title: Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells.
abstract: We examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. It has been shown that Wnt signaling in mature osteoblasts needs to be downregulated to enable the formation of a mineralized matrix. Using RNA interference, we showed that this is, at least in part, accomplished by upregulation of the Wnt antagonists Dickkopf-1 and -2.
  
  
  BACKGROUND
  The role of Wnt signaling in the initiation of osteoblast differentiation has been well studied. However, the role during late-stage differentiation is less clear. We have examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation.
  
  
  METHODS
  We treated murine bone marrow and mesenchymal stem cell-like KS483 cells with either LiCl or Wnt3A during several stages of osteoblast differentiation. In addition, we generated stable KS483 cell lines silencing either the Wnt antagonist Dkk-1 or -2
  
  
  RESULTS
  Activation of Wnt signaling by LiCl inhibits the formation of a mineralized bone matrix in both cell types. Whereas undifferentiated KS483 cells respond to Wnt3A by inducing nuclear beta-catenin translocation, differentiated cells do not. This is at least in part accomplished by upregulated expression of Dkk-1 and -2 during osteoblast differentiation. Using RNA interference, we showed that Dkk-1 plays a crucial role in blunting the BMP-induced alkaline phosphatase (ALP) response and in the transition of an ALP+ osteoblast in a mineralizing cell. In contrast, Dkk-2 plays a role in osteoblast proliferation and the initiation of osteoblast differentiation.
  
  
  CONCLUSIONS
  Our data suggest that Wnt signaling in maturing osteoblasts needs to be downregulated to enable the formation of a mineralized bone matrix. Furthermore, they suggest that Dkk-1 and Dkk-2 may have distinct functions in osteoblast differentiation.
authors.affiliation: Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Geertje
authors.initials: G
authors.lastname: van der Horst
authors.forename: Steffie M
authors.initials: SM
authors.lastname: van der Werf
authors.forename: Hetty
authors.initials: H
authors.lastname: Farih-Sips
authors.forename: Rutger L
authors.initials: RL
authors.lastname: van Bezooijen
authors.forename: Clemens W G M
authors.initials: CW
authors.lastname: Löwik
authors.forename: Marcel
authors.initials: M
authors.lastname: Karperien
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D018692:Antimanic Agents / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D018021:Lithium Chloride / Q000494:pharmacology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects*; D051153:Wnt Proteins / Q000378:metabolism / Q000494:pharmacology*; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000378:metabolism
chemical_list: D018692:Antimanic Agents; C501127:Dkk1 protein, mouse; C405060:Dkk2 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D018021:Lithium Chloride