| Objective: Hepatocyte growth factor is one kind of polypeptide cytokines generated from mesenchymal cells(such as mechanocyte and macrophage etc.). HGF has many important biological functions, such as promoting proliferation and division of cells; suppressing the formation of cell gap junctions and cell adhere lead to cell scattering ; up-regulating the expression of urokinase plasminogen activator and its receptor (uPA/uPAR), promoting the degradation of extracellular matrix; increasing the phosphorylation of cytoskeletal protein and interfering the rearrangment of cytoskeleton, which make cells apt to migration. So, HGF is also named as mitogenic factor,scattering factor,morphogen. HGF plays an important role in regulation of embryo development,organic formation,cells growth,proliferation and migration . It is also concerned with generation,development,diffusion and metastasis of tumor.The receptor of HGF is coded by proto-oncogene c-met and named as c-Met. c-Met is mono- transmembrane receptor containing tyrosine protien kinase activity and often expresses in epithelial cell(such as liver,kidney,enteron). HGF acts on target cell by paracrine after secretion from mesenchymal cells. Once conjugating with ligands, the receptors are dimerizated and autophosphorylated, which forms the phosphorylated tyrosine residues in the intracellular domain of receptors. Signal molecules containing SH2 structure bind with receptors by recognizating phosphorylated tyrosine residues, then ignite the signaling pathway in cells. It is known that, at least, there are three signaling pathways actived by HGF/c-Met, including MAPK signaling pathway, PLCγ1/DAG/PKC signaling pathway and PI3K/PDK/PKB signaling pathway. Different signaling pathway participates in the regulation of different cell function. Activation of MAPK signaling pathway promotes cell multiplicative division, the other two are relevant to deformation and migration of cells.Lipid rafts are microdomains with unique structure and function on the plasma membrane, which rich in sphingolipid and cholesterol. Lipid rafts have a lot of important biological functions, including material conveying in and out of cells, signal transmembrane transduction, cell adherency and microbic infection et al. Especially, in the filed of signal transduction, Lipid rafts are considered as a working platform for signaling in plasma membrane. The signal transmembrane transduction is a process of mutual recognizing,mutual activating of receptor with ligand and some signal molecules. So, a proper plasma membrane microenvironment is necessary. The present of lipid raft theory promotes deep study on the molecular mechanisms of receptor function. At present, it has found that the functions of many plasma membrane receptors such as EGFR,INR,PDGFR,FGFR,TCR,BCR were concerned with lipid rafts. However the effect of lipid rafts on HGF receptor mediated extracellular signal transmembrane transduction remains unclear. This study will explore the effect of lipid rafts on HGF receptor mediated signaling pathway.Method: Human hepatoma HepG2 cells were cultured in MEM medium without serum for 24 hours, then divided into group treated with MβCD and control group. After the Cells were treated with MβCD, the Cells treated with MβCD and control cells were incubated with artificial recombination hepatocyte growth factor in order to active c-Met. Then the cells were harvested, and activity of signaling pathways(PLCγ1/DAG/PKC,PI3K/PDK/PKB and MAPK) were analysed by using SDS-PAGE,Western Blotting and compute scanning technique.Result: 1) After disruption of lipid raft in HepG2 cell with MβCD ,the phosphorylation of c-Met and PLCγ1 decressed by 22% and 35% comparing with the control group; The content of PLCγ1 in the cytoplasm increased by 1.75 fold; PLCγ1 conjugated with membrane decreased by 30%. It suggested that disruption of lipid raft inhibited the translocation of PLCγ1 from the cytosol to plasma membrane and its phosphorylation. The results suggested that the treatment of MβCD can inhibit the activation of PLCγ1/DAG/PKC signaling pathway by HGF/c-Met mediated. 2) After treatment with MβCD, the content of PKB in the cytosol decreased by 38% comparing with the control group , meanwhile the phosphorylation level of PKB conjugated with membrane decreased by 14%. At the same time, the PDK translocation from the cytosol to the plasma membrane and its activation were inhibited by treatment with MβCD. The results showed that the activation of PI3K/PDK/PKB signaling pathway was inhibited by disruption of lipid raft with MβCD. 3) We also observed the effect oftreatment with MβCD on MAPK signaling pathway(ErK/MAPK,p38/MAPK and JNK/MAPK) by Western blot technology. The results showed that disruption of lipid raft has no significant effect on MAPK signaling pathway.From the finding above, we summarized that disruption of lipid raft with MβCD can inhibit the activating of PLCγ1/DAG/PKC signaling pathway and PI3K/PDK/PKB signaling pathway by HGF/cMet, but have no effect on MAPK signaling pathway. It was postulated that disruption of lipid raft by treatment with MβCD could affect the function of c-Met via two ways. One is affected receptor dimerization or the extent of dimerization, which changed the conformation of receptor intracellular domain. As a result, intracellular signal molecules with SH2 structure couldn't bind to the receptor and activate the intracellular signal transduction pathway. On the other hand, because different signal molecules identify distinct tyrosine residue phosphorylation site, disruption of lipid raft possibly inhibits the phosphorylation of certain tyrosine residue, to which downstream signal molecules can't bind , likewise, the activation of intracellular signaling pathway can be inhibited. |
