The Molecular Mechanism Underlying Tmtnf-αMediated Apoptosis Via TNFR1and The Study Of The Reverse Signaling Pathway Of TmTNF-α

Transmembrane tumor necrosis factor-alpha(tmTNF-α) is a precursor of the secretory TNF-a(sTNF-a). The tmTNF-a was expressed in the form of stable homotrimers on the surface of activated lymphocytes and macrophages and other cell types. The sTNF-a was cleaved from tmTNF-a by the TNF-converting enzyme (TACE) and bound to TNFR to play its biological functions. More and more evidences suggested that not only soluble TNF-a, but also transmembrane TNF-a mediated a variety of biological activities by binding to TNFR1 and TNFR2, including cell apoptosis and proliferation, induction of other cytokines and the modulation of local inflammation.The tmTNF-a could transmit forward signals as a ligand and reverse signals as a receptor. We previously revealed that the tmTNF-a could induce cell death through TNFR1, while tmTNF-a-expressing cancer cells constitutively activated the noncanonical NF-κB pathway and resisted apoptosis. But the signaling pathway was poorly defined. Our study investigated the mechanism of tmTNF-a induced cell death through the forward signals and tmTNF-a prevented cell death through the reverse signals. The main results are as follows:Ⅰ. The molecular mechanism underlying tmTNF-a mediated apoptosis via TNFR11. tmTNF-a could not induce the internalization of TNFR1:Flow cytometry and Laser confocal images showed that the sTNF-a could induce the decrease of TNFR1 expression on the surface and the translocation of TNFR1 to cytoplasm. After pretreated with MDC, the TNFR1 internalization induced by the sTNF-a was remarkably reduced. But the tmTNF-a could not affect the expression and location of TNFR1 neither before nor after the pretreatment of MDC. It suggested that tmTNF-a could not induce the internalization of TNFR1.2. The cytotoxicity of tmTNF-a did not depend on the internalization of TNFR1: It was found that MDC could prevent the cytotoxicity induced by sTNF-a. In contrast, tmTNF-a-mediated cytotoxicity was not prevented by MDC. Then AnnexinV-PI and Western blot assays were used to confirm that MDC could prevent the caspase-3 activation and apoptosis induced by sTNF-α., but could not affect that induced by tmTNF-a. These results suggested that the tmTNF-a-mediated apoptosis did not depend on the internalization of TNFR1.3. The tmTNF-a recruited TRADD, FADD and caspase-8 through membrane TNFR1:Co-immunoprecipitation of whole-cell proteins showed that both sTNF-a and tmTNF-a recuited TRADD, FADD, procaspase-8 and caspase-8 through TNFR1. Then, the Ip-western assays were used to confirm that DISC induced by tmTNF-a was located in membrane proteins, while that induced by sTNF-a, located in cytoplasmic proteins. And the laser confocal images also showed that FADD was translocated to plasmamembrane after the treatment of the tmTNF-a. These results implied that although sTNF-a and tmTNF-a interacted with the same receptor, they induced DISC formation at different parts of the cells.4. tmTNF-a could not recruit RIP-1, TRAF2 and cIAPl to plasmamembrane through TNFR1:Co-immunoprecipitation of membrane proteins also showed that sTNF-a stimulated TNFR1 to recruit RIP-1, TRAF2 and cIAP1 to the plasmamembrane. In contrast, TNFR1 did not recruit these molecules after the tmTNF-a was treated. The results implied that unlike the sTNF-a, the tmTNF-a could not recruit anti-apoptotic signaling molecules to plasmamembrane through TNFR1.5. tmTNF-a recruiting apoptotic signaling molecules via TNFR1 was DD-independent:His-TNFR1 and His-ADD (lack of TNFR1 death domain) plasmids were transfected into HEK293 cells. After exposed to sTNF-a or tmTNF-a for 30 min, co-immunoprecipitation of plasmamembrane proteins found that both His-TNFR1 and His-ADD could recuit TRADD, FADD and caspase-8 after tmTNF-a was treated. It suggested that TNFR1 recuiting DISC after the stimulation of tmTNF-a did not depend on the death domain of TNFR1.6. TNFRl recuited STAT1 through its non-DD after the tmTNF-a stimulation: Co-immunoprecipitation of plasmamembrane proteins found that His-TNFR1 or His-ADD could not recuit STAT1 with the untreatment or treatment of sTNF-a. But after tmTNF-a was treated, both His-TNFR1 and His-ADD could recuit STAT1. Co-immunoprecipitation of the whole-cell proteins found that His-TNFR1 recuited STAT1, TRADD and caspase-8 after sTNF-a stimulation. His-ΔDD could not recuit TRADD and caspase-8 while the recuitment of STAT1 was remarkably reduced. In contrast, after tmTNF-a stimulation, both His-TNFR1 and His-ΔDD could recuit STAT1, TRADD and caspase-8. These results indicated that TNFR1 recuited STAT1 at the plasmamembrane after the tmTNF-a stimulation, and the recuitment of STAT1 did not need the death domain of TNFR1. The results suggested that STAT1 might interact with TNFRl non-DD directly so as to participate in the DISC recuited by tmTNF-a through TNFR1.Ⅱ. The study of the reverse signaling pathway of tmTNF-a 1. tmTNF-a reuited NIK and SODD:Co-immunoprecipitation assays of the whole-cell lysates were performed from Raji cells with specific tmTNF-a monoclonal antibody. NIK and SODD could be sedimentated by the tmTNF-a antibody. Then the specific NIK and SODD antibody was used to perform the co-immunoprecipitation assays,. It was found that both NIK and SODD could immunoprecipitate tmTNF-a. The results showed that in addition to TRAF1, IKKa and NF-KBp52, NIK and SODD were members of tmTNF-a reverse signaling complexes.2. TNF-LS recuited TRAF1:TRAF1, IKKa and tmTNF-a of wild-type or mutants were co-transfected to 293T cells. The co-immunoprecipitation assays showed that both the wild-type tmTNF-a and the TNF-LS (lack of the extracellular domain) could recuit TRAF1, while ACS-tmTNF-a (lack of the intracellular domain) could not. This suggested that tmTNF-αreuited TRAF1 through its intracellular domain.3. TNF-LS could not interact with IKKa directly:TNF-LS and IKKαplasmids were co-transfected into 293 T cells to further confirm the interactions between TNF-LS and IKKα. The Ip-western assays showed that TNF-LS could not recuit IKKα. The result indicated that TNF-LS could not recuit IKKαwithout the existence of TRAF1.4. TRAF1 acted as a bridge to transmit signals from tmTNF-αto IKKα: siTRAF1 was used to silence TRAF1 expression. The Ip-western assays showed that after the knockdown of TRAF1 expression by siRNA, the amount of TRAP1 and IKKαprotein in the tmTNF-αantibody-precipitated complexes decreased remarkably. These results indicated that TRAF1 might act as a linker to recruit IKKαto tmTNF-α.5. Inhibition of the tmTNF-αphosphorylation could promote the recuitment of TRAF1/IKKα/NF-κBp52:It was previously found that the phosphorylation site for CK1 at the tmTNFαintracellular domain was important for the formation of tmTNF-αreverse signaling complexes.The specific CK1 inhibitor D4476 was used to inhibit the tmTNF-αphosphorylation. The Ip-western assays showed that the levels of TRAF1, IKKαand NF-κBp52 that was recruited to tmTNF-α, were higher than those of the untreated cells in which the tmTNF-αreverse signaling was constitutively activated. The result indicated that the inhibition of tmTNF-αphosphorylation could promote the formation of tmTNF-αreverse signaling complexes.In conclusion, the study demonstrated the molecular mechanism underlying tmTNF-α-mediated apoptosis signaling pathway through TNFR1:tmTNF-αcould directly recuit STAT1 to the non-death domain of TNFR1 on the plasmamembrane. STAT1 then recuited TRADD, FADD and caspase-8 to form DISC, which led to the activation of caspases cascades and the apoptosis of cancer cells. The molecular mechanism of the reverse signaling pathway of tmTNF-a was also clarified:tmTNF-a could recuit TRAF1, NIK and IKKa so as to activate noncanonical NF-KBp52 to prevent cancer cells from apoptosis. On the other hand, tmTNF-a could recuit SODD, which might involve in the canonical NF-κB signaling pathway.Our study investigated the mechanism of tmTNF-a-induced cell death through the forward signals and tmTNF-a-prevented cell death through the reverse signals. The work may provide new clues and targets for clinical treatment as well as chemoresistance intervention of cancer.