[The role of lipid raft in TNFR1-mediated signal transduction in osteoblasts]

To investigate the role of membrane cholesterol in TNFR1-mediated signal transduction in osteoblastic MC3T3 cells.

MCD binds cholesterol specifically and was commonly used to deplete cholesterol from cell plasma membrane. MC3T3 cells were serum-starved for 22 h, treated with MCD (10 g/L) for 60 min followed by TNF-α (10 μg/L) for 0, 5, 10, 15 or 30 min, or TNF-α plus CHX (10 mg/L) for 4 h to induce apoptosis, then TNFR1-mediated IκBα degradation, phosphorylation of AKT, ERK or p38, and processing of caspase-3 were analyzed by using SDS-PAGE/Western blotting method.

MC3T3 cell membrane cholesterol level was reduced to 35% within 60 min by MCD (10 g/L). Reduction of MC3T3 cell surface cholesterol dramatically inhibited TNFR1-mediated AKT phosphorylation, while did not affect the degradation of IκBα, activation of ERK or p38, and processing of caspase-3 induced by TNF-α.

Cholesterol depletion can destruct lipid rafts; therefore our results suggest that lipid raft is essential for TNFR1-mediated AKT phosphorylation, but is dispensable for TNFR1-mediated degradation of IκBα, activation of ERK or p38 and processing of caspase-3.