Effects Of JNK Kinase Pathway On BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective:Although BMP9is highly capable of promoting osteogenicdifferentiation of mesenchymal stem cell (MSCs),the molecular mechanisminvolved remains to be fully elucidated.Here,We sought to confirmed theeffects of JNK kinase pathway on BMP9-induced osteogenic differentiationof mesenchymal stem cells C3H10T1/2,Murine embryonic fibroblast MEFand Murine myoblastic cell line C2C12.Methods:BMP9was introduced into these cells(C3H10T1/2,MEF andC2C12)by using recombinant adenoviruses method.Phosphorylated formsof JNK kinase was assayed by Western Blot and luciferase activity assay.TheJNK activity was abolished by inhibitor SP600125and siRNA,then,earlyosteogenic marker alkaline phosphatase activity(ALP) was detected,andAlizarin red S staining was conducted to measure late osteogenic markercalcium deposition.Expression of transcription factor RUNX2andosteogenic target genes (such as ID1,ID2,ID3,Col1) were measured byRT-PCR.Moreover,activity of Smad1/5/8was tested by Western Blot andluciferase activity assay.OCN and OPN expression was examined byhistochemical staining.Animal assay was accomplished to testify that whether blockage of JNK kinase can result in inhibition of entopic boneformation induced by BMP9in vivo．Results:It was found that BMP9stimulated the activation of JNK inMSCs.BMP9-induced osteogenic differentiation(early osteogenic markerALP,late osteogenic marker calcium deposition,expression of OCN andOPN)were dramatically inhibited by JNK inhibitorSP600125.Moreover,BMP9-activated Smads signaling was decreased bySP600125treatment in C3H10T1/2.Furthermore,Expression of transcriptionfactor RUNX2and osteogenic target genes were reduced by SP600125.Theeffects of inhibitor are reproduced with adenoviruses expressing siRNAtargeted JNK.Inhibition of JNK activity was shown to decrease ALP activityand calcium deposition,and lead to significant decrease in entopic boneformation in vivo.Conclusions:Taken together,our results revealed that JNK wasactivated in BMP9-induced osteogenic differentiation of MSCs.What ismost noteworthy,however,is that inhibition of JNK activity resulted inreduction of BMP9-induced osteogenic differentiation of MSCs,implyingthat BMP9can induce osteogenic differentiation of mesenchymal stem cellsthrough JNK kinase pathway.