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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/28756
Title: Identification of the cyclic AMP responsive element (CRE) that mediates transcriptional regulation of the pyruvate carboxylase gene in HepG2 cells
Authors: Ansaya Thonpho
Chutima Sereeruk
Pinnara Rojvirat
Sarawut Jitrapakdee
Mahidol University
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 19-Mar-2010
Citation: Biochemical and Biophysical Research Communications. Vol.393, No.4 (2010), 714-719
Abstract: 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 μM of glucagon increased the expression of PC mRNA threefold within 72 h. Treatment with 1 mM 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 1 mM 8-Br-cAMP and 0.5 μM 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. © 2010 Elsevier Inc. All rights reserved.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=77949492726&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/28756
ISSN: 10902104
0006291X
Appears in Collections:Scopus 2006-2010

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