Abstract
Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K(+). Our previous data showed that exposure of cardiac myocytes to low K(+) increased the steady-state abundance of Na,K-ATPase beta1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K(+) augmented Na,K-ATPase beta1 gene expression at a transcriptional level and that this effect required extracellular Ca(2+). The stimulatory effect of low K(+) on Na,K-ATPase beta1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase beta1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta1 promoter contained a low K(+) response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta1 promoter decreased basal and low K(+)-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K(+) effect on beta1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the beta1 promoter showed enhanced binding of two complexes in response to low K(+). The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K(+) response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase beta1 subunit gene promoter mediates beta1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K(+).
Highlights
Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K؉
Na,K-ATPase 1 Gene Transcription—Our previous study [21] showed that incubation of primary cultures of neonatal rat cardiac myocytes with low extracellular Kϩ increased the steady-state level of Na,K-ATPase 1 mRNA and enhanced luciferase activity driven by the Ϫ102 to ϩ151-base pair region of the rat 1 gene
It is well established that extended incubation of mammalian cells in medium containing a low external concentration of Kϩ is associated with an up-regulation of Na,K-ATPase activity, content, and subunit mRNA abundance; the underlying mechanism(s) remains to be determined at a molecular level
Summary
Na,K-ATPase, sodium and potassium transport-dependent adenosine triphosphatase; MMTV, mouse mammary tumor virus; PCR, polymerase chain reaction; MLC-2, myosin light chain-2; BDM, 2,3-butanedione monoxime. A pretranslational mechanism has been implicated in the up-regulation of Na,KATPase activity based on the finding of either low Kϩ- or ouabain-mediated augmentation of Na,K-ATPase subunit mRNA contents in established cell lines such as Madin-Darby canine kidney and ARL-15 [18, 19] and in primary cell cultures derived from adult rat kidney [20] and neonatal rat ventricle [21]. Our previous transient transfection studies demonstrated that the region between Ϫ102 to ϩ151 base pairs of the rat Na,K-ATPase 1 subunit gene is necessary for up-regulation of 1 mRNA content in response to incubation of primary cultures of neonatal rat cardiac myocytes in low extracellular Kϩ [21]. We have further delineated the molecular mechanism underlying regulation of Na,K-ATPase 1 gene expression in response to low extracellular Kϩ in primary cultures of neonatal rat cardiac myocytes. The results of electrophoretic mobility shift and transient transfection assays show that increased binding of Sp1 and Sp3 transcription factors to these GC box sequences are required to elicit the low Kϩ response
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