Context Dependent Regulation of Human Phosphoenolpyruvate Carboxykinase Isoforms by DNA Promoter Methylation and RNA Stability

ABSTRACT Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK‐C and PEPCK‐M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post‐transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK‐C than PEPCK‐M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK‐M, absence of glucose reduced the PEPCK‐C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK‐C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK‐C but not PEPCK‐M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3′‐UTR region of PEPCK‐C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK‐C but not PEPCK‐M promoter was activated by 5‐aza‐2‐deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5′‐UTR region. Taken together, our data suggests stable PEPCK‐M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK‐C and PEPCK‐M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506–2520, 2016. © 2016 Wiley Periodicals, Inc.