INVOLVEMENT OF 3',5'-CYCLIC ADENOSINE-MONOPHOSPHATE REGULATORY ELEMENT-BINDING PROTEIN (CREB) IN BOTH BASAL AND HORMONE-MEDIATED EXPRESSIONOF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) GENE

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) and PEP CK-chloramphenicol acetyltransferase (CAT) genes is induced by cAMP an d glucocorticoids and is inhibited by insulin in H4IIE cells, as it is in liver. In contrast, PEPCK-CAT expression in HepG2 cells is not aff ected by insulin but is induced by cAMP, which in turn is repressed by glucocorticoids. Mutations were introduced into well defined transcri ption factor binding sites to investigate possible interactions betwee n the cAMP regulatory element (CRE) binding protein (CREB) and glucoco rticoid response unit (GRU) binding proteins. H4IIE rat hepatoma cells were transfected with PEPCK-CAT plasmids with or without an expressio n vector for protein kinase A (PKA). Glucocorticoid-induced CAT activi ty was dependent upon the GRU and was decreased in plasmids lacking th e CRE. To determine the direct effects of CREB, the DNA binding and di merization domain of GAL4 was substituted for that of CREB (CRG), and the PEPCK CRE was replaced with a GAL4 binding site (G4PEPCK-CAT). CRG elevated basal and glucocorticoid-induced activities of G4PEPCK-CAT e qually and restored responsiveness to PKA. The basal activity of CRG w as not diminished by concomitant treatment with PKA plus its inhibitor peptide, PKI, or by mutation of the PKA phosphorylation. Deletion of C-terminal regions of the CREB activation domain from CRG diminished b asal activation without affecting induction by PKA. The glucocorticoid -induced level of CAT activity decreased in proportion to the reduced ability of CREB to activate basal transcription. Induction by glucocor ticoid, in the absence or presence of PKA, was not affected by CRG, in dicating that interaction of GRU-bound factors with CREB is not requir ed for glucocorticoid induction of PEPCK. These results indicate that CREB is directly involved in basal and PKA-induced expression of PEPCK , and that CREB supports glucocorticoid-induced PEPCK expression throu gh its positive effect on basal transcription.