Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8511, Japan.
Am J Respir Cell Mol Biol. 2011 Jul;45(1):136-44. doi: 10.1165/rcmb.2010-0140oc.
Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-β (TGF-β)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-β control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-β family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-β receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-β1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-β-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.
Notch 是一种古老的细胞信号系统，调节细胞命运的特化。本研究探讨了 Notch 在培养的 RLE-6TN 细胞（即大鼠肺泡上皮细胞）上皮-间充质转化（EMT）和肌成纤维细胞分化中的作用。Notch 的激活，无论是通过 Notch 细胞内结构域的异位表达，还是通过将 RLE-6TN 细胞与 L-Jagged1 细胞共培养来实现，都会诱导平滑肌α-肌动蛋白（SMA）和其他间充质标记基因（I 型胶原和波形蛋白）的表达，并降低上皮标记基因（E-钙黏蛋白、occludin 和 zonula occludens-1）的表达。内源性 Notch 信号的药理学抑制显著抑制了转化生长因子-β（TGF-β）诱导的 SMA 表达。Notch 增加了细胞迁移能力。荧光素酶测定显示，CC（A/T）（6）GG（CArG）盒和 TGF-β 控制元件（TCE）是 Notch 诱导 SMA 基因转录所必需的。DNA 微阵列分析显示，TGF-β 家族成员以及 Jagged1 在 Notch 诱导的 RLE-6TN 细胞中被诱导。Western blot 分析显示，Notch 诱导 Smad3 的磷酸化，TGF-β 受体 I/激活素受体样激酶 5（ALK5）激酶抑制剂 SB431542 显著降低 Notch 诱导的 SMA 表达。酶联免疫吸附试验证实 Notch 从 RLE-6TN 细胞中产生 TGF-β1。博来霉素诱导的肺纤维化模型和特发性间质性肺炎患者肺标本的免疫组织化学染色显示，Notch 在肌成纤维细胞中强烈表达，被鉴定为 SMA 阳性细胞。这些数据表明，Notch 通过 TGF-β-Smad3 途径诱导肌成纤维细胞分化，该途径以 CArG 依赖性和 TCE 依赖性方式激活肺泡上皮细胞中的 SMA 基因转录。我们的数据还表明，Notch 诱导 EMT 表型，并在肺纤维化中增加迁移行为。
