Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression.
Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression.
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DOI：
10.1371/journal.pone.0040478
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hackett TL
中科院分区：
文献类型：
--
作者：
Leung C;Shaheen F;Bernatchez P;Hackett TL
正常的气道上皮屏障功能通过细胞-细胞接触来维持，细胞-细胞接触需要粘附蛋白通过膜囊泡运输和融合事件在细胞表面移位。Myoferlin和dysferlin是多C2结构域Ferlin超家族的成员，通过诱导膜弯曲参与膜融合过程。本研究的目的是检查dysferlin和myoferlin在人气道内的表达，并确定这些蛋白质在气道上皮稳态中的作用。采用免疫组化法检测正常人气道组织中dysferlin和myoferlin的表达，免疫印迹法检测原代人气道上皮细胞和成纤维细胞中dysferlin和myoferlin的表达。使用共聚焦显微镜确定dysferlin和myoferlin在上皮细胞中的定位。分析的功能结果包括使用人支气管上皮细胞系16 HBE在dysferlin和myoferlin siRNA敲低后的细胞粘附、蛋白表达和细胞脱离。原代人气道上皮细胞表达dysferlin和myoferlin，而从支气管和实质分离的成纤维细胞仅表达myoferlin。共聚焦显微镜观察发现dysferlin和myoferlin的表达主要分布在16 HBE细胞的高尔基体、细胞质和质膜上。用肌铁蛋白siRNA而不是dysferlin siRNA处理16 HBE细胞导致圆形细胞形态和细胞粘附的丧失。肌铁蛋白敲低后的这种细胞脱落与紧密连接分子闭合小带-1（ZO-1）的表达降低以及凋亡标志物膜联蛋白V和碘化丙啶阳性细胞数量增加相关。细胞脱落与先天性炎性细胞因子IL-6和IL-8的释放无关。本研究证实了呼吸道上皮细胞和成纤维细胞内肌钙蛋白的异质性表达。肌铁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内细胞脱离和凋亡的膜融合事件中的作用。
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. 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.
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