Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells

Sex steroid hormone receptors play a central role in modulating telomerase activity, especially in cancer cells. However, information on the regulation of steroid hormone receptors and their distinct functions on telomerase activity within the mesenchymal stem cell are largely unavailable due to low telomerase activity in the cell. In this study, the effects of estrogen (E2) treatment and function of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) on telomerase activity were investigated in human mesenchymal stem cells (hMSCs). Telomerase activity and mRNA expression of the catalytic subunit of telomerase (hTERT) were upregulated by treatment of the cells with E2. The protein concentration of ERalpha was also increased by E2 treatment, and enhancement of ERalpha accumulation in the nucleus was clearly detected with immunocytochemistry. When ERalpha expression was reduced by siRNA transfection into hMSCs, the effect of E2 on the induction of hTERT expression and telomerase activity was diminished. In contrast, the transient overexpression of ERalpha increased the effect of E2 on the expression of hTERT mRNA. These findings indicate that the activation of hTERT expression and telomerase activity by E2 in hMSCs depends on ERalpha, but not on ERbeta.     

Estrogen receptor-alpha mediates estrogen facilitation of baroreflex heart rate responses in conscious mice

Estrogen facilitates baroreflex heart rate responses evoked by intravenous infusion of ANG II and phenylephrine (PE) in ovariectomized female mice. The present study aims to identify the estrogen receptor subtype involved in mediating these effects of estrogen. Baroreflex responses to PE, ANG II, and sodium nitroprusside (SNP) were tested in intact and ovariectomized estrogen receptor-alpha knockout (ERalphaKO) with (OvxE+) or without (OvxE-) estrogen replacement. Wild-type (WT) females homozygous for the ERalpha(+/+) were used as controls. Basal mean arterial pressures (MAP) and heart rates were comparable in all the groups except the ERalphaKO-OvxE+ mice. This group had significantly smaller resting MAP, suggesting an effect of estrogen on resting vascular tone possibly mediated by the ERbeta subtype. Unlike the WT females, estrogen did not facilitate baroreflex heart rate responses to either PE or ANG II in the ERalphaKO-OvxE+ mice. The slope of the line relating baroreflex heart rate decreases with increases in MAP evoked by PE was comparable in ERalphaKO-OvxE- (-6.97 +/- 1.4 beats.min(-1).mmHg(-1)) and ERalphaKO-OvxE+ (-6.18 +/- 1.3) mice. Likewise, the slope of the baroreflex bradycardic responses to ANG II was similar in ERalphaKO-OvxE- (-3.87 +/- 0.5) and ERalphaKO-OvxE+(-2.60 +/- 0.5) females. Data suggest that estrogen facilitation of baroreflex responses to PE and ANG II is predominantly mediated by ERalpha subtype. A second important observation in the present study is that the slope of ANG II-induced baroreflex bradycardia is significantly blunted compared with PE in the intact as well as the ERalphaKO-OvxE+ females. We have previously reported that this ANG II-mediated blunting of cardiac baroreflexes is observed only in WT males and not in ovariectomized WT females independent of their estrogen replacement status. The present data suggest that in females lacking ERalpha, ANG II causes blunting of cardiac baroreflexes similar to males and may be indicative of a direct modulatory effect of the ERalpha on those central mechanisms involved in ANG II-induced resetting of cardiac baroreflexes. These observations suggest an important role for ERalpha subtype in the central modulation of baroreflex responses. Lastly, estrogen did not significantly affect reflex tachycardic responses to SNP in both WT and ERalphaKO mice.     

Estrogen receptor-alpha mediates estrogen protection from angiotensin II-induced hypertension in conscious female mice

It has been shown that the female sex hormones have a protective role in the development of angiotensin II (ANG II)-induced hypertension. The present study tested the hypotheses that 1) the estrogen receptor-alpha (ERalpha) is involved in the protective effects of estrogen against ANG II-induced hypertension and 2) central ERs are involved. Blood pressure (BP) was measured in female mice with the use of telemetry implants. ANG II (800 ng.kg(-1).min(-1)) was administered subcutaneously via an osmotic pump. Baseline BP in the intact, ovariectomized (OVX) wild-type (WT) and ERalpha knockout (ERalphaKO) mice was similar; however, the increase in BP induced by ANG II was greater in OVX WT (23.0 +/- 1.0 mmHg) and ERalphaKO mice (23.8 +/- 2.5 mmHg) than in intact WT mice (10.1 +/- 4.5 mmHg). In OVX WT mice, central infusion of 17beta-estradiol (E(2); 30 microg.kg(-1).day(-1)) attenuated the pressor effect of ANG II (7.0 +/- 0.4 mmHg), and this protective effect of E(2) was prevented by coadministration of ICI-182,780 (ICI; 1.5 microg.kg(-1).day(-1), 18.8 +/- 1.5 mmHg), a nonselective ER antagonist. Furthermore, central, but not peripheral, infusions of ICI augmented the pressor effects of ANG II in intact WT mice (17.8 +/- 4.2 mmHg). In contrast, the pressor effect of ANG II was unchanged in either central E(2)-treated OVX ERalphaKO mice (19.0 +/- 1.1 mmHg) or central ICI-treated intact ERalphaKO mice (19.6 +/- 1.6 mmHg). Lastly, ganglionic blockade on day 7 after ANG II infusions resulted in a greater reduction in BP in OVX WT, central ER antagonist-treated intact WT, central E(2) + ICI-treated OVX WT, ERalphaKO, and central E(2)- or ICI-treated ERalphaKO mice compared with that in intact WT mice given just ANG II. Together, these data indicate that ERalpha, especially central expression of the ER, mediates the protective effects of estrogen against ANG II-induced hypertension.     

P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium

Stimulation of p21-activated kinase-1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant-negative fragment or by short interference RNA markedly reduced the estrogen receptor-alpha (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the beta-lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen-activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function-2 domain of the ER at the N-terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1-mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1-ER pathway in promoting hyperplasia in mammary epithelium.     

Role of estrogen receptor-alpha in pharmacogenetics of estrogen action

PURPOSE OF REVIEW: To summarize recent data regarding the role of estrogen receptor-alpha polymorphisms in determining the response to estrogen therapy or the risk of clinical cardiovascular events. RECENT FINDINGS: Recent clinical trials of hormone replacement therapy for cardiovascular disease have yielded surprisingly negative results, shifting clinical opinions from a position of presumed cardiovascular benefit to one of confirmed harm. Understanding why hormone replacement therapy has beneficial effects on intermediate risk markers for cardiovascular disease, but produces an increase in cardiovascular events, is an important public health question with the potential to elucidate fundamentally important aspects on atherogenesis, cardiovascular disease, and the biology of estrogen action. One question concerning the cardiovascular effects of hormone replacement therapy is whether genetic factors can substantially modify individual responses to estrogen treatment. New clinical trial evidence is emerging that links the presence of particular variants in the estrogen receptor to the response of HDL and other intermediate endpoints to hormone replacement therapy. SUMMARY: One or more common variants in estrogen receptor-alpha are associated with a differential response to hormone replacement therapy in several domains of estrogen action. However, the effect of these variants on the risk of clinical cardiovascular events in the setting of hormone replacement therapy is not yet known. Additional research focusing on the clinical impact of common variants in estrogen receptor-alpha, estrogen receptor-beta and the progesterone receptor promise to improve clinical decision-making concerning the use of hormone replacement therapy and other novel estrogen agonists.     

Glycosylation of the murine estrogen receptor-alpha

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421-2428), we demonstrated that a subpopulation of the murine estrogen receptor-alpha (mER-alpha) is modified by O-GlcNAc at Thr(575). Here we mutated mER-alpha to convert Thr(575) and Ser(576) to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-alpha, at Ser(10) and Thr(50) near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-alpha turnover.     

The dynamic and elusive membrane estrogen receptor-alpha

Many studies have demonstrated the nuclear forms of steroid receptors and their activities, while fewer investigators have identified and described the membrane forms of these receptors. Our immuno-identification approaches for the qualitative and quantitative comparison of the membrane form of the estrogen receptor-alpha (mER alpha) to its nuclear counterpart now allow us to address questions about the comparative levels and regulation of these receptor forms. ER alpha-specific antisense oligonucleotides eliminate mER alpha expression, while only mildly reducing the nuclear ER alpha. Success of immuno-identification for the mER alpha is very sensitive to different fixation protocols, affecting cell permeability (and thus distinction from the intracellular form) and differential epitope preservation. All such identifications must be accompanied by proof of cell membrane integrity and focal plane assessments. The mER alpha expression on selected cells declines rapidly with cell passage number and cell density. Expression of mER alpha is enhanced by serum starvation and selection for specific phases of the cell cycle. The hinge region of the protein is sensitive to ligand-induced epitope masking and to antibody-induced changes in receptor-mediated responses. Responsive cells are often diluted within cell populations by loss of the membrane receptor form. The bimodality of the rapid estrogen action, with inhibitory doses between picomolar and nanomolar stimulatory concentrations, requires detailed dose-response curves. Finally, responsive cells can be lost from assays, as upon estrogen treatment they rapidly round up and leave the substrates to which they are attached. These regulatory phenomena demonstrate that levels of the membrane form of the estrogen receptor are very dynamic.     

Ligand-selective interdomain conformations of estrogen receptor-alpha

Selective estrogen receptor modulators (SERMs) inhibit estrogen activation of the estrogen receptor (ER) in some tissues but activate ER in other tissues. These tissue-selective actions suggest that SERMs may be identified with tissue specificities that would improve the safety of breast cancer and hormone replacement therapies. The identification of an improved SERM would be aided by understanding the effects of each SERM on the structure and interactions of ER. To date, the inability to obtain structures of the full-length ER has limited our structural characterization of SERM action to their antiestrogenic effects on the isolated ER ligand binding domain. We studied the effects of estradiol and the clinically useful SERMs 4-hydroxytamoxifen and fulvestrant on the conformation of the full-length ERalpha dimer complex by comparing, in living human breast cancer cells, the amounts of energy transfer between fluorophores attached to different domains of ERalpha. Estradiol, 4-hydroxytamoxifen, and fulvestrant all promoted the rapid formation of ERalpha dimers with equivalent interaction kinetics. The amino- and carboxyl-terminal ERalpha domains both contain activation functions differentially affected by these ligands, but the positions of only the carboxyl termini differed upon binding with estradiol, 4-hydroxytamoxifen, or fulvestrant. The association of a specific ERalpha dimer conformation with the binding of ligands of different clinical effect will assist the identification of a SERM with optimal tissue-selective estrogenic and antiestrogenic activities. These studies also provide a roadmap for dissecting important structural and kinetic details for any protein complex from the quantitative analysis of energy transfer.     

Extracellular matrix mediates epithelial effects on chondrogenesis in vitro

It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.     

Estrogen receptor-alpha predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Although 17-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)- or ER- and which signaling pathways are involved in such 17-estradiol effects. Utilizing ER-- or ER--specific agonists, this study examined the role of ER- and ER- in 17-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF- production and activation of MAPK and NF-B were measured. Macrophage IL-6 and TNF- production and MAPK activation were decreased, whereas NF-B activity was increased, following trauma-hemorrhage. PPT or 17-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER- plays the predominant role in mediating the salutary effects of 17-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-B; propyl pyrazole triol; diarylpropionitrile     

cGMP-dependent ADP depolymerization of actin mediates estrogen increase in cervical epithelial permeability

Estrogen increasessecretion of cervical mucus in women, and the effect depends onfragmentation of the cytoskeleton. The objective of the present studywas to understand the molecular mechanism of estrogen action. Treatmentof human cervical epithelial cells with 17-estradiol, sodiumnitroprusside (SNP), or 8-bromoguanosine 3',5'-cyclic monophosphate(8-Br-cGMP) increased cellular monomeric G-actin and decreasedpolymerized F-actin. The effects of estradiol were blocked bytamoxifen, by the guanylate cyclase inhibitor LY-83583, and by thecGMP-dependent protein kinase inhibitor KT-5823. The effects of SNPwere blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMPwere blocked only by KT-5823. Treatment with phalloidin decreasedparacellular permeability and G-actin. Treatment with 17-estradiol,SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of[³²P]adenylate NAD in vitro: tamoxifen blocked the effectof estrogen; LY-83583 blocked the effect of SNP but not that of8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP.These results indicate that estrogen, nitric oxide (NO), and cGMPstimulate actin depolymerization. A possible mechanism is NO-induced,cGMP-dependent protein kinase augmentation of ADP-ribosylation ofmonomeric actin.

     

JNK mediates TGF-beta1-induced epithelial mesenchymal transdifferentiation of mouse transformed keratinocytes

In this study we analyzed the role of the c-Jun N-terminal kinases (JNK) pathway in the TGF-beta1 stimulation of urokinase-type plasminogen activator (uPA), initial stages of epithelial-mesenchymal transdifferentiation (EMT) and cell migration. TGF-beta1 induces JNK phosphorylation, c-Jun transactivation and AP1 activation. The involvement of JNK was evaluated using dominant negative mutants SEK-1 AL, JNK and cJun, depletion of JNK1,2 proteins by treatment of cells with antisense oligonucleotides, as well as the chemical inhibitor SP600125. Our results demonstrated that the JNK pathway is required in the TGF-beta1 enhancement of uPA, fibronectin, E-cadherin delocalization, actin re-organization and vimentin expression, concomitant with the induction of cell migration. These results allow us to suggest a role of JNK in the TGF-beta1 induction of EMT in relation with the stimulation of malignant properties of mouse transformed keratinocytes.