Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells

Cell Physiol Biochem. 2015;35(1):326-38. doi: 10.1159/000369699. Epub 2015 Jan 12.

Abstract

Background/aim: Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC) secretion after neutrophil elastase (NE) attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process.

Methods: NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab.

Results: We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added.

Conclusions: These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes.

Publication types

  • Research Support, Non-U.S. Gov't
  • Retracted Publication

MeSH terms

  • Aged
  • Antibodies / immunology
  • Bronchi / cytology
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Cytoskeletal Proteins / antagonists & inhibitors
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Female
  • Gene Expression Regulation / drug effects
  • Humans
  • Leukocyte Elastase / metabolism*
  • Male
  • Middle Aged
  • Mucin 5AC / metabolism*
  • Neomycin / pharmacology
  • Phosphatidylinositol 4,5-Diphosphate / immunology
  • Phosphorylation / drug effects
  • Protein Binding
  • Pulmonary Disease, Chronic Obstructive / metabolism
  • Pulmonary Disease, Chronic Obstructive / pathology*
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Vesicular Transport Proteins / antagonists & inhibitors
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism*

Substances

  • Antibodies
  • Cytoskeletal Proteins
  • Exoc1 protein, human
  • Mucin 5AC
  • Phosphatidylinositol 4,5-Diphosphate
  • RNA, Small Interfering
  • Vesicular Transport Proteins
  • ezrin
  • Leukocyte Elastase
  • Neomycin