Hepatocyte Nuclear Factor‐4 Phosphorylation Mediated by Inflammatory Response

Abstract

Hepatocyte nuclear factor (HNF)‐4, a liver‐enriched transcription factor, regulates a large number of liver‐specific genes and plays an important role in liver development and maintenance of liver phenotype. While HNF‐4 function is regulated by phosphorylation, only a limited number of phosphorylation sites have been identified, and its roles and dynamic changes in response to inflammatory stimulation are not explored. Utilizing the cytokine treated HNF‐4 enriched HepG2 cells, we have quantitatively analyzed HNF‐4 phosphorylation by using the combined stable isotopic variants of dithiothreitol with mass spectrometry. Twelve serine/threonine phosphorylation sites in the diverse functional domains of HNF‐4 were identified and quantified after cytokine exposure. Cytokine treatment (IL‐6, IL‐1β and TNF‐α) causes a dynamic change in HNF‐4 phosphorylation in a peptide‐and time‐dependent manner. Protein kinase A (PKA) significantly reduces HNF‐4 binding activity, which mimics the repressive effect of cytokines, and a PKA inhibitor partially restores the reduced HNF‐4 binding activity induced by cytokines. These results suggest that cytokines mediate the phosphorylation/dephosphorylation levels of HNF‐4, and a PKA signaling pathway involves in the modification of HNF‐4 function during the inflammatory response.