Ovarian Actions of Estrogen Receptor-β

An Update

Ann E. Drummond, Ph.D.; Peter J. Fuller, B.Med.Sci., M.B.B.S., Ph.D., F.R.A.C.P.

Disclosures

Semin Reprod Med. 2012;30(1):32-38. 

In This Article

Estrogen Receptors

ERα and ERβ share 95% amino acid homology in the DNA binding domain and 55% homology in the ligand-binding domain (LBD).[2] This level of identity is also seen between the LBDs of the androgen, glucocorticoid, mineralocorticoid, and progesterone receptors (PRs), and it is associated with both unique and shared ligand binding. The N-terminal, hinge, and C-terminal regions of the ER have the greatest sequence diversity.[2]

Multiple isoforms of the ERβ subtype have now been described,[14–19] although it is not clear if these forms are all biologically active. Chu and colleagues reported the existence of a 54 nucleotide insert in the LBD of rat ERβ.[14] Termed ER-β2, this isoform, present only in rodents,[14,20] acts as a dominant negative regulator of ERβ- and ERα-mediated transcription.[21] Although this isoform has not been detected in humans, shortened transcripts and alternatively spliced forms of ERβ have been reported in normal ovary and ovarian tumors.[21–23] These forms, designated ERβ1, ERβ2 (also known as ERβ cx), ERβ3, ERβ4, and ERβ5,[17,23,24] each produce a full-length transcript. Initially it was thought that ERβ4 and ERβ5 existed only as truncated transcripts, but this has proven not to be the case.[17,25]

The affinity of ligands for the respective receptor subtypes and isoforms differs.[15] The response to estrogen in a given tissue is defined by the ER expressed and the matrix of ER-interacting proteins present within the cells. These co-regulatory molecules may influence the response in both a ligand- and promoter-dependent context, which in turn may be influenced by other signaling pathways. Nuclear hormone receptors interact with co-regulatory proteins, either coactivators that enhance transcription or corepressors that repress transcription. ERs contain two activation functions (AFs) that interact with coactivators. AF-1, which is ligand independent, lies within the N-terminal domain; AF-2 lies in the LBD, and its activity depends on ligand-induced conformational changes. The relative contribution of each AF is cell and promoter dependent. Transcription of the human ERβ genes occurs from at least two promoters, 0N and 0K,[19] with the same transcript produced.

Signaling via Estrogen Receptor-β

ERs mediate transcription as dimers. Both homodimers and heterodimers of the ER activate transcription of reporter gene constructs containing estrogen response elements.[15,26] It has been suggested that ERβ activity is compromised in the absence of ERα,[4] further supporting the heterodimer as the functional form of ERs. Studies in other tissues suggest that ERβ may antagonize/oppose the effects of ERα, thereby serving to limit cellular proliferation, promote differentiation (luteinization), and modulate apoptosis (atresia).[27] Although a biological role for ERβ2 has not yet been elucidated, the studies of Maruyama et al suggested that ER-β2 may be a negative regulator of estrogen action, given that it dose dependently suppressed ERα and ERβ1-mediated transcriptional activation.[21] Thus the formation of dimers containing ERβ2 may well induce very different effects on gene expression relative to those induced by receptor dimers that do not contain ERβ2.

ERβ plays a direct role in follicle development and is required for antrum formation and preovulatory follicle maturation.[27,28] Ovulatory defects have been linked with polymorphisms of human ERβ.[29] Hemorrhagic and cystic follicles of ERα and LHβ C-terminal peptide transgenic mice (mice that express elevated levels of LH in the absence of ERβ) require ERβ for development.[30] Polyovular follicles were induced by both ERα and ERβ agonists in neonatal mice.[31] However, mice lacking ERβ do not produce polyovular follicles when challenged with genistein or diethylstilbestrol (DES),[32,33] whereas ERα knockout mice do, suggesting that ERβ is directly involved in polyovular follicle formation. In human corpora lutea (CL), estrogenic activity is mediated by ERβ with both protein and mRNA localized to luteal cells, perivascular cells, and fibroblasts within the CL.[34] ERβ1 and ERβ2 mRNAs were differentially expressed across the luteal phase with ERβ1 maximally expressed in the midluteal phase and ERβ2 maximally expressed in the early luteal phase.[35] Co-localization of the two forms was noted but not obligatory.

Localization and Regulation of Estrogen Receptor-β

ERβ is present in the ovaries of a wide number of species, including mouse, rat, rabbit, sheep, cow, baboon, hamster, pig, and human.[4,20,36–45] Whereas ERβ is predominantly expressed by the granulosa cells, theca cells, surface epithelium, and CL, although oocytes have also been reported to express the receptor.[34,36,39,46–50]

Definitive information on the expression of the respective ER mRNAs and proteins in granulosa cells of different follicle sizes is lacking. In situ hybridization and reverse transcriptase polymerase chain reaction studies in the rat indicate there is more ERβ than ERα mRNA in the ovary, and further analysis revealed more ERβ2 than ERβ1 in ovarian RNA collected from postnatal rats.[37] Messenger RNA transcripts for ERα and ERβ1 and ERβ2 are present in granulosa cells of follicles with at most two to three layers of granulosa cells, and ERβ1 and ERβ2 proteins are present in rat granulosa cells.[27,36,51,52]

A convergence between gonadotropin signaling and ERβ-mediated transcription in the ovary has been noted, unlike ERα. Gonadotropins are important regulators of ovarian function, and thus it makes sense for them to regulate ERβ expression if indeed ERβ is important for ovarian function. The LH surge was found to downregulate ERβ mRNA in the ovaries of rats and hamsters, and gonadotropin-induced cofactor-4 induced by FSH coactivated ERβ in granulosa cells.[36,38,53]

Genes Regulated by Estrogen Receptor-β

Studies to identify genes regulated by ERβ are difficult to find for normal tissues; the few undertaken to date have used cancer cell lines.[19] Chang and colleagues investigated the effect of ERβ on gene regulation by MCF-7 cells expressing ERα.[54] Microarray analyses revealed that genes regulating signal transduction pathways, cell cycle progression, and apoptosis were modulated by ERβ. These included members of the transforming growth factor-β superfamily (which are normally associated with suppression of breast cancer cell growth), class 3 and 4 semaphorin pathways, FOXM1 (member of the forkhead box transcription factor family, only expressed in proliferating cells), CDC25A (cell division cycle 25 homologue A), E2F1 (transcription factor), survivin (member of the inhibitor of apoptosis protein family that acts as a suppressor of apoptosis and plays a central role in cell division), and p21WAF1 (cyclin-dependent kinase inhibitor).[54] Proliferation of MCF-7 cells declined when ERβ was present, consistent with the repression of FOXM1, CDC25A, E2F1, and survivin mRNAs and the upregulation of p21WAF1, an inhibitor of cell proliferation and SEMA3B, a tumor suppressor.[54]

In the presence of estradiol, ERβ enhanced the repression of thrombospondin 1, reduced the repression of integrin 6 and bone morphogenetic protein-7, and downregulated stromal cell derived factor (SDF)-1.[54,55] SDF-1, which has previously been shown to act as an autocrine growth factor for breast cancer cell, has also interestingly been shown to interfere with semaphoring signaling.[54,55] We are currently undertaking microarray analyses of our granulosa cell tumor cell lines and hope in the near future to report on genes regulated by ERβ in reproductive cells. These recent studies make clear that it is the relative levels of ERβ and ERα in a cell line/tissue that will determine its response to estrogen.

Estrogen Receptor-β Knockout Mice

Despite normal levels of gonadotropins and ovaries that contain follicles of all stages of development and CL, ERβ knockout mice (βERKO) are subfertile, producing fewer pups and litters and yielding fewer oocytes following superovulation.[5,7,9,56,57] Investigators have suggested that a disruption in communication between the theca and granulosa cell layers leads to inhibition of vascularization, preventing the increase in permeability and hyperemia that facilitates expulsion of the ovum.[58] Wedge resection of ERβ knockout ovaries, with its presumed effects on improving vascularization, restored fertility to 100%.[58]

Furthermore, the βERKO mouse displays a granulosa cell-specific phenotype.[5] Ovaries of the βERKO mice contain fewer large antral follicles and CL, and apoptosis in large follicles is increased.[27] It is clear that ERβ is important for follicle maturation from the antral stage of development to follicle rupture.[27] ERβ also appears to play a role in the expression of genes that are important for ovarian differentiation, with βERKO mice demonstrating decreased aromatase, LH receptor, and prostaglandin synthase (Ptgs)2 mRNA levels and increased androgen receptor expression within antral follicles.[27,59] Follicles from these mice also produce significantly less estradiol compared with wild-type mice in vitro, indicating an attenuated response to FSH.[57] ERβ recently was shown to be required by preovulatory follicles for the production of cyclic adenosine monophosphate (cAMP), and inadequate levels of cAMP may account for the reduced levels of estradiol produced by these follicles.[60,61]

It is apparent from the ERα knockout (ERKO) and βERKO ovarian phenotypes that ERα and ERβ have different roles to play in folliculogenesis. It has been hypothesized that the proliferative action of estrogen is transmitted preferentially via ERα, whereas the differentiating effects of estrogen are mediated principally by ERβ.[62] This hypothesis is supported by the differentiation of granulosa cells into male-type Sertoli cells in the estrogen-deficient ArKO.[63] These Sertoli cells disappear from the ovaries of mice treated with estradiol or phytoestrogens, principally genistein,[63] an ERβ-selective ligand.[26] However, interpreting the consequences of ERα and ERβ deletion in these models is complicated by the inability of these receptors to form heterodimers of ERα and ERβ. Homodimers of these transcription factors may induce very different effects on gene expression compared with ER heterodimers.

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