Abstract

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-β1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10–sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

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Change history

  • Corrected online 20 November 2017

    In the version of this article initially published online, the positions of the colored boxes in the key of Figure 5f were inverted. The treatment group is represented by the red line of the graph and the control group by the blue line. The error has been corrected in the print, PDF and HTML versions of this article.

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References

  1. 1.

    , , & Fibrosis—a lethal component of systemic sclerosis. Nat. Rev. Rheumatol. 10, 390–402 (2014).

  2. 2.

    & Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18, 1028–1040 (2012).

  3. 3.

    , & Pulmonary fibrosis: patterns and perpetrators. J. Clin. Invest. 122, 2756–2762 (2012).

  4. 4.

    & Mechanisms of alveolar epithelial injury, repair, and fibrosis. Ann. Am. Thorac. Soc. 12 (Suppl. 1), S16–S20 (2015).

  5. 5.

    Cellular and molecular mechanisms in kidney fibrosis. J. Clin. Invest. 124, 2299–2306 (2014).

  6. 6.

    et al. Accelerated variant of idiopathic pulmonary fibrosis: clinical behavior and gene expression pattern. PLoS One 2, e482 (2007).

  7. 7.

    et al. Gene expression profiling reveals novel TGFβ targets in adult lung fibroblasts. Respir. Res. 5, 24 (2004).

  8. 8.

    et al. EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 143, 145–155 (2010).

  9. 9.

    et al. Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly. Cell 124, 161–173 (2006).

  10. 10.

    & Mechanisms and functions of Eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3, 475–486 (2002).

  11. 11.

    Eph/ephrin signalling during development. Development 139, 4105–4109 (2012).

  12. 12.

    , , , & Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc. Natl. Acad. Sci. USA 101, 5583–5588 (2004).

  13. 13.

    , & Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741–753 (1998).

  14. 14.

    , , , & EphrinB2 reverse signaling protects against capillary rarefaction and fibrosis after kidney injury. J. Am. Soc. Nephrol. 24, 559–572 (2013).

  15. 15.

    et al. Enhanced expression of ephrins and thrombospondins in the dermis of patients with early diffuse systemic sclerosis: potential contribution to perturbed angiogenesis and fibrosis. Rheumatology (Oxford) 50, 1494–1504 (2011).

  16. 16.

    , , & Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol. Cell 4, 403–414 (1999).

  17. 17.

    et al. Efnb1 and Efnb2 proteins regulate thymocyte development, peripheral T cell differentiation, and antiviral immune responses and are essential for interleukin-6 (IL-6) signaling. J. Biol. Chem. 286, 41135–41152 (2011).

  18. 18.

    et al. Competition amongst Eph receptors regulates contact inhibition of locomotion and invasiveness in prostate cancer cells. Nat. Cell Biol. 12, 1194–1204 (2010).

  19. 19.

    , , & Ephrin-B2-induced cleavage of EphB2 receptor is mediated by matrix metalloproteinases to trigger cell repulsion. J. Biol. Chem. 283, 28969–28979 (2008).

  20. 20.

    et al. EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1. Nat. Commun. 5, 3516 (2014).

  21. 21.

    , , & Presenilin-dependent intramembrane cleavage of ephrin-B1. Mol. Neurodegener. 1, 2 (2006).

  22. 22.

    , & Regulated cleavage of a contact-mediated axon repellent. Science 289, 1360–1365 (2000).

  23. 23.

    et al. Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer. Exp. Cell Res. 333, 136–146 (2015).

  24. 24.

    et al. Crystal structure of an Eph receptor–ephrin complex. Nature 414, 933–938 (2001).

  25. 25.

    Eph–ephrin promiscuity is now crystal clear. Nat. Neurosci. 7, 417–418 (2004).

  26. 26.

    et al. ADAMs 10 and 17 represent differentially regulated components of a general shedding machinery for membrane proteins such as transforming growth factor alpha, L-selectin, and tumor necrosis factor alpha. Mol. Biol. Cell 20, 1785–1794 (2009).

  27. 27.

    et al. Endothelin 1 contributes to the effect of transforming growth factor beta1 on wound repair and skin fibrosis. Arthritis Rheum. 62, 878–889 (2010).

  28. 28.

    & Transforming growth factor beta in tissue fibrosis. N. Engl. J. Med. 331, 1286–1292 (1994).

  29. 29.

    et al. Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb. Chem. High Throughput Screen. 8, 161–171 (2005).

  30. 30.

    et al. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 123, 291–304 (2005).

  31. 31.

    , & Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis. Am. J. Pathol. 147, 352–361 (1995).

  32. 32.

    et al. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, 319–328 (1999).

  33. 33.

    et al. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am. J. Respir. Cell Mol. Biol. 24, 591–598 (2001).

  34. 34.

    , , & The C-terminus of ephrin-B1 regulates metalloproteinase secretion and invasion of cancer cells. J. Cell Sci. 120, 2179–2189 (2007).

  35. 35.

    , & Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs? PLoS Med. 5, e62 (2008).

  36. 36.

    , & Age-driven developmental drift in the pathogenesis of idiopathic pulmonary fibrosis. Eur. Respir. J. 48, 538–552 (2016).

  37. 37.

    et al. Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands. PLoS One 8, e81445 (2013).

  38. 38.

    et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat. Med. 14, 45–54 (2008).

  39. 39.

    et al. Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation. Arthritis Rheum. 64, 1653–1664 (2012).

  40. 40.

    et al. Loss of peroxisome proliferator-activated receptor gamma in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis. Arthritis Rheum. 60, 2822–2829 (2009).

  41. 41.

    et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am. J. Respir. Crit. Care Med. 183, 788–824 (2011).

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Acknowledgements

Authors would like to thank P. Datta, S. Nakamura, H. Endisha and J. Rockel (all from the University Health Network) for their technical assistance with mouse breeding and genotyping. The authors gratefully acknowledge funding support by University of Montreal Hospital Research Centre and University of Montreal (M.K.); Campaign to Cure Arthritis via the Toronto General and Western Foundation, University Health Network, Toronto (M.K.); an American Thoracic Society Foundation and Pulmonary Fibrosis Foundation Research Grant and the Marie A. Coyle Research Grant from the Scleroderma Foundation (D.L.), and by the National Institutes of Health, HL108975 and a grant from the Scleroderma Research Foundation (A.M.T).

Author information

Author notes

    • David Lagares
    •  & Parisa Ghassemi-Kakroodi

    These authors contributed equally to this work.

    • David Lagares
    • , Andrew M Tager
    •  & Mohit Kapoor

    These authors jointly directed this work.

Affiliations

  1. Division of Pulmonary and Critical Care Medicine, Fibrosis Research Center and Center for Immunology and Inflammatory Diseases, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • David Lagares
    • , Alba Santos
    • , Clemens K Probst
    • , Alicia Franklin
    • , Daniela M Santos
    • , Paula Grasberger
    • , Neil Ahluwalia
    • , Sydney B Montesi
    • , Barry S Shea
    • , Katharine E Black
    • , Rachel Knipe
    •  & Andrew M Tager
  2. Department of Medicine, University of Montreal Hospital Research Centre (CRCHUM), Montreal, Québec, Canada.

    • Parisa Ghassemi-Kakroodi
    • , Caroline Tremblay
    • , Meryem Blati
    • , Hassan Fahmi
    • , Jiangping Wu
    • , Jean-Pierre Pelletier
    • , Johanne Martel-Pelletier
    •  & Mohit Kapoor
  3. Division of Rheumatology, Jewish General Hospital, McGill University, Montreal, Québec, Canada.

    • Murray Baron
  4. The Arthritis Program, University Health Network, Toronto, Ontario, Canada.

    • Brian Wu
    • , Rajiv Gandhi
    •  & Mohit Kapoor
  5. Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico.

    • Annie Pardo
  6. Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Mexico City, Mexico.

    • Moisés Selman
  7. Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Ontario, Canada.

    • Mohit Kapoor
  8. Departments of Surgery and of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

    • Mohit Kapoor

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Contributions

D.L. designed most of the experiments, performed in vitro and in vivo mouse experiments, analyzed the data and generated the figures. P.G.-K. and M.B. were involved in the generation of Ephrinb2-CKO mice. A.S., P.G., N.A. and D.M.S. performed and analyzed in vitro experiments related to the ADAM10–ephrin-B2–EphB3/4 pathway in fibroblasts. C.K.P. and A.F. performed in vivo studies with ADAM10 inhibitor. C.T. was involved in histological characterization of mouse experiments in skin fibrosis model. M.S., A.P., S.B.M., R.K., K.E.B. and B.S.S. provided human lung fibroblasts, plasma and bronchoalveolar lavage fluid from individuals with IPF and healthy controls. M.B. and R.G. provided intellectual input on project design and troubleshooting. B.W. performed protein expression studies in mouse samples. J.W., H.F., J.-P.P. and J.M.-P. were involved in the characterization of mouse phenotype and troubleshooting with experiments related to ephrin biology. M.K. designed the original concept and led the entire team during the course of this study. D.L., A.M.T. and M.K. designed the study experiments, supervised the project and took overall responsibility for writing the manuscript with the help of all the authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David Lagares or Mohit Kapoor.

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    Supplementary Figures 1–8 and Supplementary Tables 1–2

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