Andrea Morani

Metabolic Actions of Estrogen Receptor Beta (ERβ) are Mediated by a Negative Cross-Talk with PPARγ

Estrogen receptors (ER) are important regulators of metabolic diseases such as obesity and insulin resistance (IR). While ERα seems to have a protective role in such diseases, the function of ERβ is not clear. To characterize the metabolic function of ERβ, we investigated its molecular interaction with a master regulator of insulin signaling/glucose metabolism, the PPARγ, in vitro and in high-fat diet (HFD)-fed ERβ -/- mice (βERKO) mice. Our in vitro experiments showed that ERβ inhibits ligand-mediated PPARγ-transcriptional activity. That resulted in a blockade of PPARγ-induced adipocytic gene expression and in decreased adipogenesis. Overexpression of nuclear coactivators such as SRC1 and TIF2 prevented the ERβ-mediated inhibition of PPARγ activity. Consistent with the in vitro data, we observed increased PPARγ activity in gonadal fat from HFD-fed βERKO mice. In consonance with enhanced PPARγ activation, HFD-fed βERKO mice showed increased body weight gain and fat mass in the presence of improved insulin sensitivity. To directly demonstrate the role of PPARγ in HFD-fed βERKO mice, PPARγ signaling was disrupted by PPARγ antisense oligonucleotide (ASO). Blockade of adipose PPARγ by ASO reversed the phenotype of βERKO mice with an impairment of insulin sensitization and glucose tolerance. Finally, binding of SRC1 and TIF2 to the PPARγ-regulated adiponectin promoter was enhanced in gonadal fat from βERKO mice indicating that the absence of ERβ in adipose tissue results in exaggerated coactivator binding to a PPARγ target promoter. Collectively, our data provide the first evidence that ERβ-deficiency protects against diet-induced IR and glucose intolerance which involves an augmented PPARγ signaling in adipose tissue. Moreover, our data suggest that the coactivators SRC1 and TIF2 are involved in this interaction. Impairment of insulin and glucose metabolism by ERβ may have significant implications for our understanding of hormone receptor-dependent pathophysiology of metabolic diseases, and may be essential for the development of new ERβ-selective agonists.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERα and ERβ. ERα mediates the proliferative effect of estrogen in breast cancer cells, whereas ERβ seems to be antiproliferative. We engineered ERα-positive T47D breast cancer cells to express ERβ in a Tet-Off–regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERβ. The presence of ERβ resulted in a reduction in tumor growth. Comparison of the ERβ-expressing and non-ERβ–expressing tumors revealed that the expression of ERβ caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ). In cell culture, with the Tet-Off–regulated ERβ-expressing cells, expression of ERβ decreased expression of VEGF and PDGFβ mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFβ proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFβ promoter constructs revealed a repressive effect of ERβ at the promoter level of these genes. Taken together, these data show that introduction of ERβ into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

Biological functions and clinical implications of oestrogen receptors alfa and beta in epithelial tissues

For the past 10 years it is known that oestrogen functions through the activation of two oestrogen receptors (ERα and ERβ). To the great surprise of endocrinologists, ERβ was found to be widely distributed in tissues throughout the body including tissues previously considered as ‘oestrogen insensitive’. The epithelium of the ventral prostate and lung as well as ovarian granulosa cells are ERα‐negative but ERβ‐positive and in these tissues ERβ seems to be involved in important physiological processes, like differentiation, extracellular matrix organization and stromal–epithelial communication. In tissues where both ERs are expressed, the two receptors seem to counteract each other. In the uterus, mammary gland and immune system, ERα promotes proliferation whereas ERβ has pro‐apoptotic and pro‐differentiating functions. The challenge of the future will be to develop specific agonists, which can selectively activate/inactivate either ERα or ERβ. These pharmaceuticals are likely to be of clinical importance in the prevention or treatment of various diseases.

Participation of ERα and ERβ in glucose homeostasis in skeletal muscle and white adipose tissue

Glucose uptake and homeostasis are regulated mainly by skeletal muscle (SM), white adipose tissue (WAT), pancreas, and the liver. Participation of estradiol in this regulation is still under intense investigation. We have demonstrated that, in SM of male mice, expression of the insulin-regulated glucose transporter (GLUT)4 is reduced by estrogen receptor (ER)beta agonists. In the present study, to investigate the relative contributions of ERalpha and ERbeta in glucose homeostasis, we examined the effects of tamoxifen (Tam) on GLUT4 expression in SM and WAT in wild-type (WT) and ER-/- mice. ERbeta-/- mice were characterized by fasting hypoglycemia, increased levels of SM GLUT4, pancreatic islet hypertrophy, and a belated rise in plasma insulin in response to a glucose challenge. ERalpha-/- mice, on the contrary, were hyperglycemic and glucose intolerant, and expression of SM GLUT4 was markedly lower than in WT mice. Tam had no effect on glucose tolerance or insulin sensitivity in WT mice. In ERalpha-/- mice, Tam increased GLUT4 and improved insulin sensitivity. i.e., it behaved as an ERbeta antagonist in SM but had no effect on WAT. In ERbeta-/- mice, Tam did not affect GLUT4 in SM but acted as an ERalpha antagonist in WAT, decreasing GLUT4. Thus, in the interplay between ERalpha and ERbeta, ERbeta-mediated repression of GLUT4 predominates in SM but ERalpha-mediated induction of GLUT4 predominates in WAT. This tissue-specific difference in dominance of one ER over the other is reflected in the ratio of the expression of the two receptors. ERalpha predominates in WAT and ERbeta in SM.

Role of estrogen receptor β in uterine stroma and epithelium: Insights from estrogen receptor β−/− mice

In this study, we compared the uterine tissue of estrogen receptor (ER)β−/− mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERβ−/− mice, but E-cadherin and cytokeratin 18 expression was lower in ERβ−/− mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERβ−/− mice. Sixteen hours after injection of 17β-estradiol (E2), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERβ−/− mice. Although ERα was abundant in intact mice, after ovariectomy, ERα could not be detected in the luminal epithelium of either WT or ERβ−/− mice. In both untreated and E2-treated mice, ERα and ERβ were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E2 + progesterone treatment, ERα and ERβ were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERβ−/− mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E2 treatment, EGFR on cell membranes was down-regulated in WT but not in ERβ−/− mice. These findings reveal an important role for ERβ in response to E2 and in the organization, growth, and differentiation of the uterine epithelium.

Insulin resistance of pregnancy involves estrogen-induced repression of muscle GLUT4.

Pregnancy is accompanied by hyperestrogenism, however, the role of estrogens in the gestational-induced insulin resistance is unknown. Skeletal muscle plays a fundamental role in this resistance, where GLUT4 regulates glucose uptake. We investigated: (1) effects of oophorectomy and estradiol (E2) on insulin sensitivity and GLUT4 expression. E2 ( approximately 200nM) for 7 days decreased sensitivity, reducing approximately 30% GLUT4 mRNA and protein (P<0.05) and plasma membrane expression in muscle; (2) the expression of ERalpha and ERbeta in L6 myotubes, showing that both coexpress in the same nucleus; (3) effects of E2 on GLUT4 in L6, showing a time- and dose-dependent response. High concentration (100nM) for 6 days reduced approximately 25% GLUT4 mRNA and protein (P<0.05). Concluding, E2 regulates GLUT4 in muscle, and at high concentrations, such as in pregnancy, reduces GLUT4 expression and, in vivo, decreases insulin sensitivity. Thus, hyperestrogenism may be involved in the pregnancy-induced insulin resistance and/or gestational diabetes.

Estrogen Receptor-Beta Regulates Epithelial Cell Differentiation in the Mouse Ventral Prostate

The epithelium of the ventral prostate expresses high levels of ERβ (but no ERα). ERα is expressed in the stroma. Thus, in addition to the indirect effects of estrogen on the epithelium which are reported to be mediated by ERα, there is a direct estrogenic influence on prostatic epithelium mediated by ERβ. We have reported that loss of ERβ results in epithelial hypercellularity in the ventral prostate. In ERβ–/– mouse prostates, there is overexpression of the androgen receptor and of the antiapoptotic factor Bcl-2 in the prostate. Bcl-2 is an estrogen-regulated gene. It is normally expressed only in the basal cells in the prostate. This apparent expansion of the ‘stem cell-like population’ in the ERβ–/– mouse prostate has been further examined. We found a higher expression of cytokeratin 5 in ERβ–/– mouse prostates so that the ratio of cytokeratin 5 to that of 19 is much higher in ERβ–/– than in wild-type littermates. In addition, labeling of DNA with BrdU showed a 3.5-fold higher proliferation rate in ERβ–/– mouse prostate. Despite these clear differences, the piling up of epithelial cells never progressed to high-grade PIN-like lesions. Hyperplastic foci in ERβ–/– mice show accumulation of cells without signs of atypia, resembling low-grade PIN in humans. The reason for this appeared to be a high rate of cellular detachment and subsequent fall off into the lumen in ERβ–/– mice. The fall off phenomenon is possibly related to the finding that the expression of the cell adhesion molecule E-cadherin was very reduced. We conclude that in ERβ–/– mouse prostates, the epithelial cell population contains more epithelial cells in the intermediate stage of differentiation, possessing both the ability to proliferate as the basal cells and the ability to secrete as the highly differentiated luminal epithelium.

Estrogen receptor β-deficient female mice develop a bladder phenotype resembling human interstitial cystitis

Interstitial cystitis/painful bladder syndrome is a disease seen mostly in women, and symptoms tend to be worse premenopausally or during ovulation. The four cardinal symptoms of interstitial cystitis/painful bladder syndrome are bladder pain, urgency, frequency, and nocturia. Estrogen has been implicated in the etiology of this disease, but the role of the two estrogen receptors (ER), ERα and ERβ, has not been investigated. We found that, in the bladders of WT mice, ERβ is expressed in the basal cell layer of the urothelium. Bladders of male ERβ−/− mice were intact and morphologically indistinguishable from those of their WT littermates. However, in female ERβ−/− mice, there was ulceration and atrophy of bladder urothelium concomitant with infiltration of γδ T cells concentrated in the areas of atrophy and shedding of urothelium. The data support the idea that activated γδ T cells are causing the damage to the urothelium. The hyperactivity of T cells may be because of an imbalance between ERα and ERβ signaling in female ERβ−/− mice. Our data suggest that reduced ERβ signaling might have a role in the pathogenesis of interstitial cystitis, and ERβ could be a candidate for a target of medical therapy.

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.