Repression Of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation Into Insulin-Producing Cells

Objective: Diabetes mellitus(DM)is mainly divided into two types: type 1 diabetes(T1DM)and type 2 diabetes(T2DM).Type I diabetes is an autoimmune disease whose cause is due to pancreatic beta cell damage.T2 DM presents with a relative deficiency of insulin in the blood caused by insulin resistance.Previous studies have shown that there is a reducion in the number of ?-cells caused by increased apoptosis of ?-cells in T2 DM.Therefore,regardless of T1 DM or T2 DM,loss or dysfunction of islet ?-cells is an important pathogenic factor,and replacement or supplementation of these cells has become the main goal of diabetes research.Although other therapeutic strategies,such as pancreatic islet transplantation,have been used in clinical practice,there is still challenging the lack of cell source.Recently,stem cell therapies have shown promise as novel treatments for cell replacement therapy.Bone marrow-derived mesenchymal stem cell(bmMSC)can be induced into insulin-producing cells(IPCs)in vitro.However,it is unclear to identify the differentiation mechanism.The transcriptional activators NeuroD/BETA2,MafA,Ngn3 and Pdx1,bind to special sites within 300–400 bp from the transcription start site of the insulin promoter and interact synergistically.Previous reports have shown that bmMSC can be differentiated into IPCs by single or combined transfection of these transcriptional activators.However,most differentiated cells cannot secrete sufficient amounts of insulin.MafA is a member of the large Maf family of transcription factors.It has been shown to be a key regulator of tissue-specific expression of Insulin 2(Ins2)in ?-cells,but its effect is weak unless it is coexpressed with other key transcription factors.Therefore,we hypothesize that there are factors that inhibit MafA induction in bmMSC.In simple animals it is easy to start the regeneration process,but along evolution of the organism being inhibited,it becomes more and more difficult to initiate the regeneration process.Looking for these inhibitory factors will certainly promote the regeneration process.Little is currently known about transcription inhibitors.In non-insulin-producing cells,such as bmMSC,the reseason of failure of differentiation into pancreatic ?-cells,there is some transcriptional repression factors which negatively regulate the insulin gene and inhibit regeneration.Therefore,the discovery and study of these transcriptional repression factors will definitely help promote the differentiation of stem cells into IPCs,thereby advancing the use of stem cell therapies for diabetes.In this study,we compared the nuclear proteins of mouse bmMSC and the pancreatic islet ?-cells line Min6,which binds to Ins2 gene promoter,and found that novel binding protein COUP-TFI in bmMSC.Chicken ovalbumin upstream promoter transcriptional factors(COUP-TFs)belong to the steroid/thyroid hormone receptor superfamily.There are two major homologues,COUP-TFI and COUP-TFII,which bind to similar DNA binding domain.COUP-TFI is essential for neural,optical,and cardiac development.It is also as an important regulator in differentiation of oligodendrocytes,retinal progenitor cells,and hair cells.COUP-TFI directly binds to the DR1 elements to repress gene expression.It is unclear whether the DR1 element is present in the mouse insulin promoter and/or involved in transcriptional regulation.There may be a novel mechanism of islet ?-cells differentiation,and a new method of differentiation of bmMSC into IPCs may be found.Therefore,we propose that repressing the expression of COUP-TFI in bmMSC,plus overexpressing MafA,will promote the differentiation of bmMSC into IPCs and research on the ways and molecular mechanisms of regulation of islet ?-cells differentiation.The application of bmMSC to differentiate into islet ?-cells for the treatment of diabetes will be well investigated.Methods: 1.New binding protein COUP-TFI on Ins2 in bone marrow mesenchymal stem cellsA.bmMSC and Min6 cell culture and identification of biological characteristics.B.Through the DNA affinity precipitation,we found a new protein binding on Ins2 promoter.C.The expression of COUP-TFI in Min6 and bmMSC cells were detected by RT-PCR and Western Blot.D.The distribution of COUP-TFI in pancreatic tissue were detected by immunofluorescence.2.Silencing COUP-TFI in bmMSC promotes the differentiation of bMSC into IPCsA.Through real-time quantitative PCR,the impact of COUP-TFI on Ins2 expression in Min6 cells was investigated.B.The effect of COUP-TFI on Ins2 promoter activity was detected by dual luciferase reporter assay.C.Through real-time quantitative PCR,the effect of siCOUP-TFI on Ins2 expression in bm MSC was detected.D.Detect the effect of MafA and siCOUP-TFI on Ins2 promoter activity by dual luciferase reporter assay.E.Dithizone staining and glucose stimulation experiments in differentiated cell.F.Animal experiments to detect the function of differentiated cells in vivo.3.Molecular mechanism of regulation of Ins 2 as transcriptional repressor COUP-TFIA.Through bioinformatics method to find the COUP-TFI binding sites in mouse Ins2 gene promoter region.B.Detect COUP-TFI binding status by ChIP and EMSA.C.Co-IP and ChIP detect whether COUP-TFI form transcriptional repression complexes with NCoR,SMRT and HDAC3.Results: 1.The differential binding protein COUP-TFI on the promoter region of the Ins2 gene was scaned in mouse bmMSC and Min6 cellsA.It is consistent with the characteristics of bmMSC that flow cytometry analysis showed that the expression of Sca-1,CD44 and CD29 was positive and the expression of CD117,CD31 and CD45 was negative.B.BmMSC is pluripotent and can be induced into cartilage,bone,and adipocytes.C.COUP-TFI is a novel binding protein on the Ins2 gene promoter.D.COUP-TFI was expressed in bmMSC cells,but not in Min6 cells.E.COUP-TFI is widely distributed in brain tissue,but no positive staining was found in pancreatic tissues.2.Silencing COUP-TFI in bmMSC plus overexpressing MafA,can promote the differentiation of bmMSC into IPCs.A.COUP-TFI can reduce the activity of Ins2 promoter.B.Overexpression of MafA while interfering with COUP-TFI can significantly enhance the activity of the Ins2 promoter.C.Repressing COUP-TFI,coupled with overexpressing MafA,can trigger bmMSC express specific markers of islet endocrine cells,such as Ins1,Ins2,Pdx1,Isl-1,Glut2 and Pax6,to differentiate into IPCs.D.Differentiated cells can release C-peptide and Insulin in response to glucose stimulation,and dithizone staining is positive.E.Differentiated cells can reduce blood glucose level in diabetic model mice.3.Through forming transcriptional corepressor complex with NcoR,SMRT and HDAC3,COUP-TFI inhibits the expression of Ins2,binding to the DR1 site in Ins2 promoter.A.There is a DR1 site in the promoter region of mouse Ins2 gene from-53 bp to-40 bp.B.Through Ch IP and EMSA,COUP-TFI could specifically bind to DR1.C.Through ChIP,NCoR,SMRT,and HDAC3 are also enriched in this region.D.Co-IP results showed that COUP-TFI can form transcriptional corepressor complexes with NCoR,SMRT and HDAC3.Conclusion:COUP-TFI is a mouse Ins2 gene promoter binding protein.It is expressed in mouse bmMSC,but not expressed in Min6 and pancreatic islet ?-cells.It may be an inhibitor of Ins2.Inhibition of COUP-TFI synergistic overexpression of MafA facilitates the differentiation of bmMSC into IPCs in vitro.The IPCs can significantly reduce blood glucose level in diabetic model mice.COUP-TFI specifically binds to the DR1 site by forming a complex with NcoR,SMRT and HDAC3,inhibiting the expression of the Ins2 gene.