GSK3β is a checkpoint for TNF-α-mediated impaired osteogenic differentiation of mesenchymal stem cells in inflammatory microenvironments

Background: The fate and differentiation of mesenchymal stem cells (MSCs) depend on various microenvironmental cues. In chronic inflammatory bone disease, bone regeneration is inhibited. The present study therefore sought to identify the underlying molecule mechanisms. Methods: We isolated periodontal ligament stem cells (PDLSCs), a new population of MSCs, from the periodontal ligament tissues of periodontitis patients and healthy controls (p-PDLSCs and h-PDLSCs). The secretion of inflammatory cytokines, like TNF-alpha, IL-1 beta, IL-6 and IL-8, after LPS stimulation was measured by ELISA. The expressions of p-GSK3 beta and GSK3 beta in two types of PDLSCs were detected by Western blot. TOPFlash was used to assay the Tcf/Lef transcriptional activity. Knockdown of GSK3 beta by siRNA and over-expression of GSK3 beta by adenoviruses were performed to confirm the role of GSK3 beta in the impaired osteogenic differentiation of PDLSCs under inflammatory microenvironment. Results: We demonstrated that p-PDLSCs displayed impaired osteogenic capacity than h-PDLSCs. Upon inflammatory stimulation, monocytes, but not PDLSCs, released inflammatory cytokines among which TNF-alpha directly act on PDLSCs and suppressed their osteogenic differentiation. TNF-alpha induced the phosphorylation of GSK3 beta, the deactivated form of GSK3 beta, which increased nuclear p-catenin and Lef-1 accumulation, and eventually reduced the Runx2-associated osteogenesis in PDLSCs. Over-expression of GSK3 beta rescued osteogenesis in TNF-alpha-stimulated PDLSCs, whereas inactivation of GSK3 beta was sufficient to liberate the beta-catenin/Lef-1/Runx2 pathway. Conclusion: GSK3 beta plays an obligatory role in the TNF-alpha-mediated inhibition of osteogenesis in MSCs. General significance: The strategy to target GSK3 beta may provide a potential approach,to bone regeneration in inflammatory microenvironments. (C) 2013 Elsevier B.V. All rights reserved.