Effect of GSK126 on the chondrogenic differentiation of mesenchymal stem cells

Abstract

Objective To investigate the effect of enhancer of Zeste homolog (EZH2) inhibitor GSK126 on the chondrogenic differentiation of the bone marrow-derived mesenchymal stem cells (BMSCs). Methods BMSCs were isolated by ficoll density-gradient centrifugation. The cells at passage 3 were used in this study. The optimal concentration of GSK126 was confirmed by methyl thiazol tetrazolium (MTT). On reaching confluence, cells were digested, suspended and then centrifuged at a density of 5×106/ml to form pellets. The pellets were cultured for 3 weeks with CM supplemented with GSK126 or not. The expression of type Ⅱ collagen and glycosaminoglycan (GAG) was detected by immunohistochemistry and toluidine blue staining, respectively. Real-time quantitative polymerase chain reaction (Real-time PCR) was performed to detect the content of type Ⅱ collagen, sex determining region Y box 9 (SOX9) and aggrecan mRNA, and the amount of H3K27me3 protein was assayed by Western blotting. Results 5 μmol/L GSK126 was regarded as the optimal concentration since the cytotoxic effect has not been observed. The cell volumn in the experiment group was larger than that in the control group, with lower cell density. The histological staining showed that type Ⅱ collagen and GAG were synthesized and secreted in all the pellets with stronger deposit in the experiment group. The absorbance (A) of experiment group and control group was 0.368 5±0.040 5 and 0.132 2±0.021 1 respectively. Real-time PCR showed that the mRNA expression of type Ⅱ collagen and aggrecan in the experiment group was significantly higher than that in the control group. High level of H3K27me3 protein was noted in the control group, with the A value 0.6742±0.056 2. Treatment with 10 μmol/L GSK126 obviously decreased the amount of H3K27me3, with the A value of 0.326 5±0.030 2. Conclusion GSK126 obviously promoted the chondrogenic differentiation of BMSCs by demethylation of H3K27me3. Key words: GSK126; H3K27me3; Chondrogenic differentiation; Bone mesenchymal stem cells