Effect of sex hormones on renal estrogen and angiotensin type 1 receptors in female and male rats

Although the mechanisms are not understood, evidence suggests that 17beta-estradiol (E2) confers protection from cardiovascular and renal complications in many diseases. We have reported that E2 decreases angiotensin type 1 receptors (AT1Rs) in different tissues and hypothesize that E2 exerts tonic inhibition on AT1Rs, reducing effects of ANG II. This study determined the effects of E2 and dihydrotestosterone (DHT) on cortical estrogen receptors (ERs) and glomerular AT1R binding in rats. Animals underwent sham operation, ovariectomy (Ovx) or orchidectomy (Cas) and were treated (Ovx +/- E2; Cas +/- DHT) for 3 wk. Cortical ERalpha protein was 2.5 times greater, and ERbeta was 80% less in females vs. males (P < 0.01). Glomerular AT1R binding was lower in females than males [4,657 +/- 838 vs. 7,457 +/- 467 counts per minute (cpm), P < 0.01]. Ovx reduced ERalpha protein by 50%, whereas E2 increased ERalpha expression after Ovx. The decrease in cortical ERalpha in Ovx rats was associated with a significant increase in AT1R binding (6,908 +/- 609 cpm), and E2 prevented this increase. There was no change in ERalpha or AT1R binding following Cas +/- DHT (25 mg) treatment, although Cas did elevate cortical ERbeta (P < 0.01). Interestingly, the high dose DHT (200 mg) elevated ERalpha 150% above intact levels and profoundly decreased AT1R binding (1,824 +/- 705 cpm, P < 0.001 vs. intact male). This indicates that under normal conditions, glomerular AT1R binding is significantly greater in male than female animals, which may be important in development of cardiovascular and renal disease in males. Furthermore, E2 regulates ERalpha and is inversely associated with glomerular AT1R binding, supporting our hypothesis that E2 tonically suppresses AT1Rs and suggesting a potential mechanism for the protective effects of estrogen.     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.     

Activities of a non-classical estrogen,Z-bis-dehydrodoisynolic acid,with ERalpha and ERbeta

(+/-)-Z-bis-Dehydrodoisynolic acid [(+/-)-Z-BDDA] is highly estrogenic in vivo, yet binds to estrogen receptor (ER) poorly. This paradox has raised the possibility of alternative ERs and/or molecular mechanisms. To address the possibility of high activities of Z-BDDA with ERbeta, we determined the activities of (+)-Z-BDDA and (-)-Z-BDDA, in cell culture and in vitro, comparing ERbeta to ERalpha. Transfectional analysis in Hela cells showed (-)-Z-BDDA is an agonist for gene activation with both ERalpha (EC(50) congruent with 0.3nM) and ERbeta (EC(50) congruent with 5nM), while little to no activity was observed with (+)-Z-BDDA. Similarly, in gene repression assays, (-)-Z-BDDA was active (EC(50) congruent with 0.2nM), but again minimal activity was exhibited by (+)-Z-BDDA. Binding to ERalpha and ERbeta in vitro used both competition and a direct binding assay. For ERalpha, the relative affinity of (-)-Z-BDDA was approximately 6% by competition and 1.7% by direct binding versus 17beta-estradiol (E2; 100%), while (+)-Z-BDDA also demonstrated binding, but with relative affinities of only 0.08% by competition and 0.3% by the direct assay. For ERbeta, the affinity of (-)-Z-BDDA was approximately 7% by competition and 1.5% by the direct assay relative to E2 (100%), while (+)-Z-BDDA had lower affinity, approximately 0.2% that of E2 by both assays.The paradox of potent in vivo activity but lower activity in receptor binding and in cell culture reporter gene assays, previously seen with ERalpha is now also associated with ERbeta. The failure of ERbeta to explain the activity-binding paradox indicates the need for additional in vivo metabolic and pharmacokinetic studies and continued consideration of alternative mechanisms.     

In vitro fluorescence anisotropy analysis of the interaction of full-length SRC1a with estrogen receptors alpha and beta supports an active displacement model for coregulator utilization

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry).

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.     

白细胞介素2对大鼠垂体前叶细胞雌激素受体蛋白含量和基因表达的影响

采用原代无血清细胞培养技术结合免疫组织（细胞）化学和半定量反转录－ＰＣＲ方法,观察白细胞介素２（ＩＬ－２）对大鼠垂体前叶雌激素受体（ＥＲ）蛋白含量和基因表达的影响,以探讨ＩＬ－２和ＥＲ在大鼠重体前叶的相互关系。结果显示：在大鼠垂体前叶细胞有ＩＬ－２受体表达。在无血清培养条件下,ＩＬ－２能增加ＥＲα蛋白含量,促进ＥＲα基因表达,而对ＥＲβ的作用正好相反,ｒｈＩＬ－２（１０μｇ／Ｌ）作用４８ｈ后,ＥＲ     

Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.