Notch Signaling Negatively Regulates BMP9-induced Osteogenic Differentiation Of Mesenchymal Progenitor Cells By Inhibiting JunB Expression

Objiective:To investigate the effect and mechanism of Notch signaling pathway on osteogenic differentiation of mouse mesenchymal stem cells(MSCs)C3H10T1/2 induced by Bone Morphogenetic Protein 9(BMP9).Methods:The NICD over-expression plasmid was transfected into C3H10T1/2 cells.Real-time fluorescence quantitative RT-qPCR was used to detect the endogenous expression level of plasmid NICD in cells.The protein expression of NICD was detected by Western Blot(WB).NICD plasmid and BMP9 were then used together to treat C3H10T1/2,Alkaline phosphatase(ALP)activity,staining and alizarin red S staining confirmed the effect of NICD on BMP9-induced mesenchymal stem cell osteogenic differentiation,RT-qPCR was used to detect the expression changes of the osteogenic-related genes Runx2,OPN,OCN,JunB,Smadl,Idl,and OSX,WB was used to detect the proteins expression levels of Runx2,OPN,OCN,JunB,Smad 1/5/8,and p-Smad 1/5/8.The agonist of JunB 12-O-tetradecanoylphorbol-13-acetate(TPA)and the inhibitor of Notch pathway ?-secretase(DAPT)were used together with BMP9 or NICD to treat with C3H10T1/2.ALP staining,activity and alizarin red S staining were used to detect osteogenic differentiation,the expression of osteogenic-related genes were detected by WB again.In addition,WB detected the effects of NICD on the expression levels of proliferation-associated proteins PCNA and Cyclin D1,colony formation assays were used to detect cell self-renewal capacity,and flow cytometry was used to detect cell cycle changes.Inverted fluorescence microscopy was used to observe the infection efficacy of BMP9 in C3H10T1/2 infection,and BMP9,and the athymic nude mice was injected with NICD or TPA treated cells at the dorsal subcutaneous,then MicroCT was used to detect the parameter of ectopic osteogenesis,including bone volume(BV/TV),trabecular thickness(BV/TV),trabecular spacing(Tb/Sp),trabecular number(TbN)and bone mineral density(BMD),the expression of osteogenic-related proteins Runx2,OPN,OCN,JunB,and adipogenic differentiation-associated proteins CCAAT enhancer-binding protein(C/EBP)and peroxisome proliferator activated receptor y(PPAR y)in heterotopic bone were detected by the immunohistochemistry,hematoxylin-eosin staining,trichrome staining,and alcian blue staining for detection of bone and cartilage in heterotopic bone,oil red O staining was used to detect the formation of adipose tissue in the heterotopic bone.Results:RT-qPCR and WB results showed that over-expression of NICD plasmid was highly expressed in C3H10T1/2 cells;NICD could inhibit the expression of osteogenic-related genes,including Runx2,OPN,OCN,OSX,and JunB,and ALP formation and calcium salt deposition,and also inhibit the expression of adipogenic differentiation-related proteins C/EBP and PPAR,without affecting the expression of Smadl/5/8,p-Smad 1/5/8;JunB's agonist TPA can reverse the negative effect of NICD on BMP9-induced osteogenic differentiation and promotes the expression of Runx2,OPN,OCN,and JunB;Notch pathway inhibitors DAPT promotes the expression of p-Smadl/5/8,Runx2,OPN,OCN and JunB,inhibite the formation of ALP in early osteogenic stage,but promotes calcium deposition of late osteogenic markers;the expression of proliferation-associated proteins PCNA and Cyclin D1 can be increased by NICD,colony formation assay and flow cytometry shows that NICD significantly promots cell proliferation;ectopic osteogenesis showed that NICD significantly inhibited BMP9-induced bone formation,and immunohistochemistry results showed that NICD inhibits the expression of Runx2,OPN,OCN,JunB in ectopic bone,special stain such as HE,trichrome,axin blue,oil red O staining shows that NICD inhibit the formation of bone tissue,cartilage tissue,and adipose tissue in heterotopic bone.In addition,TPA can reversed the negative effect of NICD on BMP9 induced osteogenic differention.Conclusion:The result shows that NICD play an important role in regulating the osteogenic and adipogenic differentiation of mesenchymal stem cells C3H10T1/2 induced by BMP9,which can inhibit BMP9-induced osteogenic and adipogenic differentiation and promote cell proliferation.Our findings demonstrate that Notch signaling significantly enhances cell proliferation but inhibits MSC osteogenic differentiation induced by BMP9 via JunB protein suppression rather than by BMP/Smadl/5/8 signaling regulation.