Effect Of ERS On Tumor Necrosis Factor- Related Apoptosis-inducing Ligand(TRAIL) Expression Inmacropages And And Its Molecular Mechanism

Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), is a new member of the family of tumor necrosis factor(TNF), which includes cytokines such as TNF and Fas L, and plays a role of biology by binding with its death receptors in cell membranes. In the immune system, immune cell apoptosis is an important way of immune regulation, and is from the body to maintain the immune system stability, avoiding the important mechanism of autoimmune disease. Importantly, many studies have reported that TRAIL is expressed on natural killer(NK) cells, neutrophils, T lymphocytes, monocytes, macrophages and dendritic cells(DC). TRAIL has important significance in regulating immunological surveillance and immune cell apoptosis, natural immunity, virus infection, immunologic tolerance mechanism and so on.The endoplasmic reticulum(ER) is a critical intracellular organelle and plays a major role in the process of protein synthesis and transport. Agents or conditions adversely influencing ER protein folding, resulting in accumulation of misfolded or unfolded proteins in the ER, a state known as ER stress(ERS). Our previous research determined that hepatic macrophages(kupffer cells) could induce activated hepatic stellate cells(HSCs) apoptosis through the upregulation of TRAIL expression and other apoptosis molecules thereby reverse liver fibrosis. In addition, ERS response participated in liver fibrosis and ralated to the outcome of liver inflammation and fibrosis. Whether ERS are involved in the expression of TRAIL in macrophages and promote the HSCs apoptosis,reversing hepatic fibrosis were still remain unclear. To explore the role of ERS in TRAIL released by macrophages, we choose three different sources of macrophages as the targets for the pharmacological, molecular, and other novel therapeutics. One focus of this review is the various aspects expression of TRAIL in ERS-induced macrophages in vitro. The main contents are divided into four sections, as follows:1. Effect of ERS on TRAIL expression in macrophagesTo determine the effects of ERS on TRAIL expression, we treated macrophages with TG, an inhibitor of ER Ca2+ ATPase, or TM, an inhibitor of glycosylation, to stimulate the ERS response in murine macrophages RAW264.7 cells, THP-1 and murine peritoneal macrophage in vitro. The expression of TRAIL m RNA and intracellular protein in ER stressors-treated cells were determined by RT-PCR and Western blot. In addition, the expression of souble TRAIL(s TRAIL) protein was performed by ELISA, and the level of surface protein was measured by FCM. These results suggest that both of the ERS inducers(TG and TM) remarkably increased TRAIL m RNA and protein. However, there was no change in surface protein expression was found neither after stimulation with TG nor TM in all experiments. These results collectively demonstrate that ERS increased TRAIL expression in macrophages.2.MAPK invovled in ERS-mediated TRAIL upregulation in macrophagesTo determine the potential signaling pathway involved in ERS-mediated TRAIL upregulation in RAW264.7 cells, we examined the involvement of MAPKs activation. The phosphorylation levels of ERK, JNK and p38 MAPK by Western blot. The results showed that ERS promoted MAPKs activation in a time dependent manner. To further distinguish MAPK signaling pathways, which are necessary for ERS-induced TRAIL upregulation, pharmacological MAPK specific inhibitors were used. We found that ERS-induced TRAIL m RNA and protein expression was both inhibited by JNK inhibitor SP600125, indicating that JNK MAPK invovled in ERS-mediated TRAIL upregulation in macrophages.We then further determined whether ERS stimulation also enhances the association of JNK with AP-1. Nuclear AP-1 binding activity was analysed using ELISA-based Trans AM kit after isolation of nuclear extract, RT-q PCR and Western blot. We observed that both of the ERS inducers remarkably increased AP-1 expression and activation. Next, we examined the potential of AP-1 to modulate TRAIL gene expression in RAW264.7 cells using AP-1 inhibitor SR 11302. RT-q PCR, Western Blot and ELISA analysis showed that SR 11302 blocked ERS-induced TRAIL m RNA and protein level. And deletion of AP-1 element reduced ER stressors-induced TRAIL promoter activity by luciferase activity assay. These findings indicate that ERS-induced TRAIL expression is mediated by the JNK/AP-1 signaling pathway.3. SOCS3 negatively modulate TRAIL induction under ERS in RAW264.7 macrophagesTo confirm whether SOCS3 acted as a regulator of TRAIL expression in RAW264.7 cells under ERS. Ransfecting with the SOCS3 RNAi and p CMV-SOCS3 plasmid into RAW264.7 cells using LipofectamineTM 2000 to observe the change of SOCS3 and TRAIL in TG/TM treatment of RAW264.7 cells. RAW264.7 transfected with p CMV-SOCS3 significantly suppressed ER stressors-induced TRAIL expression compared with the model group. Instead transfection of SOCS3 si RNA into RAW264.7 can obviously increase expression of TRAIL treated by TG/TM.4. SOCS3 regulated ERS induced-TRAIL expression through the JNK/AP-1 PathwayActivity of AP-1 was markedly decreased in activated RAW264.7 cells transfected with p CMV-SOCS3 after TG/TM stimulation compared with compared with the model group, while the TRAIL protein level was inhibited by SP600125 and SR 1130. Instead, transfection SOCS3 si RNA into RAW264.7 can produce a significant elevated in the activity of AP-1, but a descrease in the secretion of TRAIL expression treated with SP600125 and SR 1130. These results indicated that SOCS3 negatively controled the production of TRAIL by inhibiting serine phosphorylation of c-Jun via the JNK pathway.