Department of Spinal Surgery, Orthopaedic Medical Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, P.R. China.
Department of Orthopedics, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550001, P.R. China.
Int J Mol Med. 2021 Jun;47(6). doi: 10.3892/ijmm.2021.4927. Epub 2021 Apr 13.
Mesenchymal stem cells (MSCs) have the ability of differentiating into osteoblasts. Elucidating the molecular mechanisms of MSC differentiation into osteoblasts may provide novel therapeutic strategies for bone‑related diseases. Increasing evidence has confirmed that Wnt signaling plays the key role in osteoblast differentiation; however, the role of individual Wnt proteins in osteogenesis needs to be investigated. The present study thus aimed to explore the role of Wnt7a in bone formation. For this purpose, human bone‑derived MSCs were identified by flow cytometry and the cell differentiation potential, including osteogenic and adipogenic differentiation was examined. In order to explore the role of Wnt7a in MSC osteogenic differentiation, Wnt7a expression was measured at the mRNA and protein level following treatment with the osteogenic inducer, bone morphogenetic protein (BMP)4/7, and following the induction of osteogenic or adipogenic differentiation. The ectopic expression of Wnt7a in MSCs was confirmed and its influence on MSC osteogenic differentiation was detected using osteocyte markers and by Alizarin Red S staining. Mechanistically, the influence of Wnt7a on Runt‑related transcription factor 2 (RUNX2) expression was examined at the mRNA and protein level. The regulatory effects of Wnt7a on RUNX2 promoter activities were examined by promoter reporter assay, and by examining the binding of TCF1, a downstream target of Wnt, to the RUNX2 promoter by ChIP assay. The results revealed that the knockdown of Wnt7a in MSCs decreased the expression of osteocyte markers and inhibited osteogenic differentiation. In accordance, the overexpression of Wnt7a in MSCs increased the expression of osteocyte markers and promoted osteogenic differentiation. Mechanistically, the knockdown of Wnt7a in MSCs reduced RUNX2 expression and the overexpression of Wnt7a in MSCs promoted RUNX2 expression. Furthermore, it was confirmed that Wnt7a regulated RUNX2 promoter activities by promoter report assay, and by examining the binding of TCF1 to the RUNX2 promoter by ChIP assay. On the whole, the present study demonstrates that Wnt7a plays a key role in MSC differentiation into osteoblasts and the findings presented herein may provide a promising therapy target for bone‑related diseases.
间充质干细胞（MSCs）具有分化为成骨细胞的能力。阐明 MSC 分化为成骨细胞的分子机制可能为骨相关疾病提供新的治疗策略。越来越多的证据证实 Wnt 信号通路在成骨分化中发挥关键作用；然而，需要研究单个 Wnt 蛋白在成骨中的作用。本研究旨在探讨 Wnt7a 在骨形成中的作用。为此，通过流式细胞术鉴定人源性骨髓间充质干细胞，并检测其细胞分化潜能，包括成骨和成脂分化。为了探讨 Wnt7a 在 MSC 成骨分化中的作用，在使用成骨诱导剂骨形态发生蛋白 4/7 以及诱导成骨或成脂分化后，测量 Wnt7a 在 mRNA 和蛋白水平的表达。通过碱性磷酸酶染色和茜素红 S 染色检测 Wnt7a 在 MSC 中的异位表达及其对 MSC 成骨分化的影响。通过检测 Runt 相关转录因子 2（RUNX2）的表达，探讨 Wnt7a 对 RUNX2 表达的影响。通过启动子报告基因检测、ChIP 检测 Wnt 下游靶点 TCF1 与 RUNX2 启动子的结合，探讨 Wnt7a 对 RUNX2 启动子活性的调控作用。结果表明，MSC 中 Wnt7a 的敲低降低了成骨细胞标志物的表达并抑制了成骨分化。相反，MSC 中 Wnt7a 的过表达增加了成骨细胞标志物的表达并促进了成骨分化。机制上，MSC 中 Wnt7a 的敲低降低了 RUNX2 的表达，而 MSC 中 Wnt7a 的过表达促进了 RUNX2 的表达。此外，通过启动子报告基因检测和 ChIP 检测 TCF1 与 RUNX2 启动子的结合，证实 Wnt7a 通过调节 RUNX2 启动子活性来调控 RUNX2 的表达。综上所述，本研究表明 Wnt7a 在 MSC 向成骨细胞分化中起关键作用，本研究结果可为骨相关疾病提供有前景的治疗靶点。
