Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition with β-catenin signaling in inflammatory microenvironments

Abstract

Inflammation can influence multipotency and self-renewal of mesenchymal stem cells (MSCs), resulting in their awakened bone-regeneration ability. Human periodontal ligament tissue-derived MSCs (PDLSCs) have been isolated, and their differentiation potential was found to be defective due to β-catenin signaling indirectly regulated by inflammatory microenvironments. Nuclear factor-κB (NF-κB) is well studied in inflammation by many different groups. The role of NF-κB needs to be studied in PDLSCs, although genetic evidences have recently shown that NF-κB inhibits osteoblastic bone formation in mice. However, the mechanism as to how inflammation leads to the modulation of β-catenin and NF-κB signaling remains unclear. In this study, we investigated β-catenin and NF-κB signaling through regulation of glycogen synthase kinase 3β activity (GSK-3β, which modulates β-catenin and NF-κB signaling) using a specific inhibitor LiCl and a phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002. We identified that NF-κB signaling might be more important for the regulation of osteogenesis in PDLSCs from periodontitis compared with β-catenin. BAY 11-7082 (an inhibitor of NF-κB) could inhibit phosphorylation of p65 and partly rescue the differentiation potential of PDLSCs in inflammation. Our data indicate that NF-κB has a central role in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. Given the molecular mechanisms of NF-κB in osteogenic differentiation governed by inflammation, it can be said that NF-κB helps in improving stem cell-mediated inflammatory bone disease therapy.

Figures

Figure 1

Inhibition of GSK-3β rescues the osteogenic differentiation of P-PDLSCs but decreases osteogenic differentiation of H-PDLSCs. H-PDLSCs and P-PDLSCs were treated with or without LiCl along with osteogenic differentiation medium for 7 days. (a) The expression of p-GSK-3β and GSK-3β was examined by western blot analysis. (b) Quantification of ALP activity staining. (c,d) Real-time RT-PCR and western blot analysis of the osteoblast marker gene (Osterix, normalized to β-actin) on day 7. Data represent the means±S.D. *P<0.05 (n=3)

Figure 2

Effects of GSK-3β activity on NF-κB and WNT signaling in PDLSCs. (a) The expression of NF-κB (p65) and β-catenin was examined by Real-time RT-PCR (n=3). (b) The activation of NF-κB (phosphorylated p65, pNF-κB) and β-catenin (specific for the active form of β-catenin, dephosphorylated on Ser37 or Thr41) and actin was examined by western blot analysis

Figure 3

Increased GSK-3β activity blocked osteogenic differentiation of PDLSCs. H-PDLSCs, P-PDLSCs and H-PDLSCs treated with TNF-α were grown in the presence of osteogenic medium with or without LY 294002. (a) Osteoblastic differentiation was determined by ALP staining and activity at day 7. (b) Real-time RT-PCR and western blot analysis of the osteoblast marker gene (Osterix, normalized to β-actin) on day 7. Data represent the means±S.D. *P<0.05 (n=3). (c) Cytoplasmic p-GSK-3β, β-catenin, p-IκBα and p65 levels and nuclear β-catenin and p65 levels were tested after 7 days of culture in osteogenic medium by western blot analysis. β-Actin and HDAC1were used as the internal control

Figure 4

Inhibition of NF-κB restored osteogenesis of P-PDLSCs. H-PDLSCs and P-PDLSCs were treated with BAY 11-7082 (H-PDLSCs as a control), and then were cultured with osteoblastic differentiation medium for additional 7 days. (a) Osteoblastic differentiation was determined by ALP staining and activity at day 7. (b,c) The PDLSCs were treated with another inhibitor PDTC, and the expression of the osteoblast-related gene Osterix was measured by Real-time RT-PCR and western blot analysis at day 3. The expression levels were normalized to β-actin. Data were shown as means±S.D. *P<0.05, n=3

Figure 5

Inhibition of NF-κB did not affect the activity of β-catenin but increased the β-catenin expression in P-PDLSCs. To further confirm the relationship between NF-κB and β-catenin signaling in osteogenic differentiation, H-PDLSCs and P-PDLSCs had been treated with BAY 11-7082 or with DMSO (as control). (a) The expression of NF-κB (p65) and β-catenin was examined by real-time RT-PCR (n=3). *P<0.05. (b) The expression of pNF-κB p-p65, NF-κB (p65), active-β-catenin, β-catenin and actin was examined by western blot analysis

Figure 6

Schematic of GSK-3β-mediated NF-κB signaling suppression of osteoblastic differentiation governed by inflammation. In P-PDLSCs, NF-κB can be activated directly, but it can also be activated through inhibition of PI3K, which blocks the phosphorylation of GSK-3β, thereby leading to an increase of p65 and decrease of β-catenin in the nucleus. BAY 11-7082, an inhibitor of NF-κB, can promote osteogenesis of PDLSCs by interfering with p65 nuclear translocation