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.
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