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Am J Physiol Renal Physiol 297: F63-F70, 2009. First published May 6, 2009; doi:10.1152/ajprenal.00039.2009
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Sp1 trans-activates the murine H+-K+-ATPase {alpha}2-subunit gene

Zhiyuan Yu,1 Mei Li,2 Dongyu Zhang,1 William Xu,2 and Bruce C. Kone1

1Departments of Medicine and of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida; and 2Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas

Submitted 23 January 2009 ; accepted in final form 5 May 2009

The H+-K+-ATPase {alpha}2 (HK{alpha}2) 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-{kappa}B 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 HK{alpha}2 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 HK{alpha}2 gene in mIMCD3 cells in vivo. HK{alpha}2 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 HK{alpha}2 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 HK{alpha}2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HK{alpha}2 promoter without altering the binding or regulatory influence of NF-{kappa}B p65 or CREB-1 on the proximal HK{alpha}2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HK{alpha}2 gene expression in mIMCD3 cells in vivo and in vitro.

chromatin; transcription; collecting duct; transcription factor; gene expression


Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and of Biochemistry and Molecular Biology, Univ. of Florida College of Medicine, Box 100224, 1600 SW Archer Rd., Rm. CG-98, Gainesville, FL 32610 (e-mail: bkone{at}ufl.edu)






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