| The role of NR4A1 in apoptosis is controversial. Pancreatic β-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in β-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated (3-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (-1872 bp to-1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as"positive and negative regulation."Part I the relevance between NR4A1 and endoplasmic reticulum stress in islet β-cells Aim:Impact of expression of NR4A1 by ER-stress inducers and the effect of NR4A1 on apoptosis induced by ER-stress in islet β-cells. Methods:1. MIN6 cells were treated with ER-stress inducers 0.5 μM TG or 0.4 mM PA for different time points respectively, the samples were collected, the RNA and protein were extracted, the expression of NR4A1 at mRNA and protein levels were determined with quantitative PCR and western blot as well as CHOP, a molecular marker for ER-stress.2.16-week-old C57BL/6J mice were sacrificed by cervical dislocation, the mouse islets were extracted, purified and randomly divided into groups. The mouse islets were treated with 0.5 μM TG or 0.4 mM PA for different times, the expression of NR4A1 in different treatment was determined as well as BIP, CHOP at mRNA level by quantitative PCR.3.16-week-old C57BL/6J mice were sacrificed by cervical dislocation, the mouse islets were extracted and purified, and treated with 0.5μM TG or control solvent DMSO for 6 hours. The treated islets were double stained with insulin antibody and NR4A1 antibody to test the expression of NR4A1 in islet β-cells.4. MIN6 cells were infected with lentivirus encoding NR4A1 cDNA or control lentivirus, and by drug selection, over-expression NR4A1 stable cell clones (designated as OV) or control cell clones (designated as NC) were obtained. OV cells and NC cells were treated with 0.5 μM TG or 0.4 mM PA for different times, the cell viability was analyzed with MTT; NC and OV cells were treated with 0.5 μM TG or 0.4 mM PA for 24 hours, and the ratio of apoptotic cells were analyzed by TUNEL.5. NC and OV cells were treated with 0.5μM TG or 0.4 mM PA, the protein samples were collected in 0 h,3 h,6 h,9 h,12 h,24 h respectively, the expression of NR4A1 and active form of Caspase3 were detected with western blot.6. MIN6 cells were infected with Ad-NR4A1 and Ad-GFP for 48 hours, then treated with 0.5 μM TG or 0.4 mM PA, the protein samples were collected at 0 h,6 h,12 h,24 h, the protein expression of NR4A1 and active form of Caspase3 were detected with western blot.7. MIN6 cells were infected with lentivirus encoding siRNA targeting NR4A1 and control lentivirus, after drug selection NR4A1 knock-down clones (KD) and control clones (CON) were generated. Both KD cells and CON cells were treated with 0.5 μM TG or 0.4 mM PA for different times and cell viability was detected with MTT.8. CON cells and KD cells were treated with 0.5 μM TG or 0.4 mM PA, protein samples were collected at 0 h,6 h,12 h,24 h respectively, the active form of Caspase3 was detected with western blot.9.16-week-old C57BL/6J mice were sacrificed by cervical dislocation, the mouse islets were extracted and purified. The purified islets were infected with Ad-NR4A1 or Ad-GFP as control for 48 hours, then treated with 0.5 μM TG or 0.4 mM PA for 20 hours, TUNEL assay was exploited to detect the ratio of apoptotic cells in these two adino-viral infected islets.10.16-week-old NR4A1 knockout (KO) mice and wild-type (WT) mice were sacrificed by cervical dislocation, mouse islets were purified and randomly dividied into groups, the mRNA expression of NR4A1 was detected by RT-PCR in KO or WT mouse islets. The remaining islets were treated with 0.5 μM TG or 0.4 mM PA for 20 hours, thereafter, TUNEL assay was exploited to detect the ratio of apoptotic cells in the islets.11. OV cells were treated with 0.5 μM TG, samples were collected at 0 h,3 h,6 h respectively, nuclear-cytoplasmic proteins were separated with an isolation kit and the localization of NR4A1 in OV cells were detected by western blot.Results:1. After treatment with 0.5 μM TG, the expression of inducible NR4A1 was increased in MIN6 cells, the mRNA level was significantly higher at one hour and reached the peak at two hour, protein levels was gradually increased, CHOP significantly increased; MIN6 cells treated with 0.4 mM PA, the expression of NR4A1 was also induced and peaked at 16 hours, while CHOP significantly increased. These data exihibited that 0.5 μM TG or 0.4 mM PA successfully induced ER-stress occurs and induced the expression of NR4A1 simultaneously.2. Purified mouse islets treated with 0.5μM TG significantly increased the expression of NR4A1 in 0.5 h, as well as ER-stress markers of BIP and CHOP; treated with 0.4 mM PA, also significantly increased the expression of NR4A1 in 6 hours as well as CHOP.3. TG-induced NR4A1 expression was colocalizated with insulin expression.4. We successfully selected MIN6 cell clones stably over-expressing NR4A1 (OV) and the control cell clones (NC). MTT results showed that with 0.5 μM TG or 0.4 mM PA treatment, cell viability of OV cell was significantly higher than NC cells; TUNEL results showed that after TG or PA treatment, positive apoptotic cells in NC cells was significantly higher than that in OV cells.5. Western blot results showed that with the treatment of 0.5 μM TG or 0.4 mM PA, the expression level of NR4A1 in OV cells was significantly higher than in NC cells, but activated form of Caspase3 (17KDa), a marker of apoptosis, was significantly lower in OV cells than that in NC cells.6. Our data showed that with the treatment of 0.5 μM TG or 0.4 mM PA, the expression level of NR4A1 in MIN6 cells infected with Ad-NR4A1 was significantly higher than that in control cells infected with Ad-GFP, but activated form of Caspase3 (17KDa) were significantly lower than that in control cells.7. We successfully generated MIN6 cell clones knockdown NR4A1 expression (KD cells), MTT results showed after treatment with TG and PA, the cell viability in NR4A1 knockdown cells was significantly lower than that in control cells.8. Western blot results showed that with the treatment of 0.5 μM TG or 0.4 mM PA, the expression level of activated form of Caspase3 (17KDa) in KD cells were significantly higher than that in control cells (CON cells).9. NR4A1 was transiently over-expressed in mouse islets by Ad-NR4A1 infection, TUNEL results showed that after the treatment with TG or PA, the positive rate of apoptotic islet cells over-expressing NR4A1 was significantly lower than that of the control virus-infected islet cells.10. RT-PCR results showed NR4A1 was expressed in wild-type mice islets, while knockout mice islets had no NR4A1 expression detected; TUNEL results showed that after the treatment with 0.5 μM TG or 0.4 mM PA, the positive rate of apoptosis in islet cells of KO mice was significantly higher than that in WT mice.11. Western blot results showed that after the separation of the nuclear-cytoplasmic proteins in the OV cells, NR4A1 was detected in the nucleus, even after treatment with 0.5 μM TG, NR4A1 still mainly located in the nucleus.Conclusions:1. ER-stress inducers TG or PA can induce expression of NR4A1 in MIN6 cells as well as in pancreatic β-cells.2. Over-expression NR4A1 is resistant to apoptosis induced by TG or PA in β-cells, and knockdown or knockout NR4A1 is more likely to cause apoptosis.Part Ⅱ the influence of NR4A1 on UPR pathwaysAim:Figure out which UPR pathway is influenced by NR4A1 to resist apoptosis by ER-stress.Methods:1. NC and OV cells were treated with 0.5 μM TG for different time points, the samples of cells were collected. The ratio of sXBPl/tXBP1 in IRE1 pathway, the expression of ATF4 in PERK pathway and the expression of ATF6 in ATF6 pathway were detected by QPCR.2. NC and OV cells were treated with 0.5 μM TG for different time points, the samples were collected and the RNAs and proteins were prepared, the expressions of CHOP, eif2a and P-eif2α in PERK pathway were detected.3. CON and KD cells were treated with 0.5 μM TG for different time points, protein smaples were prepared and the expression of CHOP, P-eif2a and 17KDa Caspase3 were detected by western blot.4. Extracted RNA and protein of NC and OV cells, the expressions of protein phosphatase la (PP1α) and growth arrest and DNA damage-inducible protein 34 (GADD34) were detected.Results:1. XBP1 was spliced at 3 hours in NC and OV cells treated with 0.5 μM TG, and there is no significant difference of the ratio of sXBP1/tXBP1 in NC and OV cells; the expression of ATF4 was increased significantly after treated with TG, but the increased folds in OV cells was significantly lower than that in NC cells; and there was no significant difference of ATF6 expression between the two cells.2. Expression of CHOP in both RNA and protein levels were significantly increased in NC and OV cells treated with 0.5 μM TG, but the expression in OV cells was significantly lower than that in NC cells; there was no significant difference of total eif2a in the two cells, eif2a continued phosphorylation in NC cells after treated with TG, but some dephosphorylation happened in OV cells from 12 hours after treated with TG.3. The Expressions of CHOP and 17KDa Caspase3 were significantly higher in NR4A1 knockdown cells than that in control cells; the expression of phosphorylated eif2a was also significantly higher in KD cells than that in CON cells.4. There is no significant difference in expression of PPla in NC and OV cells at mRNA or protein level, while the expression of GADD34 in OV cells is significantly higher than that in NC cells; we found there are two potential binding sites of NR4A1 (TGACCT) on the promoter of GADD34.Conclusions:1. NR4A1 has no significant effect on the IRE1 and ATF6 pathway, but can change the PERK pathway.2. Over-expression NR4A1 can make P-eif2a dephosphorylation, we speculate that NR4A1 may influence eif2a phosphorylation by altering GADD34.Part Ⅲ the impact of NR4A1 on anti-apoptotic genesAims:To analyse the anti-apoptotic genes regulated by NR4A1 to resist apoptosis induced by ER-stress.Methods:1. Total RNAs and proteins were extracted from NC and OV cells, the expression of apoptosis-related genes such as NF-κB, BCL-2, SURVIVIN was detected by QPCR and western blot.2. MIN6 cells were infected with Ad-NR4A1 or Ad-GFP for 48 hours, the protein expression of NF-κB, BCL-2, SURVIVIN was detected by western blot.3.16-week-old C57BL/6J mice were sacrificed by cervical dislocation, mouse islets were purified, and infected with Ad-NR4A1 or Ad-GFP as control for 48 hours, the expression changes of NF-κB, BCL-2, SURVIVIN was detected by QPCR.4. Protein samples were collected from KD and CON cells, the expression of NF-κB, BCL-2, SURVIVIN was detected by western blot.5. The luciferase reporters of NF-κB, BCL-2, SURVIVIN were constructed, each reporter and Renilla expression plasmid as internal control were co-transfected into NC and OV cells,24 hours later, luciferase assay was carried out by using a luciferase reporter gene kit.6. A serial of SURVIVIN luciferase reporters with different lengths of SURVIVIN promoter were constructed. Each reporter and Renilla expression plasmid were co-transfected into NC and OV cells,24 hours later, luciferase assay was accomplished with a luciferase reporter gene kit.7. MIN6 cells were infected with Ad-NR4A1-HA and Ad-GFP for 48 hours, ChIP assay was carried out by exploiting a HA monoclonal andibody to precipitate choramotin fragments capable to bind NR4A1 protein, and then amplified fragment contains NR4A1 binding sites on SURVIVIN promoter by RT-PCR.Results:1. In OV cells, the expression of NF-κB, BCL-2 or SURVIVIN at the RNA and protein levels was significantly higher than that in NC cells.2. The expression of NR4A1, NF-κB, BCL-2 and SURVIVIN was significantly higher in MIN6 cells infected with Ad-NR4A1 than that in Ad-GFP-infected MIN6 cells.3. The expression of NF-κB, BCL-2, SURVIVIN at the RNA level were significantly higher in mouse islet cells infected Ad-NR4A1 than that infected with Ad-GFP.4. In KD cells, protein levels of BCL-2 and SURVIVIN were significantly lower than that in CON cells.5. Dual luciferase assays showed that NR4A1 can enhance the transcription of SURVIVIN promoter, but can not change transcription activity of promoter of NF-κB or BCL-2, by software analysis, we found there is a NR4A1 binding site on SURVIVIN promoter(-1872 bp to-1866 bp).6. Dual luciferase assays showed that, remove NR4A1 binding site from SURVIVIN promoter (-1865 bp,-1500 bp,-100 bp) lost their enhanced transactivation by NR4A1 over-expression, but -500 bp promoter still had its transcriptional activity by NR4A1 over-expression; promoter deletion from -501 bp to -100 bp but contains NR4A1 binding site still had an enhanced transcription activity by NR4A1 over-expression.7. PCR results showed that after ChIP assay, the Ad-NR4A1-HA samples can be amplified the specific fragment on SURVIVIN promoter, while Ad-GFP can not.Conclusions:NR4A1 modulates the expression of anti-apoptotic genes such as NF-κB, BCL-2 and SURVIVIN, the modulation of NF-κB and BCL-2 is no clear and might be indirect, while regulation of SURVIVIN is direct. |
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