Actions of Estrogen on Uterine Artery Endothelium

描述（由申请人提供）：本更新资助的假设是，在与质膜上的雌激素受体（即ERa和ER(3)）结合时，雌激素迅速激活受体亚型特异性细胞内信号通路（即g蛋白），导致内皮型一氧化氮（NO）合成酶（eNOS）和细胞外信号调节激酶（ERK2/1）激活。活化的ERK2/1易位刺激细胞核内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caveolin-1的表达。利用定义明确的绵羊子宫动脉内皮细胞培养模型和人脐带静脉内皮细胞，研究五个具体目标。目的1：确定近端膜内质网信号事件（g蛋白激活），以及ERa和ERb在雌激素刺激下g蛋白激活、eNOS-NO生成和ERK2/1通路中是否有不同的作用。目的2：确定膜核ERa和ERb是否都参与雌激素对eNOS和caveolin-1 mRNA和蛋白表达的相互调节。目的3：探讨c-Jun/Jun-B AP-1在雌激素调节eNOS和caveolin-1表达中的作用。目的4：确定雌激素刺激内皮细胞eNOS表达的转录机制，即通过ERa和ERb与AP-1相互作用激活eNOS启动子。目的5：确定雌激素下调内皮细胞caveolin-1表达的机制，即通过ERa/ERb与AP-1相互作用和DNA甲基化调控caveolin-1启动子。这些研究在生物学上是至关重要的，因为它们是第一个专门为全面了解膜内质网启动的信号通过AP-1与经典核内质网相互作用，调节eNOS和caveoln-1基因激活与内皮细胞一氧化氮产生有关而设计的研究。这些研究具有重要的临床意义，因为妊娠期间雌激素的生物合成和子宫血流量的增加对母亲和胎儿之间的双向交换至关重要，而血液供应不足会导致早期胚胎丢失、宫内生长受限、先兆子痫、子宫内胎儿规划的成人疾病和新生儿出生体重减少，而这些反过来又与围产期/新生儿发病率/死亡率和母亲的心血管健康呈负相关。这些研究有助于揭示雌激素对心血管保护作用的困境。

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.