Characterization of a Sodium-Response Transcriptional Mechanism

Abstract

Abstract 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 α-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 μmol/L monensin) upregulation of α ₁ -, α ₂ -, and β ₁ -subunit Na,K-ATPase RNA levels. Control β-actin and α-tropomyosin RNA levels did not change. With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat α ₁ [−1288]CAT and human α ₂ [−798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 μmol/L)–treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat α ₁ [−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 α ₂ Na,K-ATPase 5′ flanking region. Analysis of transgenic TgHα ₂ [−798]CAT rats demonstrated sodium activation of human α ₂ [−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 μmol/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 (α ₁ and α ₂ 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.