Molecular mechanism of transcriptional regulation of the phosphoenolpyruvate carboxykinase (GTP) gene by cyclic AMP

The gene for phoshoenolpyruvate carboxykinase (GTP) (PEPCK) encodes a rate limiting enzyme in the gluconeogenic pathway. In this study, the 563 base pair promoter from -490 to +73 of the PEPCK gene was ligated to the structural gene for bacterial chloramphenicol acetyltransferase (CAT). The hormonal regulation of PEPCK gene transcription was studied by transient transfection into hepatoma cells in culture. This region of the PEPCK promoter can be regulated by cAMP, the cAMP-dependent kinase (PKA), glucocorticoids, thyroid hormones, and insulin. The ten protein binding domains previously detected by footprinting analysis were changed by site-directed mutagenesis. These replacements disrupted the binding of proteins from rat liver nuclei at each specific domain. The changes in function caused by the introduction of specific mutations were examined by transfecting these mutant plasmids into HepG2 cells in the presence of 8-Br-cAMP or by co-transfection with an expression vector for the catalytic subunit of cAMP-dependent kinase A (C subunit of PKA). Two primary regions, the cAMP responsive element at -96 to -77 (cAMP responsive element 1, CRE-1), and another at -242 to -248 (P3(I)), were found to cooperatively medi ate the induction of transcription by cAMP. Another two regions, -249 to -259 (P3(II)) and -270 to -285 (P4) also contributed to such cooperativity. Furthermore, induction of transcription is mediated through the TATA box. Mutation of the TATA box resulted in a change in the start site of transcription-24 in the PEPCK promoter and a significant decrease in the extent of induction of transcription by 8-Br-cAMP. CRE-1 and P3(I) bind the CCAAT/Enhancer Binding Protein (C/EBP) which is present in the liver. Interestingly, both the CRE-1 and P3(I) sites are required for the induction of transcription by C/EBP. P3(I) is uniquely a C/EBP binding site, while CRE-1 can bind to several transcription factors including C/EBP, Fos/Jun, and CREB (cAMP Responsive Element Binding Protein). The CRE-1 site was changed to an optimal C/EBP binding site. This modified promoter maintained C/EBP binding but had a significantly decreased binding affinity for CREB. The induction of transcription by 8-Br-cAMP or the catalytic subunit of PKA from this modified promoter was the same as from the native PEPCK promoter. These results suggest that C/EBP like protein(s) is involved in mediating the effect of cAMP on transcription of the PEPCK gene