Protein O-N-acetylglucosaminylation modulates promoter activities of cyclic AMP response element and activator protein 1 and enhances E-selectin expression on HuH-7 human hepatoma cells.
High glucose accelerates O-N-acetylglucosaminylation (O-GlcNAcylation) of proteins and causes diabetic complications. In the present study, we found that treatment of HuH-7 human hepatoma cells with high glucose or the protein O-N-acetylglucosaminidase (O-GlcNAcase) inhibitor O-(2-acetoamide-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) increased the cell surface expression of E-selectin. A dual luciferase reporter assay indicated that high glucose and PUGNAc suppressed promoter activities of the cyclic AMP response element (CRE) and enhanced those of activator protein 1 (AP-1). Enhanced CRE promoter activities in HuH-7 cells treated with dibutyryl cAMP or co-transfected with a protein kinase A expression vector pFC-PKA that enhances the phosphorylation of CRE binding protein (CREB) were suppressed by PUGNAc. In contrast, PUGNAc further increased the enhanced AP-1 promoter activity in cells transfected with a mitogen-activated protein kinase kinase kinase expression vector pFC-MEKK that enhances c-Jun phosphorylation. Immuno-blotting using an anti-O-GlcNAc antibody revealed that high glucose and PUGNAc accelerated protein O-GlcNAcylation and that there were substantial differences in the O-GlcNAcylated proteins in the cytoplasmic and nuclear fractions. In addition, PUGNAc increased the nuclear import of O-GlcNAcylated CREB. These results suggest that protein O-GlcNAcylation modulates the promoter activities of E-selectin gene, suppression of CRE and enhancement of AP-1, and enhances E-selectin protein expression on hepatocytes.
Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba247-8510, Japan. yutaro@phar.toho-u.ac.jp
Yutaro
Y
Azuma
Kana
K
Miura
Koji
K
Higai
Kojiro
K
Matsumoto
D000117:Acetylglucosamine / Q000378:metabolism*; D050882:CREB-Binding Protein / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D051786:Cyclic AMP Response Element Modulator / Q000096:biosynthesis* / Q000235:genetics; D019040:E-Selectin / Q000096:biosynthesis*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D053583:Interleukin-1beta / Q000494:pharmacology; D008113:Liver Neoplasms / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D011956:Receptors, Cell Surface; D018808:Transcription Factor AP-1 / Q000096:biosynthesis* / Q000235:genetics
D019040:E-Selectin; D053583:Interleukin-1beta; D011956:Receptors, Cell Surface; D018808:Transcription Factor AP-1; D051786:Cyclic AMP Response Element Modulator; D008156:Luciferases; D050882:CREB-Binding Protein; D005947:Glucose; D000117:Acetylglucosamine