TLR activation inhibits the osteogenic potential of human periodontal ligament stem cells through Akt signaling in a Myd88- or TRIF-dependent manner.

Background This study investigated the effects of Toll-like receptors (TLRs) on human periodontal ligament stem cells (hPDLSCs) osteogenic differentiation and the associated mechanisms. Methods TLR1, TLR3, TLR4, and TLR6 expression in hPDLSCs was evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry, whereas their functional roles were assessed based on nuclear factor (NF)-kappa B activation and proinflammatory cytokine expression. The osteogenic effects of these TLRs were analyzed by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. The roles of Myd88, TRIF, and downstream molecules mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) in TLR-mediated impaired osteogenic differentiation were examined by real-time RT-PCR and western blotting using specific small interfering RNA siRNA and pharmacologic inhibitors. The involvement of Akt activation in restoring TLR1-, 4-, and 6-mediated osteogenic suppression was verified using the Akt activator SC-79. Results TLR1, TLR3, TLR4, and TLR6 were highly expressed functionally in hPDLSCs and high doses of TLR ligands inhibited osteogenic potential. Furthermore, blocking Myd88 partly rescued the decrease in osteogenesis mediated by TLR1, TLR4, and TLR6 activation by enhancing Akt phosphorylation; likewise, TRIF suppression partially rescued lipopolysaccharide (LPS)-mediated osteogenic inhibition through ERK and Akt activation. Moreover, Akt activation restored the TLR-mediated inhibition of hPDLSC osteogenic differentiation. Conclusions High doses of TLR1, TLR4, and TLR6 ligands suppress hPDLSC osteogenic differentiation by inhibiting Akt activation through Myd88- or TRIF-dependent signaling pathways. Blocking these adaptors or reactivating Akt could restore the TLR-mediated decrease in hPDLSC osteogenesis, and might be an ideal strategy for periodontitis treatment.