ESTROGEN RECEPTOR REGULATION OF NO SYNTHASES

正如本SCOR应用的介绍中所述，现在认识到雌激素通过两种主要方式直接影响脉管系统：雌激素引起血管快速舒张，雌激素激活血管细胞的基因表达和长期变化。雌激素的快速和长期作用被认为对激素的心血管保护作用很重要。该SCOR项目4基于大量未发表的初步数据，并探索了与整体SCOR假设直接相关的特定假设：雌激素受体介导一氧化氮合酶（NOS）酶的快速（非基因组）激活和长期（基因组）表达，为缺血性心血管疾病及其后遗症提供保护。在Specific Aim 1中，基于新的数据，研究了雌激素快速（非基因组）激活内皮细胞NOS的机制，这些数据表明雌激素在内皮细胞中激活eNOS是由雌激素受体erα的一种全新功能介导的，而erα是一种迄今为止被认为仅作为转录因子发挥作用的蛋白质。内皮细胞和COS细胞研究了erα介导eNOS快速激活的信号事件(a) erα结构域和残基介导eNOS被雌激素快速激活的结构-功能；(b) erα通过MAP激酶途径介导eNOS活化；(c) Src激酶参与介导erα刺激eNOS。在Specific Aim 2中，通过研究雌激素介导的血管iNOS基因表达的增加，探讨了雌激素对血管损伤的长期、基因组作用。这些研究基于广泛的新数据，这些数据有力地支持ERbeta诱导VSMC iNOS对雌激素的血管保护作用至关重要。这一假设在Specific Aim 2中得到了验证，通过研究(a) ERbeta对WT、ERalpha KO和ERbeta KO小鼠培养VSMC中iNOS基因表达的调控；(b) WT、ERalphaKO、ERbetaKO和iNOS KO小鼠血管环内皮剥离后的血管舒缩；(c)雌激素对iNOS - KO小鼠血管损伤反应的影响。通过研究雌激素受体在雌激素对血管eNOS和iNOS的快速、非基因组和长期基因组效应中的作用，项目4反映了该SCOR提案的总体假设，并直接补充了SCOR项目1、2、3和5中提出的研究。

As described in the Introduction to this SCOR application, it is now recognized that estrogen directly affects the vasculature in two principal ways: estrogen causes rapid vasodilation, and estrogen activates gene expression and longer-term changes in blood vessel cells. Both the rapid and the longer-term effects of estrogen are thought to be important to the cardiovascular protective effects of the hormone. Project 4 of this SCOR is based on extensive unpublished preliminary data and explores a specific hypothesis related directly to the overall SCOR hypothesis: estrogen receptors mediate both rapid (non-genomic) activation and longer-term (genomic) expression of nitric oxide synthase (NOS) enzymes, affording protection against ischemic cardiovascular diseases and their sequelae. In Specific Aim 1, the mechanism of rapid (non- genomic) endothelial NOS activation by estrogen is explored, based on new data demonstrating that eNOS activation by estrogen in endothelial cells is mediated by an entirely novel function of the estrogen receptor ERalpha, a protein thought until now to function solely as a transcription factor. Signaling events mediating the rapid activation of eNOS by ERalpha are explored in endothelial and COS cell studies of (a) structure- function of ERalpha domains and residues mediating rapid activation of eNOS by estrogen; (b) ERalpha-mediated activation of eNOS via the MAP kinase pathway; and (c) Involvement of Src kinase in mediating ERalpha stimulation of eNOS. In Specific Aim 2, the longer-term, genomic actions of estrogen to protect against vascular injury are explored by studies of estrogen-mediated increases in vascular iNOS gene expression. These studies are based on extensive new data that strongly support that ERbeta induction of VSMC iNOS is critical to estrogen's vascular protective effects. This hypothesis is tested in Specific Aim 2 with studies of (a) ERbeta regulation of iNOS gene expression in cultured VSMC from WT, ERalpha KO and ERbeta KO mice; (b) vasomotion in endothelium-denuded mouse vascular rings from WT, ERalphaKO, ERbetaKO and iNOS KO mice; and (c) Estrogen effects on the vascular injury response to iNOS KO mice. By examining the role of estrogen receptors in both the rapid, non-genomic and longer-term, genomic effects of estrogen on vascular eNOS and iNOS, respectively, Project 4 reflects the overall hypothesis for this SCOR proposal and directly complements studies proposed in SCOR Projects 1, 2, 3 and 5.