Associations between the proliferation of palatal mesenchymal cells, Tgfβ2 promoter methylation, Meg3 expression, and Smad signaling in atRA-induced cleft palate

All-trans retinoic acid (atRA) is a teratogen that can induce cleft palate formation. During palatal development, murine embryonic palate mesenchymal (MEPM) cell proliferation is required for the appropriate development of the palatal frame, with Meg3 serving as a key regulator of the proliferative activity of these cells and the associated epithelial-mesenchymal transition process. DNA methylation and signaling via the TGFβ/Smad pathway are key in regulating embryonic development. Here, the impact of atRA on MEPM cell proliferation and associations between Tgfβ2 promoter methylation, Meg3, and signaling via the Smad pathway were explored using C57BL/6N mice treated with atRA (100mg/kg) to induce fetal cleft palate formation. Immunohistochemistry and BrdU assays were used to detect MEPM proliferation and DNA methylation assays were performed to detect Tgfβ2 promoter expression. These analyses revealed that atRA suppressed MEPM cell proliferation, promoted the upregulation of Meg3, and reduced the levels of Smad2 and Tgfβ2 expression phosphorylation, whereas Tgfβ2 promoter methylation was unaffected. RNA immunoprecipitation experiments indicated that the TgfβI receptor is directly targeted by Meg3, suggesting that the ability of atRA to induce cleft palate may be mediated through the Tgfβ/Smad signaling pathway.