title: CCAAT/enhancer binding protein regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells.
abstract: The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPalpha began to decrease after 1 h of hepatocyte culture, and C/EBPbeta was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPalpha and C/EBPbeta resulted in 9.1-fold and 3. 8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPalpha and C/EBPbeta regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Genetic Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea.
authors.forename: J W
authors.initials: JW
authors.lastname: Kim
authors.forename: Y H
authors.initials: YH
authors.lastname: Ahn
mesh_terms: D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D051273:Glucose Transporter Type 2; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase