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RESEARCH REPORT |
1 Departments of Orthodontics and
2 Biochemistry, Hiroshima University Faculty of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;
*corresponding author, shigebon{at}hiroshima-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3 when these steroids increased alkaline phosphatase (ALP) activity. Furthermore, exogenous RGD-CAP suppressed the ALP activity and bone nodule formation of cultured PDL cells. These findings suggest that RGD-CAP in the PDL modulates the mineralization which affects adjacent alveolar bone metabolism.
KEY WORDS: periodontal ligament • RGD-CAP/ßig-h3 • alkaline phosphatase • mineralization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The characteristic four repetitive structures similar to RGD-CAP/ßig-h3 without the RGD motif were found in insect fasciclin I, as well as osteoblast specific factor 2 (OSF-2)/periostin (Skonier et al., 1992; Wang et al., 1993; Horiuchi et al., 1999). These proteins were also shown to have similar functions in cell adhesion (Takeshita et al., 1993; Sugiura et al., 1995; Horiuchi et al., 1999; Ohno et al., 1999), and have been categorized as members of the fasciclin family.
Recently, it was demonstrated that the mRNA level of RGD-CAP/ßig-h3 was decreased in human bone marrow stromal cells (BMSC) treated with Dex, which promotes osteogenic differentiation of BMSC (Dieudonné et al., 1999), and that RGD-CAP/ßig-h3 inhibited bone nodule formation of mouse osteoblasts in vitro (Kim et al., 2000). Furthermore, our recent studies have shown that recombinant RGD-CAP inhibited the mineralization of hypertrophic chondrocytes (Ohno et al., 2002). These findings suggest that RGD-CAP/ßig-h3 functions as a negative regulator of osteogenesis.
We previously hypothesized that RGD-CAP/ßig-h3 expressed in the PDL plays a role in the maintenance of the structural equilibrium of the PDL by inhibiting mineralization. In the present study, we investigated the expression of RGD-CAP/ßig-h3 in the human PDL and its effects on the mineralization of the PDL.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Cells
Human PDL cells were obtained from healthy human teeth indicated for extraction for orthodontic treatment following the methods described in detail in a previous study (Somerman et al., 1989). Prior to the experiment, informed consent was obtained from all patients regarding the extraction of their teeth. The experimental protocols were approved in advance by the Ethics Committee on Experimental Studies with Human Subjects, Hiroshima University Faculty of Dentistry. For all experiments, passage 4-5 cells were used.
Cultures were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; GIBCO), 100 units penicillin, and 100 µg/mL of streptomycin (GIBCO) (Medium A) in an atmosphere of 5% CO2 in a humidified incubator.
For investigation of the RGD-CAP mRNA level and ALP activity in the PDL cell cultures, the cells seeded in 10-cm dishes in Medium A containing 50 µg/mL ascorbic acid were treated with 10-8 M dexamethasone (Dex) or 10-8 M 1,25-dihydroxyvitamin D3 (vitamin D) for 11 days after confluence.
For investigation of the effects of RGD-CAP on the mineralization of PDL cells, the cells seeded in 35-mm dishes were maintained in mineralizing medium (DMEM containing 10% FCS, 50 µg/mL ascorbic acid, 100 nM ß-glycerophosphate, and 10-8 M Dex) for 21 days.
Western Blot Analysis
RGD-CAP is so tightly attached to the insoluble collagen fibers that this protein is resistant to protease and homogenization (Hashimoto et al., 1997). Therefore, we solubilized the samples of human PDL in Laemmli buffer in 4 M urea and boiled them for 10 min. Samples of 1 mg were separated by SDS-PAGE in a 4-20% polyacrylamide gradient gel, in the presence of ß-mercaptoethanol (0-5.0%) that breaks aggregates stabilized by disulfide bonds. Proteins were blotted onto polyvinylidene difluoride membranes by means of a semi-dry electroblotter. After being blocked, the membranes were incubated in phosphate-buffered saline (PBS, pH 7.4) containing anti-human RGD-CAP/ßig-h3 monoclonal antibody overnight, and then in PBS containing 125I-irradiated sheep anti-rat IgG (Fab')2 fragments (Amersham, Aylesburg, UK) for 3 hrs at room temperature. The membrane was exposed to x-ray film.
Polymerase Chain-reaction (PCR) Analysis
Total RNA was isolated from cultured PDL cells by means of a Total RNA Extraction Kit (Pharmacia Biotech Quick PrepR, Tokyo, Japan) according to the manufacturer's instructions.
Single-stranded cDNA was synthesized from 1 µg of total RNA with the use of Oligo (dT)20 primer (Toyobo, Osaka, Japan) and a Rever Tra Ace- first-strand cDNA synthesis kit (Toyobo).
Quantitative real-time polymerase chain-reaction (PCR) was performed for examination of the RGD-CAP mRNA level with the use of an automated fluorometer (ABI Prism 7700 Sequence Detection System, PE Biosystems, Foster, CA, USA), as described previously (Leutenegger et al., 1999). The Table (A)
shows the sequences of the primers and probes for RGD-CAP and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). We performed comparative quantification of the RGD-CAP signals by normalizing the RGD-CAP signals relative to those of G3PDH.
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. The signal intensity-detected ethidium bromide was analyzed by scanning density (NIH image, version 1.59). We determined the relative mRNA expression of type I collagen or bone sialoprotein by dividing the densitometric value of RT-PCR products of each transcript by that of G3PDH.
Measurement of ALP Activity in Cultured PDL Cells
The PDL cells were washed three times with PBS, and a 0.2-mL quantity of 10 mM Tris-HCI containing 5 mM MgCl2 was added. The cells were then sonicated for 1 min. The sonicates were centrifuged for 10 min at 3000 g, and the supernatants were used for the enzyme assay. ALP activity was assayed with p-nitrophenylphosphate used as a substrate, according to the method described previously (Piche et al., 1989). We measured the amount of p-nitrophenol produced spectrophotometrically at 410 nm and normalized it by dividing the quantity by the cell number in each dish.
Alizarin Red Staining
Recombinant RGD-CAP (20 µg/mL) in the solution buffer (PBS containing 4 M urea) or solution buffer was added to the PDL cell cultures maintained in mineralizing medium every 2 days for 21 days. The cells were rinsed twice with PBS and incubated in 40 mM alizarin red solution (Sigma, St. Louis, MO, USA) at room temperature for 30 min. After being washed twice with PBS, the cells were observed by light microscopy. The number of bone nodules was counted for the central area (3.1 x 102 mm2) in three separate dishes. Student's t test was used to determine statistical significance of the effects of RGD-CAP on the inhibition of bone nodule formation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). Treatment of the cells with 10-8 M Dex or 10-8 M vitamin D3 resulted in progressive increases in the ALP activity compared with the controls. On the other hand, the mRNA level of RGD-CAP markedly decreased in the cultures after the addition of Dex or vitamin D3 (Figs. 2B, 2C).
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). The treatment of RGD-CAP on PDL cells maintained in mineralizing medium inhibited the decrease of the type I collagen mRNA level, and resulted in the reduction of the bone sialoprotein mRNA level (Fig. 3B). To ensure the reproducibility of this experiment, we conducted two independent studies with different batches of chondrocytes and obtained similar results. The treatment with recombinant RGD-CAP on cultured PDL cells maintained in mineralizing medium showed a decrease in the intensity of alizarin red staining. The number of bone nodules was also significantly lower (p < 0.01) in the cultures treated with recombinant RGD-CAP than in control cultures (Fig. 3C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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PDL cells have a high basal ALP activity which is involved in the process of calcification in various mineralized tissues (de Bernard, 1982; Kawase et al., 1988), suggesting that PDL cells have the potential to behave like osteoblasts, given the appropriate culture conditions. Recently, it was demonstrated that RGD-CAP/ßig-h3 has a negative function on osteogenesis (Dieudonné et al., 1999; Kim et al., 2000; Ohno et al., 2002). In the present study, we showed that the RGD-CAP mRNA level markedly decreased in the PDL cell cultures, where mineralization progressed by treatment with Dex or vitamin D3. As reported previously, the down-regulation of type I collagen mRNA and up-regulation of bone sialoprotein mRNA were observed in the cultured PDL cells which were maintained in mineralizing medium (Ramakrishnan et al., 1995; Chien et al., 1999); however, recombinant RGD-CAP inhibited the down-regulation of the type I collagen mRNA level and reduced the bone sialoprotein mRNA level. Furthermore, we demonstrated that recombinant RGD-CAP suppressed the ALP activity and bone nodule formation of cultured PDL cells. These results emphasize that RGD-CAP/ßig-h3 contributes to the maintenance of the elasticity of the PDL by inhibiting mineralization.
In conclusion, RGD-CAP may play an important role in the maintenance of PDL homeostasis by regulating mineralization.
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ACKNOWLEDGMENTS |
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Received April 25, 2001; Last revision September 12, 2002; Accepted September 30, 2002
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REFERENCES |
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de Bernard B (1982). Glycoproteins in the local mechanism of calcification. Clin Orthop 162:233–244.
Dieudonné SC, Kerr JM, Xu T, Sommer B, DeRubeis AR, Kuznetsov SA, et al. (1999). Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone. J Cell Biochem 76:231–243.[Medline]
Hashimoto K, Noshiro M, Ohno S, Kawamoto T, Satakeda H, Akagawa Y, et al. (1997). Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen. Biochim Biophys Acta 1355:303–314.[Medline]
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