CCAAT-enhancer binding protein-α (C/EBPα) and hepatocyte nuclear factor 4α (HNF4α) regulate expression of the human fructose-1,6-bisphosphatase 1 (FBP1) gene in human hepatocellular carcinoma HepG2 cells

Fructose-1,6-bisphosphatase (FBP1) plays an essential role in gluconeogenesis. Here we report that the human FBP1 gene is regulated by two liver-enriched transcription factors, CCAAT-enhancer binding protein-alpha (C/EBP alpha) and hepatocyte nuclear factor 4 alpha (HNF4 alpha) in human hepatoma HepG2 cells. C/EBP alpha regulates transcription of FBP1 gene via binding to the two overlapping C/EBP alpha sites located at nucleotide-228/-208 while HNF4 alpha regulates FBP1 gene through binding to the classical H4-SBM site and direct repeat 3 (DR3) located at nucleotides-566/-554 and-212/-198, respectively. Mutations of these transcription factor binding sites result in marked decrease of C/EBP alpha- or HNF4 alpha-mediated transcription activation of FBP1 promoter-luciferase reporter expression. Electrophoretic mobility shift assays of -228/-208 C/EBP alpha or -566/-554 and -212/-198 HNF4 alpha sites with nuclear extract of HepG2 cells overexpressing C/EBP alpha or HNF4 alpha confirms binding of these two transcription factors to these sites. Finally, we showed that siRNA-mediated suppression of C/EBP alpha or HNF4 alpha expression in HepG2 cells lowers expression of FBP1 in parallel with down-regulation of expression of other gluconeogenic enzymes. Our results suggest that an overall gluconeogenic program is regulated by these two transcription factors, enabling transcription to occur in a liver-specific manner.