Effects Of Shp On BMP9-Induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective:To elucidate the effects of SHP (small heterodimer partner) on BMP9 (Bone morphogenetic protein9) induced osteogenic differentiation of MSCs (mesenchymal stem cells).Methods:Firstly, we constructed and chracterized the recombinant adenovirus overexpressing SHP.(Ad-SHP), and the adenovirus expressing small interfering RNA targeting mouse SHP (Ad-siSHP). The SHP expression upon BMP9 treatment in MSCs was detected by RT-PCR and Western Blot. C3H10T1/2 and C2C12 cells were infected by Ad-SHP or Ad-siSHP, and sitmulated with BMP9, then the effect of SHP on BMP9-indcued early osteogenic marker Alkaline phosphotase(ALP) was assessed by quantitative assay and staining assay. The late osteogenic marker calcium deposition was evaluated by Alizarin Red S staining. Other late osteogenic indicators including OCN (Osteocalcin) and OPN (Osteopontin) were measured by RT-PCR. In addition, the expressiong of osteogenic-related genes Runx2, Idl, Id2, Id3 and CTGF were determined by RT-PCR. Runx2 and Dlx5 protein expression was detected by Western Blot. The effects of SHP on BMP9-induced classic Smad signaling pathway were detected by luciferase reporter assay and Western Blot.Results:We found that the recombinant adenovirus Ad-SHP can effectively overexpress exogenous SHP, whereas Ad-siSHP can silence SHP expression in MSCs. We demonstrated that exogenous BMP9 can up-regulate the expression of SHP in C3H10T1/2 and C2C12 cells at both gene and protein level. Overexpression of SHP can increase ALP activity, promote calcium deposition, as well as induce OPN and OCN expression. However, knockdown of SHP can inhibit BMP9-induced osteogenic differentiaiton. We evidenced that Idl,Id2, CTGF, Runx2 and Dlx5 were also elevated by overexpression of SHP in BMP9-mediated osteogenic differentiation process, whereas reduced by knockdown of SHP. Although SHP had no obvious effect on the phosphorylation of Smadl/5/8 induced by BMP9, knockdown of SHP can inhibit BMP9-induced SBE luciferase activity.Conclusions:SHP may modulate BMP9-induced osteogenic differentiation of MSCs possibly through affect Smad/1/5/8 transcriptional activity.