Ruiz-Opazo N, Cloix J F, Melis M G, Xiang X H, Herrera V L
Whitaker Cardiovascular Institute, Evans Department of Medicine, Boston University School of Medicine, Mass 02118, USA.
Hypertension. 1997 Aug;30(2 Pt 1):191-8. doi: 10.1161/01.hyp.30.2.191.
On the basis of paradigms in development wherein discrete transcriptional events are pivotal regulatory steps, we tested the hypothesis that transcriptional sodium (Na+)-response mechanisms are involved in in vivo Na+-induced responses relevant to normal (homeostatic) and pathophysiological (salt-sensitive hypertension) conditions. We used Na,K-ATPase alpha-subunit genes as molecular probes and the Na+ ionophore monensin to induce a dose-specific incremental increase in [Na+]i in rat A10 embryonic aortic smooth muscle cells. RNA blot analysis of rat A10 cells revealed a dose-specific (0.022 to 30 micromol/L monensin) upregulation of alpha1-, alpha2-, and beta1-subunit Na,K-ATPase RNA levels. Control beta-actin and alpha-tropomyosin RNA levels did not change. With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat alpha1[-1288]CAT and human alpha2[-798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 micromol/L)-treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat alpha1[-1288]CAT defined a positive Na+-response regulatory region within -358 to -169 that is distinct from the basal transcriptional activation region of -155 to -49 previously defined. Similarly, a positive Na+-response regulatory region is delimited to within -301 in the human alpha2 Na,K-ATPase 5' flanking region. Analysis of transgenic TgH alpha2[-798]CAT rats demonstrated sodium activation of human alpha2[-798]CAT transgene expression in aorta parallel to observations made in rat A10 aortic tissue culture cells. Southwestern blot analysis of nuclear extracts from monensin (10 micromol/L)-treated and control untreated A10 cells revealed a nuclear DNA binding protein (approximately 95 kD) that is upregulated by increased [Na+]i. These data provide initial characterization of a transcriptional Na+-response mechanism delimiting a positive Na+-response regulatory region in two target genes (alpha1 and alpha2 Na,K-ATPase) as well as detection of a Na+-response nuclear DNA binding protein. The in vitro data are corroborated by in vivo experimental and transgenic promoter expression studies, thus validating the biological relevance of the observations.
基于发育过程中离散转录事件是关键调控步骤的范例，我们检验了以下假设：转录性钠（Na⁺）反应机制参与了与正常（稳态）和病理生理（盐敏感性高血压）状况相关的体内Na⁺诱导反应。我们使用钠钾ATP酶α亚基基因作为分子探针，并使用Na⁺离子载体莫能菌素在大鼠A10胚胎主动脉平滑肌细胞中诱导[Na⁺]i呈剂量特异性的逐步增加。对大鼠A10细胞的RNA印迹分析显示，α1-、α2-和β1-亚基钠钾ATP酶RNA水平呈剂量特异性（0.022至30 μmol/L莫能菌素）上调。对照β-肌动蛋白和α-原肌球蛋白RNA水平未发生变化。以氯霉素乙酰转移酶（CAT）作为报告基因，对大鼠α1[-1288]CAT和人α2[-798]CAT启动子构建体进行的CAT分析显示，与未处理的A10细胞相比，在经莫能菌素（10 μmol/L）处理的A10细胞中CAT活性受到诱导。大鼠α1[-1288]CAT的启动子缺失构建体确定了一个位于-358至-169之间的阳性Na⁺反应调控区域，该区域不同于先前确定的-155至-49的基础转录激活区域。同样，人α2钠钾ATP酶5'侧翼区域中的一个阳性Na⁺反应调控区域被限定在-301以内。对转基因TgHα2[-798]CAT大鼠的分析表明，主动脉中人类α2[-798]CAT转基因表达的钠激活与在大鼠A10主动脉组织培养细胞中的观察结果相似。对经莫能菌素（10 μmol/L）处理的A10细胞和未处理的对照A10细胞的核提取物进行的蛋白质印迹分析显示，一种核DNA结合蛋白（约95 kD）在[Na⁺]i增加时上调。这些数据提供了转录性Na⁺反应机制的初步特征，界定了两个靶基因（α1和α2钠钾ATP酶）中的阳性Na⁺反应调控区域，并检测到一种Na⁺反应核DNA结合蛋白。体外数据得到了体内实验和转基因启动子表达研究的证实，从而验证了这些观察结果的生物学相关性。
