The Role And Mechanism Of CCN3 In Osteogenic Differentiation Of MSCs

Notch signaling pathway is one of the highly conserved signaling pathways in the process of biological evolution and plays an important role in bone regeneration.The current studies found that in addition to the classical membrane protein ligands(DLL1,DLL3,DLL4,Jagged1,Jagged2),there is also a secreted ligand for the Notch signaling pathway: nephroblastoma overexpressed(NOV/CCN3).Our previous researches have confirmed that the Notch signal activated by classical membrane protein ligand DLL1(DLL1/Notch)can promotes bone morphogenetic proteins(BMPs)(BMP2,4,6,7,9)induced Osteogenic differentiation of mesenchymal stem cells(MSCs).However,unlike DLL1,CCN3 acts as a secreted ligand for Notch signaling,and how does it affect osteogenic differentiation of MSCs? How does it interact with membrane protein ligand DLL1 to influence osteogenic differentiation of MSCs? The aims of this study were to explore the role and mechanisms of CCN3 in the osteogenic differentiation of MSCs,so as to provide theoretical basis for the clinical application of CCN3 and related proteins and factors in bone tissue engineering.Part one: Objective: Explore the role of CCN3 in osteogenic differentiation of MSCs.Methods: The over-express CCN3 adenoviruses(Ad-CCN3)and small interfering CCN3 adenoviruses(Ad-SiCCN3)were used to infect MEFs,the cell line with MSCs characteristics.The early osteogenic index alkaline phosphatase(ALP)was detected by cytochemical staining and activity determination;The late osteogenic index calcium deposits was detected by Alizarin Red S staining;The expressions of Runt-related transcription factor 2(Runx2),the early osteogenic indicator,and Osteopontin(OPN),Osteocalcin(osteocalcin,OCN),the late osteogenic indicators were detected by q RT-PCR,Western blot and immunohistochemistry;The effects of CCN3 on subcutaneous heterotopic osteogenesis in nude mice were detected by Micro-CT scanning and analysis,H&E Staining and Masson’s Trichrome Staining.Results: Compared with the control group,CCN3 significantly inhibited ALP expression,calcium deposition,expression of the osteogenic markers Runx2,OPN and OCN and the subcutaneous ectopic osteogenesis of MEFs in nude mice.Conclusion: CCN3 exerts a negative regulatory effect on the osteogenic differentiation of MSCs.Part two: Objective: To explore the mechanism of CCN3 inhibited osteogenic differentiation of MSCs.Methods:The over-express CCN3 adenoviruses(Ad-CCN3)and small interfering CCN3 adenoviruses(Ad-SiCCN3)were used to infect MEFs,the cell line with MSCs characteristics.qRT-PCR was used to detect the effect of CCN3 on the mRNA level of BMP9(the classical ligand of BMP signaling pathway);Western blot was used to detect the effect of CCN3 on the total protein levels and phosphorylation levels of Smad1/5/8,Erk1/2,p38 and JNK in BMP/Smads and BMP/MAPK signaling pathways;q RT-PCR was used to determine the effects of CCN3 on the mRNA levels of classical ligands,receptors and target gene(Hey1)of Notch signal pathway;ALP staining and activity assay were used to measure the role of Hey1 in CCN3-inhibited early osteogenic differentiation of MEFs;Micro-CT scaning and analysis,H&E staining,Masson’s Trichrome staining were used to detect the role of Hey1 in CCN3-inhibited subcutaneous ectopic osteogenesis of MEFs in nude mice.Flow cytometry(FCM)was used to detect the effect of CCN3 on the proliferation and apoptosis of MEFs.Results: CCN3 could obviously down-regulate the mRNA level of BMP9 and the phosphorylation of Smad1/5/8,Erk1/2,p38 and JNK in BMP/Smads and BMP/MAPK signaling pathways,and up-regulated the mRNA levels of Notch1,Notch2,Notch3 and Notch4 in classical Notch signaling pathway;On the other hand,CCN3 could also inhibit the expression of DLL1 and Hey1;ALP staining and activity assay,Micro-CT scaning and analysis,H&E staining and Masson’s Trichrome staining’s results showed that Hey1 could partially reverse the inhibitory effect of CCN3 on the early osteogenic differentiation and subcutaneous ectopic osteogenesis of MEFs in nude mice.FCM’s results showed that CCN3 had no effect on the proliferation of MEFs,but could inhibit their apoptosis.Conclusion: CCN3 could significantly inhibit the osteogenic differentiation of MSCs.The inhibitory effect of CCN3 is mainly through the inhibition of BMP signal and the mutual inhibition with DLL1(one of the Notch signal membrane ligands),and thereby inhibiting the expression of Hey1,the target gene shared by BMP signal pathway and Notch signal pathway.