The James Hogg Research Centre, Institute for Heart + Lung Health, St Paul's Hospital, Vancouver, British Columbia, Canada.
PLoS One. 2012;7(7):e40478. doi: 10.1371/journal.pone.0040478. Epub 2012 Jul 10.
Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.
The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.
Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.
CONCLUSIONS/SIGNIFICANCE: This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.
正常气道上皮屏障功能由细胞-细胞连接维持，这需要粘附蛋白在细胞表面通过膜小泡运输和融合事件进行易位。肌球蛋白和畸形肌球蛋白，多 C2 结构域 Ferlin 超家族的成员，通过诱导膜曲率参与了膜融合过程。本研究的目的是研究畸形肌球蛋白和肌球蛋白在人呼吸道中的表达，并确定这些蛋白质在气道上皮稳态中的作用。
通过免疫组织化学法评估正常人类气道切片中畸形肌球蛋白和肌球蛋白的表达，并通过免疫印迹法评估原代人呼吸道上皮细胞和成纤维细胞中的表达。使用共聚焦显微镜确定畸形肌球蛋白和肌球蛋白在上皮细胞中的定位。使用人支气管上皮细胞系 16HBE，分析包括细胞粘附、蛋白表达和细胞脱落在内的功能结果，这些结果是在肌球蛋白和肌球蛋白 siRNA 敲低后得出的。
原代人呼吸道上皮细胞表达畸形肌球蛋白和肌球蛋白，而从支气管和实质分离的成纤维细胞仅表达肌球蛋白。使用共聚焦显微镜进一步将畸形肌球蛋白和肌球蛋白的表达定位在 16HBE 细胞的高尔基体、细胞质和质膜内。用肌球蛋白 siRNA 处理 16HBE 细胞，而不是用畸形肌球蛋白 siRNA 处理，会导致细胞形态变圆和细胞粘附丧失。肌球蛋白敲低后细胞脱落与紧密连接分子封闭蛋白-1 (ZO-1) 的表达减少和 Annexin V 和碘化丙啶阳性细胞数量增加有关。细胞脱落与先天炎症细胞因子 IL-6 和 IL-8 的释放无关。
结论/意义：本研究表明肌球蛋白在呼吸道上皮细胞和成纤维细胞中的表达具有异质性。肌球蛋白对气道上皮细胞中 ZO-1 表达的影响表明其在调节气道上皮细胞中细胞脱落和凋亡的膜融合事件中的作用。
