Involvement of ADAM12 in Chondrocyte Differentiation by Regulation of TGF-β1–Induced IGF-1 and RUNX-2 Expressions

  • Masahiro Horita
  • Keiichiro NishidaEmail author
  • Joe Hasei
  • Takayuki Furumatsu
  • Miwa Sakurai
  • Yuta Onodera
  • Kanji Fukuda
  • Donald M. Salter
  • Toshifumi Ozaki
Original Research


A disintegrin and metalloproteinase 12 (ADAM12) is known to be involved in chondrocyte proliferation and maturation; however, the mechanisms are not fully understood. In this study, expression and localization of ADAM12 during chondrocyte differentiation were examined in the mouse growth plate by immunohistochemistry. Adam12 expression during ATDC5 chondrogenic differentiation was examined by real-time PCR and compared with the expression pattern of type X collagen. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to generate Adam12-knockout (KO) ATDC5 cells. Adam12-KO and Adam12 overexpressing cells were used for analyses of ADAM12 expression with or without TGF-β1 stimulation. ADAM12 was identified predominantly in chondrocytes of the proliferative zone in mouse growth plates by immunohistochemistry. Adam12 was upregulated prior to Col10a1 during chondrogenic differentiation in wild-type ATDC5 cells. In Adam12-KO ATDC5 cells, following initiation of chondrogenic differentiation, we observed a reduction in Igf-1 expression along with an upregulation of hypertrophy-associated Runx2, Col10a1, and type X collagen protein expressions. In ATDC5 wild-type cells, stimulation with TGF-β1 upregulated the expressions of Adam12 and Igf-1 and downregulated the expression of Runx2. In contrast, in Adam12-KO ATDC5 cells, these TGF-β1-induced changes were suppressed. Adam12 overexpression resulted in an upregulation of Igf-1 and downregulation of Runx2 expression in ATDC5 cells. The findings suggest that ADAM12 has important role in the regulation of chondrocyte differentiation, potentially by regulation of TGF-β1-dependent signaling and that targeting of ADAM12 may have a role in management of abnormal chondrocyte differentiation.


Chondrogenic differentiation ADAM12 IGF-1 RUNX2 Type X collagen 



This work was supported by the Japan Society for the Promotion of Science (Nos. 16K20055, 17K11010). The authors thank Ms. Aki Yoshida and Ami Maehara for their technical cooperation.

Author Contributions

MH: Study design, Data collection, Data analysis and interpretation, Manuscript preparation. KN: Study design, Data analysis and interpretation, Manuscript preparation, Final approval of paper. JH: Study design, Data analysis and interpretation. TF: Data interpretation, Critical revision of the article. MS: Data collection. YO: Data collection. KF: Data interpretation, Critical revision of the article. DMS: Data analysis and interpretation, Critical revision of the article. TO: Data interpretation, Critical revision of the article. All authors revised the paper critically for intellectual content and approved the final version. All authors agree to be accountable for the work and to ensure that any questions relating to the accuracy and integrity of the paper are investigated and properly resolved.

Compliance with Ethical Standards

Conflict of interest

Masahiro Horita, Keiichiro Nishida, Joe Hasei, Takayuki Furumatsu, Miwa Sakurai, Yuta Onodera, Kanji Fukuda, Donald M. Salter and Toshifumi Ozaki declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

All procedures and protocols performed in studies were approved by the Animal Care and Use Committee of Okayama University (approval number: OKU-2018582). This article does not contain any studies with human participants performed by any of the authors.


  1. 1.
    Campbell JT, Kaplan FS (1992) The role of morphogens in endochondral ossification. Calcif Tissue Int 50(3):283–289CrossRefGoogle Scholar
  2. 2.
    Kamekura S, Hoshi K, Shimoaka T, Chung U, Chikuda H, Yamada T, Uchida M, Ogata N, Seichi A, Nakamura K, Kawaguchi H (2005) Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthr Cartil 13(7):632–641. CrossRefGoogle Scholar
  3. 3.
    Kawaguchi H (2009) Regulation of osteoarthritis development by Wnt-beta-catenin signaling through the endochondral ossification process. J Bone Miner Res 24(1):8–11. CrossRefGoogle Scholar
  4. 4.
    Provot S, Schipani E (2005) Molecular mechanisms of endochondral bone development. Biochem Biophys Res Commun 328(3):658–665. CrossRefGoogle Scholar
  5. 5.
    Gilpin BJ, Loechel F, Mattei MG, Engvall E, Albrechtsen R, Wewer UM (1998) A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo. J Biol Chem 273(1):157–166CrossRefGoogle Scholar
  6. 6.
    Loechel F, Gilpin BJ, Engvall E, Albrechtsen R, Wewer UM (1998) Human ADAM 12 (meltrin alpha) is an active metalloprotease. J Biol Chem 273(27):16993–16997CrossRefGoogle Scholar
  7. 7.
    Kurisaki T, Masuda A, Osumi N, Nabeshima Y, Fujisawa-Sehara A (1998) Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo. Mech Dev 73(2):211–215CrossRefGoogle Scholar
  8. 8.
    Verrier S, Hogan A, McKie N, Horton M (2004) ADAM gene expression and regulation during human osteoclast formation. Bone 35(1):34–46. CrossRefGoogle Scholar
  9. 9.
    Kveiborg M, Albrechtsen R, Rudkjaer L, Wen G, Damgaard-Pedersen K, Wewer UM (2006) ADAM12-S stimulates bone growth in transgenic mice by modulating chondrocyte proliferation and maturation. J Bone Miner Res 21(8):1288–1296. CrossRefGoogle Scholar
  10. 10.
    Loechel F, Fox JW, Murphy G, Albrechtsen R, Wewer UM (2000) ADAM12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3. Biochem Biophys Res Commun 278(3):511–515. CrossRefGoogle Scholar
  11. 11.
    Okada A, Mochizuki S, Yatabe T, Kimura T, Shiomi T, Fujita Y, Matsumoto H, Sehara-Fujisawa A, Iwamoto Y, Okada Y (2008) ADAM-12 (meltrin alpha) is involved in chondrocyte proliferation via cleavage of insulin-like growth factor binding protein 5 in osteoarthritic cartilage. Arthritis Rheum 58(3):778–789. CrossRefGoogle Scholar
  12. 12.
    Atsumi T, Miwa Y, Kimata K, Ikawa Y (1990) A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. Cell Differ Dev 30(2):109–116CrossRefGoogle Scholar
  13. 13.
    Shukunami C, Shigeno C, Atsumi T, Ishizeki K, Suzuki F, Hiraki Y (1996) Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol 133(2):457–468CrossRefGoogle Scholar
  14. 14.
    Choi HJ, Nepal M, Park YR, Lee HK, Oh SR, Soh Y (2011) Stimulation of chondrogenesis in ATDC5 chondroprogenitor cells and hypertrophy in mouse by Genkwadaphnin. Eur J Pharmacol 655(1–3):9–15. CrossRefGoogle Scholar
  15. 15.
    Sato E, Ando T, Ichikawa J, Okita G, Sato N, Wako M, Ohba T, Ochiai S, Hagino T, Jacobson R, Haro H (2014) High molecular weight hyaluronic acid increases the differentiation potential of the murine chondrocytic ATDC5 cell line. J Orthop Res 32(12):1619–1627. CrossRefGoogle Scholar
  16. 16.
    Onodera Y, Teramura T, Takehara T, Fukuda K (2013) c-Jun N-terminal kinase (JNK) mediates Rho/ROCK induced Sox9 diminution in chondrocytes. Acta Med Kinki Univ 38(2):91–100Google Scholar
  17. 17.
    Hougaard S, Loechel F, Xu X, Tajima R, Albrechtsen R, Wewer UM (2000) Trafficking of human ADAM 12-L: retention in the trans-Golgi network. Biochem Biophys Res Commun 275(2):261–267. CrossRefGoogle Scholar
  18. 18.
    Kojima I, Iikubo M, Kobayashi A, Ikeda H, Sakamoto M, Sasano T (2008) High serum levels of IGF-I contribute to promotion of endochondral ossification in mandibular condyle and cause its specific elongation in acromegaly-like rats. Horm Metab Res 40(8):533–538. CrossRefGoogle Scholar
  19. 19.
    Higashikawa A, Saito T, Ikeda T, Kamekura S, Kawamura N, Kan A, Oshima Y, Ohba S, Ogata N, Takeshita K, Nakamura K, Chung UI, Kawaguchi H (2009) Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type X collagen gene. Arthritis Rheum 60(1):166–178. CrossRefGoogle Scholar
  20. 20.
    Chen H, Ghori-Javed FY, Rashid H, Adhami MD, Serra R, Gutierrez SE, Javed A (2014) Runx2 regulates endochondral ossification through control of chondrocyte proliferation and differentiation. J Bone Miner Res 29(12):2653–2665. CrossRefGoogle Scholar
  21. 21.
    van der Kraan PM, van den Berg WB (2007) Osteophytes: relevance and biology. Osteoarthr Cartil 15(3):237–244. CrossRefGoogle Scholar
  22. 22.
    Shintani N, Siebenrock KA, Hunziker EB (2013) TGF-ss1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy. PLoS ONE 8(1):e53086. CrossRefGoogle Scholar
  23. 23.
    Leboy P, Grasso-Knight G, D’Angelo M, Volk SW, Lian JV, Drissi H, Stein GS, Adams SL (2001) Smad-Runx interactions during chondrocyte maturation. J Bone Joint Surg Am 83(1):S15–22CrossRefGoogle Scholar
  24. 24.
    Kempf H, Ionescu A, Udager AM, Lassar AB (2007) Prochondrogenic signals induce a competence for Runx2 to activate hypertrophic chondrocyte gene expression. Dev Dyn 236(7):1954–1962. CrossRefGoogle Scholar
  25. 25.
    Furumatsu T, Tsuda M, Taniguchi N, Tajima Y, Asahara H (2005) Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment. J Biol Chem 280(9):8343–8350. CrossRefGoogle Scholar
  26. 26.
    Kobayashi T, Lyons KM, McMahon AP, Kronenberg HM (2005) BMP signaling stimulates cellular differentiation at multiple steps during cartilage development. Proc Natl Acad Sci USA 102(50):18023–18027. CrossRefGoogle Scholar
  27. 27.
    Li TF, Darowish M, Zuscik MJ, Chen D, Schwarz EM, Rosier RN, Drissi H, O’Keefe RJ (2006) Smad3-deficient chondrocytes have enhanced BMP signaling and accelerated differentiation. J Bone Miner Res 21(1):4–16. CrossRefGoogle Scholar
  28. 28.
    Kawamura I, Maeda S, Imamura K, Setoguchi T, Yokouchi M, Ishidou Y, Komiya S (2012) SnoN suppresses maturation of chondrocytes by mediating signal cross-talk between transforming growth factor-β and bone morphogenetic protein pathways. J Biol Chem 287(34):29101–29113. CrossRefGoogle Scholar
  29. 29.
    Deheuninck J, Luo K (2009) Ski and SnoN, potent negative regulators of TGF-beta signaling. Cell Res 19(1):47–57. CrossRefGoogle Scholar
  30. 30.
    Solomon E, Li H, Duhachek Muggy S, Syta E, Zolkiewska A (2010) The role of SnoN in transforming growth factor beta1-induced expression of metalloprotease-disintegrin ADAM12. J Biol Chem 285(29):21969–21977. CrossRefGoogle Scholar
  31. 31.
    Sun Y, Liu X, Ng-Eaton E, Lodish HF, Weinberg RA (1999) SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signalling. Proc Natl Acad Sci USA 96(22):12442–12447CrossRefGoogle Scholar
  32. 32.
    Tokumasu Y, Iida A, Wang Z, Ansai S, Kinoshita M, Sehara-Fujisawa A (2016) ADAM12-deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects. Dev Growth Differ 58(4):409–421. CrossRefGoogle Scholar
  33. 33.
    Kurisaki T, Masuda A, Sudo K, Sakagami J, Higashiyama S, Matsuda Y, Nagabukuro A, Tsuji A, Nabeshima Y, Asano M, Iwakura Y, Sehara-Fujisawa A (2003) Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis. Mol Cell Biol 23(1):55–61CrossRefGoogle Scholar
  34. 34.
    Horiuchi K, Zhou HM, Kelly K, Manova K, Blobel CP (2005) Evaluation of the contributions of ADAMs 9, 12, 15, 17, and 19 to heart development and ectodomain shedding of neuregulins beta1 and beta2. Dev Biol 283(2):459–471. CrossRefGoogle Scholar
  35. 35.
    Ramdas V, McBride M, Denby L, Baker AH (2013) Canonical transforming growth factor-beta signaling regulates disintegrin metalloprotease expression in experimental renal fibrosis via miR-29. Am J Pathol 183(6):1885–1896. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Masahiro Horita
    • 1
  • Keiichiro Nishida
    • 1
    Email author
  • Joe Hasei
    • 2
  • Takayuki Furumatsu
    • 1
  • Miwa Sakurai
    • 3
  • Yuta Onodera
    • 4
  • Kanji Fukuda
    • 4
  • Donald M. Salter
    • 5
  • Toshifumi Ozaki
    • 1
  1. 1.Department of Orthopaedic SurgeryOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  2. 2.Department of Sports MedicineOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  3. 3.Laboratory of Molecular Life ScienceInstitute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe (FBRI)KobeJapan
  4. 4.Division of Cell Biology for Regenerative MedicineInstitute of Advanced Clinical Medicine, Kindai University Faculty of MedicineOsakaJapan
  5. 5.Centre for Genomic and Experimental MedicineIGMM - University of EdinburghEdinburghUK

Personalised recommendations