Guojun Cheng

Update on estrogen signaling

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor β (ERβ). In many contexts ERβ appears to antagonize the actions of ERα (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERβ knockout mice have shown that ERβ exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERβ‐specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERβ.

Estrogen receptors and proliferation markers in primary and recurrent breast cancer

To elucidate the clinical importance of estrogen receptor (ER) β in breast cancer, 29 archival primary breast cancer specimens, six locally recurrent cancers, and five benign mammary tumors were examined histochemically for ERα, ERβ and the proliferation markers Ki67 and cyclin A. In benign tumors, most epithelial cells contained ERβ, but ERα was rare. In primary cancers, both ERα and ERβ occurred in epithelial cells, the presence of ERβ being associated with elevated expression of Ki67 and cyclin A, and ERα with decreased levels. Thus, the highest content of proliferation markers was seen in primary cancers that were ERα− ERβ+. Most Ki67-containing cells coexpressed ERβ, but few showed ERα. In locally recurring cancers, ERα, ERβ, and Ki67 were more highly expressed than in the corresponding primary tumors, and many cells containing ERβ, but few with ERα, expressed Ki67. Surprisingly, ERβ, but not ERα, was seen in the stromal cells of both primary and recurrent cancers. Because the response of breast cancers to tamoxifen therapy is correlated with the presence of ERα, cancer cells that lack ERα but contain ERβ and proliferation markers represent a novel population of apparently proliferating cells that probably are not targeted by the current antiestrogens. Thus, appropriate ERβ-specific ligands, perhaps in combination with tamoxifen, may be useful in improving the treatment of breast cancers.

Regulation of Postnatal Lung Development and Homeostasis by Estrogen Receptor β

ABSTRACT Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor α (ERα) 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 ERβ is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERβ gene (ERβ−/−) revealed decreased numbers of alveoli in adult female ERβ−/− mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived 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 ERβ−/− mice, and direct transcriptional regulation of these genes by ERβ was demonstrated. This suggests that estrogens act via ERβ 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 ERβ as a previously unknown regulator of postnatal lung development and homeostasis.

A Role for the Androgen Receptor in Follicular Atresia of Estrogen Receptor Beta Knockout Mouse Ovary

Abstract Estrogen receptor beta (ERβ) is highly expressed, but ERα is not detectable in granulosa cells in the mouse ovary. In ERβ knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERβ there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.

Isoflavone treatment for acute menopausal symptoms.

Objective:The onset of climacteric symptoms (hot flashes and night sweats) is the primary reason for perimenopausal women to start hormone therapy. The association of a lower incidence of postmenopausal symptoms with high intake of soybeans in Asian women suggests that phytoestrogens are an alternative to estrogen therapy. The main effective compounds in soybean are isoflavones, which have a higher binding affinity to estrogen receptor &bgr; than to estrogen receptor &agr;. The aim of present study was to evaluate the effects of isoflavone treatment in postmenopausal women. Design:This was a double-blind prospective study. Sixty healthy postmenopausal women were randomly assigned by computer into two groups to receive 60 mg isoflavones or placebo daily for 3 months. Before and after treatment, climacteric symptoms were recorded; serum was collected to measure the levels of lipoprotein lipids, estradiol, and follicle-stimulating hormone; and biopsy specimens from endometrium and breast were analyzed to investigate the expression level of steroid receptors and proliferation. Endometrial thickness was measured by ultrasound. Results:Fifty-one women finished the 12-week study. In women receiving 60 mg isoflavones daily, hot flashes and night sweats were reduced by 57% and 43%, respectively. The treatment did not change the levels of circulating estradiol or follicle-stimulating hormone. Immunohistochemical staining of endometrial and breast biopsy specimens revealed that isoflavones did not affect expression levels of steroid receptors; estrogen receptors &agr;, &bgr;, and &bgr;cx; progesterone receptors A and B; or the proliferation marker Ki67. No side effects on body weight or lipoprotein lipids were observed. Conclusions:This short-term prospective study implies that isoflavones could be used to relieve acute menopausal symptoms.

Estrogen receptors ER alpha and ER beta in proliferation in the rodent mammary gland.

Cell proliferation in the mammary gland is under multihormonal control. Classical endocrine ablation/hormone replacement studies have demonstrated that ovarian estradiol (E2) is critical for the two major phases of mammary development, ductal elongation during puberty, and lobuloalveolar development during pregnancy (1-3). E2 acts directly on the mammary gland to stimulate ductal morphogenesis during puberty, whereas progesterone is the major stimulator of mammary epithelial DNA synthesis and alveolar development (1, 4). Although E2 elicits proliferation of the mammary gland epithelium and the antiestrogen, tamoxifen inhibits proliferation of ERα-positive breast cancer (5), the mechanism of E2-induced proliferation is a subject of much debate and investigation. One of the most confounding observations is that in the mammary gland, either normal (4, 6-10) or malignant (11) cells that express proliferation markers do not express ERα. One school of thought holds that the proliferative effects of E2 on epithelium are indirect, i.e., E2 is thought to act on ERα in stromal cells inducing the release of growth factors, which then stimulate proliferation of epithelial cells (12-14). One corollary of this reasoning would be that ERα-containing cells are protected from growth factor-stimulated proliferation. Another hypothesis to explain the dissociation between steroid receptor expression and proliferation in the normal breast is that steroid receptors are normally expressed in fully differentiated resting cells, and it is only in malignancy that proliferating cells express these receptors (6). Recently, it was shown that in mature rats that have had a pregnancy but not in age-matched virgins, ERα does colocalize with proliferation markers (15). Yet another school of thought maintains that progesterone, not E2, is the proliferative hormone in the mammary epithelium (16-20). The strongest support for this idea is that proliferation in the mammary gland occurs during the luteal phase of the estrus cycle when progesterone levels are high (17). A clear distinction has to be made between lobular growth, which is progesterone-mediated, and ductal growth, which is E2-mediated (21-23). During the estrus cycle and in preparation for pregnancy, it is lobular growth that occurs (17). The functions of stromal steroid receptors in stimulating epithelial proliferation in mammary gland have been studied in ER knockout mice (14). There is very limited ductal growth in ERα knockout mice (ERα-/-) (12, 24), whereas the mammary glands of virgin ERβ knockout mice (ERβ-/-) are morphologically indistinguishable from those of WT littermates (25). In ERα-/- mice, the mammary gland phenotype results from abnormal pituitary function. A reduction in prolactin secretion from the pituitary leads to reduced mammary gland development, and excessive luteinizing hormone secretion results in hemorrhagic follicles and lack of corpora lutea in ovary (26). Ductal elongation and lobuloalveolar development are restored in intact ERα-/- mice on receipt of a normal pituitary and in ovariectomized ERα-/- mice on estrogen/progesterone treatment (1). These results indicate that the effect of loss of ERα on mammary gland growth is indirect, via the pituitary, and this conclusion is further supported by experiments where tissue recombinants (mammary stromal/epithelial) between WT and ERα-/- mice were used. These experiments showed that epithelial growth occurs when either epithelial or stromal cells are from ERα-/- as long as mice are supplemented with E2 and progesterone (12). In both rodent and human mammary glands, the dominant ER in the stroma is ERβ, not ERα (3, 4, 7, 27, 28), indicating that E2-stimulated growth factor release from the stroma is very likely ERβ-mediated. This finding is surprising for two reasons: (i) the overwhelming evidence that ERα is the receptor controlling E2-mediated proliferation, and (ii) the apparently normal development of the mammary gland in ERβ-/- mice. Clearly, the mammary epithelium in ERβ-/- mice does not depend on stromal ERβ for E2-stimulated growth. To clarify the roles of the two ERs in E2-induced proliferation, we have examined the effects of E2 and tamoxifen on the mammary glands in WT and ERβ-/- mice and of a selective ERβ agonist in WT mice. We conclude that proliferation in the mammary epithelium is triggered by direct action of E2 on ER in epithelial cells and can be mediated by both ERα and ERβ. Once the proliferation signal is received by the cell, ERα is down-regulated, which is why ERα is never colocalized with proliferation markers.

Estrogen Receptor Beta

When the discovery of ERβ was reported in 1996 [1], many endocrinologists thought that, since this receptor had gone unnoticed for so long, it must be some sort of vestigial receptor with no function in the endocrinology of estrogen. This idea was quickly dispelled when ERβ-/- mice were created and found to have severely compromised ovarian function and prostatic hyperplasia [2]. Later, two other ERβ-/- mouse lines were created independently in two other laboratories and all three mouse lines were shown to exhibit severe ovarian dysfunction [3–5]. Moreover since the ovarian phenotype in ERβ-/- mice is distinctly different from that in ERα-/- mice [3, 6], it became clear that ERα and ERβ have distinct roles in the body. There is, at present, some argument between different labs concerning other aspects of the ERβ-/- phenotype. If biology was as straight forward as physics, where for each action there is an equal and opposite reaction, understanding knock out phenotypes would be easy. Unfortunately, in endocrinology, it is impossible to make a single alteration without eliciting a cascade of responses.

Estrogen receptors ERα and ERβ in proliferation in the rodent mammary gland

Cell proliferation in the mammary gland is under multihormonal control. Classical endocrine ablation/hormone replacement studies have demonstrated that ovarian estradiol (E2) is critical for the two major phases of mammary development, ductal elongation during puberty, and lobuloalveolar development during pregnancy (1-3). E2 acts directly on the mammary gland to stimulate ductal morphogenesis during puberty, whereas progesterone is the major stimulator of mammary epithelial DNA synthesis and alveolar development (1, 4). Although E2 elicits proliferation of the mammary gland epithelium and the antiestrogen, tamoxifen inhibits proliferation of ERα-positive breast cancer (5), the mechanism of E2-induced proliferation is a subject of much debate and investigation. One of the most confounding observations is that in the mammary gland, either normal (4, 6-10) or malignant (11) cells that express proliferation markers do not express ERα. One school of thought holds that the proliferative effects of E2 on epithelium are indirect, i.e., E2 is thought to act on ERα in stromal cells inducing the release of growth factors, which then stimulate proliferation of epithelial cells (12-14). One corollary of this reasoning would be that ERα-containing cells are protected from growth factor-stimulated proliferation. Another hypothesis to explain the dissociation between steroid receptor expression and proliferation in the normal breast is that steroid receptors are normally expressed in fully differentiated resting cells, and it is only in malignancy that proliferating cells express these receptors (6). Recently, it was shown that in mature rats that have had a pregnancy but not in age-matched virgins, ERα does colocalize with proliferation markers (15). Yet another school of thought maintains that progesterone, not E2, is the proliferative hormone in the mammary epithelium (16-20). The strongest support for this idea is that proliferation in the mammary gland occurs during the luteal phase of the estrus cycle when progesterone levels are high (17). A clear distinction has to be made between lobular growth, which is progesterone-mediated, and ductal growth, which is E2-mediated (21-23). During the estrus cycle and in preparation for pregnancy, it is lobular growth that occurs (17). The functions of stromal steroid receptors in stimulating epithelial proliferation in mammary gland have been studied in ER knockout mice (14). There is very limited ductal growth in ERα knockout mice (ERα-/-) (12, 24), whereas the mammary glands of virgin ERβ knockout mice (ERβ-/-) are morphologically indistinguishable from those of WT littermates (25). In ERα-/- mice, the mammary gland phenotype results from abnormal pituitary function. A reduction in prolactin secretion from the pituitary leads to reduced mammary gland development, and excessive luteinizing hormone secretion results in hemorrhagic follicles and lack of corpora lutea in ovary (26). Ductal elongation and lobuloalveolar development are restored in intact ERα-/- mice on receipt of a normal pituitary and in ovariectomized ERα-/- mice on estrogen/progesterone treatment (1). These results indicate that the effect of loss of ERα on mammary gland growth is indirect, via the pituitary, and this conclusion is further supported by experiments where tissue recombinants (mammary stromal/epithelial) between WT and ERα-/- mice were used. These experiments showed that epithelial growth occurs when either epithelial or stromal cells are from ERα-/- as long as mice are supplemented with E2 and progesterone (12). In both rodent and human mammary glands, the dominant ER in the stroma is ERβ, not ERα (3, 4, 7, 27, 28), indicating that E2-stimulated growth factor release from the stroma is very likely ERβ-mediated. This finding is surprising for two reasons: (i) the overwhelming evidence that ERα is the receptor controlling E2-mediated proliferation, and (ii) the apparently normal development of the mammary gland in ERβ-/- mice. Clearly, the mammary epithelium in ERβ-/- mice does not depend on stromal ERβ for E2-stimulated growth. To clarify the roles of the two ERs in E2-induced proliferation, we have examined the effects of E2 and tamoxifen on the mammary glands in WT and ERβ-/- mice and of a selective ERβ agonist in WT mice. We conclude that proliferation in the mammary epithelium is triggered by direct action of E2 on ER in epithelial cells and can be mediated by both ERα and ERβ. Once the proliferation signal is received by the cell, ERα is down-regulated, which is why ERα is never colocalized with proliferation markers.

Gonadotropin-positive pituitary tumors accompanied by ovarian tumors in aging female ERβ−/− mice

At 2 years of age, 100% (23/23) of ERβ−/− female mice have developed large pituitary and ovarian tumors. The pituitary tumors are gonadotropin-positive and the ovarian tumors are sex cord (less differentiated) and granulosa cell tumors (differentiated and estrogen secreting). No male mice had pituitary tumors and no pituitary or ovarian tumors developed in ERα−/− mice or in ERαβ−/− double knockout mice. The tumors have high proliferation indices, are ERα-positive, ERβ-negative, and express high levels of nuclear phospho-SMAD3. Mice with granulosa cell tumors also had hyperproliferative endometria. The cause of the pituitary tumors appeared to be excessive secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus resulting from high expression of NPY. The ovarian phenotype is similar to that seen in mice where inhibin is ablated. The data indicate that ERβ plays an important role in regulating GnRH secretion. We suggest that in the absence of ERβ, the proliferative action of FSH/SMAD3 is unopposed and the high proliferation leads to the development of ovarian tumors. The absence of tumors in the ERαβ−/− mice suggests that tumor development requires the presence of ERα.

Ovarian wedge resection restores fertility in estrogen receptor β knockout (ERβ−/−) mice

Ovulation rarely occurs in mice in which the estrogen receptor β (ERβ) gene has been inactivated (ERβ−/− mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ERβ−/− mice. We transplanted ERβ−/− ovaries into WT mice and WT ovaries into ERβ−/− mice. Upon mating with ERβ−/− males, fertility increased from 20% in control intact ERβ−/− group to 40% in the WT recipients with ERβ−/− ovaries. The transplantation procedure was not efficient, and when WT ovaries were transplanted into WT mice, fertility was only 36%. Surgical ovarian wedge resection, a procedure which induces ovulation in anovulatory women with polycystic ovarian syndrome, resulted in 100% fertility of ERβ−/− mice. In ERβ−/− mice, as the follicles enlarged, the thecal layer remained very compact (revealed by H&E and collagen staining), and there was no increase in vascularization (measured as smooth muscle actin). In addition, there was an increase in PDGF receptor α (PDGFRα) and a decrease in PDGFβ expression in the granulosa cells, similar to what has been found in follitropin receptor knockout mice. After wedge resection, expression of both smooth muscle actin and PDGFRs was normalized. During normal follicular development, increased vascularization of the thecal layer is a prerequisite for further follicular growth. We suggest that the defect in ERβ−/− mouse ovaries is a failure of communication between the granulosa and thecal layers. The follicles do not mature because of insufficient blood supply. This problem is overcome by stimulating neovascularization by simple wedge resection of the ovaries.