Margaret Warner

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.

Targeting estrogen receptor β in microglia and T cells to treat experimental autoimmune encephalomyelitis

A therapeutic goal in the treatment of certain CNS diseases, including multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson disease, is to down-regulate inflammatory pathways. Inflammatory molecules produced by microglia are responsible for removal of damaged neurons, but can cause collateral damage to normal neurons located close to defective neurons. Although estrogen can inactivate microglia and inhibit the recruitment of T cells and macrophages into the CNS, there is controversy regarding which of the two estrogen receptors (ERs), ERα or ERβ, mediates the beneficial effects in microglia. In this study, we found that ERβ, but not ERα, is expressed in microglia. Using the experimental autoimmune encephalomyelitis (EAE) model in SJL/J mice, we evaluated the benefit of an ERβ agonist as a modulator of neuroinflammation. Treatment of EAE mice with LY3201, a selective ERβ agonist provided by Eli Lilly, resulted in marked reduction of activated microglia in the spinal cord. LY3201 down-regulated the nuclear transcription factor NF-κB, as well as the NF-κB–induced gene inducible nitric oxide synthase in microglia and CD3+ T cells. In addition, LY3201 inhibited T-cell reactivity through regulation of indoleamine-2,3-dioxygenase. In the EAE model, treatment with LY3201 decreased mortality in the first 2 wk after disease onset, and also reduced the severity of symptoms in mice surviving for 4 wk. Our data show that ERβ-selective agonists, by modulating the immune system in both microglia and T cells, offer promise as a useful class of drugs for treating degenerative diseases of the CNS.

The role of estrogen receptor β (ERβ) in malignant diseases—A new potential target for antiproliferative drugs in prevention and treatment of cancer

The discovery of ERbeta in the middle of the 1990s represents a paradigm shift in our understanding of estrogen signaling. It has turned out that estrogen action is not mediated by one receptor, ERalpha, but by two balancing factors, ERalpha and ERbeta, which are often antagonistic to one another. Excitingly, ERbeta has been shown to be widespread in the body and to be involved in a multitude of physiological and pathophysiological events. This has led to a strong interest of the pharmaceutical industry to target ERbeta by drugs against various diseases. In this review, focus is on the role of ERbeta in malignant diseases where the anti proliferative activity of ERbeta gives hope of new therapeutic approaches.

Estrogen receptors in breast carcinogenesis and endocrine therapy.

Excessive exposure to estrogen has long been associated with an increased risk for developing breast cancer and anti-estrogen therapy is the gold standard of care in the treatment of estrogen receptor (ER) α-positive breast cancers. However, there are several mysteries concerning both anti-estrogen, tamoxifen, and estrogen. The most important of these are: (1) some ERα-positive breast cancers do not respond to tamoxifen; (2) some ERα-negative breast cancers do respond to tamoxifen; (3) initial or acquired resistance to tamoxifen occurs with recurrent tumors; (4) estrogen can cause marked tumor regression in long-term tamoxifen-resistant ERα-positive breast cancer. These mysteries indicate that we do not know enough about estrogen signaling to understand the effects of targeting these receptors in cancer. The discovery of ERβ, the second estrogen receptor, has added another level of complexity to estrogen signaling. This review summarizes recent publications and makes an updated portrait of ERα and ERβ in breast carcinogenesis and endocrine cancer therapy.

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

Significance Whether breast cancer will respond to the antiestrogen tamoxifen is determined by whether cellular proliferation is estrogen receptor (ER) α-mediated. As opposed to early ductal cancer, which is an ERα-rich, proliferating disease, in early lobular cancer both ERα and ERβ are abundantly expressed and proliferation is rare. In advanced lobular cancer, ERβ is lost, ERα is retained, and proliferation is high. Thus, tamoxifen may be an effective pharmaceutical in late but not early lobular cancer. The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.

Insight into the mechanisms of action of estrogen receptor β in the breast, prostate, colon, and CNS

Estrogen and its receptors (ERs) influence many biological processes in physiology and pathology in men and women. ERs are involved in the etiology and/or progression of cancers of the prostate, breast, uterus, ovary, colon, lung, stomach, and malignancies of the immune system. In estrogen-sensitive malignancies, ERβ usually is a tumor suppressor and ERα is an oncogene. ERβ regulates genes in several key pathways including tumor suppression (p53, PTEN); metabolism (PI3K); survival (Akt); proliferation pathways (p45(Skp2), cMyc, and cyclin E); cell-cycle arresting factors (p21(WAF1), cyclin-dependent kinase inhibitor 1 (CDKN1A)), p27(Kip1), and cyclin-dependent kinases (CDKs); protection from reactive oxygen species, glutathione peroxidase. Because they are activated by small molecules, ERs are excellent targets for pharmaceuticals. ERα antagonists have been used for many years in the treatment of breast cancer and more recently pharmaceutical companies have produced agonists which are very selective for ERα or ERβ. ERβ agonists are being considered for preventing progression of cancer, treatment of anxiety and depression, as anti-inflammatory agents and as agents, which prevent or reduce the severity of neurodegenerative diseases.

Liver X receptor β protects dopaminergic neurons in a mouse model of Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative disease whose progression may be slowed, but at present there is no pharmacological intervention that would stop or reverse the disease. Liver X receptor β (LXRβ) is a member of the nuclear receptor super gene family expressed in the central nervous system, where it is important for cortical layering during development and survival of dopaminergic neurons throughout life. In the present study we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of LXRβ as a target for prevention or treatment of PD. The dopaminergic neurons of the substantia nigra of LXRβ−/− mice were much more severely affected by MPTP than were those of their WT littermates. In addition, the number of activated microglia and GFAP-positive astrocytes was higher in the substantia nigra of LXRβ−/− mice than in WT littermates. Administration of the LXR agonist GW3965 to MPTP-treated WT mice protected against loss of dopaminergic neurons and of dopaminergic fibers projecting to the striatum, and resulted in fewer activated microglia and astroglia. Surprisingly, LXRβ was not expressed in the neurons of the substantia nigra but in the microglia and astroglia. We conclude that LXR agonists may have beneficial effects in treatment of PD by modulating the cytotoxic functions of microglia.

ERβ in CNS: New Roles in Development and Function

Estrogen acting through two estrogen receptors (ERs), ERalpha and ERbeta, regulates multiple functions in the central nervous system. Studies in rodent brains have revealed that ERalpha is the predominant ER in the hypothalamus and controls reproduction. ERbeta influences on non-reproductive processes and appears to be the main ER subtype expressed in the cerebral cortex, the hippocampus, the cerebellum and the dorsal raphe. During embryogenesis, estrogen plays an important role in brain development regulating maturation of distinct brain structures, thereby contributing to modulation of the function of these structures. Studies on the brains of ERbeta knockout mice revealed that, during embryonic brain development, ERbeta affects cortical layering and interneuron migration, thus playing a key role in brain morphogenesis. This chapter will focus on the roles of ERbeta in several aspects of the development and function of the mammalian central nervous system.

Action mechanisms of Liver X Receptors.

The two Liver X Receptors, LXRα and LXRβ, are nuclear receptors belonging to the superfamily of ligand-activated transcription factors. They share more than 78% homology in amino acid sequence, a common profile of oxysterol ligands and the same heterodimerization partner, Retinoid X Receptor. LXRs play crucial roles in several metabolic pathways: lipid metabolism, in particular in preventing cellular cholesterol accumulation; glucose homeostasis; inflammation; central nervous system functions and water transport. As with all nuclear receptors, the transcriptional activity of LXR is the result of an orchestration of numerous cellular factors including ligand bioavailability, presence of corepressors and coactivators and cellular context i.e., what other pathways are activated in the cell at the time the receptor recognizes its ligand. In this mini-review we summarize the factors regulating the transcriptional activity and the mechanisms of action of these two receptors.

Estrogen Receptor β as a Pharmaceutical Target

A major issue in clinical endocrinology today is how to use hormones to achieve the health benefits that they clearly can provide but avoid the negative side effects, that is, how to develop more precise medicines. This problem of how to use hormones is pervasive in clinical endocrinology. It is true for estrogen, progesterone, androgen, vitamin D, and thyroid hormone, and the problem is amplified in the case of new ligands for the more recently discovered nuclear receptors. Selective targeting of hormone receptor subtypes is one attractive way to harness the beneficial effects of hormones while reducing unwanted side effects. Here, we focus on estrogen receptor (ER)β, which has promise as a selective target in hormone replacement therapy, and in breast and prostate cancers.