Epigenetic Mechanism of TGF-β1 Promoting Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Osteoarthritis
Abstract Objectives: It had been proved that TGF-β1 was correlated with onset of osteoarthritis in vitro and vivo. Here, This study was to elucidate the epigenetic mechanism of TGF-β1 promoting osteogenic differentiation in osteoarthritis. Methods: hBMSCs surface antigens were assayed by flow cytometry tests. qRT-PCR was performed to detect hBMSCs mRNA levels of RUNX2, PPARγ and SOX9. hBMSCs were stained by osteoalkaline phosphatase and alizarin red. The qRT-PCR and Western blot were both used to detect the expression levels of methylases, demethylases and osteogenic transcription factor RUNX2 after hBMSCs were cultrued in osteogenic medium coincubated with TGF-β1 solution. Results: hBMSCs were identified by over expressions of CD90, CD105 and CD44, as well as the positive multi-diffenentiation potential tests. hBMSCs bone alkaline phosphatase and alizarin red staining were observed to deepen in TGF-β1 group compared with the osteogenic culture group. The mRNA expression levels of EZH1, KDM2B, KDM4A/4B/4C/4D, and KDM6A /6B were increased in hBMSCs cultured in osteogenic medium. The expression levels of KDM6A/6B were shown increasement when TGF-β1 was co-incubated with osteogenic medium. Furthermore, the mRNA and protein levels of KDM6A/6B were significantly decreased after SB431542 was added in the medium. RUNX2 was significantly inhibited by the addition of GSK-J4 solution, while KDM6A/6B expression level did not change significantly. Conclusion: The osteogenic differentiation of hBMSCs was related to the enhanced expressions of EZH1, KDM2B, KDM4A-4D, KDM6A/6B. The expression levels of demethylase KDM6A/6B were positively regulated by the TGF-β/Smad signaling pathway, which promoted the osteogenic differentiation of hBMSCs.