Study Of TNF-α-mediated Apoptotic Signaling Via TNFR2

Tumor necrosis factor-alpha(TNF-α) is a pleiotropic cytokine involved in a variety of cellular activities,including induction of other cytokines,cell proliferation,differentiation and apoptosis.TNF-αwas first synthesized 26 kDa membrane-binding factor (transmembrane tumor necrosis factor- alpha,transmembrane TNF-α,tmTNF-α).It can be cut from the membrane as a 17 kDa soluble molecule(secreted tumor necrosis factoralpha,, sTNF-α) under TACE.Two types of TNF-αhave different affinities with the TNFR1 and TNFR2 and play biological functions.cytoskeletal proteins—Actin have been found to play an important regulatory role in a variety of signal transduction pathway,including the sTNF-α-mediated cell death.However, the interaction between Actin and tmTNF-α-mediated cytotoxicity is unclear.Our laboratory first discovered tmTNF-αimmunoprecipitated with Actin while tmTNF-αkill the target cells—HL-60.Therefore,the first chapter of this study is to explore tmTNF-αctin relationship with HL-60 as targets.In addition,TNFR2 signal transduction pathways of apoptosis activated by tmTNF-αhas also not been reported,the second chapter of this study is to explore TNFR2 apoptotic signaling complex formation inducing by tmTNF-α,using HepG2 cells as a model,in order to intervene in clinical oncology and explore new molecular targets.Chapter One cytoskeleton protein-Actin involved in tmTNF-αmediated cytotoxicityThe main results are as follows:一.CytD on Actin Polymerization:Laser confocal images shows that Actin is mainly distributed around the nucleus and the cell membrane in the control group cells while dealing with the target cells HL-60 using non-toxic dose CytD(2μM) at different time points.Actin significant spread in the cytoplasm and fluorescence intensity is higher than that of control group. 二.Action time of CytD on tmTNF-αkilling effect:cytotoxic effect of tmTNF-αreduced while going with extension of time pretreatment with CytD.CytD could have the most inhibitory effect on Actin Polymerization and tmTNF-α-mediated cytotocity at the time point 120 min.Therefore,the following experiments are used 2μM CytD 2 hours for pretreatment,specific antibody of tmTNF-αcould inhibit the cytotoxicity of tmTNF-α.三.Actin depolymerization inhibits tmTNF-α-mediated apoptosis:Compared with the control group,HL-60 cells are resistant to sTNF-αmediated cytotoxicity,but sensitive to tmTNF-α-mediated apoptosis and the apoptotic rate is about 50%by AnnexinV-PI assay.In contrary,Actin depolymerized by CytD significantly inhibites sensitivity of HL-60 cells to tmTNF-α.These data show that Actin and tmTNF-α-mediated cytotoxicity are closely linked.四.The identification of cross-reactivity of TNFR1 and TNFR2 antibody:the monoclonal antibody of TNFR1 could only detected sTNFR1 and TNFR1 in HL-60,while the monoclonal antibody of TNFR2 could only detected sTNFR2 and TNFR1 in HL-60.This could imply that there is no cross reactivity of TNFR1 and TNFR2 antibody. Therefore,the antibodies could be used in the following immunoprecipation.五.Actin depolymerization has great effectiveness on tmTNF-αmediated signaling transduction:1.Effect on TNFR1 mediated signal transduction:after Actin depolymerization, SODD collected on TNFR1 significantly reduced after stimulated.While the other treatment groups had no significant changes of SODD.tmTNF-αand sTNF-αcould significantly promote TNFR1 to raise TRAF1 and RIP1,by CytD there is no significant changes after Actin depolymerization.2.Effect on TNFR2 mediated signal transduction:TNFR2.does not raise SODD through co-immunoprecipitation assay,tmTNF-αcould significantly promote the TNFR2 to raise RIP1,but Inhibit TRAF1 to raise.Instead,sTNF-αcould promote TNFR2 to raise TRAF1,but inhibit TNFR2 to aggregate RIP.Actin depolymerization by CytD could reverse tmTNF-αsignal,promote TNFR2 to raise TRAF1 and inhibit RIP1 aggregation to TNFR2.六.tmTNF-α-mediated interaction of TNFR2 with the Actin can be blocked by CytD: Actin may be co-immunoprecipitated with a little bit of TNFR1,and there is no effect on the results with all the treatments.On the contrary,tmTNF-αsignificantly increased the Actin and TNFR2 amount of precipitation.Actin depolymerization by CytD completely blocked the combination of Actin and TNFR2 induced by tmTNF-α.All the data implied that Actin participated in tmTNF-α-mediated apoptotic signal transduction through TNFR2, not TNFR1.七.Effect on expressions of TNF and its receptors after Actin depolymerization:The pretreatment of CytD causes TNFR2 expression on this kind of cell reduced obviously,but no function on TNFR1 expression.There is no influence on TNFR2 total amount after Actin depolymerization.Actin may affect the TNFR2 translocation to the cell surface,but it does not affect the synthesis of TNFR2.At the same time,CytD pretreatment can make tmTNF-αexpression increase significantly on cell surfaces.八.Actin depolymerization can block tmTNF-α-induced dissociation of TRAF2 with TNFR2:After tmTNF-αstimulation,the combination of TRAF2 with TNFR2 reduced. However,CytD pretreatment completely blocked this effect of tmTNF-α.Instead,it promoted TNFR2 and TRAF2 collection.Laser confocal images showed that Actin was distributed under the cell membrane and cytoplasm surrounding the nucleus,and TRAF2 was dispersed in the cytoplasm whether there is tmTNF-αor not.CytD led to diffuse distribution of Actin and promoted TRAF2 translocation from the cytoplasm to the membrane in tmTNF-αstimulated cells.Yet,CytD alone can not lead to TRAF2 translocation.That shows dynamics of Actin can promote dissociation of TRAF2 with TNFR2 induced by tmTNF-α.九.Actin depolymerization inhibited TNFR2 and RIP1 collection induced by tmTNF-α:tmTNF-αpromoted RIP1 to raise on the TNFR2 complex,detected by co-immunoprecipitation.On the contrary,CytD completely blocked this change.Confocal image displayed,tmTNF-α-induced RIP1 translocated from the cytoplasm to the membrane, as well as RIP1 and the Actin are co-localized.However,Actin depolymerization by CytD led to RIP1 dispersing in the cell cytoplasm.These results indicate that the dynamic structure of Actin promote RIP1 to raise to TNFR2 complex induced by tmTNF-α.十.Actin depolymerization could reverse of NF-κB inactivation induced by tmTNF-α: the level of IκB-αin the cell cytoplasm increased,induced by tmTNF-α.Maybe the constitutive degradation of IκB-αcould be inhibited by tmTNF-α.However,CytD pretreatment blocked this function of tmTNF-α,tmTNF-αsignificantly inhibited constitutive NF-κB activation of HL-60 cells with ELISA assay.The CytD blocked this effect of tmTNF-αand restored the NF-κB activation.In addition,CytD alone had no effect on the NF-κB activity.十一.Actin depolymerization blocked tmTNF-α-induced activation of Caspase-8 and its impact on RIP1 Shear:1 Caspase-8 Activity changes in TNFR2-mediated apoptotic cells:Caspase-8 activation was observed as early as 3h and to the top at 12 h obviously seen under stimulation of tmTNF-α,detected by western blotting assay2.Effects on tmTNF-α-induced activation of Caspase-8 and RIP1 trim after Actin depolymerization:RIP1 can be cut by tmTNF-αactivation of Caspase-8,detected using Western Blotting.With this contrast,CytD pretreatment significantly inhibited the activation of Caspase-8 caused by tmTNF-αand,therefore,reducing trim of RIP1.3.Effects on tmTNF-α-induced activation of Caspase-8 and RIP1 trim using Z-IETD-FMK:Caspase-8 inhibitor Z-IETD-FMK completely blocked tmTNF-α-induced activation of Caspase-8 and its impact on RIP1 trim by Western blot.4.Z-IETD-FMK on tmTNF-α-mediated apoptosis:Z-IETD-FMK completely blocked tmTNF-α,inhibited degradation of IκB-αand tmTNF-α-mediated cytotoxicity.十二.Actin depolymerization blocked the inhibition of the expression of cFLIP and raise coused by tmTNF-α:1.Inhibitory effects on the raise of cFLIP and TNFR2 caused by tmTNF-αafter Actin depolymerization:co-immunoprecipitation showed that tmTNF-αstimulation reduced TNFR2 and cFLIP combination.In contrast,CytD pretreatment completely blocked tmTNF-αin this role,increased the combination of cFLIP and TNFR2.2.Inhibitory effects on the expression of cFLIP caused by tmTNF-αafter Actin depolymerization:cFLIP expression was almost completely inhibited in 12 h after tmTNF-αstimulation and The CytD completely blocked tmTNF-αin this role,returned the expression of cFLIP to the basic level.Chapter Two TNFR2-mediated apoptosis signal transduction induced by tmTNF-α.The main results are as follows:一.The expression of TNFR and tmTNF-αon the surfaces of nine kinds of tumor cells: FCM showed that HeLa cell line mainly expressed TNFR1 while K562,Raji and HepG2 cell lines mainly expressed TNFR2.and the TNFR and tmTNF-αexpression rates were low of No.1 MCF-7,T24,and 239 cell lines and no significant differences,the TNFR and tmTNF-αexpression rates were high of No.3 MCF-7 cell line and no significant difference.二.Caspase inhibitor on tmTNF-α-mediated apoptosis:Caspase-8 inhibitor Z-VAD-十FMK could inhibit cytotoxicity of MCF-7,HeLa,HepG2,and T24 cell lines induced by tmTNF-α.However,the inhibitor had no significant effect on No.3 MCF-7 cell line.It indicated that tmTNF-αkilling effect needed to rely on caspase,but also executed by caspase-independent pathway.三.Caspase-8 and Caspase-3 Activation in tmTNF-α-mediated apoptosis1.The expression of caspase in tmTNF-α-mediated apoptosis of MCF-7 cells: There was no significant changes in the content of Caspase-2,Caspase-3,Caspase-8, Caspase-9 and Caspase-12 under the stimulation of tmTNF-αto MCF-7,detected by western blot assay.2.Caspase activation of tmTNF-α-mediated apoptosis in HeLa cells:Caspase-8 was activated as early as 6 h and its activation gradually increased as time went by under the stimulation of tmTNF-αto HeLa,mainly expressed TNFR1,detected by western blot assay.3.Caspase activation of tmTNF-α-mediated apoptosis in HepG2 cells:Caspase-8 was activated as early as 24 h,under the stimulation of tmTNF-αto HeLa,mainly expressed TNFR1,detected by western blot assay.At the same time,Caspase-3 levels are also decreasing.Basically no other significant change in caspase levels.4.Caspase activation of tmTNF-α-mediated apoptosis in T24 cells:Caspase-8 was activated as early as 24 h,under the stimulation of tmTNF-αto T24,detected by western blot assay.At the same time,Caspase-3 levels are also decreasing.Basically no other significant change in caspase levels.These results above suggest that tmTNF-α-mediated apoptosis is related to Caspase-8 and Caspase-3.The difference of TNFR expression patterns may affect the Caspase-8 activation time.We chose HepG2 cell line,mainly expressing TNFR2,to carry out our following experiments.四.Cytotoxicity on HepG2 cells induced by Two types of TNF-α:tmTNF-αmay effectively kill target cells and apoptosis rate is about 40%.However,sTNF-αhad no killing effect on HepG2 cells by MTT assay.Actinomycin D may be part of the increase in sTNF-α-mediated cytotoxicity sensitivity to HepG2 cells.These data showed that two types of TNF-αcould induce different biological effects on the same target cells.五.Roles of tmTNF-αeffected for 12 h on the HepG2 cell apoptosis:PI staining test results confirmed that,after 12 h stimulation,tmTNF-α-mediated apoptosis rate was about 50%.and HepG2 cells were significantly resistant to sTNF-αeffect.These data showed that two types of TNF-α-mediated apoptosis varied significantly.六.Two types of TNF-αon HepG2 cell cycle effects:PI staining test results showed that after 12 h stimulation,tmTNF-αtreatment group turned out an apparent sub-G1 peak, while cells in G1 phase had a reduction of about 40%,indicated that tmTNF-αmainly induced cells in G1 phase to apoptosis,while sTNF-αhad resulted in S phase cells to increase.七.tmTNF-αrecruited pro-apoptotic signaling molecules through TNFR2: co-immunoprecipitation results showed that,after 15 mins' stimulation,TNFR2 recruited FADD,RIP1,and Caspase-8,while TNFR2 only recruited FADD and RIP1 after 25 mins' stimulation.In contrast,sTNF-αcompletely inhibited TNFR2 to recruit the above-mentioned molecules.At the same time,we did not detect TNFR2 to recruit TRADD.八.tmTNF-αinhibited TNFR2 to recruit anti-apoptotic signaling molecules: co-immunoprecipitation results showed that tmTNF-αinhibited the recruitment of TRAF1, TRAF2,TRAF3,cIAP1,cIAP2,and cFLIP to TNFR2.In contrast,sTNF-αenhanced TNFR2 to recruit these six signal molecules,sTNF-αcould recruit NIK to to TNFR2 complex possibly through TRAF2,while tmTNF-αdoes not have this effect.九.tmTNF-α-mediated NF-κB inactivation and sTNF-α-mediated NF-κB activation: ELISA assay found,constitutively activated NF-κB was significantly inhibited,caused by tmTNF-αin HepG2 cells,while sTNF-αwill enable the further NF-κB activation.In summary,two types of TNF-αto switch on TNR2 different signaling pathway in HepG2 cells,tmTNF-αmainly induced the formation of TNFR2 apoptotic signaling complex(FADD/RIP1/Caspase-8) while sTNF-αmainly induced the formation of TNFR1 apoptotic signaling complex(TRAF1-3/cIAP1-2/cFLIP) then led respective target cells to apoptosis or survival.In addition,Actin mainly involved in tmTNF-αstart TNFR2 signaling pathway of apoptosis,to promote TNFR2 to recruit RIP1,leading to TRAF2, and cFLIP and TNFR2 dissociation,thus contributing to Caspase-8 activation and its impact on RIP1 trim,leading to NF-κB inactivation and mediating tmTNF-α-induced apoptosis through CascadeThe study not only provided experimental basis for clinical biological characteristics of the two types of TNF,but also provided new clues to the molecular target and intervention for anti-tumor treatment.