Up-regulation of Na,K-ATPase beta 1 transcription by hyperoxia is mediated by SP1/SP3 binding.
The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,K-ATPase beta(1) gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase beta(1) transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5'-deletions of the rat Na,K-ATPase beta(1) promoter-luciferase fusion gene demonstrated promoter activation by hyperoxia between -102 and +151. The hyperoxia response was localized to a 7-base pair region between -62 and -55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at -55 had higher affinity for SP proteins.
Wendt
C H
CH
University of Minnesota, Department of Medicine, Minneapolis, Minnesota 55455, USA. wendt005@tc.umn.edu
Gick
G
G
Sharma
R
R
Zhuang
Y
Y
Deng
W
W
Ingbar
D H
DH
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D004285:Dogs; D018496:Hyperoxia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D000254:Sodium-Potassium-Exchanging ATPase