REGULATION OF TISSUE FACTOR PATHWAY INHIBITOR EXPRESSION

这些研究的主要目的是调查 诱导型组织因子途径抑制物(TFPI)基因的表达 单核细胞和成纤维细胞。使用Northern blotting分析和TEPI 化验，申请人以前曾证明内皮细胞是 TFPI合成的主要生理部位以及单核细胞和 成纤维细胞通常不表达或很少表达TFPI。然而， 申请人的初步数据表明，TFPI的表达大量 在贴壁的单核细胞和单核细胞U93细胞中上调。在这 建议书中，申请人将定义附着力反应要素 TFPI基因上调单核细胞和U937细胞中TFPI的表达。这个 申请人还发现，成纤维细胞显著上调TFP I 表达对血清的反应，在这项提案中，她计划定义 在成纤维细胞中上调其表达的血清反应元件。 初步数据还表明，贴壁的单核细胞和U937细胞， 血清刺激的成纤维细胞和几个转化的细胞系 表达TFPI还表达GATA-2转录因子。在凝胶流动性方面 肝癌细胞(转化株)细胞核提取液中S因子的位移分析 同时表达TFPI和GATA-2转录因子的细胞系)出现 结合TFPI启动子区域的DNA片段，该DNA片段包含 推定的GATA主题。此外，GATA-2转录因子反义 寡聚体显著降低TFPI和GATA-2的表达 贴壁UJ937细胞中转录因子mRNA的表达。因此，GATA-2 转录因子可能是TFPI基因表达所必需的。这 这种可能性将在单核细胞和成纤维细胞中进一步测试 正义和反正义的策略。 初步数据是在发起人保罗的指导下产生的 他是凝血和血栓领域的专家。这个 申请者将继续在Paul Bajaj的实验室工作 建议进行的研究。她还将接受合作伙伴的持续指导- 赞助商，Joel Eissenberg，他是一位经验丰富和公认的分子 生物学家。申请人将经常与咨询委员会举行会议。 (Sly、Huang、Hyers和Payvar博士)寻求指导并讨论她 进步。因此，充足的资源和丰富的专业知识可供 申请人须进行拟进行的研究。据预计， 研究将在诱导物的监管方面产生重要的新信息 炎症反应过程中TEPI基因的表达。

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.