Hepatocyte Growth Factor Activates Ccaat Enhancer Binding Protein and Cell Replication Via Pi3–Kinase Pathway

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Abstract
Hepatocyte growth factor (HGF), a ligand of c–Met receptor, stimulates activation of cellular kinases via phosphatidylinositol 3–kinase (PI3–kinase). CCAAT/enhancer binding protein (C/EBP) controls cell cycle progression. The present study was designed to determine whether HGF activates C/EBP in association with the S–phase entrance for cell replication and whether PI3–kinase contributes to the activation of C/EBP. Treatment of H4IIE cells, a hepatocyte–derived cell line, with HGF increased protein binding to the C/EBP binding site at an early time. Immunodepletion, subcellular fractionation, and confocal microscopic analyses showed that the HGF–induced C/EBP DNA binding activity depended on nuclear translocation of C/EBPβ. Whereas stable transfection of the p110 catalytic subunit of PI3–kinase enhanced HGF–mediated nuclear translocation of C/EBPβ and DNA binding, stable transfection of p85 subunit or chemical inhibition of PI3–kinase completely blocked C/EBP activation. HGF increased luciferase reporter activity in cells transfected with a mammalian cell expression vector containing -1.65 kilobase rGSTA2 promoter comprising C/EBP response element (pGL–1651). Whereas transfection with pCMV500, a control vector, allowed pGL–1651 to respond to HGF, expression of dominant negative mutant C/EBP completely inhibited the ability of HGF to stimulate the reporter gene expression. Flow cytometric analysis showed that HGF caused an increase in the area of S phase with a reciprocal decrease in that of G1 phase, suggesting that HGF promoted cell cycle progression to S phase. In conclusion, HGF induces nuclear translocation of C/EBPβ via the PI3–kinase pathway and stimulates C/EBP DNA binding and gene transcription and that the PI3–kinase-mediated C/EBP activation by HGF may contribute to cell replication.

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