title: Transactivation of the human renin promoter by the cyclic AMP/protein kinase A pathway is mediated by both cAMP-responsive element binding protein-1 (CREB)-dependent and CREB-independent mechanisms in Calu-6 cells.
abstract: We examined the DNA sequence and transcription factor requirements for cAMP-induced transactivation of the human renin promoter using Calu-6 cells that express human renin mRNA endogenously. A series of constructs containing 896 base pairs of human renin 5'-flanking DNA fused to the luciferase gene and containing either the native, a consensus, or a nonfunctional cAMP response element (CRE) were used to assess DNA sequence requirements mediating the cAMP response. Expression vectors encoding the CREB-1 transcription factor, a dominant negative mutant form of CREB-1, and the catalytic subunit of protein kinase A (PKA) were used to assess transcription factor requirements mediating the cAMP response. Forskolin treatment alone only caused a 2-3-fold activation of the HREN promoter in Calu-6 cells, but nearly a 10-fold activation in JEG-3 cells, which do not express renin but are highly responsive to cAMP. Gel shift assays revealed the binding of five specific DNA-protein complexes consisting of the ATF-1 and CREB-1 transcription factors, one of which was an ATF-1.CREB-1 heterodimer suggesting the potential for regulation of CREB-1 activity by ATF-1. However, over-expression of CREB-1 did not significantly enhance forskolin-induced human renin transcriptional activity. Transfection of both Calu-6 and JEG-3 cells with a PKA expression vector resulted in a 10-fold induction of human renin transcriptional activity in constructs containing the native or consensus CRE and 5-fold activation in a construct containing a nonfunctional CRE. We confirmed that the PKA response has both a CREB-dependent and CREB-independent component by demonstrating that the PKA response was abolished by co-transfection of a dominant negative mutant form of CREB-1 into cells containing the native or consensus CRE construct but not in cells containing the nonfunctional CRE construct. We therefore conclude that the human renin promoter can be transcriptionally activated in a renin expressing cell line through the cAMP-PKA pathway and is mediated by both a CREB-dependent and CREB-independent mechanism.
authors.affiliation: Departments of Internal Medicine and Physiology & Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
authors.forename: L
authors.initials: L
authors.lastname: Ying
authors.forename: B J
authors.initials: BJ
authors.lastname: Morris
authors.forename: C D
authors.initials: CD
authors.lastname: Sigmund
mesh_terms: D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D016350:Leucine Zippers; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012083:Renin / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D051697:Activating Transcription Factor 1; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D012260:Ribonucleases; D012083:Renin