Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.
The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.
Yu
Zhiyuan
Z
Departments of Medicine and of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Li
Mei
M
Zhang
Dongyu
D
Xu
William
W
Kone
Bruce C
BC
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004330:Drosophila; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D023421:Models, Animal; D016328:NF-kappa B / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014162:Transfection
D002843:Chromatin; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D016328:NF-kappa B; D021122:Protein Subunits; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D017506:H(+)-K(+)-Exchanging ATPase