TRAIL/TRAIL受体介导炎症反应及其信号传递途径的研究

【作者】 唐万虎；

【导师】 郑德先； 刘彦信；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 肿瘤坏死因子相关的凋亡诱导配体(TRAIL)发现于1995年,是肿瘤坏死因子(TNF)超家族的成员。目前,在人类中已发现5种TRAIL受体,分别是:DR4、DR5、DcR1、DcR2和OPG。在这些受体中,DR4和DR5的胞内区拥有完整的死亡结构域,因此能够介导相关信号途径的激活。而DcR1却没有死亡结构域,DcR2也只有一个截短的死亡结构域,OPG是这五种受体中唯一的可溶性蛋白,这三种受体均不能介导细胞凋亡。TRAIL能够诱导肿瘤细胞的凋亡而对大部分正常细胞没有毒害,因此TRAIL被视为有着临床应用前景的肿瘤治疗药物。与TRAIL处理一样,TRAIL受体过表达同样可以在很多细胞系中诱导细胞凋亡。一旦与TRAIL结合以后,TRAIL受体能够募集一系列接头分子,从而引发相应的信号传递途径。TRAIL及其受体相关的研究工作大部分都集中在细胞凋亡上,至今人们对其生理学功能仍知之甚少。与其它TNF超家族成员相比,TRAIL在免疫系统广泛表达,包括激活的T细胞,B细胞,NK细胞,树突状细胞,中性粒细胞和单核细胞。并且DR4和DR5也广泛表达于许多正常组织,例如脾,外周血淋巴细胞和胸腺。虽然TRAIL及其受体同时表达于众多正常细胞,但是并不诱导这些细胞的凋亡。这些事实表明TRAIL和TRAIL受体除了诱导细胞凋亡以外,还有更为复杂的生理功能。为了探寻TRAIL受体的生理功能,我们分别在293T,MDA-MB-231和HCT-116中过表达DR4和DR5,以检测TRAIL受体过表达能否激活炎症反应。我们发现DR4或DR5过表达能够诱导IL-8、TNF-α、CCL20、MIP-2和MIP-1β等炎症因子的分泌,而且这一过程依赖于NF-κB的激活;DR4或DR5过表达激活炎症炎症因子的分泌是死亡受体胞外区非依赖的;TRAF2-NIK-IKKα/β和FADD-Caspases信号途径都与TRAIL受体过表达引起的炎症反应和NF-κB激活有关。我们进一步利用人重组可溶性TRAIL处理293T和HCT-116细胞,发现TRAIL上调IL-8,CCL20,MIP-2,MIP-1β和MCP-1的表达,这一过程依赖于NF-κB的激活;RIP对TRAIL诱导的炎性趋化因子分泌是不可缺少的,但TRADD和TRAF2与此过程关系不大:p38 MAPK在TRAIL诱导趋化因子的过程中起重要调节作用;我们还在HCT116的皮下移植瘤模型中证实TRAIL同样可以在体内激活炎性趋化因子的分泌。本项研究报告了TRAIL及其受体可介导一系列炎性细胞因子的释放,并揭示了其信号转导途径,为TRAIL及其受体的生理功能研究和临床应用提供了新的资料。更多还原

【Abstract】 Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL),which is also designated as Apo-2 ligand,is a typical member of the structurally related TNF family.To date five TRAIL receptors have been identified in human:TRAIL-R1(DR4),TRAIL-R2 (DR5),TRAIL-R3(DcR1,TRID),TRAIL-R4(DcR2,TRUNDD) and OPG (osteoprotegerin).Among these receptors,DR4 and DR5 possess an intracellular tail containing the death domain,which mediated cell death by apoptosis.In contrast,DcR2 possesses a truncated death domain and DcR1 have no death domain.Osteoprotegerin is a soluble TRAIL receptor,which is known to participate in regulation of bone density.TRAIL has been known to induce apoptosis in a variety of tumor cells and some vitally infected cells,but not in most normal cells.Thus TRAIL and TRAIL death receptor specific agonistic antibodies have attracted considerable attention for their potential use in cancer therapy.TRAIL receptor overexpression as well as TRAIL treatment induced apoptosis in different cell lines.Once engaged,TRAIL receptors recruit a number of adaptor proteins and subsequently a signaling cascade is activated.Most investigations about TRAIL focused on its ability to induce apoptosis in cancer cells;however,the physiological function of TRAIL and TRAIL receptors is much less well understood.Compared with other members of TNF superfamily,TRAIL are widely expressed in the immune system,including activated T cells,B cells,natural killer cells, dendritic cells,neutrophils,and monocytes.DR4 and DR5 are detected in most human tissues,such as spleen,peripheral blood leukocytes,and thymus.Although TRAIL and its receptors are both expressed in many normal cells,TRAIL seldom initiates apoptosis in these cells.The fact suggests that the physiological roles of TRAIL and TRAIL receptors are more complex than just inducing apoptosis of cancer cells.In order to shed a light on the physiological roles of TRAIL receptors,we transfected 293T cells with expression plasmids of DR4 or DR5 to determine whether TRAIL receptors overexpression activated inflammatory cytokines release.To our knowledge,this is the first demonstration that TRAIL receptors overexpression activates inflammatory cytokine release in a NF-κB dependent signaling pathway.We described that DR4 and DR5-mediated inflammation and apoptosis was extra cellular domain independent. Furthermore,TRAF2-NIK-IKKα/βsignaling cascade,which plays an essential role in TNF-induced NF-κB activation,is involved in TRAIL receptors overexpression-activated signaling pathway.And FADD-Caspases signaling pathway,which was reported to be mostly related to apoptosis,was elucidated to be essential for TRAIL receptors-mediated inflammation.To present further evidence,we treated 293T and HCT-116 cells with rsTRAIL to determine whether TRAIL induced inflammatory cytokines release.We elucidated that TRAIL induced IL-8,CCL20,MIP-2,MIP-1βand MCP-1 secretion in a NF-κB dependent manner.RIP was proved to be essential for TRAIL-induced chemokines release,while TRADD and TRAF2 were not required.It was also demonstrated that p38 MAPK was involved in TRAIL-induced chemokines release without any effect on NF-κB activity, which suggested that some other transcription factors were also activated by TRAIL. Furthermore,using xenografi tumor model,we illustrated that TRAIL induced chemokines release in vivo.These data provide novel evidences for the physiological functions of TRAIL and TRAIL receptor,and throw some light on their clinical application.更多还原