ARTICLE |
Correspondence to: Yasuyuki Sasano, Div. of Oral Molecular Biology, Tohoku U. Graduate School of Dentistry, Sendai 980-8575, Japan. E-mail: sasano@anat.dent.tohoku.ac.jp
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Summary |
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Matrix metalloproteinases (MMPs) 8 and 13 comprise the collagenase subfamily in rats and mice, and only MMP13 has been implicated in degradation of the collagenous matrices during development of bone and cartilage. On the hypothesis that MMP8 is also involved in bone and cartilage development, the present study was designed to investigate gene expression of MMP8 in rat embryonic mandibles and hind limbs. Expression of MMP8 was examined with in situ hybridization and RT-PCR and was compared with that of MMP13. Osteoblastic and chondrocytic cells expressing collagenous matrix molecules were identified using in situ hybridization for collagen Types I and II. The results demonstrated that MMP8 is expressed by osteoblastic progenitors, differentiated osteoblasts, osteocytes, and chondrocytes in the growth plate for the first time. Furthermore, the expression of MMP8 is much broader than that of MMP13, for which expression is confined to differentiated phenotypes of osteoblastic and chondrocytic lineage.
(J Histochem Cytochem 50:325332, 2002)
Key Words: MMP8, MMP13, collagenase, osteoblasts, osteocytes, chondrocytes, type I collagen, type II collagen, in situ hybridization
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Introduction |
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The major constituents of bone and cartilage extracellular matrices are collagens, i.e., Type I for bone and Type II for cartilage (
The matrix metalloproteinases (MMPs) are believed to play a central role in the breakdown of ECM, which is essential for embryonic development, morphogenesis, and tissue remodeling (
MMP13 is the only collagenase that has been implicated in degradation of the collagenous matrices during development of bone and cartilage (
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Materials and Methods |
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Preparation of Tissue
The principles of laboratory animal care (NIH publication no. 8623, revised 1985) were followed as well as specific national laws. The preparation was performed according to animal protocols that were institutionally approved by the Tohoku University. Wistar rat embryos 14 to 20 days post coitum and Wistar rats 1 week post natum were used. At least three embryos or postnatal animals at each stage were examined. Heads and hind limbs were resected and immediately fixed by immersion in 4% paraformaldehyde with 0.5% glutaraldehyde (GA) in 0.1 M phosphate buffer, pH 7.4, at 4C overnight. Some of the fixed specimens were decalcified in 10% EDTA in 0.01 M phosphate buffer, pH 7.4, for 34 days for embryos and 2 weeks for 1-week-old rats at 4C. The EDTA solution was autoclaved before use. After dehydration through a graded series of ethanol solutions, the tissues were embedded in paraffin (
Preparation of Riboprobes
Digoxigenin (DIG)-labeled single-strand riboprobes were prepared using the DIG RNA labeling kit (Roche; Mannheim, Germany) according to the manufacturer's instruction.
Fragments encoding rat MMP8 (1711253 bp:GenBank
AJ007288) and rat MMP13 (15472411 bp:GenBank
M60616,
M36452) were obtained from the total RNA of embryonic rat limbs using reverse transcription followed by polymerase chain reaction (RT-PCR) and subcloned into the PCR II TOPO (Invitrogen; Carlsbad, CA). A fragment encoding rat pro-1(I) collagen (28384329 bp:GenBank
Z78279) was obtained from the total RNA of rat skin using RT-PCR and subcloned into the pT7/T3-18 plasmid (Life Technologies; Grand Island, NY). A 545-bp fragment encoding mouse pro-
1(II) collagen was obtained from the total RNA of embryonic mouse limbs using RT-PCR as described previously (
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In Situ Hybridization
The protocol used in the present study has been reported elsewhere (
RT-PCR
The total RNA was extracted from embryonic rat mandibles and hind limbs using the RNeasy Mini Kit (QIAGEN; Hilden, Germany) and processed as follows (
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After amplification, equal amounts of PCR products were size-separated by electrophoresis through a 1.5% agarose gel and visualized by ethidium bromide staining and UV transillumination. All samples were amplified at least twice on different occasions to control for any variations in the PCR technique. Negative controls (no reverse transcription and no template RNA) did not produce bands visible on gels. Images of the stained gels were captured using the Electrophoresis Documentation and Analysis System (Kodak; Rochester, NY).
The PCR products in bands corresponding to MMP8 and MMP13 were extracted using the Gel Extraction Kit (QIAGEN) and the sequence was confirmed by dideoxynucleotide sequencing.
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Results |
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In Situ Hybridization
Mandibles.
Expression of pro-1(I) collagen (COL I) transcripts was demonstrated in mesenchymal cell condensation of the putative osteogenic region in E14 mandibles (Fig 1a). MMP8 was expressed in the mesenchymal cell condensation and chondrocytes in Meckel's cartilage (Fig 1b). In contrast, no expression of MMP13 was identified in E14 mandibles (Fig 1c).
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Cuboidal osteoblasts differentiated and expressed COL I actively in E15 (Fig 2a). MMP8 transcripts were demonstrated in embryonic periosteal cells as well as chondrocytes of Meckel's cartilage but not in cuboidal osteoblasts (Fig 2b). No expression of MMP13 was identified in E15 mandibles (Fig 2c).
Flat osteoblasts and osteocytes appeared in addition to cuboidal osteoblasts, and all of these cell types expressed COL I at E16 (Fig 3a). MMP8 was continuously shown in chondrocytes in Meckel's cartilage and periosteal cells around cuboidal osteoblasts (Fig 3b). MMP13 expression was demonstrated in flat osteoblasts and osteocytes strongly at E16 (Fig 3c). Chondrocytes in Meckel's cartilage expressed MMP13 weakly.
Cuboidal osteoblasts, flat osteoblasts, and osteocytes expressed COL I at E18 (Fig 4a) and E20 (data not shown). MMP8 was demonstrated in chondrocytes, periosteal cells around cuboidal osteoblasts, and flat osteoblasts at E18 (Fig 4b, and Fig 5) and E20 (data not shown). The strong expression of MMP13 was confined to flat osteoblasts and osteocytes at E18 (Fig 4c) and E20 (data not shown).
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Osteocytes in 1-week postnatal mandibles expressed COL I, MMP8, and MMP13 (Fig 6a6c). The pattern of gene expressions for COL I, MMP8, and MMP13 in osteogenic cells is summarized in Table 4, where the hybridization signal is graded as not detectable, weak, or strong according to the intensity.
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In addition to the osteogenic and chondrogenic phenotypes, muscle cells expressed MMP8 strongly (Fig 7a) and MMP13 weakly (Fig 7b).
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Hind Limbs. Cuboidal osteoblasts, flat osteoblasts, and osteocytes expressed COL I in growing rat hind limbs at E18 (data not shown), E20 (Fig 8a), and 1-week postnatum (data not shown), while periosteal cells showed weak expression. MMP8 transcripts were demonstrated in flat osteoblasts and osteocytes and very weakly in periosteal cells (Fig 8b). MMP13 expression was localized in flat osteoblasts and osteocytes (Fig 8c). The pattern of gene expressions for COL I, MMP8, and MMP13 in osteogenic cells was comparable to that in mandibles, as shown in Table 4.
MMP8 was expressed in almost all chondrocytes (Fig 9b) which expressed pro-1(II) collagen (COL II) (Fig 9a) in developing tibias and femurs, whereas MMP13 expression was localized in hypertrophic chondrocytes (Fig 9c). The pattern of gene expressions for MMP8, MMP13, and COL II in differentiating chondrocytes is summarized in Table 5, where the intensity of hybridization signals is graded as not detectable, weak, or strong.
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Controls for In Situ Hybridization. No hybridization signal was identified in the adjacent sections processed with control sense riboprobes (Fig 4d4f and Fig 9d9f).
RT-PCR
RT-PCR demonstrated that the gene of MMPs 8 and 13 is expressed in developing mandibles and hind limbs (Fig 10).
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Discussion |
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Collagenases are the only members of the MMP family that degrade native fibrillar collagens of Types I, II, and III (
The present study demonstrated that MMP8 is expressed in the mesenchymal cell condensation of osteoblastic progenitors (
MMP13 is the only collagenase that has been implicated in degradation of the collagenous matrices during development of bone (
The present study suggested that osteocytes express both MMP8 and MMP13. Our previous studies demonstrated that osteocytes express ECM molecules, such as Type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin (
Cuboidal osteoblasts engaged in production of ECM molecules such as Type I collagen may not be involved in expression of MMPs in rat embryos. In contrast, flat osteoblasts whose production of matrix molecules is less than the cuboidal osteoblasts could participate in degradation of the collagenous matrix with synthesis of MMP8 and MMP13 (
Chondrocytes expressed MMPs 8 and 13. MMP13 was confined to hypertrophic chondrocytes, as previously reported (
Muscle cells expressed MMP8 strongly and MMP13 weakly. These collagenases may be involved in remodeling of the ECM during myogenesis, along with other MMPs, such as MMPs 2 and 9 (
The previous study indicated that MMP13 is expressed during fracture healing of bone (
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Acknowledgments |
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Supported in part by grants-in-aid (12671986, 13671893, 13672076, 13470454) from the Ministry of Education, Science, Sports and Culture of Japan.
We wish to thank Mr Masami Eguchi and Mr Yasuto Mikami, Division of Oral Molecular Biology, Tohoku University Graduate School of Dentistry, for excellent assistance in this study.
Received for publication July 31, 2001; accepted October 18, 2001.
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