Rescue of Functional F508del Cystic Fibrosis Transmembrane Conductance Regulator by Vasoactive Intestinal Peptide in the Human Nasal Epithelial Cell Line JME/CF15

  1. Sara Rafferty,
  2. Nicole Alcolado,
  3. Caroline Norez,
  4. Frederic Chappe,
  5. Siegried Pelzer,
  6. Frederic Becq and
  7. Valerie Chappe
  1. Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada (S.R., N.A., F.C., S.P.,V.C.); and Institut de Physiologie et Biologie Cellulaire, Université de Poitiers, Centre National de la Recherche Scientifique, Poitiers, France (C.N., F.B.).
  1. Address correspondence to:
    Dr. Valérie Chappe, Department of Physiology and Biophysics, 5850 College Street, Halifax, NS, Canada B3H 1X5. E-mail: valerie.chappe{at}dal.ca

Abstract

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC1 receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC1 receptor. At reduced temperature (27°C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37°C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTRinh-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37°C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Gαs stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the Gi/o protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

Footnotes

  • This work was supported by the Canadian Institutes of Health Research/Nova Scotia Health Research Foundation Regional Partnership; the Dalhousie Medical Research Foundation; the Canadian Cystic Fibrosis Foundation, and Vaincre La Mucoviscidose. Some equipment used for this study was obtained through a Canadian Foundation for Innovation Leaders Opportunity Fund award and Dalhousie Medical Research Foundation Capital equipment grant.

  • S.R. was supported by a Canadian Cystic Fibrosis Foundation summer studentship and by the Nova Scotia Economic Development Cooperative Employment Program.

  • N.A. is supported by a Nova Scotia Health Research Foundation Master award.

  • V.C. is a Canadian Institutes of Health Research Scholar.

  • A preliminary report of this work has been presented previously: Rafferty S, Alcolado N, Chappe F, Pelzer S, Becq F, and Chappe V (2008) VIP stimulates DF508-CFTR trafficking and membrane insertion in human nasal JME/CF15 cells. Pediatr Pulmonol Suppl 43 (Suppl 31):218. 22nd Annual North American Cystic Fibrosis Conference; 23-25 Oct 2008; Orlando, FL.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • ABBREVIATIONS:

    • Received April 20, 2009.
    • Accepted July 6, 2009.
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  1. Published online before print July 7, 2009, doi: 10.1124/jpet.109.155341 JPET October 2009 vol. 331 no. 1 2-13
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