Role Of Smad7 In Sox9-Enhanced BMP2 Inducing Chondrgenic Differentiation

Objective:Bone morphogenetic protein 2 (BMP-2) has been confirmed as one of the most powerful chondrogenic inducer.Our previous studies have shown expression of Sox9 can enhance BMP2-induced chondrogenic marker and inhibit ossification markers.However,Smad7,induced by BMP2,seem to be potential target of Sox9 for its feature of chondrgenic differentiation inhibiting and Endochondral Ossification adjusting. This studies aim to show the role of Smad7 in Sox9-enhanced BMP2 inducing chondrgenic differentiation and Endochondral Ossification. The study try to provide now target for gene-enhanced cartilage engineering.Methods:(1)BMP2 Smad7 and Sox9 gene expression were mediated by recombinant adenoviruses. AdBMP2 AdSmad7 and AdSox9, And AdGFP was used as a control. After amplified in HEK 293 cells,AdGFP, AdBMP2 AdSmad7 and AdSox9were used to infect C3H10T1/2 cells. The gene expression of aimed protein were detected by Western-blot.(2)C3H10Tl/2 cells infected by AdGFP, AdBMP2+ AdSox9 and AdSox9 were planted in micromass culture for 14 days in vitro.(3) C3H10T1/2 cells transduced with AdGFP, AdBMP2 and/or AdSox9 were cultured, Smad7 and its related factors(OPN and MMP13) were measured at certain time points.(4) Ectopic cartilage formation research was used to show the function of different level of Smad7 during BMP2 induced C3H10T1/2 cells chondrogenic and endochondral ossification.(5) Infected mouse fetal limbs were used to evaluate the effect of the function of different level of Smad7 on BMP2 induced chondrocyte proliferation, hypertrophy and ossification.(6) Flow cytometry test apoptosis rate of BMP2-induced C3H10T1/2 cells in different level of Smad7.Results:(1) C3H10T1/2 cells could be steadily infected by AdBMP2, AdSmad7, AdSox9. The express of the target gene were proved to be effective respectively.(2) Overexpression of BMP2 lead by Ad-BMP2 can induce Smad7 increase,however,Overexpressed-Sox9 can restrain Smad7expression induced by BMP2 in sequential time points in micromass culture in vitro.(3) Exogenous overexpression of Sox9 inhibited BMP2 induced Smad7 related factors MMP13 and OPN in vitro.(4) Ectopic cartilage/bone formation assay showed that high level of Smad7 inhibit cartilage formation,however when Smad7 was surpressed by Sox9, the cartilage formation was increased and the BMP2-induced endochondral ossification was inhibited.(5) Mouse limb results showed that increase of Smad7 acted synergistically to chondrocytes proliferation and chondrocytes hypertrophy and ossification induced by BMP2.And when suppressed by Sox9,the chondrocytes proliferation was promoted and chondrocytes hypertrophy and ossification was inhibited(6) Flow cytometry test shows the apoptosis rate of BMP2-induced C3H10T1/2 cells increase in high level of Smad7.And when Sox9 can reduce the rate of apoptosis.Conclusion:All the data present that Sox9 enhance BMP2-induced MSCs chondrogenic differentiation and block the endochondral ossification by inhibiting Smad7 expression.Decrease of Smad7 increase chondrogenic differentiation and inhibit terminal maturation of chondrocyte. Thus, increase the expression of Sox9 in BMP2-induced chondrogenic differentiation can be a new way for cartilage tissue engineering.