Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased β1-integrin signaling and delayed wound repair.
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ~60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated β1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated β1-integrin, pFAK, and pCAS to near control levels and partially restored (~40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses β1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.
Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, Minnesota;
Yutaka
Y
Itokazu
Richard E
RE
Pagano
Andreas S
AS
Schroeder
Scott M
SM
O'Grady
Andrew H
AH
Limper
David L
DL
Marks
D002460:Cell Line; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency* / Q000235:genetics; D015536:Down-Regulation; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D034622:RNA Interference; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine; D014945:Wound Healing
C493841:BCAR1 protein, human; C505032:CFTR protein, human; D050719:Crk-Associated Substrate Protein; D019012:Integrin beta1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005677:G(M1) Ganglioside; D014443:Tyrosine; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human
cell attachment; chloride channels; pulmonary epithelial cells; sialo-lipids