VIP regulates CFTR membrane expression and function in Calu-3 cells by increasing its interaction with NHERF1 and P-ERM in a VPAC1- and PKCε-dependent manner.
Vasoactive intestinal peptide (VIP) is a topical airway gland secretagogue regulating fluid secretions, primarily by stimulating cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride secretion that contributes to the airways innate defense mechanism. We previously reported that prolonged VIP stimulation of pituitary adenylate cyclase-activating peptide receptors (VPAC1) in airway cells enhances CFTR function by increasing its membrane stability. In the present study, we identified the key effectors in the VIP signaling cascade in the human bronchial serous cell line Calu-3. Using immunocytochemistry and in situ proximity ligation assays, we found that VIP stimulation increased CFTR membrane localization by promoting its colocalization and interaction with the scaffolding protein Na(+)/H(+) exchange factor 1 (NHERF1), a PDZ protein known as a positive regulator for CFTR membrane localization. VIP stimulation also increased phosphorylation, by protein kinase Cε of the actin-binding protein complex ezrin/radixin/moesin (ERM) and its interaction with NHERF1 and CFTR complex. On the other hand, it reduced intracellular CFTR colocalization and interaction with CFTR associated ligand, another PDZ protein known to compete with NHERF1 for CFTR interaction, inducing cytoplasmic retention and lysosomal degradation. Reducing NHERF1 or ERM expression levels by specific siRNAs prevented the VIP effect on CFTR membrane stability. Furthermore, iodide efflux assays confirmed that NHERF1 and P-ERM are necessary for VIP regulation of the stability and sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.
Departments of Physiology and Biophysics, Dalhousie University, Nova Scotia, Canada and.
Walaa
W
Alshafie
Departments of Physiology and Biophysics, Dalhousie University, Nova Scotia, Canada and.
Frederic G
FG
Chappe
Departments of Physiology and Biophysics, Dalhousie University, Nova Scotia, Canada and.
Mansong
M
Li
Departments of Physiology and Biophysics, Dalhousie University, Nova Scotia, Canada and Obstetrics and Gynecology, Dalhousie University, Nova Scotia, Canada.
Younes
Y
Anini
Departments of Physiology and Biophysics, Dalhousie University, Nova Scotia, Canada and valerie.chappe@dal.ca.
Valerie M
VM
Chappe
D048868:Adaptor Proteins, Signal Transducing; D001980:Bronchi / Q000166:cytology / Q000201:enzymology*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology*; D000077614:Golgi Matrix Proteins; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D034622:RNA Interference; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000378:metabolism*; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*; D000099149:Ezrin
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003598:Cytoskeletal Proteins; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D051744:Protein Kinase C-epsilon; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D017923:Sodium-Hydrogen Exchangers; D014660:Vasoactive Intestinal Peptide; D000099149:Ezrin; C505032:CFTR protein, human; C434484:GOPC protein, human; C093376:sodium-hydrogen exchanger regulatory factor; C071058:moesin; C071776:radixin; C497255:PRKCE protein, human
Na+/H+ exchange factor 1; cystic fibrosis associated ligand; cystic fibrosis transmembrane conductance regulator; ezrin/radixin/moesin; protein kinase C epsilon; vasoactive intestinal peptide