Smad Signaling Mechanism In AtRA-induced Cleft Palate

Cleft palate (CP) is one of the most common birth defects in humans, which brings huge suffering to patients and their families. Though People have been looking forward to finding the reason of CP, it is hard to tell the exact mechanism because many factors are involved.all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. One commonly studied defect produced by excessive atRA is cleft palate. atRA regulates the expression of transforming growth factor-βs (TGF-βs) in a variety of systems, including embryonic cells and tissues. The crosstalk between the atRA and TGF-βsignaling pathways is important for cellular proliferation and differentiation.The effect of TGF-βs can be achived by Smad protein in cytoplasm. Smads, the only substrates for TGF-βreceptors known to have a signalling function, were first identified as the products of the Drosophila Mad and C. elegans Sma genes. The receptor-regulated Smads, Smad2 and Smad3, ,can be phosphorylated by TGF-βreceptors , then translocates to and accumulates in the nucleus with TGF-βsignal, where they regulate the expressxion of the target gene. Smad7 function as negative regulators of the TGF-βsignaling pathway can inhibit the phosphorylation of Smad2 and Smad3 by binding to TGF-βreceptors .There are several lines of experimental evidence which indicate that TGF-βs are molecules which regulate these processes in the developing palate. However, these interaction between the atRA and Smad signaling pathways, for our understanding of mechanisms of cleft palate, is poorly understood. To investigated the effect of atRA on palatal fusion and the crosstalk between the atRA and Smad signaling pathways for palatal fusion , an in vitro organ culture model and an in vivo CP model induced by atRA were established.Methods:1. The grouping of experimentation Mouse strain Kunming was used in this study. The females were mated overnight, the presence of a vaginal plug established day 0 and hour 0. Totally 48 pregnant females were obtained. All the pregnant mice were randomly divided into two groups: organ culture group (n=24) and animal model group (n=24).2. Organ culture: atRA was disolved in dimethylsulfoxide (DMSO) with different concentration.The experimental groups were treated with atRA (1μM,5μM,10μM), while the control group was treated with 0.1% DMSO. The mice of organ culture group were sacrificed at GD13. The palatal shelves were dissected from the fetal murine heads under sterile conditions and placed in pairs on Millipore filters with correct anterior-posterior orientation and with their medial edges in contact. The palatal shelves were cultured at the air-fluid interface in BGJb medium using Grobstein organ culture dishes (GIBCO, Grand Island, NY) at 37°C in 5%CO₂ . Cell death were detected by TUNEL and laminin immunohistochemistry were detected on the same slice at vehicle control group and the atRA-treated group (5μM) in organ culture for 72 h;Western blot analyzed the phosphorylation of Smad2 and Smad3 and the expression of Smad7 in MEE in organ culture for 24 h.2. Established animal model: The pregnant mice of animal model group were randomly divided into two groups: control group (n=12) and atRA-treated group (n=12). Pregnant mice in atRA-treated group received by gavage a single dose of atRA at 100mg/kg body weight at 1 P.M. on GD10. Pregnant mice in control group received 0.2ml corn oil at the same time. The Pregnant mice in control group and atRA-treated group were killed on GD12, GD13, GD14, GD14.5, GD15 and GD16 for histological study ,and immunolocalization of phospho-Smad2 in the palate shelf of the control mice and the atRA-induced mice at GD14 and GD14.5 was detected. The expression level of P-Smad2 in MEE cells was compared between control group and atRA-treated group to make sure what kind of effect atRA had on Smad signaling and to make it clear how atRA can result in CP.Results:1. The effect of atRA on the palate fusionIn organ culture group,the palate of vehicle control group fused complately,and at 1μM and 5μM atRA-treated group, the opposing shelves were contact, but did not fused, and at 10μM atRA-treated group, the opposing shelves were not contact at the end of the culture (72 h) in vitro.In animal model group, the palate of vehicle control group fused complately, and the palate shelves of atRA-induced mice failed to fuse with each other,the lift of palate shelves was delayed and small palate shelves developed.2. The effect of atRA on the apoptosis of MEE cells and degradation of basal laminaIn organ culture group, migration and apoptosis of MEE cells and degradation of basal lamina within midline epithelial seam (MES) markedly characterized in vehicle control palatal shelves in organ culture for 72h, while atRA (5μM) induced apoptosis in mesenchyme and inhibited apoptosis of MEE cells and degradation of basal lamina within MES.3. The effect of atRA on Smad signaling pathways in MEE cellIn organ culture group,Western blot analysis revealed that Smad2 and Smad3 were endogenously activated and expression of Smad7 was inhibited during the fusion process. In contrast, atRA treatment abrogated phosphorylation of Smad2 and Smad3 and stimulated expression of Smad7 in MEE.In animal model group,the inhibitory effect of atRA on the expression of P-Smad2 protein in MEE cell at GD14 and GD14.5 was detected by immunohistochemistry.The animal experiment in vivo also showed the similar results.At GD14,the palatal shelf of the contral mice was remodeled to a horizontal position above the tongue. The distribution of P-Smad2 protein spreaded in the palatal epithelium. At GD14.5, the midline seam of the contral mice was disrupted as palate fusion progresses. The distribution of P-Smad2 in the midline was discontinuous and accompanied by the intense staining concentrated in the oral and nasal epithelial triangle areas. At GD14 and GD14.5, the palate shelf of atRA-induced mice was still vertical along the lateral sides of the tongue . No P-Smad2 was detected in the palate shelf.Conclusion:1. During the process of palatal fusion, atRA treatment inhibits the apoptosis of MEE cells and basal lamina degradation,which results in CP.2. At the moment of palatal horizontal development, atRA treatment inhibits the apoptosis of MEE cells and basal lamina degradation, which is involved in the inhibition of Smad signaling pathways.