The Role Of MSX2in Bone Morphogenetic Protein9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Understanding the interactions between MSX2and bone morphogenicproteins (BMPs) signaling remains a crucial issue to optimize the use ofmesenchymal stem cells (MSCs) and BMPs in bone tissue engineering.Herein, we explore the possible involvement and detail role of MSX2inBMP9-induced osteogenic differentiation of MSCs.Firstly, C3H10T1/2cells,MC3T3-E1cells,C2C12cells were infected with recombinantadenovirus Ad-BMP9,the expression of endogenous MSX2were tested byRT-PCR and Western blot.we demonstrate that BMP9can increaseendogenous Msx2mRNA levels and protein level. Then,C3H10T1/2cells、 MC3T3E1cells、 C2C12cells were infected by recombinantadenovirus Ad-MSX2and BMP9, Alkalinephosphatase (ALP) activityassay and Western blotting was used to investigate whether MSX2couldeffect BMP9induce osteogenic differentiation of mesenchymal stemcells.And it indicate that MSX2could enhance BMP9induced earlyosteogenic maker Alkalinephosphatase (ALP) activity but inhibit lateosteogenic maker-opn and ocn. RT-PCR and Western blotting were accomplished to testify that MSX2dramatically inhibited osteogenticrelated genes-Id1,Id2,Id3,coll1a and BMP crucial transcription factorRunx2increased by BMP9via blocking BMP9-induced Smads signalingand subsequently reducing Smads dependent up-regulation of ALK1andALK2in MSCs. Furthermore,the activation of canonical Smad signalingwas also inhibited by MSX2according to Luciferase activity.However, theactivation of MAKP signaling was not effected by MSX2.In order tofarther confirm the effect MSX2on BMP9-induced osteogenic differen-tiation,the interference adenovirus of MSX2(si-MSX2) is structured byAd-easy system.Firstly，the cDNA of siMSX2gene was cloned into theshuttle plasmid pSES-1，and then the linearized plasmid was electroporatedinto E. coli BJ5183cells which had backbone plasmidpAdEasy-1.Secondly,the identified recombinant plasmid was digested withPac Ⅰ and transfected into293cells via liposome to package theadenovirus.In the end,the recombinant adenovirus was gained, Redfluorescence protein expression in293cells was detected under fluorescentmicroscope after transfection.Our results discovered a clue to explain themechanism involved in the doubleaction effect of MSX2on BMP9-induced osteogenic differentiation of MSCs. This crosstalk between MSX2and BMP9should be emphasized in the future use of BMP9in therapeuticpurpose of fracture repair.