Adherens Junction Integrity and Barrier Function

粘附连接完整性和屏障功能

• 批准号: 6469442
• 负责人: FRED L MINNEAR
• 金额: $ 30.15万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469442
• 关键词: actins; cadherins; cyclic AMP; cytoskeletal proteins; human tissue; immunofluorescence technique; immunoprecipitation; intercellular connection; microfilaments; phorbols; phosphorylation; protein localization; protein protein interaction; protein structure function; sphingosine; tumor necrosis factor alpha; vascular endothelium permeability; vinculin; western blottings

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.

描述（由申请人提供）：血管内皮控制流量 从血管间隙到间质的大分子。理解 维持和收紧内皮屏障的机制将有助于 阐明应对屏障丧失的治疗干预措施 活动和组织水肿。 1-磷酸鞘氨醇 (S1P) 和 cAMP 增强剂可增强屏障功能。拟议的机制 收紧屏障包括增加细胞-细胞或细胞-基质粘附， 和/或收缩活动减少。屏障活性增加 S1P 独立于 cAMP 和蛋白激酶 A 和 G，并且不受 抑制肌球蛋白轻链 (MLC) 激酶和细胞外 信号调节激酶。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力纤维。我们建议 S1P 和 cAMP 的屏障增强活性针对细胞间质 路口。或者，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，纽蛋白是一种 α-连环蛋白/肌动蛋白连接蛋白，在细胞与细胞接触处并增加 α-连环蛋白与肌动蛋白的关联。初步数据表明S1P 减少 IQGAP1 的外周定位，它与 α-连环蛋白与 β-连环蛋白结合，增加 Rac1 的活性，并且 增加 IQGAP1 与 Rac1 的相互作用。这些发现表明 S1P 通过增强粘附连接完整性来增加屏障活性。 初步数据还表明 S1P 和 cAMP 逆转了势垒下降 肿瘤坏死因子（TNF）的作用。我们建议粘附连接是 S1P 和 cAMP 的共同目标。这些介质通过以下方式增强屏障功能 通过添加接头蛋白来加强粘附连接，例如 纽蛋白并通过激活的 Rac1 去除 IQGAP1。三个具体目标 是：S1P 和 cAMP 1) 通过增强 通过添加连接蛋白将 α-连环蛋白转化为肌动蛋白； 2）增加壁垒 通过激活 Rac1 从 β-连环蛋白中去除 IQGAP1 来发挥作用；和 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 通过免疫荧光显微镜、免疫沉淀和免疫印迹； 粘附连接完整性和内皮屏障的功能分析 功能;和腺病毒重组体感染以评估重要性 连接蛋白（如纽蛋白）、α-连环蛋白、IQGAP1 和 Rac1。这 生物学意义是识别激活的细胞靶标 S1P 和 cAMP 的作用是加强细胞之间的连接。