CXCL12/CXCR4Signal Axis Plays An Important Role In Mediating Bone Morphogeneic Protein9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Pluripotent mesenchymal stem cells (MSCs) are a group of bonemarrow stromal progenitor cells processing osteogenic, chondrogenic,adipogenic and myogenic lineages differentiations. Previous studies havedemonstrated that bone morphogeneic protein9(BMP9) is one of the mostosteogenic BMPs both in vitro and in vivo, however, the underlyingmolecular mechanism of osteogenesis induced by BMP9is needed to bedeep explored. Here, in order to elucidate the role of SDF-1/CXCR4signalaxis during BMP9-incuced osteogenic differentiation in MSCs, weconstructed and identified a recombinant adenovirus overexpressing SDF-1,and then used the recombinant adenoviruses assay to introduce BMP9intoC3H10T1/2cells to elucidate the mRNA and protein expression levels ofSDF-1and CXCR4induced by Ad-BMP9. At the same time, therecombinant adenovirus and neutralizing antibody were used to perturbingthe SDF-1/CXCR4signal axis in C3H10T1/2cells and HS5cells before orafter the addition of BMP9to make sure the fuction of CXCR4in the signal. The alkaline phosphatase (ALP) quantitative assay and fast blue RR saltstaining were used to determine the expression of ALP.Immunocytochemistry was used to determine the expression of osteocalcin(OCN), while calcium deposition was determined by alizarin red S staining.The expression of the osteogenic transcription factor Runx2, Osx, Plzf andDlx5were detected by real time PCR. Western blot was used to detect thechange of osteogenic differentiation signaling pathway Smad and MAPK,and the luciferase reporter gene assay was used to detect the change of Smad,too. The results showed that Ad-SDF-1was successfully obtained. SDF-1and CXCR4expressions were down-regulated at the stage of BMP9-inducedosteogenic differentiation, in a dose-and time-dependent. Pretreatment ofC3H10T1/2cells with SDF-1/CXCR4could significantly affect the earlyand mid osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN),the transcription factors of Runx2, Osx, Plzf and Dlx5expression, throughactivating the Smad, MAPK signaling pathway. Addition of exogenousSDF-1did not affect the changes of the late osteogenic marker calciumdeposition. The result of ALP quantitative assay of HS5cells is similar toC3H10T1/2cells. Thus, our findings suggest a co-requirement of theSDF-1/CXCR4signal axis in BMP9-induced the early-and mid-process ofosteogenic differentiation of MSCs.