Wang Gang, Kawakami Kiyoshi, Gick Gregory
Department of Biochemistry, Center for Cardiovascular and Muscle Research, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.
J Cell Physiol. 2007 Oct;213(1):167-76. doi: 10.1002/jcp.21107.
Na,K-ATPase expression is upregulated in mammalian cells as a consequence of persistent inhibition of Na,K-ATPase enzymatic activity by low external K(+). We previously demonstrated that exposure of neonatal rat cardiac myocytes to low K(+) increased Na,K-ATPase alpha1 subunit mRNA content and promoter activity. In this work, we utilized transient transfection studies with rat Na,K-ATPase alpha1 subunit 5'-flanking region deletion plasmids to identify DNA sequences required for low K(+)-mediated stimulation of alpha1 subunit promoter expression in cardiac myocytes. Maximal low K(+)-responsiveness of the alpha1 promoter was found to be dependent on nucleotides from -102 to -62 and a downstream region from +53 to +261. Further analysis of the upstream low K(+)-responsive region using mutant constructs revealed that a CRE/ATF site at -70 to -63 and a GC box motif at -57 to -48 were both required for the effect of low K(+) on alpha1 subunit gene transcription. Electrophoretic mobility shift assays revealed that low K(+) increased binding of transcription factors to the GC box and, to a lesser extent, to the CRE/ATF site. Western blot analysis demonstrated that exposure of cardiac myocytes to low K(+) resulted in increased nuclear content of Sp1, Sp3 and CREB-1. Finally, a selective increase in phosphorylation of Sp1 was found in nuclear extracts from low K(+)-treated cells. We conclude that low K(+)-mediated upregulation of Na,K-ATPase alpha1 subunit gene expression in neonatal rat cardiac myocytes is dependent, in part, on CRE/ATF- and GC box-binding transcription factors.
由于细胞外低钾（K+）持续抑制钠钾ATP酶的酶活性，哺乳动物细胞中钠钾ATP酶（Na，K-ATPase）的表达上调。我们之前证明，将新生大鼠心肌细胞暴露于低钾环境会增加钠钾ATP酶α1亚基的mRNA含量和启动子活性。在这项研究中，我们利用大鼠钠钾ATP酶α1亚基5'-侧翼区缺失质粒进行瞬时转染研究，以确定低钾介导的心肌细胞α1亚基启动子表达刺激所需的DNA序列。发现α1启动子的最大低钾反应性依赖于-102至-62位的核苷酸以及+53至+261位的下游区域。使用突变体构建体对上游低钾反应区域进行进一步分析表明，-70至-63位的CRE/ATF位点和-57至-48位的GC盒基序对于低钾对α1亚基基因转录的影响都是必需的。电泳迁移率变动分析表明，低钾增加了转录因子与GC盒的结合，在较小程度上也增加了与CRE/ATF位点的结合。蛋白质印迹分析表明，将心肌细胞暴露于低钾环境会导致Sp1、Sp3和CREB-1的核含量增加。最后，在低钾处理细胞的核提取物中发现Sp1的磷酸化选择性增加。我们得出结论，新生大鼠心肌细胞中低钾介导的钠钾ATP酶α1亚基基因表达上调部分依赖于CRE/ATF和GC盒结合转录因子。
