The primary goal of these studies is to investigate the regulation of inducible tissue factor pathway inhibitor (TFPI) gene expression in monocytes and fibroblasts. Using Northern blotting analysis and TEPI assays, the applicant had previously demonstrated that endothelium is the primary physiologic site of TFPI synthesis and that monocytes and fibroblasts normally express none to very little TFPI. However, the applicant's preliminary data indicate that TFPI expression is vastly upregulated in adherent monocytes and monocytic U93 cells. In this proposal, the applicant will define the adhesion response elements in the TFPI gene that upregulate TFPI expression in monocytes and U937 cells. The applicant has also found that fibroblasts significantly upregulate TFPI expression in response to serum and in this proposal she plans to define the serum response elements that upregulate its expression in fibroblasts. preliminary data also suggest that adherent monocytes and U937 cells, serum-stimulated fibroblasts and several transformed cell lines that express TFPI also express GATA-2 transcription factor. In gel mobility shift assays, factor(s) from nuclear extracts of HepG2 cells (a transformed cell line that expresses both TFPI and GATA-2 transcription factor) appear to bind to a DNA fragment from the TFPI promoter region containing a putative GATA motif. Additionally, GATA-2 transcription factor antisense oligomers significantly decreased the expression of TFPI and GATA-2 transcription factor mRNA in adherent UJ937 cells. Thus GATA-2 transcription factor may be required for TFPI gene expression. This possibility will be further tested in monocytes and fibroblasts by the use of sense and antisense strategies. Preliminary data was generated under the guidance of the sponsor, Paul Bajaj, who is an expert ina the field of coagulation and thrombosis. The applicant will continue to work in the laboratory of Paul Bajaj for the proposed studies. She will also receive constant guidance from the co- sponsor, Joel Eissenberg who is an experienced and recognized molecular biologist. The applicant will meet frequently with the advisory committee (Drs. Sly, Huang, Hyers, and Payvar) to seek guidance and discuss her progress. Thus ample resources and vast expertise is available to the applicant to perform the proposed studies. It is anticipated that the studies will yield important new information on the regulation of inducible TEPI gene expression during an inflammatory response.
