P-selectin, an adhesion receptor for leukocytes, is constitutively synthesized by megakaryocytes and endothelial cells, where it is sorted into membranes of secretory granules. Upon cellular activation by agonists such as thrombin, P-selectin is rapidly redistributed from secretory granules to the cell surface, where it is then internalized. Inflammatory mediators such as TNFalpha, LPS, IL-3, IL-4, IL-6, and oncostatin M also increase synthesis of P-selectin over its basal levels. We have identified a short 5' flanking region of the human P-selectin gene that confers specific expression of a reporter gene in cultured endothelial cells. This sequence contains several regulatory domains that include a functional GATA motif, a kappaB element, sequences that bind members of the STAT protein family, and at least three elements that bind unknown nuclear proteins. Unlike kappaB elements in genes encoding proteins such as E-selectin, the kappaB element in the P-selectin gene binds p50 and p52 homodimers but not p65 homodimers or p65/p50 heterodimers. We propose to (1) Compare the effects of agonists on expression of P- and E-selectin in a panel of endothelial and megakaryocytic cells, (2) Determine the effects of agonists on expression of reporter genes containing P-selectin 5' flanking sequences in transfected cells, (3) Correlate the sequence requirements for nuclear protein binding with those for reporter gene expression for each putative regulatory element in the P-selectin gene, (4) Clone and characterize cDNAs for transcription factors that bind to these regulatory elements, (5) Determine whether the purified transcription factors function cooperatively through protein-protein or protein-DNA interactions, and (6) Determine the expression patterns of reporter genes containing P- selectin regulatory elements in transgenic mice. Because P-selectin mediates the adhesion of leukocytes to both activated platelets and endothelial cells, it links the hemostatic and inflammatory responses to tissue injury. These studies should clarify how the expression of P-selectin is controlled by inflammatory mediators, under physiological conditions and in pathological conditions such as thrombosis and atherogenesis. The findings may also contribute to our understanding of how gene expression is restricted to megakaryocytes and endothelial cells.
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