Abstract：
Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) principally contributes to the pathogenesis of fibrotic cataract. Sprouty (Spry) and Spred proteins are receptor tyrosine kinase (RTK) antagonists that can regulate RTK-mediated signaling pathways, such as the MAPK/ERK1/2-signaling pathway. The present study examines the ability of Spry and Spred to inhibit TGFβ-induced EMT in LECs. LECs explanted from postnatal-day-21 Wistar rats were transduced with adenoviral vectors coding for Spry1, Spry2 or Spred2, and subsequently treated with or without TGFβ2. Immunofluorescent labeling of explants for the epithelial membrane marker β-catenin, and the mesenchymal marker alpha-smooth muscle actin (α-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of α-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGFβ, including a block to cell elongation, β-catenin delocalization and α-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGFβ treatment but did not impair the earlier activation of ERK1/2 at 20 min. These findings suggest that Spry and Spred may not directly impact ERK1/2-signaling activated by the serine/threonine kinase TGFβ receptor, but may selectively target later ERK1/2-signaling driven by downstream RTK-mediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGFβ-induced EMT that can regulate ERK1/2-signaling in a temporal manner. A greater understanding of how Spry and Spred regulate the complex signaling interactions that underlie TGFβ-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract.
摘要：
晶状体上皮细胞（LECs）的上皮-间质转化（EMT）主要参与纤维化白内障的发病。Sprouty（Spry）和Spred蛋白是受体酪氨酸激酶（RTK）拮抗剂，可以调节RTK介导的信号通路，如MAPK/ERK1/2-信号通路。本研究检查了Spry和Spred抑制LEC中TGFβ诱导的EMT的能力。用编码Spry1，Spry2或Spred2的腺病毒载体转导从出生后21天Wistar大鼠移植的LEC，然后用或不用TGFβ2处理。上皮膜标记β-catenin的外植体的免疫荧光标记，和间充质标记α-平滑肌肌动蛋白（α-sma），用于表征EMT的进展。蛋白质印迹用于定量α-sma和ERK1/2-信号的水平。在LEC中Spry或Spred的过表达足以抑制响应TGFβ的EMT，包括块到细胞的延伸，β-连环蛋白离域和α-sma积累。Spry和Spred也显示出显着阻断ERK1/2磷酸化长达18小时的TGFβ处理，但在20分钟时并未损害ERK1/2的早期激活。这些发现表明Spry和Spred可能不会直接影响丝氨酸/苏氨酸激酶TGFβ受体激活的ERK1/2信号，但可以选择性地靶向由下游RTK介导的信号驱动的后期ERK1/2信号。一起来看,我们的数据证实Spry和Spred拮抗剂是TGFβ诱导的EMT的有效负调节因子,可以以时间方式调节ERK1/2信号传导.更深入地了解Spry和Spred如何调节TGFβ诱导的EMT背后的复杂信号相互作用，对于促进EMT驱动的不同病理的新疗法的开发至关重要。包括白内障的纤维化形式。
