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Effect of glypican-1 gene on the pulp cells during thereparative 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, Japan1

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 ourhypothesis thatgpc-1may 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, -3and -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-1expression resulted in a 3.9-fold increase in tgf-b1expression in pulp cells and a 0.3-

fold decrease indspp(dentine sialophosphoprotein) expression compared with control. These results suggested thatgpc-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-1expression 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,collagena1 type 1,

nestinand osteocalcin, as well as gpc-1 (glypican-1), which was

not detected in the intact pulp cDNA library (Matsuki et al., 1995).

Gpc-1was detected with similar frequency astgf-b1(transforming

growth factor beta-1).

GPC-1 is a member of a family of glycosylphosphatidylinositol-

anchored 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 develop-

ment, 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 (Liet 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

ofgpc-1, -2, -3and -4in pulp cells, as well as the effect ofgpc-1

on odontoblast-like cell differentiation using siRNA (small inter-

fering RNA) to down-regulate gpc-1 expression.

1To whom correspondence should be addressed (email [email protected]).

Abbreviations: DSPP, dentine sialophosphoprotein; GPC-1, glypican-1; RT, reverse transcription; siRNA, small interfering RNA; TGF-b1, transforming growthfactor beta-1.

Cell Biol. Int. (2010) 34, 10691074 (Printed in Great Britain)

Short Communication

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2. Materials and methods

2.1. Animals

After appr oval by the Uni ve rsity Ani mal Car e 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 asdescribed elsewhere (Murakami et al., 2002). GPC-1 was

detected immunohistochemically using a goat anti-GPC-1

a n t ibo dy (1 : 1 00 di l u ti o n ; Sa n t a C r u z B i o te c h n ol o g y).

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.

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-glyceropho-

sphate, 50 mg/ml ascorbic acid and antibiotics].

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

polymerase and 10 pmoles of each specific primer set. The

nucleotide sequences of the primer pairs for gpc-1 are 59-

CATGCCCTGAGCACATTCAC-39 (sense) and 59-AGGCACTCG-

TTGATGCCAGA-39 (antisense), for gpc-2 are 59-TTCGAGCTGG-

CTGCTGAGTC-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-GAC-

TCATCGTACTCCTGCTTGCTG-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, -3and -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-GTGCTAA-

TGGTGGACCGCAAC-39 (sense) and 59-TCCCGAATGTCTGA-

CGTATTGAAG-39 (antisense) for tgf-b1, 59-TCAATGGCGGGTG-

CTTTAGA-39 (sense) and TGCTCACTGCACAACATGAAGA-39

(antisense) for dspp (dentine sialophosphoprotein), 59-AGACTC-

CGGCGCTACCTCAA-39 (sense) and 59-CGTCCTGGAAGCCA-

ATGTG-39 (antisense) for osteocalcin and 59-GACAACTTTGGCA-

TCGTGGA-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,dsppandosteocalcinwere normalized to thegapdh

mRNA level.

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, wasconstructed as a negative control. Pulp cells in a well of a 12-well

plate that had been cultured for 7 days were transfected with 1mg

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

forgpc-1,tgf-b1,dsppandgapdh. 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

WhitneyUtest.

Effect of glypican-1 gene on the pulp cells

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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, immunohisto-

chemical 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 ofgpc-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

ofgpc-3 mRNA was increased at day 8, although the expressionpattern 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 a nd -4 were comparatively weak

(Figure 2B).

3.3. Real-time PCR analysis (serial changes of therelative quantity of mRNA)

Figure 3 shows the patterns of serial changes of the relative

quantity of mRNA ofgpc-1, tgf-b1, dspp a nd osteocalcin duringthe process of odontoblast-like cell differentiation. The express-

ion of gpc-1 mRNA gradually increased by day 15 and then

gradually decreased by days 22 and 28. The expression oftgf-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 ofgpc-1expression)

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).

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 reparat-

ive dentine process has been under investigation. In this study,

we show that GPC-1 was observed in pulp cells entrapped by

Figure 1 Immunohistochemical staining of rat mandibular incisors for GPC-1

and TGF-b1Sections 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-b1was 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 primaryantibody). B, bone; D, dentine; Os, osteodentine; P, pulp cells; Pdl, periodontium; Fi,

fibrodentine. Small arrow head, epithelial cells of the rests of Malassez. Large arrowhead, the surrounding Pdl fibroblasts and their matrix.

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Figure 2 Expression patterns ofgpc-1, -2, -3and -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 ofgpc-1, tgf-b1, dsppand osteocalcin during odontoblast-like cell differentiation in pulp cell

culture using real-time PCRData are the meanS.E.M. of five experiments (P,0.05).



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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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