Nishioka Michiyoshi, Venkatesan Narayanan, Dessalle Kevin, Mogas Andrea, Kyoh Shigenori, Lin Ting-Yu, Nair Parameswaran, Baglole Carolyn J, Eidelman David H, Ludwig Mara S, Hamid Qutayba
Meakins-Christie Laboratories, Research Institute-McGill University Health Centre, 1001 Decarie Boulevard, Block E, Montreal, QC, H4A 3J1, Canada.
Department of Thoracic Medicine, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taipei, Taiwan.
Respir Res. 2015 Jun 18;16(1):72. doi: 10.1186/s12931-015-0232-4.
Epithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT.
Primary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot.
Basal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells.
Our results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD.
上皮-间质转化（EMT）涉及高度极化的上皮细胞的细胞形态变化以及获得具有迁移和侵袭能力的间充质细胞表型，与吸烟相关的慢性阻塞性肺疾病（COPD）有关。然而，成纤维细胞与上皮细胞的相互作用以及成纤维细胞在COPD的EMT过程中的参与情况尚不清楚。在此，我们研究了以下假设：EMT在COPD患者的人支气管上皮（HBE）细胞中活跃，并且COPD患者肺成纤维细胞分泌的介质可诱导EMT。
从LONZA购买正常受试者和COPD患者的原代HBE细胞。HLF来自从正常（N）和COPD（D）受试者切除的肺，其条件培养基（CM）在无血清培养基中培养2天后收集。通过免疫组织化学、qRT-PCR和蛋白质印迹法评估肺活检组织以及用正常人肺成纤维细胞（NHLF）和COPD人肺成纤维细胞（DHLF）的CM刺激后的HBE细胞中上皮和间充质标志物以及EMT相关转录因子的表达。
与正常人支气管上皮细胞（NHBE）以及COPD肺组织相比，DHBE细胞中间充质标志物和EMT相关转录因子的基础mRNA表达增加。NHLF的CM显著诱导NHBE和COPD人支气管上皮细胞（DHBE）中波形蛋白的表达，但仅增加DHBE细胞中N-钙黏蛋白的表达。NHLF的CM显著诱导NHBE细胞中Twist1和Twist2的表达，并增加DHBE细胞中Snai2（Slug）的表达。虽然NHLF的CM对这些EMT标志物没有影响，但与对照相比，DHLF的CM显著增加了NHBE细胞中E-钙黏蛋白和波形蛋白的蛋白表达。NHBE细胞中N-钙黏蛋白的表达上调程度高于DHBE细胞。仅DHLF的CM显著增加了DHBE细胞中E-/N-钙黏蛋白的比率。
我们的结果表明，DHBE细胞在基线条件下已部分经历EMT。DHLF-CM促进NHBE中的EMT，表明成纤维细胞与上皮细胞之间的相互作用可能在COPD的EMT过程中起重要作用。
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