Abstract
BackgroundNormal 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.MethodsThe 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.ResultsPrimary 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/SignificanceThis 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.
Highlights
The airway epithelium forms a continuous, highly regulated physical barrier, which lines the airway lumen, separating the underlying tissue from inhaled environmental antigens
Since dysferlin and myoferlin are membrane repair proteins that are crucial for maintaining membrane integrity, we sort to determine whether these proteins have a functional role in the homeostasis of human airway epithelium
All airway epithelial cells and fibroblasts express myoferlin at similar expression levels to that found in bovine aorta endothelial cells (BAECs), which we have previously reported to express both myoferlin and dysferlin (Figure 2B)
Summary
The airway epithelium forms a continuous, highly regulated physical barrier, which lines the airway lumen, separating the underlying tissue from inhaled environmental antigens. Fusion of cytoplasmic vesicles containing adhesion molecules to the plasma membrane is essential for the formation of selectively permeable membrane barriers. Since dysferlin and myoferlin are membrane repair proteins that are crucial for maintaining membrane integrity, we sort to determine whether these proteins have a functional role in the homeostasis of human airway epithelium. The purpose of this study was to examine the expression of dysferlin and myoferlin within the human airway and determine the effects of myoferlin and dysferlin knockdown on airway epithelial functions. 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. 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
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