[Effects of lipid rafts on signal transmembrane transduction mediated by c-Met].

OBJECTIVE: To study the effects of lipid rafts on cell signal transmembrane transduction mediated by c-Met. METHODS: After HepG2Cells were treated with MbCD to disrupt the lipid rafts and were treated with artificial recombination hepatocyte growth factor to activate c-Met, the activities of PLCr1/PKC, PI3K/Akt and MAPK signaling pathways in HepG2 cells were analyzed using Western blot. RESULTS: (1) After disruption of lipid rafts with MbCD, phosphorylation of PLCr1 decreased by 35% (P = 0.022); the content of PLCr in the cytoplasm increased by 1.75 fold (P = 0.017); PLCr1 conjugated with membrane decreased by 30% (P = 0.037). (2) The content of PKB in the cytosol decreased by 38% (P = 0.028), and the phosphorylation level of PKB conjugated with membrane decreased by 14% (P = 0.041). At the same time, PDK translocation from cytosol to the plasma membrane and its activation were inhibited by treatment with MbCD. (3) Treatment with MbCD had no significant effect on ErK/MAPK, p38/MAPK and JNK/MAPK signaling pathways. CONCLUSION: Disruption of lipid rafts with MbCD inhibits the activation of PLCr1/PKC and PI3K/PKB signaling pathways by HGF/cMet, but has no effect on MAPK signaling pathway.

[Overexpression of PEMT2 inhibits the phosphorylation and translocation of PLC gamma 1].

OBJECTIVES: To explore the mechanism of CBRH-7919 cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2). METHODS: The effects of PEMT2 transfection on phosphorylation and translocation from cytosol to plasma membrane of PLC gamma 1 in cells were studied using SDS-PAGE and Western blot techniques. The phosphorylation and activity of c-Met were determined. RESULTS: After transfection of pemt2, the PLC gamma 1 and phosphorylated PLC gamma 1 conjugated with plasma membrane were decreased by 45% and 27% of that of control cells respectively, and the phosphorylated c-Met was decreased to 32% of that of control cells. CONCLUSION: Transfection of phosphatidylethanolamine N-methyltransferase 2 gene can inhibit the phosphorylation and translocation from cytosol to plasma membrane of PLC gamma 1 in cells. At the same time, the autophosphorylation of c-Met was decreased, which suggests that transfection of phosphatidylethanolamine N-methyltransferase 2 gene can downregulate the c-Met/PLC gamma 1 signaling pathway in CBRH-7919 cells.

[Effects of overexpression of PEMT2 on expression and translocation of different PKC isoforms in rat hepatoma cells].

OBJECTIVE: To explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2). METHODS: The expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique. RESULTS: Transfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection. CONCLUSION: Effects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.

Effects of ganglioside GM3 on phospholipid turnover of human leukemic J6-2 cells.

Ganglioside GM3 was reported to induce the differentiation of HL-60 cells to differentiate along the macrophage-monocytic route. We used human monocytoid leukemia J6-2 cells and successfully induced differentiation by GM3. Because differentiation is accompanied by retarded growth rate and cell cycle is intimately related to phospholipid metabolism, so we explored how GM3 was related to phospholipid metabolism. By using [32P]Pi, [3H-CH3]choline, [3H-CH3]SAM, and [3H]inositol as radioactive tracers, we studied the turnover changes of phospholipids and their metabolites induced by GM3. For the morphological changes of differentiation to occur, the cells had to be treated with GM3 at a concentration of 50 microM for 5-6 days, but the phospholipid changes occurred at a very early stage of GM3 treatment (only 1 h). Our results indicate that GM3 stimulated PE methylation pathway inhibited both CDP-choline pathway and PI cycle. The phospholipid changes may constitute the early events in differentiation induced by GM3.

Overexpression of PEMT2 downregulates the PI3K/Akt signaling pathway in rat hepatoma cells.

Phosphatidylethanolamine N-methyltransferase 2 (PEMT2) is an isoform of PEMT that converts phosphatidylethanolamine to phosphatidylcholine in mammalian liver. Overexpression of PEMT2 led to inhibition of proliferation of hepatoma cells [J. Biol. Chem. 269 (1994) 24531]. The present study aims to unravel the molecular mechanism of the reduced proliferation, especially the signaling transducer proteins involved in this process. Thus, we chose PI3K/Akt pathway that is initiated by growth factors and leads to cell survival and proliferation. Rat hepatoma CBRH-7919 cells transfected with pemt2-cDNA showed that: (1) signaling proteins including c-Met, PDGF receptor, PI3K, Akt and Bcl-2 all had reduced expression as shown by Western blotting studies; (2) flow cytometric and DNA ladder assays showed that 22.9% of the pemt2-transfected cells were undergoing apoptosis; (3) the activity of Akt was decreased as shown by Western blotting using antibody directed against p-Akt (Thr308); (4) wortmannin and PD98059, inhibitors of PI3K and MEK, respectively, both inhibited Akt activity, indicating that PI3K and MAPK pathways were merging at Akt in CBRH-7919 cells. The above results suggest that overexpression of PEMT2 strongly downregulated the PI3K/Akt signaling pathway at multiple sites and induced apoptosis. This, at least partly, explains the molecular mechanism of impaired proliferation induced by pemt2 transfection.