Actions of Estrogen on Uterine Artery Endothelium

描述(申请人提供)：这项续期拨款的假设是，在与质膜上的雌激素受体(即Era和ER(3))结合后，雌激素迅速激活受体亚型特有的细胞内信号通路(即G蛋白)，导致内皮型一氧化氮合酶(ENOS)和细胞外信号调节激酶(ERK2/1)的激活。激活的ERK2/1易位刺激核内的AP-1(Jun/Fos二聚体)转录因子与ERA和ERP相互作用，通过雌激素相互调节eNOS和小窝蛋白-1的表达。利用明确的绵羊子宫动脉内皮细胞培养模型和人脐静脉内皮细胞培养模型，将研究五个特定的目标。目的：探讨雌激素受体信号转导事件(G蛋白激活)以及ERa和ERb在G蛋白激活和雌激素刺激下eNOS-NO产生及ERK2/1通路中的不同作用。目的：探讨膜、核Era和ERb是否共同参与雌激素对内皮型一氧化氮合酶和小窝蛋白-1mRNA和蛋白表达的相互调节。目的：探讨c-jun/jun-B AP-1在雌激素调节eNOS和小窝蛋白-1表达中的作用。目的：探讨雌激素刺激内皮细胞eNOS表达的转录机制(S)，即通过ERA激活eNOS启动子以及ERb与AP-1的相互作用。目的：探讨雌激素下调血管内皮细胞小窝蛋白-1表达的机制，即通过ERA/Erb与AP-1相互作用和DNA甲基化来调节小窝蛋白-1启动子的表达。这些研究在生物学上具有至关重要的意义，因为它们是首次专门设计用于全面了解膜ER启动的信号，通过AP-1与经典的核ER相互作用，调节eNOS和aveoln-1基因的激活，与类固醇受体和内皮生物学特有的雌激素产生内皮NO有关。这些研究具有重要的临床意义，因为怀孕期间雌激素的显著生物合成和子宫血流的增加对于母亲和胎儿之间的双向交换是必不可少的，而血液供应不足会导致早期胚胎丧失、宫内生长受限、先兆子痫、胎儿宫内程序化、成人疾病和新生儿出生体重降低，而这反过来又与围产期/新生儿发病率/死亡率和母亲的心血管健康状况呈负相关。这些研究有助于揭示雌激素对心血管的保护作用。

DESCRIPTION (provided by applicant): The hypothesis of this renewal grant is that, on binding to estrogen receptors (i.e., ERa and ER(3) on the plasma membranes, estrogen rapidly activates receptor subtype-specific intracellular signaling pathways (i.e., G-proteins) leading to endothelial nitric oxide (NO) synthase (eNOS) and extracellular signal-regulated kinases (ERK2/1) activation. Translocation of activated ERK2/1 stimulates the AP-1 (Jun/Fos dimers) transcription factors in the nucleus where they interact with ERa and ERp to reciprocally regulate eNOS and caveolin-1 expression by estrogen. Five specific aims will be studied by using the well-defined sheep uterine artery endothelial cell culture model and human umbilical cord vein endothelial cells. Aim 1: to determine the proximal membrane ER signaling events (G-protein activation) and if ERa and ERb play different roles in G-protein activation and eNOS-NO production and ERK2/1 pathway on estrogen stimulation. Aim 2: to determine if membrane and nuclear ERa and ERb are both involved in the reciprocal regulation of eNOS and caveolin-1 mRNA and protein expression by estrogen. Aim 3: to determine the role of c-Jun/Jun-B AP-1 in regulating eNOS and caveolin-1 expression by estrogen. Aim 4: to determine the transcriptional mechanism(s) underlying estrogen stimulation of endothelial eNOS expression, i.e., activation of eNOS promoter via ERa and ERb interaction with AP-1. Aim 5: to determine the mechanisms underlying down-regulation of endothelial caveolin-1 expression by estrogen, i.e., regulation of caveolin-1 promoter via ERa/ERb interaction with AP-1 and DNA methylation. These studies are of critical importance biologically because they are the first designed specifically for a comprehensive understanding of membrane ER initiated signaling cross-talk with classical nuclear ER via AP-1 to the regulation of eNOS and caveoln-1 gene activation in relation to endothelial NO production by estrogen unique to steroid receptor and endothelial biology. These studies are clinically important as dramatic estrogen biosynthesis and rise in uterine blood flow during pregnancy are essential for the bi-exchange between the mother and fetus and insufficient blood supply causes early embryonic loss, intrauterine growth restriction, preeclampsia, in utero fetal programming of adult diseases and reduced neonatal birthweight, which in turn inversely correlate to perinatal/neonatal morbidity/mortality and cardiovascular well-being of the mother. These studies are relevant to uncover the dilemma of the cardiovascular protective effects of estrogen.