Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells.
Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis. Here we investigated the effect of various hormones including cAMP, dexamethasone and insulin on the abundance of PC mRNA in the human hepatocyte cell line, HepG2. Treatment of HepG2 cells with 1 microM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1mM 8-Br-cAMP caused the abundance of PC mRNA to increase by 2-3-fold by 48 h, peak at fourfold at 72 h, and remain unchanged to 96 h. This is in contrast to phosphoenolpyruvate carboxykinase (PEPCK) for which expression was decreased after 72 h, suggesting a distinct difference in the control of these two enzymes in the long term. Dexamethasone or insulin alone did not affect the abundance of PC mRNA whereas treatment of HepG2 cells with the combination of 1mM 8-Br-cAMP and 0.5 microM dexamethasone further increased the abundance of PC mRNA, suggesting the predominant role of 8-Br-cAMP over dexamethasone. Transient transfection of the luciferase reporter construct driven by a 1.95 kbp 5'-flanking sequence of the mouse PC gene and a plasmid encoding the human cAMP-responsive element binding protein increased luciferase reporter activity to 7-fold similar to that observed with a PEPCK promoter-luciferase reporter construct. Deletion of the 5'-flanking region of the PC gene to 781 bp resulted in the complete loss of CREB-mediated induction of reporter gene, suggesting the presence of the cAMP-responsive unit is located between 1.95 kbp and 781 bp upstream of the mouse PC gene. Electrophoretic mobility shifted and chromatin immunoprecipitation assays demonstrated that CREB bind to -1639/-1631 CRE of mouse PC gene in vitro and in vivo, respectively.
Thonpho
Ansaya
A
Molecular Metabolism Research Group, Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Sereeruk
Chutima
C
Rojvirat
Pinnara
P
Jitrapakdee
Sarawut
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D005934:Glucagon / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D011766:Pyruvate Carboxylase / Q000235:genetics*; D020218:Response Elements; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D007328:Insulin; D003907:Dexamethasone; D005934:Glucagon; D000242:Cyclic AMP; D011766:Pyruvate Carboxylase