Abstract
In this report we have studied insulin regulation of malic enzyme (ME) gene transcription in rat H-35 hepatoma cells and localized the insulin-responsive region of the ME promoter between positions -177 and -102. This region contains a putative insulin response element (IRE-II). When nuclear extracts from untreated or insulin-treated H-35 cells were incubated with IRE-II, transcription factors Sp1 and Sp3 were observed to bind constitutively to this element, whereas insulin induces the quick and transient binding of an insulin response factor. This induction requires de novo protein synthesis. Competition and supershift assays demonstrated that the insulin response factor is the immediate-early gene Egr-1. In vitro assays revealed that Egr-1 displaces Sp1 from its binding site in IRE-II. Insulin induces Egr-1 mRNA, with a time course pattern that corresponds perfectly to the Egr-1 binding 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 cotransfecting the wild-type or a dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras inhibits ME promoter activity. Furthermore, overexpression of Egr-1 in H-35 cells represses the ME gene promoter in a dose-dependent manner. These results suggest that insulin induces a quick, transient, and Ras/MAP kinase-dependent activation of Egr-1 which leads to a transient repression of ME gene transcription. On a late phase, insulin would activate a different, Egr-1-independent pathway, which would result in activation of the ME gene.
Highlights
Apart from a well reported role in metabolism, insulin behaves as a mitogen [1, 2] and differentiation factor [3, 4] for a great number of cells and tissues
In a previous work [27] we described the existence of two putative Insulin response elements (IREs) within the malic enzyme (ME) promoter: IRE-I, which resembles the negative IRE described in the phosphoenolpyruvate carboxykinase promoter [10], and IRE-II, which is similar to the positive IRE-A element of the glyceraldehyde-3-phosphate dehydrogenase gene protrophoretic mobility shift assay; translation system (TNT), transcription/translation; MEK, mitogen-activated protein (MAP)/ERK kinase
The insulin response is completely lost with a subsequent deletion of 75 base pairs, indicating that a positive IRE of the rat ME gene promoter is located between positions Ϫ177 and Ϫ102
Summary
Apart from a well reported role in metabolism, insulin behaves as a mitogen [1, 2] and differentiation factor [3, 4] for a great number of cells and tissues. Because IRE-II contains putative binding sites for Sp1 (Fig. 2A), we examined if any of the complexes detected in H-35 cells corresponded to this transcription factor or a related protein.
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