Insulin-induced early growth response gene (Egr-1) mediates a short term repression of rat melic enzyme gene transcription

In this report we have studied insulin regulation of malic enzyme (ME) gene transcription in rat H-35 hepatoma cells and localized the insulin-respons ive region of the ME promoter between positions -177 and -102. This region contains a putative insulin response element (IRE-II), When nuclear extract s from untreated or insulin-treated H-35 cells were incubated with IRE-II!, transcription factors Spl and Sp3 were observed to, bind constitutively to this element, whereas insulin induces the quick and transient binding of a n insulin response factor. This induction requires de novo protein synthesi s, Competition and supershift assays demonstrated that the insulin response factor is the immediate-early gene Egr-1. In vitro assays revealed that Eg r-1 displaces Spl from its binding site in IRE-II. Insulin induces Egr-1 mR NA,with a time course pattern that corresponds perfectly to the Egr-1 bindi ng to IRE-II. This induction depends on the activation of mitogen-activated protein (MAP) kinase, and it is phosphatidylinositol 3-kinase-independent, as demonstrated with specific inhibitors for both pathways. By cotransfect ing the wild-type or a dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras inhibits ME promoter activity. Furthermore, overex pression of Egr-1 in H-35 cells represses the ME gene promoter in a dose-de pendent manner. These results suggest that insulin induces a quick, transie nt, and Ras/MAP kinase-dependent activation of Egr-1 which leads to a trans ient repression of ME gene transcription. On a late phase, insulin would ac tivate a different, Egr-1-independent pathway, which would result in activa tion of the ME gene.