Skip to main content
SpringerLink
Log in

Dynamic compression alters NFκB activation and IκB-α expression in IL-1β-stimulated chondrocyte/agarose constructs

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Objective and design

Determine the effect of IL-1β and dynamic compression on NFκB activation and IκB-α gene expression in chondrocyte/agarose constructs.

Methods

Constructs were cultured under free-swelling conditions or subjected to dynamic compression for up to 360 min with IL-1β and/or PDTC (inhibits NFκB activation). Nuclear translocation of NFκB-p65 was analysed by immunofluoresence microscopy. Gene expression of IκB-α, iNOS, IL-1β and IL-4 was assessed by real-time qPCR.

Results

Nuclear translocation of NFκB-p65 was concomitant with an increase in nuclear fluorescence intensity which reached maximum values at 60 min with IL-1β (p < 0.001). Dynamic compression or PDTC reduced nuclear fluorescence and NFκB nuclear translocation in cytokine-treated constructs (p < 0.001 and p < 0.01 respectively). IL-1β increased IκB-α expression (p < 0.001) at 60 min and either induced iNOS (p < 0.001) and IL-1β (p < 0.01) or inhibited IL-4 (p < 0.05) expression at 360 min. These time-dependent events were partially reversed by dynamic compression or PDTC (p < 0.01) with IL-1β. Co-stimulation by dynamic compression and PDTC favoured suppression (IκB-α, iNOS, IL-1β) or induction (IL-4) of gene expression.

Conclusions

NFκB is one of the key players in the mechanical and inflammatory pathways, and its inhibition by a biophysical/therapeutic approach could be a strategy for attenuating the catabolic response in osteoarthritis.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

IL-1β:

Interleukin-1β

NFκB:

Nuclear factor-kappa B

IκB-α:

Inhibitory κB-α

iNOS:

Inducible nitric oxide synthase

IL-4:

Interleukin-4

References

  1. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002;39(1/2):237–46.

    CAS  PubMed  Google Scholar 

  2. Berenbaum F. Signaling transduction: target in osteoarthritis. Curr Opin Rheumatol. 2004;16(5):616–22.

    Article  PubMed  Google Scholar 

  3. Malemud CJ. Protein kinases in chondrocyte signalling and osteoarthritis. Clin Orthop Relat Res. 2004;427(Suppl):145–51.

    Article  Google Scholar 

  4. Griffin TM, Guilak F. The role of mechanical loading in the onset and progression of osteoarthritis. Exerc Sport Sci Rev. 2005;33:195–200.

    Article  PubMed  Google Scholar 

  5. Millward-Sadler SJ, Salter DM. Integrin-dependent signal cascades in chondrocyte mechanotransduction. Ann Biomed Eng. 2004;32(3):435–46.

    Article  CAS  PubMed  Google Scholar 

  6. Guilak F, Fermor B, Keefe FJ, Kraus VB, Olson SA, Pisetsky DS, et al. The role of biomechanics and inflammation in cartilage repair and injury. Clin Orthop Relat Res. 2004;423:17–26.

    Article  PubMed  Google Scholar 

  7. Vincent TL, Saklatvala J. Is the response of cartilage to injury relevant to osteoarthritis? Arthritis Rheum. 2008;58(5):1207–10.

    Article  CAS  PubMed  Google Scholar 

  8. Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol. 2007;213(3):626–34.

    Article  CAS  PubMed  Google Scholar 

  9. Saklatvala J. Inflammatory signalling in cartilage. MAPK and NFκB pathways in chondrocytes and the use of inhibitors for research into pathogenesis and therapy of OA. Curr Drug Targets. 2007;8:305–13.

    Article  CAS  PubMed  Google Scholar 

  10. Malemud CJ. Inhibitors of stress-activated protein/mitogen-activated protein kinase pathways. Curr Opin Pharmacol. 2007;7(3):339–43.

    Article  CAS  PubMed  Google Scholar 

  11. Kobayashi M, Squires GR, Mousa A, Tanzer M, Zukor DJ, Antoniou J, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum. 2005;52(1):128–35.

    Article  CAS  PubMed  Google Scholar 

  12. Auron PE. The interleukin 1 receptor: ligand interactions and signal transduction. Cytokine Growth Factor Rev. 1998;9(3/4):221–37.

    Article  CAS  PubMed  Google Scholar 

  13. Newton R, Stevens DA, Hart LA, Lindsay M, Adcock IM, Barnes PJ. Superinduction of COX-2 mRNA by cycloheximide and interleukin-1beta involves increased transcription and correlates with increased NF-kappaB and JNK activation. FEBS Lett. 1997;418(1/2):135–8.

    Article  CAS  PubMed  Google Scholar 

  14. Fan Z, Yang H, Bau B, Söder S, Aigner T. Role of mitogen-activated protein kinases and NFkappaB on IL-1beta-induced effects on collagen type II, MMP-1 and 13 mRNA expression in normal articular human chondrocytes. Rheumatol Int. 2006;26(10):900–3.

    Article  CAS  PubMed  Google Scholar 

  15. Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of IL-1 stimulated MAP kinases, AP-1 and NF-kappa B transcription factors down-regulates MMP gene expression in articular chondrocytes. Matrix Biol. 2002;21:251–62.

    Article  CAS  PubMed  Google Scholar 

  16. Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. IL-1 induction of MMP-13 gene expression in chondrocytes requires p38 c-Jun N-terminal kinase and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 2000;43:801–11.

    Article  CAS  PubMed  Google Scholar 

  17. Tsutsumi R, Ito H, Hiramitsu T, Nishitani K, Akiyoshi M, Kitaori T, et al. Celecoxib inhibits production of MMP and NO via down-regulation of NF-kappaB and JNK in a PGE2 independent manner in human articular chondrocytes. Rheumatol Int. 2008;28(8):727–36.

    Article  CAS  PubMed  Google Scholar 

  18. Roman-Blas JA, Jimenez SA. NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis. Osteoarthr Cartil. 2006;14(9):839–48.

    Article  CAS  PubMed  Google Scholar 

  19. Aigner T, McKenna L, Zien A, Fan Z, Gebhard PM, Zimmer R. Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes—a molecular analysis based on chondrocytes isolated from one donor. Cytokine. 2005;31(3):227–40.

    Google Scholar 

  20. Brown K, Park S, Kanno T, Franzoso G, Siebenlist U. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl Acad Sci USA. 1993;90(6):2532–6.

    Article  CAS  PubMed  Google Scholar 

  21. Zabel U, Henkel T, Silva MS, Baeuerle PA. Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus. EMBO J. 1993;12(1):201–11.

    CAS  PubMed  Google Scholar 

  22. Bottero V, Imbert V, Frelin C, Formento JL, Peyron JF. Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression. Mol Diagn. 2003;7(3/4):187–94.

    Google Scholar 

  23. Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nat Immunol. 2002;3(3):221–7.

    Article  CAS  PubMed  Google Scholar 

  24. Rodriguez MS, Thompson J, Hay RT, Dargemont C. Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation. J Biol Chem. 1999;274(13):9108–15.

    Article  CAS  PubMed  Google Scholar 

  25. Agarwal S, Deschner J, Long P, Verma A, Hofman C, Evans CH, et al. Role of NF-kappaB transcription factors in anti-inflammatory and pro-inflammatory actions of mechanical signals. Arthritis Rheum. 2004;50:3541–8.

    Article  CAS  PubMed  Google Scholar 

  26. Deschner J, Hofman CR, Piesco NP, Agarwal S. Signal transduction by mechanical strain in chondrocytes. Curr Opin Clin Nutr Metab Care. 2003;6:289–93.

    Article  CAS  PubMed  Google Scholar 

  27. Chowdhury TT, Bader DL, Lee DA. Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes. Biochem Biophys Res Commun. 2006;339(1):241–7.

    Article  CAS  PubMed  Google Scholar 

  28. Doi H, Nishida K, Yorimitsu M, Komiyama T, Kadota Y, Tetsunaga T, et al. Interleukin-4 downregulates the cyclic tensile stress-induced matrix metalloproteinases-13 and cathepsin B expression by rat normal chondrocytes. Acta Med Okayama. 2008;62(2):119–26.

    CAS  PubMed  Google Scholar 

  29. Madhavan S, Anghelina M, Rath-Deschner B, Wypasek E, John A, Deschner J, et al. Biomechanical signals exert sustained attenuation of proinflammatory gene induction in articular chondrocytes. Osteoarthr Cartil. 2006;14:1023–32.

    Article  CAS  PubMed  Google Scholar 

  30. Chowdhury TT, Arghandawi S, Brand J, Akanji OO, Bader DL, Salter DM, et al. Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs. Arthritis Res Ther. 2008;10(2):R35.

    Article  CAS  PubMed  Google Scholar 

  31. Lee DA, Bader DL. Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J Orthop Res. 1997;15:181–8.

    Article  PubMed  Google Scholar 

  32. Lee DA, Knight MM. Mechanical loading of chondrocytes embedded in 3D constructs: in vitro methods for assessment of morphological and metabolic response to compressive strain. Methods Mol Med. 2004;100:307–24.

    CAS  PubMed  Google Scholar 

  33. Chowdhury TT, Salter DM, Bader DL, Lee DA. Signal transduction pathways involving p38 MAPK, JNK, NFkappaB and AP-1 influences the response of chondrocytes cultured in agarose constructs to IL-1beta and dynamic compression. Inflamm Res. 2008;57(7):306–13.

    Article  CAS  PubMed  Google Scholar 

  34. Meyer M, Schreck R, Baeuerle PA. H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 1993;12(5):2005–15.

    CAS  PubMed  Google Scholar 

  35. Marks-Konczalik J, Chu SC, Moss J. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both AP-1 and NF-κB binding sites. J Biol Chem. 1998;273:22201–8.

    Article  CAS  PubMed  Google Scholar 

  36. Chowdhury TT, Bader DL, Shelton JC, Lee DA. Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression. Arch Biochem Biophys. 2003;417(1):105–11.

    Article  CAS  PubMed  Google Scholar 

  37. Raveenthiran SP, Chowdhury TT. Dynamic compression inhibits fibronectin fragment induced iNOS and COX-2 expression in chondrocyte/agarose constructs. Biomech Model Mechanobiol. 2008. doi: 10.1007/s10237-008-0134-1

  38. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group wise comparison and statistical analysis of relative expression results in real time PCR. Nucl Acids Res. 2002;30:e3.

    Article  Google Scholar 

  39. Clancy RM, Gomez PF, Abramson SB. Nitric oxide sustains nuclear factor kappaB activation in cytokine-stimulated chondrocytes. Osteoarthr Cartil. 2004;12(7):552–8.

    Article  CAS  PubMed  Google Scholar 

  40. Delhase M, Hayakawa M, Chen Y, Karin M. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science. 1999;284(5412):309–13

    Google Scholar 

  41. Schmitz ML, dos Santos Silva MA, Baeuerle PA. Transactivation domain 2 (TA2) of p65 NF-kappa B similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. J Biol Chem. 1995;270(26):15576–84.

    Article  CAS  PubMed  Google Scholar 

  42. Carlotti F, Chapman R, Dower SK, Qwarnstrom EE. Activation of nuclear factor kappaB in single living cells. Dependence of nuclear translocation and anti-apoptotic function on EGFPRELA concentration. J Biol Chem. 1999;274(53):37941–9.

    Article  CAS  PubMed  Google Scholar 

  43. Ding GJ, Fischer PA, Boltz RC, Schmidt JA, Colaianne JJ, Gough A, et al. Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha: development and use of a high capacity fluorescence cytometric system. J Biol Chem. 1998;273(44):28897–905.

    Article  CAS  PubMed  Google Scholar 

  44. Goldring MB, Otero M, Tsuchimochi K, Ijiri K, Li Y. Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism. Ann Rheum Dis. 2008;67(Suppl 3):iii75–82.

    Article  CAS  PubMed  Google Scholar 

  45. Scherle PA, Pratta MA, Feeser WS, Tancula EJ, Arner EC. The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes. Biochem Biophys Res Commun. 1997;230:573–7.

    Article  CAS  PubMed  Google Scholar 

  46. Kracht M, Saklatvala J. Transcriptional and post-transcriptional control of gene expression in inflammation. Cytokine. 2002;20(3):91–106.

    Article  CAS  PubMed  Google Scholar 

  47. Mendes AF, Carvalho AP, Caramona MM, Lopes MC. Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS II expression in articular chondrocytes. Inflamm Res. 2002;51:369–75.

    Article  CAS  PubMed  Google Scholar 

  48. Malinin NL, Boldin MP, Kovalenko AV, Wallach D. MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1. Nature. 1997;385(6616):540–4.

    Article  CAS  PubMed  Google Scholar 

  49. Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science. 1996;271(5252):1128–31.

    Article  CAS  PubMed  Google Scholar 

  50. Chowdhury TT, Salter DM, Bader DL, Lee DA. Integrin-mediated mechanotransduction processes in TGFβ stimulated monolayer-expanded chondrocytes. Biochem Biophys Res Commun. 2004;318:873–81.

    Article  CAS  PubMed  Google Scholar 

  51. Chowdhury TT, Appleby RN, Salter DM, Bader DL, Lee DA. Integrin-mediated mechanotransduction in IL-1β stimulated bovine chondrocytes cultured in agarose constructs. Biomech Model Mechanobiol. 2006;5(2/3):192–201.

    Article  CAS  PubMed  Google Scholar 

  52. Millward-Sadler SJ, Khan NS, Bracher MG, Wright MO, Salter DM. Roles for the interleukin-4 receptor and associated JAK/STAT proteins in human articular chondrocyte mechanotransduction. Osteoarthr Cartil. 2006;14(10):991–1001.

    Article  CAS  PubMed  Google Scholar 

  53. Salter DM, Millward-Sadler SJ, Nuki G, Wright MO. Integrin-interleukin-4 mechanotransduction pathways in human chondrocytes. Clin Orthop Relat Res. 2001;391(Suppl):S49–60.

    Google Scholar 

  54. Vincent TL, McLean CJ, Full LE, Peston D, Saklatvala J. FGF-2 is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer. Osteoarthr Cartil. 2007;15(7):752–63.

    Article  CAS  PubMed  Google Scholar 

  55. Geng Y, Lotz M. Increased intracellular Ca2+ selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability. J Cell Biol. 1995;129(6):1651–7.

    Article  CAS  PubMed  Google Scholar 

  56. Chowdhury TT, Knight MM. Purinergic pathway suppresses the release of NO and stimulates proteoglycan synthesis in chondrocyte/agarose constructs subjected to dynamic compression. J Cell Phys. 2006;209(3):845–53.

    Article  CAS  Google Scholar 

  57. Millward-Sadler SJ, Wright MO, Flatman PW, Salter DM. ATP in the mechanotransduction pathway of normal human chondrocytes. Biorheology. 2004;41:567–75.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This project was funded by the Welcome Trust (project grant: 073972).

Author information

Authors and Affiliations

  1. School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, E1 4NS, London, UK

    O. O. Akanji, P. Sakthithasan & T. T. Chowdhury

  2. Queens Medical Research Institute, Edinburgh University, Edinburgh, UK

    D. M. Salter

Authors
  1. O. O. Akanji
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Sakthithasan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. M. Salter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. T. Chowdhury
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. T. Chowdhury.

Additional information

Responsible Editor: J. Di Battista.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Akanji, O.O., Sakthithasan, P., Salter, D.M. et al. Dynamic compression alters NFκB activation and IκB-α expression in IL-1β-stimulated chondrocyte/agarose constructs. Inflamm. Res. 59, 41–52 (2010). https://doi.org/10.1007/s00011-009-0068-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-009-0068-9

Keywords

Search

Navigation