Institute for Stem Cells and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, USA.
J Periodontal Res. 2012 Jun;47(3):309-19. doi: 10.1111/j.1600-0765.2011.01433.x. Epub 2011 Dec 11.
BACKGROUND AND OBJECTIVE: Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/βcatenin 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/β-catenin pathway. MATERIAL AND METHODS: SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/β-catenin pathway activation assessed by western blotting, β-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 β-catenin. RESULTS: WNT3A induced β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-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/β-catenin signaling. CONCLUSION: These data suggest that stabilization of β-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.
背景与目的:骨形态发生蛋白 2(BMP2)诱导的成骨分化是通过经典 Wnt/β-catenin 途径发生的,而在成牙骨质细胞中促进经典 Wnt 信号的因素抑制细胞分化并促进体外细胞增殖。本研究旨在探讨牙囊细胞(鼠 SVF4 细胞)在受到 BMP2 刺激时,其与 Wnt/β-catenin 通路相关的基因/蛋白是否会发生变化。
材料与方法:用 BMP2 刺激 SVF4 细胞,并进行以下检测:(i)通过 Western blot、β-catenin/转录因子(TCF)报告基因检测和淋巴增强结合因子 1(Lef1)、转录因子 7(Tcf7)、Wnt 抑制剂因子 1(Wif1)和轴蛋白 2(Axin2)基因的表达来评估 Wnt/β-catenin 通路的激活;(ii)通过体外矿化和定量 PCR 检测 runt 相关转录因子 2(Runx2)、骨形成蛋白 4(Osx)、碱性磷酸酶(Alp)、骨钙素(Ocn)和骨涎蛋白(Bsp)的 mRNA 水平,来评估成牙骨质细胞/成骨细胞的分化,在 WNT3A 处理和β-catenin 敲低后进行。
结果:WNT3A 诱导β-catenin 核易位并上调经典 Wnt 反应报告基因的转录活性,提示 Wnt/β-catenin 通路在 SVF4 细胞中发挥作用。用 WNT3A 激活 Wnt 信号会抑制 BMP2 诱导的 SVF4 细胞成牙骨质细胞/成骨细胞成熟。然而,β-catenin 敲低表明,BMP2 诱导的成牙骨质细胞/成骨细胞分化标志物的表达需要内源性β-catenin。与未处理的细胞相比,WNT3A 下调了 SVF4 细胞中 Runx2、Alp 和 Ocn 的转录本。相比之下,BMP2 诱导 Bsp 转录本的表达不依赖于 Wnt/β-catenin 信号。
结论:这些数据表明,WNT3A 通过稳定β-catenin 抑制 SVF4 细胞中 BMP2 介导的成牙骨质细胞/成骨细胞分化,尽管 BMP2 需要内源性 Wnt/β-catenin 信号来促进细胞成熟。
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