Excessive retinoic acid inhibit mouse embryonic palate mesenchymal cell growth through involvement of Smad signaling

Huanhuan Zhang,Xiaozhuan Liu,Zhan Gao,Zhitao Li,Zengli Yu,Jun Yin,Yuchang Tao,Lingling Cui

doi:10.1080/19768354.2016.1165287

Abstract

ABSTRACTAll-trans retinoic acid (atRA), the oxidative metabolite of retinoic acid (RA), is essential for palatogenesis. Overdose RA is capable of inducing cleft palate in mice and humans. Normal embryonic palatal mesenchymal (EPM) cell growth is crucial for shelf growth. Smad signaling is involved in many biological processes. However, it is not much clear if atRA could affect Smad signaling during EPM cells growth. In this study, the timed pregnant mice with maternal administration of 100 mg/kg body weight of RA by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by hematoxylin and eosin (H&E) staining. At the same time, a primary mouse EPM (MEPM) cell culture model was also established. MEPM cells were treated with atRA (0.1, 0.5, 1, 5 and 10 μM) for 24, 48 and 72 h. The results indicated that the sizes of the shelves were smaller than those in control. AtRA inhibited MEPM cell growth with both increasing concentration and increasing incubation time, especially at 72 h in vitro. Moreover, atRA significantly increased the mRNA and protein expression levels of Smad7 (P < .05), but the mRNA and protein expression levels of PCNA were reduced (P < .05). We also found atRA inhibited phosphorylation of Smad2 compared with untreated group (P < .05). However, the protein and mRNA levels of Smad2 did not change both in atRA-treated and untreated group (P > .05). We demonstrated that RA induced inhibition of MEPM cell growth that could cause cleft palate partly by down-regulation of Smad pathway.

Highlights

In mammals, the palatal shelf is an important structure and plays an important role in the essential functions of breathing and feeding
In order to investigate the effect of All-trans retinoic acid (atRA) on the growth of mouse embryonic palatal mesenchymal (MEPM) cells, cells were exposed to the vehicle or indicated concentrations (0.1, 0.5, 1, 5 and 10 μM) of atRA for 24, 48 and 72 h, and the quantification of MEPM cell growth and viability were tested by MTT assay
At the time of palatal shelf outgrowth, overdose of Retinoic acid (RA) resulted in growth retardation of bilateral palate shelves in mice (Yoshikawa et al 1987)

Summary

Introduction

The palatal shelf is an important structure and plays an important role in the essential functions of breathing and feeding. The secondary palate develops from bilateral outgrowth of the maxillary processes, it increases in size mainly depends on condensation of mesenchymal cells which results in palatal growth, elevation and fusion. Previous studies demonstrated that atRA could induce mouse embryonic palatal mesenchymal (MEPM) cell cycle arrest (Yu et al 2005). Both atRA and Smad protein are implicated in a wide range of cellular functions in craniofacial development (Opperman et al 1997; Hu et al 2013). TGF-β/Smad pathway played a crucial role in the regulation of cell proliferation and growth (Zhu et al 2012; Parada et al 2013).

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Conclusion