cAMP activation of phosphoenolpyruvate carboxykinase transcription in renal 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 the cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) for 8 h causes a 21-fold increase in PCK mRNA. This response is very rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfected with PCK(-490)CAT exhibit an 8- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity when treated with cAMP for 24 h. The addition of okadaic acid, a protein phosphatase inhibitor, both stimulated the CAT activity and potentiated the cAMP effect by twofold, suggesting that phosphorylation may contribute to the transcriptional activation. Assays using a series of PCK-CAT constructs containing specific deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experiments using dominant negative expression vectors indicated that a CCAAT enhancer binding protein (C/EBP) transcription factor, and not CREB, mediates cAMP activation of transcription in LLC-PK1-F+ cells.
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523, USA.
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D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D018374:LLC-PK1 Cells; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D012333:RNA, Messenger; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)