Smad2Δexon3 and Smad3 have distinct properties in signal transmission leading to TGF-β-induced cell motility.

In mammalian cells, Smad2 and Smad3, two receptor-regulated Smad proteins, play crucial roles in the signal transmission of transforming growth factor-β (TGF-β) and are involved in various cell regulatory processes, including epithelial–mesenchymal transition–associated cell responses, that is, cell morphological changes, E-cadherin downregulation, stress fiber formation, and cell motility enhancement. Smad2 contains an additional exon encoding 30 amino acid residues compared with Smad3, leading to distinct Smad2 and Smad3 functional properties. Intriguingly, Smad2 also has an alternatively spliced isoform termed Smad2Δexon3 (also known as Smad2β) lacking the additional exon and behaving similarly to Smad3. However, Smad2Δexon3 and Smad3 signaling properties have not yet been compared in detail. In this study, we reveal that Smad2Δexon3 rescues multiple TGF-β–induced in vitro cellular responses that would become defective upon SMAD3 KO but does not rescue cell motility enhancement. Using Smad2Δexon3/Smad3 chimeric proteins, we identified that residues Arg-104 and Asn-210 in Smad3, which are not conserved in Smad2Δexon3, are key for TGF-β–enhanced cell motility. Moreover, we discovered that Smad2Δexon3 fails to rescue the enhanced cell motility as it does not mediate TGF-β signals to downregulate transcription of ARHGAP24, a GTPase-activating protein that targets Rac1. This study reports for the first time distinct signaling properties of Smad2Δexon3 and Smad3.

在哺乳动物细胞中，Smad2和Smad3这两种受体调节型Smad蛋白在转化生长因子-β（TGF -β）的信号传递中起着关键作用，并参与多种细胞调控过程，包括上皮 - 间质转化相关的细胞反应，即细胞形态变化、E - 钙黏蛋白下调、应力纤维形成以及细胞运动性增强。与Smad3相比，Smad2包含一个额外的外显子，编码30个氨基酸残基，这导致Smad2和Smad3具有不同的功能特性。有趣的是，Smad2还有一种可变剪接异构体，称为Smad2Δexon3（也称为Smad2β），它缺少这个额外的外显子，其行为与Smad3相似。然而，Smad2Δexon3和Smad3的信号特性尚未得到详细比较。在本研究中，我们发现Smad2Δexon3可挽救多种TGF -β诱导的体外细胞反应，这些反应在SMAD3基因敲除时会出现缺陷，但不能挽救细胞运动性增强。通过使用Smad2Δexon3/Smad3嵌合蛋白，我们确定了Smad3中的精氨酸 - 104和天冬酰胺 - 210残基（在Smad2Δexon3中不保守）是TGF -β增强细胞运动性的关键。此外，我们发现Smad2Δexon3无法挽救增强的细胞运动性，因为它不介导TGF -β信号来下调ARHGAP24的转录，ARHGAP24是一种靶向Rac1的GTP酶激活蛋白。本研究首次报道了Smad2Δexon3和Smad3不同的信号特性。