Article | Published:

Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition

Nature Cell Biology volume 12, pages 863875 (2010) | Download Citation

Abstract

Accumulation of unwanted/misfolded proteins in aggregates has been observed in airways of patients with cystic fibrosis (CF), a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show how the defective CFTR results in defective autophagy and decreases the clearance of aggresomes. Defective CFTR-induced upregulation of reactive oxygen species (ROS) and tissue transglutaminase (TG2) drive the crosslinking of beclin 1, leading to sequestration of phosphatidylinositol-3-kinase (PI(3)K) complex III and accumulation of p62, which regulates aggresome formation. Both CFTR knockdown and the overexpression of green fluorescent protein (GFP)-tagged-CFTRF508del induce beclin 1 downregulation and defective autophagy in non-CF airway epithelia through the ROS–TG2 pathway. Restoration of beclin 1 and autophagy by either beclin 1 overexpression, cystamine or antioxidants rescues the localization of the beclin 1 interactome to the endoplasmic reticulum and reverts the CF airway phenotype in vitro, in vivo in Scnn1b-transgenic and CftrF508del homozygous mice, and in human CF nasal biopsies. Restoring beclin 1 or knocking down p62 rescued the trafficking of CFTRF508del to the cell surface. These data link the CFTR defect to autophagy deficiency, leading to the accumulation of protein aggregates and to lung inflammation.

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Acknowledgements

We thank Noboru Mizushima for the gift of the pEGFP–LC3 and pcDNA3-HA–beclin 1 expression vectors; Ron Kopito for the gift of the pGFP–F508del-CFTR expression vector; Michael Bownlee for the gift of the adenoviral vectors; Gian Maria Fimia for the gift of the TG2 plasmid; Dieter C. Gruenert for the gift of CFBE41o and 16HBE14o cell lines; Maria Carla Panzeri for support in electron microscopy and in the analysis of the data; Rosarita Tatè for technical support in confocal microscopy; and Ilaria Russo for technical support in histology. Cftrtm1EUR (F508del (FVB/129) mice were obtained from Bob Scholte under European Economic Community European Coordination Action for Research in Cystic Fibrosis program EU FP6 LSHM-CT-2005-018932. This work was supported by the European Institute for Research in Cystic Fibrosis, Cancer Research UK, Rothschild Trust, Coeliac UK and Regione Campania (L. 229/99).

Author information

Author notes

    • Alessandro Luciani
    •  & Valeria Rachela Villella

    These authors contributed equally to this work

Affiliations

  1. European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan 20132, Italy.

    • Alessandro Luciani
    • , Manuela Gavina
    • , Laura Pulze
    •  & Luigi Maiuri
  2. Department of Chemical Engineering, Federico II University, Naples 80125, Italy.

    • Alessandro Luciani
    • , Valeria Rachela Villella
    • , Speranza Esposito
    •  & Stefano Guido
  3. Institute of Pediatrics, University of Foggia, Foggia 71100, Italy.

    • Speranza Esposito
    • , Massimo Pettoello-Mantovani
    • , Maria D'Apolito
    •  & Luigi Maiuri
  4. Telethon Institute of Genetics and Medicine (TIGEM), Naples 80131, Italy.

    • Nicola Brunetti-Pierri
    • , Diego Medina
    • , Carmine Settembre
    •  & Andrea Ballabio
  5. Department of Pediatrics, Federico II University, Naples 80131, Italy.

    • Nicola Brunetti-Pierri
  6. Department of Laboratory Medicine, University of Foggia, Foggia 71100, Italy.

    • Ida Giardino
  7. Departments of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624, USA.

    • Eliezer Masliah
    •  & Brian Spencer
  8. Cancer Research UK Oncology Unit, University of Southampton, Southampton, SO16 6YD, U.K.

    • Sonia Quaratino
  9. Cystic Fibrosis Unit, Department of Pediatrics, Federico II University, Naples 80131, Italy.

    • Valeria Raia
  10. Medical Genetics, Department of Pediatrics, Federico II University, Naples 80131, Italy.

    • Andrea Ballabio

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Contributions

A.L. co-designed the research concept, planned the overall experimental design, performed organ culture and confocal microscopy studies and wrote the manuscript. V.R.V. co-designed the research concept, planned the overall experimental design and performed immunoblot and immunoprecipitation experiments, cell cultures and transfections. S.E. contributed to the study design, interpretation and analysis of the data and performed immunoblot and immunoprecipitation experiments, cell cultures and transfections. N.B. contributed to the study design, provided scientific knowledge, contributed to the interpretation and analysis of the data, performed experiments on mice and wrote the manuscript. D.M. contributed to the study design, provided scientific knowledge, contributed to the interpretation and analysis of the data and performed the analysis of mitochondrial function. C.S. provided expression vectors and scientific knowledge and contributed to the analysis of the data. M.G. and L.P. performed experiments on mice and contributed to the interpretation and analysis of the data. I.G., M.P.M. and M.D. performed PCR and contributed to the interpretation and analysis of the data. S.G. contributed to the discussion of the data. E.M. and B.S. provided the lentiviral vectors and scientific knowledge. S.Q. contributed to the interpretation and analysis of the data and provided scientific knowledge. A.B. co-designed the research concept and co-supervised the project. V.R. and L.M. designed the research concept, planned the overall experimental design, supervised the study and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Valeria Raia or Andrea Ballabio or Luigi Maiuri.

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DOI

https://doi.org/10.1038/ncb2090

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