Identification of an XXXX -responsive element in the 5'-flanking sequence of the @entity35 cytosolic phosphoenolpyruvate carboxykinase-1 gene, modulating its glucagon-dependent activation.

@entity268 @entity23887 @entity10298 @entity2235 @entity7954 @entity26 @entity35 @entity2755 @entity4095
The glucagon-stimulated transcription of the cytosolic @entity7954 carboxykinase-1 ( @entity23887 ) gene is mediated by @entity268 and positively modulated by @entity26 in primary hepatocytes. @entity35 hepatocytes were transfected with constructs containing the first 2500, 493 or 281 bp of the @entity23887 5'-flanking region in front of the @entity2755 acetyltransferase (CAT) reporter gene. With all three constructs glucagon induced CAT activity with decreasing efficiency maximally under arterial @entity4095 and to about 65% under venous @entity4095 . @entity35 hepatocytes were then transfected with constructs containing the first 493 bp of the @entity23887 5'-flanking region in front of the luciferase (LUC) reporter gene, which were block-mutated at the CRE1 ( @entity268 -response element-1; -93/-86), putative CRE2 (-146/-139), promoter element (P) 1 (-118/-104), P2 (-193/-181) or P4 (-291/-273) sites. Glucagon induced LUC activity strongly when the P1 and P2 sites were mutated and weakly when the P4 site was mutated; induction of the @entity10298 mutants was positively modulated by the @entity4095 . Glucagon also induced LUC activity strongly when the putative CRE2 site was altered; however, induction of the CRE2 mutant was not modulated by the @entity4095 . Glucagon did not induce LUC activity when the CRE1 site was modified. These experiments suggested that the CRE1 but not the putative CRE2 was an essential site necessary for the @entity268 -mediated @entity23887 gene activation by glucagon and that the putative CRE2 site was involved in the @entity26 -dependent modulation of @entity23887 gene activation. To confirm these conclusions @entity35 hepatocytes were transfected with @entity2235 (SV40)-promoter-driven LUC-gene constructs containing three CRE1 sequences (-95/-84), three CRE2 sequences (-148/-137) or three CRE1 sequences plus two CRE2 sequences of the @entity23887 gene in front of the SV40 promoter. Glucagon induced LUC activity markedly when the CRE1, but not when the CRE2, sites were in front of the SV40-LUC gene; however, induction of the (CRE1)3SV40-LUC constructs was not modulated by the @entity4095 . Glucagon also induced LUC activity very strongly when the CRE1 and CRE2 sites were combined; induction of the (CRE1)3(CRE2)2SV40-LUC constructs was positively modulated by the @entity4095 . These findings corroborated that sequences of the putative CRE2 site were responsible for the modulation by @entity26 of the CRE1-dependent induction by glucagon of @entity23887 gene transcription.
