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Role of Matrix Metalloproteinases in Extracellular Matrix Disintegration of Meckel's Cartilage in Mice
J. Oral Biosci. 52 （2）：143−149, 2010
REVIEW（The Specificity of Meckel’s Cartilage）
Role of Matrix Metalloproteinases in Extracellular Matrix Disintegration of Meckel’s Cartilage in Mice Yasunori Sakakura§ Division of Anatomy, Department of Oral Growth & Development, School of Dentistry, Health Sciences University of Hokkaido 1757 Kanazawa, Ishikari−Tobetsu, Hokkaido 061−0293, Japan 〔Received on February 9, 2010；Accepted on February 24, 2010〕 Key words：matrix metalloproteinases／gelatinolysis／ECM degradation／chondroclasts／Meckel’s cartilage Abstract：Meckel’s cartilage is a temporary supporting tissue that forms during the embryonic period. Unlike the distal（anterior）, posterior, and proximal portions, the middle portion degenerates with the death of chondrocytes as well as resorption of the cartilaginous matrix by chondroclasts without giving rise to ossification. Perichondral cells initially differentiate into osteoblasts and subsequently form periosteal bone on the lateral surface of Meckel’s cartilage closest to the incisor teeth, and then chondrocytes become hypertrophied in the restricted position of Meckel’s cartilage. Thereafter, the calcified periosteal bone and cartilage are resorbed by osteoclasts and chondroclasts；however, little is known regarding the mechanisms by which the middle portion is disintegrated during the embryonic period. Based on immunohistochemical and in situ zymographic findings in mice, we discuss the role of matrix metalloproteinases in the disintegration of the middle portion of Meckel’s cartilage.
Introduction Meckel’s cartilage is a supporting tissue found in the embryonic mandible of mammals. In rodents, it initially forms as a pair of rod−shaped cartilaginous bars within the first branchial arch and then, as development and growth of the mandible proceeds, three regions of Meckel’s cartilage determine its ultimate fate. In the distal（anterior）region, the cartilage bars fuse and undergo endochondral ossification to form intra−mandibular symphysis, while the proximal region converts to inner ear ossicles, such as the malleus and incus. The intermediate region occupying §
Corresponding author E−mail：yasaka@hoku−iryo−u.ac.jp
the major area of Meckel’s cartilage is further subdivided into the middle and posterior portions. The middle portion（anterior part of the intermediate region） degenerates with the death of chondrocytes as well as resorption of the cartilaginous matrix by chondroclasts, whereas the posterior portion is replaced by fibrous tissue, e. g. the sphenomandibular ligament. Especially in the middle portion, Meckel’s cartilage ；howdisappears without giving rise to ossification1,2） ever, how proteolytic enzymes contribute to degradation of the middle portion remains unknown. In this review, the mechanisms involved in the degradation of the middle portion of Meckel’s cartilage are discussed on the basis of immunolocalization of matrix metalloproteinases（MMPs）and in situ localization of gelatinolytic activity.
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Resorption Process in the Middle Portion of Meckel’s Cartilage On embryonic day 15（E15）, perichondral cells differentiate into osteoblasts in the middle portion of Meckel’s cartilage in mice and subsequently form periosteal bone on the lateral surface closest to the incisor teeth（Fig. 1A）. Consequently, nearly all of the chondrocytes become hypertrophied on E15, though early hypertrophic chondrocytes are found in the outermost periphery on the lateral side. A marker for the initiation of cartilage resorption is the appearance of multinuclear cells responsible for the resorption of bone and cartilage on the lateral side1）. At this time, dental follicular cells of the incisor teeth moderately express the receptor activator of nuclear factor−κB ligand（RANKL）and osteoprotegerin（OPG） , whereas osteoblasts derived from perichondral cells on the lateral surface do not1）. Thus, the development of incisor teeth might contribute to the recruitment of multinuclear cells at the appropriate time and in the correct position. On E16, the periosteal bone and cartilaginous matrix between chondrocytes become calcified, and the calcified bone and cartilage are eroded by osteoclasts and chondroclasts. Thereafter, this disintegrative process suddenly commences in the restricted position of the cartilage, and expands toward the medial or anterior and posterior directions within the middle portion on E16（Fig. 1B）. Thus, a local site−specific environmental cue is involved in the resorption of Meckel’s cartilage. Involvement of MMPs in the Initiation of Cartilage Resorption on E15 Although fragmentary, many studies have reported that MMPs and tissue inhibitor of metalloproteinase （TIMP）−2 are strongly involved in the development and resorption of Meckel’s cartilage3―7）. MMP−2 （gelatinase A）immunoreactivity was detected in the bone matrix under periosteal osteoblasts in mice on E151）. In addition, MMP−13（collagenase 3）immunoreactivity was found preferentially localized in bone matrix deposited immediately under periosteal
osteoblasts. However, MMP−9 （gelatinase B） and MMP−14（membrane type 1−MMP）immunoreactivity was scarcely detected in the deposited bone matrix and periosteal osteoblasts, whereas these proteinases showed intense immunolocalization in the peripheral chondrocytes of Meckel’s cartilage. These results indicate that the uncalcified periosteal bone matrix undergoes remodeling via a proteolytic process prior to calcification. On the other hand, in situ gelatinolytic zymography with application of DQ−gelatin to unfixed non−decalcified frozen sections has indicated that the difference in fluorescence intensity shows localization of MMP− 2 and MMP−9, as well as gelatinolytic MMPs other than the gelatinases, and gelatinolytic enzymes other than MMPs, which is dependent on the absence or presence of CTT（a selective inhibitor of MMP−2 and MMP−9）or EDTA（a general broad−spectrum MMP inhibitor）8）. During the degradation process, gelatinolytic activity has been found to predominate in the periosteal bone matrix covering the lateral surface and the intercellular septa between chondrocytes. On E15, CTT slightly attenuated gelatinolytic activity, whereas it almost completely disappeared as a result of EDTA treatment, indicating that gelatinolytic MMPs, other than MMP−2 and MMP−9, also play a pivotal role in ECM remodeling of the periosteal bone and intercellular septa. The most likely MMP candidates are considered to be MMP−1, MMP−13, and MMP−14, which have already been found in Meckel’s cartilage2,5,6） and have activities capable of degrading gelatin9）. In addition, gelatinolytic activity in the intercellular septa was more intense in Meckel’s cartilage on E15 than E14. ECM degradation by proteinases is essential for chondrocyte enlargement, which occurs before the onset of calcification10）. Moreover, MMP− 2, MMP−8, MMP−9, MMP−13, and MMP−14 play integral roles in chondrocyte hypertrophy, as well as the initiation of calcification through the activation of pro−MMPs and reconstitution of the ECM, including proteoglycans and typeⅡ and Ⅹ collagens in endochondral bone formation 11―18）. Therefore, these results implicate the activities of gelatinases and the other gelatinolytic MMPs in ECM degradation during hypertrophy of Meckel’s chondrocytes, prior to subse-
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Fig. 1 Histology images of the middle portion of Meckel’s cartilage from mouse mandibles on E15（A）and E16（B） Osteoclasts and chondroclasts（arrows）can be seen on the lateral side facing the incisor tooth（It）in both embryonic stages. Bars＝10 μm.
quent calcification of the intercellular septa between chondrocytes. MMP−2 and MMP−9 cleave denatured collagens, typeⅣ and Ⅴ collagens, and elastin. MMP−2 is the most widespread of all the MMPs and can activate pro−MMP−1319,20）. Interestingly, the activities of these MMPs are regulated at the transcription, translation, and proenzyme activation levels, and via TIMPs, which bind to MMPs. TIMP−2 can bind to pro−MMP−2 via its C−terminal region, while it also binds to the active site of MMP−14, implying the formation of an MMP−14／TIMP−2／pro−MMP−2 ternary complex21,22）. Bound pro−MMP−2 is activated by neighboring TIMP−2−free MMP−14, and active MMP−2 is released into the extracellular space, while it can also promote the activation of pro−MMP− 923,24）. Thus, MMP−2, MMP−9, MMP−13, and MMP− 14 form part of an activation cascade that involves TIMP−2. Taken together, an activation cascade of MMPs by an MMP−14／TIMP−2／pro−MMP−2 ternary complex may be involved in disintegration of the uncalcified bone matrix covering the lateral surface through face−to−face crosstalk between periosteal osteoblasts and peripheral chondrocytes of Meckel’s cartilage （Fig. 2）. In these processes, MMPs may release and activate a latent form of transforming g ro w t h f a c t o r−β （T G F−β） f ro m E C M−b o u n d stores15,25,26）. In addition, TGF−β may be involved in the differentiation and recruitment of osteoclasts
through regulation of RANK, a receptor for RANKL, RANKL, and OPG, a decoy receptor against RANKL, on the lateral surface of Meckel’s cartilage on E151,27―29）. Role of MMPs in Resorption Process on E16 MMPs expressed by chondroclasts clearly play a role in E16 Meckel’s cartilage. Although the immunoreactivity of MMP−2 is faint, opened chondrocytic lacunae and connective tissue were positive in the resorption area, whereas hypertrophic chondrocytes were negative. MMP−9 immunoreactivity was especially intense in the chondroclasts, similar to reports showing that MMP−9 is highly expressed in multinucleated osteoclasts3,11,30）. In addition, MMP−14 immunolocalization was also recognized in chondroclasts. Unexpectedly, intense immunostaining for MMP−13 was found in the ruffled border of chondroclasts adjacent to chondrocytes. In rat proximal tibiae, MMP−13 immunoreactivity is observed on the bone surface under the clear zone and ruffled border of resorbing osteoclasts, as well as in osteocytes adjacent to osteoclasts and bone lining cells31）. The enzyme seems to be derived from osteocytes adjacent to osteoclasts and translocated onto bone surfaces under osteoclasts through the osteocytic lacunae−canaliculi channel. However, osteoblasts and osteocytes with alkaline phosphatase activity were not detected in the resorp-
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Fig. 2 Schematic diagram of hypothesis regarding the role of matrix metalloproteinases （MMPs）in the initiation of Meckel’s cartilage disintegration on E15 Filled arrows indicate activation of MMPs and transforming growth factor（TGF）−β, dotted arrows show the release of MMPs, latent TGF−β, and RANKL／OPG, and the open arrow the action of TGF−β.
tion area of E16 Meckel’s cartilage and in the remnants of calcified cartilaginous matrix 2）. Thus, Meckel’s cartilage does not participate in mandibular bone formation by endochondral ossification, and MMP−13−positive hypertrophic chondrocytes may play a key role in the degradation of Meckel’s cartilage through the supply of MMP−13 to chondroclasts. Calcification of ECM is essential for resorption by osteoclasts and chondroclasts, and the cartilaginous matrix has already been calcified by E16 in Meckel’s cartilage. In the resorption area, dot−like fluorescence was often observed in intercellular septa that were not yet calcified, regardless of CTT treatment. Also, at a position anterior to the resorption area, reactivity was detected in intercellular septa that had just started the process of calcification. On the other hand, EDTA treatment clearly inhibited fluorescence in the calcified cartilaginous matrix and dot−like fluorescence in the intercellular septa. These results indicate that gelatinolytic activity is closely related to calcification of the intercellular septa between chondrocytes. In endochondral bone formation, authentic matrix vesicles isolated from the hypertro-
Fig. 3 Schematic diagram of hypothesis regarding the role of matrix metalloproteinases in chondrocyte differentiation, and calcification and degradation of the extracellular matrix Filled arrows indicate activation of MMPs and dotted arrows show the release of MMPs.
phic growth plate contain MMP−2, MMP−9, and MMP−13（major MMPs and integral components present in matrix vesicles） for activation of TGF−β15）, which indicates the potential role of MMPs in the initiation of calcification. Furthermore, MMP−2, MMP− 9, and MMP−13 immunoreactivity was detected in hypertrophic chondrocytes of resorbing cartilage. These findings strongly suggest the large contribution by MMPs to the initiation of calcification in focal sites in the intercellular septa of hypertrophic chondrocytes. In addition, intense fluorescence associated with gelatinolytic activity was detected in the cytoplasm of some hypertrophic chondrocytes facing the resorption front, not in the intercellular septa, in the presence of EDTA on E16, indicating the existence of gelatinolytic proteinases other than MMPs in the cytoplasm of chondrocytes. MMP−13 can degrade both typeⅠ collagen of bone and typeⅡ collagen of cartilage at neutral pH32）, whereas the cysteine cathepsin B has an extracellular role to increase MMP levels by proteolytic inhibition of TIMPs33）. Moreover, the gelatinolytic activity of cathepsins B, L, S, and F can degrade heat−denatured or MMP−1−predigested typeⅠ collagen （gelatin）34）. Interestingly, no significant differ-
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ences were found in the gelatinolytic activities of cathepsins in the presence or absence of chondroitin− 4 sulfate. Lysosomal cysteine and aspartic cathepsins （e. g., B, D, H, L, and S）are localized in hypertrophic chondrocytes of growing cartilage35―37）. Thus, gelatinolytic proteinases other than MMPs may play an important role in the degradation of Meckel’s cartilage at the terminal stage of chondrocytes near the resorption front. In Fig. 3, hypothetical mechanisms by which MMPs play roles in the remodeling of ECM during chondrocyte differentiation and in the resorption of calcified ECM are presented. MMP−2 in opened chondrocytic lacunae and connective tissue may be associated with the activation of other MMPs regulated by MMP−14 from chondroclasts. In addition, activated MMP−2 may act toward the activation of MMP−9 secreted by chondroclasts and MMP−13 from vacuolated hypertrophic chondrocytes. Therefore, chondrocytes adjacent to chondroclasts appear to be indispensable partners for the resorption of calcified Meckel’s cartilage by chondroclasts. Concluding Remarks Chondrocytes of Meckel’s cartilage are largely responsible for the development, calcification, and resorption of Meckel’s cartilage through ECM degradation by gelatinolytic activity of MMPs and other proteinases.
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Maskos, K.：Cristal 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 ―5248, 1998. 22）Morgunova, E., Tuuttila, A., Bergmann, U. and Tryggvason, K.：Structural insight into the complex formation of latent matrix metalloproteinase 2 with tissue inhibitor of metalloproteinase 2. Proc. Natl. Acad. Sci. USA 99：7414―7419, 2002. 23）Bernardo, M. M. and Fridman, R.：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―745, 2003. 24）Toth, M., Chvyrkova, I., Bernardo, M. M., Hernandez−Barrantes, S. and Fridman, R.：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―395, 2003. 25）Yu, Q. and Stamenkovic, I.：Cell surface−localized matrix metalloproteinase−9 proteolytically activates TGF−β and promotes tumor invasion and angiogenesis. Genes Dev. 14：163―176, 2000. 26）Karsdal, M. A., Larsen, L., Engsig, M. T., Lou, H., Ferreras, M., Lochter, A., Delaissé, J.−M. and Foged, N. T.：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―44067, 2002. 27）Takai, H., Kanematsu, M., Yano, K., Tsuda, E., Higashio, K., Ikeda, K., Watanabe, K. and Yamada, Y.：Transforming growth factor−β stimulates the production of osteoprotegerin／osteoclastogenesis inhibitory factor by bone marrow stromal cells. J. Biol. Chem. 273：27091―27096, 1998. 28）Thirunavukkarasu, K., Miles, R. R., Halladay, D. L., Yang, X., Galvin, R. J. S., Chandrasekhar, S., Martin, T. J. and Onyia, J. E.：Stimulation of osteoprotegerin （OPG）gene expression by transforming growth factor−β（TGF−β）. J. Biol. Chem. 276：36241―36250, 2001. 29）Karsdal, M. A., Hjorth, P., Henriksen, K., Kirkegaard, T., Nielsen, K. L., Lou, H., Delaissé, J.−M. and Foged, N. T.：Transforming growth factor−β controls human osteoclastogenesis through the p38 MAPK
149 and regulation of RANK expression. J. Biol. Chem. 278：44975―44987, 2003. 30）Werb, Z. and Chin, J. R.：Extracellular matrix remodeling during morphogenesis. Ann. NY Acad. Sci. 857：110―118, 1998. 31）Nakamura, H., Sato, G., Hirata, A. and Yamamoto, T.：Immunolocalization of matrix metalloproteinase− 13 on bone surface under osteoclasts in rat tibia. Bone 34：48―56, 2004. 32）Kahari, V. M. and Saarialho−Kere, U.：Matrix metalloproteinases and their inhibitors in tumor growth and invasion. Ann. Med. 31：34―45, 1999. 33）Kostoulas, G., Lang, A., Nagase, H. and Baici, A.： Stimulation of angiogenesis through cathepsin B inactivation of the tissue inhibitors of matrix metalloproteinases. FEBS Lett. 455：286―290, 1999. 34）Li, Z., Yasuda, Y., Li, W., Bogyo, M., Katz, N., Gordon, R. E., Fields, G. B. and Brömme, D.：Regulation of collagenase activities of human cathepsins by
glycosaminoglycans. J. Biol. Chem. 279：5470― 5479, 2004. 35）Ohsawa, Y., Nitatori, T., Higuchi, S., Kominami, E. and Uchiyama, Y.：Lysosomal cysteine and aspartic proteinases, acid phosphatase, and an endogenous cysteine proteinase inhibitor, cystatin−β, in rat osteoclasts. J. Histochem. Cytochem. 41：1075― 1083, 1993. 36）Söderström, M., Salminenm, H., Glumoff, V., Kirschke, H., Aro, H. and Vuorio, E.：Cathepsin expression during skeletal development. Biochim. Biophys. Acta 1446：35―46, 1999. 37）Nakase, T., Kaneko, M., Tomita, T., Myoui, A., Ariga, K., Sugamoto, K., Uchiyama, Y., Ochi, T. and Yoshikawa, H.：Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human. Histochem. Cell Biol. 114：21― 27, 2000.
REVIEW（The Specificity of Meckel’s Cartilage）
Role of Matrix Metalloproteinases in Extracellular Matrix Disintegration of Meckel’s Cartilage in Mice Yasunori Sakakura§ Division of Anatomy, Department of Oral Growth & Development, School of Dentistry, Health Sciences University of Hokkaido 1757 Kanazawa, Ishikari−Tobetsu, Hokkaido 061−0293, Japan 〔Received on February 9, 2010；Accepted on February 24, 2010〕 Key words：matrix metalloproteinases／gelatinolysis／ECM degradation／chondroclasts／Meckel’s cartilage Abstract：Meckel’s cartilage is a temporary supporting tissue that forms during the embryonic period. Unlike the distal（anterior）, posterior, and proximal portions, the middle portion degenerates with the death of chondrocytes as well as resorption of the cartilaginous matrix by chondroclasts without giving rise to ossification. Perichondral cells initially differentiate into osteoblasts and subsequently form periosteal bone on the lateral surface of Meckel’s cartilage closest to the incisor teeth, and then chondrocytes become hypertrophied in the restricted position of Meckel’s cartilage. Thereafter, the calcified periosteal bone and cartilage are resorbed by osteoclasts and chondroclasts；however, little is known regarding the mechanisms by which the middle portion is disintegrated during the embryonic period. Based on immunohistochemical and in situ zymographic findings in mice, we discuss the role of matrix metalloproteinases in the disintegration of the middle portion of Meckel’s cartilage.
Introduction Meckel’s cartilage is a supporting tissue found in the embryonic mandible of mammals. In rodents, it initially forms as a pair of rod−shaped cartilaginous bars within the first branchial arch and then, as development and growth of the mandible proceeds, three regions of Meckel’s cartilage determine its ultimate fate. In the distal（anterior）region, the cartilage bars fuse and undergo endochondral ossification to form intra−mandibular symphysis, while the proximal region converts to inner ear ossicles, such as the malleus and incus. The intermediate region occupying §
Corresponding author E−mail：yasaka@hoku−iryo−u.ac.jp
the major area of Meckel’s cartilage is further subdivided into the middle and posterior portions. The middle portion（anterior part of the intermediate region） degenerates with the death of chondrocytes as well as resorption of the cartilaginous matrix by chondroclasts, whereas the posterior portion is replaced by fibrous tissue, e. g. the sphenomandibular ligament. Especially in the middle portion, Meckel’s cartilage ；howdisappears without giving rise to ossification1,2） ever, how proteolytic enzymes contribute to degradation of the middle portion remains unknown. In this review, the mechanisms involved in the degradation of the middle portion of Meckel’s cartilage are discussed on the basis of immunolocalization of matrix metalloproteinases（MMPs）and in situ localization of gelatinolytic activity.
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Y. Sakakura：MMPs in Meckel’s Cartilage Disintegration
Resorption Process in the Middle Portion of Meckel’s Cartilage On embryonic day 15（E15）, perichondral cells differentiate into osteoblasts in the middle portion of Meckel’s cartilage in mice and subsequently form periosteal bone on the lateral surface closest to the incisor teeth（Fig. 1A）. Consequently, nearly all of the chondrocytes become hypertrophied on E15, though early hypertrophic chondrocytes are found in the outermost periphery on the lateral side. A marker for the initiation of cartilage resorption is the appearance of multinuclear cells responsible for the resorption of bone and cartilage on the lateral side1）. At this time, dental follicular cells of the incisor teeth moderately express the receptor activator of nuclear factor−κB ligand（RANKL）and osteoprotegerin（OPG） , whereas osteoblasts derived from perichondral cells on the lateral surface do not1）. Thus, the development of incisor teeth might contribute to the recruitment of multinuclear cells at the appropriate time and in the correct position. On E16, the periosteal bone and cartilaginous matrix between chondrocytes become calcified, and the calcified bone and cartilage are eroded by osteoclasts and chondroclasts. Thereafter, this disintegrative process suddenly commences in the restricted position of the cartilage, and expands toward the medial or anterior and posterior directions within the middle portion on E16（Fig. 1B）. Thus, a local site−specific environmental cue is involved in the resorption of Meckel’s cartilage. Involvement of MMPs in the Initiation of Cartilage Resorption on E15 Although fragmentary, many studies have reported that MMPs and tissue inhibitor of metalloproteinase （TIMP）−2 are strongly involved in the development and resorption of Meckel’s cartilage3―7）. MMP−2 （gelatinase A）immunoreactivity was detected in the bone matrix under periosteal osteoblasts in mice on E151）. In addition, MMP−13（collagenase 3）immunoreactivity was found preferentially localized in bone matrix deposited immediately under periosteal
osteoblasts. However, MMP−9 （gelatinase B） and MMP−14（membrane type 1−MMP）immunoreactivity was scarcely detected in the deposited bone matrix and periosteal osteoblasts, whereas these proteinases showed intense immunolocalization in the peripheral chondrocytes of Meckel’s cartilage. These results indicate that the uncalcified periosteal bone matrix undergoes remodeling via a proteolytic process prior to calcification. On the other hand, in situ gelatinolytic zymography with application of DQ−gelatin to unfixed non−decalcified frozen sections has indicated that the difference in fluorescence intensity shows localization of MMP− 2 and MMP−9, as well as gelatinolytic MMPs other than the gelatinases, and gelatinolytic enzymes other than MMPs, which is dependent on the absence or presence of CTT（a selective inhibitor of MMP−2 and MMP−9）or EDTA（a general broad−spectrum MMP inhibitor）8）. During the degradation process, gelatinolytic activity has been found to predominate in the periosteal bone matrix covering the lateral surface and the intercellular septa between chondrocytes. On E15, CTT slightly attenuated gelatinolytic activity, whereas it almost completely disappeared as a result of EDTA treatment, indicating that gelatinolytic MMPs, other than MMP−2 and MMP−9, also play a pivotal role in ECM remodeling of the periosteal bone and intercellular septa. The most likely MMP candidates are considered to be MMP−1, MMP−13, and MMP−14, which have already been found in Meckel’s cartilage2,5,6） and have activities capable of degrading gelatin9）. In addition, gelatinolytic activity in the intercellular septa was more intense in Meckel’s cartilage on E15 than E14. ECM degradation by proteinases is essential for chondrocyte enlargement, which occurs before the onset of calcification10）. Moreover, MMP− 2, MMP−8, MMP−9, MMP−13, and MMP−14 play integral roles in chondrocyte hypertrophy, as well as the initiation of calcification through the activation of pro−MMPs and reconstitution of the ECM, including proteoglycans and typeⅡ and Ⅹ collagens in endochondral bone formation 11―18）. Therefore, these results implicate the activities of gelatinases and the other gelatinolytic MMPs in ECM degradation during hypertrophy of Meckel’s chondrocytes, prior to subse-
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Fig. 1 Histology images of the middle portion of Meckel’s cartilage from mouse mandibles on E15（A）and E16（B） Osteoclasts and chondroclasts（arrows）can be seen on the lateral side facing the incisor tooth（It）in both embryonic stages. Bars＝10 μm.
quent calcification of the intercellular septa between chondrocytes. MMP−2 and MMP−9 cleave denatured collagens, typeⅣ and Ⅴ collagens, and elastin. MMP−2 is the most widespread of all the MMPs and can activate pro−MMP−1319,20）. Interestingly, the activities of these MMPs are regulated at the transcription, translation, and proenzyme activation levels, and via TIMPs, which bind to MMPs. TIMP−2 can bind to pro−MMP−2 via its C−terminal region, while it also binds to the active site of MMP−14, implying the formation of an MMP−14／TIMP−2／pro−MMP−2 ternary complex21,22）. Bound pro−MMP−2 is activated by neighboring TIMP−2−free MMP−14, and active MMP−2 is released into the extracellular space, while it can also promote the activation of pro−MMP− 923,24）. Thus, MMP−2, MMP−9, MMP−13, and MMP− 14 form part of an activation cascade that involves TIMP−2. Taken together, an activation cascade of MMPs by an MMP−14／TIMP−2／pro−MMP−2 ternary complex may be involved in disintegration of the uncalcified bone matrix covering the lateral surface through face−to−face crosstalk between periosteal osteoblasts and peripheral chondrocytes of Meckel’s cartilage （Fig. 2）. In these processes, MMPs may release and activate a latent form of transforming g ro w t h f a c t o r−β （T G F−β） f ro m E C M−b o u n d stores15,25,26）. In addition, TGF−β may be involved in the differentiation and recruitment of osteoclasts
through regulation of RANK, a receptor for RANKL, RANKL, and OPG, a decoy receptor against RANKL, on the lateral surface of Meckel’s cartilage on E151,27―29）. Role of MMPs in Resorption Process on E16 MMPs expressed by chondroclasts clearly play a role in E16 Meckel’s cartilage. Although the immunoreactivity of MMP−2 is faint, opened chondrocytic lacunae and connective tissue were positive in the resorption area, whereas hypertrophic chondrocytes were negative. MMP−9 immunoreactivity was especially intense in the chondroclasts, similar to reports showing that MMP−9 is highly expressed in multinucleated osteoclasts3,11,30）. In addition, MMP−14 immunolocalization was also recognized in chondroclasts. Unexpectedly, intense immunostaining for MMP−13 was found in the ruffled border of chondroclasts adjacent to chondrocytes. In rat proximal tibiae, MMP−13 immunoreactivity is observed on the bone surface under the clear zone and ruffled border of resorbing osteoclasts, as well as in osteocytes adjacent to osteoclasts and bone lining cells31）. The enzyme seems to be derived from osteocytes adjacent to osteoclasts and translocated onto bone surfaces under osteoclasts through the osteocytic lacunae−canaliculi channel. However, osteoblasts and osteocytes with alkaline phosphatase activity were not detected in the resorp-
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Y. Sakakura：MMPs in Meckel’s Cartilage Disintegration
Fig. 2 Schematic diagram of hypothesis regarding the role of matrix metalloproteinases （MMPs）in the initiation of Meckel’s cartilage disintegration on E15 Filled arrows indicate activation of MMPs and transforming growth factor（TGF）−β, dotted arrows show the release of MMPs, latent TGF−β, and RANKL／OPG, and the open arrow the action of TGF−β.
tion area of E16 Meckel’s cartilage and in the remnants of calcified cartilaginous matrix 2）. Thus, Meckel’s cartilage does not participate in mandibular bone formation by endochondral ossification, and MMP−13−positive hypertrophic chondrocytes may play a key role in the degradation of Meckel’s cartilage through the supply of MMP−13 to chondroclasts. Calcification of ECM is essential for resorption by osteoclasts and chondroclasts, and the cartilaginous matrix has already been calcified by E16 in Meckel’s cartilage. In the resorption area, dot−like fluorescence was often observed in intercellular septa that were not yet calcified, regardless of CTT treatment. Also, at a position anterior to the resorption area, reactivity was detected in intercellular septa that had just started the process of calcification. On the other hand, EDTA treatment clearly inhibited fluorescence in the calcified cartilaginous matrix and dot−like fluorescence in the intercellular septa. These results indicate that gelatinolytic activity is closely related to calcification of the intercellular septa between chondrocytes. In endochondral bone formation, authentic matrix vesicles isolated from the hypertro-
Fig. 3 Schematic diagram of hypothesis regarding the role of matrix metalloproteinases in chondrocyte differentiation, and calcification and degradation of the extracellular matrix Filled arrows indicate activation of MMPs and dotted arrows show the release of MMPs.
phic growth plate contain MMP−2, MMP−9, and MMP−13（major MMPs and integral components present in matrix vesicles） for activation of TGF−β15）, which indicates the potential role of MMPs in the initiation of calcification. Furthermore, MMP−2, MMP− 9, and MMP−13 immunoreactivity was detected in hypertrophic chondrocytes of resorbing cartilage. These findings strongly suggest the large contribution by MMPs to the initiation of calcification in focal sites in the intercellular septa of hypertrophic chondrocytes. In addition, intense fluorescence associated with gelatinolytic activity was detected in the cytoplasm of some hypertrophic chondrocytes facing the resorption front, not in the intercellular septa, in the presence of EDTA on E16, indicating the existence of gelatinolytic proteinases other than MMPs in the cytoplasm of chondrocytes. MMP−13 can degrade both typeⅠ collagen of bone and typeⅡ collagen of cartilage at neutral pH32）, whereas the cysteine cathepsin B has an extracellular role to increase MMP levels by proteolytic inhibition of TIMPs33）. Moreover, the gelatinolytic activity of cathepsins B, L, S, and F can degrade heat−denatured or MMP−1−predigested typeⅠ collagen （gelatin）34）. Interestingly, no significant differ-
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ences were found in the gelatinolytic activities of cathepsins in the presence or absence of chondroitin− 4 sulfate. Lysosomal cysteine and aspartic cathepsins （e. g., B, D, H, L, and S）are localized in hypertrophic chondrocytes of growing cartilage35―37）. Thus, gelatinolytic proteinases other than MMPs may play an important role in the degradation of Meckel’s cartilage at the terminal stage of chondrocytes near the resorption front. In Fig. 3, hypothetical mechanisms by which MMPs play roles in the remodeling of ECM during chondrocyte differentiation and in the resorption of calcified ECM are presented. MMP−2 in opened chondrocytic lacunae and connective tissue may be associated with the activation of other MMPs regulated by MMP−14 from chondroclasts. In addition, activated MMP−2 may act toward the activation of MMP−9 secreted by chondroclasts and MMP−13 from vacuolated hypertrophic chondrocytes. Therefore, chondrocytes adjacent to chondroclasts appear to be indispensable partners for the resorption of calcified Meckel’s cartilage by chondroclasts. Concluding Remarks Chondrocytes of Meckel’s cartilage are largely responsible for the development, calcification, and resorption of Meckel’s cartilage through ECM degradation by gelatinolytic activity of MMPs and other proteinases.
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