C/EBP beta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK1-F+ cells

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in ren al gluconeogenesis. Activation of various PEPCK-(2300)Luc reporter construc ts in LLC-PK1-F+ cells, a gluconeogenic line of porcine renal proximal tubu le-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK1-F+ cells prod uced multiple bands, all of which were blocked by antibodies that are speci fic for C/EBP beta but not for C/EBP alpha or C/EBP delta. Treatment of cel ls with cAMP did not affect the expression of C/EBP beta, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal -4 binding site reduced the PKA-dependent activation of PEPCK-(2300)Luc to 40% of that observed with the wild-type construct. Coexpression of a chimer ic protein containing a Gal-4 binding domain and the transactivation domain of C/EBP beta, but not of C/EBP alpha or CRE binding protein (CREB), resto red full activation by PKA. A deletion construct that lacks the activation domain of C/EBP beta functions as a dominant negative inhibitor. Thus the b inding of C/EBP beta to the CRE-1 may contribute to the cAMP-dependent acti vation of the PEPCK promoter in kidney cells.