Cell and Tissue Research

, Volume 328, Issue 1, pp 137–151

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies, including comparisons with developing endochondral bones

  • Yasunori Sakakura
  • Yoichiro Hosokawa
  • Eichi Tsuruga
  • Kazuharu Irie
  • Masanori Nakamura
  • Toshihiko Yajima
Regular Article


The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.


Matrix metalloproteinases Galectin-3 Meckel’s cartilage Endochondral ossification Immunohistochemistry Mouse (ddY strain) 


  1. Akahani S, Nangia-Makker P, Inohara H, Kim HR, Raz A (1997) Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res 57:5272–5276PubMedGoogle Scholar
  2. Aubin JE, Gupta AK, Bhargava U, Turksen K (1996) Expression and regulation of galectin 3 in rat osteoblastic cells. J Cell Physiol 169:468–480PubMedCrossRefGoogle Scholar
  3. Bae J-W, Takahashi I, Sasano Y, Onodera K, Mitani H, Kagayama M, Mitani H (2003) Age-related changes in gene expression patterns of matrix metalloproteinases and their collagenous substrates in mandibular condylar cartilage in rats. J Anat 203:235–241PubMedCrossRefGoogle Scholar
  4. Barondes SH, Cooper DNW, Gitt MA, Leffler H (1994) Galectins. Structure and functions of a large family of animal lectins. J Biol Chem 269:20807–20810PubMedGoogle Scholar
  5. Bernardo MM, Fridman R (2003) TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP. Biochem J 374:739–745PubMedCrossRefGoogle Scholar
  6. Bernick S, Patek PQ (1969) Postnatal development of the rat mandible. J Dent Res 48:1258–1263PubMedGoogle Scholar
  7. Bhaskar SN, Weinmann JP, Schour I (1953) Role of Meckel’s cartilage in the development and growth of the rat mandible. J Dent Res 32:398–410PubMedGoogle Scholar
  8. Blavier L, Delaissé JM (1995) Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones. J Cell Sci 108:3649–3659PubMedGoogle Scholar
  9. Chin JR, Werb Z (1997) Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 124:1519–1530PubMedGoogle Scholar
  10. Colnot C, Sidhu SS, Balmain N, Poirier F (2001) Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones. Dev Biol 229:203–214PubMedCrossRefGoogle Scholar
  11. Cowell S, Knäuper V, Stewart ML, d’Ortho M-P, Stanton H, Hembry RM, López-Otín C, Reynolds JJ, Murphy G (1998) Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3. Biochem J 331:453–458PubMedGoogle Scholar
  12. Dallas SL, Rosser JL, Mundy GR, Bonewald LF (2002) Proteolysis of latent transforming growth factor-β (TGF-β)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-β from bone matrix. J Biol Chem 277:21352–21360PubMedCrossRefGoogle Scholar
  13. D’Angelo M, Billings PC, Pacifici M, Leboy PS, Kirsch T (2001) Authentic matrix vesicles contain active metalloporoteinases (MMP). A role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-β. J Biol Chem 276:11347–11353PubMedCrossRefGoogle Scholar
  14. Dreier R, Grässel S, Fuchs S, Schaumburger J, Bruckner P (2004) Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade. Exp Cell Res 297:303–312PubMedCrossRefGoogle Scholar
  15. Engsig MT, Chen Q-J, Vu TH, Pedersen A-C, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaissé J-M (2000) Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151:879–889PubMedCrossRefGoogle Scholar
  16. Fernandez-Catalan C, Bode W, Huber R, Turk D, Calvete JJ, Lichte A, Tschesche H, Maskos K (1998) Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinase-2, the soluble progelatinase A receptor. EMBO J 17:5238–5248PubMedCrossRefGoogle Scholar
  17. Fosang AJ, Last K, Knäuper V, Murphy G, Neame PJ (1996) Degradation of cartilage aggrecan by collagenase-3 (MMP-13). FEBS Lett 380:17–20PubMedCrossRefGoogle Scholar
  18. Freije JMP, Díaz-Itza I, Balbín M, Sánchez LM, Blasco R, Tolivia J, López-Otín C (1994) Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem 269:16766–16773PubMedGoogle Scholar
  19. Frommer J, Margolies MR (1971) Contribution of Meckel’s cartilage to ossification of the mandible in mice. J Dent Res 50:1260–1267PubMedGoogle Scholar
  20. Guévremont M, Martel-Pelletier J, Boileau C, Liu F-T, Richard M, Fernandes J-C, Pelletier J-P, Reboul P (2004) Galectin-3 surface expression on human adult chondrocytes: a potential substrate for collagenase-3. Ann Rheum Dis 63:636–643PubMedCrossRefGoogle Scholar
  21. Haeusler G, Walter I, Helmreich M, Egerbacher M (2005) Localization of matrix metalloproteinases (MMPs), their tissue inhibitors, and vascular endothelial growth factor (VEGF) in growth plates of children and adolescents indicates a role for MMPs in human postnatal growth and skeletal maturation. Calcif Tissue Int 76:326–335PubMedCrossRefGoogle Scholar
  22. Harada Y, Ishizeki K (1998) Evidence for transformation of chondrocytes and site-specific resorption during the degeneration of Meckel’s cartilage. Anat Embryol 197:439–450PubMedCrossRefGoogle Scholar
  23. Hayami T, Endo N, Tokunaga T, Yamagiwa H, Hatono H, Uchida M, Takahashi HE (2000) Spatiotemporal change of rat collagenase (MMP-13) mRNA expression in the development of the rat femoral neck. J Bone Miner Metab 18:185–193PubMedCrossRefGoogle Scholar
  24. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, Birkedal-Hansen H (1999) MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99:81–92PubMedCrossRefGoogle Scholar
  25. Holmbeck K, Bianco P, Chrysovergis K, Yamada S, Birkedal-Hansen H (2003) MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth. J Cell Biol 163:661–671PubMedCrossRefGoogle Scholar
  26. Holmbeck K, Bianco P, Pidoux I, Inoue S, Billinghurst RC, Wu W, Chrysovergis K, Yamada S, Birkedal-Hansen H, Poole AR (2005) The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone. J Cell Sci 118:147–156PubMedCrossRefGoogle Scholar
  27. Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, López-Otín C, Krane SM (2004) Critical roles for collagenase-3 (MMP-13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci USA 101:17192–17197PubMedCrossRefGoogle Scholar
  28. Irie K, Tsuruga E, Sakakura Y, Muto T, Yajima T (2001) Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo. Tissue Cell 33:478–482PubMedCrossRefGoogle Scholar
  29. Ishizeki K, Nawa T (2000) Further evidence for secretion of matrix metalloproteinase-1 by Meckel’s chondrocytes during degradation of the extracellular matrix. Tissue Cell 32:207–215PubMedCrossRefGoogle Scholar
  30. Ishizeki K, Saito H, Shinagawa T, Fujiwara N, Nawa T (1999) Histochemical and immunohistochemical analysis of the mechanism of calcification of Meckel’s cartilage during mandible development in rodents. J Anat 194:265–277PubMedCrossRefGoogle Scholar
  31. Karsdal MA, Larsen L, Engsig MT, Lou H, Ferreras M, Lochter A, Delaissé J-M, Foged NT (2002) Matrix metalloproteinase-dependent activation of latent transforming growth factor-β controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem 277:44061–44067PubMedCrossRefGoogle Scholar
  32. Karsdal MA, Hjorth P, Henriksen K, Kirkegaard T, Nielsen KL, Lou H, Delaissé J-M, Foged NT (2003) Transforming growth factor-β controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem 278:44975–44987PubMedCrossRefGoogle Scholar
  33. Kinoh H, Sato H, Tsunezuka Y, Takino T, Kawashima A, Okada Y, Seiki M (1996) MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A), is expressed with its substrate in mouse tissue during embryogenesis. J Cell Sci 109:953–959PubMedGoogle Scholar
  34. Knäuper V, López-Otin C, Smith B, Knight G, Murphy G (1996a) Biochemical characterization of human collagenase-3. J Biol Chem 271:1544–1550PubMedCrossRefGoogle Scholar
  35. Knäuper V, Will H, López-Otin C, Smith B, Atkinson SJ, Stanton H, Hembry RM, Murphy G (1996b) Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase A (MMP-2) are able to generate active enzyme. J Biol Chem 271:17124–17131PubMedCrossRefGoogle Scholar
  36. Knäuper V, Cowell S, Smith B, López-Otin C, O’Shea M, Morris H, Zardi L, Murphy G (1997a) The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction. J Biol Chem 272:7608–7616PubMedCrossRefGoogle Scholar
  37. Knäuper V, Smith B, López-Otin C, Murphy G (1997b) Activation of progelatinase B (proMMP-9) by active collagenase-3 (MMP-13). Eur J Biochem 248:369–373PubMedCrossRefGoogle Scholar
  38. Mackay AR, Hartzler JL, Pelina MD, Thorgeirsson UP (1990) Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen. J Biol Chem 265:21929–21934PubMedGoogle Scholar
  39. Mattot V, Raes MB, Henriet P, Eeckhout Y, Stehelin D, Vandenbunder B, Desbiens X (1995) Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis. J Cell Sci 108:529–535PubMedGoogle Scholar
  40. Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ (1996) Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 97:761–768PubMedCrossRefGoogle Scholar
  41. Morgunova E, Tuuttila A, Bergmann U, Tryggvason K (2002) Structural insight into the complex formation of latent matrix metalloproteinase 2 with tissue inhibitor of metalloproteinase 2. Proc Natl Acad Sci USA 99:7414–7419PubMedCrossRefGoogle Scholar
  42. Nagase H, Woessner JF Jr (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494PubMedCrossRefGoogle Scholar
  43. Nakamura M, Yagi H, Ishii T, Kayaba S, Soga H, Gotoh T, Ohtsu S, Ogara M, Itoh T (1997) DNA fragmentation is not the primary event in glucocorticoid-induced thymocyte death in vivo. Eur J Immunol 27:999–1004PubMedCrossRefGoogle Scholar
  44. Nakamura H, Sato G, Hirata A, Yamamoto T (2004) Immunolocalization of matrix metalloproteinase-13 on bone surface under osteoclasts in rat tibia. Bone 34:48–56PubMedCrossRefGoogle Scholar
  45. Ochieng J, Fridman R, Nangia-Makker P, Kleiner DE, Liotta LA, Stetler-Stevenson WG, Raz A (1994) Galectin-3 is a novel substrate for human matrix metalloproteinases-2 and -9. Biochemistry 33:14109–14114PubMedCrossRefGoogle Scholar
  46. Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, Okada Y (1997) Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 272:2446–2451PubMedCrossRefGoogle Scholar
  47. Ortega N, Behonick DJ, Colnot C, Cooper DNW, Werb Z (2005) Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation. Mol Biol Cell 16:3028–3039PubMedCrossRefGoogle Scholar
  48. Reponen P, Sahlberg C, Huhtala P, Hurskainen T, Thesleff I, Tryggvason K (1992) Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development. J Biol Chem 267:7856–7862PubMedGoogle Scholar
  49. Reponen P, Sahlberg C, Munaut C, Thesleff I, Tryggvason K (1994) High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development. J Cell Biol 124:1091–1102PubMedCrossRefGoogle Scholar
  50. Roach HI, Clarke NMP (2000) Physiological cell death of chondrocytes in vivo is not confined to apoptosis. J Bone Joint Surg Br 82-B:601–613CrossRefGoogle Scholar
  51. Sakakura Y, Tsuruga E, Irie K, Hosokawa Y, Nakamura H, Yajima T (2005) Immunolocalization of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in Meckel’s cartilage compared with developing endochondral bones in mice. J Anat 207:325–337PubMedCrossRefGoogle Scholar
  52. Sasano Y, Zhu J-X, Tsubota M, Takahashi I, Onodera K, Mizoguchi I, Kagayama M (2002) Gene expression of MMP8 and MMP13 during embryonic development of bone and cartilage in the rat mandible and hind limb. J Histochem Cytochem 50:325–332PubMedGoogle Scholar
  53. Savostin-Asling I, Asling CW (1973) Resorption of calcified cartilage as seen in Meckel’s cartilage of rats. Anat Rec 176:345–360PubMedCrossRefGoogle Scholar
  54. Shimada M, Yamamoto M, Wakayama T, Iseki S, Amano O (2003) Different expression of 25-kDa heat-shock protein (Hsp25) in Meckel’s cartilage compared with other cartilages in the mouse. Anat Embryol 206:163–173PubMedGoogle Scholar
  55. Shimo T, Kanyama M, Wu C, Sugito H, Billings PC, Abrams WR, Rosenbloom J, Iwamoto M, Pacifici M, Koyama E (2004) Expression and roles of connective tissue growth factor in Meckel’s cartilage development. Dev Dyn 231:136–147PubMedCrossRefGoogle Scholar
  56. Soga H, Nakamura M, Yagi H, Kayaba S, Ishii T, Gotoh T, Itoh T (1997) Heterogeneity of mouse thymic macrophages. I. Immunohistochemical analysis. Arch Histol Cytol 60:53–63PubMedGoogle Scholar
  57. Ståhle-Bäckdahl M, Sandstedt B, Bruce K, Lindahl A, Jiménez MG, Vega JA, López-Otín C (1997) Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. Lab Invest 76:717–728PubMedGoogle Scholar
  58. Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z (2004) Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 131:5883–5895PubMedCrossRefGoogle Scholar
  59. Stock M, Schäfer H, Stricker S, Gross G, Mundlos S, Otto F (2003) Expression of galectin-3 in skeletal tissues is controlled by Runx2. J Biol Chem 278:17360–17367PubMedCrossRefGoogle Scholar
  60. Takai H, Kanematsu M, Yano K, Tsuda E, Higashio K, Ikeda K, Watanabe K, Yamada Y (1998) Transforming growth factor-β stimulates the production of osteoprotegerin/osteoclastogenesis inhibitory factor by bone marrow stromal cells. J Biol Chem 273:27091–27096PubMedCrossRefGoogle Scholar
  61. Thirunavukkarasu K, Miles RR, Halladay DL, Yang X, Galvin RJS, Chandrasekhar S, Martin TJ, Onyia JE (2001) Stimulation of osteoprotegerin (OPG) gene expression by transforming growth factor-β (TGF-β). J Biol Chem 276:36241–36250PubMedCrossRefGoogle Scholar
  62. Toth M, Chvyrkova I, Bernardo MM, Hernandez-Barrantes S, Fridman R (2003) Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes. Biochem Biophy Res Commun 308:386–395CrossRefGoogle Scholar
  63. Vu TH, Werb Z (2000) Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 14:2123–2133PubMedCrossRefGoogle Scholar
  64. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z (1998) MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422PubMedCrossRefGoogle Scholar
  65. Werb Z, Chin JR (1998) Extracellular matrix remodeling during morphogenesis. Ann NY Acad Sci 857:110–118PubMedCrossRefGoogle Scholar
  66. Yu Q, Stamenkovic I (2000) Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes Dev 14:163–176PubMedGoogle Scholar
  67. Zhou Z, Apte SS, Soininen R, Cao R, Baaklini GY, Rauser RW, Wang J, Cao Y, Tryggvason K (2000) Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci USA 97:4052–4057PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Yasunori Sakakura
    • 1
  • Yoichiro Hosokawa
    • 2
  • Eichi Tsuruga
    • 1
  • Kazuharu Irie
    • 1
  • Masanori Nakamura
    • 3
  • Toshihiko Yajima
    • 1
  1. 1.Department of Oral Anatomy, School of DentistryHealth Sciences University of HokkaidoHokkaidoJapan
  2. 2.Department of Dental Radiation, School of DentistryHealth Sciences University of HokkaidoHokkaidoJapan
  3. 3.Department of Oral AnatomyShowa University School of DentistryTokyoJapan

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