Endothelial Redox Signaling Mediated by PECAM-1

存在于血液和血管细胞表面的细胞黏附分子介导细胞-细胞和细胞-基质的相互作用，它们的相互作用导致许多信号转导通路的激活。通过最近的研究，对参与这些信号通路的许多因素进行了阐明，启动细胞激活的生理事件的范围以及调节这些过程的许多成分的身份仍未完全确定。血小板内皮细胞黏附分子-1(PECAM-1，CD31)是免疫球蛋白(Ig)超家族的一个130 kDa的成员，是内皮细胞间连接的主要组成部分，聚集着多达10/6的分子，也表达在循环中的血小板、单核细胞、中性粒细胞和某些T细胞上。除了其亲和性结合特性外，PECAM-1也是免疫球蛋白样抑制性受体家族的成员，每个受体在其胞浆结构域中都含有一个基于酪氨酸的免疫受体抑制基序(ITIM)。当细胞激活时，PECAM-1 ITIM被磷酸化，导致蛋白质酪氨酸磷酸酶SHP-2的募集和激活。研究表明，PECAM-1/SHP-2信号复合体可以调节T和B细胞内钙离子的动员，抑制FcGammaRIIa介导的吞噬作用，并控制血小板对胶原的激活反应。我们实验室最近的观察表明，氧化剂H_2O_2是能够“激活”PECAM-1的更有效的试剂之一，然而，其他生物和生理上相关的活性氧物种(ROS)启动PECAM-1SHP-2信号复合体的形成的能力，以及该复合体调节细胞对氧化应激的反应的可能性，尚不完全清楚。因此，项目4的目标是测试这一假设，即PECAM-1在血管细胞中充当氧化还原传感器，既是活性氧和氮物种的靶标，也是减缓细胞对活性氧和氮物种反应的靶标。特定目的1检测选定的生理相关ROS诱导PECAM-1酪氨酸磷酸化和募集SHP-2的能力，以及细胞内自由基清除剂调节PECAM-1激活程度的能力。这些研究不仅确定了氧化应激对PECAM-1功能的影响，而且也可以作为氧化损伤对其抑制受体功能影响的模型。具体目的2建立在最近观察到的PECAM-1在酪氨酸残基上被硝化的基础上，并探索PECAM-1酪氨酸硝化抑制其磷酸化并干扰其抑制功能的性质和程度。具体目标3探索在几种体内氧化应激模型中PECAM-1的自由基修饰的生理学后果。终于来了。特定目的4使用基因阵列技术来评估暴露于氧化应激后细胞转录谱的变化，并确定PECAM-12对这一反应的影响。总之，这些目标包括一个及时、协调和有重点的研究计划，旨在提高我们对细胞在炎症、动脉粥样硬化和缺血/再灌注诱导的心脏损伤等病理生理条件下对氧化损伤做出反应的分子机制，以及PECAM-1介导的信号转导在调节这种反应中所起的作用。

Cell adhesion molecules present on the surface of blood and vascular cells mediate cell-cell and cell-matrix interactions, and their engagement leads to the subsequent activation of numerous signal transduction cascades. Through recent studies have resulted in the elucidation of many of the elements that participate in these signaling pathways, the range of physiological events that initiate cellular activation, as well as the identity of the many of the components that regulate these processes, remain to be fully defined. Platelet Endothelial Cell Adhesion Molecule- 1 (PECAM-1, CD31) is a 130 kDa member of the Immunoglobulin (Ig) superfamily that is a major constituent of the endothelial cell intercellular junction, where up to 10/6 molecules are concentrated, and is also expressed on circulating platelets, monocytes, neutrophils, and certain T-cells. In addition to its homophilic binding properties, PECAM-1 is also a member of a family of Ig-like inhibitory receptors, each of which harbors an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) within its cytoplasmic domain. Upon cellular activation, the PECAM-1 ITIM becomes phosphorylated, resulting in recruitment and activation of the protein-tyrosine phosphatase, SHP-2. The PECAM- 1/SHP-2 signaling complex has been shown to regulate intracellular calcium mobilization in T and B cells, inhibit FcgammaRIIa-mediated phagocytosis, and control platelet activation responses to collagen. Recent observations in our laboratory suggest that the oxidant H2O2 is among the more potent agents capable of "activating" PECAM-1, however the ability of other biologically and physiologically relevant reactive oxygen species (ROS) to initiate formation of the PECAM- 1SHP-2 signaling complex, and the potential for this complex to regulate cellular responses to oxidative stress, is incompletely understood. The goal of Project 4, therefore, is to test the hypothesis that PECAM-1 serves as a redox sensor in vascular cells, serving both as a target for, and moderating cellular responses to reactive oxygen and nitrogen species. Specific Aim 1 examines the ability of selected physiologically relevant ROS to induce PECAM-1 tyrosine phosphorylation and recruit SHP-2, as well as the ability of intracellular free radical scavengers to regulate the degree of PECAM-1 activation. These studies not only define the consequences of oxidant stress for PECAM-1 function, but may serve as a model for the effect of oxidative damage on the function of their inhibitory receptors as well. Specific Aim 2 builds on recent observations that PECAM-1 an become nitrated on tyrosine residues, and explores both the nature and the extent to which PECAM-1 tyrosine nitration inhibits its phosphorylation and interferes with its inhibitory function. Specific Aim 3 explores both the physiologic consequences of free radical modification of PECAM-1 in several in vivo models of oxidative stress. Finally. Specific Aim 4 employs gene array technology to assess alterations in the cellular transcription profile following exposure to oxidative stress, and determines the effect of PECAM-12 on this response. Together, these aims comprise a timely, coordinated, and focused research program designed to improved our understanding of the molecular mechanisms by which cells respond to oxidative injury in pathophysiological conditions such as inflammation, atherosclerosis, and ischemia/reperfusion-induced cardiac injury, and the role that PECAM- 1-mediated signal transduction plays in regulating this response.