Aberrant hypertrophy in Smad3-deficient murine chondrocytes is rescued by restoring transforming growth factor β–activated kinase 1/activating transcription factor 2 signaling: A potential clinical implication for osteoarthritis
Yrjö T. Konttinen
Helsinki University Central Hospital, Helsinki, ORTON Orthopaedic Hospital of the ORTON Foundation, Helsinki, and COXA Hospital for Joint Replacement, Tampere, Finland
Search for more papers by this authorJennifer H. Jonason
University of Rochester, Rochester, New York
Search for more papers by this authorCorresponding Author
Regis J. O'Keefe
University of Rochester, Rochester, New York
Center for Musculoskeletal Research, University of Rochester, 601 Elmwood Avenue, Box 665, Rochester, NY 14642Search for more papers by this authorYrjö T. Konttinen
Helsinki University Central Hospital, Helsinki, ORTON Orthopaedic Hospital of the ORTON Foundation, Helsinki, and COXA Hospital for Joint Replacement, Tampere, Finland
Search for more papers by this authorJennifer H. Jonason
University of Rochester, Rochester, New York
Search for more papers by this authorCorresponding Author
Regis J. O'Keefe
University of Rochester, Rochester, New York
Center for Musculoskeletal Research, University of Rochester, 601 Elmwood Avenue, Box 665, Rochester, NY 14642Search for more papers by this authorAbstract
Objective
To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor β (TGFβ) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.
Methods
Joint disease in Smad3-knockout (Smad3−/−) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3−/− mice.
Results
In Smad3−/− mice, an end-stage OA phenotype gradually developed. TGFβ-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3−/− mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3−/− cells restored the normal p38 response to TGFβ. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3−/− chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms α, β, and γ, but not δ.
Conclusion
Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3–phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.
REFERENCES
- 1 Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol 2007; 213: 626–34.
- 2 Hardingham T. Extracellular matrix and pathogenic mechanisms in osteoarthritis. Curr Rheumatol Rep 2008; 10: 30–6.
- 3 Blaney Davidson EN, van der Kraan PM, van den Berg WB. TGF-β and osteoarthritis. Osteoarthritis Cartilage 2007; 15: 597–604.
- 4 Drissi H, Zuscik M, Rosier R, O'Keefe R. Transcriptional regulation of chondrocyte maturation: potential involvement of transcription factors in OA pathogenesis. Mol Aspects Med 2005; 26: 169–79.
- 5 Hashimoto M, Nakasa T, Hikata T, Asahara H. Molecular network of cartilage homeostasis and osteoarthritis. Med Res Rev 2008; 28: 464–81.
- 6 Kobayashi T, Lyons KM, McMahon AP, Kronenberg HM. BMP signaling stimulates cellular differentiation at multiple steps during cartilage development. Proc Natl Acad Sci U S A 2005; 102: 18023–7.
- 7 Li TF, O'Keefe RJ, Chen D. TGF-β signaling in chondrocytes. Front Biosci 2005; 10: 681–8.
- 8 Feng XH, Derynck R. Specificity and versatility in TGF-β signaling through Smads. Annu Rev Cell Dev Biol 2005; 21: 659–93.
- 9 Massague J, Seoane J, Wotton D. Smad transcription factors. Genes Dev 2005; 19: 2783–810.
- 10 Grimm OH, Gurdon JB. Nuclear exclusion of Smad2 is a mechanism leading to loss of competence. Nat Cell Biol 2002; 4: 519–22.
- 11 Matsuura I, Denissova NG, Wang G, He D, Long J, Liu F. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature 2004; 430: 226–31.
- 12 Alarcon C, Zaromytidou AI, Xi Q, Gao S, Yu J, Fujisawa S, et al. Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-β pathways. Cell 2009; 139: 757–69.
- 13 Wang G, Matsuura I, He D, Liu F. Transforming growth factor-β-inducible phosphorylation of Smad3. J Biol Chem 2009; 284: 9663–73.
- 14 Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 1999; 400: 687–93.
- 15 Lin X, Liang M, Feng XH. Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling. J Biol Chem 2000; 275: 36818–22.
- 16 Gao S, Alarcon C, Sapkota G, Rahman S, Chen PY, Goerner N, et al. Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling. Mol Cell 2009; 36: 457–68.
- 17 Serra R, Johnson M, Filvaroff EH, LaBorde J, Sheehan DM, Derynck R, et al. Expression of a truncated, kinase-defective TGF-β type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis. J Cell Biol 1997; 139: 541–52.
- 18 Yang X, Chen L, Xu X, Li C, Huang C, Deng CX. TGF-β/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J Cell Biol 2001; 153: 35–46.
- 19 Li TF, Darowish M, Zuscik MJ, Chen D, Schwarz EM, Rosier RN, et al. Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation. J Bone Miner Res 2006; 21: 4–16.
- 20 Moustakas A, Heldin CH. Non-Smad TGF-β signals. J Cell Sci 2005; 118: 3573–84.
- 21 Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, et al. Identification of a member of the MAPKKK family as a potential mediator of TGF-β signal transduction. Science 1995; 270: 2008–11.
- 22 Shim JH, Greenblatt MB, Xie M, Schneider MD, Zou W, Zhai B, et al. TAK1 is an essential regulator of BMP signalling in cartilage. EMBO J 2009; 28: 2028–41.
- 23 Gunnell LM, Jonason JH, Loiselle AE, Kohn A, Schwarz EM, Hilton MJ, et al. TAK1 regulates cartilage and joint development via the MAPK and BMP signaling pathways. J Bone Miner Res 2010. E-pub ahead of print.
- 24 Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, et al. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-β-induced gene expression. J Biol Chem 1999; 274: 27161–7.
- 25 Moriguchi T, Kuroyanagi N, Yamaguchi K, Gotoh Y, Irie K, Kano T, et al. A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J Biol Chem 1996; 271: 13675–9.
- 26 Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 1996; 16: 1247–55.
- 27 Stanton LA, Underhill TM, Beier F. MAP kinases in chondrocyte differentiation. Dev Biol 2003; 263: 165–75.
- 28 Zhang R, Murakami S, Coustry F, Wang Y, de Crombrugghe B. Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation. Proc Natl Acad Sci U S A 2006; 103: 365–70.
- 29 Namdari S, Wei L, Moore D, Chen Q. Reduced limb length and worsened osteoarthritis in adult mice after genetic inhibition of p38 MAP kinase activity in cartilage. Arthritis Rheum 2008; 58: 3520–9.
- 30 Ionescu AM, Schwarz EM, Zuscik MJ, Drissi H, Puzas JE, Rosier RN, et al. ATF-2 cooperates with Smad3 to mediate TGF-β effects on chondrocyte maturation. Exp Cell Res 2003; 288: 198–207.
- 31 Sano Y, Harada J, Tashiro S, Gotoh-Mandeville R, Maekawa T, Ishii S. ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-β signaling. J Biol Chem 1999; 274: 8949–57.
- 32 Reimold AM, Grusby MJ, Kosaras B, Fries JW, Mori R, Maniwa S, et al. Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice. Nature 1996; 379: 262–5.
- 33 Beier F, Lee RJ, Taylor AC, Pestell RG, LuValle P. Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. Proc Natl Acad Sci U S A 1999; 96: 1433–8.
- 34 Breitwieser W, Lyons S, Flenniken AM, Ashton G, Bruder G, Willington M, et al. Feedback regulation of p38 activity via ATF2 is essential for survival of embryonic liver cells. Genes Dev 2007; 21: 2069–82.
- 35 Dickinson RJ, Keyse SM. Diverse physiological functions for dual-specificity MAP kinase phosphatases. J Cell Sci 2006; 119: 4607–15.
- 36 Zhu M, Chen M, Zuscik M, Wu Q, Wang YJ, Rosier RN, et al. Inhibition of β-catenin signaling in articular chondrocytes results in articular cartilage destruction. Arthritis Rheum 2008; 58: 2053–64.
- 37 Grimaud E, Heymann D, Redini F. Recent advances in TGF-β effects on chondrocyte metabolism: potential therapeutic roles of TGF-β in cartilage disorders. Cytokine Growth Factor Rev 2002; 13: 241–57.
- 38 Van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB. Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-β injections. Osteoarthritis Cartilage 2000; 8: 25–33.
- 39 Beier F, Taylor AC, LuValle P. Activating transcription factor 2 is necessary for maximal activity and serum induction of the cyclin A promoter in chondrocytes. J Biol Chem 2000; 275: 12948–53.
- 40 Vale-Cruz DS, Ma Q, Syme J, LuValle PA. Activating transcription factor-2 affects skeletal growth by modulating pRb gene expression. Mech Dev 2008; 125: 843–56.
- 41 Owens DM, Keyse SM. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene 2007; 26: 3203–13.
- 42 Tong XK, Hamel E. Transforming growth factor-β1 impairs endothelin-1-mediated contraction of brain vessels by inducing mitogen-activated protein (MAP) kinase phosphatase-1 and inhibiting p38 MAP kinase. Mol Pharmacol 2007; 72: 1476–83.
- 43 Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, et al. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 1996; 97: 761–8.
- 44 Little CB, Barai A, Burkhardt D, Smith SM, Fosang AJ, Werb Z, et al. Matrix metalloproteinase 13–deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development. Arthritis Rheum 2009; 60: 3723–33.
- 45 Neuhold LA, Killar L, Zhao W, Sung ML, Warner L, Kulik J, et al. Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. J Clin Invest 2001; 107: 35–44.
- 46 Leivonen SK, Chantry A, Hakkinen L, Han J, Kahari VM. Smad3 mediates transforming growth factor-β-induced collagenase-3 (matrix metalloproteinase-13) expression in human gingival fibroblasts: evidence for cross-talk between Smad3 and p38 signaling pathways. J Biol Chem 2002; 277: 46338–46.
- 47 Stanton LA, Sabari S, Sampaio AV, Underhill TM, Beier F. p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation. Biochem J 2004; 378: 53–62.
- 48 Korb A, Tohidast-Akrad M, Cetin E, Axmann R, Smolen J, Schett G. Differential tissue expression and activation of p38 MAPK α, β, γ, and δ isoforms in rheumatoid arthritis. Arthritis Rheum 2006; 54: 2745–56.
- 49 Schett G, Zwerina J, Firestein G. The p38 mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis. Ann Rheum Dis 2008; 67: 909–16.
