Cell Biol. Int.

(2010) 34, 10691074 (Printed in Great Britain)

Short Communication

Effect of glypican-1 gene on the pulp cells during the reparative dentine process
Yoshiko Murakami Masuda1*, Xiaogu Wang*, Satoshi Yokose{, Yoshishige Yamada*, Yuichi Kimura1, Tomohiro Okano{ and Koukichi Matsumoto*
* Department of Endodontology, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ohta-ku, Tokyo 145-8515, Japan
{ {

Department of Oral Radiology, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ohta-ku, Tokyo 145-8515, Japan Division of Restorative Dentistry, Department of Conservative Dentistry Ohu University School of Dentistry, 31-1Aza Sankakudo, Tomita, Koriyama, Fukushima 963-8611, Japan 1 Division of Endodontics, Department of Conservative Dentistry Ohu University School of Dentistry, 31-1Aza Sankakudo, Tomita, Koriyama, Fukushima 963-8611, Japan

Abstract
GPC-1 (glypican-1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors and members of the TGF-b (transforming growth factor beta-1) family. The function of cell-surface proteoglycans in the reparative dentine process has been under investigation. Gpc-1 was detected with similar frequency as tgf-b1 in the cDNA library using mRNA from the odontoblast-like cell-enriched pulp of rat incisors. The aim of this study was to test our hypothesis that gpc-1 may be related to reparative dentine formation. We examined the expression of this gene during the reparative dentine process, as well as the effect of gpc-1 on odontoblast-like cell differentiation using siRNA (small interfering RNA) to down-regulate gpc-1 expression. Immunohistological examination showed that GPC-1 was expressed in pulp cells entrapped by fibrodentine and odontoblast-like cells as well as TGF-b1. The mRNAs for gpc-1, -3 and -4, except for gpc-2, were expressed during odontoblast-like cell differentiation in pulp cells. The relative levels of gpc-1 mRNA were increased prior to the differentiation stages and were decreased during the secretory and maturation stages of pulp cells. Down-regulation of gpc-1 expression resulted in a 3.9-fold increase in tgf-b1 expression in pulp cells and a 0.3fold decrease in dspp (dentine sialophosphoprotein) expression compared with control. These results suggested that gpc-1 and tgfb-1 expression are necessary for the onset of differentiation, but should be down-regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc-1 expression in odontoblast-like cells is associated with the early differentiation but not with the formation of reparative dentine.
Keywords: glypican; odontoblast-like cell; pulp cell; RNA

1. Introduction
Dental pulp is unique in its ability to regenerate and form tertiary dentine (Tziafas et al., 2000; Goldberg and Smith, 2004). Dentineogenesis occurs after intense injury (i.e. caries, trauma and operative procedures), resulting in odontoblast death, which facilitates deposition of a protective layer of reparative dentine by odontoblast-like cells at the injured dentinepulp interface. Signalling molecules that are expressed by pulp cells could play a role in pulp healing during dental repair [reviewed in Smith and Lesot (2001)]. To increase our understanding of the molecules controlling dental repair, we prepared a cDNA library using mRNA from the odontoblast-like cell-enriched pulp of rat incisors using pulsed Nd:YAG (neodymium:yttriumaluminiumgarnet) laser irradiation (Masuda et al., 2006). Among the 200 cDNA clones from this cDNA library that we sequenced, several identical genes were detected, including amelogenin, ameloblastin, collagen a1 type 1, nestin and osteocalcin, as well as gpc-1 (glypican-1), which was not detected in the intact pulp cDNA library (Matsuki et al., 1995).

Gpc-1 was detected with similar frequency as tgf-b1 (transforming growth factor beta-1). GPC-1 is a member of a family of glycosylphosphatidylinositolanchored cell surface heparan sulfate proteoglycans. Six members of this family have been identified in mammals (GPC-1 to GPC-6). GPCs are predominantly expressed during development, and they are thought to play a role in morphogenesis (Filmus, 2001) and can either stimulate or inhibit signalling activity (Filmus et al., 2008). GPC-1 has been reported to control cellular responses to growth factors. In pancreatic cancer, investigators have reported its overexpression (Kleeff et al., 1998, 1999) and a correlation with TGF-b1 signalling (Li et al., 2004). In oral tissues, the expression and distribution of cell-surface proteoglycans have been reported only in periodontal tissues (Worapamorn et al., 2000). There are numerous molecules that are implicated in reparative dentine formation. Gpc-1 might be an additional molecule and a crucial one. To test our hypothesis, we examined the expression of gpc-1, -2, -3 and -4 in pulp cells, as well as the effect of gpc-1 on odontoblast-like cell differentiation using siRNA (small interfering RNA) to down-regulate gpc-1 expression.

To whom correspondence should be addressed (email yoshik@senzoku.showa-u.ac.jp). Abbreviations: DSPP, dentine sialophosphoprotein; GPC-1, glypican-1; RT, reverse transcription; siRNA, small interfering RNA; TGF-b1, transforming growth factor beta-1. Volume 34 (11) N pages 10691074 N doi:10.1042/CBI20090062 N www.cellbiolint.org 1069

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Effect of glypican-1 gene on the pulp cells

2. Materials and methods

2.1. Animals
After approval by the University Animal Care and Use Committee, Wistar strain male rats (approximately 350 g) were used for immunohistochemistry, and 5-week-old littermate male rats were used for cell culture. Five-week-old rats were used for the easy manipulation of anaesthetization and removal of mandibles and pulps. The methods of Yokose et al. (2000), which established the culture system for rat pulp cells, were used as reference.

2.2. Histology and immunohistochemistry

The pulp of incisors was selected at 6 and 10 days after laser irradiation, during which time reparative dentine formation had occurred and odontoblast-like cells could be found (Murakami et al., 2002). The histological procedure was performed as described elsewhere (Murakami et al., 2002). GPC-1 was detected immunohistochemically using a goat anti-GPC-1 antibody (1:100 dilution; Santa Cruz Biotechnology). Peroxidase activity was visualized with a DAB (diamino benzidine tetrahydrochloride) substrate kit (Nichirei). As a positive control, TGF-b1 was detected using a monoclonal mouse anti-human TGF-b1 antibody (500 mg/ml; R&D Systems) (Murakami et al., 2002). Negative control specimens were treated in the same manner, but were incubated with normal calf serum instead of primary antibody.

polymerase and 10 pmoles of each specific primer set. The nucleotide sequences of the primer pairs for gpc-1 are 59CATGCCCTGAGCACATTCAC-39 (sense) and 59-AGGCACTCGTTGATGCCAGA-39 (antisense), for gpc-2 are 59-TTCGAGCTGGCTGCTGAGTC-39 (sense) and 59-AGGCGTCCCACATTCCTGA-39 (antisense), for gpc-3 are 59-CTCTGGTGACGGCATGATGAA-39 (sense) and 59-GCATCGTCCACATCCAGATCATA-39 (antisense), for gpc-4 are 59-CCAAGCACTGTCTGCAATGATG-39 (sense) and 59-CCTGGTTGGCTAATCCGTTTC-39 (antisense) and for b-actin are 59-GGAGATTACTGCCCTGGCTCCTA-39 (sense) and 59-GACTCATCGTACTCCTGCTTGCTG-39 (antisense). The reaction was amplified for 35 or 40 cycles, with denaturation at 94uC for 30 s, annealing at 63uC (gpc-1), 64uC (gpc-2, -3 and -4) or 65uC (b-actin) for 30 s and extension at 72uC for 90 s. The PCR products were detected on a 2% agarose gel.

2.6. Real-time PCR

Real-time PCR was performed with the SYBR Green I assay kit and the StepOneTM real-time PCR system (Applied Biosystems). The nucleotide sequences of the primer pairs were 59-GTGCTAATGGTGGACCGCAAC-39 (sense) and 59-TCCCGAATGTCTGACGTATTGAAG-39 (antisense) for tgf-b1, 59-TCAATGGCGGGTGCTTTAGA-39 (sense) and TGCTCACTGCACAACATGAAGA-39 (antisense) for dspp (dentine sialophosphoprotein), 59-AGACTCCGGCGCTACCTCAA-39 (sense) and 59-CGTCCTGGAAGCCAATGTG-39 (antisense) for osteocalcin and 59-GACAACTTTGGCATCGTGGA-39 (sense) and 59-ATGCAGGGATGATGTTCTGG-39 (antisense) for gapdh. The primer pairs for gpc-1 were the same as for RT-PCR. The reaction was performed as follows: 10 min at 95uC and then 40 cycles (15 s at 95uC, 1 min at 60uC and 15 s at 95uC), followed by 1 min at 60uC and 15 s at 95uC. We plotted a standard curve for each primer pair by applying known quantities of the PCR products of each sample. The expression levels of gpc-1, tgf-b1, dspp and osteocalcin were normalized to the gapdh mRNA level.

2.3. Cell culture

The pulp cell culture was performed according to a previous study (Yokose et al., 2000). Five independent primary cultures were performed for each experiment. After reaching subconfluency, the pulp cells were removed from the dish and added to six-well plates for real-time PCR and to 12-well plates for siRNA transfection (Falcon Labware) at a density of 104 cells/cm2. After 24 h, the cells were grown with or without mineralizing medium [10% heat-inactivated CS (calf serum), 300 mg/ml b-glycerophosphate, 50 mg/ml ascorbic acid and antibiotics].

2.7. siRNA transfection

Synthetic oligonucleotides were inserted between the human U6 promoter and terminator sequences of the pBAsi-hU6 vector (Takara Bio., Inc.) to generate a stem-loop type siRNA in transfected cells. pBAsi-Gpc1#1414, which targeted nucleotides 1414-GGACACTGTGTAGTGAGAA-1432 of the gpc-1 gene, was constructed and pBAsi-NC, which targeted a T7 stop, was constructed as a negative control. Pulp cells in a well of a 12-well plate that had been cultured for 7 days were transfected with 1 mg of pBAsi-Gpc1#1414 using the TransITj-LT1 transfection reagent (Mirus Bio. Co., Ltd). After 48 h, total RNA was extracted, and real-time PCR assays were performed with the primer pairs for gpc-1, tgf-b1, dspp and gapdh. All values are expressed as the meansS.E.M., and statistical comparisons were made (n55) with the MannWhitney U test. *P,0.05 and **P,0.01 were considered statistically significant with the KruskalWallis test. P,0.01 was considered statistically significant with Mann Whitney U test.

2.4. Total RNA preparation and reverse transcription

Total RNA was extracted from the pulp cells using an RNaid kit (BIO 101, Inc.). One microgram of total RNA extracted on days 3, 6, 10, 15, 22 and 28 for real-time PCR and on days 4, 8, 14, 21 and 27 for RT (reverse transcription)-PCR was converted into cDNA using reverse transcriptase and a SYBR RT-PCR kit (Takara) with random hexamer primers.

2.5. RT-PCR
PCR amplification was performed in a 20-ml PCR reaction mixture containing target cDNA, 10 mM dNTPs, 2.5 mm of rTaq DNA

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3. Results
3.1. Immunohistochemical examination
Figure 1 shows immunolocalization of GPC-1. After 6 days, staining of GPC-1 was observed in pulp cells entrapped by fibrodentine and in odontoblast-like cells. Weak staining was observed in the fibrodentine (Figure 1a). After 10 days, staining of GPC-1 was similar to that seen after 6 days in odontoblast-like cells (Figure 1b). The staining of TGF-b1 was similar to that of GPC-1 (Figures 1c, 1d). In the positive control, immunohistochemical staining of GPC-1 was found in epithelial cells in the rest of Malassez, and the surrounding fibroblasts of the periodontal ligaments and their matrix (Figure 1e) (Worapamorn et al., 2000).

3.2. RT-PCR
Figure 2 shows the expression of gpc-1, -2, -3 and -4 during the process of odontoblast-like cell differentiation in pulp cell culture with mineralizing medium (Figure 2A). The expression of gpc-1 mRNA increased on day 14 and then decreased. The expression of gpc-3 mRNA was increased at day 8, although the expression pattern was weak, and the change was minor. mRNA for gpc-4 was observed throughout the time period studied. After 27 days, the expression of gpc-3 and -4 was decreased. Expression of gpc-2 mRNA was not observed at any time point studied (Figures 2A, 2B). In the absence of mineralizing medium, the expression of gpc-1, -3 and -4 were comparatively weak (Figure 2B).

3.3. Real-time PCR analysis (serial changes of the relative quantity of mRNA)
Figure 3 shows the patterns of serial changes of the relative quantity of mRNA of gpc-1, tgf-b1, dspp and osteocalcin during the process of odontoblast-like cell differentiation. The expression of gpc-1 mRNA gradually increased by day 15 and then gradually decreased by days 22 and 28. The expression of tgf-b1 mRNA gradually increased by day 10 and then gradually decreased by days 15, 22 and 28. The expression of dspp mRNA was low up to day 15 and then increased at days 22 and 28. The expression of osteocalcin mRNA was first observed at day 15, was low up to day 22 and then rapidly increased by day 28.

3.4. Real-time PCR analysis (down-regulation of gpc-1 expression)

Figure 4 shows the comparison of the levels of mRNA expression of gpc-1, dspp and tgf-b1 in pulp cells with real-time PCR following down-regulation of gpc-1 expression with its specific siRNA. The mRNA levels for gpc-1 in pulp cells transfected with pBAsi-Gpc1#1414 averaged 0.17-fold compared with that in untransfected control cells, and without gpc-1 siRNA (control plasmid pBAsi-NC) averaged 0.6-fold compared with that in untransfected control cells. The reduction of gpc-1 mRNA was between 66% and 90%. Down-regulation of gpc-1 expression resulted in a 3.9-fold increase in tgf-b1 expression (mean of five experiments) in pulp cells compared with control (P,0.05) and a 0.3-fold decrease in dspp expression (mean of five experiments) compared with control (P,0.05).

Immunohistochemical staining of rat mandibular incisors for GPC-1 and TGF-b1 Sections were lightly counterstained with Mayers haematoxylin. (a, b) GPC-1 staining. GPC-1 was expressed in odontoblast-like cells (arrow). (c, d) TGF-b1 staining. TGF-b1 was expressed in odontoblast-like cells (arrow). (a, c) 6 days after laser irradiation. (b, d) 10 days after laser irradiation. (e) Positive control. (f) Negative control (no primary antibody). B, bone; D, dentine; Os, osteodentine; P, pulp cells; Pdl, periodontium; Fi, fibrodentine. Small arrow head, epithelial cells of the rests of Malassez. Large arrow head, the surrounding Pdl fibroblasts and their matrix.

Figure 1

4. Discussion
The main function of membrane-attached GPC is to regulate the signalling of Wnts, Hedgehogs, fibroblast growth factors and BMPs (bone morphogenetic proteins) (Filmus, 2001; Fico et al., 2007). The function of cell-surface proteoglycans in the reparative dentine process has been under investigation. In this study, we show that GPC-1 was observed in pulp cells entrapped by

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Effect of glypican-1 gene on the pulp cells

Figure 2

Expression patterns of gpc-1, -2, -3 and -4 mRNA during odontoblast-like cell differentiation in pulp cell culture using RT-PCR (A) Pulp culture grown in mineralizing medium. (B) Control; without mineralizing medium.

Figure 3

Comparison of the levels of mRNA expression of gpc-1, tgf-b1, dspp and osteocalcin during odontoblast-like cell differentiation in pulp cell culture using real-time PCR Data are the meanS.E.M. of five experiments (P,0.05).

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Figure 4

Comparison of the levels of mRNA expression of gpc-1, tgf-b1, and dspp after gpc-1 siRNA transfection Data are the meansS.E.M. from five experiments. Bars, S.D. MannWhitney U test: *P,0.05 and **P,0.01 were considered statistically significant. KruskalWallis test: P,0.01 was considered statistically significant.

fibrodentine and odontoblast-like cells and the mRNAs for gpc-1, -3 and -4 were expressed during odontoblast-like cell differentiation in pulp cells, and gpc-2 was not detected in this experiment. According to the patterns of serial changes of the relative quantity of mRNA, the expression of gpc-1 mRNA gradually increased by day 15 and then gradually decreased by days 22 and 28. The expression of the dspp gene has been shown to begin in secretory odontoblasts and to increase with the gradient of cytodifferentiation in rodent teeth (MacDougall et al., 1997; Begue-Kirn et al., 1998; Bleicher et al., 1999). The expression of dspp mRNA suggested that on day 22, odontoblast-like cells are in the secretory stage. gpc-1 mRNA expression begins prior to the differentiation and secretory stages. On days 22 and 28, gpc-1 mRNA expression was decreased. On day 28, the expression of osteocalcin mRNA was distinctly increased. Osteocalcin mRNA is expressed in fully mature odontoblasts at a high level during dentineogenesis (Bidder et al., 1998). This suggests that in our culture on day 28, many fully mature odontoblast-like cells were present and that gpc-1 mRNA expression was decreased in these mature odontoblast-like cells. The location of the expression of GPC-1 was similar to that of TGF-b1 (Figure 1) in the pulp of rat incisor. Rodent incisors are continuously growing teeth. In the incisor, the cells are always young, and it is a model for accelerated aging process. After laser irradiation, the eruption of the developing tooth was disturbed for a short period only. We selected the pulp at 6 and 10 days after irradiation for the formation of a layer of odontoblast-like cells between fibrodentine and the pulp cells at 6 days and the formation of large amounts of osteodentine and thickened tubular dentine at 10 days (Murakami et al., 2002). According to the patterns of serial changes of the relative quantity of mRNA, the peak of gpc-1 mRNA expression was slightly later than that of tgf-b1. It has been reported that TGF-b1

stimulates odontoblast differentiation and dentineogenesis [reviewed in Smith and Lesot (2001)]. The correlation of GPC-1 expression with TGF-b1 has been investigated in pancreatic cancer cells (Li et al., 2004). Down-regulation of gpc-1 expression resulted in decreased anchorage-dependent and -independent cell growth in pancreatic cancer cells and attenuated TGF-b1-mediated cell growth inhibition, as gpc-1 is essential for efficient TGF-b1 signalling in pancreatic cancer cells (Li et al., 2004). It was previously reported that the teeth of transgenic mice overexpressing active TGF-b1 in odontoblasts showed a reduction in tooth mineralization. The expression of the dspp gene is significantly down-regulated in the teeth of these mice (Thyagarajan et al., 2000). Our results are consistent with this report. The pattern of tgf-b1 mRNA expression was approximately opposite to that of dspp mRNA: gpc-1 down-regulation resulted in increased tgf-b1 expression and decreased dspp expression. These results suggested that gpc-1 expression decreased before the differentiation of pulp cells into odontoblast-like cells and maturation of odontoblast-like cells, as shown by dspp and osteocalcin expression. This showed that gpc-1 and tgf-b1 expression are necessary for the onset of differentiation, but should (at least for gpc-1) be down-regulated before other molecules are implicated in the formation of reparative dentine. In conclusion, gpc-1 expression in odontoblast-like cells is associated with the early differentiation but not with the formation of reparative dentine.

Author contribution
Yoshiko Masuda and Satoshi Yokose were involved in planning and analysis of the data. Xiaogu Wang, Yoshishige Yamada and Yuichi Kimura were involved in analysis of the data. Tomohiro

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Effect of glypican-1 gene on the pulp cells

Okano and Koukichi Mastumoto were involved in the organization of the experiments.

Funding
This study was supported in part by a Grant-In-Aid for Scientific Research (12771150, 10297038) from the Ministry of Education, Science, and Culture of Japan.

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Received 21 March 2009/ 26 January 2010; accepted 1 June 2010 Published as Immediate Publication 1 June 2010, doi 10.1042/CBI20090062

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