Journal of Cell Communication and Signaling

, Volume 13, Issue 1, pp 113–118 | Cite as

CCN2/CTGF binds the small leucine rich proteoglycan protein Tsukushi

  • Kunimasa OhtaEmail author
  • Eriko Aoyama
  • Shah Adil Ishtiyaq Ahmad
  • Naofumi Ito
  • Mohammad Badrul Anam
  • Satoshi Kubota
  • Masaharu TakigawaEmail author
Research Article


Extracellular molecules coordinate the multiple signaling pathways spatiotemporally to exchange information between cells during development. Understanding the regulation of these signal molecule-dependent pathways elucidates the mechanism of intercellular crosstalks. CCN2/CTGF is one of the CCN family members that binds BMP2, fibronectin, aggrecan, FGFR2 - regulating cartilage and bone formation, angiogenesis, wound repair etc. Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family, binds nodal/Vg1/TGF-β1, BMP4/chordin, Delta, FGF8, Frizzled4, and is involved in the early body formation, bone growth, wound healing, retinal stem cell regulation etc. These two secreted molecules are expressed in similar tissues and involved in several biological events by functioning as extracellular signaling modulators. Here, we examine the molecular interaction between CCN2 and TSK biochemically. Co-precipitation assay and Surface Plasmon Resonance measurement showed their direct binding with the Kd value 15.3 nM. Further, the Solid-phase Binding Assay indicated that TSK binds to IGFBP and CT domains of CCN2. Our data suggest that CCN2 and TSK exert their function together in the body formation.


CCN2/CTGF Tsukushi Soluble molecule SLRP Vertebrate development 



The authors thank Mitsue Kumamaru and Megumi Takiguchi. We also thank all members of our labs for their technical assistance and for valuable helps. This study was supported by the Kumamoto University International Research Core for Stem Cell-based Developmental Medicine and HIGOprogram and in part by grants from the programs Grants-in-Aid for Scientific Research (B) to MT (#JP15H05014) and for Challenging Exploratory Research to MT (#JP17K19757) from Japan Society for the Promotion of Sciences, Japan.

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.


  1. Abreu JG, Ketpura NI, Reversade B, De Robertis EM (2002) Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4:599–604CrossRefGoogle Scholar
  2. Ahmad S a I, Anam MB, Ito N, Ohta K (2018) Involvement of Tsukushi in diverse developmental processes. J Cell Commun Signal 12:205–210CrossRefGoogle Scholar
  3. Aoyama E, Takigawa M (2017) Evaluation of molecular interaction between CCN2 protein and its binding partners by surface Plasmon resonance (SPR). Methods Mol Biol 1489:169–176CrossRefGoogle Scholar
  4. Aoyama E, Hattori T, Hoshijima M, Araki D, Nishida T, Kubota S, Takigawa M (2009) N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan. Biochem J 420:413–420CrossRefGoogle Scholar
  5. Aoyama E, Kubota S, Khattab HM, Nishida T, Takigawa M (2015) CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG. Bone 73:242–248CrossRefGoogle Scholar
  6. Arnott JA, Lambi AG, Mundy C, Hendesi H, Pixley RA, Owen TA, Safadi FF, Popoff SN (2011) The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis. Crit Rev Eukaryot Gene Expr 21:43–69CrossRefGoogle Scholar
  7. Bell E, Muñoz-Sanjuán I, Altmann CR, Vonica A, Brivanlou AH (2003) Cell fate specification and competence by coco, a maternal BMP, TGFbeta and Wnt inhibitor. Development 130:1381–1389CrossRefGoogle Scholar
  8. Grotendorst GR, Rahmanie H, Duncan MR (2004) Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation. FASEB J 18:469–479CrossRefGoogle Scholar
  9. Harrington AE, Morris-Triggs SA, Ruotolo BT, Robinson CV, Ohnuma S-I, Hyvönen M (2006) Structural basis for the inhibition of activin signalling by follistatin. EMBO J 25:1035–1045CrossRefGoogle Scholar
  10. Igarashi A, Okochi H, Bradham DM, Grotendorst GR (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4:637–645CrossRefGoogle Scholar
  11. Iozzo RV, Schaefer L (2015) Proteoglycan form and function: a comprehensive nomenclature of proteoglycans. Matrix Biol 42:11–55CrossRefGoogle Scholar
  12. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130:2779–2791CrossRefGoogle Scholar
  13. Khattab HM, Aoyama E, Kubota S, Takigawa M (2015) Physical interaction of CCN2 with diverse growth factors involved in chondrocyte differentiation during endochondral ossification. J Cell Commun Signal 9:247–254CrossRefGoogle Scholar
  14. Kubota S, Takigawa M (2007) CCN family proteins and angiogenesis: from embryo to adulthood. Angiogenesis 10:1–11CrossRefGoogle Scholar
  15. Kuriyama S, Lupo G, Ohta K, Ohnuma S-I, Harris WA, Tanaka H (2006) Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity. Development 133:75–88CrossRefGoogle Scholar
  16. Lim, W., Mayer, B., and Pawson, T. (2014). Cell signaling : principles and mechanisms (Garland science)Google Scholar
  17. Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG et al (2004) Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279:55958–55968CrossRefGoogle Scholar
  18. Mercurio S, Latinkic B, Itasaki N, Krumlauf R, Smith JC (2004) Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex. Development 131:2137–2147CrossRefGoogle Scholar
  19. Niimori D, Kawano R, Niimori-Kita K, Ihn H, Ohta K (2014) Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors. J Cell Commun Signal 8:173–177CrossRefGoogle Scholar
  20. Ohta, K. (2014). The role of Tsukushi as an extracellular signaling coordinator. In New principles in developmental processes, H. and K. Kondoh, ed. (Tokyo: Springer), pp. 227–238Google Scholar
  21. Ohta K, Lupo G, Kuriyama S, Keynes R, Holt CE, Harris WA, Tanaka H, Ohnuma S-I (2004) Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin. Dev Cell 7:347–358CrossRefGoogle Scholar
  22. Ohta K, Kuriyama S, Okafuji T, Gejima R, Ohnuma S, Tanaka H (2006) Tsukushi cooperates with VG1 to induce primitive streak and Hensen’s node formation in the chick embryo. Development 133:3777–3786CrossRefGoogle Scholar
  23. Ohta K, Ito A, Kuriyama S, Lupo G, Kosaka M, Ohnuma S, Nakagawa S, Tanaka H (2011) Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4. Proc Natl Acad Sci U S A 108:14962–14967CrossRefGoogle Scholar
  24. Perbal B (2018) The concept of the CCN protein family revisited: a centralized coordination network. J Cell Commun Signal 12:3–12CrossRefGoogle Scholar
  25. Piccolo S, Agius E, Leyns L, Bhattacharyya S, Grunz H, Bouwmeester T, De Robertis EM (1999) The head inducer Cerberus is a multifunctional antagonist of nodal, BMP and Wnt signals. Nature 397:707–710CrossRefGoogle Scholar
  26. Shi-Wen X, Leask A, Abraham D (2008) Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 19:133–144CrossRefGoogle Scholar
  27. Song JJ, Aswad R, Kanaan RA, Rico MC, Owen TA, Barbe MF, Safadi FF, Popoff SN (2007) Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation. J Cell Physiol 210:398–410CrossRefGoogle Scholar
  28. Takashi N, Emura K, Kubota S, Lyons KM, Takigawa M (2011) CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell-specific transmembrane protein (DC-STAMP). J. Bone Miner. Res. 26 (2):351–363Google Scholar
  29. Takigawa M (2013) CCN2: a master regulator of the genesis of bone and cartilage. J Cell Commun Signal 7:191–201CrossRefGoogle Scholar
  30. Takigawa M (2017) The CCN proteins: An overview. CCN Proteins: Methods and Protocols, in: M. Takigawa (Ed), Methods in Molecular Biology, Springer Nature, New York, vol.1489 pp. 1–8Google Scholar
  31. Takigawa M (2018) An early history of CCN2/CTGF research: the road to CCN2 via hcs24, ctgf, ecogenin, and regenerin. J Cell Commun Signal 12:253–264CrossRefGoogle Scholar
  32. Vial C, Gutiérrez J, Santander C, Cabrera D, Brandan E (2011) Decorin interacts with connective tissue growth factor (CTGF)/CCN2 by LRR12 inhibiting its biological activity. J Biol Chem 286:24242–24252CrossRefGoogle Scholar
  33. Yano K, Washio K, Tsumanuma Y, Yamato M, Ohta K, Okano T, Izumi Y (2017) The role of Tsukushi (TSK), a small leucine-rich repeat proteoglycan, in bone growth. Regenerative Therapy 7:98–107CrossRefGoogle Scholar

Copyright information

© The International CCN Society 2018

Authors and Affiliations

  1. 1.Department of Developmental Neurobiology, Graduate School of Life SciencesKumamoto UniversityKumamotoJapan
  2. 2.Program for Leading Graduate Schools “HIGO Program”Kumamoto UniversityKumamotoJapan
  3. 3.Global COE Cell Fate Regulation Research and Education UnitKumamoto UniversityKumamotoJapan
  4. 4.Japan Agency for Medical Research and Development (AMED)TokyoJapan
  5. 5.Advanced Research Center for Oral and Craniofacial SciencesOkayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesOkayamaJapan
  6. 6.Department of Biotechnology and Genetic EngineeringMawlana Bhashani Science and Technology UniversityTangailBangladesh

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