The Reverse Signaling Pathway Of TmTNF-α And The Molecular Mechanism Underlying Its Protection Of Tumor From Apoptosis

transmembrane TNF-a(tmTNF-a) can not only interact directly with TNFR and transduce forward signaling into TNFR bearing cells to play effects, this signaling pathway is also called forward signaling pathway. Meanwhile, tmTNF-a can also work as receptors to accept membrane bound TNFR or soluble TNFR stimulation, and transdues signaling into tmTNF-a bearing cells, this signaling pathway is called reverse signaling pathway, which mediate several biological effects. The reverse signaling of tmTNF-a can upregulate activated T cells to secret IFN-y and downregulate generation of IL-4; it can also inhibit induced monocytes by LPS to secret proapoptotic factors and proinflammation factors, such as TNF-α,IL-1,IL-6 generation; but it upregulate IL-12 secretion. Furthermore, reverse signaling pathway induced by tmTNF-a can induce HTLV infected T cells or PHA activated CD4+T cells expression of E-selectin. We previously found that tumor cells with high expression of tmTNF-a can protect tumor cells from secretory TNF-a (sTNF-a) cytotoxicity. However, the reverse signaling pathway is still unknown.We previously prepared a specific monoclonal tmTNF-a antibody, and we used this specific antibody to immunoprecipitate, and we had got IKKα,TRAF1 and NF-κBp52 to interact with the intracellular domain of tmTNF-a. This research is mainly studying the relationship of the member of the reverse signaling pathway of tmTNF-a, and their relationship with mitochondria. We also worked on the relationship between tmTNF-a expression on tumor cells and chemoresistance, and we also further study into the molecular mechanism of the chemoresistance induced by tmTNF-a, so as to provide new target molecular and theory basis for clinical intervention. Main results as below:I. Construction and identification of prokaryotic,eukaryotic and fluorescence fusion expression vectors of TNF-LS,IKKαand TRAF11. Construction and identification of prokaryotic,eukaryotic and fluorescence fusion expression vectors of TNF-LS,IKKαand TRAF1:PCR and molecular cloning techniques were used to construct prokaryotic fusion vectors of pTriEx-4C-TNF-LS,pTriEx-4C-TRAF1,pTrieX-4C-IKKα,pET28a-his-TRAF1 and pET28a-his-IKKa; fluorescence expression vectors of pEGFPNl-TNF-LS,pDsRedN1-TNF-LS,pCFPN1-TNF-LS and pEGFPC1-IKKa; eukaryotic fusion vectors of pTriEx-4C-TNF-LS,pTriEx-4C-TRAF,pTriEx-4C-IKKa and pcDNA3.1-IKKa. And they were successfully constructed by sequencing and blasting.2. Construction and identification of the mutated TNF-LS in eukaryotic and fluorescence fusion expression vectorspcDNA3.1/V5-His-TOPO-TNF-LS was taken as a template, PCR amplification of a fragment about 208 bp, restriction to connect to pcDNA3.1, transformation of the ligated product into competent DH5a bacteria, choosing positive clones, restriction enzyme digestion and sequencing. And then the correct sequenced pcDNA3.1-△-75--69-TNF-LS and pcDNA3.1-△-68～-62-TNF-LS digested, recycling the fragment, after digestion, connected to digested pEGFPNl, the identification of the positive clones by digesttion.II. Identification of the major members of the reverse signaling pathway of tmTNF-αTRAFl and IKKa and the relationship among them1. IKKα,TRAF-1 and NF-κBp52 can be immunoprecipitated by tmTNF-αmonoclonal antibodyIn order to investigate into the reverse signaling pathway of tmTNF-α, specific tmTNF-αmonoclonal antibody was used to IP-Wetern. The results show that TRAFl, IKK-αand NF-κBp52 can be sedimentated by tmTNF-αantibody. However, TNFR1/2, TRAF2/3, NIK, SODD, FADD and NF-κBp65 can not be immunoprecipitated. Conversely, using TRAF1, IKKa and NF-κBp52 antibody to re-IP, all of them can immunoprecipitated tmTNF-α. The results imply that TRAFl and IKKa might participitate in the formation of reverse signaling complexes of tmTNF-αso as to activate NF-κB.2. TNF-LS directly interact with TRAFlFirst of all, we intend to explore the relationship between TNF-LS and TRAFl, direct or indirect interactions. Prokaryotic expression with his label with TRAF1 and prokaryotic expression of the non-his tagged TNF-LS in vitro Pull down, we confirmed that TNF-LS and TRAFlinteract directly. In order to further confirm this direct interaction, we transfected TRAF1 and TNF-LS plasmid alone or co-transfected into 293T cells to overexpress, and then by IP-Western to verify their interaction in vivo and found that tmTNF-αwith monoclonal antibodies can pull down significantly higher TRAFl in cells cotransfected with TNF-LS and TRAF1 than that of the single-TRAFl transgenic group or the blank control group. Further, we build TRAFl and TNF-LS fluorescence fusion vectors with different corresponding fluorescence to study their cellular localization, found that over-expression of TRAFl in 293T cells showed the limited point-like distribution, and TNF-LS distributed near the membrane. Co-transfected TRAF1 and TNF-LS resulted in gathering of TNF-LS to TRAF1 localization, the confocal results showed that the fluoresence of the TRAF1 and the fluorescence of the TNF-LS merged. Direct interaction of TNF-LS with TRAF1 confirmed in Three-pronged approach.3.-75～-69 amino acids of the N terminal of TNF-LS determined the direct interaction of TNF-LS and TRAFlThe intracellular domain of TNF-LS has only 30 amino acids, and we produced mutation of TNF-LS deletion of the N-terminal -75～-69 or -68～-61 amino acids. Mutants or wild type tmTNF-αor TNF-LS together with TRAF1 were co-transfected into 293T cells and found that the TNF-LS deletion of the N-terminal of -75～-69 amino acids influenced greatly on the interaction of TRAF1 and TNF-LS, suggesting that the N-terminal -75～-69 amino acids of TNF-LS plays a decisive role in TRAF1 and TNF-LS interaction.4. Dephosphorylation of the intracellular domain of TNF-LS caused the recruitment of the reverse signaling complex, leading to NF-κB activation, resulting in the resistance of tmTNF-αhigh expression tumors resistance to ADM and sTNF-αThe N-terminal-75 to -69 amino acids decides TRAFl and TNF-LS interaction, we found that the N-terminal-75 to -69 amino acids of tmTNF-αis the site for CKI phosphorylation. Using specific inhibitors of CKI inhibitor D4476 to inhibit the activity of CKI, and then tmTNF-a monoclonal antibody was used to IP, compared with untreated Raji cell group, treatment group IP to TRAF1, IKKa and NF-κBp52 significantly more than untreated group. The results implies that phosphorylation of tmTNF-a by CKI inhibited the reverse transmission signal pathway. CKI inhibitor with Raji cells inhibited the activity of CKI can lead to NF-κB activation and further lead to Raji cells resistant to sTNF-a and chemotherapy drug doxorubicin. Suggesting that tmTNF-a may be involved in a reverse signal of NF-κB activation and resistance to chemotherapy drugs.5. TRAFl and IKKa interacted directlyWe also confirmed direct interaction of TRAF1 and IKKa from three-pronged approaches. First of all, we prokaryotically expressed TRAF1 and IKKa, Pull down assay was done in vitro and confirmed that TRAF1 directly interacted with IKKa. In addition, we use IP in the body to further give evidence that TRAF1 directly interacted with IKKa. Finally, TRAF1 and IKKa with different color fluorescent protein fusion expressed in 293T cells, after co-transfection of TRAF1 and IKKa, TRAF1 resulted in partial aggregation of IKKa protein and the fluorescence of TRAFl and IKKa merged.6. TNF-LS can not interact directly with IKKaProkaryotically expressed TNF-LS and IKKa, and in vitro pull down assay was done, we found that TNF-LS can not directly interact with IKKaIII. The close relationship of mitochondrial with the reverse signaling complex molecular TNF-LS,TRAF1 and IKKα1. TRAFl co-localized with mitochondriaIn 293T cells, mitochondria showed the mesh point distribution, after transfection of TRAF1, the distribution of mitochondria changed into point-like distribution, and co-localized with TRAF1.2. Members of the reverse signaling of tmTNF-a all exist in the mitochondriamitochondira was extracted, and western blot was taken to indentify if there was any relationship between members of the reverse signaling complex members of TRAF1, IKKa and NF-κBp52 and mitochondria. We found that TRAF1,IKKαand NF-κB p52 all could localize in the mitochondria. Co-transfection of 293T cells with TNF-LS and TRAF1 could induce mitochondria aggregation, and TRAF1 changed the TNF-LS distribution on the membrane which assembled to the position of TRAF1. IP was further taken to immunoprecipitated mitochondria proteins with monoclonal tmTNF-αantibody and NF-KBp52 can be precipitated, implying that the reverse signaling pathway might occur in the mitochondria.3. tmTNF-αand related reverse signaling complex members can be found in the nucleusnuclear proteins were prepared, and western blot was used and we identified the nuclear localization of tmTNF-a and related molecular TRAF1,IKKa and NF-κB p52, but there is no report about their relationship until now.IV. TNFLS stably high expression in MCF-7 induced the tolerance of such cells to sTNF-α1. High expression of tmTNF-a protects cells from the sTNF-a induced cytotoxicitySources of the same breast cancer tumor, some sensitive to sTNF-a induced cytotoxicity,some resistant, we found that high expression of tmTNF-a in the MDA-MB-231 induced sTNF-αtolerance, while low tmTNF-a expression in the MCF-7 cells were sensitive to sTNF-α. Prompt, tmTNF-a expression may be the protection effect from sTNF-αinduced cytotoxicity.2. Down-regulation of tmTNF-a in MDA-MB-231 cells reduced NF-κB activity with a reversal of the cells on the sensitivity to sTNF-αInterestingly, down-regulation of tmTNF-a expression in MDA-MB-231 cells with TNF siRNA resulted in the recovery of MDA-MB-231 cells sensitive to sTNF-αto some extent.3. TNF-LS expressed on the membrane of MCF-7 cellsAfter transfection of pIRES2-EGFP/TNF-LS into MCF-7 cells, the expression of EGFP and TNF-LS were separate. tmTNF-a monoclonal antibody were used for staining, and then adding the second TRITC labeled antibody. Indirect fluorescence under the microscope shows that the diffuse cytoplasmic green fluorescence with TNF-LS distribution in the cell membrane, which shows a red aperture under the fluorescence microscope.4. High expression of TNFLS in MCF-7 cells caused the cells tolerant to sTNF-αand persistent NF-κB activationEstablishment of TNF-LS stably expressed in MCF-7 cells, we found that TNF-LS/MCF-7 cells are tolerant to sTNF-a and persistent NF-κB activation, showing the same features like another breast cancer cell line MDA-MB-231 cell with high tmTNF-a expression. Prompt, TNF-LS has some tmTNF-a properties.We speculated that the reverse signaling pathway of tmTNF-a may determined by TNF-LS.5. NF-κB inhibitor PDTC inhibited stably transfected MCF-7 cells with TNF-LS NF-κB activity, resulting in reversal of the stable cell lines TNF-LS/MCF-7 sensitive to sTNF-αPDTC can selectivly inhibited the activity of NF-κB. Selective inhibition of NF-κB activity in TNF-LS/MCF-7 cells and found that the sensitivity of TNF-LS/MCF-7 cells to sTNF-a increased, suggesting that TNF-LS lead to the stability of the TNF-LS/MCF-7 cells to sTNF-a for NF-κB activation.6. Anti-apoptotic gene expression leading to TNF-LS/MCF-7 cells resistant to sTNF-a induced cytotoxicityNF-κB is an important factor in determining cell death and survival, and cIAPl is one of the target gene of NF-κB which is an antiapoptotic factor. comparing before and after STNF-a stimulation, the expression of cIAPl was significantly higher in MCF-7 cells stably transfected with TNF-LS than MCF-7 cells untranfected or transfected empty vectors.sTNF-a stimulation led to further cIAPl gene expression. The results suggest that regulation of antiapoptotic gene expression by NF-κB activation may be the reason for TNF-LS/MCF-7 cells resistant to sTNF-αinduced cytotoxicity. 7. The intracellualr domain of tmTNF-a induced durable NF-κB activation in MCF-7 cellsHigh tmTNF-a expression in breast cancer cell line MDA-MB-231 cells can lead to NF-κB activation,while, TNF-LS stabily expressed on MCF-7 cells also results in a persistent activation of NF-κB. Comparison transfection of Acs-tmTNF-α,TNF-LS and tmTNF-a into MCF-7 cell lines, we only could find durable NF-κB activation in cells instant transfection of tmTNF-a and TNF-LS. Cells transfected with Acs-tmTNF-a could not activate NF-κB when compared with untransfected or transfected empty vector groups. Our results implied that high tmTNF-a expression in breast cancer cells induced durable NF-κB activation possibly by TNF-LS.8. Endocytosis inhibitor MDC pretreatment TNF-LS/MCF-7 cellS induced surface expression of TNF-LSInhibition of TNF-LS endocytosis, TNF-LS/MCF-7 increased cell surface expression of TNF-LS may be a further increase in NF-κB activity in TNF-LS/MCF-7 cells.9. Signal peptide peptidase inhibitor ZLL pretreatment increased TNF-LS/MCF-7 cell surface TNF-LS expression, resulting in further NF-κB B activationInhibition TNF-LS shear from membrane by signal peptide peptidase inhibitor increased TNF-LS/MCF-7 cell surface TNF-LS expression, and this may be a reason for further increase in NF-κB activity in TNF-LS/MCF-7 cells.V. Reverse signaling of tmTNF-a induced the resistance of breast cancer cells to chemotherapy and related molecular mechanisms1. High expression of tmTNF-a on breast cancers induced carboplatin resistanceComparing the sensitivity difference of breast cancer cells with different tmTNF-αexpression to carboplatin, we found that MDA-MB-435 cells with high level of tmTNF-αexpression were much more resistant to carboplatin than T47D cells with low level of tmTNF-αexpression did, and found that MDA-MB-435 cells with high tmTNF-αexpression showed durable NF-κB activation, while no obvious NF-κB activity was observed in T47D cells with low tmTNF-αexpression.2. Upregulation of tmTNF-αexpression on T47D cells induced their resistance to carboplatin with the phenomenon that the cleavage of caspase-9 decreased, and NF-κB inhibitor PDTC can reverse the resistanceAfter transfection of T47D with tmTNF-α, the cells are resistant to carboplatin cytotoxicity, the cleavage of caspase-9 decreased. NF-κB inhibitor can reverse the resistance to carboplatin at a certain level.3. MCF-7 cells stably transfected with TNF-LS were resistant to carboplatin cytotoxicityIt has been confirmed that stable transfection of TNF-LS into MCF-7 cells resulted in durable NF-κB activation and tolerance to sTNF-α. In this, we have further found that TNF-LS/MCF-7 cells are more resistant to carboplatin chemotherapy treatment than untransfected or transfected with empty vectors.4. MCF-7 cells with TNF-LS inhibited the sensitivity of MCF-7 cells to carboplatinTreatment of transfected and untransfected MCF-7 cells with different concentration of carboplatin, we found that the IC50 of carboplatin to untransfected MCF-7 cells was just 231μg/ml, however, the IC50 of transfected MCF-7 cells were much higher than 400μg/ml.5. The proliferation of MCF-7 cells with high expression of tmTNF-αis quicker than MCF-7 cells with low tmTNF-αexpression after treatment of MCF-7 cells with carboplatinWe got two lines of MCF-7 cells with high tmTNF-αexpression and low tmTNF-αexpression, and we compared these two lines sensitivity to carboplatin. We found that high expression of tmTNF-αin MCF-7 cells proliferated significantly faster than the speed of low tmTNF-αexpression MCF-7 cells treated with carboplatin. And extended over time, the inhibitory effect of carboplatin to the MCF-7 cells with high tmTNF-αexpression becomes weaker and weaker.6. Inhibition NF-κB activation in MCF-7 cell line 2-E8 stably transfected with TNF-LS could reverse sensitivity of 2-E8 to carboplatin at a certain extentAs has reported in part four that stably transfected MCF-7 cells with TNF-LS induced durable NF-κB activation in these cell lines, however, inhibiting the NF-κB activity in these cells can reverse the sensitivity of these cells to carboplatin.7. Comparing the ERK phosphorylation status of T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatinERK phosphorylation was not very clear for untransfected T47D cells.After carboplatin treatment, as early as five minutes after treatment with carboplatin can resulted in ERK phosphorylation, and the ERK phosphorylation level reached peak at 10 minutes with significant decrease at 30 minutes, suggesting that stimulation of carboplatin resulted in a transient ERK phosphorylation. Instant transfection of tmTNF-a into MCF-7 cells resulted in durable ERK phosphorylation to certain degree, and activation was not affected by treatment with carboplatin.8. Comparing the JNK phosphorylation status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatinBoth transfected and untransfected T47D cells have no JNK phosphorylation without stimulation. After treatment with carboplatin, the JNK phosphorylation level of untransfected T47D cells were significantly higher than T47D cells transfected with tmTNF-α, and both JNK phosphorylation status reduced after 30 min stimulation.9. Comparing the PI3K expression status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatin T47D cells transfected with tm TNF-α, we can see continuous PI3K p85 expression, and carboplatin treatment has little effect on the expression level of PI3K. However, no observed PI3K p85 expression could be found in non-transfected T47D cells in the resting state. Carboplatin treatment of untransfected T47D cells led to a small amount of PI3K p85 expression, suggesting that PI3K may be involved in a high tmTNF-a expression in tumor cells resistant to chemotherapeutic drugs.lO.Comparing the expression of p53 in T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatinComparing the expression status of p53, we found a very interesting results. We found that after treatment of untransfected T47D cells with carboplatin, the expression of p53 in cells gradually upregulated as time went by. While, we could see p53 expression even after transfection of cells with tmTNF-αwithout treatment of carboplatin, and expression of p53 in T47D cells transfected with tmTNF-awas independent on carboplatin treatment.