Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines.
To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPalpha) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPalpha and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPalpha upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPalpha. Actinomycin D was used to analyze half-life of C/EBPalpha mRNA. IGF-II up-regualted C/EBPalpha by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPalpha. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPalpha while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase.
Tomizawa
Minoru
M
Department of Medicine and Clinical Oncology, Chiba University Graduate School of Medicine, Chiba City, Chiba 260-8670, Japan. nihminor-cib@umin.ac.jp
Saisho
Hiromitsu
H
D000730:Androstadienes / Q000494:pharmacology; D023081:CCAAT-Binding Factor / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018197:Hepatoblastoma; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D008113:Liver Neoplasms; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D020871:RNA Stability; D015854:Up-Regulation; D000077191:Wortmannin
D000730:Androstadienes; D023081:CCAAT-Binding Factor; D002867:Chromones; D004791:Enzyme Inhibitors; D007335:Insulin-Like Growth Factor II; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one