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Regulation of Na,K‐ATPase α1 subunit gene transcription in response to low K + : Role of CRE/ATF‐ and GC box‐binding proteins
Author(s) -
Wang Gang,
Kawakami Kiyoshi,
Gick Gregory
Publication year - 2007
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.21107
Subject(s) - microbiology and biotechnology , protein subunit , transcription (linguistics) , promoter , biology , gene expression , myocyte , transcription factor , transfection , downregulation and upregulation , creb , electrophoretic mobility shift assay , atpase , gene , biochemistry , enzyme , philosophy , linguistics
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 α1 subunit mRNA content and promoter activity. In this work, we utilized transient transfection studies with rat Na,K‐ATPase α1 subunit 5′‐flanking region deletion plasmids to identify DNA sequences required for low K + ‐mediated stimulation of α1 subunit promoter expression in cardiac myocytes. Maximal low K + ‐responsiveness of the α1 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 α1 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 α1 subunit gene expression in neonatal rat cardiac myocytes is dependent, in part, on CRE/ATF‐ and GC box‐binding transcription factors. J. Cell. Physiol. 213: 167–176, 2007. © 2007 Wiley‐Liss, Inc.