Selectins initiate adhesion to the vessel wall during inflammation. P- selectin on activated platelets and endothelial cells binds to P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In vitro studies suggest that engagement on PSGL-1 transmits signals through the cytoplasmic domain. However, the adhesive and designing functions of PSGL-1 in vivo are poorly understood. In mice, TNF-alpha, IL-1beta, LPS, and oncostatin M increase synthesis of P-selectin in endothelial cells. In humans, only oncostatin M increase synthesis of P-selectin. The biological significance of this specifies difference is not known. Oncostatin M and other IL-6 family members bind to receptors that include gp130. In vitro studies suggest that signaling via gp130 induces synthesis of adhesion receptors and chemokines in endothelial cells. However, many cells express gp130, making it difficult to determine the function of gp130 signaling in endothelial cells in vivo. We will use gene targeting to study the expression and function of P-selectin, gp130 and PSGL- in vivo. First, we will make mice with a chimeric P-selectin gene in which we replace part of the 5' flanking region of the murine gene with the corresponding region from the human gene. We will determine whether the chimeric P-selectin gene responds to cytokines as in humans, and if so, will examine how P-selectin expressed in this manner contributes to models of inflammation and atherosclerosis. Second, we will use Cre-loxP methods to delete gp130 specifically in endothelial cells of mice. We will determine if oncostatin M fails to augment expression of P- and E-selectin and chemokines inflammation and atherosclerosis. Third, we will generate PSGL-1-null mice and mice expressing PSGL-1 without the cytoplasmic domain. We will use the mice to determine whether PSGL-12 couples adhesion to cytoplasmic- dependent signaling in models of inflammation and arterial injury. These studies will clarify the relationships between adhesion and signaling in the vasculature during inflammation.
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