Involvement of Notch2 in all-trans retinoic acid-induced inhibition of mouse embryonic palate mesenchymal cell proliferation

Cleft palate is among the most common congenital disorders, and can be induced by exposure to all-trans retinoic acid (atRA) during mice and human embryogenesis. However, the mechanism underlying the implication of atRA in the development of cleft palate has yet to be elucidated. In the present study, atRA administered by gavage resulted in formation of a cleft palate in 99% of treated C57BL/6 mice. Notch2 was revealed to be upregulated in mouse embryonic palate mesenchymal (MEPM) cells in the atRA-treated group compared with untreated control mice between embryonic day (E)12.5 and E14.5. In addition, atRA was demonstrated to mediate Notch2 expression via the activation of RA receptors (RARs). Since Notch2 activation has previously been reported to inhibit the proliferation of MEPM cells, the expression levels of extracellular signal-regulated kinase (ERK), p21, cyclin D1 and Ki-67 were assessed in samples from atRA-treated and control mouse embryos between E12.5 and E14.5. It was demonstrated that Notch2 silencing partially reversed the atRA-induced inhibition of ERK phosphorylation in MEPM cells. In addition, the atRA-induced cyclin D1 downregulation and p21 upregulation were partially reversed following Notch2 silencing, whereas the atRA-induced inhibition of cellular proliferation was also attenuated. Furthermore, it was revealed that Notch2 expression was upregulated, whereas Ki-67 expression was downregulated following atRA exposure, as assessed using resin bead-released atRA in MEPM cells. The present findings suggested that during embryonic development, atRA may enhance the expression of Notch2, which may inhibit cellular proliferation, possibly through ERK signaling.