The overall goal of this proposal is to elucidate regulatory pathways that control the in vivo expression of tissue factor (TF) in Gram-negative septic shock. Although tumor necrosis factor (TNF) and interleukin 1 (IL- 1) are important polypeptide mediators in the pathogenesis of this disease, recent studies have shown that inhibition of TF activity in a baboon model attenuated the coagulopathy and protected against lethality. Within the vasculature, TF expression is strictly regulated because contact of this receptor with blood is sufficient to initiate the coagulation protease cascades. Peripheral blood monocytes are the only circulating cell type that can be induced to express TF in response to various inflammatory agents including bacterial endotoxin (lipopolysaccharide [LPS]). Cellular responses to physiological levels of LPS in the bloodstream depend on binding to the serum protein LBP (LPS binding protein). The three specific aims of this proposal will determine the transcriptional control mechanisms that regulate expression of the human TF gene in monocytes. First, regulation of the endogenous TF gene will be studied in monocytic cells exposed to LPS alone, following engagement of the CD14 receptor by the LPS-LBP complex, and after activation of the protein kinase C signal transduction pathway by phorbol esters (PMA). Second, the roles of the transcription factors AP-1 and NF-kappaB in the regulation of the TF promoter will be determined by in vitro analysis of the protein-DNA interactions, as well as protein-protein interactions. Recently, a 56 bp LPS response element (LRE) within the TF promoter was shown to mediate induction of the TF gene in LPS-treated monocytic cells by binding AP-1 and NF-kappaB. In addition, functional studies on the TF promoter, cloned upstream of a luciferase reporter gene, will be performed in monocytic cells exposed to PMA, and in cells cotransfected with plasmids which express AP-1 at a high level. Finally, an in vitro transcription assay for monocytic cells will be established to analyze the role of AP-1 and NF- kappaB in the constitutive and induced expression of the TF promoter. Transcription of the TF promoter will be analyzed using nuclear extracts specifically depleted of AP-1 and NF-kappaB. These studies will be performed using monocytes and two human monocytic cell lines, THP-1 and Mono-Mac-6. Understanding the transcriptional control of the TF gene should allow the development of effective strategies for the prevention and treatment of thrombotic and vascular diseases.
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