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RESEARCH REPORT |
1 Department of Periodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
2 Taisho Laboratory of Functional Genomics, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan
* corresponding author, ipshinya{at}dent.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: PLAP-1 • FGF-2 • BMP-2 • periodontal ligament cells • mineralization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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We recently reported the gene expression profile describing quantitative aspects of the genes active in the human PDL and identified a novel gene, PLAP-1 (periodontal-ligament-associated protein-1), which is frequently and predominantly expressed in the PDL tissue (Yamada et al., 2001). Other groups have discovered an identical gene (Henry et al., 2001; Lorenzo et al., 2001). They named this gene Asporin, due to its unique aspartic stretch at the N terminus of the translated open reading frame. The PLAP-1/Asporin gene encoded a novel SLRP (small leucine-rich repeat proteoglycan) protein, which resembled Decorin and Biglycan. Interestingly, expression of the PLAP-1 gene was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized-tissue-forming cells (Yamada et al., 2001). This suggests the possible involvement of PLAP-1 in the process of mineralized matrix formation in PDL tissue. PLAP-1 has no glycosaminoglycan attachment site in its predicted amino acid sequence (Yamada et al., 2001), implying that PLAP-1 is not a proteoglycan and may function differently in PDL tissue compared with other SLRP proteins such as Decorin and Biglycan in the PDL.
In this study, we hypothesized that PLAP-1 expression is regulated by the mineralization-related cytokines in human PDL cells.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture
Human PDL cells were isolated in accordance with the method described previously (Takayama et al., 1997). The cells were cultured in 
-MEM supplemented with 10% FCS, 50 units/mL penicillin G, and 50 µg/mL streptomycin (standard medium) at 37°C in 5% CO2.
Stimulation of Human PDL Cells with Cytokines
Human PDL cells were cultured in standard medium. The next day, the medium was replaced with FCS-free -MEM. After serum deprivation for 48 hrs, the cells were stimulated with FGF-2 (Kaken Pharmaceutical, Tokyo, Japan) (100 ng/mL), platelet-derived growth factor BB (PDGF-BB) (SIGMA, St. Louis, MO, USA) (50 ng/mL), BMP-2 (Genzyme/Techne, Minneapolis, MN, USA) (100 ng/mL), BMP-4 (Genzyme/Techne) (100 ng/mL), hepatocyte growth factor (HGF) (SIGMA) (100 ng/mL), or epidermal growth factor (EGF) (SIGMA) (100 ng/mL) and incubated for another 48 hrs. The stimulated cells were then harvested, and total RNA was isolated for RT-PCR analysis.
RT-PCR Analysis
Primers for real-time RT-PCR analysis were designed with Perfect Real Time Primer Design software (TAKARA, Shiga, Japan). Primers for PLAP-1 were: (sense) 5'-GGGTGACGGTGTTCCATATCAG-3' and (antisense) 5'-TGAAGCTCCAATAAAGTTGGTGGTA-3'. Primers for Biglycan were: (sense) 5'-CAACCAGATCAGGATGATCGAGAA-3' and (antisense) 5'-CCCATGGGACAGAAGTCGTTG-3'. Primers for Decorin were: (sense) 5'-GGGAGCTTCACTTGGACAACAAC-3' and (antisense) 5'-GGGCAGAAGTCACTTGATCCAAC-3'. Primers for hypoxanthine-guanine phosphoribosyl transferase (HPRT) were: (sense) 5'-CCAGACAAGTTTGTTGTAGG-3' and (antisense) 5'-TCCAAACTCAACTTGAACTC-3'. Real-time RT-PCR reaction was carried out with a SYBR RT-PCR Kit (TAKARA) and performed with Smart Cycler version II (TAKARA). The amount of mRNA was calculated for each sample from the standard curve via the instrument software.
Semi-quantitative RT-PCR was performed according to the procedures described previously (Yamada et al., 2001). RT-PCR primers for Osteonectin were: (sense) 5'-GGAAGAAACTGTGGCAGAGGTGAC-3' and (antisense) 5'-TGTTGTCCTCATCCCTCTCATACAG-3'. Primers for Osteopontin were (sense) 5'-CCAAGTAAGTCCAACGAAAG-3' and (antisense) 5'-GGTGATGTCCTCGTCTGTA-3'. Primers for Osteocalcin were: (sense) 5'-CATGAGAGCCCTCACA-3' and (antisense) 5'-AGAGCGACACCCTAGAC-3'. Primers for Bone sialoprotein were: (sense) 5'-GCCTGTGCTTTCTCAATG-3' and (antisense) 5'-TTCCTTCCTCTTCCTCCTC-3'. Primers for HPRT were: (sense) 5'-CGAGATGTGATGAAGGAGATGGG-3' and (antisense) 5'-GCCTGACCAAGGAAAGCAAAGTC-3'.
In vitro Assay for Alkaline Phosphatase (ALPase) Activity
Human PDL cells were cultured with the standard medium (see above) in the presence of 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid (mineralization medium). Cellular DNA content and ALPase activity in the PDL cells were determined according to the procedures described previously (Takayama et al., 1997).
Production of Anti-PLAP-1 Antibody
A peptide (EPRSHFFPFD) homologous to human PLAP-1 was synthesized with a Model 430 A peptide synthesizer (Applied Biosystems). The peptide was conjugated to keyhole limpet hemocyanin (KLH) and used for the immunization of rabbits. This animal experiment was carried out in accordance with the guidelines for animal experimentation approved by the Japanese Association for Laboratory Animal Science.
Immunocytochemical Staining
Human PDL cells grown to confluence in a 60-mm poly-L-lysine-coated glass-bottomed dish (Matsunami Glass, Osaka, Japan) were stimulated with BMP-2 for 48 hrs. Cells were washed with PBS 3 times and incubated with anti-PLAP-1 polyclonal antibody or pre-immune rabbit serum as a control at 4°C for 15 min. Cells were then incubated with biotinylated goat anti-rabbit IgG (H+L) antibody (Vector Laboratories, Burlingame, CA, USA) at 4°C for 15 min, and, finally, streptavidin-Alexa Fluor 488 (Molecular Probe, Engene, OR, USA) was added for the detection of immunoreactivity. Cells were washed 3 times with PBS after each step.
For the pre-incubation assay, we pre-incubated the anti-PLAP-1 polyclonal antibody with the antigenic KLH-conjugated PLAP-1 peptide, KLH alone, recombinant human Decorin (R&D Systems, Minneapolis, MN, USA), or recombinant human Biglycan (ABNOVA, Taipei, Taiwan) at room temperature for 15 min before staining BMP-2-stimulated PDL cells. In each pre-incubation assay, a 30-mg quantity of KLH-conjugated peptide or recombinant proteins was added to 1 mL of the anti-PLAP-1 antibody. The amount of peptide and recombinant proteins (30 mg/1 mL of anti-PLAP-1 antibody) was equivalent to the amount of total protein in the anti-PLAP-1 polyclonal antibody serum.
Statistical Analysis
Data are expressed as means ± standard deviations. The statistical significance of differences between 2 means was examined by the Mann-Whitney U test, and P values less than 0.05 were considered to indicate a significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The PDL cells simultaneously differentiated into hard-tissue-forming cells, and calcified nodule formation was finally observed in the culture (data not shown). We also confirmed that the addition of FGF-2 in the late stage of this long-term culture resulted in down-regulation of ALPase activity (Takayama et al., 1997) (Fig. 1A) and inhibited the calcified nodule formation (data not shown). We performed real-time RT-PCR analysis of PLAP-1 using RNAs isolated from the PDL cells harvested on day 0 and day 20, with or without FGF-2 stimulation (Fig. 1B). Expression of PLAP-1 was increased during the culture in mineralization media, and strong expression was detected on day 20. The transcription of PLAP-1 was clearly decreased in the PDL cells cultured in the presence of FGF-2 from day 15 (Fig. 1B), accompanying the down-regulation of their ALPase activity (Fig. 1A.). The transcription of Biglycan and Decorin also tended to be suppressed by stimulation with FGF-2. However, the expression of Biglycan mRNA and Decorin mRNA during the culture was changed only slightly, compared with that of PLAP-1 mRNA (Fig. 1B).
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). We used the optimum concentration for each cytokine, which induced proliferation of PDL cells equally (data not shown). The real-time RT-PCR analysis showed that FGF-2 and PDGF-BB significantly down-regulated the expression of PLAP-1 mRNA in PDL cells. In contrast, BMP-2 and BMP-4, which strongly induced mineralization of PDL cells, significantly up-regulated PLAP-1 transcription. In contrast, neither HGF nor EGF changed the expression of PLAP-1 mRNA in PDL cells.
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We then confirmed the effects of BMP-2 on the induction of the mineralization and cytodifferentiation of PDL cells by investigating the gene expression of mineralization-related proteins (Fig. 3). PDL cells were stimulated with BMP-2 (100 ng/mL) for 48 hrs, and RNA was isolated from the cells. RT-PCR analysis revealed that the transcription of PLAP-1 was induced along with Osteopontin, Osteocalcin, and Bone sialoprotein transcripts by the stimulation of BMP-2 (Fig. 3).
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). Fig. 4A shows the peptide sequences which we selected for the immunization of rabbits. We cultured human PDL cells for 48 hrs in the presence or absence of BMP-2, and stained the cells with the polyclonal antibody (Fig. 4B). During the whole staining process, the PDL cells were alive and not fixed. The cell surface of BMP-2-stimulated PDL cells was stained with the anti-PLAP-1 polyclonal antibody in a dose-dependent manner (Fig. 4Ba~c). No staining was observed in the negative control, in which pre-immune rabbit serum was used as the primary antibody (Fig. 4Bd). We then pre-incubated the antibody before staining the BMP-2-stimulated PDL cells with different amounts of the PLAP-1 antigenic peptide, which we utilized for immunization. Pre-incubation of the PLAP-1 antigenic peptide inhibited the binding of the antibody to BMP-2-stimulated PDL cells in a dose-dependent manner (Fig. 4Be~g). No inhibition was observed in the negative control in which only KLH carrier protein was used for the pre-incubation with the antibody (data not shown). Moreover, we pre-incubated the anti-PLAP-1 antibody with either recombinant human Decorin or recombinant human Biglycan before staining the BMP-2-stimulated PDL cells (Fig. 4Bh~k). No inhibition was observed by the pre-incubation with either recombinant Decorin or recombinant Biglycan.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). This suggests that PLAP-1 expression is closely associated with the process of cytodifferentiation of PDL cells. FGF-2 and PDGF-BB, which have mitogenic and chemotactic activities (Rifkin and Moscatelli, 1989; Hoch and Soriano, 2003), clearly suppressed the expression of PLAP-1 in PDL cells (Fig. 2). Since PDL cells proliferate after stimulation with FGF-2 or PDGF-BB, PLAP-1 may not be essential for cell proliferation. In contrast, BMP-2 and BMP-4 clearly induced the expression of PLAP-1 (Fig. 2). BMP-2 is one of the most potent cytokines that stimulate osteoblast differentiation and bone formation (Hogan, 1996; Canalis et al., 2003). BMP-2 has also been reported to stimulate osteoblastic differentiation in human PDL cells (Kobayashi et al., 1999), and to promote dental follicle cells, putative progenitor cells for the periodontium, to induce differentiation into a cementoblastic/osteoblastic phenotype (Zhao et al., 2002). Given that BMP-2 induced the cytodifferentiation and mineralization of PDL cells, PLAP-1 is closely associated with the cytodifferentiation of PDL cells into hard-tissue-forming cells. BMP-2 induced the expression of PLAP-1 in a dose-dependent manner. PLAP-1 was then transcribed along with the induction of other BMP-2-induced mineralization-related genes (Fig. 3). These results suggest that PLAP-1 is a mineralization-related gene, and that its transcription might be driven by the BMP-2 signaling pathway.
Recently, it was demonstrated that topical application of recombinant FGF-2 or PDGF-BB enhances the healing process and accelerates periodontal tissue regeneration (Takayama et al., 2001; Murakami et al., 2003; Nevins et al., 2003). In the in vivo process of periodontal tissue regeneration, FGF-2 is likely to generate a suitable micro-environment in the FGF-2-applied sites by regulating the production of extracellular matrix (Takayama et al., 1997; Shimabukuro et al., 2005). Interestingly, we found that both FGF-2 and PDGF-BB strongly suppressed transcription of PLAP-1 in PDL cells (Fig. 2). Thus, the effects of FGF-2 and PDGF-BB on periodontal tissue regeneration might be partly associated with suppression of PLAP-1 expression in PDL. Temporal reduction of PLAP-1 during the early phase of wound healing might be suitable for periodontal tissue regeneration in terms of the creation of an ideal micro-environment at the site.
PLAP-1 is categorized into the same subclass of SLRP proteoglycan families as Biglycan and Decorin (Yamada et al., 2001). Biglycan and Decorin were dominantly expressed in bone and connective tissue of skin, respectively (Ameye and Young, 2002). In general, both proteoglycans exist ubiquitously in mineralized tissues and connective tissues. In oral tissues, Biglycan is expressed in odontoblasts and ameloblasts and regulates the cytodifferentiation of those cells (Iozzo, 1997). Decorin is expressed ubiquitously in tissues of the periodontium (Hakkinen et al., 1993). However, we could not detect the PLAP-1 transcript in bone tissue by Northern blot analysis, but its expression was specifically revealed in PDL by in situ hybridization (Yamada et al., unpublished observations). Changes in Decorin and Biglycan were slight in the course of cytodifferentiation of PDL cells, compared with PLAP-1 expression (Fig. 1). These findings suggest that PLAP-1 has unique function(s) in the PDL, compared with those of Decorin and Biglycan, and may play different roles in the process of the cytodifferentiation of the PDL cells.
The present findings demonstrated that the PLAP-1 transcript is tightly regulated by FGF-2 and BMP-2 and closely associated with the process of cytodifferentiation and mineralization of human PDL cells. Thus, PLAP-1 is expected to be useful for our understanding of the molecular basis of periodontal ligament functions.
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ACKNOWLEDGMENTS |
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Received July 20, 2004; Last revision June 22, 2005; Accepted January 11, 2006
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REFERENCES |
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