C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells.

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK(-2300)Luc reporter constructs in LLC-PK(1)-F+ cells, a gluconeogenic line of porcine renal proximal tubule-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-PK(1)-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta. Treatment of cells with cAMP did not affect the expression of C/EBPbeta, 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 chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta, but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.