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

1.在肾脏系统，应用体外培养的肾小管上皮细胞和血管平滑肌细胞，分析了皮质类固醇激素、高渗和血清刺激对Na，K-ATP酶基因表达的影响。nortehrn印迹分析表明，a和B亚基基因均被诱导了数倍。将Na，K-ATP酶a1和b1亚基基因的启动子区与荧光素酶基因融合，转染大鼠心脏，观察到Na，K-ATP酶α 3亚基基因的转录诱导，表明该基因的转录是在转录水平上进行的。以大鼠心肌细胞为模型系统，我们鉴定了该基因的顺式元件和反式作用因子。它们是一个NF-Y位点和两个Sp1/Sp3位点。这三个元件作为正调控元件，并表现出协同激活。通过体内足迹分析，观察这些元件上的蛋白质占有率。3.在-84 ~-44区域鉴定了β 1亚基基因对高氧反应的调控元件。该区域的两个Sp1/Sp3位不是共振单元。为了分析as亚单位基因在出生期的诱导机制，我们建立了成人和胚胎肺细胞核提取物a1亚单位基因的体外转录体系。我们鉴定了α 1亚基基因-375 ~-201的负调控元件。在胚胎期，全转录活性所需的顺式元件为-155 ~+31，而在成体期，全转录活性所需的顺式元件为+66 ~+92。

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.