Regulation of Na,K-ATPase alpha1 subunit gene transcription in response to low K(+): role of CRE/ATF- and GC box-binding proteins.
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.
Wang
Gang
G
Department of Biochemistry, Center for Cardiovascular and Muscle Research, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203, USA.
Kawakami
Kiyoshi
K
Gick
Gregory
G
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; C499075:Atp1a1 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium