Molecular Mechanisms On TNFRSF10B And CFLAR Regulation In The Apoptosis In Human Lung Cancer Cells

BackgroundTNFRSF10B (also named DR5), one of TRAIL receptors, belongs to the tumor necrosis factor receptor superfamily. In apoptotic cells, it locates on the cell surface, and becomes trimerized by binding to its ligand TRAIL and recruits adapter proteins such as fas-associated death domain (FADD) and then forms the death inducing signaling complex (DISC), eventually activates CASP cascades. Nowadays, many studies have demonstrated that up-regulation the expression of TNFRSF10B contributes to certain cancer therapeutic agents-induced apoptosis and enhances TRAIL-induced apoptotic pathway.Various disturbances caused aggregation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) trigger ER stress. DDIT3 (DNA-damage-inducible transcript 3), a downstream component of ER stress pathways, can be mediated by EIF2AK3-, ATF6- and ERN1-induced signal transduction pathways. Many studies have documented that DDIT3 promotes apoptosis through up-regulation TNFRSF10B expression caused by many agents, such as lonafarnib, MG132, CDDO-Me in a variety of cancer cells. Activating transcription factor 4 (ATF4) and ATF3, are stress responsive proteins which can be induced by ER stress. ATF4 has proved induces DDIT3 expression when there are severe ER stress signal. Some earlier studies have found that ATF3 can suppress DDIT3 gene transcription, while DDIT3 inhibits ATF3 protein function. However, some data show that ATF3 promotes DDIT3 expression.MAPK1 executes its function as anti-apoptotic or pro-apoptotic effect dependent on the specific cell type and the nature of the stimuli. Studies have shown that MAPK1 activation enhances cisplatin and celecoxib induced apoptosis in multiple cancer cells. RPS6KA3 is a known direct downstream effector of MAPKl and it plays dual roles in regulating cell survival, proliferation and apoptosis. RPS6KA3 belongs to the Ser/Thr kinases, recent studies have shown that MAPK1 and RPS6KA3 regulate ATF4 and DDIT3 expression and then increase TNFRSF10B expression.The protein kinase C (PKC) family is important in regulation of cell proliferation and apoptosis. PKC8 is generally considered to be a pro-apoptotic protein. It can be cleaved between the regulatory domain and catalytic domain when cells are exposed to diverse stimuli. Consequently, a 41kD fragment is generated which has constitutively bioactivity and is responsible for apoptosis. PKCa plays a positive function in cell survival, but studies also show it induces apoptosis, for example, PKCa expression increased camptothecin-induced apoptosis. However, the mechanism whether PKCa palys a role in PS-341 mediated apoptosis is unknown.Many preclinical experiments have shown that the proteasome inhibitor PS-341 alone or combination with other agents have anti-tumor effect in various cancers, including lung cancer. However, the molecular mechanism underlying PS-341 mediated apoptosis is not very clear now. Our previous studies have shown that TNFRSF10B expression contributes to PS-341-mediated apoptosis in human NSCLC cells. So this research is further characterize the mechanism by which PS-341 up-regulates TNFRSF10B expression in NSCLC cells.Lysine acetylation is important in diverse biological processes, including cell signaling, cytoskeleton dynamics, metabolism, DNA replication and so on. The balance between protein acetylation and deacetylation is maintained with acetyltransferases (HATs) and deacetylases (HDACs). An increasing number of non-histone proteins have been identified to be acetylated now, for example TP53, PKM2, c-MYC, NF-κB, α-tubulin. The acetylation of non-histone proteins often accompanied with the diverse function of the proteins. Acetylation is one of the most important alterations in many cancers and is a potential contributing element to tumorigenesis, so acetylation could function as a key target for the therapy of cancer in the futher.Protein methylation is catalyzed by methyltransferases. It is implicated in many cellular pathways including protein-protein interactions, signal transduction, DNA repair, transcription and so on. The protein arginine deiminases 4 (PADI4) is one of demethylases which was first found in 2004, demethyliminates peptidyl monomethyl arginine into citrulline in H3 and H4. It also proficiently removes the methyl group on the non-histone proteins, such as the nuclear coactivator EP300, nucleophosmin/B23, the 40S ribosomal protein S2 (RPS2) and inhibitor of growth 4 (ING4).CFLAR functions as a specific inhibitor of CASP8/10. It can be recruited to the DISC and inhibit the cleavage of PROCASP8/10. CFLAR is expressed in many human tumor cells, including hepatocellular, colon, melanoma, ovarian and so on. A number of small molecule drugs induce apoptosis through inhibition its expression. So CFLAR might be a promising target for cancer therapy. Studies have revealed CFLARL interacted with XRCC6 enhances CFLARL protein stability; the acetylation of XRCC6 disrupted the CFLARL/XRCC6 complex and triggered CFLARL polyubiquitination and degradation by the proteasome; but whether CFLARL could be acetylated is unknown. Mass spectrometry has predicted that CFLARL has a methylation site, but it has not been confirmed by experiment in vivo now. Our purpose is to determine whether CFLARL is acetylated or methylated and futher study the molecular mechanisms on the modification of CFLARL.Methods1. Silencing gene expression using small interfering RNA or over-expression a certain protein by plasmid transient transfection are used to study the role of the protein in signal transduction pathway.2. Cell survival assay is estimated by use of sulforhodamine B (SRB) assay.3. The expression of relevant proteins in cells is detected by western blot assay.4. Immunoprecipitation and GST pull down assay are used to detect the interactions between proteins and analysis the modification of proteins.5. Apoptosis is evaluated by Annexin V staining using Annexin V-PE/7-AAD apoptosis detection kit.6. The location of certain proteins is estimated by fluorescence microscopy.Results1. PS-341 strongly promoted DDIT3 expression in NSCLC cells in a dose- and time-dependent fashion.2. PS-341-induced TNFRSF10B expression is dramatically decreased in DDIT3 siRNA transfected cells, and weaker cleavage of CASP8, CASP9, CASP3 and PARP1 was examined in DDIT3 knockdown cells. Moreover, the lower apoptosis was detected in DDIT3 siRNA-transfected cells by Annexin V staining-flow cytometry.3. The expression of ATF4, ATF3, HSPA5, p-EIF2S 1 and ERNla, which were regarded as key protein markers of ER stress were all elevated in a dose- and time-dependent manner.4. Western blot analysis showed that the levels of TNFRSF10B and the cleaved forms of CASP8, CASP9, CASP3 and PARP1 were all decreased in ATF3 siRNA-transfected cells. The percentage of apoptotic cells induced by PS-341 in control siRNA-transfected cells was 41%, whereas only 26% in ATF3 siRNA-transfected cells by Annexin V staining-flow cytometry.5. Inhibition of ATF4 up-regulation decreased the expression of TNFRSF10B and CASP cascades after exposed to PS-341 in H460, A549 and H157 cells. Accordingly, the percentage of apoptotic cells induced by PS-341 decreased from 29% to 19% when H157 was transfected with ATF4 siRNA.6. In H460, A549 and H157 cells, ATF4 expression up-regulated the levels of ATF3 and DDIT3. In H157 and Calu-1 cells, when ATF3 expression was suppressed, DDIT3 expression was weakly inhibited while ATF4 expression was unaltered; when DDIT3 was knocked down, ATF3 level was lightly depressed while ATF4 was unaffected. Immunoprecipitation experiment was conducted to further investigate the physical relationship between ATF3 and DDIT3 and found ATF3 associated with DDIT3.7. Along with the increased dose and time, the phosphorylation of MAPK1 and RPS6KA3 was augmented when cells were exposed to PS-341. The induction of TNFRSF10B was diminished by using MEK inhibitor U0126 to suppress MAPK1 and RPS6KA3 phosphorylation.8. Using western blot analysis, the 41kDa fragment of PKCδ was detected in all tested cells and the levels were increased along with the increased concentration and time.9. After inhibition of PKC8 expression by small interfering RNA, we found that TNFRSF10B expression was decreased. Furthermore, the cleaved forms of CASP8, CASP9, CASP3 and PARP1 were all reduced. On the other hand, when PKC8 was over-expressed in cells, PS-341-induced TNFRSF10B expression and CASP cascades were both elevated. The percentage of apoptotic cells induced by PS-341 decreased from 41% in control cells to 23% in PKC8 siRNA-transfected cells.10. The phosphorylation of MAPK1 and RPS6KA3 induced by PS-341 in PKCδ siRNA-transfected cells was suppressed; and the induction of ATF4, ATF3 and DDIT3 were also attenuated in PKC8 abolished cells.11. PKC8 translocated to nucleus when cells were transfected with pEGFP-PKCδ plasmid in H157 cells.12. After inhibition of PKCa expression by small interfering RNA, the expression of TNFRSF10B and CASP cascades induced by PS-341 were decreased, and the up-regulation of ATF4, ATF3 and DDIT3 was also suppressed.13. The plasmids containing PKCa was transfected in H157 cells, and we found that PKCa was located in cytoplasm and cannot translocate to nucleus.14. The expression of CFLAR1 dramatically decreased in PADI4-siRNA transfected cells.15. Cells were transfected with PADI4 siRNA and pcDNA3.1-CFLARL-Flag or pEBG-CFLARL plasmids in the same time for 20h, then immunoprecipitation or GST pull down assay were conducted and found that inhibition of PADI4 expression increased the interaction between CFLAR1 and XRCC6.16.293FT cells were transfected with pEBG-CFLARLplasmid and GST pull down assay identified that CFLARL was arginine methylated.17. We cloned pEBG-CFLARL-R122A plasmid with mutation of Arg122 to Ala. GST pull down assay found the interaction between CFLARL and XRCC6 was decreased in cells transfected with pEBG-CFLARL-R122A plasmid.18. The degradation speed of CFLARL was lower in CFLARL-R122A over-expressed cells than the speed in CFLARL over-expressed cells.19. The cleaved forms of CASP8, CASP3 and PARP1 induced by SAHA were all reduced in CFLARL-R122A over-expressed cells compared to that in CFLARL over-expressed cells.20. CFLARLwas identified to be acetylated by immunoprecipitation.Conclusion1. PS-341 strongly promotes DDIT3 expression and DDIT3 expression contributes to PS-341-induced TNFRSF10B up-regulation and apoptosis.2. PS-341 activates ER stress in NSCLC cells.3. ATF3 enhances TNFRSF10B expression and apoptosis induced by PS-341.4. ATF4 increases PS-341-mediated TNFRSF10B expression and apoptosis.5. ATF4 regulates the expression of ATF3 and DDIT3, while ATF3 and DDIT3 form a complex, and they together regulate TNFRSF10B expression.6. MAPK1/RPS6KA3 regulates TNFRSF10B expression in PS-341-induced apoptosis7. PS-341 activates PKC5 and induces PKC8 translocated to nucleus, PKCδ regulates TNFRSF10B expression and apoptosis induced by PS-341 through MAPK1/RPS6KA3 signaling pathway and subsequent ER stress.8. PKCa is contributed to PS-341-mediated TNFRSF10B up-regulation and cell apoptosis through ER stress pathway.9. Inhibition the expression of PADI4 decreases the expression of CFLARL and promotes the interaction between CFLARL and XRCC6.10. CFLARL is methylated.11. Inhibition of CFLARL methylation decreases the interaction between CFLARL and XRCC6, enhances the stability of CFLARL and inhibits cell apoptosis induced by SAHA.12. CFLARL is acetylated.Collectively, we show that PS-341 induces PKCδ-dependent TNFRSF10B expression through phosphorylation of MAPK1/RPS6KA3 signaling pathway and activation of ER Stress. In addition, PKCa enhances PS-341-mediated TNFRSF10B up-regulation and apoptosis through ER stress pathway. On the other hand, CFLARL is identified to be acetylated and arginine methylated for the first time. Inhibition of CFLARL methylation decreases the interaction between CFLARL and XRCC6, enhances the stability of CFLARL and inhibits cell apoptosis induced by SAHA.