Periostin promotes migration and osteogenic differentiation of human periodontal ligament mesenchymal stem cells via the Jun amino-terminal kinases (JNK) pathway under inflammatory conditions

Abstract

Objectives: Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism.

Materials and methods: PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways.

Results: After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs.

Conclusions: Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration.

Figure 1

The knockdown of POSTN attenuated the migration in PDLSCs after TNF‐α treatment. (A, B) Real‐time RT‐PCR (A) and Western blot (B) results revealed that 10 ng/mL TNF‐α decreased the expression of POSTN. (C) Quantitative analysis of POSTN based on Western blot results for PDLSCs. (D‐F) PDLSCs were infected with POSTN shRNA, Western blot (D), quantitative analysis results (E) and real‐time RT‐PCR (F) results showed the decreased POSTN expression after depletion of POSTN. (G, H) PDLSCs were treated with 10 ng/mL TNF‐α, Scratch migration assay (G) and quantitative analysis (H) results revealed that the POSTNsh group showed a significantly decreased rate of wound closure compared with the CONTsh group. GAPDH was used as an internal control. The Student's t test was performed to determine statistical significance. The error bars represent the SD (n = 3). *P ≤ .05. **P ≤ .01. Bar: 200 μm

Figure 2

POSTN knockdown inhibited the osteogenic differentiation in PDLSCs after TNF‐α treatment. PDLSCs were treated with 10 ng/mL TNF‐α. (A) Alkaline phosphatase (ALP) activity. (B) Alizarin Red staining. (C) Calcium quantitative analysis. (D‐G) Real‐time RT‐PCR results showed the expressions of RUNX2 (D), OSX (E), OCN (F) and OPN (G). GAPDH was used as an internal control. (H) Western blot results and (I) quantitative analysis showed the expressions of RUNX2, OSX, OCN and OPN. β‐Actin was used as an internal control. Student's t test was performed to determine statistical significance. The error bars represent the SD (n = 3). *P ≤ .05. **P ≤ .01

Figure 3

POSTN promoted the osteogenic differentiation and migration in PDLSCs after TNF‐α treatment. PDLSCs were treated with 10 ng/mL TNF‐α. (A) The results of the ALP activity by treated with different concentrations of rhPOSTN. (B) Alizarin Red staining. (C) Calcium quantitative analysis. (D‐G) Real‐time RT‐PCR results showed the expressions of RUNX2 (D), OSX (E), OCN (F) and OPN (G). GAPDH was used as an internal control. (H) Western blot results and (I) quantitative analysis showed the expressions of RUNX2, OSX, OCN and OPN. β‐Actin was used as an internal control. (J, K) Scratch migration assay (J) and quantitative analysis (K) results showed that 75 ng/mL rhPOSTN displayed a statistically significant enhancement in cell migration. The Student's t test or one‐way ANOVA was performed to determine statistical significance. The error bars represent the SD (n = 3). *P ≤ .05. **P ≤ .01. Bar: 200 μm

Figure 4

POSTN activated Jun amino‐terminal kinases (JNK) signalling pathway in PDLSCs after TNF‐α treatment. (A) Western blot results showed that knockdown of POSTN inhibited phosphorylation of JNK at 2 h after treatment with 10 ng/mL TNF‐α in PDLSCs. (B) PDLSCs were pre‐treated with 75 ng/mL rhPOSTN for 30 min, and then treated with 10 ng/mL TNF‐α. Western blot results showed that 75 ng/mL rhPOSTN increased phosphorylation of JNK at 1 h after treated with 10 ng/mL TNF‐α. β‐Actin was used as an internal control. (C, D) Quantitative analysis of p‐JNK based on Western blot results for PDLSCs. The Student's t test was performed to determine statistical significance. The error bars represent the SD (n = 3). *P ≤ .05. **P ≤ .01

Figure 5

JNK inhibition repressed the POSTN‐enhanced migration and osteogenic differentiation of PDLSCs after TNF‐α treatment. (A) PDLSCs were treated with the JNK inhibitor, 20 μmol/L SP600125 for 48 h, and then treated with 75 ng/mL rhPOSTN for 30 min. Western blot showed reduction in the phospho‐JNK in POSTN‐stimulated PDLSCs. β‐Actin was used as an internal control. (B) Quantitative analysis of p‐JNK based on Western blot results for PDLSCs. (C, D) PDLSCs were pre‐treated with 20 μmol/L SP600125 for 48 h, and then treated with 75 ng/mL rhPOSTN for 30 min, and then PDLSCs were cultured in routine medium with 20 μmol/L SP600125, 75 ng/mL rhPOSTN and 10 ng/mL TNF‐α. The scratch migration assay (C) and quantitative analysis (D) results revealed that SP600125 significantly restrained POSTN‐enhanced scratch closure. (E‐G) PDLSCs were pre‐treated with 20 μmol/L SP600125 for 48 h, next treated with 75 ng/mL rhPOSTN for 30 min, then PDLSCs were cultured in osteogenic‐inducing medium with 20 μmol/L SP600125, 75 ng/mL rhPOSTN and 10 ng/mL TNF‐α. ALP activity (E), Alizarin Red staining (F) and quantitative analysis (G). The one‐way ANOVA was performed to determine statistical significance. The error bars represent the SD (n = 3). *P ≤ .05. **P ≤ .01. Bar: 200 μm