Modulation of Leukocyte Adhesion by P-selectin/PSGL-1 Signaling Leukocyte recruitment entails a cascade of cellular events, including rolling and firm adhesion of responding cells. P-selectin (CD62P), P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and beta2-integrins are endothelial and leukocyte cell adhesion molecules essential for innate immunity and inflammation. The interaction of P-selectin with PSGL-1 mediates leukocyte rolling, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for beta2-integrin-mediated firm adhesion. However, the mechanisms for feedback regulation of P-selectin adhesion activity and P- selectin-induced beta2-integrin activation remain undetermined. Nef-associated factor 1 (Nafl) is an endogenous inhibitor for NF-kappaB activation. Serum amyloid P component (SAP) is an acute phase protein synthesized and secreted rapidly by hepatocytes in response to various inflammatory mediators. In the preliminary studies, we found that Naf1 formed a stable complex with the cytoplasmic tail of PSGL-1. Engagement of PSGL-1 by P-selectin phosphorylated the Y552PPM motif of Naf1 for recruiting p85 subunit of phosphatidylinositol-3 kinase (PI3K) and triggering the signaling cascade that culminated in activation of alphaMbeta2 (CD11bCD18, Mac-1). In addition, we observed that SAP interacted with P-selectin and acted as an endogenous inhibitor of PSGL-1 for P-selectin recognition. In this grant application, we propose 1) to define the functional importance of P-selectin-induced activation of alphaMbeta2; 2) to determine the biological significance of the PSGL-1-Naf1-p85 signaling pathway for modulation of alphaMbeta2 activity; and 3) to explore the regulation of P-selectin adhesion activity by SAP. Overall, this study will not only enhance our understanding of molecular mechanisms that precisely determine leukocyte fates in the multi- step paradigm of leukocyte recruitment, but also discover novel therapeutic targets for prevention and treatment of inflammatory disorders. ? ? ?
