CREB trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene

Despite its key role in potassium homeostasis, transcriptional control of the H(+)-K(+)-ATPase alpha(2)-subunit (HKalpha(2)) gene in the collecting duct remains poorly characterized. cAMP increases H(+)-K(+)-ATPase activity in the collecting duct, but its role in activating HKalpha(2) transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKalpha(2) promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca(2+)-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKalpha(2) transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKalpha(2) mRNA levels, and CREB overexpression stimulated the activity of HKalpha(2) promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKalpha(2) promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKalpha(2) promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKalpha(2) transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKalpha(2) promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at -86/-60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HKalpha(2) promoter. In contrast, mutation of the neighboring -104/-94 kappabeta element did not alter CREB-VP16 trans-activation of the HKalpha(2) promoter. Thus CREB-1, binding to one or more CRE-like elements in the -86/-60 region, trans-activates the HKalpha(2) gene and may represent an important link between rapid and delayed effects of cAMP on HKalpha(2) activity.