Endoplasmic Reticulum Stress Inducible MANF Relocalizes And Negatively Regulates NF-κB Signaling Pathway

Inflammation can cause endoplasmic reticulum stress (ERS) and therefore activate the unfolded-protein response (UPR). Crosstalk between inflammatory responses and ER stress-induced UPR probably influences pathogenesis and progression of diseases. The ER stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death and be critical for dopaminergic neural cells to survive. Here, we analyzed the role of MANF during ER stress using primary fibroblast-like synoviocytes (FLSs) and neural cells. ER stress can trigger expression of MANF, which translocates and accumulates in the nucleus. Previous studies have demonstrated that inflammation induced ER stress and MANF expression in rheumatoid arthritis rats and MANF plays a protective role in controlling inflammation through inhibiting synoviocytes proliferation and inflammatory cytokines production. However, detailed mechanisms are not fully understood. Objective:To investigate the molecular mechanism of MANF inhibiting inflammation under endoplasmic reticulum stress and it’s involved signaling pathway. Methods:Immunofluorescence microscopy reveals MANF is significantly increased and translocates in the nucleus under ER stress. RT-PCR reveals induction of MANF in inflammatory cells under ER stress. Nuclear and cytoplasmic lysates were immunoblotted with anti-MANF antibody, the results also indicates nuclear translocation of MANF in inflammatory cells under ER stress. We performed a yeast two-hybrid to screen a Human Fetal cDNA library using MANF as the bait and found that MANF interact with p65 in eukaryotic cell. Using immunofluorescent analysis, we observed colocalization of p65 and MANF in FLSs and neural cells following tunicamycin treatment. To test if MANF forms a cognate complex with p65 under ER stress, we carried out an immunoprecipitation assay. To explore the MANF-binding region within p65, we constructed a series of p65 deletion mutants and carried out an in vitro GST pull-down assay and found that MANF binds to the DNA binding domain of p65. To study the effect of MANF on NF-κB-mediated gene activation, double luciferase reporter assays were carried out in N2A cells. To further examine the possibility that MANF might act on processes other than directly on NF-κB itself, we asked whether MANF would affect the degradation of IκBα.293T cells transfected with MANF were treated with 10 ng/ml TNF-α and western blot was performed on the cell extracts to check the degradation of IκB-α or on the nuclear and cytoplasmic extracts to examine the nuclear translocation of p65. To explore whether MANF regulates NF-κB DNA-binding activity, we performed EMSA assay to testify NF-κB DNA-binding activity was influenced by MANF in vitro. To further examine whether MANF has the ability to regulate the DNA binding activity of p65 in vivo, we performed ChIP assays to examine the binding of p65 to the promoters of endogenous NF-κB-regulated genes. Results:1. Nuclear translocation of MANF in FLS induced by TM or LPSTo determine whether inflammation regulated nuclear lcalization of endogenous MANF, primary FLSs were isolated from AIA joint synovium and treated with chemical ER stress inducer tunicamycin. MANF was mainly present in the cytoplasm in control cells. When cells were treated with tunicamycin(2.5μg/ml)for 16 hrs, however, MANF accumulated in the nucleus. To mimic inflammatory conditions in vivo, murine peritonel macrophages were treated with LPS (10μg/ml) for 48 hrs, supernatants were harvested and incubated with FLSs for 16 hrs, in untreated cells, endogenous MANF localized in the cytoplasm while LPS induce endogenous MANF to accumulate in the nucleus. Meanwhile, western blotting also showed that endogenous MANF localized to the nucleus following tunicamycin treatment.These data suggest that inflammation can induce MANF relocalize to nuclei.2. Nuclear translocation of MANF in neurons under ER stressImmunofluorescence microscopy reveals nuclear translocation of MANF under ER stress in primary neurons,SH-SY5Y,N2A and BV2 cell lines. RT-PCR reveals induction of MANF in N2A cells under ER stress (time course). N2A cells were cultured with or without TM (5μg/ml) for 12 hrs, Cytoplasmic and nuclear fractions were prepared for Western blot analysis, which reveals nuclear translocation of MANF in N2A cells under ER stress.3. Identification of p65 interacts with MANF in the nucleus during ER stressTo explore the potential function of MANF in cellular processes, we performed a yeast two-hybrid to screen a Human Fetal cDNA library using MANF as the bait. Among the positive clones, one interesting candidate is the p65 subunit of NF-κB (RelA). To verify the interaction between p65 and MANF, we co-transformed AH109 yeast competent cell with pGADT7-p65 and pGBKT7-MANF. The co-transformation assay confirmed that p65 interacts with MANF. Using immunofluorescent analysis, we observed colocalization of p65 and MANF in normal and inflammatory FLS following tunicamycin treatment.This colocalization was also found in neural cells, such as SH-SY5Y, N2A and BV2 cell lines. To test if MANF forms a cognate complex with p65 under ER stress, we carried out an immunoprecipitation in which p65 antibody and control IgG were preconjugated to the protein A/G Sepharose beads and incubated with N2A cell lysate upon tunicamycin stimulation. Cells extracts were prepared, immunoprecipitated with anti-p65 antibody and subjected to western blotting analysis. Indeed, we found that endogenous MANF interacts with p65 only under ER stress. All these results show that MANF interacts with p65 in the nucleus during ER stress.4. DNA binding domain of p65 mediates its interaction with MANFThe p65 subunit of NF-κB harbors an N-terminal conserved region (300 amino acid residues) known as RHD and a C-terminal transactivation domain. To explore the MANF-binding region within p65, we constructed a series of p65 deletion mutants. GFP-tagged p65 and its deletion mutant proteins were expressed in N2A cells to obtain soluble p65 proteins. Aliquots of N2A cells lysates containing GFP-p65 and its deletion mutants were incubated with GST-MANF. It was found that the loss of amino acids 1-190 at the N terminus of p65 resulted in its complete inability to interact with MANF. To explore whether MANF could directly interact with p65, we carried out an in vitro GST pull-down assay using recombinant GST-MANF and 6His-p65-N2 (amino acids 1-190) proteins expressed in E.coli. The result suggests GST-MANF, but not GST, was able to pull down 6His-p65-N2, demonstrating that MANF binds to the DNA binding domain of p65 directly.5. Suppression of cytokine-trigged NF-kB activation by MANFTo study the effect of MANF on NF-κB-mediated gene activation, double luciferase reporter assays were carried out in N2A cells. Cells were transiently transfected with a luciferase reporter construct containing three copies of the NF-κB binding site together with MANF-flag. Twenty-four hours post-transfection, cells were either left untreated or treated with TNF-α for 6hrs, followed by luciferase assays. Reporter assays showed that overexpression of MANF inhibited TNF-α-induced NF-κB activation. To determine whether endonuclear MANF regulates NF-κB reporter gene activity, MANF-NLS (including nuclear localization signal profile:RRGGCGSVGRRRQRR) was constructed and used in luciferase report assay. The result showed that overexpression of MANF-NLS dramatically inhibited TNF-α induced NF-κB activation. Then we turned to investigate the effect if endogenous MANF expression was reduced via SiRNAs. Three MANF siRNAs were fished out, and we confirmed the effectiveness of them against MANF by monitoring the protein level. The data shows knock-down of endogenous MANF significantly enhanced TNF-α stimulated reporter gene activity, which is consistent with the overexpression studies and suggests that MANF is a negative regulator of the NF-κB signaling pathway. To further confirm the role of MANF in the regulation of the NF-κB signaling pathway, we increased the amounts of MANF-flag and examined the effect of MANF on the activation of the NF-κB induced by TNF-α. The proper expression of MANF-flag in cells was confirmed by Western blot analysis. The data indicated TNF-a-induced NF-κB activation was inhibited by MANF in a dose-dependent manner. These results suggest that MANF is an inhibitor of the NF-κB signaling pathway.6. MANF interacts with p65 in the nucleus and represses its DNA-binding activityTo further examine the possibility that MANF might act on processes other than directly on NF-κB itself, we asked whether MANF would affect the degradation of IκBα.293T cells transfected with MANF were treated with 10 ng/ml TNF-α and western blotting was performed on the cell extracts to check the degradation of IκB-α, or on the nuclear and cytoplasmic extracts to examine the nuclear translocation of p65.We did not find any obvious differences in this regard between MANF over-expressed cells and normal cells during TNF-α stimulation. The fact that MANF interacts with p65, but does not affect its nuclear translocation, together with the above finding that MANF significantly blocks p65-mediated transcriptional activation, strongly suggests that MANF probably regulates NF-κB at the level of p65 in the nucleus. Indeed, we found that endogenous MANF interacted with p65 only in the nucleus but not in the cytoplasm.Thus, we hypothesized that MANF performed its function by serving as a potential molecular regulator of NF-κB inside the nucleus. To explore this hypothesis, we investigated whether the nuclear presence of p65 was influenced by MANF. Therefore, 293 T cells were transfected with the MANF siRNAs or MANF-flag and stimulated with or without TNF-α. Interestingly, the amount of nuclear p65 was markedly diminished in cells that were over-expressed with MANF-flag even though these cells were stimulated with TNF-α. In contrast, the amount of nuclear p65 was increased in cells that were treated with siRNA against MANF. Notably, the control cells displayed no such effect on the nuclear presence of p65. The transcription factor Spl as a negative control was expressed constitutively inside the nucleus and remained intact in response to MANF knockdown or overexpression.293 T Cells were transfected with pFR reporter gene, Ga14-BD-p65-TAD fusion protein plus MANF-NLS or MANF siRNA, respectively. After 24 hrs, cells were lysed and luciferase activities were determined. MANF repressed the activation of κB-Luc reporter gene by p65-DBD-VP16-TD, But MANF did not induce any considerable change on Gal4-BD-p65-TAD-mediated activation of pFR reporter gene, which suggest MANF represses p65 DNA-binding activity.To further examine whether MANF has the ability to regulate the DNA binding activity of p65 in vivo, we performed ChIP assays to examine the binding of p65 to the promoters of endogenous NF-κB regulated genes, such as A20 and IκBα. Consistently, deficiency of the endogenous MANF level resulted in considerable increases in the amount of p65 associated with its endogenous cognate promoters upon TNF-α stimulation. In addition, this was also true for other components of the NF-κB transcriptional enhanceosome such as RNA polymerase Ⅱ. Understandably, deficiency of the endogenous MANF level had no effect on the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcriptional complex. Collectively, these results further support the conclusion that MANF inhibits the activity of NF-κB by interfering with the recruitment of p65 to the promoters of NF-κB-regulated genes. Conclusions:This study identifies MANF as a novel repressor of NF-κB, which reveals a negative feedback mechanism for NF-κB signaling under ER stress.