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Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/?-catenin signaling pathway.

ABSTRACT: OBJECTIVES:Polydatin (PD), extracted from Polygonum cuspidatum, has shown potential therapeutic applications due to its antiosteoporotic and anti-inflammatory activities. Our previous study suggested that PD promotes the osteogenesis of human bone marrow stromal cells (hBMSCs) via the BMP2-Wnt/?-catenin pathway. The aim of our present study was to further explore the role of PD-mediated regulation of Tafazzin (TAZ), a transcriptional coactivator with a PDZ-binding motif, in osteogenesis. MATERIALS AND METHODS:hBMSCs were isolated and treated with PD at various concentrations. Alizarin red staining and RT-qPCR were performed to identify calcium complex deposition in hBMSCs as well as the expression of specific osteoblast-related markers, respectively, in each group. Next, TAZ-silenced hBMSCs were generated by lentivirus-produced TAZ shRNA. After treatment with PD, the osteogenic abilities of the TAZ-silenced and control hBMSCs were estimated by ALP activity assay, and expression of the TAZ protein was detected by Western blot analysis and immunofluorescence staining. In vitro, an ovariectomized (OVX) mouse model was established and used to evaluate the effect of PD on bone destruction by micro-CT, immunohistochemistry, and ELISA. RESULTS:In vitro, 30??M PD significantly improved the proliferation and calcium deposition of hBMSCs and markedly stimulated the expression of the mRNAs RUNX2, Osteopontin, DLX5, ?-catenin, TAZ, and Osteocalcin (OCN). Osteogenic differentiation induced by PD was blocked by lentivirus-mediated TAZ shRNA. Furthermore, Noggin (a regulator of bone morphogenic protein 2 (BMP2)) and DKK1 (an inhibitor of the Wnt/?-catenin pathway) were found to inhibit the increase in TAZ expression induced by PD. In vivo, PD prevented estrogen deficiency-induced bone loss in the OVX mouse model. CONCLUSION:Taken together, our findings suggest that PD improved the osteogenic differentiation of hBMSCs and maintained the bone matrix in the OVX mouse model through the activation of TAZ, a potential target gene of the BMP2-Wnt/?-catenin pathway.

SUBMITTER: Shen YS

PROVIDER: S-EPMC7251742 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/β-catenin signaling pathway.

Shen Ying-Shan YS Chen Xiao-Jun XJ Wuri Sha-Na SN Yang Fan F Pang Feng-Xiang FX Xu Liang-Liang LL He Wei W Wei Qiu-Shi QS

Stem cell research & therapy 20200527 1

<h4>Objectives</h4>Polydatin (PD), extracted from Polygonum cuspidatum, has shown potential therapeutic applications due to its antiosteoporotic and anti-inflammatory activities. Our previous study suggested that PD promotes the osteogenesis of human bone marrow stromal cells (hBMSCs) via the BMP2-Wnt/β-catenin pathway. The aim of our present study was to further explore the role of PD-mediated regulation of Tafazzin (TAZ), a transcriptional coactivator with a PDZ-binding motif, in osteogenesis. ...[more]

PMID: 32460844

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Project description:Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/?catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/?-catenin pathway.SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/?-catenin pathway activation assessed by western blotting, ?-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of ?-catenin.WNT3A induced ?-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/?-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, ?-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous ?-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/?-catenin signaling.These data suggest that stabilization of ?-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/?-catenin signaling to promote cell maturation.

Project description:BackgroundManagement of fracture healing with a large bone defect remains a tricky subject in orthopedic trauma. Enhancing osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) is one of the useful therapeutic strategies for fracture healing. Previous studies have revealed that Apelin may play an important role in bone metabolism. However, its function in the osteogenesis of hBMSCs remains unclear. Therefore, in this study, we investigated the effects and mechanism of Apelin on osteogenic differentiation.MethodsWe investigated the osteogenesis effects of hBMSCs by both exogenous Apelin protein and overexpression Apelin in vitro. Cell proliferation assay was used to assess the effect of Apelin on the proliferation of hBMSCs. ALP staining and Alizarin Red staining were used to evaluate ALP activity and mineral deposition respectively. qPCR and Western blotting analysis were used to detect the expression of target genes and proteins. In vivo, a rat tibial osteotomy model was established; radiographic analysis and histological evaluation were used to confirm the therapeutic effects of Apelin in fracture healing. Statistical significance was determined by two-tailed Student's t test when 2 groups were compared. When more than 2 groups were compared, one-way ANOVA followed by Bonferroni's post-hoc test was used. And two-way ANOVA, followed by Bonferroni multiple comparisons post-hoc test, was performed when the treatment groups at different time points were compared.ResultsThe addition of exogenous Apelin protein or overexpression of Apelin promoted osteoblast differentiation of hBMSCs in vitro. Increased mineral deposits were observed after treatment with extracellular Apelin protein or after the upregulation of Apelin. Moreover, β-catenin levels were upregulated by Apelin. The enhancement of osteogenic differentiation induced by Apelin was attenuated by specific Wnt/β-catenin signaling pathway inhibitors. In a rat tibial osteotomy model, local injection of exogenous Apelin protein improved bone healing, as demonstrated by imaging and histological analyses.ConclusionsTaken together, these findings indicate that Apelin regulates osteogenic differentiation of hMSCs partly via the Wnt/β-catenin signaling pathway and effectively promotes fracture healing.

Dataset Information

Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/?-catenin signaling pathway.

Publications

Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/β-catenin signaling pathway.

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