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.
VIT32, a vasopressin-induced transcript, inhibits Na(+) 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 approximately 1.5-kb fragment of the 5'-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 site-directed 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 promoter-reporter 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.
Thomas
Christie P
CP
Department of Internal Medicine, Graduate Program in Molecular Biology, University of Iowa College of Medicine, Iowa City, Iowa, USA. christie-thomas@uiowa.edu
Loftus
Randy W
RW
Liu
Kang Z
KZ
D024506:5' Flanking Region / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D012076:Renal Agents / Q000494:pharmacology*; D020545:Respiratory Mucosa / Q000166:cytology; D015533:Transcriptional Activation / Q000187:drug effects*
C490681:AVPI1 protein, human; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D012076:Renal Agents; C490686:VIT32 protein, mouse; D000242:Cyclic AMP; D003894:Deamino Arginine Vasopressin