Abstract
Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and we have reported that protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homologue of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. To our knowledge, these results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.
Highlights
From the ‡University of Illinois College of Medicine at Chicago and Chicago Area Veterans Health Care System (West Side Division), Chicago, Illinois 60612 and the §Department of Biochemistry, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
To determine whether FKHR may influence the expression of genes that are regulated by insulin through an insulin response sequence (IRS), we first performed transient transfection studies in HepG2 cells with a luciferase reporter gene construct containing the insulin-like growth factor-binding protein-1 (IGFBP-1) promoter (BP1.Luc) and a -galactosidase expression vector driven by the CMV promoter
Constructs that contain the IRS found in the phosphoenolpyruvate carboxykinase (PEPCK) or apolipoprotein CIII (apoCIII) gene (⌬IRS.1 m2 and ⌬IRS.1 m8) are responsive to both insulin and FKHR, suggesting that FKHR may stimulate the activity of the PEPCK and apoCIII promoters through related IRSs. These results indicate that FKHR and the endogenous factor(s) responsible for mediating the effects of insulin and protein kinase B (PKB) on promoter activity interact with the IRS with similar sequence specificity and suggest the possibility that endogenous FKHR or closely related proteins may contribute to the regulation of IGFBP-1 promoter activity through an IRS
Summary
From the ‡University of Illinois College of Medicine at Chicago and Chicago Area Veterans Health Care System (West Side Division), Chicago, Illinois 60612 and the §Department of Biochemistry, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Insulin suppresses the expression of a number of genes that contain a conserved insulin response sequence (IRS) (CAAAA(C/T)AA), including insulin-like growth factor-binding protein-1 (IGFBP-1), apolipoprotein CIII (apoCIII), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase [2,3,4,5,6] This observation suggests that insulin may regulate the expression of multiple hepatic genes through a common mechanism. Mutation of daf-16, which codes for a forkhead/winged-helix transcription factor, restores a normal life span and prevents entry into the dauer stage [28, 30, 31] These observations have suggested that daf-16 promotes entry into the dauer phase and enhanced longevity and that signaling via the insulin/IGF-I receptor-PI3K-PKB pathway may disrupt these effects of daf-16 [28, 30, 31]
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