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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Department of Internal Medicine and Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, and Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, Texas 77030

Submitted 5 February 2004 ; accepted in final form 21 May 2004

Despite its key role in potassium homeostasis, transcriptional control of the H⁺-K⁺-ATPase ₂-subunit (HK₂) gene in the collecting duct remains poorly characterized. cAMP increases H⁺-K⁺-ATPase activity in the collecting duct, but its role in activating HK₂ transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HK₂ promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca²⁺-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HK₂ transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HK₂ mRNA levels, and CREB overexpression stimulated the activity of HK₂ promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HK₂ promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HK₂ promoter-luciferase activity, suggesting that constitutive CREB participates in basal HK₂ transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HK₂ 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 HK₂ promoter. In contrast, mutation of the neighboring –104/–94 element did not alter CREB-VP16 trans-activation of the HK₂ promoter. Thus CREB-1, binding to one or more CRE-like elements in the –86/–60 region, trans-activates the HK₂ gene and may represent an important link between rapid and delayed effects of cAMP on HK₂ activity.

transcription; promoter; cAMP; potassium

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