Characterization of elements mediating regulation of phosphoenolpyruvate carboxykinase gene transcription by protein kinase A and insulin - Identification of a distinct complex formed in cells that mediate insulin inhibition

The in vivo pattern of induction of phosphoenolpyruvate carboxykinase (PEPC K) gene transcription by cAMP and its inhibition by insulin is reproduced i n H4IIe cells and is mediated by a bipartite cAMP/insulin response unit (C/ IRU) consisting of a cAMP response element (-95/-87) and an upstream enhanc er, AC (-271/-225), Studies in HepG2 cells showed that binding of AP-1 and CAAT/enhancer-binding protein (C/EBP) to AC is required for induction by cA MP, but insulin did not inhibit cAMP-induced PEPCK expression in HepG2 cell s. Binding of H4IIe nuclear proteins to an AC element probe was inhibited b y antibodies or a consensus site for C/EBP, but not AP-1, Transfection with dominant negative bZIP factors, which prevent endogenous factors from bind ing to DNA, showed that elimination of cAMP regulatory element-binding prot ein CREB or C/EBP activity blocked induction by protein kinase A (PRA), whe reas elimination of AP-1 activity had no effect. In addition, promoters wit h multiple CREB sites, or a single CREB site and multiple C/EBP sites, medi ated PKA induction, but this was inhibited to no greater extent than basal activity was by insulin. These results indicate that an AC factor other tha n C/EBP must mediate insulin inhibition. An A-site probe (-265/-247) or a p robe across the middle of the AC element (-256/-237) competed for complexes formed by factors other than AP-1 or C/EBP, However, analysis of competito r oligonucleotides and antibodies for candidate factors failed to identify other factors. Scanning mutations throughout the AC element interfered with induction but allowed us to define five overlapping sites for regulatory f actors in AC and to design probes binding just one or two factors, Comparis on of the protein-DNA complexes formed on these smaller probes revealed tha t a specific complex present in rat liver and H4IIe cell nuclear extracts d iffered from those formed by HepG2 cell nuclear extracts. Our results sugge st that multiple factors binding the AC element of the C/IRU interact with each other and CREB to regulate PEPCK induction by cAMP and inhibition by i nsulin and that the unique factor expressed in H4IIe cells is a candidate f or involvement in insulin regulation of PKA-induced PEPCK gene transcriptio n.