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.
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.
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