Erythropoietin enhances osteogenic differentiation of human periodontal ligament stem cells via Wnt/β-catenin signaling pathway

De-Hua Zheng, Xu-Xia Wang, Dan Ma, Li-Na Zhang, Qing-Fang Qiao, Jun Zhang, De-Hua Zheng, Xu-Xia Wang, Dan Ma, Li-Na Zhang, Qing-Fang Qiao, Jun Zhang

Abstract

Objectives: The aim of this study is to examine the roles of erythropoietin (EPO) in regulating proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and analyze the underlying signaling of these processes.

Materials and methods: PDLSCs were isolated and characterized. The PDLSCs were transfected with β-catenin shRNA. qRT-PCR and Western blot analysis were used to examine the osteogenic effects of EPO on the expression of osteogenic-related genes and protein (Runx2, OCN and Osterix) in PDLSCs. Alizarin Red-S staining was used to detect mineralized nodule formation. In addition, the relationship between the Wnt/β-catenin pathway and the effect of EPO on the osteogenesis of PDLSCs was investigated.

Results: The results suggested that EPO exerts positive osteogenic effects on PDLSCs. The results showed that EPO decreased the growth of PDLSCs slightly and increased alkaline phosphatase activity and calcium deposition in a dose-dependent manner. The expression of Runx2, Osterix and OCN was increased after EPO administration. EPO increases β-catenin and Cyclin D1 in PDLSCs. After transfected with β-catenin shRNA, the osteogenic effect of EPO on PDLSCs was attenuated.

Conclusion: EPO promotes osteogenic differentiation of PDLSCs. The underlying mechanism may be activating Wnt/β-catenin signaling pathway.

Keywords: Wnt/β-catenin; erythropoietin; osteogenesis; periodontal ligament stem cell.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characterization of Peridontal ligament stem cells (PDLSCs) by flow cytometric analysis. Flow cytometric analysis of PDLSCs reve aled expression of CD34 (A, B), CD90 (C, D) and CD105 (E, F).
Figure 2
Figure 2
Morphological observation of Peridontal ligament stem cells (PDLSCs) and multilineage differentiation assay. (A) PDLSCs exhibited thin and long fibroblastic spindle morphology. (B) PDLSCs were under adipogenic differentiation induction and stained with Oil Red O. (C) PDLSCs were under osteogenic differentiation induction and stained with Alizarin Red. Scale bars: 200 μm.
Figure 3
Figure 3
Effect of Erythropoietin (EPO) on the proliferation of Periodontal ligament stem cells (PDLSCs). Note:*P<0.05.
Figure 4
Figure 4
The ALP activity of Peridontal ligament stem cells (PDLSCs) under different concentration of Erythropoietin (EPO) treatment. (*P<0.05, compared with 0 U/mL EPO group. #P<0.05, compared with 50 U/mL EPO group).
Figure 5
Figure 5
Effect of Erythropoietin (EPO) on osteogenic genes expression of Periodontal ligament stem cells (PDLSCs) under different conditions. (A) The mRNA expression of Osterix after 7 and 14 day(s). (B) The mRNA expression of Runx2 after 7 and 14 days. (C). The mRNA expression of OCN after 7 and 14 day(s). (*P<0.05, compared with control group. #P<0.05, compared with EPO+β-catenin shRNA+group).
Figure 6
Figure 6
Effect of Erythropoietin (EPO) on protein expression of Osterix, Runx2 and OCN in Periodontal ligament stem cells (PDLSCs) under different treatment (Osteogenic induction group, EPO group, EPO+NCshRNA group and EPO+β-catenin shRNA group).
Figure 7
Figure 7
(A) ARS staining of mineralized nodule formation. (B) Amount of calcium deposition on day 21, quantified by absorbance at 562nm. Periodontal ligament stem cells (PDLSCs) were treated with osteogenic induction (OI), Erythropoiethin (EPO)+OI, EPO+OI+NCshRNA and EPO+OI+β-catenin shRNA. Scale bars: 200 µm.
Figure 8
Figure 8
Effect of EPO on Wnt/β-catenin pathway. (A) ARS staining of mineralized nodule formation. Scale bars: 200 µm. (B) Amount of calcium deposition on day 21, quantified by absorbance at 562 nm. Note: *P<0.05. Abbreviation: OI, osteogenic induction group.
Figure 9
Figure 9
EPO can upregulate the expression of β-catenin and CyclinD1 in the process of osteogenesis in a concentration-dependent manner.
Figure 10
Figure 10
The protein level of β-catenin and CyclinD1 was assessed by Western blot analysis. (Osteogenic induction group, EPO group, EPO+NCshRNA group and EPO+β-catenin shRNA group).
Figure 11
Figure 11
β-catenin was detected by immunostaining. Nuclei were stained with DAPI (blue staining). (Osteogenic induction group, EPO group, EPO+NCshRNA group and EPO+β-catenin shRNA group). Scale bars: 200 µm.

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