Influence Of Lipid Rafts On Bidirectional Signaling Of TmTNF-α

Part 1:Construction and Function Research of Transmembrane TNF-αMutants Deleted Catalytic SiteTransmembrane tumor necrosis factor-α(tmTNF-α) is the precursor of sTNF-αand themolecular weight is 26kD,which is mainly expressed in activated monocytes and immunecells.As a transmembrane protein tmTNF has a 76 amino signal peptide which composesof a hydrophobic domain compared to sTNF-α.The molecule expressed on the cell surfacecan be catalyzed by TNF-converting enzyme (TACE/ADAM17) to release sTNF-α.When we study the biological function of tmTNF-α,we get the integrated effects ofboth tmTNF-αand sTNF-αbecause it is often difficult to avoid the catalysis of tmTNF-α.Itis necessary to generate a mutant which can not release sTNF-αbecause the digested site ismutated.Although the mutant deleted the site of 1 to 12 lost the ability to be catalyzed,thecytotoxicity of which were also affected.Our lab found that the mutant also had defects inreverse signal transduction.So this mutant is not an ideal mutant for research.ThetmTNF-αmutant deleted the sites of 1 to 9 and mutated in the site of 11 from K to E caninhibit the function of TACE,and its cytotoxicity is same to wild-type tmTNF-α.It isunclear whether this mutant effects the reverse signal transduction.In this study,we aim tomutate the digestion site of TACE using site-directed mutagenesis method and to study theforward and reverse signal transduction of the mutants.1 We successfully constructed mutants lack catalysis site for TACE asΔ1-12 tmTNF-αandΔ1-9,K11E tmTNF-αby using overlap PCR technology.It was confirmed thatthere were absence of any other point mutations,frame-shift and deletion mutations butexpected mutations by restriction enzyme digestion,PCR and sequencingidentification.2 Hela cells were transfected with tmTNF-αand mutants and total proteins wereextracted.A specific band about 26kD was detected in wide type tmTNF-αgroup byWestern blot.A specific band about 25kD was detected in mutants groups. 3 The supernatant of Hela transfected cells with wild type tmTNF-αand mutants wascollected to detect the content of sTNF-α.We found that wt tmTNF-αgroup cansecrete large amount of tmTNF-αand there was not detected sTNF-αcompletely in themutants transfected grouPs.Both of the mutants can inhibitor the digestion of TACE.4 Hela cells tranfected with wt tmTNF-αand mutants were fixed with paraformaldehydeto kill the T24 cells.We found that groups both tranfected with wt tmTNF-αandΔ1-9,K11E tmTNF-αcan significantly induce cell death and the cytotoxicity ofΔ1-12tmTNF-αwas reduced nearly half of other groups.It was shown thatΔ1-9,K11EtmTNF-αmutant has retained the ability to induce cytotoxicity.5 Western blot was used to detect the IκB-αexpression of Hela cells transfected withtmTNF-αand its mutants.IκB-αdegraded in the cells transfected with wt tmTNF-αandΔ1-9,K11E tmTNF-α,meanwhileΔ1-12 tmTNF-αcan not degrade IκB-αtoactivate NF-κB.The results showed thatΔ1-9,K11E tmTNF-αnot only lost the ablity to releasesTNF-αbut also can retain the ability to transduce forward and reverse signals.It's a betterand power tool to research the bio-function of tmTNF-αthanΔ1-12 tmTNF-α.Part 2:Lipid Rafts and the Bidirectional Signaling of tmTNF-αRecent years,Lipid rafts have been found as the existence of the micro-structure in themembrane.They are distinct from classical lipid bilayer membrane and rich of cholesteroland phosphatidylinositol.Many receptor molecules and their associated signalingmolecules can be located in lipid rafts.Because lipid rafts in the plasma membranedistributes scattered and can lateral drift to gather to form a platform for signal transductionwhich is involved in activation of receptor signal transduction and the intracellular signaltransmission.tmTNF-αnot only can bind to TNFR of target cells to induce signal transduction whichis termed forward signal,but also can act as a receptor itself to accept external signals andtransmit intracellular signals reversely.It has been reported that -47 site is palmitoylated.We speculated that tmTNF-αmay be partitionated to lipid rafts and the lipid rafts may be related to the location-related signal transduction.In addition it was reported that TNFR canlocalized to lipid rafts,but the relationship between tmTNF-αand the lipid raftslocalization of TNFR is not clear now.In order to clarify the relationship between tmTNF-αand the lipid rafts,we confirm thelipid rafts localization of tmTNF-αusing sucrose density gradient centrifugation.We studythe functions of lipid rafts in tmTNF-αforward and reverse signal transduction via twoways including effector cells expressed tmTNF-αand the target cells expressed TNFR.1 Lipid rafts and reverse signaling of tmTNF-α1.1 tmTNF-αcan localize in the lipid rafts.The lipid rafts of Raji cells which have highexpression of tmTNF-αwere separated using the mothod of sucrose density gradientcentrifugation and were detected by Western blot.A part of tmTNF was within the lipidrafts.Raji cells were treated with 10mM MCD for 45min to destroy lipid rafts,alltmTNF-αwere distributed outside of lipid rafts.1.2 Disruption of lipid rafts enhanced the cytotoxicity of sTNF-αsensitivity.The highexpression of tmTNF-αon Raji cells can induce sTNF-αtolerance.After lipid rafts weredisrupted,the cytotoxicity of sTNF-αincreased markedly.T24 ceils which have noneexpression of tmTNFs were sensitive to sTNF-αand sTNF-αcytotoxicity also enhancedafter lipid rafts were damaged,but the increasement was significantly lower than Rajicells1.3 Destruction of lipid rafts resulted in decreased activity of NF-κB.When Raji cellswere treated with MCD the level of IκB-αsignificantly become higher which indicatedthe inhibition of NF-κB signal pathway.But it was not change significantly for T24cells.It was shown that the destruction of lipid rafts can weaken reverse signaltransduction of tmTNF-α.1.4 Construction the mutants only in or outside of the lipid rafts and establish stablecell lines.We constructed a mutant named cav-tmTNF-c which can locate only in lipidrafts and another mutant C-47A tmTNF-αwhich only partitionate out of lipid rafts.T24cells were stably transfected with wild type tmTNF-αand the mutants and theirlipid rafts were extracted to confirm the location. 1.5 tmTNF-αwithin lipid rafts induced sTNF-αtolerance.We observed thecytotoxicity of stable cell lines induced by sTNF-α.Results showed that parental T24cells,the cells transfected with empty vector and C-47A tmTNF-αwere induced about50% of cell death by sTNF-α.The cytotoxicity of stable cells transfected with wttmTNF-αwas significantly decreased.The results showed that only tmTNF-αin lipidrafts may resist to sTNF-αinduced death death.1.6 NF-κB pathway can be constructively activated by tmTNF-αin the lipid rafts.Itwas found that the level of IκB-αwas degraded and the level of phosphorylated p65was increased constructively in cell lines stable transfected with wt tmTNF-α.Thelevel of IκB-αand p-p65 had no significant changes for the C-47A tmTNF-αcompared with the control groups.1.7 tmTNF-αin lipid rafts can up-regulate the expression of cIAP1,down-regulatethe expression of Bax.Realtime PCR was used to detect gene expressions.It wasfound that in wt tmTNF-αgroup anti-apoptosis genes cIAP1 expression increasedsignificantly and the pro-apoptotic gene Bax gene transcription were obviouslyinhibited compared with the control group.But these two genes of C-47A are nosignificant difference from that of the control group.1.8 CKI inhibitor D4476 is helpful for tmTNF-αin lipid rafts to resist tosTNF-αinduced cytotocity.CKI-specific inhibitor can inhibit the phosphorylation oftmTNF-αand enhance the reverse signal transduction.It was observed that wttmTNF-αtransfected cells can resist to sTNF-αinduced cytotoxicity.CKI inhibitorD4476 can enhance the role of the resistance,but it had no significant effect to C-47AtmTNF-αtransfected cell.1.9 Lipid rafts as a platform to transmit tmTNF-αreverse signals.The reverse signalof tmTNF-αcan be activated by sTNFR2 stimulation and transfection of tmTNF-αinthe lipid rafts,at the same time related signal molecules can be recruited to lipid rafts.The activation of reverse signal can induced the aggregation of tmTNF-αTRAF1IKK-αand p52 to lipid rafts.The molecules translocation from outside to inside of lipid rafts can not been seen for the C-47A tmTNF-αtransfected cells.It is suggestedthat Lipid rafts as a platform to transmit tmTNF-αreverse signals.2 Lipid rafts and forward signal of tmTNF-α2.1 Effect of lipid rafts to biological functions tmTNF-αmediated by forward signal.2.1.1 Lipid rafts location of tmTNF-αhas no relationship to its cytotoxicity.ThetmTNF-αcytotoxicity to T24 cells was almost completely blocked as inhibited withTNF-αAb when Raji cells were treated with MCD to destroy lipid rafts.In order torule out the possibility of non-specific effects of MCD,we use Hela cells transientlytransfected with mutants which only locate in or out of lipid rafts to kill the T24 cells.The results showed that the effector cells transfected with wild-type and mutanttmTNF-αhave similar cytotoxicity and there's no significant difference among thethree mutants.It was shown that tmTNF-αwithin and outside lipid rafts both hadcell-killing ability,the tmTNF-αcytotoxicity has no relation to lipid rafts location.2.1.2 Destruction of ICAM-1 location in lipid rafts led to decline of adhesionbetween target and effetor cells and blocked the cytotoxicity of tmTNF-α.Becausethe cytotoxicity of tmTNF-αis dependent on cell-cell contact which is often mediatedby adhesion molecules.We further observed the relationship between lipid rafts,ICAM-1 and anti-tumor effect of tmTNF-α.The results showed that some of ICAM-1distributes in lipid rafts in resting state and all ICAM-1 target to non-rafts when lipidrafts were destroyed by MCD.Also the cytotoxicity to t24 ceils was almost completelyobstructed and the adhesion between target and effector cells was decrease.ICAM-1antibodies only partially inhibited the adhesion between the target and effector cellsand partially blocked the cytotoxicity of tmTNF-α.2.1.3 Destruction of lipid rafts resulted in increased secretion of sTNF-α.We furtherobserved the effect of lipid rafts destruction on the generation of sTNF-α.lipid raftsdamagement of Raji cells with MCD can lead to TACE,some of which have beenfound in lipid rafts,to the out of lipid rafts,and sTNF-αincreased significantly.2.2 Lipid rafts affected the bio-function of tmTNF-αmediated by forward signals2.2.1 tmTNF-αcaused TNFR1 of target cells recruit to lipid rafts.It was found that a small part of TNFR1 of T24 cells located in lipid rafts in resting state.A large numberof TNFR1 from outside to lipid rafts when we engaged fixed Raji cells to T24 cells.This result suggested that TNFR recruitment to lipid rafts may affect the forward signalof tmTNF-α.2.2.2 sTNF-αand tmTNF-αinside or outside of lipid rafts have the different effectto TNFR1 inside or outside of lipid rafts.Cytotoxicity of sTNF-αincreasedsignificantly when lipid rafts were destroyed by MCD,but that of tmTNF-αsignificantly decreased and there was no significant difference among the three mutants.These results suggest that lipid rafts played the different roles in sTNF-αand tmTNF-αinduced cytotoxicityOur study revealed that tmTNF-αcan target to lipid rafts which rely on cysteine on thesite of -47.Reverse signal tranduction of tmTNF-αdepends on its lipid raft localization.For effector cells,tmTNF in or out of lipid rafts have no difference in forward signaltransduction.But lipid rafts of target cells play an important role in forward signaltransduction of tmTNF-α.