TY - JOUR
T1 - Up-regulation of Na,K-ATPase β1 transcription by hyperoxia is mediated by SP1/SP3 binding
AU - Wendt, Christine H.
AU - Gick, Greg
AU - Sharma, Renuka
AU - Zhuang, Yong
AU - Deng, Wenlian
AU - Ingbar, David H.
PY - 2000/12/29
Y1 - 2000/12/29
N2 - 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 β1 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 β1 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 β1 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.
AB - 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 β1 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 β1 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 β1 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.
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U2 - 10.1074/jbc.M004759200
DO - 10.1074/jbc.M004759200
M3 - Article
C2 - 10988288
AN - SCOPUS:0034731495
SN - 0021-9258
VL - 275
SP - 41396
EP - 41404
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 52
ER -