Centre for Digestive Diseases, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, 4 Newark St., London E1 2AT, UK.
Am J Physiol Gastrointest Liver Physiol. 2012 Dec 1;303(11):G1270-8. doi: 10.1152/ajpgi.00458.2011. Epub 2012 Sep 13.
Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/β-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1β-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1β-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1β-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1β stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1β-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1β-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.
黏膜下肌成纤维细胞参与肠道上皮修复的启动和协调，但分子信号通路在很大程度上尚不清楚。修复过程中发生的细胞适应包括去分化、迁移、增殖和再分化，这强烈地让人联想到正常的隐窝-绒毛上皮成熟过程。因此，我们假设 Wnt/β-catenin 信号通路可能在肠道上皮损伤修复中起关键作用。我们使用已建立的 Caco-2 细胞和非转化的 NCM460 细胞划痕实验方法，监测 IL-1β 刺激的结肠肌成纤维细胞 (CCD-18co) 对肠道上皮修复的影响，并用免疫印迹和免疫耗竭来检测条件培养基。与未经处理的肌成纤维细胞相比，IL-1β 刺激的肌成纤维细胞可使 Caco-2 伤口在 24 小时内的闭合增加两倍。IL-1β 刺激的肌成纤维细胞下调了 Caco-2 细胞中的分化标志物蔗糖酶-异麦芽糖酶，而增殖标志物 c-myc 则上调。基因表达谱分析表明，Wnt-5a 是最受 IL-1β 刺激的 CCD 上调的 Wnt 相关基因（上调 28 倍）。重组 Wnt-5a 增强了 Caco-2 和 NCM460 细胞的增殖。在划痕实验中，它增加了两种细胞系中前沿的迁移。用 Wnt-5a 免疫耗竭 IL-1β-CCD 条件培养基可消除增强修复的能力。Wnt-5a 通常通过非经典信号转导途径发挥作用。进一步的实验支持了该途径在肠道上皮损伤愈合中的作用：IL-1β-CCD 介导的修复不受经典 Wnt 拮抗剂 Dickkopf-1 的影响。此外，刺激肌成纤维细胞的培养基（而非 Wnt-5a 耗尽的培养基）增加了 Caco-2 细胞核提取物中的 c-jun。因此，肌成纤维细胞介导的非经典 Wnt-5a 信号在肠道上皮损伤修复的去分化和迁移阶段非常重要。
