NR4A1 Protects Pancreatic ? Cells Via Quenching JNK Activation Caused By Cellular Stresses

Type 2 diabetes(T2DM)is a metabolic disease.Its basic pathophysiological mechanisms include insulin resistance,progressive impairment or failure of islet function.Under these continuous actions of metabolic load(such as excess glucose and lipid),insulin resistance or chronic inflammation,reactive oxygen species(ROS)are over-produced,then cellular stress occurred in pancreatic ? cells.At the same time,it is accompanied by various adaptive responses and compensations under stress state.But,the imbalance of impairment and compensations can lead to the dysfunction of pancreatic ? cells and even cell apoptosis.Impairment of pancreatic ?cells are the key factors in the genesis of type 2 diabetes.C-Jun N-terminal kinase(JNK)activation is often associated with apoptosis induced by cellular stress.JNK is often activated by various cellular stresses,and activated JNK is involved in many signaling pathways.JNK is closely related to the formation and development of diseases,such as diabetes,neurodegenerative diseases,and chronic inflammation.In our previous study,islet ? cells treated with hydrogen peroxide(H2O2)or Thapsigargin(TG,endoplasmic reticulum stress inducer)for a certain period of time resulted in apoptosis accompanied with increased activated JNK level.NR4A1(Nuclear receptor subfamily 4 group A)is a stress response factor,and plays a role as a transcription factor.NR4A1 participates in the regulation of various cellular life activities by promoting the transcription expression of related genes.Previous studies in our laboratory showed that NR4A1 expression was increased in pancreatic ? cells subjected to oxidative stress or er stress,and NR4A1 could resist pancreatic ? cell apoptosis by enhancing anti-apoptotic molecules or antioxidant molecules.However,the regulatory role of NR4A1 on JNK activation is not clear.Therefore,the aim of this study is to reveal the regulatory role of NR4A1 on JNK activation induced by oxidative stress or ER stress in pancreatic ? cells,so as to find the new the protective mechanism of NR4A1 on pancreatic ? cells.In this study,we found that NR4A1 can reduce JNK activation and pancreatic ?cell apoptosis induced by oxidative stress and ER stress,and NR4A1 down-regulates JNK activation through two pathways.Namely,NR4A1 can enhance Casitas Blineage lymphoma B(cbl-b)to degrade Mitogen activated protein kinase kinase 4(MKK4),and ultimately reduce JNK activation;NR4A1 also enhances the transcriptional level of Dual specific phosphatase 16(MKP7)to enhance the dephosphorylation of activated JNK(p-JNK).This study is divided into three parts as follows:Part ? NR4A1 can negatively regulate JNK activation to protect pancreatic ?cells under cellular stresses[Aim]The effects of NR4A1 expression on JNK activation under stresses were verified by using NR4A1-KO mice with high-fat diet and MIN6 cell lines with overexpression of NR4A1.[Methods]1.NR4A1-KO mice was constructed by using the technology of CRISPR/Cas9mediated gene editing from C57BL/6N mice and the genotypes were confirmed by PCR and Western blot.2.The metabolic phenotypes,including fasting blood glucose and serum insulin content were detected to NR4A1-KO mice and WT mice fed with high-fat diet.3.The islets or pancreas of NR4A1-KO mice and WT mice were isolated and purified to observe the morphological changes of islets and the ratio of ? cells by light microscopy and immunofluorescence(IF)experiments.? cells apoptosis in islet were detected by TUNEL assay.4.Western blot was used to detect the JNK activation level(p-JNK level)in islet of NR4A1-KO mice and WT mice fed with high-fat diet.5.Based on MIN6 cell line,OV cells(overexpressing NR4A1 cells)and NC cells(control cells)were constructed,and the level of NR4A1 was detected.6.OV or NC cells were treated with hydrogen peroxide(H2O2)to establish an oxidative stress model,and were treated with thapsigargin(TG)to establish an ER stress model.The effect of NR4A1 on the survival of pancreatic ? cells under stresses was detected.The effect of NR4A1 on apoptosis or JNK activation in pancreatic ?cells under stresses was detected.7.MIN6 cells were treated with SP600125(JNK inhibitor)combined with H2O2 or TG,and the relevance between JNK activation level and cell apoptosis was detected with Western blot.[Results]1.PCR and Western blot confirmed that the NR4A1-KO mice used in this study were homozygote mice of NR4A1 knockout.2.The results showed that after high-fat diet,NR4A1-KO mice had higher fasting glucose levels,impaired pancreatic ? cell function and reduced insulin secretion.3.In NR4A1-KO mice,the number of large islet and the ratio of ?/? cells were higher,and apoptosis is more likely to occur.4.Western blot assay showed that the activation level of JNK was increased in the islet of NR4A1-KO mice.5.OV cells(MIN6 cells stably over-expressed NR4A1)and NC cells(control cells)were successfully generated.6.CCK8 assay showed that the survival rate of OV cells increased compared with NC cells after treatment with H2O2 or TG.Western blot showed that the Cleaved caspase3 level and p-JNK level were decreased in OV cells compared with NC cells after treatment with H2O2 or TG,but the protein level of total JNK was no significant difference in two cells.7.After treatment with H2O2 or TG,Western blot showed that the expression of Cleaved Caspase-3 decreased in the SP600125-treated cells compared with the cells without SP600125 treatment.[Conclusions]1.With high-fat diet,NR4A1 deficiency leads to impaired insulin secretion in mice,and increased JNK activation and the occurrence of apoptosis in islet.2.NR4A1 reduces the apoptosis and JNK activation in pancreatic ? cells induced by cellular stresses.3.NR4A1 does reduce the level of JNK activation by some unknown mechanisms,rather than reducing the expression of total JNK protein.Part ? NR4A1 up-regulates cbl-b expression to attenuate JNK activation induced by cellular stresses[Aim]To explored the mechanism of NR4A1 negative regulation of JNK activation from upstream kinases of JNK,including MKK4 and MKK7.[Methods]1.After OV and NC cells were treated with H2O2 or TG,the effects of NR4A1 on the expression of MKK4 and MKK7 were detected.2.Flag-MKK4 and HA-Ub plasmids were co-transfected into OV and NC cells.The exogenous MKK4 was first immunoprecipitated with Flag-antibody,and then the ubiquitination level of MKK4 was detected.3.OV and NC cells were treated with proteasome inhibitor MG132,and the association between the ubiquitination of MKK4 and NR4A1 was detected.4.OV and NC cells were treated with H2O2 or TG,and the effect of NR4A1 on the expression of cbl-b was detected by qPCR and Western blot.5.Infection with shRNA targeting cbl-b or scramble lentivirus in OV cells to construct stable KD-cblb cells or CON-cblb cells;KD-cblb and CON-cblb cells were treated with H2O2 or TG,and the changes of JNK activation level were detected by Western blot.6.Four plasmids of cbl-b promoter reporter gene were constructed and co-transfected into OV cells with TK plasmids.Dual-luciferase reporter gene assay was used to detect whether NR4A1 could enhance the transcriptional activity of cbl-b promoters.7.MIN6 cell was infected with Ad-NR4A1-HA or Ad-GFP adenovirus,and ChIP assay was used to detect the physical binding of NR4A1 to the cbl-b promoter.8.Islets or pancreases of NR4A1-KO mice and WT mice were isolated and purified.The effect of NR4A1 deficiency on the level of cbl-b was detected.[Results]1.Western blot showed that NR4A1 decreased the protein level of MKK4;However,the qPCR showed that there was no significant difference in the RNA level of MKK4 in two cells.2.Immunoprecipitation assay showed that NR4A1 increased the ubiquitination level of exogenous MKK4 in OV cells.3.Western blot showed that the protein expression of MKK4 in OV cells increased after treatment with MG132,which was similar to that in NC cells.4.QPCR and Western blot showed that the expression of cbl-b was increased in OV cells than NC cells.5.Western blot showed that the expression of cbl-b was significantly decreased in KD-cblb cells than CON-cblb cells,while the protein level of MKK4 was increased;After treatment with H2O2 or TG,the level of JNK activation increased in KD-cblb cells than CON-cblb cells.6.Dual-luciferase reporter gene assay showed that NR4A1 could enhance the transcriptional activity of cbl-b promoters of different lengths,and the enhancement effect was most significant in the range from-499bp to-14bp.7.ChIP assay showed that NR4A1 could physically bind to the predicted binding site at-198bp on the cbl-b promoter.8.QPCR,Western blot and immunofluorescence assay showed that the expression of cbl-b was significantly decreased in the islet of NR4A1-KO mice than WT mice.[Conclusions]1.NR4A1 negatively regulates MKK4 to reduce JNK activation.2.NR4A1 enhanced the expression of cbl-b(E3 ligase).3.MKK4 protein degradation is ubiquitination dependent and associates with cbl-b.4.NR4A1 can up-regulate the transcriptional activity of cbl-b gene promoter and bind to it physically.Part ? NR4A1 up-regulates MKP7 expression to attenuate JNK activation induced by cellular stress[Aim]In this part,we investigated MKPs(p-JNK phosphatase)expression to explore other possible mechanism that NR4A1 negatively regulates JNK activation.[Methods]1.OV and NC cells were treated with H2O2 or TG,and the effects of NR4A1 on the expression of MKP2 and MKP7(phosphatase targeted p-JNK)were detected by qPCR and Western blot.2.MKP7 knockdown cells(KD-MKP7)or the control cells(CON-MKP7)were constructed by infecting ?-TC6 cells(pancreatic ? cells)with the shRNA lentivirus targeting MKP7 or its scramble shRNA lentivirus.After the cells were treated with H2O2 or TG,the JNK activation was detected by Western blot.3.MKP7 promoter reporter gene plasmids were constructed,and then co-transfected with TK plasmid into NC and OV cells.The dual-luciferase reporter gene assay was used to detect whether NR4A1 could enhance the transcriptional activity of MKP7 promoter of different lengths.4.?-TC6 cells were infected with Ad-NR4A1-HA adenovirus or Ad-GFP adenovirus.Chromatin immunoprecipitation(ChIP)assay was used to detect whether NR4A1 physically binds to the putative binding sites of the MKP7 promoter.5.Islets of NR4A1-KO mice and WT mice with high-fat diet were extracted,and the effect of NR4A1 deficiency on the expression of MKP7 were detected by qPCR and Western blot.[Results]1.The expression of MKP7 was increased in OV cells than NC cells after treatment with H2O2 or TG by qPCR and Western blot.2.The protein expression of MKP7 was significantly decreased in KD-MKP7 cells than CON-MKP7 cells.After treatment with H2O2 or TG,the JNK activation was increased in KD-MKP7 cells.3.Dual-luciferase reporter gene assay showed that NR4A1 could enhance the transcriptional activity of MKP7 promoters of different lengths,and the enhancement effect was most significant in these ranges of-500bp to 0bp and-2000bp to 0bp.4.ChIP assay showed that NR4A1 could physically bind to the predicted binding sites at-55bp and-1637bp on the MKP7 promoter.5.QPCR and Western blot showed that the expression of MKP7 was decreased in the islet of NR4A1-KO mice than WT mice.[Conclusions]1.NR4A1 reduces JNK activation level(p-JNK)by enhancing the expression of MKP7.2.NR4A1 up-regulates the transcriptional activity of MKP7 promoter and physically bind to the two predicted binding sites.