Role Of Notch Signaling Pathway In Bone Morphgenetic Protein9-induced Ostegenic Differentiation Of Marrow-derived Mesenchymal Stem Cells And Its Mechanism

Mesenchymal stem cells are pluripotent cells with tissue-specificdiffierentiation and self-renewed capability. Bone morphogentic protein9(BMP9) is one of the least studied BMPs and have identified as a mostpotent inducer of ostergenic diffierentiation in MSCs. Notch signalingregulates the proliferation and diffierentition of cells and has been showento act synergistically in BMP9induced MSCs ostegensis diffieretiation.The reseach aims to study the effect of BMP9on mesenchymal stem cellsosteogenic diffierentiaion (Part1) and the role of Notch signaling pathwayin BMP9induced osteogenic diffierentiaion of marrow-derivedmesenchymal stem cells and its mechanism (Part2and3).PART1THE EFFECT OF BMP9ON MESENCHYMAL STEM CELLSOSTEOGENIC DIFFIERENTIAIONObjective To prove the role of BMP9on MSCs osteogenicdiffierentiaion. Method After C2C12cells were infected by recombinantadenovirus GFP and BMP9(AdGFP/AdBMP9), the early osteogenicmarker Alkaline phosphatase (ALP) activity was detected by staining assay,later osteogenic marker calcium deposition, OPN and OCN weredetermined by Alizarin Red S staining and RT-PCR.The total protein leveland phosphorylated form of Smad1/5/8, P38, Erk1/2and JNK MAPKs were determined by Western blot. Results AdBMP9is not only increasedthe activity of ALP on days3,5,7d, but also matrix mineralization on7dand14d in a dose-dependent manner. BMP9increased OPN and OCNmRNA level and the protein expression of phosphorylated form ofSmad1/5/8, P38,Erk1/2and JNK MAPKs, not the total protein level.Conclusion BMP9may involve in osteoblast commitment of C2C12mesenchymal stem cells via the activity of BMPs-Smad1/5/8and MAPKsignaling pathway.PART2THE ROLE OF NOTCH SIGNALING ON BMP9-INDUCEDC2C12CELLS OSTEOGENIC DIFFIERENTIAIONObjective To analysis the role of Notch signaling pathway in BMP9-induced osteogenic diffierentiaion of C2C12cells. Methods Aftertreatment C2C12cells with diffierent cell density and DAPT,the earlyosterogenic marker,ALP activity were detected by quantitative andstaining assay, later ostergenic marker OCN was detected by RT-PCR.Results The ALP activity of BMP9-induced high cell density group ishigher than low cell density group, but the expression of OCN was notmarkedly changed. Notch specific inhibitor DAPT decreased ALP activityand the expression of OCN. Conclusion Notch signaling pathwayincreases BMP9induced osteoblast commitment of C2C12mesenchymalstem cells.PART3THE MECHANISIM OF NOTCH SIGNALING ON BMP9INDUCED C2C12CELLS OSTEOGENIC DIFFIERENTIAIONObjective To study the mechansim of signaling on BMP9-inducedC2C12cells osteogenic differentiation. Methods After treatment ofC2C12cells at5day, The receptor of BMP9, osteogenic marker Runx2,Colla-1and BMPs target gene Id1, Id2and Id3were detected byRT-PCR. Furthermore, the protein expression of Smad1/5/8, P38, JNK,Erk1/2were detected by Western blot. Results DAPT markedlydecreased expression level of ALK2, Hey1and ostegenic differentiationmarker Runx2and Col1a-1induced by BMP9of C2C12cells. Moreover, italso decreased BMPs target gene Id1，Id2, and Id3at1d. DAPT did notchange total protein level of Smad1/5/8, P38, JNK, Erk1/2, however, itdecreased the phosphorlated form of Smad1/5/8, P38, Erk1/2. ConclusionNotch signaling pathway may promote the ostegenic transcript factorsRunx2and Colla-1to regulate BMP9-induced MSCs ostegenicdifferentiation via the activity of BMPs-Smad and BMPs-MAPK signalingpathway.