Role of PI3-Kinase in Estrogen-induced eNOS Activation

描述（由申请人提供）：绝经后和切除卵巢的妇女与绝经前妇女相比，患心血管疾病的风险更高。这种降低的风险归因于雌激素对绝经前妇女和绝经后妇女雌激素替代疗法的有益作用。因此，了解雌激素如何预防心血管疾病是一个重要的问题，不仅对女性，对男性也是如此。雌激素是一种有效的血管扩张剂，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血管壁。事实上，最近的研究表明，雌激素的一些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。我们最近报道了雌激素通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B （Akt）通路刺激eNOS活性。有趣的是，我们发现雌激素受体（ER）异构体erα与PI3K的p85alpha调节亚基以配体依赖的方式相互作用。基于这些初步发现，我们现在建议用共聚焦显微镜确定ERa的亚细胞定位；使用荧光共振能量转移（FRET）共定位ERa和p85a；并确定ERalpha和p85alpha的推定相互作用域。然后，我们提出通过敲入突变的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，然后确定雌激素是否会损害这些小鼠的eNOS激活。这一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PI3K联系起来，提示了雌激素非核作用的一个潜在关键步骤，并且由于已知PI3K介导多种细胞功能，因此ER的作用大大扩大。这些结果可能解释选择性雌激素受体调节剂（SERMs）的一些作用，并为使用雌激素治疗心血管疾病提供治疗依据。

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.