Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression.
BACKGROUND
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.


METHODS
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.


RESULTS
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
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.
The James Hogg Research Centre, Institute for Heart + Lung Health, St Paul's Hospital, Vancouver, British Columbia, Canada.
Cleo
C
Leung
Furquan
F
Shaheen
Pascal
P
Bernatchez
Tillie-Louise
TL
Hackett
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D051242:Caveolin 1 / Q000378:metabolism; D002448:Cell Adhesion; D016923:Cell Death; D002460:Cell Line; D048430:Cell Shape / Q000235:genetics; D015536:Down-Regulation; D000073939:Dysferlin; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology*; D019108:Tight Junctions / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D051242:Caveolin 1; C114839:DYSF protein, human; D000073939:Dysferlin; C415584:MYOF protein, human; D008565:Membrane Proteins; D009124:Muscle Proteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D040301:Vascular Endothelial Growth Factor Receptor-2