SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.

Protein Phosphatase 2A Activation Via ApoER2 in Trophoblasts Drives Preeclampsia in a Mouse Model of the Antiphospholipid Syndrome.

[Figure: see text].

Supplementation With the Sialic Acid Precursor N-Acetyl-D-Mannosamine Breaks the Link Between Obesity and Hypertension.

BACKGROUND: Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. METHODS: Involvement of IgG was studied using IgG µ heavy chain-null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell-specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). RESULTS: Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow-fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. CONCLUSIONS: Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.

Antiphospholipid antibodies induce thrombosis by PP2A activation via apoER2-Dab2-SHC1 complex formation in endothelium.

In the antiphospholipid syndrome (APS), antiphospholipid antibody (aPL) recognition of ß2 glycoprotein I promotes thrombosis, and preclinical studies indicate that this is due to endothelial nitric oxide synthase (eNOS) antagonism via apolipoprotein E receptor 2 (apoER2)-dependent processes. How apoER2 molecularly links these events is unknown. Here, we show that, in endothelial cells, the apoER2 cytoplasmic tail serves as a scaffold for aPL-induced assembly and activation of the heterotrimeric protein phosphatase 2A (PP2A). Disabled-2 (Dab2) recruitment to the apoER2 NPXY motif promotes the activating L309 methylation of the PP2A catalytic subunit by leucine methyl transferase-1. Concurrently, Src homology domain-containing transforming protein 1 (SHC1) recruits the PP2A scaffolding subunit to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits that mediate inhibitory dephosphorylation of Akt and eNOS. In mice, the coupling of these processes in endothelium is demonstrated to underlie aPL-invoked thrombosis. By elucidating these intricacies in the pathogenesis of APS-related thrombosis, numerous potential new therapeutic targets have been identified.

Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice.

Steroid hormone receptors including estrogen receptors (ER) classically function as ligand-regulated transcription factors. However, estrogens also elicit cellular effects through binding to extra-nuclear ER (ERα, ERß, and G protein-coupled ER or GPER) that are coupled to kinases. How extra-nuclear ER actions impact cardiac ischemia-reperfusion (I/R) injury is unknown. We treated ovariectomized wild-type female mice with estradiol or an estrogen-dendrimer conjugate (EDC), which selectively activates extra-nuclear ER, or vehicle interventions for two weeks. I/R injury was then evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol significantly decreased infarct size and improved post-ischemic contractile function. Similarly, EDC treatment significantly decreased infarct size and increased post-ischemic functional recovery compared to vehicle-treated hearts. EDC also caused an increase in myocardial protein S-nitrosylation, consistent with previous studies showing a role for this post-translational modification in cardioprotection. In further support of a role for S-nitrosylation, inhibition of nitric oxide synthase, but not soluble guanylyl cyclase blocked the EDC mediated protection. The administration of ICI182,780, which is an agonist of G-protein coupled estrogen receptor (GPER) and an antagonist of ERα and ERß, did not result in protection; however, ICI182,780 significantly blocked EDC-mediated cardioprotection, indicating participation of ERα and/or ERß. In studies determining the specific ER subtype and cellular target involved, EDC decreased infarct size and improved functional recovery in mice lacking ERα in cardiomyocytes. In contrast, protection was lost in mice deficient in endothelial cell ERα. Thus, extra-nuclear ERα activation in endothelium reduces cardiac I/R injury in mice, and this likely entails increased protein S-nitrosylation. Since EDC does not stimulate uterine growth, in the clinical setting EDC-like compounds may provide myocardial protection without undesired uterotrophic and cancer-promoting effects.

Bazedoxifene and conjugated estrogen prevent diet-induced obesity, hepatic steatosis, and type 2 diabetes in mice without impacting the reproductive tract.

Despite the capacity of estrogens to favorably regulate body composition and glucose homeostasis, their use to combat obesity and type 2 diabetes is not feasible, because they promote sex steroid-responsive cancers. The novel selective estrogen receptor modulator (SERM) bazedoxifene acetate (BZA) uniquely antagonizes both breast cancer development and estrogen-related changes in the female reproductive tract. How BZA administered with conjugated estrogen (CE) or alone impacts metabolism is unknown. The effects of BZA or CE + BZA on body composition and glucose homeostasis were determined in ovariectomized female mice fed a Western diet for 10-12 wk. In contrast to vehicle, estradiol (E2), CE, BZA, and CE + BZA equally prevented body weight gain by 50%. In parallel, all treatments caused equal attenuation of the increase in body fat mass invoked by the diet as well as the increases in subcutaneous and visceral white adipose tissue. Diet-induced hepatic steatosis was attenuated by E2 or CE, and BZA alone or with CE provided even greater steatosis prevention; all interventions improved pyruvate tolerance tests. Glucose tolerance tests and HOMA-IR were improved by E2, CE, and CE + BZA. Whereas E2 or CE alone invoked a uterotrophic response, BZA alone or CE + BZA had negligible impact on the uterus. Thus, CE + BZA affords protection from diet-induced adiposity, hepatic steatosis, and insulin resistance with minimal impact on the female reproductive tract in mice. These combined agents may provide a valuable new means to favorably regulate body composition and glucose homeostasis and combat fatty liver.

Coupling of Fcγ receptor I to Fcγ receptor IIb by SRC kinase mediates C-reactive protein impairment of endothelial function.

RATIONALE: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fcγ receptor IIB (FcγRIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5'-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. OBJECTIVE: How CRP activates FcγRIIB in endothelium is not known. We determined the role of Fcγ receptor I (FcγRI) and the basis for coupling of FcγRI to FcγRIIB in endothelium. METHODS AND RESULTS: In cultured endothelial cells, FcγRI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via FcγRI. CRP-induced increases in FcγRIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5'-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the γ subunit of FcγRI (FcRγ(-/-)) or FcγRIIB(-/-) mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcRγ(-/-); TG-CRP and FcγRIIB(-/-); TG-CRP mice. CONCLUSIONS: CRP antagonism of eNOS is mediated by the coupling of FcγRI to FcγRIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5'-phosphatase 1, and consistent with this mechanism, both FcγRI and FcγRIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. FcγRI and FcγRIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.

Endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle.

The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.

Non-nuclear estrogen receptor signaling in the endothelium.

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.

C-reactive protein inhibits insulin activation of endothelial nitric oxide synthase via the immunoreceptor tyrosine-based inhibition motif of FcgammaRIIB and SHIP-1.

Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcgammaRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcgammaRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain-containing inositol 5'-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcgammaRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity.

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.

Estrogen modulation of endothelial nitric oxide synthase.

Over the past decade, clinical and basic research has demonstrated that estrogen has a dramatic impact on the response to vascular injury and the development of atherosclerosis. Further work has indicated that this is at least partially mediated by an enhancement in nitric oxide (NO) production by the endothelial isoform of NO synthase (eNOS) due to increases in both eNOS expression and level of activation. The effects on eNOS abundance are primarily mediated at the level of gene transcription, and they are dependent on estrogen receptors (ERs), which classically serve as transcription factors, but they are independent of estrogen response element action. Estrogen also has potent nongenomic effects on eNOS activity mediated by a subpopulation of ERalpha localized to caveolae in endothelial cells, where they are coupled to eNOS in a functional signaling module. These observations, which emphasize dependence on cell surface-associated receptors, provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae. Estrogen binding to ERalpha on the SRFC in caveolae leads to G(alphai) activation, which mediates downstream events. The downstream signaling includes activation of tyrosine kinase-MAPK and Akt/protein kinase B signaling, stimulation of heat shock protein 90 binding to eNOS, and perturbation of the local calcium environment, leading to eNOS phosphorylation and calmodulin-mediated eNOS stimulation. These unique genomic and nongenomic processes are critical to the vasoprotective and atheroprotective characteristics of estrogen. In addition, they serve as excellent paradigms for further elucidation of novel mechanisms of steroid hormone action.

High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I.

Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-I(-/-) mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI(-/-) mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.

Direct interactions with G α i and G ßγ mediate nongenomic signaling by estrogen receptor α .

Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor alpha (ER alpha). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ER alpha binds directly to G alpha i and G betagamma. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251-260 and 271-595 of ER alpha, respectively. Studies of ER alpha complexed with heterotrimeric G proteins further show that estradiol causes the release of both G alpha i and G betagamma without stimulating GTP binding to G alpha i. Moreover, in COS-7 cells, the disruption of ER alpha-G alpha i interaction by deletion mutagenesis of ER alpha or expression of blocking peptide, as well as G betagamma sequestration with beta-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ER alpha-G alpha i interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ER alpha mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.

Selective Nonnuclear Estrogen Receptor Activation Decreases Stroke Severity and Promotes Functional Recovery in Female Mice.

Estrogens provide neuroprotection in animal models of stroke, but uterotrophic effects and cancer risk limit translation. Classic estrogen receptors (ERs) serve as transcription factors, whereas nonnuclear ERs govern numerous cell processes and exert beneficial cardiometabolic effects without uterine or breast cancer growth in mice. Here, we determined how nonnuclear ER stimulation with pathway-preferential estrogen (PaPE)-1 affects stroke outcome in mice. Ovariectomized female mice received vehicle, estradiol (E2), or PaPE-1 before and after transient middle cerebral artery occlusion (tMCAo). Lesion severity was assessed with MRI, and poststroke motor function was evaluated through 2 weeks after tMCAo. Circulating, spleen, and brain leukocyte subpopulations were quantified 3 days after tMCAo by flow cytometry, and neurogenesis and angiogenesis were evaluated histologically 2 weeks after tMCAo. Compared with vehicle, E2 and PaPE-1 reduced infarct volumes at 3 days after tMCAo, though only PaPE-1 reduced leukocyte infiltration into the ischemic brain. Unlike E2, PaPE-1 had no uterotrophic effect. Both interventions had negligible effect on long-term poststroke neuronal or vascular plasticity. All mice displayed a decline in motor performance at 2 days after tMCAo, and vehicle-treated mice did not improve thereafter. In contrast, E2 and PaPE-1 treatment afforded functional recovery at 6 days after tMCAo and beyond. Thus, the selective activation of nonnuclear ER by PaPE-1 decreased stroke severity and improved functional recovery in mice without undesirable uterotrophic effects. The beneficial effects of PaPE-1 are also associated with attenuated neuroinflammation in the brain. PaPE-1 and similar molecules may warrant consideration as efficacious ER modulators providing neuroprotection without detrimental effects on the uterus or cancer risk.

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial FcγRIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via FcγRIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of FcγRIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial FcγRIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.

Molecular basis of estrogen-induced cyclooxygenase type 1 upregulation in endothelial cells.

Estrogen upregulates cyclooxygenase-1 (COX-1) expression in endothelial cells. To determine the basis of this process, studies were performed in ovine endothelial cells transfected with the human COX-1 promoter fused to luciferase. Estradiol (E2) caused activation of the COX-1 promoter with maximal stimulation at 10(-8) mol/L E2, and the response was mediated by either ERalpha or ERbeta. Mutagenesis revealed a primary role for a putative Sp1 binding motif at -89 (relative to the ATG codon) and lesser involvement of a consensus Sp1 site at -111. Electrophoretic mobility shift assays yielded a single complex with the site at -89, and supershift analyses implicated AP-2alpha and ERalpha, and not Sp1, in protein-DNA complex formation. In endothelial cells with minimal endogenous ER, the transfection of ERalpha mutants lacking the DNA binding domain or primary nuclear localization signals caused 4-fold greater stimulation of promoter activity with E2 than wild-type ERalpha. In contrast, mutant ERalpha lacking the A-B domains was inactive. Thus, estrogen-mediated upregulation of COX-1 in endothelium is uniquely independent of direct ERalpha-DNA binding and instead entails protein-DNA interaction involving AP-2alpha and ERalpha at a proximal regulatory element. In addition, the process may be initiated by cytoplasmic ERalpha, and critical receptor elements reside within the amino terminus.

Estrogen causes dynamic alterations in endothelial estrogen receptor expression.

Estrogen receptor (ER)alpha mediates many of the effects of estrogen on the vascular endothelium. The purpose of the present study was to determine whether estrogen modifies endothelial ERalpha expression. In experiments in cultured ovine endothelial cells, physiological concentrations of 17beta-estradiol (E2, 10(-10) to 10(-8) mol/L) caused an increase in ERalpha protein abundance that was evident after 6 hours of hormone exposure. Shorter (2-hour) E2 treatment caused ERalpha downregulation. In contrast to the upregulation in ERalpha after long-term E2, the expression of the other ER isoform, ERbeta, was downregulated. Both nonselective ER antagonism with ICI 182,780 and the inhibition of gene transcription with actinomycin D blocked the increase in ERalpha with E2. In studies using the human ERalpha gene promoter P-1 coupled to luciferase, an increase in ERalpha gene transcription was evident in endothelial cells within 4 hours of E2 exposure. The transcriptional activation was fully blocked by ICI 182,780, whereas the specific ERbeta antagonist RR-tetrahydrochrysene yielded partial blockade. Overexpression of ERalpha or ERbeta caused comparable 10- and 8-fold increases, respectively, in ERalpha promoter activation by E2. Thus, long-term exposure to E2 upregulates ERalpha expression in endothelial cells through the actions of either ERalpha or ERbeta on ERalpha gene transcription; in contrast, E2 causes ERbeta downregulation in the endothelium. We postulate that E2-induced changes in ERalpha and ERbeta expression modify the effects of the hormone on vascular endothelium.

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.