by enlargement of the lumen, increase in volume and protein content of uterine secretion, induction of the luminal epithelial secretory protein, complement C3, and its regulatory cytokine IL-1beta, and induction of vascular endothelial growth factor and insulin-like growth factor 1 but not its receptor. As expected, E(2) increased PR in the stroma and decreased it in the luminal epithelium of wild-type mice. In the BERKO uterus, E(2) induced PR in the stroma but did not down-regulate it in the epithelium. Increased cell proliferation and exaggerated response to E(2) in BERKO suggest that ERbeta plays a role in modulation of the effects of ERalpha and in addition (or as a consequence of this) has an antiproliferative function in the immature uterus.

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor ␤ (ER␤). In rat ventral prostate, 530-and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ER␤ does not contain the same protein chaperones that are associated with ER␣. Estradiol (E2) binding and ER␤ immunoreactivity coincide on the gradient, with no indication of ER␣. In prostates from mice in which the ER␤ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5␣-androstane-3␤,17␤-diol (3␤Adiol). This compound, which competes with E 2 for binding to ER␤ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ER␤ knockout (BERKO) mice. Thus ER␤, probably as a complex with 3␤Adiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ER␤ may be useful in the prevention and͞or clinical management of prostatic hyperplasia and neoplasia. E pidemiological and experimental studies indicate that estrogenic hormones are involved in both the induction and prevention of prostatic cancer (1-7), but their precise role is not well understood. Excessive exposure to estrogens during critical stages of development or long-term treatment of adult animals with estrogens or androgens leads to prostatic neoplasia (8, 9). In apparent contrast, diets rich in phytoestrogens, particularly soy products, are associated with a low risk of prostate cancer (10-12) and have chemopreventive properties in experimental tumor models (12, 13). Some of these conflicting observations may be explained by the fact that there are two distinct estrogen receptors, ER␣ and ER␤, which have unique and sometimes opposing roles (14). For example, recent studies have demonstrated that, in the rodent uterus, ER␤ acts to restrain the stimulatory action of ER␣ (15).Early studies, using both ligand-binding and immunochemical techniques, detected two types of estrogen-binding substances in human prostate (16), one of which is the classical estrogen receptor now known as ER␣. Low levels of this receptor are present in the stroma of rodent prostates, but none is detectable in the epithelium (17, 18). Because of this difference in the levels of this receptor, it was proposed that the effects of estrogen on the epithelium are indirect via an initial interaction with the stroma (18). But after the discovery of ER␤ in rat prostatic epithelium ...

Epithelial proliferation of the ventral prostate in rodents peaks between 2 and 4 weeks of age, and by week 8, proliferating cells are rare. We have used ER␤ ؊/؊ and CYP7B1 ؊/؊ mice to investigate the role of ER␤ and one of its ligands, 5␣-androstane-3␤,17␤-diol (3␤Adiol), in growth of the ventral prostate. Before puberty, ER␤ was found in quiescent but not in proliferating cells, and proliferating cells occurred more frequently in ventral prostates of ER␤ ؊/؊ mice than in wild-type littermates. Treatment with 3␤Adiol decreased proliferation in wild-type but not in ER␤ ؊/؊ mice. In rats, treatment with 3␤Adiol from postnatal day 2 to 28 resulted in reduction in growth of ventral prostates. The prostates of CYP7B1 ؊/؊ mice were hypoproliferative before puberty and smaller than those of their wild-type littermates after puberty. Because CYP7B1 represents the major pathway for inactivating 3␤Adiol in the prostate, we suggest that ER␤, 3␤Adiol, and CYP7B1 are the components of a pathway that regulates growth of the rodent ventral prostate. In this pathway, ER␤ is an antiproliferative receptor, 3␤Adiol is an ER␤ ligand, and CYP7B1 is the enzyme that regulates ER␤ function by regulating the level of 3␤Adiol.

We have known for many years that estrogen is more than the female hormone. It is essential in the male gonads, and in both sexes, estrogen has functions in the skeleton and central nervous system, on behavior, and in the cardiovascular and immune systems. An important aspect of the discovery of estrogen receptor (ER) beta is that the diverse functions of estrogen can now be divided into those mediated by ERalpha and those mediated by ERbeta. Pharmacological exploitation of this division of the labors of estrogen is facilitated by the ligand-binding specificity and selective tissue distribution of the two ERs. Because the ligand binding domains of ERalpha and ERbeta are significantly different from each other, selective ligands can be (and have been) developed to target the estrogenic pathway that is malfunctioning, without interfering with the other estrogen-regulated pathways. Because of the absence of ERbeta from the adult pituitary and endometrium, ERbeta agonists can be used to target ERbeta with no risk of adverse effects from chemical castration and uterine cancer. Some of the diseases in which there is hope that ERbeta agonists will be of benefit are prostate cancer, autoimmune diseases, colon cancer, malignancies of the immune system, and neurodegeneration.

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