Participation of ERalpha and ERbeta 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.

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.

Metabolic actions of estrogen receptor beta (ERbeta) are mediated by a negative cross-talk with PPARgamma.

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

Estrogen receptor beta-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), ERalpha and ERbeta, has not been investigated. We found that, in the bladders of WT mice, ERbeta is expressed in the basal cell layer of the urothelium. Bladders of male ERbeta(-/-) mice were intact and morphologically indistinguishable from those of their WT littermates. However, in female ERbeta(-/-) mice, there was ulceration and atrophy of bladder urothelium concomitant with infiltration of gammadelta T cells concentrated in the areas of atrophy and shedding of urothelium. The data support the idea that activated gammadelta T cells are causing the damage to the urothelium. The hyperactivity of T cells may be because of an imbalance between ERalpha and ERbeta signaling in female ERbeta(-/-) mice. Our data suggest that reduced ERbeta signaling might have a role in the pathogenesis of interstitial cystitis, and ERbeta could be a candidate for a target of medical therapy.

Ovarian wedge resection restores fertility in estrogen receptor beta knockout (ERbeta-/-) mice.

Ovulation rarely occurs in mice in which the estrogen receptor beta (ERbeta) gene has been inactivated (ERbeta-/- mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ERbeta-/- mice. We transplanted ERbeta-/- ovaries into WT mice and WT ovaries into ERbeta-/- mice. Upon mating with ERbeta-/- males, fertility increased from 20% in control intact ERbeta-/- group to 40% in the WT recipients with ERbeta-/- 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 ERbeta-/- mice. In ERbeta-/- 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 alpha (PDGFRalpha) and a decrease in PDGFbeta 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 ERbeta-/- 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.

Estrogen receptor beta 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 ERalpha and ERbeta. ERalpha mediates the proliferative effect of estrogen in breast cancer cells, whereas ERbeta seems to be antiproliferative. We engineered ERalpha-positive T47D breast cancer cells to express ERbeta 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 ERbeta. The presence of ERbeta resulted in a reduction in tumor growth. Comparison of the ERbeta-expressing and non-ERbeta-expressing tumors revealed that the expression of ERbeta 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 beta (PDGFbeta). In cell culture, with the Tet-Off-regulated ERbeta-expressing cells, expression of ERbeta decreased expression of VEGF and PDGFbeta mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFbeta proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFbeta promoter constructs revealed a repressive effect of ERbeta at the promoter level of these genes. Taken together, these data show that introduction of ERbeta into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis.

Role of estrogen receptor beta in uterine stroma and epithelium: Insights from estrogen receptor beta-/- mice.

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

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice.

Estrogen receptor beta (ERbeta) is highly expressed in both type I and II pneumocytes as well as bronchiolar epithelial cells. ERalpha is not detectable in the adult lung. Lungs of adult female ERbeta knockout (ERbeta-/-) mice have already been reported to have fewer alveoli and reduced elastic recoil. In this article, we report that, by 5 months of age, there are large areas of unexpanded alveoli in lungs of both male and female ERbeta-/- mice. There is increased staining for collagen and, by EM, abnormal clusters of collagen fibers are seen in the alveolar septa of ERbeta-/- mice. Immunohistochemical analysis and Western blotting with lung membrane fractions of ERbeta-/- mice revealed down-regulation of caveolin-1, increased expression of membrane type-1 metalloproteinase, matrix metalloproteinase 2 (active form), and tissue inhibitors of metalloproteinases 2. Hypoxia, measured by immunohistochemical analysis for hypoxia-inducible factor 1alpha and chemical adducts (with Hypoxyprobe), was evident in the heart, ventral prostate, periovarian sac, kidney, liver, and brain of ERbeta-/- mice under resting conditions. Furthermore, both male and female adult ERbeta-/- mice were reluctant to run on a treadmill and tissue hypoxia became very pronounced after exercise. We conclude that ERbeta is necessary for the maintenance of the extracellular matrix composition in the lung and loss of ERbeta leads to abnormal lung structure and systemic hypoxia. Systemic hypoxia may be responsible for the reported left and right heart ventricular hypertrophy and systemic hypertension in ERbeta-/- mice.

Estrogen receptor beta regulates epithelial cellular differentiation in the mouse ventral prostate.

We have previously reported epithelial cellular hyperplasia in ventral prostates (VP) of mice lacking estrogen receptor beta (ER beta). To investigate the causes of this phenomenon, we measured cellular proliferation and apoptosis in VP of ER beta(-/-) and WT mice. With BrdUrd labeling, the number of proliferating cells was 3.6-fold higher in ER beta(-/-) mice. There was also a decrease in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and an increase in expression of the anti-apoptotic bcl-2. The state of differentiation of the epithelial cells of the VP was studied by immunohistochemical staining, Western blotting, and fluorescence-activated cell sorting (FACS). In ER beta(-/-) mouse VP, the number of p63-positive cells (basal phenotype) was 2.6-fold higher, and expression level of cytokeratin (CK) 8, a luminal cell marker, was lower. FACS analysis with p63 showed that in WT mice the ratio of basal to intermediate/luminal cell populations expressing p63 was 1:2.5, whereas in ER beta(-/-) mice it was 1:9. The expression of basal/intermediate marker CK 19 in three FACS areas, g1, g2, and g3, gated according to cellular size and granularity, was 1:0.6:2 in WT and 1:4:6.7 in ER beta(-/-) mice, showing a shift of CK 19-positive cells toward a cell population of intermediate size and granularity. We conclude that, in ER beta(-/-) mouse VP, there is increased epithelial proliferation, decreased apoptosis, and accumulation of incompletely differentiated cells in an intermediate pool. The continued proliferation of intermediate cells leads to the prostatic epithelial hyperplasia observed in the absence of ER beta signaling.