Sp1 trans-activates the murine H+-K+-ATPase α2-subunit gene

Yu Z, Li M, Zhang D, Xu W, Kone BC. Sp1 trans-activates the murine H+-K+-ATPase alpha(2)-subunit gene. Am J Physiol Renal Physiol 297: F63-F70, 2009. First published May 13, 2009; doi:10.1152/ajprenal.00039.2009.-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.