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

Abstract

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)‐κB 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.

Transforming Growth Factor-β3 Chitosan Sponge (TGF-β3/CS) Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

“…By comparison of our results with those reported previously, we found that PDLSCs undergo osteogenic differentiation through the mitogen-activated protein kinase (MAPK) signaling pathway [32][33][34], and that signaling transduction mediated by TGF-β3 in osteogenic differentiation and bone regeneration [35] specifically occurs through both canonical Smad-dependent pathways (TGF-β ligands, receptors, and Smads) and non-canonical Smad-independent signaling pathway (e.g., p38…”

“…Studies have shown that bioactive molecules promote the proliferation, migration, and osteogenic differentiation of PDLSCs by activating the MAPK pathway in vitro [32][33][34]. Through contrastive analysis of the mechanism of osteogenic differentiation of TGF-β3 and hPDLSCs, we…”

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis

“…Additionally, it is the only study that evaluated both osteoclastogenesis and osteogenesis on human periodontal cells, and more specifically GF. There are a few studies ( 34 , 42 , 43 ) that have studied the osteogenic potential of human periodontal ligament cells (hPDL) exposed to TLR agonists, sometimes with conflicting results. In two independent studies ( 42 , 43 ), hPDL cells were infected with E. coli LPS (TLR4 agonist) and it was found that the osteogenic capacity of the cells was reduced significantly.…”

“…Others found that TLR4 has an inhibitory effect on osteogenesis in murine bone marrow mesenchymal stem cells ( 33 ). Recently, it was shown that in vitro TLR4 activation in high doses (10 μg/mL) inhibits the osteogenic potential of human periodontal ligament cells ( 34 ).…”

Synergistic effects triggered by simultaneous Toll‐like receptor‐2 and ‐3 activation in human periodontal ligament stem cells

“…There is only limited knowledge about the influence of TLR‐3 activation in hPDLSCs. Stimulation with TLR‐3 agonist polyinosinic:polycytidylic acid (Poly I:C) leads to downregulation of the osteogenic capacity of hPDLSCs . Furthermore, a conference paper by Klincumhom et al.…”