Regulation of Endothelial Cell Isometric Tension

内皮细胞等长张力的调节

• 批准号: 6436380
• 负责人: Robert B. Wysolmerski
• 金额: $ 33.21万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6436380
• 关键词: actins; calcium flux; cell morphology; cell motility; cytoskeleton; enzyme activity; fibroblasts; guanosinetriphosphatases; laboratory mouse; laboratory rabbit; myosin light chain kinase; myosins; phosphorylation; protein isoforms; protein kinase; tissue /cell culture; vascular endothelium permeability; vasomotion

The overall aim of the proposed research is to define the events that initiate and regulate endothelial cell contraction in an effort to elucidate the basis of increased vascular permeability. The primary hypothesis underlying the proposed work dictates that exposure to inflammatory mediators activates the endothelial cell actomyosin based contractile system. This hypothesis states that phosphorylation of myosin II in concert with assembly of the filamentous cytoskeleton (actin, myosin IIA and IIB) is essential for activation of endothelial cell contractile activity. Calcium (Ca2+) stimulated phosphorylation of the myosin II regulatory light chain (RLC) by myosin light chain kinase (MLCK) and Ca2+ independent phosphorylation of myosin II RLC by p-21 activated kinase (PAK2) have been shown to activate endothelial cell contraction. In nonmuscle cells, MLCK-catalyzed phosphorylation results in mono- and diphosphorylation of RLC at site Ser19 or sites Ser19 and Thr18, respectively (36). Within the last 10 years additional pathways of myosin II activation have been discovered and are currently being defined (5,13,19,20,35,48,). In endothelial cells, we have shown that the small GTPase dependent enzyme, PAK2 catalyzes Ca2+ independent activation of nonmuscle myosin II by phosphorylation of the RLC that is restricted to site Ser19 (19). This PAK2-mediated monophosphorylation results in a less forceful contractile response than when the RLC is diphosphorylated by MLCK. More recent studies have shown that PAK2 also phosphorylates unactivated MLCK which results in inhibition of MLCK activation by Ca2+ (35). Collectively, these findings have clearly established a role for PAK2 in mediating and regulating myosin II activity, and thus contractile activity, in endothelial cells. The goal of the proposed studies is to biochemically and morphologically characterize myosin II activation by Ca2+ - dependent and Ca2+ -independent signaling pathways in endothelial cells. The working hypothesis is that specific pools of myosin II, determined by the heavy chain isoform specificity (IIA vs IIB), are activated by enzyme specific (MLCK210, PAK2, Rho- kinase) modifications (monophosphorylation or diphosphorylation). Functionally, regulated differential phosphorylation of myosin II would allow the endothelial cell to react to a variety of physiological signals with graded contractile responses.

拟议研究的总体目的是定义启动和调节内皮细胞收缩的事件，以阐明血管渗透性增加的基础。 拟议工作的基本假设表明，暴露于炎症介质会激活基于内皮细胞肌动蛋白的收缩系统。 该假设指出，肌球蛋白II的磷酸化与丝状细胞骨架（肌动蛋白，肌球蛋白IIA和IIB）的组装一致，对于激活内皮细胞收缩活性至关重要。 钙（Ca2+）通过肌球蛋白光链激酶（MLCK）（MLCK）和Ca2+独立的肌球蛋白II RLC刺激肌球蛋白II调节光链（RLC），P-21活化激酶（PAK2）已表明肌球蛋白II RLC（PAK2）已表明可以激活内皮细胞构造。 在非肌肉细胞中，MLCK催化的磷酸化分别在SER19或SER19或SER19和THR18的位置上导致RLC的单磷酸化和双磷酸化（36）。 在过去的十年中，已经发现了肌球蛋白II激活的其他途径，目前正在定义（5,13,​​19,20,35,48）。 在内皮细胞中，我们已经表明，较小的GTPase依赖性酶，Pak2通过磷酸化的RLC磷酸化限于位点Ser19（19）（19）。该PAK2介导的单磷酸化导致的收缩反应较小，而RLC被MLCK二次磷酸化时。 最近的研究表明，PAK2还磷酸化了未激活的MLCK，从而导致Ca2+抑制MLCK激活（35）。 总的来说，这些发现清楚地确定了PAK2在内皮细胞中介导和调节肌球蛋白II活性，从而调节收缩活性的作用。拟议的研究的目的是在生化和形态学上表征肌球蛋白II通过Ca2+ - 依赖性和CA2+非依赖性信号通路的激活。 工作假设是，由重链同工型特异性（IIA VS IIB）确定的肌球蛋白II的特定池被酶特异性（MLCK210，PAK2，Rho-kinase）修饰（单磷酸化或双磷酸化）激活。在功能上，肌球蛋白II的调节差磷酸化将使内皮细胞对各种具有分级收缩反应的生理信号反应。