title: Wnt/β-catenin pathway regulates bone morphogenetic protein (BMP2)-mediated differentiation of dental follicle cells.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Stem Cells and Regenerative Medicine, University of Washington, School of Medicine, Seattle, WA, USA. kgsilverio@fop.unicamp.br
authors.forename: K G
authors.initials: KG
authors.lastname: Silvério
authors.forename: K C
authors.initials: KC
authors.lastname: Davidson
authors.forename: R G
authors.initials: RG
authors.lastname: James
authors.forename: A M
authors.initials: AM
authors.lastname: Adams
authors.forename: B L
authors.initials: BL
authors.lastname: Foster
authors.forename: F H
authors.initials: FH
authors.lastname: Nociti
authors.forename: M J
authors.initials: MJ
authors.lastname: Somerman
authors.forename: R T
authors.initials: RT
authors.lastname: Moon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D060466:Axin Protein / Q000032:analysis; D055396:Bone Morphogenetic Protein 2 / Q000187:drug effects / Q000502:physiology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D003739:Dental Cementum / Q000187:drug effects / Q000502:physiology; D003795:Dental Sac / Q000166:cytology* / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000032:analysis; D055785:Gene Knockdown Techniques; D051538:Hepatocyte Nuclear Factor 1-alpha; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000032:analysis; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology; D015675:Osteocalcin / Q000032:analysis; D010012:Osteogenesis / Q000502:physiology; D053495:Osteopontin / Q000032:analysis; D000076182:Sp7 Transcription Factor; D051822:T Cell Transcription Factor 1 / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000502:physiology*; D060509:Wnt3A Protein / Q000494:pharmacology; D016335:Zinc Fingers; D051176:beta Catenin / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D060466:Axin Protein; C111729:Axin2 protein, mouse; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C497574:Lef1 protein, mouse; D051821:Lymphoid Enhancer-Binding Factor 1; C493716:Runx2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; C506896:Spp1 protein, mouse; D051822:T Cell Transcription Factor 1; D014157:Transcription Factors; C118544:Wif1 protein, mouse; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D015675:Osteocalcin; D053495:Osteopontin; D000469:Alkaline Phosphatase