Adherens Junction Integrity and Barrier Function

描述(申请人提供)：血管内皮细胞控制流量 从血管间隙到间质的大分子。理解 维持和加强内皮屏障的机制将有助于 阐明对抗屏障丧失的治疗干预措施 活跃性和组织水肿。1-磷酸鞘氨醇(S1P)和 CAMP增强剂增加屏障功能。建议的机制 加强屏障包括增加细胞-细胞或细胞-基质的粘附力， 和/或收缩活动减少。增加的屏障活性 S1P不依赖于cAMP和蛋白激酶A和G，不受 肌球蛋白轻链(MLC)激酶和细胞外的抑制 信号调节的激酶。此外，S1P和cAMP对MLC有相反的作用 磷酸化、细胞张力和肌动蛋白应激纤维。我们建议， S1P和cAMP的屏障增强活性是针对细胞间的 交汇点。另外，S1P和cAMP可能通过不同的机制发挥作用。 初步数据表明，S1P增加了纽蛋白的定位，并 α-连环蛋白/肌动蛋白连接蛋白，在细胞-细胞接触，并增加 α-连环蛋白与肌动蛋白的结合。初步数据显示，S1P 减少IQGAP1的外周本地化，后者与 与β-连环蛋白结合的α-连环蛋白，增加rac1的活性，以及 增加IQGAP1与rac1的相互作用。这些发现表明，S1P 通过增强粘结剂的结合点完整性来增加屏障活性。 初步数据还表明，S1P和cAMP逆转了屏障的减少 肿瘤坏死因子(TNF)的作用。我们认为粘附结是 S1P和CAMP的共同目标。这些调节剂通过以下方式增强屏障功能 通过添加连接蛋白来加强粘连连接，例如 纽蛋白和活化的rac1通过去除IQGAP1。三个具体目标 它们是：S1P和cAMP 1)通过增强细胞的黏附而增强屏障功能 α-连环蛋白通过连接蛋白增加到肌动蛋白；2)增加屏障 通过激活rac1从β-连环蛋白中去除IQGAP1的功能；以及3) 挽救佛波酯、PMA和细胞因子的屏障破坏作用， 肿瘤坏死因子。实验方法包括蛋白质相互作用的演示。 免疫荧光显微镜、免疫沉淀和免疫印迹； 黏附分子连接完整性和内皮屏障的功能分析 功能；以及用腺病毒重组体感染来评估重要性 连接蛋白(如纽蛋白)、α-连环蛋白、IQGAP1和rac1。这个 生物学意义在于识别被激活的细胞靶标 通过S1P和cAMP，起到加强细胞间连接的作用。

DESCRIPTION (provided by applicant): The vascular endothelium controls the flux of macromolecules from the vascular space to the interstitium. Understanding the mechanisms that maintain and tighten the endothelial barrier would aid in the elucidation of therapeutic interventions to counter a loss of barrier activity and tissue edema. Both sphingosine 1-phosphate (S1P) and cAMP-enhancing agents increase barrier function. Proposed mechanisms for tightening the barrier include increases in cell-cell or cell-matrix adhesion, and/or a decrease in contractile activity. The increased barrier activity of S1P is independent of cAMP and protein kinases A and G and is not affected by inhibition of myosin light chain (MLC) kinase and extracellular signal-regulated kinase. Furthermore, S1P and cAMP have opposing effects on MLC phosphorylation, cellular tension, and actin stress fibers. We propose that the barrier-enhancing activity of S1P and cAMP is directed at the intercellular junctions. Alternatively, S1P and cAMP may function via different mechanisms. Preliminary data indicate that S1P increases the localization of vinculin, an alpha-catenin/actin linker protein, at cell-cell contacts and increases the association of alpha-catenin with actin. Preliminary data indicate that S1P decreases the peripheral localization of IQGAP1, which competes with alpha-catenin for binding to beta-catenin, increases the activity of Rac1, and increases the interaction of IQGAP1 with Rac1. These findings suggest that S1P increases barrier activity by enhancing adherens junction integrity. Preliminary data also indicate that S1P and cAMP reverse the barrier-decreasing effect of tumor necrosis factor (TNF). We propose that the adherens junction is a common target for S1P and cAMP. These mediators increase barrier function by strengthening the adherens junction via addition of linker proteins such as vinculin and via removal of IQGAP1 by activated Rac1. The three Specific Aims are: S1P and cAMP 1) increase barrier function by enhancing the attachment of alpha-catenin to actin via addition of linker proteins; 2) increase barrier function by removal of IQGAP1 from beta-catenin via activation of Rac1; and 3) rescue the barrier-disrupting effect of a phorbol ester, PMA, and a cytokine, TNF. Experimental approaches include the demonstration of protein interactions by immunofluorescence microscopy and immunoprecipitation and immunoblotting; functional analyses of adherens junctional integrity and endothelial barrier function; and infections with adenoviral recombinants to assess the importance of linker proteins (such as vinculin), alpha-catenin, IQGAP1, and Rac1. The biological significance is the identification of the cellular targets activated by S1P and cAMP that function to tighten the junctions between cells.