Department of Orthodontics and Craniofacial Biology, Radboud University Medical Center, Philips van Leydenlaan 25, Nijmegen, the Netherlands.
Department of Oral Health Sciences, University KU Leuven. Herestraat 49, Leuven, Belgium.
Int J Biochem Cell Biol. 2019 Nov;116:105597. doi: 10.1016/j.biocel.2019.105597. Epub 2019 Aug 31.
The skull bones are formed by osteoblasts by intramembranous ossification. WNT signaling is a regulator of bone formation. Retinoic Acid (RA) act as a teratogen affecting craniofacial development. We evaluated the effects of RA on the differentiation and mineralization of MC-3T3 cells, and on the expression of WNT components. MC-3T3 were cultured with or without 0.5 μM RA in osteogenic medium and mineralization was assessed by alizarin red staining. The expression of osteogenic marker genes and WNT genes was evaluated at several time points up to 28 days. RA significantly inhibited MC-3T3 mineralization (p < 0.01), without affecting ALP activity or Alp gene expression. Both parameters gradually increased in time. During culture, RA stimulated Runx2 expression at 14 and 28 days compared to the respective controls (p < 0.05). Also, RA significantly reduced Sp7 expression at days 14 and 21 (p < 0.05). Simultaneously, RA significantly reduced the expression of the WNT genes cMyc, Lef1, Lrp5, Lrp6 and Wnt11 compared to the controls (p < 0.05). In contrast, RA increased the expression of the WNT inhibitors Dkk1 at day 21 and Dkk2 at days 14 and 21 (p < 0.01). Our data indicate that RA disrupts osteogenic differentiation and mineralization by inhibiting WNT signaling.
颅骨由成骨细胞通过膜内成骨形成。WNT 信号是骨形成的调节剂。维甲酸（RA）作为一种致畸剂影响颅面发育。我们评估了 RA 对 MC-3T3 细胞分化和矿化的影响，以及对 WNT 成分表达的影响。MC-3T3 在成骨培养基中培养，或在成骨培养基中添加或不添加 0.5μM RA，并通过茜素红染色评估矿化情况。在多达 28 天的时间内评估成骨标记基因和 WNT 基因的表达。RA 显著抑制 MC-3T3 的矿化（p<0.01），而不影响 ALP 活性或 Alp 基因表达。两个参数随时间逐渐增加。在培养过程中，RA 在第 14 天和第 28 天与相应的对照相比，刺激 Runx2 的表达（p<0.05）。同时，RA 在第 14 天和第 21 天显著降低 Sp7 的表达（p<0.05）。同时，RA 与对照组相比，显著降低了 WNT 基因 cMyc、Lef1、Lrp5、Lrp6 和 Wnt11 的表达（p<0.05）。相反，RA 在第 21 天增加了 WNT 抑制剂 Dkk1 的表达，在第 14 天和第 21 天增加了 Dkk2 的表达（p<0.01）。我们的数据表明，RA 通过抑制 WNT 信号干扰成骨分化和矿化。
