LPS‑induced upregulation of the TLR4 signaling pathway inhibits osteogenic differentiation of human periodontal ligament stem cells under inflammatory conditions

Abstract

Toll‑like receptor 4 (TLR4) is a transmembrane receptor responsible for the activation of a number of signal transduction pathways. Despite its involvement in inflammatory processes, the regulation of TLR4 signaling in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions remains to be fully elucidated. The present study aimed to clarify the regulatory mechanisms of the TLR4 signaling pathway and its role in the differentiation of hPDLSCs under inflammatory conditions. hPDLSCs from the periodontal tissues of healthy subjects and patients with periodontitis were identified by analyzing their cell surface marker molecules, and their osteogenic and adipogenic differentiation abilities. To determine the effect of TLR4 signaling on osteogenic and adipogenic differentiation under inflammatory conditions, cells were challenged with TLR4 agonist and antagonist under pluripotent differentiation conditions. Cell proliferation, apoptosis and migration were then determined using appropriate methods. The alkaline phosphatase (ALP) activity, Alizarin Red staining, Oil red O staining and relative gene and protein levels expression were also determined. The results showed that lipopolysaccharide (LPS)‑induced inflammation inhibited cell proliferation and migration, promoted cell apoptosis and affected the cell cycle. Under inflammatory conditions, the activation of TLR4 decreased the activity of ALP and the expression of osteogenic markers, including osteocalcin, Runt‑related transcription factor 2 and collagen I, compared with the control group, but increased the expression of adipogenesis‑related genes poly (ADP‑ribose) polymerase γ and lipoprotein lipase. The activation of TLR4 also induced the expression of proinflammatory cytokines interleukin‑1β, tumor necrosis factor‑α, nuclear factor‑κBP65 and TLR4, compared with that in the control group and the TLR4 antagonist group. The findings showed that LPS‑induced upregulation of the TLR4 signaling pathway inhibited osteogenic differentiation and induced adipogenesis of the hPDLSCs under inflammatory conditions. The present study provided a novel understanding of the physiopathology of periodontitis, and a novel avenue for targeted treatments based on stem cell regeneration.

Figure 1

Isolation and culture of hPDLSCs from tissue blocks. Tissue blocks were cultured for (A) 5 days and (B) 7 days. Primary hPDLSCs sub-cultured for (C) P1 and (D) P2. hPDLSCs, human periodontal ligament stem cells; D, day; P, passage.

Figure 2

Identification and purification of hPDLSCs by flow cytometry. hPDLSCs, human periodontal ligament stem cells.

Figure 3

Immunofluorescence and immunohistochemistry of human periodontal ligament stem cells. (A) Positive staining of Stro-1. (B) Negative staining of keratin. (C) Positive staining of TLR4. TLR4, Toll-like receptor 4.

Figure 4

Effect of inflammation on human periodontal ligament stem cells. (A) Cell proliferation detected using an MTT assay; (B) cell cycle analysis; (C) distribution of cell cycle detected by flow cytometry; (D) pluripotency induction (osteogenic differentiation and adipogenic differentiation); (E) relative content of ALP; (F) relative content of osteogenic induction genes (OCN, Runx2 and COL1) and adipogenesis-related genes (PPARγ and LPL). ^*P<0.05, vs. Control group. LPS, lipopolysaccharide; ALP, alkaline phosphatase; OCN, osteocalcin; Runx2, Runt-related transcription factor 2; Col1, collagen I; PPAR, poly (ADP-ribose) polymerase; LPL, lipoprotein lipase; D, day.

Figure 5

Functions of the TLR4 agonist and antagonist on hPDLSCs. Effects of the TLR4 agonist and antagonist on (A) cell proliferation and (B) cell migration. (C) Expression levels of IL-1β and TNF-α. (D) Apoptosis of hPDLSCs detected by flow cytometry. ^*P<0.05 vs. Control group; ^#P<0.05 vs. TLR4 Agonist group. hPDLSCs, human periodontal ligament stem cells; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4.

Figure 6

Effects of the TLR4 agonist and antagonist on the osteogenic differentiation of human periodontal ligament stem cells. (A) Expression of NF-κBp65 and TLR4 (1, Control; 2, TLR4 agonist; 3, TLR4 antagonist). (B) Quantification of levels of NF-κBp65 and TLR4. (C) Osteogenic differentiation ability was detected by staining with Alizarin Red. ^*P<0.05 vs. Control group; ^#P<0.05 vs. TLR4 agonist group. NF-κBp65, nuclear factor-κBp65; TLR4, Toll-like receptor 4; LPS, lipopolysaccharide.

Figure 7

Expression levels of osteogenic induction genes (OCN, Runx2 and COL1), adipogenesis-related genes (PPARγ and LPL), ALP, NF-κBp65 and TLR4. ^*P<0.05 vs. C group; ^#P<0.05 vs. TA group. TLR4, Toll-like receptor 4; C, Control; TA, TLR4 agonist; Tan, TLR4 antagonist; OIC, osteogenic induction control; OITA, osteogenic induction TLR4 agonist; OITAn, osteogenic induction TLR4 antagonist; ALP, alkaline phosphatase; OCN, osteocalcin; Runx2, Runt-related transcription factor 2; Col1, collagen I; PPAR, poly (ADP-ribose) polymerase; LPL, lipoprotein lipase.