tmTNF-α通过TNFR1介导细胞凋亡的分子机制及tmTNF-α反向信号通路的研究
发布时间:2018-06-17 07:46
本文选题:tmTNF-α + TNFR1 ; 参考:《华中科技大学》2012年博士论文
【摘要】:跨膜型TNF-a (transmembrane tumor necrosis factor-alpha, tmTNF-a)是分泌型TNF-a (secretory TNF-a, sTNF-a)的前体,它以稳定的同源三聚体的形式表达于活化的巨噬细胞、淋巴细胞或其他类型细胞的表面。sTNF-a被TNF转化酶(TNF-a converting enzyme,TACE)从tmTNF-a上剪切释放到胞外,与其相应的受体结合并发挥生物学功能。越来越多的证据显示,不仅是sTNF-a, tmTNF-a亦可通过TNFR1和TNFR2介导多种生物学效应,包括细胞的凋亡与增殖、细胞因子的产生以及局部炎症反应的调节等。 tmTNF-α既可作为配体传递正向信号,也可作为受体来传递反向信号。本室前期工作证实tmTNF-α可通过TNFR1介导细胞凋亡;然而该分子如表达在肿瘤细胞则可通过反向信号激活NF-κB,导致抗凋亡。但其具体的分子机制尚不清楚。本研究主要探讨tmTNF-a作为配体通过TNFR1转导正向信号介导肿瘤细胞凋亡及其作为受体转导反向信号保护肿瘤细胞抵抗凋亡的分子机制。其主要结果如下: 一、tmTNF-a通过TNFR1介导细胞凋亡的分子机制 1. tmTNF-a不能介导TNFR1的内化:用流式与激光共聚焦显微镜证实sTNF-a导致细胞表面TNFR1表达降低,TNFR1胞浆移位明显增加,用MDC预处理则可明显抑制sTNF-a诱导的TNFR1向胞浆内的内化;而tmTNF-a则对细胞表面TNFR1的表达无明显影响,且不诱导受体向胞浆内移位。提示tmTNF-a不能诱导TNFR1内化。 2. tmTNF-a的胞毒效应不依赖TNFR1的内化:首先用MTT证实用MDC抑制TNFR1的内化,可明显降低sTNF-a的胞毒效应,却对tmTNF-a胞毒效应无影响。进一步用AnnexinV-PI凋亡检测试剂盒和WB证实用MDC可明显降低sTNF-a诱导的caspase-3的活化及细胞凋亡,但不影响tmTNF-a诱导的caspase-3的活化及细胞凋亡。上述结果提示,与sTNF-a胞毒效应依赖TNFR1内化的特征相反,tmTNF-a诱导靶细胞凋亡为TNFR1内化非依赖性的。 3. tmTNF-a通过细胞膜上的TNFR1募集TRADD,FADD和caspase-8:分别提取细胞总蛋白,IP-western证实:tmTNF-a与sTNF-a均能通过TNFR1募集TRADD、FADD和caspase-8,形成DISC信号复合物。进一步分离胞膜蛋白和胞浆蛋白,用IP证实tmTNF-a诱导TNFR1形成的DISC信号复合物位于膜蛋白中,且可用激光共聚焦显微镜观察到tmTNF-a诱导DISC信号复合物的成员之一FADD从胞浆转位到细胞膜;而sTNF-a诱导TNFR1形成的DISC信号复合物则位于胞浆蛋白中。提示两型TNF-a尽管作用于同一受体,但二者分别在细胞不同部位诱导DISC信号复合物的形成。 4.tmTNF-a不能通过TNFR1在细胞膜上募集RIP-1,TRAF2和cIAPl:提细胞膜蛋白进行IP证实:sTNF-a可通过TNFR1在细胞膜上募集RIP-1, TRAF2和cIAP1等激活NF-κB的信号复合物;而tmTNF-a则无明显类似效应。提示与sTNF-a不同,tmTNF-a不能通过TNFR1在细胞膜上募集抗凋亡信号分子。 5. tmTNF-a不依赖于TNFR1的死亡结构域募集死亡信号分子:分别将野生型TNFR1和缺失DD的TNFR1突变体瞬时转染HEK293细胞,两型TNF刺激30分钟后提取细胞膜蛋白进行IP。结果发现tmTNF-a不但可通过野生型TNFR1,也能够通过缺失DD结构域的TNFR1募集TRADD, FADD和caspase-8等DISC信号复合物。提示与sTNF-a不同,tmTNF-a通过TNFR1募集DISC信号复合物不依赖于受体的死亡结构域。 6. tmTNF-a通过TNFR1非DD结构域在细胞膜上募集STAT1,形成DISC信号复合物:为探讨tmTNF-a诱导TNFR1募集DISC复合物分子的机制,用胞膜蛋白进行IP,证实:tmTNF-a可通过野生型和缺失DD的TNFR1募集STAT1,但sTNF-a则不能。提取细胞总蛋白进行IP,进一步证实sTNF-a诱导TNFR1形成的DISC复合物中有大量STAT1参与,但TNFR1缺失DD则不能被诱导形成DISC复合物,且无STAT1共沉淀,提示STAT1参与sTNF-a诱导的DISC信号复合物,且依赖TNFR1的DD结构域;然而,tmTNF-a通过野生型和缺失DD的TNFR1形成的DISC复合物中均有明显STAT1的共沉淀,提示STAT1可能通过与TNFR1非DD结构域直接结合,参与tmTNF-a通过TNFR1募集DISC信号复合物。 二、tmTNF-α反向信号通路的研究 1. NIK, SODD参与tmTNF-α反向信号复合物:用tmTNF-a单抗对高表达tmTNF-a的Raji细胞进行IP,证实tmTNF-a可与NIK、SODD发生免疫共沉淀。反之,我们用NIK或SODD的特异性抗体分别进行反向IP,证实均有tmTNF-a共沉淀。提示除了前期工作发现的TRAF1,IKKa和NF-KBp52以外,NIK和SODD也是tmTNF-a反向信号复合物中的成员。 2. tmTNF-a通过胞浆段(TNF-LS)募集TRAF1:我们将TRAF1, IKKa和tmTNF-a野生型或突变体质粒共转染于293T细胞中,提取总蛋白,用tmTNF-a单抗进行IP。证实:野生型tmTNF-a和缺失胞外段的TNF-LS均可募集TRAF1,而缺失胞浆段的ACS-tmTNF-a则不能募集TRAF1。提示tmTNF-a通过其胞浆段募集TRAF1。 3. tmTNF-a胞浆段(TNF-LS)不能直接与IKKa结合:本研究为了验证细胞内TNF-LS和IKKa的相互作用,将二者共转到293T细胞中,IP-Western显示TNF-LS并未与IKKa共沉淀。提示TNF-LS不能直接结合IKKa,可能通过TRAF1间接募集该分子。 4. TRAF1是连接tmTNF-a与IKKa的接头分子:使用siTRAF1下调Raji细胞表达TRAF1,导致与tmTNF-a免疫共沉淀的IKKa明显减少。提示TRAF1作为衔接分子可将IKKa募集到tmTNF-a反向信号复合物中。 5.抑制tmTNF-α磷酸化可促进其反向信号复合物TRAF1/IKKa/NF-KBp52的形成:tmTNF-a胞浆段含有CKI磷酸化酶的作用位点,故我们用CKI的特异性抑制剂D4476抑制tmTNF-a的磷酸化,结果发现tmTNF-a反向信号复合物(TRAF1/IKKa/NF-KBp52)形成明显增加。提示抑制tmTNF-a的磷酸化能促进其反向信号复合物的形成。 综上所述,本课题阐明了tmTNF-a通过TNFR1介导正向信号导致靶细胞凋亡的信号通路,证实tmTNF-a可能直接通过细胞膜上TNFR1的非DD结构域与STAT1结合,进而募集TRADD、FADD和caspase-8,形成DISC信号复合物,导致caspases的级联活化,进而诱导细胞凋亡。同时在前期工作的基础上,我们进一步阐明了tmTNF-a反向信号复合物的组成及其成员之间的相互关系,tmTNF-a一方面通过其胞浆段募集TRAF1、NIK和IKKa,进而激活NF-KBp52(非经典途径),另一方面可能通过其胞浆段与TNFR1竞争SODD,参与激活NF-κB经典途径,导致tmTNF-a(+)的肿瘤细胞抵抗凋亡。 本研究深入探讨了tmTNF-a通过TNFR1介导正向信号诱导靶细胞凋亡以及通过反向信号促进tmTNF-a(+)的肿瘤细胞抵抗凋亡的分子机制,为临床肿瘤治疗及干预肿瘤耐药提供新的思路和靶点。
[Abstract]:Tumor necrosis factor - alpha ( tmTNF - a ) is a precursor of secreted TNF - a ( sTNF - a ) , which is expressed in activated macrophages , lymphocytes or other types of cells in the form of stable homotrimer . sTNF - a is cleaved from tmTNF - a by TNF - a converting enzyme ( TACE ) to the extracellular domain , and plays a biological function . More and more evidence shows that not only sTNF - a , tmTNF - a can also mediate various biological effects through TNFR1 and TNFR2 , including apoptosis and proliferation of cells , production of cytokines and regulation of local inflammatory response .
tmTNF - 伪 can be used both as a ligand to deliver a positive signal , and can be used as a receptor to transmit a reverse signal . The previous work of the chamber proves that tmTNF - alpha can mediate apoptosis through TNFR1 ;
However , if the molecule is expressed in tumor cells , NF - 魏B can be activated by the reverse signal , which leads to anti - apoptosis . However , the specific molecular mechanism is not clear . The main results of this study are as follows :
One , tmTNF - a molecule mechanism of cell apoptosis mediated by TNFR1
1 . tmTNF - a did not mediate the internalization of TNFR1 : the expression of TNFR1 in the cell surface was decreased by the flow cytometry and the laser confocal microscope , and the expression of TNFR1 was significantly increased , and the amount of TNFR1 induced by sTNF - a was significantly inhibited by MDC pretreatment , and the internalization of TNFR1 induced by sTNF - a was inhibited significantly ;
However , tmTNF - a did not significantly affect the expression of TNFR1 in the cell surface , and did not induce the translocation of the receptor to the cytoplasm .
2 . The cytotoxic effect of tmTNF - a did not depend on the internalization of TNFR1 : firstly , MTT was used to confirm that MDC could inhibit the internalization of TNFR1 and reduce the cytotoxic effect of sTNF - a .
3 . tmTNF - a raised TRADD , FADD and caspase - 8 through TNFR1 on the cell membrane : the total protein of the cells was extracted , and IP - western confirmed that both tmTNF - a and sTNF - a could raise TRADD , FADD and caspase - 8 through TNFR1 to form DISC signal complexes .
The DISC signal complex induced TNFR1 induced by sTNF - a is located in the cytoplasm protein . It is suggested that the two types of TNF - a induce the formation of DISC signal complexes at different sites of the cell , although they act on the same receptor .
4 . tmTNF - a was unable to recruit RIP - 1 , TRAF2 and cIAPl on the cell membrane by TNFR1 : the membrane protein was confirmed by IP : sTNF - a could induce the activation of NF - 魏B signal complexes on the cell membrane via TNFR1 , TRAF2 and cIAP1 .
The results suggested that tmTNF - a could not recruit anti - apoptotic signal molecules on the cell membrane by TNFR1 , unlike sTNF - a .
5 . tmTNF - a did not rely on the death domain of TNFR1 to recruit death signal molecules : the wild type TNFR1 and the deleted DD TNFR1 mutant were transiently transfected into 293 cells and the two types of TNF stimulated 30 minutes to extract the membrane protein . The results showed that tmTNF - a not only can raise DISC signal complexes such as TRADD , FADD and caspase - 8 through TNFR1 with the DD domain .
6 . tmTNF - a raised STAT1 on the cell membrane via TNFR1 non - DD domain to form DISC signal complex . In order to investigate the mechanism of tmTNF - a - induced TNFR1 - raised DISC complex molecule , it was confirmed that tmTNF - a was able to raise STAT1 through wild - type and missing DD - induced TNFR1 , but sTNF - a could not be induced to form DISC complex , but sTNF - a could not be induced to form DISC complex , but without STAT1 co - precipitation , STAT1 was involved in sTNF - a - induced DISC signal complex and depended on the DD domain of TNFR1 ;
However , tmTNF - a is co - precipitated with STAT1 in DISC complexes formed by wild - type and missing DD TNFR1 , suggesting STAT1 may be directly bound to the TNFR1 non - DD domain and participate in tmTNF - a to raise DISC signal complexes through TNFR1 .
Study on the reverse signal pathway of di , tmTNF - 伪
1 . NIK and SODD were involved in tmTNF - a reverse signaling complexes : the presence of tmTNF - a monoclonal antibody against the high expression of tmTNF - a . The results showed that tmTNF - a could co - precipitate with NIK and SODD . On the contrary , we used NIK or SODD - specific antibodies to reverse IP respectively to confirm that tmTNF - a co - precipitated .
2 . tmTNF - a raised TRAF1 through the cytoplasmic segment ( TNF - LS ) : we co - transfected TRAF1 , IKKa and tmTNF - a wild - type or mutant plasmid into 293 cells , extracted the total protein and used tmTNF - a monoclonal antibody for IP . It was confirmed that both wild - type tmTNF - a and TNF - LS of the absent extracellular segment could raise TRAF1 , whereas the ACS - tmTNF - a in the missing cell segment could not raise TRAF1 . It was suggested that tmTNF - a raised TRAF1 through its cytoplasmic segment .
3 . The TNF - LS was not directly bound to IKKa . In order to verify the interaction between TNF - LS and IKKa in the cells , TNF - LS was not co - precipitated with IKKa . It was suggested that TNF - LS could not directly bind IKKa and could indirectly raise the molecule via TRAF1 .
4 . TRAF1 is a linker molecule linked to tmTNF - a and IKKa : Down - regulation of TRAF1 with siTRAF1 results in a significant decrease in IKKa co - precipitated with tmTNF - a . It is suggested that TRAF1 can be used as a linking molecule to raise IKKa to the tmTNF - a reverse signal complex .
5 . Inhibition of tmTNF - a phosphorylation can promote the formation of reverse signal complexes TRAF1 / IKKa / NF - KBp52 : the tmTNF - a cytoplasmic segment contains the action site of CKI phosphorylase , so we use the specificity inhibitor D4476 of CKI to inhibit the phosphorylation of tmTNF - a .
In conclusion , we have clarified that tmTNF - a can induce apoptosis of target cells through TNFR1 - mediated positive signals . It is confirmed that tmTNF - a may directly bind to STAT1 through the non - DD domain of TNFR1 in cell membrane , which leads to the cascade activation of caspases , thus inducing apoptosis . On the other hand , we can compete with TNFR1 to activate NF - KBp52 ( non - classical pathway ) . On the other hand , it is possible to activate NF - 魏B classical pathway through its cytoplasmic segment , which leads to the tumor cells of tmTNF - a ( + ) to resist apoptosis .
In this study , the molecular mechanism of tmTNF - a mediated by TNFR1 to induce apoptosis of target cells and to promote the apoptosis of the tumor cells of tmTNF - a ( + ) by reverse signal is discussed in detail , which provides new ideas and targets for clinical tumor therapy and intervention of tumor drug resistance .
【学位授予单位】:华中科技大学
【学位级别】:博士
【学位授予年份】:2012
【分类号】:R363
【参考文献】
相关期刊论文 前2条
1 石文芳,李卓娅,龚非力,熊平,徐勇;跨膜型与分泌型TNF-α细胞毒效应的比较[J];中华微生物学和免疫学杂志;1998年06期
2 石文芳,李卓娅,龚非力;跨膜型和分泌型TNF-α对TNF受体作用的比较[J];中国免疫学杂志;1998年04期
,本文编号:2030279
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