The Study On Mechanisms Of Cell Proliferation And Migration Induced By Hepatocyte Growth Factor

Hepatocyte growth factor (HGF) is a multifunctional polypeptide which play important roles in the regulation of cell functions including stimulating proliferation and migation, enhancing survival of various cell types. Its various effects depends on the different signal pathways. SIP, which play important roles in diverse biological processes such as cell proliferation, migration and survival, is produced by metabolism of the membrane phospholipid sphingomyelin. Sphingosine kinase (SK) is a key enzyme catalyzing the formation of SIP and also functions as an important signal molecule. However, whether SK is involved in HGF signaling remains unclear. In this study, the role of SK in the signal cascade triggered by HGF was investigated in human hepatoma cell lines HepG2 and SMMC-7721.The expression of c-met, SK and the receptors of SIP (EDG-1, EDG-3, EDG-5) was detected by RT-PCR and immunohistochemical analysis. HepG2 and SMMC-7721 express these molecules. So, both of cell lines are the ideal models to assay the interactions of SK and HGF. Recombinant HGF (rhHGF) induces proliferation mildly of hepatoma cells while it was a strong stimulator for the migration of these cells. To determine whether HGF could activate the SK, we established an assay method of cellular activitiy of SK by use of y-32PATP incorporation. HGF could activate SK in a concentration-dependent manner. It is reported that binding of c-met by HGF leads to activation of several signals such as PI-3K, MAPK and Rho signal. Both LY294002, a specific inhibitor of PI-3K, and PD98059, a specific inhibitor of MAPK blocked the activation of SK stimulated by HGF. It is suggested that SK is a downstream molecule of PI-3K and MAPK in the HGF signal pathway.To determine whether the activation of SK is involved in the biological functions of HGF, we used DMS, a competitive inhibitor of SK, to block the SK pathway. Theblockage of SIP production by DMS significantly decreased the HGF induced migration and proliferation of hematoma cells. In additon, LY294002, a specific inhibitor of PI-3K, and PD98059, a specific inhibitor of MAPK have the same effects, while Y27632, a specific inhibitor of Rho has no effects. These results suggest that SK is involved and plays an important role in the proliferation and migration of hepatoma cells induced by HGF.To establish a novel approach to trigger c-Met signal cascade without ligands' binding by chemical reagents, we expressed a fusion protein of intracellular domain of c-met with FKBP in cells and induced the dimerization of this fusion protein by a synthetic drug, AP20187. An intracellular domain of human c-met was amplified by RT-PCR and cloned to T easy vector, then this gene was sequenced, fused with FKBP and subcloned into pIRESneo expression vector. The expression vector was transfected into Mo7e and SMMC-7721 cells by Lipofectamine. The expression of fusion protein was determined by RT-PCR and Western Blot. Treatment the SMMC-7721 cells expressing this fusion gene with AP20187 results in activation of SK. Thus, this study will supply a pilot beginning for chemical activated c-Met signaling.