Role of PI3-Kinase in Estrogen-induced eNOS Activation

描述（由申请人提供）：与绝经前女性相比，绝经后和卵巢切除女性患心血管疾病的风险更高。 这种风险的降低归因于雌激素对绝经前妇女的有益作用和绝经后妇女的雌激素替代疗法。 因此，了解雌激素如何预防心血管疾病是一个重要的问题，不仅在女性，而且在男性。 雌激素是一种强有力的血管扩张剂，以往的研究表明，雌激素的血管扩张作用是由内皮源性一氧化氮（NO）介导的。 NO抑制血小板聚集、血管平滑肌增殖和白细胞粘附于血管壁。 事实上，最近的研究表明，一些雌激素的心血管保护作用是通过其对内皮型一氧化氮合酶（eNOS）的刺激作用介导的。 因此，了解雌激素如何激活eNOS可能会为雌激素的一些快速分子作用提供重要的新见解。 我们最近报道雌激素通过磷脂酰肌醇3-激酶（PI 3 K）/蛋白激酶B（Akt）途径刺激eNOS活性。 有趣的是，我们发现雌激素受体（ER）亚型，ER α，与PI 3 K的p85 α调节亚基以配体依赖性方式相互作用。 基于这些初步的发现，我们现在建议确定ER α的亚细胞定位的共聚焦显微镜;共定位ER α和p85 α使用荧光共振能量转移（FRET）;并确定推定的相互作用域（S）的ER α和p85 α。 然后，我们建议通过“敲入”突变的ER α或p85 α来研究这种相互作用的生理意义，这些突变的ER α或p85 α不能相互作用，然后确定这些小鼠中雌激素对eNOS的激活是否受损。 该途径的相关性将在血管损伤和缺血性中风模型中进一步测试，其中雌激素和NO已被证明具有保护作用。 这些小鼠的产生也可能有助于研究雌激素在其他非血管组织（如骨）中的“核”与“非核”效应。 所提出的研究的意义在于，通过将ER与PI 3 K联系起来，提示了雌激素的非核作用中的潜在关键步骤，并且由于已知PI 3 K介导多种细胞功能，因此ER的作用大大拓宽。 这些结果可以解释选择性雌激素受体调节剂（SERM）的某些作用，并为雌激素在心血管疾病中的应用提供了治疗依据。

DESCRIPTION (provided by applicant): Post-menopausal and ovariectomized women have a higher risk for cardiovascular disease compared to pre-menopausal women. This decreased risk has been attributed to the beneficial effects of estrogen in premenopausal women and estrogen replacement therapy in post-menopausal woman. Therefore, understanding how estrogen protects against cardiovascular disease is an important problem, not only in women but also in men. Estrogen is a potent vasodilator, and previous studies indicate that estrogen's vasodilatory effect is mediated by endothelium-derived nitric oxide (NO). NO inhibits platelet aggregation, vascular smooth muscle proliferation, and leukocyte adhesion to the vascular wall. Indeed, recent studies suggest that some of estrogen's cardiovascular protective effects are mediated by its stimulatory effects on endothelial nitric oxide synthase (eNOS). Therefore, understanding how estrogen activates eNOS may provide important new insights into some of the rapid molecular actions of estrogen. We have recently reported that estrogen stimulates eNOS activity via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Interestingly, we found that the estrogen receptor (ER) isoform, ERalpha, interacts with the p85alpha regulatory subunit of PI3K in a ligand-dependent manner. Based upon these initial findings, we now propose to determine the subcellular localization of ERa by confocal microscopy; colocalize ERa and p85a using fluorescent resonance energy transfer (FRET); and to identify the putative interaction domain(s) of ERalpha and p85alpha. We then propose to study the physiological significance of this interaction by "knocking in" the mutated ERalpha or p85alpha, which cannot interact with each other, and then to determine whether eNOS activation by estrogen is impaired in these mice. The relevance of this pathway will be further tested in models of vascular injury and ischemic stroke where estrogen and NO have been shown to be protective. The generation of these mice may also be useful in studying the "nuclear" versus "non-nuclear" effects of estrogen in other non-vascular tissues such as bone. The significance of the proposed studies is that by linking the ER to PI3K, a potential critical step in the non-nuclear action of estrogen is suggested and the role of ER is considerably broadened since PI3K is known to mediate diverse cellular functions. These results may explain some of the actions of selective estrogen receptor modulators (SERMs) and provide a therapeutic basis for using estrogens in cardiovascular disease.