Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression

MARÍA E. SCASSA, ALEJANDRA S. GUBERMAN, JULIETA M. CERUTI, EDUARDO TOMAS CANEPA

JOURNAL OF BIOLOGICAL CHEMISTRY

The American Society for Biochemistry and Molecular Biology Inc

Lugar: Bethesda, EEUU; Año: 2004 vol. 279 p. 28082 - 28092

0021-9258

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5´-regulatory region allowed us to identify an insulin-responsive region located at –459 to –354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropiate expression of ALAS gene. Insulin overrides the HNF3b or HNF3b plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and southwestern blotting indicate that HNF3 binds to ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggest that HNF3b is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, southwestern blotting assay shows that at least two polypeptides other than HNF3b can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3b binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors, support this hypothesis. Do to this event, NF1 would lose accessibility to the promoter. The postranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.