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.

本研究旨在探讨TGF-β激活的Smad1/5在牙上皮中的分子机制和生物学功能。采用免疫组化法检测小鼠磨牙胚中TGF-β信号相关基因的表达。培养原代牙齿上皮细胞并用浓度为 0.5 或 5 ng/mL 的 TGF-β1 处理。小分子抑制剂 SB431542 和 ML347 分别用于抑制 ALK5 和 ALK1/2。使用小干扰 RNA 来敲除 Smad1/5 或 Smad2/3。通过EdU测定评估细胞的增殖率。在牙上皮芽的基底层，TGF-β1和p-Smad1/5高表达，在上皮芽内部，TGF-β1低表达，而p-Smad2/3高表达。在原代培养的牙上皮细胞中，低浓度的 TGF-β1 激活 Smad2/3 但不激活 Smad1/5，而高浓度的 TGF-β1 能够激活 Smad2/3 和 Smad1/5。SB431542 而不是 ML347 能够阻断 TGF-β1 对 Smad2/3 的磷酸化。SB431542 或 ML347 都能够阻断 TGF-β1 对 Smad1/5 的磷酸化。EdU染色显示高浓度TGF-β1促进牙上皮细胞增殖，通过沉默Smad1/5逆转，而低浓度TGF-β1抑制细胞增殖，通过沉默Smad2/3逆转。总之，TGF-β通过两种途径在调节牙齿上皮细胞增殖中发挥双重作用。一方面，TGF-β 通过 ALK5 激活经典的 Smad2/3 信号，抑制内部牙上皮细胞的增殖。另一方面，