Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.     

Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERalpha domains with known nuclear functions

Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) alpha. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERalpha upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERalpha domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17beta (E2) caused PM-associated ERalpha to form dimers. However, eNOS activation by E2 was unaltered for a dimerization-deficient mutant ERalpha (ERalphaL511R). In contrast, ERalpha mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERalphaDelta250-274) or the DNA binding domain (ERalphaDelta185-251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERalpha interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E2 activation of Src or erk, but ligand-induced PI3 kinase-ERalpha binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERalpha coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERalpha and PI3 kinase.     

Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells

Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.     

Rapid activation of endothelial NO synthase by estrogen: evidence for a steroid receptor fast-action complex (SRFC) in caveolae

Estrogen has important atheroprotective and vasoactive properties related to its capacity to stimulate nitric oxide (NO) production by endothelial NO synthase. Previous work has shown that these effects are mediated by estrogen receptor (ER) alpha functioning in a nongenomic manner via calcium-dependent, MAP kinase-dependent mechanisms. Recent studies have demonstrated that estradiol (E(2)) activates eNOS in isolated endothelial plasma membranes in the absence of added calcium, calmodulin or eNOS cofactors. Studies of blockade by ICI 182,780 and by ER alpha antibody, and also immunoidentification experiments indicate that the process is mediated by a subpopulation of plasma membrane-associated ER alpha. Fractionation of endothelial cell plasma membranes has further revealed that ER alpha protein is localized to caveolae, and that E(2) causes stimulation of eNOS in isolated caveolae which is ER-dependent and calcium-dependent, whereas noncaveolae membranes are insensitive. Furthermore, in intact endothelial cells the activation of eNOS by E(2) is prevented by pertussis toxin, and exogenous GDP beta S inhibits the response in isolated plasma membranes. Coimmunoprecipitation studies have shown that E(2) exposure causes interaction between ER alpha and G(alpha i) on the plasma membrane, and eNOS activation by E(2) is enhanced by overexpression of G(alpha i) and attenuated by expression of a protein regulator of G protein signaling (RGS), RGS4. Thus, a subpopulation of ER alpha is localized to caveolae in endothelial cells, where they are coupled via G(alpha i) to eNOS in a functional signaling module. Emphasizing the dependence on cell surface-associated receptors, these observations provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

8-Prenylnaringenin, inhibits estrogen receptor-alpha mediated cell growth and induces apoptosis in MCF-7 breast cancer cells

The discovery that the hop constituent 8-prenylnaringenin (8PN) shows potent estrogenic activity, higher than that of the known phytoestrogens coumestrol, genistein and daidzein, has spurred an intense activity aimed at elucidating its biological profile and its dietary relevance connected with the consumption of beer. We have investigated if 8PN can induce signal transduction pathways via rapid estrogen receptor (ER) activation. Under conditions of estrogen-dependent growth, treatment of MCF-7 human breast cancer cells with 8PN induced a rapid and transient activation of the MAP kinase Erk-1 and Erk-2, with kinetics similar to those induced by 17beta-estradiol (E2). 8PN could trigger the MAP kinase pathway via dual c-Src kinase activation and association with ERalpha. Co-treatment with the ER antagonist ICI 182,780 blocked each step of this transduction pathway, confirming its ER dependence. However, and in striking contrast with E2, 8PN could not induce the PI3K/Akt pathway, resulting in altered kinetics and levels of cyclin D1 expression. In accordance with these observations, flow cytometric and biochemical analysis showed that 8PN inhibited cell cycle progression and induced apoptosis in MCF-7 cells. Interference with an ER associated PI3K pathway is proposed as a possible mechanism underlying the inhibition of survival and proliferation of estrogen responsive cells by 8PN. Taken together, our finding show that 8PN is an interesting new chemotype to explore the biology of ERs.     

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.     

Expression of endothelial NO synthase, inducible NO synthase, and estrogen receptors alpha and beta in placental tissue of normal, preeclamptic, and intrauterine growth-restricted pregnancies.

In the physiology of placental blood circulation, nitric oxide (NO) synthases seem to play important roles, although their expression in pathological placentas and their role is still unclear. In addition, NO synthase activation seems to be related to estrogen receptor expression. Therefore, the aims of this study were to investigate the expression of estrogen receptors alpha (ERalpha), estrogen receptor beta (ER and the endothelial NO synthase (eNOS), and inducible NO synthase (iNOS) in intrauterine growth-restricted (IUGR) placentas, preeclamptic placentas, and in normal healthy control placentas. Slides of paraffin-embedded placental tissue were obtained after delivery from patients diagnosed with IUGR, preeclampsia, and normal term placentas and analyzed for eNOS, iNOS as well as ERalpha and ERbeta expression. Intensity of immunohistochemical reaction was analyzed using a semiquantitative score and statistical analysis was performed. In addition, Western blot experiments were performed for comparison of staining intensities obtained by immunohistochemistry and western blot. Expression of eNOS, iNOS, and ERbeta is significantly reduced in trophoblast cells of placentas with IUGR. However, preeclamptic placentas demonstrated a significant elevated expression intensity of these proteins compared with normal controls. A different expression of eNOS, iNOS, ERalpha, and ERbeta by human trophoblast cells seems to results in lower NO output and impaired trophoblast invasion. Results obtained in our study provide evidence that reduced expression of the investigated proteins is related to IUGR.     

Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release

Estradiol (E(2)) causes endothelium-dependent vasodilation, mediated, in part, by enhanced nitric oxide (NO) release. We have previously shown that E(2)-induced activation of endothelial nitric oxide synthase (eNOS) reduces its calcium dependence. This pathway of eNOS activation is unique to a limited number of stimuli, including shear stress, the response to which is herbimycin-inhibitable. Consistent with this, herbimycin and geldanamycin pretreatment of human umbilical vein endothelial cells (HUVEC) abrogated E(2)-stimulated NO release and cGMP production, respectively. These benzoquinone ansamycins are potent inhibitors of Hsp90 function, which has recently been shown to play a role in stimulus-dependent eNOS activation. As in response to shear, E(2) induced an Hsp90-eNOS association, peaking at 30 min and completely inhibited by the conventional estrogen receptor antagonist ICI 182,780. These findings suggest that Hsp90 plays an important role in the rapid, estrogen receptor-mediated modulation of eNOS activation by estrogen.     

The isoflavone Equol mediates rapid vascular relaxation: Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells

We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nM) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nM, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER)alpha and ERbeta. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.     

Proximal events in signaling by plasma membrane estrogen receptors

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.     

Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta

Estrogen has been shown to affect vascular cell and arterial function in vitro and in vivo. Here we examined the ability of estradiol (E(2)) to cause rapid arterial dilation of elastic and muscular arteries in vivo and the mechanisms involved. E(2) administration caused a rapid increase in the outer wall diameter of both types of arteries in ovariectomized female mice. This resulted from estrogen receptor (ER)-mediated stimulation of nitric oxide production, demonstrated by preinjecting the mice arteries with a soluble inhibitor of nitric oxide (monomethyl l-arginine) and by showing the absence of E(2) action in eNOS-/- mice. Rapid activation of both ERK/MAP kinase and phosphatidylinositol 3-kinase activity was found in the E(2)-exposed arteries, and inhibiting either kinase prevented the vasodilatory action of E(2). Kinase activation and vasodilator responses to E(2) were absent in either ERalpha or ERbeta knock-out mice, implicating both receptor subtypes as mediating this E(2) action. These results indicate that E(2) modulation of arterial tonus through plasma membrane ER and rapid signaling could underlie many previously observed actions of estrogen reported to occur in women.     

Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase

Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC). Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-(32)P]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro (32)P phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.