Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells.
cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit. HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit. HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.
Eggers
A
A
Department of Biochemistry, University of Barcelona, School of Pharmacy, E-08028 Barcelona, Spain.
Caudevilla
C
C
Asins
G
G
Hegardt
F G
FG
Serra
D
D
D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements; D013004:Somatostatin / Q000235:genetics; D015533:Transcriptional Activation
D017362:Cyclic AMP Response Element-Binding Protein; D013004:Somatostatin; D000242:Cyclic AMP; D006904:Hydroxymethylglutaryl-CoA Synthase; D017868:Cyclic AMP-Dependent Protein Kinases