Skip to main content

Methods for the Assessment of Active Transforming Growth Factor-β in Cells and Tissues

  • Protocol
  • First Online:
Fibrosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1627))

Abstract

The potent and pluripotent cytokine TGFβ has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFβ exists in both latent and functionally active forms. Large amounts of latent TGFβ are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFβ activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFβ expression as small changes in TGFβ activity levels could be masked by the large amounts of latent TGFβ available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFβ in cells and tissues. This chapter includes methods for the assessment of TGFβ activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dubois CM, Laprise MH, Blanchette F et al (1995) Processing of transforming growth factor beta 1 precursor by human furin convertase. J Biol Chem 270(18):10618–10624. doi:10.1074/jbc.270.18.10618

    Article  CAS  PubMed  Google Scholar 

  2. Saharinen J, Taipale J, Keski-Oja J (1996) Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. EMBO J 15(2):245–253. doi:10.1074/jbc.271.47.29891

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Annes JP, Munger JS, Rifkin DB (2003) Making sense of latent TGFbeta activation. J Cell Sci 116(Pt 2):217–224. doi:10.1242/jcs.00229

    Article  CAS  PubMed  Google Scholar 

  4. Tatler AL, Jenkins G (2012) TGF-beta activation and lung fibrosis. Proc Am Thorac Soc 9(3):130–136. doi:10.1513/pats.201201-003AW

    Article  CAS  PubMed  Google Scholar 

  5. Schultz-Cherry S, Murphy-Ullrich JE (1993) Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J Cell Biol 122(4):923–932. doi:10.1083/jcb.122.4.923

    Article  CAS  PubMed  Google Scholar 

  6. Jenkins RG, Su X, Su G et al (2006) Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury. J Clin Invest 116(6):1606–1614. doi:10.1172/JCI27183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wipff PJ, Rifkin DB, Meister JJ et al (2007) Myofibroblast contraction activates latent TGF- 1 from the extracellular matrix. J Cell Biol 179(6):1311–1323. doi:10.1083/jcb.200704042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tatler AL, John AE, Jolly L et al (2011) Integrin alphavbeta5-mediated TGF-beta activation by airway smooth muscle cells in asthma. J Immunol 187(11):6094–6107. doi:10.4049/jimmunol.1003507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang Z, Mu Z, Dabovic B et al (2007) Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice. J Cell Biol 176(6):787–793. doi:10.1083/jcb.200611044

  10. Henderson NC, Arnold TD, Katamura Y et al (2013) Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19(12):1617–1624. doi:10.1038/nm.3282

    Article  CAS  PubMed  Google Scholar 

  11. Tatler AL, Goodwin AT, Gbolahan O et al (2016) Amplification of TGFbeta Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis. PLoS One 11(8):e0158047. doi:10.1371/journal.pone.0158047

    Article  PubMed  PubMed Central  Google Scholar 

  12. Shi M, Zhu J, Wang R et al (2011) Latent TGF-beta structure and activation. Nature 474(7351):343–349. doi:10.1038/nature10152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xu MY, Porte J, Knox AJ et al (2009) Lysophosphatidic Acid Induces {alpha}v{beta}6 Integrin-Mediated TGF-{beta} Activation via the LPA2 Receptor and the Small G Protein G{alpha}q. Am J Pathol 174(4):1264–1279. doi:10.2353/ajpath.2009.080160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. John AE, Wilson MR, Habgood A et al (2016) Loss of epithelial Gq and G11 signaling inhibits TGFbeta production but promotes IL-33-mediated macrophage polarization and emphysema. Sci Signal 9(451):ra104. doi:10.1126/scisignal.aad5568

    Article  PubMed  Google Scholar 

  15. Abe M, Harpel JG, Metz CN et al (1994) An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem 216(2):276–284. doi:10.1006/abio.1994.1042

    Article  CAS  PubMed  Google Scholar 

  16. Munger JS, Huang X, Kawakatsu H et al (1999) The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96(3):319–328

    Article  CAS  PubMed  Google Scholar 

  17. Tatler AL, Porte J, Knox A et al (2008) Tryptase activates TGFbeta in human airway smooth muscle cells via direct proteolysis. Biochem Biophys Res Commun 370(2):239–242. doi:10.1016/j.bbrc.2008.03.064

    Article  CAS  PubMed  Google Scholar 

  18. Bedke N, Sammut D, Green B et al (2012) Transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response. PLoS One 7(9):e44580. doi:10.1371/journal.pone.0044580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Porte J, Jenkins G (2014) Assessment of the effect of potential antifibrotic compounds on total and alphaVbeta6 integrin-mediated TGF-beta activation. Pharmacol Res Perspect 2(4):e00030. doi:10.1002/prp2.30

    Article  PubMed  PubMed Central  Google Scholar 

  20. Jolly L, Stavrou A, Vanderstoken G et al (2014) Influenza Promotes Collagen Deposition via alphavbeta6-integrin Mediated Transforming Growth Factor beta Activation. J Biol Chem. doi:10.1074/jbc.M114.582262

    PubMed  PubMed Central  Google Scholar 

  21. Tatler AL, Barnes J, Habgood A et al (2016) Caffeine inhibits TGFbeta activation in epithelial cells, interrupts fibroblast responses to TGFbeta, and reduces established fibrosis in ex vivo precision-cut lung slices. Thorax 71(6):565–567. doi:10.1136/thoraxjnl-2015-208215

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Amanda L. Tatler .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

John, A.E., Porte, J., Jenkins, G., Tatler, A.L. (2017). Methods for the Assessment of Active Transforming Growth Factor-β in Cells and Tissues. In: Rittié, L. (eds) Fibrosis. Methods in Molecular Biology, vol 1627. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7113-8_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7113-8_23

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7112-1

  • Online ISBN: 978-1-4939-7113-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics