AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5′-flanking region of the VIT32 gene

Abstract

. AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5Ј-flanking region of the VIT32 gene. Am J Physiol Renal Physiol 287: F460 -F468, 2004. First published May 12, 2004 10.1152/ajprenal.00107.2004.-VIT32, a vasopressin-induced transcript, inhibits Na ϩ transport when coexpressed with the epithelial sodium channel in Xenopus laevis oocytes (EMBO J 21: 5109 -5117, 2002). To understand the mechanism of VIT32 gene regulation, we examined the effect of DDAVP and cAMP stimulation on VIT32 expression in M-1 mouse collecting duct cells and in H441 human airway epithelial cells. Elevation of cAMP with forskolin and IBMX increased VIT32 gene expression with a peak effect at 2 h. The increase in gene expression was abolished by H89 and by actinomycin D, suggesting that cAMP stimulates VIT32 mRNA expression by a PKA-mediated increase in gene transcription. An ϳ1.5-kb fragment of the 5Ј-flanking region of VIT32 was cloned and was able to confer cAMP-stimulated reporter gene activity when transfected into M-1 and H441 cells. By deletion analysis and sitedirected mutagenesis, a cAMP response element (CRE) was identified within the proximal promoter region that was sufficient to account for the increase in VIT32 gene expression seen with DDAVP and elevation of cAMP. Furthermore, DDAVP-stimulated VIT32 promoterreporter activity was inhibited by H89 and by a dominant negative CREB construct. Finally, we were able to identify CREB as a nuclear protein that bound to the VIT32 CRE in gel mobility shift assays. In summary, DDAVP stimulates transcription of VIT32 via a CRE within the proximal promoter region of the VIT32 gene.PP5395; epithelial sodium channel; sodium transport; airway epithelia; adenosine 3Ј,5Ј-cyclic monophosphate response element binding protein IN THE COLLECTING duct, AVP binds the V2 receptor and regulates water homeostasis by activation of the water channel, aquaporin-2 (34). AVP also regulates Na ϩ handling in the distal nephron by its effects on Na ϩ transporters (30). The principal effect is a stimulation of benzamil-sensitive Na ϩ transport, which has been noted in the toad bladder, in the isolated rat cortical collecting duct, in primary cultures of collecting duct cells, and in many distal nephron cell lines. The early effects of AVP may be mediated by cAMP and result in an increase in surface expression of the epithelial Na ϩ channel (ENaC) (24,31,35). Others have reported an increase in Na ϩ -K ϩ -ATPase activity in microdissected mouse and rat collecting ducts and in a mouse collecting duct cell line, mpkCCD14 (6,8,13). In A6 cells, an amphibian cell line used as a model of collecting duct epithelia, the early effects of AVP effect on Na ϩ transport appear to require phosphatidylinositol-3 kinase and be mediated by the regulatory kinase sgk1 (2,11,12).Although the earliest effects of AVP on Na ϩ transport are independent of transcription, chronic effects are likely to require the transcription of target genes (18). DDAVP, a selective V2 receptor agoni...

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