Excess vitamin a might contribute to submucous clefting by inhibiting WNT-mediated bone formation

Objectives Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). Setting and Sample Population MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. Materials and Methods MEPM cells were cultured with and without 0.5 mu M ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. Results The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% +/- 2%, 59% +/- 7%, 79% +/- 12% and 57% +/- 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 +/- 0% (P < .05) and 23 +/- 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 +/- 18% (P < .001), at day 7. Conclusion Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.