Orphan Nuclear Receptor Nr4a Expression Indhoed By Endoplasmic Reticuium Stress Sensitive Reagents And Its Possible Role In Modulating Insulin Expression In Pancreatic Beta Cells

In eukaryotic cells, the endoplasmic reticulum (ER) is the important place for protein synthesis, folding, processing and quality control as well as Ca2+store. When cytoplasmic calcium concentration abnormally changes, or the endoplasmic reticulum protein folding is overloaded, the unfolded protein response (UPR) will be initiated to reduce this ER sress. Cells regulate the expression of downstream genes to enhance Ca2+transportation or protein folding in the endoplasmic reticulum through three key UPR signaling pathways:ATF6, PERK and IRE1. Therefore, UPR pathway plays an important role in the homeostasis of the cell. In liver, adipose tissue, islets and hypothalamus, ER stress modulates the metabolic activities.As we all know, with the improvement of people’s living conditions, diabetes (especially type2diabetes) exhibited a rapid increase in the incidence trends. The end pathology result of type2diabetes is apoptotic pancreatic β cell death. Sustained high glucose and free fatty acids (FFA) can lead to pancreatic β cells ER stress. In pancreatic β cells, the balance between ER stress and activation of UPR determines the fate of these cells. If ER stress can not be effectively alleviated or eliminated, it will possibly initiate the pancreatic β cell apoptosis.Nuclear receptors make up a large family of ligand-dependent transcription factors. They have common structural motifs:the N-terminal activation domain called AF, the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD). Based on the DBD sequence homology, a number of orphan nuclear receptors were found, whose natural or synthetic ligand were not been identified initially. The NR4A subfamily is true orphan receptor because its ligand binding pocket (LBP) is entirely filled by hydrophobic amino acid side chains, and lacks a cavity for ligand binding.NR4A1(also known as Nur77, TR3, or NGFI-B), NR4A2(also known as Nurrl, RNR-1, or TONOR), and NR4A3(also known as NOR-1or MINOR) comprise the NR4A subfamily of orphan nuclear receptors. The three members of the NR4A subfamily have high levels of homology in the DBD domain (-91to95%) and modest levels of homology in the LBD domain (-60%), whereas they are very divergent in the activation domain (AF). It was regarded as one of the rapid but transient response genes that sense and respond to a variety of stimuli in the cellular environment. The correlation between NR4A subfamily expression and ER stress in cells remains unknown. The role of NR4A subfamily in modulating insulin expression in pancreatic β cells was considered worth exploring. Our research includes two parts:Part I The correlation between NR4A subfamily expression and ER stress in pancreatic cells or liver cellsAim:To investigate the expression of NR4A subfamily when pancreatic β cells and hepatic cells are treated with ER stress inducible reagents in vitro.Methods1. MIN6cells were treated with different dosage of thapsigargin (TG), palmitate (PA), tunicamycin (TM)and dithiothreitol (DTT). The mRNA levels of Bip/Grp78, Chop/Ddit3and NR4As were determined with real-time quantitative PCR. Two forms of Xbpl (a UPR molecule) were detected with reverse transcription PCR (RT-PCR). Optimize the dosage of various inducers which is able to activate UPR and induce NR4A subfamily expression.2. The culture medium for MIN6, HepG2, Hepal-6and3T3-L1cells were replaced with fresh full medium, the mRNA levels of NR4A were analyzed with real-time quantitative PCR after each time points(0h, lh,2h,3h,4h,6h,9h,12h). The protein levels of NR4A1were detected with western blotting after a series of time points in order to check the impact of medium change on NR4A elavation.3. MIN6, HepG2and Hepal-6cells were incubated with0.5μM TG for Oh,15min,30min,1h,2h,3h,6h,9h and12h. The cells were collected. RNA and protein of cells were extracted. Dynamic changes of UPR molecules and NR4A subfamily were analyzed with RT-PCR, real-time quantitative PCR and western blotting.4. MIN6, HepG2and Hepal-6cells were incubated with10μg/ml TM for Oh,15min,30min,1h,2h,3h,6h,9h and12h. The cells were collected. RNA and protein of cells were extracted. The dynamic changes of UPR molecules and NR4A subfamily were analyzed with RT-PCR, real-time quantitative PCR and western blotting.5. MIN6, HepG2and Hepal-6cells were incubated with0.5mM PA for6h,8h,10h,12h,16h,20h and24h. The cells were collected. RNA and protein of cells were extracted. The dynamic changes of UPR molecules and NR4A subfamily were analyzed with RT-PCR, real-time quantitative PCR and western blotting.6. MIN6, HepG2and Hepal-6cells were incubated with3mM DTT for Oh,15min,30min,1h,2h,3h,4h,6h and9h. The cells were collected and RNA of cells was extracted. The dynamic changes of UPR molecules and NR4A subfamily were analyzed with RT-PCR and real-time quantitative PCR.7. C57BL/6-ob/ob mice were fed as obesity model. The same week age mice were fed as control. All the mice were fed with control diet. The body weight of each animal was monitored once a week. The animals were sacrificed after deep anesthesia. Pancreas, liver tissues were collected for RNA extraction. The dynamic changes of UPR molecules and NR4A subfamily were analyzed with real-time quantitative PCR.8. Nuclear and cytoplasmic proteins were extracted with a special kit, the expression and localization of NR4A1/NUR77was determined with western blotting after the MIN6cells were treated with0.5μM TG for3h and6h.9. Inverted phase contrast fluorescence microscope was exploited to analyze the expression level and location of NR4A1protein in HepG2cells treated with0.5μM TG for2h or0.5mM PA for10h.Results: 1. MIN6cells had been resulted in ER stress upon treatment with0.1-1μM TG for2h、10μg/ml TM for3h、2-4mM DTT for2h or0.4-0.8mM for12h. We observed that UPR was activated and the expression of NR4As remarkably increased in these conditions. The optimal dosage for these reagents is0.5μM TG,10μg/ml TM,3mM DTT or0.5mM PA.2. The RNA and protein levels of NR4A subfamily increased significantly after changing medium during6hours, peaked on2hours in MIN6, HepG2, Hepal-6and3T3-L1cells. The levels fall back to basic lines after6hours. Based on these information, we add ER stress reagents stock (1mM TG,5mg/ml TM,1M DTT) directly to medium after replacing medium12h in order to eleminiate the impact of medium change.3. In MIN6, HepG2and Hepal-6cells, UPR were activated and all NR4A subfamily expression was markedly induced after treatment with0.5μM TG, especially for NR4A1and NR4A2.4. Like TG treatment, three cells incubation with10μg/ml TM, UPR were activated and NR4A expression increased. However, the elevated fold of NR4A subfamily in TM treatment was far lower than that with TG treatment. TM mainly induced NR4A2expression.5. In MIN6, HepG2and Hepal-6cells upon0.5mM PA treatment, UPR molecules expression increased but with lower degree than that upon TG treatment. The elevation of NR4A subfamily expression is concomitantly lower than that upon TG treatment. PA treatment induced different NR4A member’s expression in different cells.6. In MIN6and HepG2cells, the result of3mM DTT treatment is similar to TG treatment. But in Hepal-6, the result of3mM DTT treatment is the same as TM or PA treatment.7. The body weight of10weeks’ob/ob mouse was1.7fold of control group. The UPR molecule level (Bip, Chop) from obesity animal’s pancreas and liver tissues remarkably increased. The mRNA levels of NR4A in pancreas and liver from ob/ob mice was higher than that of control (P<0.05).8. In MIN6and HepG2cells, the elevation of NR4A1protein localized in nuclei uponTG or PA treatment rather than in cytoplasm.Conclusions:1. As NR4A subfamily was one of early response genes, changing medium also can increase their expression during a short time. It is important to consider the impact of medium change on NR4A expression when the effect of ER stress reagents will be tested.2. All ER stress reagents were able to result in both the elevation of NR4A members and UPR activation.3. The UPR activation and NR4A transcription elevation were also detected in pancreas and liver from C57BJ/6-ob/ob mice.4. When cells are under ER stress, the elevation of NR4A subfamily and its localization in nuclei may indicate that NR4A subfamily modulates some genes expression as transcription factor. Part II The role of NR4A in modulating the expression of insulin genes in pancreatic beta cellsAim:To investigate the possible mechanism on NR4A subfamily modulating insulin expression in pancreatic beta cells.Method:1. The AdEasy System was used to generate recombinant adenovirus over-expressing NR4A1or NR4A3. The control adenovirus, Ad-GFP (only encoding GFP) was amplified and purified in the same way. The titers of these three kinds of recombinant adenovirus were detected with UV spectrophotometry and gradient dilution virus infection.2. After recombinant adenovirus infection for44h or treatment with0.5μM TG for1h or0.5mM PA for12h, glucose stimulated insulin secretion was assayed by radioimmunoassay (RIA) with[125] iodine using an Insulin Radioimmunoassay Kit.3. Two pancreatic beta cells (MIN6and INS1) were infected with a series of double dilutions of Ad-NR4A1-HA or Ad-NR4A3adenovirus. Additional Ad-GFP adenovirus was used for complementary infection in order to ensure each infection had an equal virus titer. Post-infection, levels of secreted insulin were assayed by RIA. mRNA levels of two insulin genes (Ins1and Ins2) were determined with reverse transcription PCR in MIN6cells infected with Ad-NR4A3/Ad-GFP.4. Post-infection with three kinds of recombinant adenovirus, the localization of over-expression NR4A1/3in MIN6cells was determined with immunofluorescence staining.5. Screen stably over-expression NR4A1MIN6cell lines with puromycin selection (6μg/ml) after lentivirus infection. Nuclear and cytoplasmic proteins were extracted with kit, the expression and location of NR4A1was determined with western blotting in stably transfected MIN6cell lines.6. Fusion cDNAs containing the full-length NR4A3coding sequence and a C-terminal-HA epitope tag were cloned into a pLenti6/V5-D-topo vector. The plasmids of each deletion cDNA (ΔAF12-288aa, ΔDBD292-364aa, and ΔLBD398-626aa) were generated by mutagenesis techniques. The four recombinant plasmids and a control GFP gene construct were transfected into MIN6cells. For stable transfection, cells were grown under blasticidin selection (3μg/ml) for over15days, then single clones of cells were picked and amplified. Western blotting analyses were performed to test the stability of the transfected cell lines. The levels of insulin gene (Insl, Ins2) mRNA in different stably cell lines were assayed with RT-PCR.7. The levels of insulin genes and its transcription factor genes (NeuroD1, Pdxl, MafA, Glis2) were assayed with RT-PCR and real-time quantitative PCR after three recombinant adenovirus post-infection in pancreatic beta cells (MIN6, INS1).Results:1. Generation of recombinant adenovirus with AdEasy system was successfully obtained. Equivalent titer of three kinds of adenovirus was0.5μl/ml Ad-NR4A1-HA,0.5μl/ml Ad-NR4A3,0.25μl/ml Ad-GFP. The infectant effecency was about over80%in MIN6cells.2. Treatment of MIN6cells with0.5μM TG for1h and0.5mM PA for12h markedly suppressed insulin secretion. The insulin secretion in MIN6was also decreased significantly after Ad-NR4A1/3adenovirus post-infection.3. Pancreatic beta cells were infected with Gradient titer dilutions of Ad-NR4A1/A3adenovirus. The secreted insulin protein and Ins1/Ins2mRNA levels of the Ad-NR4A1/3-infected cells were markedly lower than those of the control infected cells (Ad-GFP). The greater the NR4A1/3expression, the lower the level of secreted insulin detected, thus the secreted insulin and Ins1/Ins2gene transcription levels were negatively correlated with NR4A1/3expression.4. Over-expression NR4A1protein was localized in nuclei in both Ad-NR4A1/3infected MIN6cells and stably MIN6cell lines.5. MIN6stably transfected cell lines of NR4A3wild-type or its deletion mutations were screened. Ins1/Ins2expression in NR4A3wild-type cell lines and ΔLBD cell lines were predominantly lower than in control cells. In the ΔAF1and ΔDBD cell lines, the mRNA transcription was not significantly different from the control cells. The modulation of insulin expression by NR4A3was closely related to AF1and DBD.6. In order to explain the manner of NR4A3-regulated insulin expression, we analyzed the changes in transcription factor binding to the insulin gene promoter in MIN6and INS1cells infected with Ad-NR4A1-HA and Ad-NR4A3adenovirus. The mRNA levels of Ins1and Ins2and its positive transcription factors (NeuroDl, Pdxl, MafA, Glis3) in Ad-NR4A1/3-infected cells were significantly lower than in control infected cells. These data indicated that NR4A3modulates insulin gene transcription indirectly.Conclusion:1. We successfully generated adenovirus over-expression of NR4A1or A3and a variety of stably transfected MIN6cell lines with NR4A1or A3full lenth cDNA or deletions..2. Over-expression NR4A subfamily was localized in nuclei in MIN6cells, which showed that NR4A subfamily was transcription factor in modulating gene expression.3. NR4A subfamily members can down-regulate insulin expression by indirectly inhibiting insulin positive transcription factors’ expression, which in turn reduces the burden of ER and protects cells in the pancreatic beta cell...