Biochemical and Biophysical Research Communications 392 (2010) 166–170

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

The pro-a2(XI) collagen gene is expressed in odontoblasts
Yasuhide Hamada a,b, Hideaki Sumiyoshi a, Noritaka Matsuo a, Wu Yun-Feng a, Momoko Nakashima a,
Shigetaka Yanagisawa b, Hidekatsu Yoshioka a,*ab

Department of Matrix Medicine, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu City, Oita 879-5593, Japan
Department of Dentistry and Oral-Maxillo-Facial Surgery, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu City, Oita 879-5593, Japana r t i c l e

i n f oArticle history:
Received 30 December 2009
Available online 6 January 2010
Keywords:
Odontoblast
Tooth
Type XI collagen
Extracellular matrix
In situ hybridization
Immunohistochemistrya b s t r a c t
Since the dentine is analogous to bone, its extracellular matrix shares many similarities to bone tissues.
Similar to the bone, type I collagen is the major organic component in dentine. However, little is known
about minor ﬁbrillar collagens, which seem to be co-expressed such as type I or II collagen. The present
study examined the gene expression of type V and XI collagens, which play important roles in collagenﬁbril formation and skeletal morphogenesis, using RT-PCR and in situ hybridization combined withimmunohistochemistry. The transcripts of pro-a1(XI), pro-a2(XI), pro-a1(V) and pro-a2(V) chains were
present, but not pro-a3(V) and pro-a1(II) chains, of which an overglycosylated variant is pro-a3(XI)
chain, in mouse incisor tooth, using RT-PCR and in situ hybridization. The pro-a2(XI) protein, which
is mainly expressed in cartilage, were observed in the odontoblast using a speciﬁc polyclonal antibody.
Real-time RT-PCR showed that the transcripts of pro-a2(XI), pro-a1(V) and pro-a2(V) were predominant in crown and that of pro-a1(XI) in root of the tooth. Finally, the expression of pro-a2(XI) was conﬁrmed with an odontoblastic cell line transformed with human telomerase reverse transcriptase(hTERT) both in vitro and in vivo. The data suggest a new subtype of the V/XI collagen molecule containing a2(XI) chain.
Ó 2010 Elsevier Inc. All rights reserved.

Introduction
Odontoblasts are responsible for the formation of dentine, thecollagen-based mineralized tissue that forms the bulk of teeth.
Odontoblasts are derived from ectomesenchymal cells, exhibit atall columnar shape and establish a continuous single layer witha clear epithelioid appearance [1]. After dentinogenesis, they arealigned along the periphery of the dental pulp, thus playing a rolein the maintenance of the tooth integrity owing to their capacity
for depositing new layers of dentine throughout life. Since dentine
is a tissue analogous to bone, its extracellular matrix shares many
similarities with the bone matrix.
Collagen is an important class of extracellular matrix components. The structural integrity and functional properties of different tissues are inﬂuenced by the characteristic combinations andamounts of these molecules [2]. Among them, type I, II, III, V andXI collagens are included in the group of ﬁbril-forming collagens,based on their structural and functional features and are divided
Abbreviations: hTERT, human telomerase reverse transcriptase; PBS , phosphate-buffered saline, Mg2+ and Ca2+ free; DSPP, dentin sialophosphoprotein; DSP,
dentin sialoprotein; RT-PCR, reverse transcription-polymerase chain reaction; GST,
glutatione S-transferase; ELISA, enzyme-linked immunosorbent assay.
* Corresponding author. Fax: +81 97 586 5674.
E-mail address: hidey@med.oita-u.ac.jp (H. Yoshioka).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.01.001

into two subgroups, major (I, II and III) and minor (V and XI) ﬁbrillar collagen based on their contents in tissues [3]. Minor ﬁbrillar
collagens type V and XI are incorporated into the ﬁbrils of themuch more abundant collagen types I and II, respectively, andact as regulators of the sizes and shapes of the resultant heterotypic ﬁbrils [4–6].
Type I collagen is mainly expressed in odontoblast [7]. However, little is known regarding the expression of minor ﬁbrillar collagens [8]. The expression of minor ﬁbrillar collagen was examinedin the tooth. The pro-a2(XI) collagen chain, which is a componentof type XI collagen found in the ﬁbrillar network of cartilage, wasobserved to be expressed in odontoblasts.
Materials and methods
Animals. The mice and rabbits were purchased from commercial
sources (Yoshitomi, Fukuoka, Japan). The animals were treated inaccordance with the Oita University Guidelines for the Care andUse of Laboratory Animals based on the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Staging of mouse embryos and preparation of sections. The gestational age was initially determined by the date of formation of the
copulation plug and conﬁrmed by crownrump length. For in situ
hybridization and immunohistochemistry, the head portion of

Y. Hamada et al. / Biochemical and Biophysical Research Communications 392 (2010) 166–170

mouse embryo was ﬁxed overnight in fresh 4% paraformaldehydein phosphate-buffered saline (PBS), dehydrated and embedded
in parafﬁn and 10 lm consecutive sections were prepared.
RT-PCR (reverse transcription-polymerase chain reaction) analysis.
Total RNA (5 lg) from a mouse tooth was reverse transcribed using
MMLV reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA)with random hexamers at 37 °C for 1 h. The reaction sample wasthen heated at 95 °C for 5 min to stop reaction. After reverse transcription, PCR was performed in a 25 ll mixture containing 1 ll of
reverse transcription the reaction product, using Go Taq Green
Master Mix (Promega, Madison, WI, USA). Twenty-eight cycles ofdenaturation at 95 °C for 30 s, annealing at 55 °C for 1 min andextension at 72 °C for 1 min were performed for Col1a1, Col2a1,
Col5a1, Col5a2, Col5a3, Col11a1, Col11a2, Dspp (Dentin sialophosphoprotein) and b-actin genes. The gene-speciﬁc primers are listed inAppendix Table I. PCR products (12 ll) were resolved electrophoretically on 1.5% agarose gel and the gel was stained with ethidium
bromide and photographed under UV light.
Real-time RT-PCR. For a quantitative analysis of the expression
level of the mRNA, real-time PCR was performed using a LightCycler TaqMan Master (Roche, Indianapolis, IN). The thermal cycling conditions included 1 cycle at 95 °C for 10 min, and 45
cycles at 95 °C for 10 s and at 60 °C for 30 s. The relative mRNA
expression level of Dspp, Col111, Col11a2, Col5a1 and Col5a2 wasnormalized against that of the b-actin gene from the same RNA
preparations using a comparative threshold cycle method. The primer sequences are listed in Appendix Table I.
In situ hybridization. cDNAs for Col1a1, Col2a1, Col5a1, Col5a2,
Col5a3, Col11a1, Col11a2 and Dspp genes were generated by RTPCR (Appendix Table I). The ampliﬁed fragments were subcloned
into the pGEM-T Easy vector (Promega). The inserts were further
subcloned into a pBluescript vector for generating riboprobes. Allconstructs were sequenced on an ABI310 sequencer (Applied Biosystems, Foster, CA, USA). After linearization at the appropriate
restriction sites, antisense and sense probes were generated byin vitro transcription with T3 or T7 polymerases in the presenceof [35S]-dUTP. In situ hybridization was performed on 10 lm sections, as previously described [9].
Production of GST (glutathione S-transferase)-tagged fusion proteins. cDNAs encoding a portion of pro-a1(XI) , pro-a2(XI) collagenand dentin sialoprotein (Dsp), which is derived from 50 the regionof the DSPP gene, were generated by RT-PCR (Appendix Table I).
The ampliﬁed fragments were subcloned into the pGEM-T Easy
vector (Promega). Following digestion at the appropriate restriction sites, the fragment was subcloned into a pGEX-4T vector(Amersham). The other recombinant proteins, GST-tagged proa1(V) and pro-a3(V) have all have previously described [9]. The recombinant GST fusion proteins were expressed and preparedaccording to the manufacturer’s instructions.
Production of antibodies. Recombinant GST-pro-a2(XI) collagenand Dsp protein (0.5 mg) were mixed with Freund’s Complete
Adjuvant (Difco, Detroit, MI, USA) and subcutaneously injected into
female rabbits. The animals were boosted twice with the sameamount of protein mixed with Freund’s Incomplete Adjuvant (Difco) and the blood was collected 1 week after the second booster.
The antibody was puriﬁed using GST afﬁnity column chromatography. The speciﬁcity of the antibodies was conﬁrmed by ELISA (enzyme-linked immunosorbent assay) as previously described [9].
Immunohistochemistry. Immunohistochemistry was performed
as previously described [10].
Cell culture. The root of an incisor tooth was dissected fromadult mice and primary cell culture was conducted as described
before [11]. Rat ROS 17/2.8 osteosarcoma cells and rat RCS chondrosarcoma cells were also cultured.
Transfection with hTERT gene and isolating a single clone. Eighty
percent of the conﬂuent primary cells from a tooth specimen were167

transfected with the construct of hTERT (Geron Corporation, Menlo
Park, CA, USA) using lipofectamine (invitrogen, Carlsbad, CA, USA)[12]. Forty-eight hour after transfection, the cells were replated ata low density. They were further expanded into cell lines. Single
clones were passed and assayed for the speciﬁc marker, DSPP gene.
This yielded cell line, MOC-4, which expressed the DSPP gene.
Transplantation of MOC-4 cells into mice. The transformed MOC-4
cells were transplanted into immunodeﬁcient mice [12]. Brieﬂy,
the cells were injected subcutaneously into 4-week-old male
BALB/cAnNcrj-nu mice (Charles River Japan Inc., Kanagawa, Japan)
after incubating the cells (1.5  106) in a mixture of 40 mg ofhydroxyapatite granules (Apaseram, Pentax, Tokyo, Japan) and ﬁbrin clot (mixture of mouse ﬁbrinogen and thrombin, Sigma, St.
Louis, MO, USA). After one month, the transplants were retrieved
and prepared for histological analysis. The sections were decalciﬁed using Kalkitox (Wako, Tokyo, Japan).
Statistical analysis. Data were analyzed by t-test using Stat
Views J-5.0 (Abacus Concepts, Berkeley, CA, USA). A P value of<0.05 was considered to be statistically signiﬁcant.
Results
Expression of pro a-chains of V/XI collagen
RT-PCR was initially performed using mouse tissue to examine
the expression pattern of the a chains constituents of V/XI collagenmolecule in the tooth. As shown in Fig. 1A, pro-a1(XI), pro-a2(XI),
pro-a1(V) and pro-a2(V) chains were detectable in an adult mouseincisor tooth, but not pro-a3(V) and pro-a1(II). The expression patterns of ROS and RCS cells derived from bone and cartilage, respectively, were different from that of the tooth.
In situ hybridization was performed on mouse neonate, young(Fig. 1B) and embryonic (Fig. 1C) tissue specimens to determine
the precise expression of the a chains in the tooth. The DSPP gene
was strongly and restrictedly expressed in odontoblasts and preameloblasts. As shown previously [8], pro-a1(XI) chain was moderately expressed in odontoblasts as well as in bone and cartilage
tissues. The pro-a2(XI) chain was also moderately expressed inodontoblasts. The expression of pro-a2(XI) was negative in a ROS
in an RT-PCR analysis (Fig. 1A), but positive in the surrounding
bone tissue using in situ hybridization (Fig. 1B). During the developing stage (Fig. 1C), the signal of DSPP gene appeared in odontoblasts at 16.5E of mouse embryo. Very weak signals of the proa2(XI) and pro-a1(XI) chain were seen in odontoblasts and mesenchymal cells of the interstitial region as well as in bone tissue inthe 16.5E embryos (Fig. 1C and Appendix Fig. 1B). The pro-a1(II)chain was never expressed in odontoblasts in the 16.5E mouse embryo (Appendix Fig. 1B).
Distribution of pro-a2(XI) collagen chain in toothAs mentioned above, unexpectedly, we showed transcripts ofpro-a2(XI) collagen gene in odontoblasts for the ﬁrst time. To detect the protein, we prepared a speciﬁc polyclonal antibody
against pro-a2(XI) chain (Appendix Fig. 2A). The speciﬁcity of
the antibody was conﬁrmed using ELISA (Appendix Fig. 2B). The
rabbit antiserum against pro-a2(XI) polypeptide-GST fusion protein was passed in GST column to eliminate antibody against
GST protein. The antibody was not cross-reacted with GST andacidic domains of pro-a1(V), pro-a3(V) and pro-a1(XI) collagenchain. We also generated speciﬁc antibody against DSP protein(Appendix Fig. 2C).
Immunohistochemistry was performed using sections of E14.5
embryo and postnatal day 2 mouse (Fig. 2). DSP protein was observed in the dental pulp of the day 2 mouse, but not in E14.5.
These ﬁndings were consistent with those of in situ hybridization.

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Fig. 1. (A) RT-PCR analysis of pro-a1(XI), pro-a2(XI), pro-a1(II), pro-a1(V), pro-a2(V), pro-a3(V) and pro-a1(I) collagen chains and DSPP gene expression in ROS cells, RCS
cells and teeth. RNA from teeth at day 21 or older mice was used. RT-PCR was performed using speciﬁc primers. b-Actin was used as an internal control. (B) In situ
hybridization of the tissues at a 0, 2, 7 and 14 day mouse. The sections were hybridized with radioactively labeled DSPP and pro-a2(XI) collagens antisense (A–E, K–O) andsense (F–J, P–T) riboprobes. Photomicrographs are shown with brightﬁeld (days 0, 2, 7 and 14) or darkﬁeld (day 0: panel B, G, L and Q). There is no signal in control panels (F–J,
P–T). Scale bar: 100 lm. (C) In situ hybridization of the tissues from E14.5 and E16.5 mouse embryos. The sections were hybridized with radioactively labeled DSPP and proa2(XI) antisense (A–D, I–L) and sense (E–H, M–P) riboprobes. Photomicrographs are shown with brightﬁeld (panels A, C, E, G, I, K, M and O) or darkﬁeld (panels B, D, F, H, J, L, N
and P). There is no signal in control panels (E–H, M–P). Scale bar: 100 lm.

Fig. 2. Immunohistochemical localization of DSP protein and pro-a2(XI) collagen chain in E14.5 mouse embryos and day 2 mice. Photomicrographs were shown for sectionsof an E14.5 mouse embryo (A and D) and a day 2 mouse (B, C, E and F). The portions of square in B and D were magniﬁed in C and F, respectively. Parafﬁn sections were stainedwith anti-DSP (A, B and C) and anti- pro-a2(XI) antibody (D, E and F). The arrows in A and D show the cartilage tissue. Note that anti-pro-a2(XI) antibody stained the cartilage
tissue in the E14.5 mouse embryo (D). Scale bar in A and D: 50 lm, B and E: 100 lm, C and F: 10 lm.

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169

Fig. 3. The relative amount of mRNA of pro-a1(XI), pro-a2(XI), pro-a1(V) and pro-a2(V) collagen chains and DSPP in crown and root of tooth using real-time RT-PCR. (A) The
values of the relative ratio of mRNA of DSPP against that of b-actin are the mean ± SD for three independent wells. *p < 0.05 (B) The values of the relative ratio of mRNA of proa1(XI), pro-a2(XI), pro-a1(V) and pro-a2(V) collagen chains against that of b-actin are the mean ± SD for three independent wells. *p < 0.05 Note that the values of pro-a1(XI),
pro-a2(XI), pro-a1(V) and pro-a2(V) collagen chains are less than 102 times than that of DSPP.

The pro-a2(XI) chain was observed in odontoblasts. At E14.5 it wasseen in cartilage tissue (an arrow in Fig. 2D), but never in theodontoblasts.

In contrast, that of pro-a1(XI) in the crown was 0.1 times lower
than in the root.
Expression of pro-a2(XI) chain in the immortalized odontoblast

Relative amount of mRNA in toothReal-time RT-PCR was used to examine the relative amountof mRNA of each gene in tooth. The tissues specimens from atooth of 5-week-old mouse were divided into two portions,
namely the root and crown. The mean value of the relative
amount of transcripts of pro-a1(XI), pro-a2(XI), pro-a1(V),pro-a2(V) and DSPP genes against that of b-actin were
16.30  102, 0.53  102, 0.98  102, 0.16  102 and 0.98 in
the root, 1.58  102, 6.33  102, 14.70  102, 6.67  102
and 13.30 in the crown (Fig. 3), respectively. The amount oftranscripts of the pro-a2(XI), pro-a1(V) and pro-a2(V) in thecrown were 11.9, 15.0 and 41.7 times greater than in the root.

There is a quantitative and qualitative limitation to using primary culture cells from tooth. To avoid those problems, a cell line
was established that retains the nature of the odontoblasts using
hTERT. The established cell line, MOC-4, was morphologically similar to the non-transfected original cells (Appendix Fig. 4) and expressed the DSPP gene as well as the hTERT gene (Fig. 4A). The
transcript of the pro-a2(XI) chain was also expressed in MOC-4
cells. To examine in vivo, MOC-4 cells were transplanted subcutaneously into immunodeﬁcient mice. Alkaline phosphatase activity
was detectable in the growing tumor cells (Fig. 4B-b). The tumor
cells expressed the pro-a2(XI) collagen chain as well as the DSP
protein (Fig. 4B-c and B-d).

Fig. 4. The gene expression in the transformed odontoblast. (A) Expression of pro-a1(XI), pro-a2(XI), pro-a1(II), pro-a1(V) and pro-a2(V) collagen chains, hTERT and DSPP
genes in MOC-4 and original cell. b-Actin was used as an internal control. (B) Immunohistochemical localization of DSP and pro-a2(XI) collagen chain in a tumor that wastransplanted MOC-4 cell. The photomicrographs show hematoxylin and eosin (H–E) staining (a), staining for alkaline phosphatase activity (b) and immunohistochemical
staining with anti-DSP protein (c) and anti-pro-a2(XI) collagen (d) antibodies. Scale bar: 10 lm.

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Discussion
The present study examined the expression of type V/XI collagen in the tooth. Unexpectedly, a moderate expression of the
pro-a2(XI) chain was seen in the tooth using RT-PCR and in situ
hybridization combined with immunohistochemistry methods.
The data were conﬁrmed using an established cell line that possesses osteoblastic characteristics in regard to morphology andgene expression. Type XI collagen, which is composed of a1(XI),
a2(XI) and a3(XI) chain, is expressed in cartilage. However, thea1(XI) collagen chain is widely expressed in non-cartilage tissues[8]. In comparison to the a1(XI) chain, the expression of thea2(XI) and a3(XI) chains is rather restricted to cartilage.
Type XI collagen could nucleate and promote the assembly oftype II collagen into thin ﬁbrils in cartilage [13]. On the other hand,
the molecule containing a1(V) chain can form banded ﬁbrils with
type I collagen [14]. Previous biochemical studies have documented the presence of heterotypic collagen molecules consistingof types V and XI collagen chains. The [a1(XI)]2a2(V) molecule ispresent in a human rhabdomyosarcoma cell line (A204) and thebovine vitreous [15,16]. Niyibizi and Eyre have previously suggested that a1(V) chain is involved in forming heterotypic V/XI
molecules in bovine bone [17]. In the chondrodysplasia mouse(cho), which is a neonatal lethal mouse caused by absence ofa1(XI) collagen chain, Fernandes et al. hypothesized the substitution of a1(V) for a1(XI) collagen chain and expected a1(V)a2(XI)a3(XI) in the mouse [18]. The present study has showed thea1(XI) and a2(XI) chains to be expressed, but never a1(II) chain
in dentin. Therefore, type XI collagen, which contain a3(XI) chain
which is overglycosylated variant of a1(II) chain, is never present.
Instead, a homotrimer of a2(XI) chain or heterotypic V/XI moleculecontaining a2(XI) chain may therefore be present in dentin. A realtime PCR analysis showed the amount of pro-a2(XI), pro-a1(V) andpro-a2(V) mRNA to be similar in the root and crown. This may
therefore suggest the occurrence of the molecule ofa2(XI)a1(V)a2(V).
The current data suggest a new subtype of V/XI collagen molecule containing a2(XI) chain and the complexity of the molecularassembly in dentin. The different combination of type V/XI collagen chains could confer different physiological properties to the ﬁbril network in the matrix.
Acknowledgments
We thank the staff of Division of Biomolecular Medicine andMedical Imaging, and Division of Radioisotope Research, Instituteof Scientiﬁc Research. We also thank Ms. T. Kabayama and R. Ogata
for help of experiment of transplantation and Prof. K. Kawano for
encouraging our research. This work was supported by GrantsIn-Aid for Scientiﬁc Research (11470312, 14370468 and

20390402 to H.Y.) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2010.01.001.
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