Hormonal regulation of the phosphoenolpyruvate carboxykinase gene - Role of specific CCAAT/enhancer-binding protein isoforms

The CCAAT/enhancer-binding protein alpha (C/EBP) is a transcription factor that trans-activates a number of metabolically important genes. Previous work has demonstrated that C/EBP alpha and C/EBP beta have the potential to mediate the cAMP responsiveness of phosphoenolpyruvate carboxykinase (PEPCK) in liver cells. However, these studies used GAL4 fusion proteins and artificial promoter-reporter gene vectors in transfection experiments; as a result, these studies only indicated that both isoforms had the potential to mediate the hormonal response and not which isoform actually participated in vivo. To address this issue, Fee produced hepatoma cell lines that stably expressed either a dominant negative inhibitor or antisense RNA for these two main liver C/EBP isoforms, Inhibition of all C/EBP isoforms via expression of the dominant negative protein eliminated cAMP responsiveness, and reduced glucocorticoid responsiveness, of the endogenous PEPCK gene in hepatoma cells. Antisense directed against C/EBP alpha mRNA, which reduced C/EBP alpha protein levels by nearly 80%, also significantly reduced the cAMP responsiveness of the endogenous PEPCK promoter, whereas antisense directed against C/EBP beta was without effect. There was no major alteration in cAMP signaling in the C/EBP alpha antisense cells, as cAMP induction of the C/EBP beta gene was similar to that in wild-type H4IIE cells. These data suggest that the alpha-isoform of C/EBP is specifically utilized for mediating the cAMP responsiveness of the PEPCK gene.