Regulation of Na, K-ATPase in renal, cardiac, pulmonaly disease

肾脏、心脏、肺部疾病中 Na、K-ATP 酶的调节

基本信息

批准号：
07044289
负责人：
KAWAKAMI Kiyoshi
金额：
$ 2.75万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

1.In renal system, we applied cultured distal tubule cells and vascular smooth muscle cells and analyzed the response of Na, K-ATPase genes expression by trearment with corticosteroid hormones, high osmolarity or serum stimulation. Both a and b subunit gene were induced several fold by nortehrn blotting analysis. The induction was also observed when we transfected the reporter luciferase gene fused with the promote region of the Na, K-ATPase a1 and b1 subunit gene, indicating that the regulation was transcriptinal level.2.Na, K-ATPase alpha3 subunit gene is transcribed in a few weeks after birth in rat heart. Using rat cardiocyte as a model system, we identified cis elements and transacting factors responsible for the gene. They are one NF-Y site and two Sp1/Sp3 sites. All three elements act as positive regulatory element and showed synergic activation. Protein occupancy on these elements were observed by in vivo footprinting analysis in cardiocyte specific manner.3.The regulatory element of the beta1 subunit gene in response to hyperoxya was identified at -84 to -44 region. The two Sp1/Sp3 site in the region was not the resonsible element. For the analysis of the induction mechanism of the as subuunit gene during birth period, we established in vitro transcription system of the a1 subuit gene from adult and embyonic lung nuclear extracts. We identified negative regulatory element of the alpha1 subuit gene from -375 to -201. The cis element necessary for the full transcriptin activity was -155 to +31 in embryo while +66 to +92 was also necessary in adult.

1.在肾脏系统中，我们应用培养的远曲小管细胞和血管平滑肌细胞，分析了皮质类固醇激素、高渗透压或血清刺激处理后Na、K-ATPase基因表达的反应。通过nortehrn印迹分析，a和b亚基基因均被诱导数倍。转染与Na,K-ATPase a1和b1亚基基因启动子区融合的报告荧光素酶基因时也观察到了诱导作用，表明调控是转录水平的。2.Na,K-ATPase α3亚基基因被转录在老鼠出生后几周的心脏中。使用大鼠心肌细胞作为模型系统，我们鉴定了负责该基因的顺式元件和反式因子。它们是 1 个 NF-Y 位点和 2 个 Sp1/Sp3 位点。所有三个元件均充当正向调节元件并表现出协同激活作用。通过心肌细胞特异的体内足迹分析观察这些元件的蛋白质占据情况。3.在-84至-44区域鉴定了β1亚基基因响应高氧的调控元件。该区域的两个Sp1/Sp3位点不是敏感元件。为了分析出生期as亚基基因的诱导机制，我们从成体和胚胎肺核提取物中建立了a1亚基基因的体外转录系统。我们从-375到-201鉴定出了α1亚基基因的负调控元件。胚胎中完整转录蛋白活性所需的顺式元件为-155至+31，而成人中也需要+66至+92。