Actions of Estrogen on Uterine Artery Endothelium

描述（由申请人提供）：该更新授权的假设是，在与雌激素受体结合时（即，ER α和ER（3）在质膜上，雌激素迅速激活受体亚型特异性细胞内信号传导途径（即，G蛋白），导致内皮一氧化氮（NO）合酶（eNOS）和细胞外信号调节激酶（ERK 2/1）激活。活化的ERK 2/1的易位刺激细胞核中的AP-1（Jun/Fos二聚体）转录因子，在那里它们与ER α和ER β相互作用以通过雌激素间接调节eNOS和小窝蛋白-1的表达。本研究将利用羊子宫动脉内皮细胞培养模型和人脐静脉内皮细胞进行五个方面的研究。目标1：以确定近端膜ER信号传导事件（G蛋白活化）以及ER α和ER b在G蛋白活化和eNOS-NO产生以及雌激素刺激的ERK 2/1通路中是否发挥不同的作用。目标二：以确定是否膜和核ERa和ERb都参与雌激素对eNOS和小窝蛋白-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eNOS表达的转录机制，即，通过ER a和ER b与AP-1的相互作用激活eNOS启动子。目的5：确定雌激素下调内皮细胞小窝蛋白-1表达的机制，即，通过ERa/ERb与AP-1的相互作用和DNA甲基化调控小窝蛋白-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设计用于全面理解膜ER启动的信号转导串扰与经典的核ER通过AP-1调节eNOS和cavebase-1基因激活与内皮NO产生有关的雌激素独特的类固醇受体和内皮生物学。这些研究在临床上是重要的，因为妊娠期间雌激素的急剧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，并且血液供应不足导致早期胚胎丢失、子宫内生长受限、先兆子痫、子宫内胎儿编程成人疾病和新生儿出生体重降低，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呈负相关。这些研究有助于揭示雌激素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.