CAMP ACTIVATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE TRANSCRIPTION INRENAL LLC-PK1-F+ CELLS

Phosphoenolpyruvate carboxykinase (PCK) is a key regulatory enzyme in renal ammoniagenesis and gluconeogenesis. LLC-PK1-F+ cells are porcine renal proximal tubule-like cells that express significant levels of t he cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAM P) for 8 h causes a 21-fold increase in PCK mRNA. This response is ver y rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfecte d with PCK(-490)CAT exhibit an 8 to 10-fold increase in chloramphenico l acetyltransferase (CAT) activity when treated with cAMP for 24 h. Th e addition of okadaic acid, a protein phosphatase inhibitor, both stim ulated the CAT activity and potentiated the cAMP effect by twofold, su ggesting that phosphorylation may contribute to the transcriptional ac tivation. Assays using a series of PCK-CAT constructs containing speci fic deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experimen ts using dominant negative expression vectors indicated that a CCAAT e nhancer binding protein (C/EBP) transcription factor, and not CREB, me diates cAMP activation of transcription in LLC-PK1-F+ cells.