Characterization of a sodium-response transcriptional mechanism.
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.
Ruiz-Opazo
N
N
Whitaker Cardiovascular Institute, Evans Department of Medicine, Boston University School of Medicine, Mass 02118, USA.
Cloix
J F
JF
Melis
M G
MG
Xiang
X H
XH
Herrera
V L
VL
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D005786:Gene Expression Regulation; D005796:Genes; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics; D008985:Monensin / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000378:metabolism* / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014158:Transcription, Genetic; D019076:Transgenes
D004268:DNA-Binding Proteins; D007476:Ionophores; D007527:Isoenzymes; D008985:Monensin; D012964:Sodium; D015500:Chloramphenicol O-Acetyltransferase; D000254:Sodium-Potassium-Exchanging ATPase