Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Bio-MAX institute, Seoul National University, Seoul, Korea.
PLoS One. 2022 May 17;17(5):e0266409. doi: 10.1371/journal.pone.0266409. eCollection 2022.
Transforming growth factor-β (TGF-β) is an important inducer of the epithelial-to-mesenchymal transition (EMT) in various cancers. Our previous study demonstrated that prohaptoglobin (proHp) stimulates Smad1/5 activation via ALK1, a TGF-β type I receptor, in endothelial cells, suggesting that proHp plays a role in TGF-β signaling. However, the function of proHp in cellular events downstream of Smads remains unclear. The current study investigated the effects of proHp on TGF-β-mediated Smad-dependent EMT induction and cell invasion in vitro using proHp-overexpressing SK-Hep1 liver cancer cells. The results of Western blotting, quantitative real-time RT-PCR, and immunocytochemistry indicated that proHp downregulated expression of mesenchymal marker and EMT regulator such as N-cadherin, vimentin, and twist, and upregulated expression of the epithelial marker E-cadherin. Compared with control cells, proHp-overexpressing cells exhibited high levels of ALK1/2/3 receptors and markedly increased Smad1/5 phosphorylation. Interestingly, proHp attenuated TGF-β-induced expression of mesenchymal markers and Smad2/3 phosphorylation. It also significantly suppressed cell invasion and migration. Knockdown of Smad1/5 abolished the inhibitory effects of proHp on TGF-β-stimulated Smad2/3 phosphorylation and mesenchymal marker expression. These findings indicate that proHp suppresses the TGF-β-induced EMT and cell invasion in vitro by enhancing Smad1/5 activation via ALK1/2/3 receptors and thus suppressing the Smad2/3 signaling pathway in SK-Hep1 cells. This study suggests that proHp may prevent a de-differentiation of hepatic cells and induce a cell differentiation by regulating the Smad signaling pathway.
转化生长因子-β（TGF-β）是各种癌症中上皮间质转化（EMT）的重要诱导因子。我们之前的研究表明，前血影蛋白（proHp）通过 TGF-β Ⅰ型受体 ALK1 刺激 Smad1/5 的激活，提示 proHp 在 TGF-β信号转导中发挥作用。然而，proHp 在 Smads 下游细胞事件中的功能尚不清楚。本研究通过过表达前血影蛋白的 SK-Hep1 肝癌细胞，在体外研究了前血影蛋白对 TGF-β 介导的 Smad 依赖性 EMT 诱导和细胞侵袭的影响。Western blot、定量实时 RT-PCR 和免疫细胞化学结果表明，前血影蛋白下调了间质标志物和 EMT 调节剂如 N-钙粘蛋白、波形蛋白和 twist 的表达，而上调了上皮标志物 E-钙粘蛋白的表达。与对照细胞相比，过表达前血影蛋白的细胞表现出高水平的 ALK1/2/3 受体，并且 Smad1/5 磷酸化明显增加。有趣的是，前血影蛋白减弱了 TGF-β 诱导的间质标志物表达和 Smad2/3 磷酸化。它还显著抑制了细胞侵袭和迁移。Smad1/5 的敲低消除了前血影蛋白对 TGF-β 刺激的 Smad2/3 磷酸化和间质标志物表达的抑制作用。这些发现表明，前血影蛋白通过增强 ALK1/2/3 受体的 Smad1/5 激活，抑制 SK-Hep1 细胞中的 Smad2/3 信号通路，从而抑制 TGF-β 诱导的 EMT 和细胞侵袭。本研究提示，前血影蛋白可能通过调节 Smad 信号通路来防止肝实质细胞去分化并诱导细胞分化。
