The Effects Of Excessive Retinoic Acid On Chondrogenesis During The Induction Of Cleft Palate In Mice

Background and objectives:Celft palate is a common congenital birth defects, and the underlying mechanism is unclear. The abnormal form and apoptosis of cartilage are the important factors for cleft palate during the formation of palate. all-trans Retinoic acid (atRA), an oxidative metabolite of vitamin A, is known a teratogenic effector of cleft palate. In high-density cultures of craniofacial and limb bud mesenchyme, nonphysiological levels of atRA inhibit chondrogenesis, suggesting that similar effects on cartilage differentiation in vivo may be responsible for abnormalities in the developing palate and limb. Indeed, atRA has been shown to inhibit chondrogenic differentiation of embryonic mesenchymal cells and to cause loss of differentiated chondrocyte phenotype.TGFβplays a pivotal role in regulating the proliferation and differentiation of chondrocyte precursors. The cellular effects of TGFP are mediated by the classical Smads pathway. Briefly, TGFβbinds to the TGFβreceptor typeⅡ(TβRⅡ) and then activates TGFβreceptor typeⅠ(TβRⅠ), which subsequently activate the R-Smad (Smad2 and Smad3). The phosphorylated R-Smad will bind and activate co-Smad, Smad4, and translocates into the nucleus to regulate the target genes.However, the molecular mechanisms of interaction between TGFβand RA signaling remain unknown. The present study evaluated the effects of atRA on cell differentiation of mouse embryonic palate mesenchymal (MEPM) cells.Materials and Mathods:This study includes two sessions, in vivo and in vitro experiments. In vivo experiments, pregnant mice were intubated orally with either corn oil (vehicl control) or atRA (80mg/kg body weight) on GD11. The mice were killed on GD18 and fetal pups were checked for overall development. In vitro experiments, the effects of atRA were evaluating when mouse embryonic palate mesenchymal (MEPM) cells differentiated into osteo-chondrogenic through high-density micromass environment cell culture. The levels of cartilage transformation were detected by quantitative alcian blue staining. Protein levels of TGFβ1 and Smad7 and phosphorylation levels of Smad2 and Smad3 were determined by western blot analysis. All statistical analyses were performed using the Statistical product and service solutions 12.0(SPSS12.0). Dunnet-t test has been used to analyses the differentis between experimental groups and the control group. Statistical differences of the catagorical variable were evaluated by Chi-square test. The statistical significance level was 0.05.Results:By comparing with the vehicle control mice, the size of atRA-treated mice were smaller, bulging head, their eyes, ears and nose were not fully developed, limbs were short and could not extended normally, cleft palate the whole body vascular developed imperfectly. MMCs exposed to high concentrations of atRA (1μM and 5μM) exhibited a decrease in the production of sulfated glycosaminoglycans, inhibited maturation of cartilage matrix, whereas lower doses of atRA (0.1μM and 0.5μM) had no significant effect. The negative effect of atRA is consistent with previous reports showing dose-dependent inhibitory effects of atRA on embryonic limb mesenchymal micromass cultures. western blot analysis showed that atRA inhibted protein level of TGFβ1 in a dose-dependent manner. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression.Conclusions:(1) Excessive atRA has developmental toxicity, and can induce various malformations in mice, including cleft palate.(2) Excessive atRA inhibites Chondrogenesis of MEPM cells by down-regulating TGF-β/Smad expression which is incharged of its signal pathway.