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 intra cellular ion concentration, cellular volume, and ion transport and is regul ated both transcriptionally and post-transcriptionally. We previously-demon strated that hyperoxia increased Na,K-ATPase beta (1) gene expression in Ma din-Darby canine kidney (MDCK) cells. In this study, we identify a DNA elem ent necessary for up-regulation of the Na,K-ATPase beta (1) transcription b y hyperoxia and evaluate the nuclear proteins responsible for this up-regul ation. Transient transfection experiments in MOCK 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 hy peroxia 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 b ound both transcription factors SP1 and SP3 in electrophoretic mobility shi ft assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation o f this element eliminated protein binding. Other GC sites within the proxim al promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at -55 had higher affinity for SP proteins.