Cell and Tissue Research

, Volume 355, Issue 1, pp 163–171 | Cite as

TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling

  • Dennis F. G. Remst
  • Esmeralda N. Blaney Davidson
  • Elly L. Vitters
  • Ruud A. Bank
  • Wim B. van den Berg
  • Peter M. van der Kraan
Regular Article
First Online:
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Accepted:

Abstract

Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

Keywords

Lysyl hydroxylase 2b Fibrosis Osteoarthritis Synovium TGF-ß 

Abbreviations

Ad

Adenovirus

ALK

Activin receptor-like kinase

ca

Constitutively active

COL1A1

Collagen, type I, alpha 1

CTGF

Connective tissue growth factor

hSF

Human synovial fibroblasts

LH

Lysyl hydroxylase

MMP

Matrix metalloproteinases

PLOD2

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2

TGF-ß

Transforming growth factor-beta

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Notes

Acknowledgments

The authors would like to thank MJ Goumans and Peter ten Dijke (Leiden University Medical Center). This study was supported by a grant from the Dutch Arthritis Association (grant NR 09-1-403).

Supplementary material

441_2013_1740_Fig5_ESM.jpg (26 kb)
Fig. 1S

Human synovial fibroblasts (original magnification ×25) (JPEG 25 kb)

441_2013_1740_MOESM1_ESM.tif (748 kb)
High resolution image (TIFF 747 kb)
441_2013_1740_Fig6_ESM.jpg (22 kb)
Fig. 2S

Dorsomorphin inhibits caALK1-induced Smad1/5/8P. Human synovial fibroblasts were transduced with Ad-caALK1 or Ad-Luc, which was used as control and cultured for 24 h. Thereafter the cells were cultured for another 24 h in the presence of dorsomorphin or DMSO and subsequently harvested for Western blot analysis. Both the basal as well as the caALK1-induced Smad1/5/8P were inhibited by dorsomorphin (5 μM) (JPEG 21 kb)

References

  1. Arnott JA, Zhang X, Sanjay A, Owen TA, Smock SL, Rehman S, DeLong WG, Safadi FF, Popoff SN (2008) Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts. Bone 42:871–885PubMedCentralPubMedCrossRefGoogle Scholar
  2. Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, van der Kraan PM, Richards CD, van den Berg WB (2001) Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthr Cartil 9:128–136PubMedCrossRefGoogle Scholar
  3. Blaney Davidson EN, Vitters EL, Mooren FM, Oliver N, Berg WB, van der Kraan PM (2006) Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damage. Arthritis Rheum 54:1653–1661PubMedCrossRefGoogle Scholar
  4. Blaney Davidson EN, Remst DF, Vitters EL, van Beuningen HM, Blom AB, Goumans MJ, van den Berg WB, van der Kraan PM (2009) Increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice. J Immunol 182:7937–7945PubMedCrossRefGoogle Scholar
  5. Blobe GC, Schiemann WP, Lodish HF (2000) Role of transforming growth factor beta in human disease. N Engl J Med 342:1350–1358PubMedCrossRefGoogle Scholar
  6. Boergermann JH, Kopf J, Yu PB, Knaus P (2010) Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells. Int J Biochem Cell Biol 42:1802–1807PubMedCrossRefGoogle Scholar
  7. Bonniaud P, Margetts PJ, Kolb M, Haberberger T, Kelly M, Robertson J, Gauldie J (2003) Adenoviral gene transfer of connective tissue growth factor in the lung induces transient fibrosis. Am J Respir Crit Care Med 168:770–778PubMedCrossRefGoogle Scholar
  8. Bonniaud P, Margetts PJ, Kolb M, Schroeder JA, Kapoun AM, Damm D, Murphy A, Chakravarty S, Dugar S, Higgins L, Protter AA, Gauldie J (2005) Progressive transforming growth factor beta1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Med 171:889–898PubMedCrossRefGoogle Scholar
  9. DaCosta Byfield S, Major C, Laping NJ, Roberts AB (2004) SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol 65:744–752PubMedCrossRefGoogle Scholar
  10. Fuchshofer R, Birke M, Welge-Lussen U, Kook D, Lutjen-Drecoll E (2005) Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Investig Ophthalmol Vis Sci 46:568–578CrossRefGoogle Scholar
  11. Gordon KJ, Blobe GC (2008) Role of transforming growth factor-beta superfamily signaling pathways in human disease. Biochim Biophys Acta 1782:197–228PubMedCrossRefGoogle Scholar
  12. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P (2002) Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J 21:1743–1753PubMedCrossRefGoogle Scholar
  13. Hill CL, Hunter DJ, Niu J, Clancy M, Guermazi A, Genant H, Gale D, Grainger A, Conaghan P, Felson DT (2007) Synovitis detected on magnetic resonance imaging and its relation to pain and cartilage loss in knee osteoarthritis. Ann Rheum Dis 66:1599–1603PubMedCrossRefGoogle Scholar
  14. Kroening S, Solomovitch S, Sachs M, Wullich B, Goppelt-Struebe M (2009) Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells. Nephrol Dial Transplant 24:755–762PubMedCrossRefGoogle Scholar
  15. Leask A, Abraham DJ (2004) TGF-beta signaling and the fibrotic response. FASEB J 18:816–827PubMedCrossRefGoogle Scholar
  16. Liu IM, Schilling SH, Knouse KA, Choy L, Derynck R, Wang XF (2009) TGFbeta-stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFbeta switch. EMBO J 28:88–98PubMedCrossRefGoogle Scholar
  17. Mori T, Kawara S, Shinozaki M, Hayashi N, Kakinuma T, Igarashi A, Takigawa M, Nakanishi T, Takehara K (1999) Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: a mouse fibrosis model. J Cell Physiol 181:153–159PubMedCrossRefGoogle Scholar
  18. Mori Y, Chen SJ, Varga J (2003) Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts. Arthritis Rheum 48:1964–1978PubMedCrossRefGoogle Scholar
  19. Nakerakanti SS, Bujor AM, Trojanowska M (2011) CCN2 is required for the TGF-beta induced activation of Smad1-Erk1/2 signaling network. PLoS ONE 6:e21911PubMedCentralPubMedCrossRefGoogle Scholar
  20. Pannu J, Nakerakanti S, Smith E, ten Dijke P, Trojanowska M (2007) Transforming growth factor-beta receptor type I-dependent fibrogenic gene program is mediated via activation of Smad1 and ERK1/2 pathways. J Biol Chem 282:10405–10413PubMedCrossRefGoogle Scholar
  21. Pornprasertsuk S, Duarte WR, Mochida Y, Yamauchi M (2004) Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells. J Bone Miner Res 19:1349–1355PubMedCrossRefGoogle Scholar
  22. Remst DF, Blaney Davidson EN, Vitters EL, Blom AB, Stoop R, Snabel JM, Bank RA, van den Berg WB, van der Kraan PM (2013) Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression. Osteoarthr Cartil 21:157–164PubMedCrossRefGoogle Scholar
  23. Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB, Sporn MB, Thorgeirsson SS (1995) Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA 92:2572–2576PubMedCrossRefGoogle Scholar
  24. Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB (2003) Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein inhibitors during experimental osteoarthritis. Arthritis Rheum 48:3442–3451PubMedCrossRefGoogle Scholar
  25. Sciaky D, Brazer W, Center DM, Cruikshank WW, Smith TJ (2000) Cultured human fibroblasts express constitutive IL-16 mRNA: cytokine induction of active IL-16 protein synthesis through a caspase-3-dependent mechanism. J Immunol 164:3806–3814PubMedGoogle Scholar
  26. ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869PubMedCrossRefGoogle Scholar
  27. van Beuningen HM, van der Kraan PM, Arntz OJ, van den Berg WB (1993) Does TGF-beta protect articular cartilage in vivo? Agents Actions Suppl 39:127–131PubMedGoogle Scholar
  28. van der Slot AJ, Zuurmond AM, Bardoel AF, Wijmenga C, Pruijs HE, Sillence DO, Brinckmann J, Abraham DJ, Black CM, Verzijl N, DeGroot J, Hanemaaijer R, TeKoppele JM, Huizinga TW, Bank RA (2003) Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis. J Biol Chem 278:40967–40972PubMedCrossRefGoogle Scholar
  29. van der Slot AJ, Zuurmond AM, van den Bogaerdt AJ, Ulrich MM, Middelkoop E, Boers W, Karel Ronday H, DeGroot J, Huizinga TW, Bank RA (2004) Increased formation of pyridinoline cross-links due to higher telopeptide lysyl hydroxylase levels is a general fibrotic phenomenon. Matrix Biol 23:251–257PubMedCrossRefGoogle Scholar
  30. van der Slot AJ, van Dura EA, de Wit EC, De Groot J, Huizinga TW, Bank RA, Zuurmond AM (2005) Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels. Biochim Biophys Acta 1741:95–102PubMedCrossRefGoogle Scholar
  31. Vogt J, Traynor R, Sapkota GP (2011) The specificities of small molecule inhibitors of the TGFss and BMP pathways. Cell Signal 23:1831–1842PubMedCrossRefGoogle Scholar
  32. Wrighton KH, Lin X, Yu PB, Feng XH (2009) Transforming growth factor beta can stimulate Smad1 phosphorylation independently of bone morphogenic protein receptors. J Biol Chem 284:9755–9763PubMedCrossRefGoogle Scholar
  33. Wu J, Reinhardt DP, Batmunkh C, Lindenmaier W, Far RK, Notbohm H, Hunzelmann N, Brinckmann J (2006) Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts. Exp Cell Res 312:3485–3494PubMedCrossRefGoogle Scholar
  34. Zhang X, Arnott JA, Rehman S, Delong WG Jr, Sanjay A, Safadi FF, Popoff SN (2010) Src is a major signaling component for CTGF induction by TGF-beta1 in osteoblasts. J Cell Physiol 224:691–701PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Dennis F. G. Remst
    • 1
  • Esmeralda N. Blaney Davidson
    • 1
  • Elly L. Vitters
    • 1
  • Ruud A. Bank
    • 2
  • Wim B. van den Berg
    • 1
  • Peter M. van der Kraan
    • 1
  1. 1.Rheumatology Research and Advanced TherapeuticsRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  2. 2.Medical Biology Section, Stem Cell and Tissue Engineering GroupUniversity Medical Center GroningenGroningenThe Netherlands

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