Hepatocyte Nuclear Factor-4 Is a Novel Downstream Target of Insulin via FKHR as a Signal-regulated Transcriptional Inhibitor

Abstract

HNF-4, 1 a member of the steroid/thyroid nuclear receptor superfamily, is a transcriptional factor which expresses in the liver, intestine, kidney, and pancreatic ␤-cells (1, 2). It contains several functional domains: a ligand-independent activation domain (AF1), a zinc finger DNA binding domain, and a ligand-dependent activation domain (AF2) (3). HNF-4 binds to a specific DNA element as a homodimer and regulates the expression of many genes, involved in glucose, fatty acid, and cholesterol metabolisms (4 -6). The blood glucose levels are controlled by the balance of two opposing hormones, glucagon and insulin. Whereas glucagon decreases the activity of HNF-4, the effect of insulin on that of HNF-4 has not fully been understood yet (7-9).FKHR, a forkhead family member, is a transcriptional factor and regulates the expression of multiple genes, such as key enzymes of gluconeogenesis (10, 11). Insulin has a dynamic effect on the localization of FKHR, when phosphorylated by Akt at the three residues of FKHR: Thr-24, Ser-253, and Ser-316. Once phosphorylated, the cytoplasmic retention is induced, leading to inhibit the transcriptional activity. On the other hand, in the absence of insulin, FKHR is dephosphorylated and localized to the nucleus, where FKHR binds to the specific DNA element, resulting in transcriptional activation of the target genes (12-15).Recently, it has been reported that FKHR activates or represses the transactivation by nuclear receptor family members as a DNA binding-independent cofactor (16,17). In the present study, we analyzed the interaction of FKHR with HNF-4, the effect of FKHR on the transactivation mediated by HNF-4, and its molecular mechanism. FKHR associates with HNF-4 in vivo and in vitro and represses the transactivation by HNF-4 through the decrease in its DNA binding affinity. Interestingly, the inhibitory effect is canceled by insulin, resulting from the dissociation of HNF-4 from phosphorylated FKHR that subsequently translocates to the cytoplasm. This suggests the possibility that HNF-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor. EXPERIMENTAL PROCEDURESCell Culture and Transfections and Reporter Gene Assays-HepG2 cells were cultured in Dalbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfections were performed by FuGENE-6 (Roche Molecular Biochemicals). Twenty ng of pCMV-␤gal plasmid were included in each transfection experiment to control for the efficiency of transfection. To ensure equal DNA amounts, empty plasmids were added in each transfection. The luciferase activity was measured with an ARVO™SX (Wallac Berthold). The values were normalized to ␤-galactosidase activity as an internal control.Plasmids-The hHNF-4 ␣2 cDNA was subcloned into pcDNA3 tagged with the HA epitope (pcDNA3HA). A series of HNF-4 deletion fragments were generated by PCR and subcloned into pGEX 4T-1 (Amersham Biosciences) or pcDNA3HA. The pGAL4-HNF-4 vector was made by subcloning hHNF-4 ␣2 cDNA tagged with the Gal4 DN...

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