In the Penis, RhoA Activation is Enhanced by Guanine Nucleotide Exchange Factors

描述(由申请人提供)： 项目总结 美国有3000多万男性患有勃起功能障碍。阴茎血管的收缩和扩张决定了阴茎的勃起。在没有觉醒刺激的情况下，异三聚体G蛋白在配体与膜受体结合后被激活，维持海绵体小动脉和窦的收缩，保持阴茎不勃起。在异源三聚体G蛋白激活后，两条信号通路被发挥作用以引起收缩：已被很好地描述的钙依赖通路(磷脂酶C)和最近发现的被称为钙敏化的RhoA/Rho-Kinase通路。G蛋白受体与磷脂酶C活化的偶联已有很好的描述，但关于RhoA/Rho-Kinase如何偶联G蛋白活化的研究却少之又少。最近的研究表明，RhoG核苷酸交换因子(RhoGEF)正向调节RhoA的激活。对罗氏全球环境基金活动的监管也不清楚。有趣的是，一种特殊的Rhogef，PDZ-Rhogef受富含脯氨酸的酪氨酸激酶2(PYK2)的调控，PYK2是一种依赖于钙的酶。我们已经获得了初步数据支持PYK2在血管对血管紧张素II的反应中的作用。我们假设血管紧张素II激活PYK2和PDZ-Rhogef导致RhoA/Rho-Kinase增加以维持非勃起状态。此外，PYK2和PDZ-Rhogef的长期过度表达会导致勃起功能障碍。这些假说将受到3个特定目标的验证：1)证明PYK2/PDZ-RhoGlobal激活可导致血管收缩和阴茎松弛；1)确定PYK2在阴茎中的表达是否可以受到调控以改变勃起功能，以及PDZ-RhoGlobal的siRNA是否会抑制RhoA/Rho-Kinase信号通路的组成部分；2)确定PYK2/PDZ-Rhogef活性增强是否有助于血管紧张素II诱导的高血压患者的勃起功能障碍；以及3)确定PYK2/PDZ-Rhogef活性增强是否与糖尿病患者的勃起功能障碍有关。该方法将利用大鼠和小鼠的勃起模型。这些实验将确定在完整的阴茎和分离的海绵体条中，PYK2/Rhogef活性在刺激RhoA/Rho-Kinase途径方面的生化、药理学和生理学指标。过度表达PYK2的基因转移将导致一种新的勃起功能障碍模型，而PDZ-Rhogef的siRNA将减少通过RhoA/Rho-Kinase途径的信号转导。对分离的海绵体条带(完整的和通透性的)的收缩力的测量将为PYK2/PDZ-Rhogef对钙的敏化及其调节提供证据。糖尿病和高血压引起的勃起功能障碍将被用来评估这一信号通路可能的临床意义。PYK2(-/-)小鼠为研究PYK2/PDZ-Rhogef信号通路提供了重要的途径。如果得到支持，这些研究将为正常状态下平滑肌收缩的分子基础以及阴茎的长期变化如何导致勃起功能障碍提供解释。这项工作将为新的治疗方法提供洞察力。 项目叙事 美国有3000多万男性患有勃起功能障碍。尽管阴茎勃起是由促进海绵体血管收缩和扩张的细胞信号机制决定的，但目前的治疗方法侧重于刺激扩张反应。我们的研究发现了调节收缩反应的新的细胞事件，了解这些事件如何被抑制将为治疗勃起功能障碍提供另一种治疗方法。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Over 30 million men suffer from erectile dysfunction in the U.S. Constriction and dilation of the cavernosal vasculature determines penile erection. In the absence of arousal stimuli, activation of heterotrimeric G proteins following ligand binding to membrane receptors maintains constriction of the cavernosal arterioles and sinuses, keeping the penis non-erect. Subsequent to heterotrimeric G protein activation, two signaling pathways are brought into play to cause constriction: the well-characterized Ca2+-dependent pathway (phospholipase C) and the recently identified RhoA/Rho-kinase pathway known as Ca2+ sensitization. The coupling of G protein receptors to phospholipase C activation is well characterized, but much less is known about how RhoA/Rho-kinase is coupled to G protein activation. Recent evidence indicates that the RhoA activation is positively regulated by Rho guanine nucleotide exchange factors (RhoGEFs). The regulation of RhoGEF activity is also unclear. Interestingly, one particular RhoGEF, PDZ-RhoGEF is regulated by a proline- rich tyrosine kinase 2 (PYK2), a Ca2+-dependent enzyme. We have obtained preliminary data to support a role for PYK2 in vascular responses to angiotensin II. We hypothesize that angiotensin II activation of PYK2 and PDZ-RhoGEF leads to increased RhoA/Rho-kinase to maintain the non-erect state. Further, -term over- expression of PYK2 and PDZ-RhoGEF leads to erectile dysfunction. These hypotheses will be tested by 3 specific aims: 1A) to demonstrate that activation of PYK2/PDZ-RhoGEF leads to vasoconstriction and penile flaccidity; 1B) to determine if PYK2 expression in the penis can be modulated to alter erectile function and if siRNA for PDZ-RhoGEF will inhibit components of the RhoA/Rho-kinase signaling pathway; 2) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in angiotensin II-induced hypertension; and 3) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in diabetes. The approach will utilize rat and mouse models of erection. The experiments will determine the biochemical, pharmacological and physiological measures of PYK2/RhoGEF activity with respect to stimulation of the RhoA/Rho-kinase pathway in the intact penis and in isolated cavernosal strips. Gene transfer to over- express PYK2 should lead to a new model of erectile dysfunction whereas; siRNA for PDZ-RhoGEF will reduce signaling via the RhoA/Rho-kinase pathway. Contractile force measurements in isolated cavernosal strips (intact and permeabilized) will provide evidence for Ca2+ sensitization and its regulation by PYK2/PDZ- RhoGEF. Erectile dysfunction induced by diabetes and hypertension will be used to evaluate a possible clinical significance of this signaling pathway. PYK2 (-/-) mice will provide an important approach to studying the PYK2/PDZ-RhoGEF signaling pathway. If supported these studies would provide an explanation for the molecular basis for smooth muscle contraction in the normal state and how long-term changes in the penis contribute to erectile dysfunction. The work will provide insight into novel therapeutic approaches. PROJECT NARRATIVE Over 30 million men suffer from erectile dysfunction in the U.S. Whereas, penile erection is determined by cell signaling mechanisms promoting both constriction and dilation of the cavernosal vasculature, current therapeutic approaches focus on stimulating the dilatory response. Our research has identified novel cellular events regulating constrictor responses, and an understanding of how these events can be inhibited will provide an alternative therapeutic approach to treating erectile dysfunction.