Dual roles of TGF-β signaling in the regulation of dental epithelial cell proliferation.

The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-β-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-β signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-β1 at a concentration of 0.5 or 5 ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-β1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-β1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-β1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-β1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-β1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-β1. EdU staining showed that high concentration of TGF-β1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-β1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-β exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-β activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-β activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.