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

1.在肾脏系统中，我们应用培养的远端小管细胞和血管平滑肌细胞，分析了皮质类固醇激素、高渗透压和血清刺激对Na、k - atp酶基因表达的影响。通过northern blotting分析，a和b亚基基因均被诱导数倍。将报告荧光素酶基因与Na、K-ATPase a1和b1亚基基因的促进区融合后，也观察到这种诱导作用，表明其调控是在转录水平2。Na， k - atp酶α 3亚基基因在出生几周后在大鼠心脏中转录。以大鼠心肌细胞为模型系统，我们确定了该基因的顺式元件和交易因子。它们是一个NF-Y站点和两个Sp1/Sp3站点。三者均为正调控元件，并表现出协同激活。通过心肌细胞特异性的体内足迹分析，观察到这些元件上的蛋白质占用。beta1亚基基因对高氧反应的调控元件在-84至-44区域被确定。该地区的两个Sp1/Sp3位点不是合理的因素。为了分析as亚基基因在出生期的诱导机制，我们从成人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鉴定了α 1亚基基因的负调控元件。胚胎的顺式元件为-155 ~ +31，成体的顺式元件为+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.