PPAR-GAMMA REGULATION ALPHA 1 TYPE I COLLAGEN PROMOTER

PPAR-γ 调节 ALPHA 1 I 型胶原蛋白促进剂

• 批准号: 6801079
• 负责人: SHAROM YAVROM
• 金额: $ 2.49万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6801079
• 关键词: DNA binding protein; DNA footprinting; actins; alcoholism /alcohol abuse; biological signal transduction; collagen; fibrosis; flow cytometry; gel mobility shift assay; gene expression; genetically modified animals; green fluorescent proteins; immunoprecipitation; inhibitor /antagonist; laboratory mouse; liver cells; peroxisome proliferator activated receptor; predoctoral investigator; reporter genes; smooth muscle; transfection /expression vector

DESCRIPTION (provided by applicant): Induced collagen expression by hepatic stellate cells (HSC) is the hallmark of alcoholic liver fibrosis. Our laboratory recently demonstrated the potential link between depletion of PPARgamma in HSC and liver fibrogenesis and the reversal of HSC activation with PPARgamma ligands. The trainee will test the hypothesis that PPARgamma inhibits alpha1(I) collagen promoter in HSC line and liver fibrosis. Specific aims include determination of the specificity of PPARgamma-mediated inhibition of the promoter and elucidation of the mechanisms underlying the effect. The former will be achieved by transient transfection assays with the promoter-reporter gene and expression vectors for the members of nuclear receptors including PPARgamma. The latter will address PPARgamma-mediated negative cross-coupling with DNA-binding proteins for the promoter via DNase foot printing and gel shift assays and inhibition of co-activator recruitment by this nuclear protein which will be assessed by GST-fusion protein pull down assays and coimmununoprecipitation. PPARgamma-mediated suppression of the promoter will further be tested in vivo using the transgenic mice expressing GFP driven by the collagen promoter and adenoviral-mediated PPARgamma overexpression targeted to HSC by alpha-smooth muscle actin promoter. A similar approach will be taken to finally test the efficacy of PPARgamma overexpression for CCI[4] liver fibrosis in mice. These experiments will define the molecular basis for inhibition of collagen gene expression by PPARgamma and will test the potential of this mode if intervention as a novel therapy for alcoholic liver fibrosis.

描述（由申请人提供）：肝星状细胞（HSC）诱导的胶原蛋白表达是酒精性肝纤维化的标志。我们的实验室最近证明了 HSC 中 PPARgamma 的消耗和肝纤维化之间的潜在联系，以及 PPARgamma 配体逆转 HSC 激活之间的潜在联系。学员将测试 PPARgamma 抑制 HSC 系和肝纤维化中的 α1(I) 胶原蛋白启动子的假设。具体目标包括确定 PPARgamma 介导的启动子抑制的特异性并阐明该作用的机制。前者将通过启动子报告基因和核受体成员（包括 PPARgamma）表达载体的瞬时转染测定来实现。后者将通过 DNase 足迹和凝胶位移测定解决 PPARgamma 介导的与启动子 DNA 结合蛋白的负交叉偶联，并通过该核蛋白抑制共激活剂募集，这将通过 GST 融合蛋白下拉测定进行评估和免疫共沉淀。将使用由胶原蛋白启动子驱动的表达 GFP 的转基因小鼠和通过 α-平滑肌肌动蛋白启动子靶向 HSC 的腺病毒介导的 PPARgamma 过表达，进一步在体内测试 PPARgamma 介导的启动子抑制。最终将采用类似的方法来测试 PPARgamma 过表达对小鼠 CCI[4] 肝纤维化的功效。这些实验将确定 PPARgamma 抑制胶原蛋白基因表达的分子基础，并将测试这种模式作为酒精性肝纤维化新疗法的干预潜力。