Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Autophagy is defective in human and mice CF airway epithelia.
Figure 2: Sequestration of beclin 1 interactome in aggresomes drives defective autophagy in CF airway epithelia.
Figure 3: TG2-mediated crosslinking of beclin 1 induces aggresome sequestration of beclin 1 interactome and drives defective autophagy in CF airway epithelial cells.
Figure 4: Defective CFTR drives inhibition of autophagy by means of ROS-mediated TG2 SUMOylation in airway epithelial cells.
Figure 5: Restoring beclin 1 and autophagy decreases aggresome accumulation in CF epithelia.
Figure 6: Restoring beclin 1 and autophagy rescues CF phenotype in IB3-1 cells.
Figure 7: Restoring autophagy by cystamine or beclin 1 overexpression rescues CF phenotype in human and mice CF airways.
Figure 8: NAC restores autophagy and ameliorates CF lung phenotype in CF mice.

Similar content being viewed by others

References

  1. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 28, 1069–1075 (2008).

    Article  Google Scholar 

  2. Moreau, K., Luo, S. & Rubinsztein, D. C. Cytoprotective roles for autophagy. Curr. Opin. Cell Biol. 22, 206–211 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Korolchuk, V. I., Mansilla, A., Menzies, F. M. & Rubinsztein, D. C. Autophagy inhibition compromises degradation of ubiquitin–proteasome pathway substrates. Mol. Cell 33, 517–527 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kirkin, V., McEwan, D. G., Novak, I. & Dikic, I. A role for ubiquitin in selective autophagy. Mol. Cell 34, 259–269 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Bjørkøy, G. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Dohm, C. P., Kermer, P. & Bahr, M. Aggregopathy in neurodegenerative diseases: mechanisms and therapeutic implication. Neurodegen. Dis. 5, 321–338 (2008).

    Article  CAS  Google Scholar 

  7. Williams, A. et al. Aggregate-Prone proteins are cleared from the cytosol by autophagy: Therapeutic Implications. Curr. Top. Dev. Biol. 76, 89–101 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Schessl, J., Zou, Y., McGrath, M. J., Cowling, B. S. & Maiti, B. Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy. J. Clin. Invest. 118, 904–912 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Rodriguez-Gonzalez, A. et al. Role of the aggresome pathway in cancer: targeting histone deacetylase 6-dependent protein degradation. Cancer Res. 68, 2557–2560 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Maiuri, L. et al. Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARγ down-regulation. J. Immunol. 180, 7697–7705 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Ratjen, F. & Doring, G. Cystic fibrosis. Lancet 361, 681–689 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Sha, Y., Pandit, L., Zeng, S. & Eissa, N. T. A critical role of CHIP in the aggresome pathway. Mol. Cell. Biol. 29, 116–128 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Bence, N. F., Sampat, R. M. & Kopito, R. R. Impairment of the ubiquitin–proteasome system by protein aggregation. Science 292, 1552–1555 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Fu, L. & Sztul, E. ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy. Eur. J. Cell Biol. 88, 215–226 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Luciani, A. et al. SUMOylation of tissue transglutaminase as link between oxidative stress and inflammation. J. Immunol. 183, 2775–2784 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Trudel, S. et al. Peroxiredoxin 6 fails to limit phospholipid peroxidation in lung from Cftr-knockout mice subjected to oxidative challenge. PLoS One 4, e6075 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Sinha, S. & Levine, B. The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene 27, S137–S148 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Maiuri, M. C., Criollo, A. & Kroemer, G. Crosstalk between apoptosis and autophagy within the Beclin 1 interactome. EMBO J. 29, 515–516 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. He, C. & Levine, B. The Beclin 1 interactome. Curr. Opin. Cell Biol. 22, 140–149 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K. & Aliev, G. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Raia, V. et al. Inhibition of p38 mitogen activated protein kinase controls airway inflammation in cystic fibrosis. Thorax 60, 773–780 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Legssyer, R. et al. Azithromycin reduces spontaneous and induced inflammation in DF508 cystic fibrosis mice. Respir. Res. 7, 134–136 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Matsunaga, K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nature Cell Biol. 11, 385–396 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Zhong, Y. et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nature Cell Biol. 11, 468–476 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nature Cell Biol. 11, 1433–1437 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Maiuri, M. C. et al. Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1. EMBO J. 26, 2527–2539 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pattingre, S. et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 23, 927–939 (2005).

    Article  Google Scholar 

  30. Liang, C. et al. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nature Cell Biol. 10, 776–787 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Kawaguchi, Y. et al. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727–738 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Spencer, B. et al. Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in α-synuclein models of Parkinson's and Lewy body diseases. J. Neurosci. 29, 13578–13588 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Stocker, A. G. et al. Single-dose lentiviral gene transfer for lifetime airway gene expression. J. Gene Med. 11, 861–867 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Lorand, L. & Graham, R. M. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nature Rev. Mol. Cell Biol. 4, 140–156 (2003).

    Article  CAS  Google Scholar 

  35. Akar, U. et al. Tissue transglutaminase inhibits autophagy in pancreatic cancer cells. Mol. Cancer Res. 5, 241–249 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Ron, D. & Walter, P. Signal integration in the endoplasmatic reticulum unfolded protein response. Nature Rev. Mol. Cell Biol. 8, 519–529 (2007).

    Article  CAS  Google Scholar 

  37. Bartoszewski, R. et al. Activation of the unfolded protein response by ΔF508 CFTR. Am. J. Respir. Cell Mol. Biol. 39, 448–457 (2008).

    Article  CAS  Google Scholar 

  38. Moscat, J. & Diaz-Meco, M. T. p62 at the crossroads of autophagy, apoptosis, and cancer. Cell 137, 1001–1004 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mathew, R. et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Komatsu, M. et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131, 1149–1163 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Riordan, J. R. CFTR function and prospects for therapy. Annu. Rev. Biochem. 77, 701–726 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, X. et al. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127, 803–815 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Wang, X., Koulov, A. V., Kellner, W. A., Riordan, J. R. & Balch, W. E. Chemical and biological folding contribute to temperature-sensitive ΔF508 CFTR trafficking. Traffic 11, 1878–1893 (2008).

    Article  Google Scholar 

  44. Skach, W. R. CFTR: new members join the fold. Cell 127, 673–675 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Amaral, M. D. CFTR and chaperones: processing and degradation. J. Mol. Neurosci. 23, 41–48 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Pedemonte, N. et al. Small-molecule correctors of defective ΔF508-CFTR cellular processing identified by high-throughput screening. J. Clin. Invest. 115, 2564–2571 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Caohuy, H., Jozwik, C. & Pollard, H. B. Rescue of ΔF508-CFTR by the SGK1/Nedd4-2 signaling pathway. J. Biol. Chem. 284, 25241–25253 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ghavami, S. et al. S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3. Cell Res. 20, 314–331 (2010).

    Article  CAS  PubMed  Google Scholar 

  49. Karpuj, M. V. et al. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nature Med. 8, 143–149 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Sablina, A. A., Budanov, A. V., Ilyinskaya, G. V., Agapova, L. S. & Kravchenko, J. E. The antioxidant function of the p53 tumor suppressor. Nature Med. 11, 1306–1313 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Mall, M., Grubb, B. R., Harkema, J. R., O'Neal, W. K. & Boucher, R. C. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nature Med. 10, 487–493 (2004).

    Article  CAS  PubMed  Google Scholar 

  52. Frizzell, R. A. & Pilewski, J. M. Finally, mice with CF lung disease. Nature Med. 10, 452–454 (2004).

    Article  CAS  PubMed  Google Scholar 

  53. Maiuri, C., Zalckvar, E., Kimchi, A. & Kroemer, G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nature Rev Mol. Cell. Biol. 8, 741–752 (2007).

    Article  CAS  Google Scholar 

  54. Hara, T. et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441, 885–889 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Takahashi, Y. et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biol. 9, 1142–1151 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. Pickford, F. et al. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J. Clin. Invest. 118, 2190–2199 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Tebbenkamp, A. T. & Borchel, D. R. Protein aggregate characterization in models of neurodegenerative disease. Methods Mol. Biol. 566, 85–91 (2009).

    Article  CAS  PubMed  Google Scholar 

  58. Martínez, A, Portero-Otin, M., Pamplona, R. & Ferrer, I. Protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates. Brain Pathol. 20, 281–297 (2010).

    Article  PubMed  Google Scholar 

  59. Korolchuk, V. I., Menzies, F. M. & Rubinsztein, D. C. Mechanisms of cross-talk between the ubiquitin–proteasome and autophagy–lysosome systems. FEBS Lett. 584, 1393–1398 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Muma, N. A. Transglutaminase is linked to neurodegenerative disease. J. Neuropathol. Exp. Neurol. 66, 258–263 (2007).

    Article  CAS  PubMed  Google Scholar 

  61. Teichgräber, V. et al. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nature Med. 14, 382–391 (2008).

    Article  PubMed  Google Scholar 

  62. Vij, N., Mazur, S. & Zeitlin, P. L. CFTR is a negative regulator of NFκB mediated innate immune response. PLoS One 4, e4664 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Farinha, C. M. & Amaral, M. D. Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol. Cell. Biol. 25, 5242–5252 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Scott-Ward, T. S. & Amaral, M. D. Deletion of Phe508 in the first nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator increases its affinity for the heat shock cognate 70 chaperone. FEBS J. 276, 7097–7109 (2009).

    Article  CAS  PubMed  Google Scholar 

  65. Rochat, T., Lacroix, J. S. & Jornot, L. N-acetylcysteine inhibits Na+ absorption across human nasal epithelial cells. J. Cell Physiol. 201, 106–116 (2004).

    Article  CAS  PubMed  Google Scholar 

  66. Luciani, A. et al. Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa. Gut 59, 311–319 (2010).

    Article  PubMed  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Valeria Raia, Andrea Ballabio or Luigi Maiuri.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 3152 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Luciani, A., Villella, V., Esposito, S. et al. Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition. Nat Cell Biol 12, 863–875 (2010). https://doi.org/10.1038/ncb2090

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2090

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing