A Relationship Of Lipid Rafts And The Internalization Of TNFR1

Lipid rafts and caveolae are cholesterol and sphingolipid enriched membranemicrodomains, and play an important role in initiating signal pathways. Caveolae arebottleneck-like pits on the cell membrane as a subset of lipid rafts and a special formof lipid rafts.The internalization functions in translocation of recruitment of signal moleculesat the cell surface into the cytoplasm and activation of the signal cascade help signaltransduction[1-3]. Receptors and ligands can be internalized in different ways, such asa most common clathrin-mediated internalization, or lipid rafts-related internalization[4,5].A large number of evidences show that sTNF-α can promote either cell survivalby activating NF-κB or cell apoptosis by DISC signal complex via TNFR1. Ourprevious work confirmed that the cytotoxicity induced by sTNF-α is significantlyenhanced in Hela cells by MCD treatment to destroy lipid rafts（p<0.01）. Wepresumed that the destruction of lipid rafts may enhance sTNF-α-mediatedcytotoxicity through promotion of TNFR1internalization, suppressing the activationof NF-κB and increasing the formation of DISC signal complexes. This study mainlyexplores the relationship of lipid raft and TNFR1internalization.The main results are as follows:I. Determination of appropriate concentration of MCD1. According to our previous work and related articles, we did time course and dosedependent test for MCD to choose its best dose and incubation time point. We foundthat there was no significant cytotoxic effect of MCD on cells in concentrations equalto or lower than7.5mM for45minutes.2. Our previous work demonstrated that the destruction of lipid rafts could decreasethe cellular adhesion. Homologous adhesion test showed that cell adhesion could be significantly inhibited by treatment with MCD in concentrations higher than5mM.Therefore, we used5mM MCD to treat cells for45minutes to disrupt lipidrafts in the following experiments.II. Disruption of lipid rafts enhances sTNF-α-induced cell death and apoptosis.1. We transfected293T cells with wild-type TNFR1and TNFR1-Y236A(internalization-deficient mutant). We found that disruption of lipid rafts by MCDincreased sTNF-α-induced cytotoxicity toward wild-type TNFR1-transfectants by18.7%(p<0.05), while sTNF-α had no cytotoxic effect on TNFR1-Y236A-transfectants. This suggests that MCD may affect sTNF-α-induced cytotoxicitythrough promotion of TNFR1internalization.2. Disruption of lipid rafts of TNFR1expressed Hela cells not only promotedsTNF-α-induced cytotoxicity, but also increased the activation of caspase-8andcaspase-3, indicating that the disruption of lipid rafts facilitates sTNF-α-mediatedapoptosis.III. The disruption of lipid rafts decreases TNFR1expression on the cellsurface.Pretreatment of Hela cells with MCD to destroy lipid rafts, FCM was used todetect the expression of TNFR1after sTNF-α stimulation. The results showed thatthe disruption of lipid rafts decreased TNFR1expression on the cell surface by13.17%（p<0.05）, while results of western blotting revealed that the total cellular productionof TNFR1remained unchanged by MCD treatment. These data suggest the promotionof sTNF-α-induced TNFR1internalization by disruption of lipid rafts.IV. Construction of TNFR1mutant located in lipid rafts.Normally, TNFR1locates both inside and outside lipid rafts. To make allTNFR1in the lipid rafts, we linked the lipid rafts targeting domain (301-378) ofcaveolin1that is a protein only located in lipid rafts to TNFR1containing itsinternalization domain (1-729). This mutant was named as TNFR1-CAV that wasinserted to N1-EGFP plasmid. We transfected it into293T cells and the transfection efficiency was62.4%. After isolation of lipid rafts, all of TNFR1-CAV mutants werefound to be contained in lipid rafts.V. The internalization of TNFR1and its mutants detected by the laser scanningconfocal microscopy.293T cells were transfected with GFP-labeled TNFR1and its mutants(TNFR1-Y236A，TNFR1-ΔDD，TNFR1-CAV) and sTNF-induced internalizationwas observed under confocal microscope. Destruction of lipid rafts has been found topromote sTNF-α-induced internalization of wild type and DD lackinng TNFR1, butthe internalization of TNFR1lacking its internalizing domain was not observed inresponse to any stimuli. Interestingly, sTNF-α could not induce internalization ofTNFR1-CAV that were located in lipid rafts, but destroye of lipid rafts by MCDresulted in reoccurrence of ligand-induced receptor internalization, indicating thatlipid rafts can protect TNFR1from internalization.Summarized, this study revealed that lipid rafts are involved in cell response tosTNF-α through regulation of receptor internalization, deciding cell fate, apoptosis orsurviving. Thereby, lipid rafts could be as target for changing signal transduction ofcells including tumor cells in response to a given factor, which provides a new cluefor clinical treatment of related diseases.