Wnt/&beta-catenin inhibits cementoblast differentiation, in vitro

Objectives: Canonical Wnt signaling has been demonstrated to be involved in regulating bone formation by controlling osteoblast function. However, the role of Wnt pathway during the process of cementoblast differentiation is largely unknown. We investigated the effects of Wnt signaling on gene expression of extracellular matrix in cementoblasts in vitro. Methods: Lithium chloride (LiCl) or supernatants from Wnt3a-expressing L cell (ATCC) were used as stimulators for Wnt/canonical signaling. Mouse cementoblasts (OCCM-30), cultured in DMEM with 5% FBS plus ascorbic acid were stimulated with various concentrations of stimulants over a 4-day period and gene expressions were analyzed using real-time quantitative RT-PCR. To assess nuclear translocation of β-catenin, localization was analyzed by immunocytochemistry using anti-β-catenin antibody. A specific transcriptional activity was measured using the T-cell factor (TCF)/β-catenin-responsive firefly luciferase reporter plasmid pTopflash. Results: Gene expression of Wnt signaling components, β-catenin, adenomatous polyposis coli, glycogen synthase kinase-3β, TCF1-4, and low-density lipoprotein receptor-related protein-5 and -6 were detected by conventional RT-PCR (agarose gel). Data from histocytochemical analyses of untreated cells showed staining of β-catenin localized to the intercellular junctions. On the other hand, LiCl (10 mM) or Wnt3a (5% (v/v)) treatment for 24 hrs was associated with nuclear β-catenin translocation. LiCl (0-20 mM) exposure for 24 hrs resulted in a dose dependent increase in luciferase activity in cells transfected with pTopflash. LiCl (0-20 mM), as well as Wnt3a (0-10% (v/v)), inhibited expression of genes associated with cementoblast maturation, alkaline phosphatase, osteocalcin, and bone sialoprotein in dose and time dependent manners. Almost complete inhibition was observed in 20 mM LiCl or 10% (v/v) Wnt3a. Conclusion: These results suggest canonical Wnt signaling functionally operates in cementoblasts and negatively regulates cementoblast differentiation. Supported by the Grant-in-aid from JSPS (No. 19592380).