Investigation of the roles of Smad linker region phosphoryulation in epithelial-mesenchymal transition and tissue fibrosis.

在视网膜色素上皮细胞系、ARPE-19细胞系和小鼠晶状体上皮细胞系的培养中，α-TN4、TGFbeta1的加入激活了Smad2/3 C末端和中间Rinker区的磷酸化，并伴随着上皮-间充质转化(EMT)。即使在TGFbeta1缺失的情况下，Smad2/3中间体的磷酸化也被观察到，并且似乎被MAPKs的抑制剂所减弱。免疫组织化学结果显示，EMT晶状体细胞Smad3中间肾区的磷酸化程度明显高于其C末端的磷酸化程度。在平滑肌肌动阳性细胞中未观察到C末端的磷酸化。大鼠晶状体损伤实验表明，JNK和p38参与了Smad中间连接区的磷酸化。在苔藓增殖性玻璃体视网膜病变(PVR)模型中全面检测EMT相关基因的表达。免疫组织化学显示Smad3的C末端和中间连接区均有磷酸化。腺病毒过表达显性阴性p38似乎抑制了Smad3的中间连接区的磷酸化。对人类样本及其动物模型的研究以及细胞培养实验表明，SMAD3中间连接区的磷酸化参与了EMT相关的眼部纤维化的病理过程。

In cultures of retinal pigment epithelial cell line, ARPE-19 and mouse lens epithelial cell line, a-TN4, TGFbeta1 addition activates phosphorylation of Smad2/3 C-terminal and middle rinker region along with epithelial-mesenchymal transition (EMT). Phorphorylation of Smad2/3 middle lilnker was observed even under TGFbeta1-minus condition and seemed to be attenuated with inhibitors of MAPKs. Immunohistochemistry of human samples showed that EMT-lens cells exhibited phosphorylation of Smad3 middle renker region more prominently as compared with its C-terminus phosphrylation. C-terminus phosphorylation was not observed in smooth muscle action-positive cells. Rat lens injury experiments suggested that invlovement of JNK and p38 in Smad middle linker region phosphorylation. EMT-related gene expression was overall detecterd in mosue proliferative vitreoretinopathy (PVR) model. Immunohistochemistry showed both C-terminus and middle linker region phosphorylations of Smad3. Adenoviral overexpression of dominant negative p38 seemed to suppress middle linker region phosphorylation of Smad3. Investigations on human samples and its animal model, as well as cell culture experiments, suggetsed involvement of Smad3 middle linker region phosphorylation in EMT-related fibrogenic ocular pathologies.