Celastrol improves self-renewal and differentiation of human tendon-derived stem cells by suppressing Smad7 through hypoxia

Background: We aimed to evaluate the potential enhancing effect of celastrol on the stemness of human tendonderived stem cells (hTSCs) in vitro and the underlying molecular mechanisms. Methods: The capability of hTSC self-renewal was assessed by cell proliferation and colony formation as determined with the CCK-8 kit. Adipogenesis, chondrogenesis, and osteogenesis were determined by Oil Red O, Alcian Blue, and Alizarin Red staining, respectively. The relative mRNA levels of Sox9, PPAR., Runx2, Smad7, and HIF1a were determined by real-time polymerase chain reaction (PCR). The levels of Smad7 and HIF1a protein were measured by immunoblotting. The chromatin immunoprecipitation (ChIP) assay was used to assess the direct binding of HIF1a to the Smad7 promoter. Suppression of Smad7 induced by hypoxia was examined using the luciferase reporter assay. Results: We found that treatment with celastrol resulted in improvement in both the multi-differentiation potential and self-renewal capability of hTSCs. Celastrol elicited hypoxia and subsequently suppressed the expression of Smad7 through direct association with the hypoxia response element consensus sequence. Further, we demonstrated that both Smad7 and HIF1a were involved in the beneficial effects of celastrol on the differentiation and self-renewal of hTSCs. Conclusions: We demonstrated the positive effect of celastrol on the stemness of hTSCs and elucidated the essential role of the HIF1a-Smad7 pathway in this process.