Investigation Of The Signal Transduction In Ectopic Expression OfγδT Cell-ligand HMSH2in Stress And The Dynamic Expression Of HMSH2during The Cell Malignant Transformation

Human y8T cells act as a crucial population of human innate immune cells. y5T cells can directly recognize some stress-induced ligands expresssing on the surface of tumors or transformed cells, and play important roles in mediating the cytotoxicity to relative target cells and maintaining the immune homeostasis.Human MutS homologue2(hMSH2), a critical component of the highly conserved DNA mismatch repair system, normally synthesised in the cytoplasm and transported into the nucleus to participate in the maintenance of the fidelity of DNA replication. Previously, we observed an unusual and extensive expression of hMSH2on the surface of epithelial tumor cells. The surface expression hMSH2was also inducible by Epstein-barr virus (EBV) infection and could be dually recognized by T cell receptor (TCR)y5and natural killer group2member D (NKG2D), promoting the specific clearance of EBV-infected cells by γδT cells. However, there is only limited numbers of identified stress ligands recognized by γδT cells so far, and it is unclear of the mechanism that stress induces the surface expression of these ligands on tumor cells.Based on our previous study, we proposed that hMSH2might be a stress-inducible ligand of γδT cells. To test this possibility, we first examined the surface expression of hMSH2on renal carcinoma (RCC) cells in different cellular stress models because hMSH2was constitutively expressed at a low level on the surface of RCC cells. Flow cytometric (FCM) analysis showed that the ectopic expression of hMSH2was induced in stressful environments at different degrees, and the most obvious up-regualtion appeared in oxidative stress (from8～10%to40～50%). However, oxidative stress-induced ectopic expression of hMSH2could be blocked by the addition of a reactive oxygen species (ROS) scavenger N-aceryl-L-cysteine (NAC). Inducible ectopic expression of hMSH2was also observed on other tumor cells such as melanoma A375, lymphoma Jurkat and leukemia cells K562using the FCM assay, which raised the possibility of hMSH2being a stress-inducible ligand of γδT cells. Meanwhile, oxidative stress could induce the expression of hMSH2both on mRNA and total protein levels analyzed by quantitative real-time PCR (qRT-PCR) and Western blot. In addition, oxidative stress-induced ectopic expression of hMSH2showed a similar change pattern to the surface expression of MHC class I-related chains A and B (MICA/B), classical stress sensitive ligands of NKG2D. It further confirms the stress-inducible characteristic of ectopically expressed hMSH2on tumor cells, which might be a member of the danger associated molecular pattern (DAMP) family.The second point of this study was to explore the signal transduction mechanisms responsible for the ectopic expression of hMSH2in RCC cells and to identify the key factors regulating the above course. Western blot analysis showed that p38mitogen-activated protein kinase (p38-MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK-JNK) and mitogen-activated protein/extracellular signal-regulated kinase (MEK-ERK) pathways were activated in oxidative stress. The administration of specific inhibitors for p38-MAPK and SAPK-JNK pathways but not the inhibitor for MEK-ERK pathway could suppress the surface expression of hMSH2on RCC cells. Quantitative real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and total protein expression of hMSH2were also down-regulated when blocking the role of p38-MAPK or SAPK-JNK pathway in oxidative stress. We also assessed the effect of apoptosis signal-regulating kinase1(ASK1) on ectopic expression of hMSH2because it could activate both SAPK-JNK and p38-MAPK pathways in stress. The results showed that both the total and the ectopic expression of hMSH2were enhanced in RCC cells when ASK1was activated and this increase could be suppressed substantially by NAC or calyculin A, the latter could inhibit the primary activation of ASK1. The effect of ASK1on the ectopic expression of hMSH2further demonstrates the role of p38-MAPK and SAPK-JNK pathways in regulating the ectopic expression of hMSH2in RCC cells. We also found that activating transcription factor3(ATF3), a stress-sensitive transcription factor (TF) locating at the downstream of p38-MAPK and SAPK-JNK pathways, played a crucial role in the ectopic expression of hMSH2. Knockdown of ATF3in G401cells by small interference RNA (siRNA) could block the up-regulation of surface expressed hMSH2. However, there is no significant down-regulation of MICA/B in GO-treated G401cells even knockdown the expression of ATF3, suggesting that ATF3might be a specific executor promoting the ectopic expression of hMSH2regulated by p38-MAPK and SAPK-JNK pathways in oxidative environemnts.The third point of this study was to further seek key factors inducing the ectopic expression or the distribution of hMSH2in oxdiative stress. Our study showed that interleukin18(IL-18) was released increasingly into supernatants of oxidative stress-stimulated RCC cells. The expression of interelukin18receptor a (IL-18Ra) was constitutively on normal cultured tumor cells and increased in oxidative stress. The up-regulation of the surface expression of hMSH2was induced by IL-18treatment and stress-induced ectopic expression of hMSH2could be inhibited by knocking down of IL-18Ra in RCC cells. All these data suggest that IL-18released by tumor cells in oxidative stress might have a direct effect on regulating the ectopic expression of hMSH2. Using enzyme linked immunosorbent assay (ELISA) and intracellular staining assay, we found that the production of interferon (IFN)-y was induced by oxidative stress-stimulated γδT cells. Exogenous IFN-y could also promote oxidative stress-induced ectopic expression of hMSH2and this effect could be inhibited by NAC. Furthermore, the cytolysis of G401cells by γδT cells could be enhanced by oxidative stress, IL-18or IL-18plus IFN-y. These data suggest that IFN-y released by γδT cells might promote the ectopic expression of hMSH2, and IL-18or IFN-y promotes the cytolysis of RCC cells by γδT cells.The final point of this study was a primary analysis for the process of ectopic expression of hMSH2in malignant transformation of HK-2cells. HK-2cells were treated with different doses of3-methylcholanthrene (3-MCA). We analyzed the variation of the cell morphology, chromosome numbers, the concanavalin A (ConA) agglutination and the ability of soft agar colony forming, the relevant change of ectopically expressed hMSH2on HK-2cells. The FCM data showed that during the malignant transformation, there was an up-regulated trend of the ectopic expression of hMSH2on HK-2cells. Ectopic expression of hMSH2was finally stabilized at30～40%. This increased ectopic expression of hMSH2could promote the cytolysis of transformed HK-2cells by γδT cells.In conclusion, our major findings in this study include (1) the ectopic expression of hMSH2on tumor cells possesses the inducibility in stress. The stress-inducible characteristic of the ectopic expression of hMSH2makes it be a potential novel target warning the immune system in stress.(2) p38-MAPK and SAPK-JNK pathways play critical roles in controlling the ectopic expression of hMSH2on RCC cells through ASK1and ATF3in oxidative stress.(3) IL-18produced by tumor cells could induce the ectopic expression of hMSH2in oxidative stress, and γδT cells-produced IFN-y might promote above effects. These results suggest that ROS, IL-18and IFN-y might constitute a positive feedback maintaining the ectopic expression of hMSH2on tumor cells, acting as an alert signal to γδT-mediated immunosurveillance in stressful environments.