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:

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis

A Corrigendum to this article was published on 07 December 2017

This article has been updated

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.

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: Bleomycin-induced lung fibrosis is dependent on ephrin-B2 in fibroblasts.
Figure 2: Ephrin-B2 ectodomain is shed by fibroblasts upon lung injury.
Figure 3: The ephrin-B2 ectodomain directs fibroblast migration, invasion and myofibroblast differentiation in vitro and in vivo.
Figure 4: ADAM10-mediated sEphrin-B2 generation is required for TGF-β1-induced myofibroblast differentiation.
Figure 5: ADAM10 inhibition prevents ephrin-B2 shedding, myofibroblast formation and lung fibrosis in mice.
Figure 6: ADAM10-sEphrin-B2 signaling is upregulated in IPF.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

Change history

  • 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.

References

  1. Ho, Y.Y., Lagares, D., Tager, A.M. & Kapoor, M. Fibrosis—a lethal component of systemic sclerosis. Nat. Rev. Rheumatol. 10, 390–402 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Wynn, T.A. & Ramalingam, T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18, 1028–1040 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Noble, P.W., Barkauskas, C.E. & Jiang, D. Pulmonary fibrosis: patterns and perpetrators. J. Clin. Invest. 122, 2756–2762 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chambers, R.C. & Mercer, P.F. Mechanisms of alveolar epithelial injury, repair, and fibrosis. Ann. Am. Thorac. Soc. 12 (Suppl. 1), S16–S20 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Noren, N.K., Lu, M., Freeman, A.L., Koolpe, M. & Pasquale, E.B. Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc. Natl. Acad. Sci. USA 101, 5583–5588 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang, H.U., Chen, Z.F. & Anderson, D.J. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741–753 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Kida, Y., Ieronimakis, N., Schrimpf, C., Reyes, M. & Duffield, J.S. EphrinB2 reverse signaling protects against capillary rarefaction and fibrosis after kidney injury. J. Am. Soc. Nephrol. 24, 559–572 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Avouac, J. 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).

    Article  CAS  Google Scholar 

  16. Gerety, S.S., Wang, H.U., Chen, Z.F. & Anderson, D.J. Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol. Cell 4, 403–414 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Luo, H. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Lin, K.T., Sloniowski, S., Ethell, D.W. & Ethell, I.M. Ephrin-B2-induced cleavage of EphB2 receptor is mediated by matrix metalloproteinases to trigger cell repulsion. J. Biol. Chem. 283, 28969–28979 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  21. Tomita, T., Tanaka, S., Morohashi, Y. & Iwatsubo, T. Presenilin-dependent intramembrane cleavage of ephrin-B1. Mol. Neurodegener. 1, 2 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hattori, M., Osterfield, M. & Flanagan, J.G. Regulated cleavage of a contact-mediated axon repellent. Science 289, 1360–1365 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Lisle, J.E. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Le Gall, S.M. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lagares, D. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Ludwig, A. 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).

    Article  CAS  PubMed  Google Scholar 

  30. Janes, P.W. 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).

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, K., Flanders, K.C. & Phan, S.H. Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis. Am. J. Pathol. 147, 352–361 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Munger, J.S. 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).

    Article  CAS  PubMed  Google Scholar 

  33. Ramos, C. 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).

    Article  CAS  PubMed  Google Scholar 

  34. Tanaka, M., Sasaki, K., Kamata, R. & Sakai, R. The C-terminus of ephrin-B1 regulates metalloproteinase secretion and invasion of cancer cells. J. Cell Sci. 120, 2179–2189 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Selman, M., Pardo, A. & Kaminski, N. Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs? PLoS Med. 5, e62 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Selman, M., López-Otín, C. & Pardo, A. Age-driven developmental drift in the pathogenesis of idiopathic pulmonary fibrosis. Eur. Respir. J. 48, 538–552 (2016).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Tager, A.M. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kapoor, M. 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).

    Article  CAS  PubMed  Google Scholar 

  41. Raghu, G. 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).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to David Lagares or Mohit Kapoor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

Supplementary Figures 1–8 and Supplementary Tables 1–2 (PDF 5139 kb)

Life Sciences Reporting Summary (PDF 172 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lagares, D., Ghassemi-Kakroodi, P., Tremblay, C. et al. ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis. Nat Med 23, 1405–1415 (2017). https://doi.org/10.1038/nm.4419

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4419

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