Expression of Versican and ADAMTS1, 4, and 5 During Bone Development in the Rat Mandible and Hind Limb
Divisions of Oral Surgery (MN,SE), Craniofacial Development and Regeneration (MN,YS), Pediatric Dentistry (SS), and Orthodontics and Dentofacial Orthopedics (IT), Tohoku University Graduate School of Dentistry, Sendai, Japan, and Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan (IM)
Correspondence to: Yasuyuki Sasano, DDS, PhD, Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan. E-mail: sasano{at}anat.dent.tohoku.ac.jp
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Summary |
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Key Words: versican ADAMTS bone development osteoblast extracellular matrix remodeling in situ hybridization immunohistochemistry RT-PCR
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Introduction |
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ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) is a family of extracellular proteases. It has a characteristic ADAM-like protease domain, a disintegrin-like and a cysteine-rich domain, and possess some thrombospondin type 1 motifs. To date, 20 members of the ADAMTS family have been identified. Unlike the ADAMs, these proteins lack transmembrane domains and are, therefore, secreted into the extracellular environment (Tang 2000). Although a distinct proteolytic activity has been demonstrated for several members of ADAMTS (Apte 2004
), little information is available about involvement of ADAMTS in bone remodeling and development.
ADAMTS4 (aggrecanase-1) and ADAMTS5/11 (aggrecanase-2) are the proteases responsible for cleaving aggrecan, a major proteoglycan in the ECM of cartilage (Abbaszade et al. 1999; Tortorella et al. 1999
). ADAMTS1 is also capable of cleaving aggrecan (Kuno et al. 2000
). ADAMTS4 also cleaves brevican, which is a brain-specific ECM proteoglycan (Matthews et al. 2000
). Furthermore, it has been suggested that ADAMTS1, 4, and 5 also have the ability to degrade versican, which is a member of the lectican family that includes aggrecan, neurocan, and brevican. Versican is a large chondroitin sulfate proteoglycan. Its N-terminal globular domain (G1) binds to the glycosaminoglycan hyaluronan, and its carboxy-terminal globular domain (G3) consists of two epidermal growth factorlike domains, a lectin-like domain, and a complement regulatory proteinlike domain. Four splice variants (V0V3) of versican are known (Wight 2002
). Recent studies showed cleavage of versican V0 and V1 by ADAMTS1 and ADAMTS4 (Sandy et al. 2001
; Russell et al. 2003
), and cleavage of versican V2 by ADAMTS4 (Westling et al. 2004
).
Aggrecan is most abundant in cartilage, whereas versican has a rather wide tissue distribution (Zako et al. 1995). It has been suggested that a large proteoglycan presumed to be versican is present in the bone matrix during early bone formation (Lee et al. 1998
). However, the profile of expression and degradation of this proteoglycan during bone development remains unknown. We hypothesized that versican is expressed in bone and its putative degrading enzymes ADAMTS1, 4, and 5 are also expressed and involved in versican remodeling during bone development. To test our hypothesis, we investigated the expression of versican mRNA and ADAMTS1, 4, and 5 mRNAs in rat mandibles and hind limbs at various stages of bone development and growth, by RT-PCR and in situ hybridization. Immunohistochemistry was used to detect versican polypeptides.
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Materials and Methods |
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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. cDNA was synthesized using 1.0 µg of total RNA primed with 1.5 µg of random primers (Invitrogen; Carlsbad, CA) in the presence of reverse transcriptase at 100 U/µg RNA, in the reverse transcription buffer supplied by Invitrogen. For cDNA amplification, 1.0 µl of reverse transcription products was incubated in the presence of 20 pmol of two specific primers (Table 1), 1.5 mM MgCl2, 0.2 mM of the four dNTPs (Invitrogen), and 0.05 U of Taq polymerase, in the manufacturer's reaction buffer (Invitrogen). The reaction mixture was subjected to one cycle of denaturation for 3 min, 50 sec at 95C, followed by 50 cycles of an amplification sequence that consisted of denaturation for 1 min, 10 sec at 95C, annealing for 1 min, 10 sec at 64C for ADAMTS4 and versican, 62C for ADAMTS1, and 58C for ADAMTS5, and extension for 1 min, 30 sec, with an additional 8 min, 30 sec extension for the last cycle.
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Preparation of Riboprobes
Digoxigenin-labeled, single-stranded riboprobes were prepared using the DIG RNA-labeling kit (Roche; Mannheim, Germany) according to the manufacturer's instructions.
Fragments encoding rat versican (4231039 bp: GenBank AF072892), rat ADAMTS1 (76520 bp: GenBank NM024400), rat ADAMTS4 (6251226 bp: GenBank XM237904), and rat ADAMTS5 (15482305 bp: GenBank NM198761) were obtained from the total RNA of embryonic rat limbs using RT-PCR and subcloned into the plasmid pCRIITOPO (Invitrogen). Oligonucleotide primers used for the RT-PCR are shown in Table 1. The identities of the cDNAs were verified by digestion with restriction enzymes and confirmed by dideoxynucleotide sequencing. Riboprobes were generated as indicated in Table 2. An 818-bp fragment (2231040 bp: GenBank AF062402) encoding rat versican was obtained from total RNA of rat brain, and a fragment encording rat pro- 1 (I) collagen (28384329 bp: GenBank Z78279) was obtained from the total RNA of rat skin using RT-PCR, and both were subcloned into the pT7T3-a18 plasmid (Life Technologies; Grand Island, NY). The plasmid was linearized and transcribed by RNA polymerases to generate riboprobes as indicated in Table 2.
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The sections were deparaffinized and washed in PBS, pH 7.4, and then immersed in 0.2 N HCl for 20 min. After being washed in PBS, the sections were incubated in proteinase K (20 g/ml; Roche) in PBS for 30 min at 37C.
The sections were then dipped in 100% ethanol and dried in air and incubated with the antisense probe or the sense control probe (400 ng/ml) in a hybridization mixture for 16 hr at 45C.
The sections were washed and treated with RNase (Type 1a, 20 µg/ml; Sigma, St Louis, MO) for 30 min at 37C. After washing, the hybridized probes were detected immunologically using the Nucleic Acid Detection Kit (Roche), counterstained with methyl green, and mounted with a mounting medium.
At least two sections from each of five embryos or three postnatal animals at each stage were examined using the same probe. The intensity of hybridization signals was evaluated by observing at least three fields of every section.
Immunohistochemistry
The protocol used in the present study has been reported elsewhere (Sasano et al. 2001; Nakamura et al. 2004
) and is only briefly described as follows.
The sections were deparaffinized and immersed in 3.0% hydrogen peroxide in absolute methanol for 10 min at room temperature.
After being washed in PBS containing 0.025% Triton X-100 (PBS-TX), the sections were incubated with the monoclonal mouse antibody against a large chondroitin-sulfate proteoglycan from bovine sclera (5D5) for 2 hr at room temperature. The antibody 5D5 was a generous gift of Dr. Firoz Rahemtulla, Department of Oral Biology, School of Dentistry, University of Alabama. The antibody recognizes the core protein of versican from sclera, tendon, aorta, and periodontium, but does not react with aggrecan (Bratt et al. 1992; Larjava et al. 1992
; Hakkinen et al. 1993
; Lee et al. 1998
; Mizoguchi et al. 1998
). The antibodies were diluted 1:500 with 5% normal goat serum in PBS-TX (NGS-PBS-TX).
After being washed in PBS-TX, the sections were incubated in Histofine Simple Stain rat MAX-PO (MULTI) (NICHIREI Co.; Tokyo, Japan) for 30 min at room temperature.
The sections were washed and immunoreactivity was visualized by immersion in Histofine Simple Stain DAB (3, 3'- diaminobenzidine) solution (NICHIREI Co.) for 5 min at room temperature. The sections were counterstained with methyl green and mounted with a mounting medium.
Control sections were processed as described previously, except that preimmune NGS-PBS-TX was used as a substitute for the specific antibody.
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Results |
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Hind Limbs
Osteogenesis began in the middle of the cartilaginous template at E18, whereas osteoid was hardly detectable, unlike E15 mandibles described previously. Some cells in the perichondrium (the future periosteum) expressed versican mRNA (Figure 3A) and ADAMTS1, 4 (Figure 3C), and 5 mRNAs, whereas osteoblasts that expressed COL1 (Figure 3D) did not express these molecules. Versican protein was localized around some perichondrial cells, but not in osteoblasts (Figure 3B).
Woven Bone
Mandibles
Flat osteoblasts on the bone surface showed intense expression of versican mRNA and COL1 at E18 (Figures 4A and 4C). In contrast, cuboidal osteoblasts expressed COL1 but not versican mRNA (Figures 4A and 4C). Versican protein was detected in flat osteoblasts (Figure 4B), which corresponded to the mRNA expression pattern. In addition, intense immunoreactivity was seen in the bone matrix (Figure 4B). The expression of ADAMTS1 (Figure 4D), 4 (Figure 4E), and 5 (Figure 4F) mRNA was also detected in flat osteoblasts but not cuboidal osteoblasts. Some osteocytes expressed versican and these ADAMTSs.
Hind Limb
The intense hybridization signals of versican (Figure 5A and Figure 6A) and ADAMTS1, 4 (Figure 5C and Figure 6C), and 5 were identified in flat osteoblasts and in a confined population of osteocytes, whereas cuboidal osteoblasts showed little expression of these molecules at E20. Periosteal cells expressed versican and these ADAMTSs strongly. Immunoreactivity for versican in osteoblasts corresponded to the mRNA expression pattern. The bone matrix around some osteocytes also showed versican immunoreactivity. The periosteum and the bone matrix showed strong immunoreactivity (Figure 5B and Figure 6B).
Lamellar Bone
The periosteum covered the outer surface of lamellar bone, whereas the endosteum lined the bone surface facing the marrow cavity. The periosteum consisted of osteoblasts lining the bone matrix and the fibrous layer of periosteal cells overlying the osteoblasts, whereas the endosteum was composed of the osteoblastic layer with little fibrous tissue.
Mandibles
In lamellar bone of the body of the mandible at W3 and W6, endosteal osteoblasts showed hybridization signals for versican (Figure 7A) and ADAMTS1 (Figure 7D), 4 (Figure 7E), and 5 (Figure 7F). In contrast, mRNA expression of these mRNAs was weak in periosteal osteoblasts and periosteal cells. These endosteal and periosteal osteoblasts were of varied morphology: cuboidal or flat, and some intermediate. Some osteocytes expressed versican and these ADAMTSs.
The versican immunoreactivity was localized in the periosteum and, very weakly, in the bone matrix. The protein was hardly detectable in the endosteum (Figure 7B).
Hind Limbs
In lamellar bone of the femur at W3 and W6, intense hybridization signals for versican (Figure 8A) and ADAMTS1, 4 (Figure 8C), and 5 were seen in endosteal osteoblasts, whereas expression of these mRNA was weak in periosteal osteoblasts (data not shown). These endosteal and periosteal osteoblasts were heterogeneous in morphology, as they were in the mandibles.
The versican immunoreactivity was localized in the periosteum and very weakly in the bone matrix. The protein was hardly detectable in the endosteum (Figure 8B).
Periodontal Ligaments
Some cells of periodontal ligaments at W3 and W6 expressed versican mRNA (Figure 9A) and ADAMTS1, 4 (Figure 9C), and 5 mRNA. The versican protein was localized in periodontal ligaments (Figure 9B). Flat osteoblasts and some osteocytes showed strong expression of these molecules in alveolar bone. The versican protein was more abundant in the matrix of alveolar bone around periodontal ligaments than the body of the mandible.
Controls
No hybridization signal was identified in the sections processed with the sense RNA probes (Figures 2B and 2E). None of the controls for immunohistochemistry showed labeling.
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Discussion |
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We found that the mRNA expression patterns of ADAMTS1, 4, and 5 were comparable to one another during bone development. It has been reported that the ability of ADAMTS4 to cleave aggrecan is higher than that of ADAMTS5 (Tortorella et al. 2002). In addition, ADAMTS4 has been reported to be more active than ADAMTS1 toward both versican and aggrecan (Sandy et al. 2001
). These studies suggest that ADAMTS1, 4, and 5 do not contribute equally to the degradation of versican during bone development.
In this study, we divided the process of bone development into three stages: the beginning of osteogenesis, the formation of woven bone and the formation of lamellar bone. Immunoreactivity for versican in the bone matrix was intense at the stage of woven bone, and subsequently became weaker at the stage of lamellar bone. These findings suggest that extensive remodeling of the ECM occurs in the formation of lamellar bone from woven bone, including a decrease in versican levels during bone development. In addition, the present study has shown that versican may be one of the most characteristic ECM molecules that distinguish woven bone from lamellar bone, because woven bone has been indicated to contain more proteoglycan than lamellar bone (Ross et al. 2003).
The expression of the versican protein corresponded to the expression of versican mRNA in osteoblasts, except in osteoid in mandibles, in the endosteum, and in the periosteum of lamellar bone in both mandibles and hind limbs. In E15 mandibles, versican proteins were localized within osteoid, whereas versican mRNA was not detected in osteoblasts lining osteoid, but was present mesenchymal cells surrounding the osteoblasts. In E14 mandibles, versican mRNA and protein were detected in the mesenchymal cell condensate in a putative osteogenic region (Hall and Miyake 2000; Sasano et al. 2000
; Zhu et al. 2001
). Versican may therefore be expressed by mesenchymal cells, but not osteoblasts, before differentiation of osteoblasts, and accumulated in osteoid, which may facilitate subsequent osteoblast differentiation. Although mRNA expression was detected, little versican protein was found in the endosteum of lamellar bone in both mandibles and hind limbs. We assume that this is because production of versican surpasses its degradation by ADAMTS1, 4, and 5 or other proteases in the endosteum of lamellar bone. In contrast, the expression of versican mRNA was very weak in the periosteum of lamellar bone, where immunoreactivity was strong. Periosteal cells in the fibrous layer overlying the active osteogenic region expressed high level of versican mRNA in woven bone. The versican expression in the periosteal cells decreased as woven bone developed to lamellar bone. We assume that the versican protein accumulates in the periosteum because production of versican exceeds its degradation in lamellar bone. Osteoblasts may be involved in both production and degradation of versican and regulate the balance between them, possibly by secreting these ADAMTSs.
We examined the process of bone development in both mandibles and hind limbs. There were distinct differences between mandibles and hind limbs in the beginning stage of osteogenesis. The intramembranous ossification of the mandible may be characterized by formation of osteoid rich in versican, whereas the endochondral ossification of the hind limbs may be characterized by little osteoid, where osteoid matures into bone immediately after it is deposited on the surface of calcified cartilage (Sasano et al. 2000). Thereafter, both mandibles and hind limbs proceed with the common sequential process in which woven bone is formed and gradually altered to become lamellar bone.
Osteoblasts are generally considered to be cuboidal in shape, whereas we demonstrated that flat osteoblasts producing COL1 express versican and ADAMTS1, 4, and 5. Previous studies showed that these flat osteoblasts express bone sialoprotein, osteopontin (Zhu et al. 2001), MMP8, and MMP13 (Sasano et al. 2002
). Besides osteoblasts, bone lining cells are known as cells on the bone surface, which are flat in shape and quiescent in bone formation (Marks and Odgren 2002
). The flat osteoblast identified in the present study may differ from the bone lining cell, because the flat osteoblast seems to be actively involved in bone formation by expressing ECM molecules. At the stage of woven bone, both cuboidal and flat osteoblasts expressed COL1, whereas expression of versican mRNA and ADAMTS1, 4, and 5 mRNAs was detected in flat osteoblasts and barely detected in cuboidal osteoblasts. Flat osteoblasts that express versican and ADAMTS1, 4, and 5 may play an important role in remodeling proteoglycans in the woven bone matrix. Osteocytes may be also involved in production and degradation of versican by secreting ADAMTS1, 4, and 5.
It is not known whether cuboidal and flat osteoblasts are the same cells and change their morphological characteristics depending on functional requirements. Alternatively, they may be distinct phenotypes of osteoblasts and have different assignments in bone formation and degradation. Further studies will be required to clarify the functional significance of these cuboidal and flat osteoblasts.
Based on the present results, we propose the following. Versican is expressed before differentiation of osteoblasts and is localized in osteoid during intramembranous ossification. In endochondral ossification, versican is expressed in periosteal cells overlying the active osteogenic region on the surface of calcified cartilage. Thereafter, bones undergoing either type of ossification proceed with the common sequential process of bone development. The bone matrix expands and woven bone rich in versican is formed. Versican expression in the bone matrix is decreased as woven bone is altered into lamellar bone, where ADAMTS1, 4, and 5 are involved. Osteoblasts may be involved in both production and degradation of versican by secreting ADAMTSs.
Further investigation of versican and ADAMTSs may provide a better understanding of remodeling of the extracellular matrix during bone development.
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Acknowledgments |
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We wish to thank Mr. Masami Eguchi and Mr. Yasuto Mikami, Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, for their excellent assistance in this study. We also wish to thank Dr. Firoz Rahemtulla, Department of Oral Biology, School of Dentistry, University of Alabama, for generously providing the antibody. We would like to thank Dr. Paul Kretchmer for reviewing this manuscript.
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Footnotes |
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