1 Divisions of Cell Biology and Molecular Pharmacology, Niigata University
Graduate School of Medical and Dental Sciences, 5274 2-Bancho, Gakkocho-dori,
Niigata-city, Niigata 951-8514, Japan
2 Division of Periodontology, Niigata University Graduate School of Medical and
Dental Sciences, 5274 2-Bancho, Gakkocho-dori, Niigata-city, Niigata 951-8514,
Japan
3 Divisions of Anatomy and Cell Biology of the Hard Tissue, Niigata University
Graduate School of Medical and Dental Sciences, 5274 2-Bancho, Gakkocho-dori,
Niigata-city, Niigata 951-8514, Japan
4 Daiichi Pharmaceutical Co. Ltd., 1-16-13 Kita-kasai, Edogawa-ku, Tokyo
134-8630, Japan
5 Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku
University, 4-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Author for correspondence (e-mail:
kawashim{at}dent.niigata-u.ac.jp)
Accepted 13 August 2002
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Summary |
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Key words: Periodontal ligament, Cell line, Runx2/Cbfa1/Osf-2, Mineralization, BMP-2
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Introduction |
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Fibroblastic cells of the PDL have also been suggested to be a source of
osteoblasts for continued remodeling of alveolar bone of the mandibula
(Roberts et al., 1982).
Therefore, PDL fibroblastic cells have been suggested to be multipotent cells
(McCulloch and Bordin, 1991
)
or composed of heterogenous cell populations that have the capacity to
differentiate into either osteoblasts or cementoblasts depending on the
microenvironment (Gould et al.,
1980
; Roberts et al.,
1982
; McCulloch and Melcher,
1983
). Indeed, recent studies demonstrated that cells isolated
from the PDL have osteoblast-like properties, such as alkaline phosphatase
(ALPase) activity (Yamashita et al.,
1987
), being responsive to PTH
(Nojima et al., 1990
) and
producing bone sialoprotein in response to 1,25-dihydroxyvitamin D3
(Nojima et al., 1990
). In
addition, primary rat PDL cells in culture formed mineralized nodules in
vitro, although the mineralized nodules appeared to be different from those
produced by osteoblasts (Cho et al.,
1992
). A similar result was observed with human PDL cells
(Arceo et al., 1991
). However,
since it has been shown that there are subsets of fibroblastic cells in the
PDL (Roberts and Chamberlain,
1978
; Limeback et al.,
1982
; Rose et al.,
1987
), it is not clear whether these osteoblastic phenotypes and
functions were due to one type of cell or to a combination of cells in the
PDL.
To circumvent these problems, it is necessary to establish a cell line
possessing the exact nature of PDL fibroblastic cells. Therefore, we have
tried to establish immortalized cell lines from transgenic mice harboring the
temperature-sensitive (ts) Simian Virus 40 large tumor antigen (SV 40 large
T-antigen) gene (Yanai et al.,
1991). Here, we report that one of the established cell lines is
indistinguishable from the fibroblastic cells of PDL in terms of gene
expression. Furthermore, it produces mineralized nodules in the presence of
recombinant human bone morphogenetic protein (rhBMP)-2.
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Materials and Methods |
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Histochemistry
Frozen sections were treated with 0.1 M Tris-maleate buffer (pH 7.4)
containing 50 mM MgSO4 at 37°C for 1 hour, and ALPase activity
was detected according to the azo-dye method of Burstone
(Burstone, 1962).
Probe preparation
Digoxigenin UTP-labeled sense and anti-sense single-stranded RNA probes
were prepared with a DIG RNA Labeling Kit (Roche Diagnostics, Mannheim,
Germany) according to the manufacturer's instructions. For type I collagen
(Col. I) (Ac. No. X06753), osteocalcin (OCN) (Ac. No. X4142) and
bone sialoprotein (BSP) (Ac. No. L20232), each corresponding cDNA
fragment, 202, 470 and 1048 bp, respectively, was obtained by RT-PCR and
subcloned into pBluescript SK(-) (Stratagene, La Jolla,
CA). For periostin, 2.5 kb fragment of mouse cDNA
(Xiao et al., 1998) was used
in a similar manner. After transcription, 40 units of RNase-free DNase (Roche
Diagnostics) was added to the reaction and incubated for an additional 10
minutes at 37°C. Transcription products were recovered by ethanol
precipitation with 25 µg of RNase-free glycogen (Roche Diagnostics) as a
carrier, and precipitates were washed once in 70% ethanol, air-dried and
resuspended in 50 µl of diethyl pyrocarbonate-treated water. For
runt-related transcription factor-2 (Runx2)/core binding factor a1
(Cbfa1)/osteoblast-specific transcription factor-2 (Osf2), a 0.3 kb
fragment of exon I of mouse cDNA (Horiuchi
et al., 1999
) was subcloned into pBluescript
KS(-) (Stratagene). The fragment of Runx2/Osf2 is
known to detect the isoform expressed specifically in osteoblasts and
chondrocytes. The subcloned fragments were confirmed to be identical to the
respective cDNA sequence using the ABI prismTM 377 DNA Sequencing System
(PE Co., Foster City, CA).
In situ hybridization
Deparaffinized sections were mounted on silane-coated slides (Dako Japan
Co., Ltd., Kyoto, Japan), washed in PBS, treated with 0.2 N HCl for 10 minutes
and incubated with 0.5 µg/ml of proteinase K (Roche Diagnostics) in 10 mM
Tris-HCl (pH 8.0) at 37°C for 30 minutes. The sections were then refixed
with 4% paraformaldehyde in PBS at room temperature for 30 minutes and further
treated with 0.25% acetic anhydride in 0.1 M triethanolamine for acetylation.
The acetylated sections were hybridized with probes at a final concentration
of 1.0 µg/ml in a hybridization mixture containing 50% deionized formamide,
2% blocking agent (Roche Diagnostics), 5xSSC (1xSSC=0.15 M NaCl,
pH 7.5, 15 mM sodium citrate, 0.02% SDS (sodium dodecyl sulfate), 0.1%
N-lauroylsarcosine, 200 µg/ml yeast tRNA and 100 µg/ml salmon sperm DNA
at 55°C for 18 hours, then washed twice with 0.1xSSC at 60°C for
30 minutes. This was followed by RNase (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) treatment at 37°C for 30 minutes. The sections were further
washed twice with 2xSSC at 60°C for 15 minutes and then twice with
0.1xSSC at 60°C for 15 minutes. Specific transcripts were detected
with ALPase-conjugated anti-digoxigenin antibody according to the
manufacturer's protocol (DIG Detection Kit, Roche Diagnostics). The sections
were counterstained with nuclear fast red or methyl green.
Cloning of cell lines
Molars were aseptically removed from 5-week-old transgenic mice harboring
the temperature-sensitive SV40 large T-antigen gene under ether anesthesia,
and the PDL tissues were scraped off from the root surface of the mandibula.
Tissue explants were placed in 24-well tissue culture plates (Falcon, Nippon
Becton Dickinson Co., Ltd., Tokyo, Japan) containing Dulbecco's modified
Eagle's medium (DMEM) (Gibco BRL, Life Technologies Inc., Rockville, MD)
supplemented with 10% FBS (Gibco BRL), penicillin G (100 IU/ml) (Gibco BRL)
and streptomycin (100 µg/ml) (Gibco BRL). The explants were covered with a
coverslip (NUNC, Nalge Nunc International, Roskilde, Denmark) and incubated at
33°C and 5% CO2 in air. The outgrown cells from explants were
cloned according to the limited dilution method after 10 passages. Nineteen
clonal cell lines were obtained and one of them (designated as PDL-L2) was
chosen for further characterization by ALPase activity, mineralization assay
and reverse transcriptase (RT)-PCR. A mixture of uncloned cell population was
also used as PDL cells for the control. After the PDL-L2 was established, the
cell line has maintained its phenotype over 40 passages.
Cell culture
In addition to the PDL-L2 and PDL cells, four other cell lines were used in
the study. They are murine mesenchymal pluripotent cell line, C3H10T1/2 (Human
Science Research Resources Bank, Osaka, Japan), murine osteoblastic cell line,
MC3T3-E1 (Riken Gene Bank, Wako, Japan), murine fibroblastic cell line, NIH3T3
(Cell Resource Center for Biomedical Research Institute of Development, Aging
and Cancer, Tohoku University, Sendai, Japan) and rat osteoblastic cell line
ROS 17/2.8 (kindly provided from Yamanouchi Pharmaceutical Co. Ltd., Tsukuba,
Japan). C3H10T1/2, MC3T3-E1, NIH3T3, and ROS 17/2.8 cells were cultured at
37°C in basal MEM with 10% heat-inactivated FBS, MEM with 10%FBS,
DMEM with 10% FBS and DMEM/F12 with 10% FBS, respectively.
Semi-quantitative RT-PCR
Cells were plated at 5x104 cells/35 mm dish (Falcon)
containing the medium described above and cultured for the indicated number of
days at 33°C for PDL-L2 and PDL cells, and at 37°C for MC3T3-E1 and
NIH3T3 cells. Total RNA of each cell line was extracted according to the acid
guanidinium isothiocyanate-phenol-chloroform method. Then, the first-strand
cDNA was synthesized using random primers (nine-mers) (Takara Shuzo, Osaka,
Japan) and Superscript II (Gibco BRL) as a reverse transcriptase. For PCR,
aliquots of synthesized cDNA were added to PCR mixtures containing 3'
and 5' primers (0.2 µM each), dNTP mixture (0.2 mM each) (Gibco BRL)
and Taq polymerase (0.05 unit/µl) (Gibco BRL). Cycling conditions were
94°C/30 seconds, 53°C/45 seconds, and 72°C/30 seconds for 20
cycles for Col. I, 94°C/30 seconds, 55°C/45 seconds,
72°C/40 seconds for 19 cycles for periostin, 94°C/30 seconds,
58°C/45 seconds, 72°C/40 seconds for 24 cycles for BSP,
94°C/30 seconds, 62°C/45 seconds, 72°C/40 seconds for 21 cycles
for OCN, 94°C/30 seconds, 62°C/45 seconds, 72°C/30
seconds for 33 cycles for Runx2/Cbfa1/Osf2, 94°C/20 seconds,
54°C/30 seconds, 72°C/30 seconds for epidermal growth factor
receptor (EGFR) for 32 cycles and 94°C/30 seconds,
55°C/45 seconds, 72°C/30 seconds for 12 cycles for
Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH),
respectively. Primers for PCR were as follows:
5'-ACC ATC Tgg CAT CTC ATg gC-3' and 5'-gCA ACA CAA TTg CAC CTgAgg-3' for type I collagen gene, 5'-ATC CCC ATg ACT gTC TAT Ag-3' and 5'-CAA ATA AgT gAC CAT CgC CA-3' for periostin, 5'-AAC AAT CCg TgC CAC TCA-3' and 5'-ggA ggg ggC TTC ACT gAT-3' for BSP, 5'-TgC gCT CTg TCT CTC TgA CC-3' and 5'-CTg TgA CAT CCA TAC TTg Cag g-3' for osteocalcin gene, 5'-gAg ggC ACA AgT TCT ATC Tgg A-3' and 5'-ggT ggT CCg CgA TgA TCT C-3' for Runx2/Osf2, 5'-Aag gAT gTg AAg TgT gg-3' and 5'-ACT TTC TCA CCT TCT gg-3' for EGFR, and 5'-AAg ATg gTg AAg gTC ggT gT-3' and 5'-gCA Tgg ACT gTg gTC Atg Ag-3' for GAPDH.
PCR products were fractionated on a 1% agarose gel, transferred to positively charged nylon membranes, and cross-linked by ultraviolet light. Membranes were hybridized with DIG-labeled (Roche Diagnostics) DNA probes and detected with CDP-Star substrate (NEW ENGLAND BioLabs. Inc., Beverly, MA) according to manufacture's standard protocols.
Western blot analysis
To discover whether Runx2/Cbfa1/Osf2 is produced in the PDL cells, western
blot analysis was carried out. PDL-L2 and MC3T3-E1 cells were grown in 10 cm
diameter tissue culture plates, and nuclear extracts of cells at confluence
were prepared using the Nuclear Extract Kit (ACTIVE MOTIF, Carlsbad, CA). The
nuclear extract of each plate was separated by 8% SDS-PAGE and then subjected
to immunoblot analysis with a 1:8,000 dilution of anti-Runx2/Osf2 antibody
(generated against the N-terminal peptide sequence of Osf2) (kindly provided
by Dr Karsenty of Baylor College of Medicine) and
horseradish-peroxidase-conjugated anti-rabbit IgG antibody (1:250,000)
followed by ECL+Plus detection (Amersham Biosciences, Tokyo, Japan).
Alkaline phosphatase activity and mineralization assay
To assess alkaline phosphatase (ALPase) activity, cells were incubated with
normal medium (-MEM containing 5% FBS) or differentiation medium
(normal medium supplemented with 1 µM dexamethazone, 10 mM
ß-glycerophosphate and 50 µg/ml ascorbic acid). Cells were cultured
for 3 days and for an additional 3 days in the presence or absence of 250
ng/ml rhBMP-2 (kindly provided by Yamanouchi Pharmaceutical Co. Ltd., Tokyo,
Japan). The cultured cells were fixed with 2% formaldehyde for 15 minutes and
subjected to ALPase staining. For the mineralization study, cells at
confluence were cultured for 3 days in the differentiation medium in the
presence or absence of 250 ng/ml rhBMP-2 and cultured for an additional 25
days. The incubation medium was changed every 3 days without further addition
of rhBMP-2. Cells were then fixed with 2% formaldehyde for 15 minutes and
subjected to Alizarin red-S staining.
Alizarin red-S (AR-S) staining
At the end of each experiment, the cultures were rinsed with PBS and fixed
for 15 minutes at 4°C with 2% paraformaldehyde in PBS. Fixed cultures were
rinsed with PBS and Nanopure water, and stained with 40 mM AR-S (Wako Pure
Chemical Industries, Ltd.) (pH 4.2, 1 ml/35 mm dish) at room temperature with
gentle rotation. Cultures were then washed five times with water followed by a
15 minutes rinse with PBS with gentle rotation to reduce nonspecific AR-S
stain. Stained cultures were photographed and then destained by incubating in
10% (w/v) cetylpyridinium chloride (CPC) (Wako Pure Chemical Industries, Ltd)
in 10 mM sodium phosphate buffer (pH 7.0) for 1 hour at room temperature.
Aliquots of these AR-S extracts were diluted 10-fold in the 10% CPC solution,
and the AR-S concentration of each sample was determined by absorbance at 562
nm for quantification (Stanford et al.,
1995).
Expression and reporter plasmid
For the mammalian expression plasmid encoding Runx2/Cbfa1/Osf2,
the RT-PCR-amplified N-terminal portion of Osf2 (ATG-NaeI
0.26 kb) was fused to PEBP2A (NaeI-XbaI 3
kb) from pBKS2
A (kindly provided by T. Komori), and this was
cloned into the pcDNA3 (Invitrogen Corp., Carlsbad, CA, USA). To
assess the transcriptional activity of Runx2/Osf2, we generated reporter
plasmid p6OSE2-Luc, in which luciferase expression is controlled by
six copies of the Cbfa1 binding OSE2 site of osteocalcin promoter
followed by the minimal promoter, as described previously
(Ducy and Karsenty, 1995
).
Transfection and reporter assays
Cells (5x104 cells per 35 mm dish) were transfected with
various plamid DNAs using Fugene-6 (Roche Diagnostics) according to the
manufacturer's recommendations. Twenty-four hours after transfection, cells
were harvested using the Reporter Lysis Buffer (Promega Corporation, Madison,
WI, USA) for analysis of luciferase and ß-galactosidase activities. The
luciferase and ß-galactosidase assays were performed with the PicaGene
system (Wako Pure Chemical Industries, Ltd.) and AURORA Gal-XE kit (Wako Pure
Chemical Industries, Ltd.), respectively. All luciferase activities are
normalized for transfection efficiency against the corresponding
ß-galactosidase activities from the cotransfected
pCMV-SPORT-ß-gal plasmid (Gibco BRL). Each assay was performed
in duplicate or triplicate, and the same experiment was repeated at least
twice.
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Results |
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ALPase activity
Fig. 1A and 1B depict ALPase
staining of the mandibula. It is apparent that the majority of fibroblastic
cells are positive for this enzyme activity, although the intensity of the
staining of each cell varies widely.
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Type I collagen
In terms of type I collagen expression, approximately 80% of cells
in the PDL were positive (Fig.
1C); however, the expression level was lower than those in
osteoblasts.
Periostin
Periostin was isolated as one of the osteoblast-specific genes
(Takeshita et al., 1993), and
the physiological role of this gene is still unknown. Surprisingly, strong
expression of the periostin gene was detected in most of the PDL
cells, whereas expression in the osteoblasts was weaker
(Fig. 1D). A similar finding
was recently reported (Horiuchi et al.,
1999
).
Runx2/Cbfa1/Osf2
Ranx2/Cbfa1/Osf2 is known to be a transcription factor essential for
osteoblast differentiation (Ducy et al.,
1997; Komori et al.,
1997
). As expected, Runx2/Cbfa1/Osf2 expression was
detected in the osteoblasts (Fig.
1E). Similarly, intense expression was evident in a large
percentage (60 to 70%) of the PDL cells
(Fig. 1E). The expression was
higher in odontoblasts than in osteoblasts.
Osteocalcin
Osteocalcin is a marker of late stage osteoblast differentiation, and a
lack of this gene reportedly causes an increase in bone formation in mice
(Ducy et al., 1996).
Expression of the osteocalcin gene was most intense in the
odontoblasts, followed by osteoblasts and then by cementoblasts. No expression
was detected in the PDL cells (Fig.
1F).
BSP
BSP is a marker of osteoblasts at the middle stage of differentiation, and
expression of this gene is undetectable or minimal in the PDL
(MacNeil et al., 1994;
MacNeil et al., 1996
;
D'Errico et al., 2000
) in vivo.
Consistent with the previous observation, expression of this gene was not
detected in the PDL, whereas it was positive in the osteoblasts and
cementoblasts. Odontoblasts were negative for BSP
(Fig. 1G).
Cloning and selection of cell lines from the PDL explant culture
Using the dilution cloning method we obtained 19 cell lines from explant
cultures of the PDL. Most of these cells were fibroblastic in appearance,
although some looked polygonal at confluence. A cell line designated PDL-L2
was selected for further characterization on the basis of preliminary gene
expression analyses using RT-PCR (see below).
RT-PCR analysis demonstrates that gene expression in the PDL-L2 cells
is parallel with that seen in the PDL in vivo
To assess the differences between the cloned cell lines and osteoblasts,
expression of genes specific to osteoblasts was studied by RT-PCR. As shown in
Fig. 2A, PDL-L2 cells were
positive for type I collagen, periostin and
Runx2/Cbfa1/Osf2, whereas they were negative for osteocalcin
and BSP. Osteocalcin expression was detected only in the
differentiated osteoblasts. Intense BSP expression was observed in
the differentiated osteoblasts, whereas much weaker expression was seen in the
undifferentiated osteoblasts. BSP is a marker of osteoblasts at the
middle stage of differentiation, whereas osteocalcin is a marker of
osteoblasts at the late stage of differentiation. A fibroblastic NIH3T3 cell
line expressed type I collagen only at a much lower level than those
of PDL-L2 and MC3T3-E1 cells. These data, together with those in
Fig. 1, clearly demonstrate
that the established cell line PDL-L2 shares identical expression of the genes
examined. Furthermore, PDL-L2 expressed the EGFR genes (data not
shown), which have previously been shown to be present in the PDL
(Matsuda et al., 1993;
Cho and Garant, 1996
). Since
Runx2/Cbfa1/Osf2 is a transcription factor that is specifically expressed and
functions in osteogenic cells, western blot analysis was also performed to
examine whether or not this protein is produced in PDL-L2 cells. As shown in
Fig. 2, Runx2/Cbfa1 was
detected in the nuclear extracts of the PDL-L2 cells. The expression level of
Runx/Cbfa1 in PDL-L2s is approximately one-sixth of that in MC3T3-E1s, and the
difference is much smaller compared with the 60-fold difference of gene
expression (Fig. 2A). C3H10T1/2
cells, in which Runx/Cbfa1 is undetectable, were transfected with
Runx/Cbfa1 plasmid, and the cell lysates were analyzed in the same
way as the control.
|
PDL cells respond to rhBMP-2 and mineralize in vitro
When PDL-L2 cells were incubated in normal medium, they grew until they
reached confluence and remained healthy for up to 7 days. However, they became
detached from the dish as a thin sheet-like structure after 7 days and could
not grow any more (data not shown). By contrast, the osteoblastic cell line,
MC3T3-E1, grew further and eventually formed nodules that ultimately
calcified. Although PDL-L2 cells express the ALPase gene, its enzyme
activity was barely detected under the conditions shown in
Fig. 3. Since the cell line
expresses BMP receptor type I and II (data not
shown), however, stimulation by BMP may enhance alkaline ALPase activity and
induce mineralization as is the case with C3H10T1/2 cell line
(Katagiri et al., 1990). To
test this possibility, PDL-L2 cells were incubated in the presence of rhBMP-2.
As shown in Fig. 3, BMP-2
treatment enhanced ALPase activity not only in osteoblastic MC3T3-E1 cells but
also in PDL-L2 cells, whereas fibroblastic NIH3T3 cells failed to respond
(Fig. 3). BMP-2 also induced
nodule formation and mineralization in PDL-L2 cells, although the extent of
these phenomena was much less than those in MC3T3-E1 cells
(Fig. 4). NIH3T3 cells again
failed to respond to the BMP treatment. In contrast to the PDL-L2 cells, PDL
cells, a mixed cell population from the PDL, readily mineralized in the
absence of rhBMP-2, and the extent of mineralization was even higher than that
of osteoblastic MC3T3-E1 cells (Fig.
4).
|
|
PDL-L2 has a mechanism by which the Runx/Cbfa1 gene function
is suppressed
As shown in Fig. 1, the
expression level of Runx2/Cbfa1 in the PDL fibroblasts is similar to
or even higher than that in osteoblasts in vivo, but the PDL never mineralize,
indicating the presence of a mechanism that inhibits mineralization of the
tissue. This seems to be also true in vitro, since a superphysiological
concentration of BMP-2 caused only a minimal mineralization nodules. When
PDL-L2 cells were cultured in the differentiation medium without rhBMP-2,
Runx2/Osf2 expression markedly increased after 3 days, whereas
expression of osteocalcin was barely detectable and that of
BSP was not detected until 28 days of culture
(Fig. 5A). Addition of rhBMP-2
increased the expression level of Runx2/Osf2 to the maximum level,
whereas the expression of osteocalcin and BSP were delayed
and still suppressed compared to those seen in MC3T3-E1 cells
(Fig. 2). Although the
expression levels of these genes in PDL-L2 cells are lower than those in
osteoblastic MC3T3-E1 cells, the pattern and sequence of change in each cell
line was essentially the same. In contrast to those genes, the expression
level of periostin transiently increased and returned to the control
level at 28 days (Fig. 5A).
This is also similar to the change in MC3T3-E1 cells
(Fig. 2; data not shown). To
further understand the underlying mechanism, we next compared the level of
Runx2/Osf2 activity in cell lines using the cis-element OSE2, six of which are
connected in tandem to a reporter plasmid. As depicted in
Fig. 5B, transcriptional
activation by Runx2/Osf2 in the rat osteoblastic ROS 17/2.8 cells was 60-fold
more than that in C3H10T1/2; the latter is devoid of a significant amount of
Runx2/Cbfa1 (Sasaki-Iwaoka et al.,
1999). On the other hand, transcriptional activation in the PDL-L2
was minimal (Fig. 5B). When
C3H10T1/2 cells were transfected with the Runx2/Osf2 plasmid, the
transcriptional activity dramatically increased
(Fig. 5C). By contrast, the
same treatment failed to stimulate the transcriptional activity in the PDL-L2.
These data clearly indicate the presence of a mechanism responsible for
suppressing the Runx2/Osf2 activity in PDL-L2 cells.
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![]() |
Discussion |
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PDL cells have been suggested to be osteogenic in nature and to
differentiate into either osteoblasts or cementoblasts depending on the need
and the environment. Cho et al. demonstrated that PDL cells from rats formed
mineralized nodules in vitro, although the nodules differed from those formed
by osteoblasts (Cho et al.,
1992). Arceo et al. also demonstrated that human PDL cells, but
not gingival fibroblastic cells, formed mineral-like nodules in vitro
(Arceo et al., 1991
). However,
the PDL cells used in those and other studies were a mixture of heterogenous
cell populations, and it was difficult to identify which cells, alone or
interacting with other cell types, were responsible for the nodule formation.
The present study further supports the notion that the PDL cells are closely
related to the osteogenic cell lineage by demonstrating that the PDL cells
express genes thought to be specific to osteoblasts including
periostin and Runx2/Cbfa1/Osf2 and that PDL cells alone can
produce mineralized nodules in response to rhBMP, although the dose used was
in a super-physiological range.
It may be argued that the PDL-L2 cell line originated from cementoblasts
attached to the explants of PDL used for cloning. This seems unlikely,
however, since cementoblasts abundantly expressed BSP gene in vivo,
whereas fibroblastic PDL cells were negative for BSP, as shown in
Fig. 1G, a finding that is
consistent with those of previous reports
(MacNeil et al., 1994;
MacNeil et al., 1996
;
D'Errico et al., 1997
). In
addition, the Runx2/Cbfa1/Osf2 gene is expressed in PDL cells but not
in cementoblasts (Fig. 1E).
Similarly, periostin is highly expressed in PDL cells but not in
cementoblasts (Fig. 1D). The
cloned PDL-L2 cells showed an identical expression pattern for these genes to
in vivo fibroblastic PDL cells (Fig.
2). Furthermore, PDL-L2 cells did not produce mineralized nodules
in the absence of exogenous BMP-2 (Fig.
4), whereas cementoblasts have been shown to be capable of
producing mineralized nodules without BMP treatment
(D'Errico et al., 2000
). It is
also unlikely that PDL-L2 cells originated from gingival fibroblasts, since
neither of these genes is expressed in gingival fibroblasts in vivo (data not
shown). To our knowledge, this is the first demonstration of a cloned PDL cell
line that reflects the exact gene expression seen in vivo, although in a
limited number of cells, and is capable of producing mineralized nodules in
vitro.
PDL-L2 cells express genes for periostin and Runx2/Osf2
but not BSP and osteocalcin, suggesting that PDL-L2s may be
cells at a stage a little before the middle stage of osteoblast
differentiation. However, the cells do not mineralize without BMP-2 treatment,
which suggests that they do not yet appear to be committed towards
osteoblastic cells. This is consistent with the previous observation that a
pluripotent cell line C3H10T1/2 can differentiate into mature osteoblasts in
response to rhBMP-2 treatment (Katagiri et
al., 1990). However, a big difference exists between the two cell
lines: while C3H10T1/2 cells fully mineralize in response to rhBMP-2
(Katagiri et al., 1990
), the
response of PDL-L2 cells is minimal (Fig.
4). This observation suggests that there may be a regulatory
mechanism preventing differentiation of PDL-L2 cells toward osteoblasts; this
notion is consistent with the fact that the expression level of the
Runx2/Osf2 gene in PDL cells in vivo appeared to be comparable to
that in osteoblasts (Fig. 1E),
and yet the PDL cells never mineralize. Since our data suggest that PDL-L2s
may have a mechanism by which the Runx/Osf2 function in inducing BSP
and osteocalcin is suppressed, we examined this possibility
further.
We first compared the transcriptional activity of Runx2/Osf2 in
osteoblastic cells and PDL-L2 cells. The activity in ROS 17/2.8 cells was
60-fold that in C3H10T1/2 cells, which are devoid of a significant amount of
the Runx2/Osf2 gene
(Sasaki-Iwaoka et al., 1999).
By contrast, the transcriptional activity in PDL-L2 cells was not much higher
than that in C3H10T1/2 despite the fact that PDL-L2 cells expressed a
significant amount of the Runx2/Osf2 gene
(Fig. 5B). When C3H10T1/2 cells
were transfected with Runx2/Osf2 plasmid, the transcriptional
activity increased more than 30-fold, whereas the response of PDL-L2 cells was
minimal (Fig. 5C). It may be
argued that Runx2 protein production is suppressed in the PDL-L2 even though
gene expression is relatively high. This possibility, however, was excluded
since western blot analysis revealed that endogenous Runx2/Cbfa1 is detectable
in the PDL-L2 cells. These data clearly indicate that there exists a mechanism
by which the function of Runx2/Osf2 is suppressed in PDL-L2 cells.
More work is needed to clarify the exact mechanism underlying the unique
character of the PDL-L2 cells.
The hypothesis that there may be a regulatory mechanism in vivo preventing
differentiation of PDL cells toward osteoblasts is also consistent with the
observation by Lang et al. that periodontal cell populations cultured in vitro
failed to form cementum-like tissues in vivo when they were orthotopically
re-implanted, whereas cell populations from alveolar bone did form
cementum-like tissue in the same situation
(Lang et al., 1995). The
thesis that cementoblasts and osteoblasts in the periodontium commonly
originate from precursor cells residing in the alveolar bone is also supported
by previous observations (Gould et al.,
1977
; Melcher et al.,
1986
). Our data are also consistent with the data reported by
Rajishankar and colleagues (Rajishankar et
al., 1998
). Using the periodontal window wound healing model, they
demonstrated in vivo that PDL inhibited invasion of alveolar bone even when
the damaged tissues were stimulated by a large amount of BMP-7. Meanwhile the
wounds including bone and cementum as well as the PDL were completely healed
by the treatment. They concluded that PDL has a mechanism for regulating PDL
width and maintaining periodontal homeostasis that is resistant to the strong
osteogenic stimulation by BMP. Our study supports this notion and suggests
that the mechanism may be due to the suppression of the transcriptional
activity of Runx2/Osf2, since the transcription factor is necessary for
calcification of bone and cartilage (Ducy
et al., 1997
; Komori et al.,
1997
). Thus, whether or not PDL fibroblasts differentiate into
osteoblasts and/or cementoblasts in vivo is still a matter of debate, our data
on the PDL-L2, however, rather indicate that PDL fibroblastic cells is not
osteogenic. Further studies are needed to clarify the underlying
mechanism.
It should be noted that Runx2/Osf2 was expressed in odontoblasts
in addition to PDL cells and osteoblasts and that the expression level was
much higher in odontoblasts than in PDL cells and osteoblasts
(Fig. 1E). Our results were
somewhat inconsistent with those of recent studies demonstrating
Runx2/Osf2 gene expression in odontoblasts during tooth development,
but after completion of tooth development, the principal site of expression of
this gene was restricted to the PDL
(D'Souz et al., 1999;
Jiang et al., 1999
). The
reason for the discrepancies between those two studies and the present study
is not clear at present. Since the signal detected in our study was clear and
consistent with the previous observations concerning the expression of this
gene in osteoblasts and PDL cells (D'Souz
et al., 1999
; Jiang et al.,
1999
), and since the signal disappeared completely when a sense
probe was used, it is unlikely that the signals were non-specific. Differences
in the probes used and in the differentiation stage of the animals in these
studies may account for the discrepancies. Further studies are needed to
resolve this issue.
In addition to the PDL-L2 cells, we established 18 more cell lines. Most of them (approximately 70%) showed characteristics similar to those of PDL-L2. This is in agreement with in situ hybridization data showing that Runx2/Osf2-positive cells occupy 60 to 70% of PDL fibroblasts (Fig. 1). Other cell lines showed patterns of gene expression different from those of PDL-L2 (data not shown). Further characterization of these cell lines will allow us to determine whether these cell lines exhibit all the osteogenic functions proposed for the PDL tissues, whether there are really heterogenous types of fibroblastic cells in the PDL and which cell types are responsible for one or all of these functions. It is important to identify a marker(s) for PDL cells, and our cell lines will also be useful for this purpose.
In conclusion, we have established an immortalized murine PDL cell line, PDL-L2, which appears to mimic gene expression observed in PDL cells in vivo. The cell line produces a limited amount of mineralized nodules when stimulated by exogenous BMP-2 and is equipped with a mechanism for suppressing the function of Runx2/Osf2. PDL-L2 cells together with other cloned cell lines will provide new tools with which to study the biological function of the PDL and new insights into the role of the PDL in tooth development and maintenance of teeth and alveolar bone of the mandibula. Our study also confirmed that a population of PDL cells expresses the Runx2/Osf2 gene at a similar level to that in osteoblasts and demonstrated that odontoblasts express this gene at even higher levels in adult life. In addition, our study demonstrated that PDL cells express the periostin gene at a much higher level than do osteoblasts in mandibula.
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Acknowledgments |
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References |
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Arceo, N., Sauk, J. J., Moehring, J., Foster, R. A. and Somerman, M. J. (1991). Human periodontal cells initiate mineral-like nodules in vitro. J. Periodontol. 62,499 -503.[Medline]
Beertsen, W. (1975). Migration of fibroblasts in the periodontal ligament of the mouse incisor as revealed by autoradiography. Arch. Oral. Biol. 20,659 -666.[Medline]
Beertsen, W., McCulloch, C. A. G. and Sodek, J. (1997). The periodontal ligament: a unique, multifunctional connective tissue. Periodontology 13, 20-40.[Medline]
Berkovitz, B. K. B. and Shore, R. C. (1995). Cells of the periodontal ligament. In The periodontal ligament in health and disease, 2nd edn (ed. B.K.B. Berkovitz, B. J. Moxham and H. J. Newman), pp. 9-33. London: Mosby-Wolfe.
Burstone, M. S. (1962). Alkline phosphatase, naphthol AS-BI phosphate method. In Enzyme histochemistry and its application on the study of neoplasm (ed. M. S. Burstone), pp.275 -276. New York: Academic Press.
Cho, M.-I. and Garant, P.R. (1996). Expression and role of epidermal growth factor receptors during differentiation of cementoblasts, osteoblasts, and periodontal ligament fibroblasts in the rat. Anat. Rec. 245,342 -360.[Medline]
Cho, M.-I., Matsuda, N., Lin, W.-L., Moshier, A. and Ramakrishnan, P. R. (1992). In vitro formation of mineralized nodules by periodontal ligament cells from the rat. Calcif. Tissue. Int. 50,459 -467.[Medline]
D'Errico, J. A., Berry, J. E., Ouyang, H., Strayhorn, C. L., Windle, J. J. and Somerman, M. J. (2000). Employing a transgenic animal model to obtain cementoblasts in vitro. J. Periodontol. 71,63 -72.[Medline]
D'Errico, J. A., MacNeil, R. L., Takata, T., Berry, J. E., Strayhorn, C. L. and Somerman, M. J. (1997). Expression of bone associated markers by tooth root lining cells, in situ and in vitro. Bone 20,117 -126.[CrossRef][Medline]
D'Souz, R. N., Aberg, T. J., Gaikwad, T. A., Cavebder, T., Owen,
M., Karsenty, G. and Thesleff, I. (1999). Cbfa1 is required
for epithelial-mesenchymal interactions regulating tooth development in mice.
Development 126,2911
-2920.
Ducy, P. and Karsenty, G. (1995). Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol. Cell. Biol. 15,1858 -1869.[Abstract]
Ducy, P., Desbois, C., Boyce, B., Pinero, G. B., Story, G., Dunstan, C., Smith, E., Bonadio, J., Goldstein, S., Gundberg, C., Bradley, A. and Karsenty, G. (1996). Increased bone formation in osteocalcin-deficient mice. Nature 382,448 -452.[CrossRef][Medline]
Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L. and Karsenty, G. (1997). Osf2/Cbfa1: A transcriptional activator of osteoblast differentiation. Cell 89,747 -754.[Medline]
Gould, T. R. L., Melcher, A. H. and Brunette, D. M. (1977). Location of progenitor cells in periodontal ligament of mouse molar stimulated by wounding. Anat. Rec. 188,131 -141.
Gould, T. R. L., Melcher, A. H. and Brunette, D. M. (1980). Migration and division of progenitor cell populations in periodontal ligament after wounding. J. Periodont. Res. 15,20 -42.[Medline]
Horiuchi, K., Amizuka, N., Takeshita, S., Takamatsu, H., Katsuura, M., Ozawa, H., Toyama, Y., Bonewald, L. F. and Kudo, A. (1999). Identification and characterization of novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor ß. J. Bone Miner. Res. 14,1239 -1249.[Medline]
Jiang, H., Sodek, J., Karsenty, G., Thomas, H., Ranly, D. and Chen, J. (1999). Expression of core binding factor Osf2/Cbfa-1 and bone sialoprotein in tooth development. Mech. Develop. 81,169 -173.[CrossRef][Medline]
Katagiri, T., Yamaguchi, A., Ikeda, T., Yoshiki, S., Wozney, J. M., Rosen, V., Wang, E. A., Tanaka, H., Omura, S. and Suda, T. (1990). The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem. Biophys. Res. Commun. 172,295 -299.[Medline]
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S. and Kitshimoto, T. (1997). Targeted disruption of Cbfa1 results in complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89,755 -764.[Medline]
Lang, H., Schuler, N., Arnold, S., Nolden, R. and Mertens, T. (1995). Formation of differentiated tissues in vivo by periodontal cell populations culture in vitro. J. Dent. Res. 74,1219 -1225.[Abstract]
Limeback, H., Sodek, T. and Aubin, J. E. (1982). Variation in collagen expression by cloned periodontal ligament cells. J. Periodont. Res. 18,242 -248.
MacNeil, R. L., Sheng, N., Strayhorn, C. L., Fisher, L. W. and Somerman, M. J. (1994). Bone sialoprotein is localized to the root surface during cementogenesis. J. Bone Miner. Res. 9,1597 -1606.[Medline]
MacNeil, R. L., Berry, J. E., Strayhorn, C. L. and Somerman, M. J. (1996). Expression of bone sialoprotein mRNA by cells lining the mouse tooth root during cementogenesis. Arch. Oral. Biol. 41,827 -835.[CrossRef][Medline]
Magnusson, I., Stenberg, W. V., Batich, C. and Egelberg, J. (1990). Connective tissue repair in circumferential periodontal defects in dogs following use of biodegradable membrane. J. Clin. Periodontol. 17,243 -248.[Medline]
Matsuda, N., Kumar, N. M., Ramakrishnan, P. R., Lin, W.-L., Genco, R. J. and Cho, M.-I. (1993). Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro. Arch. Oral. Biol. 38,559 -569.[Medline]
McCulloch, C.A.G. and Bordin, S. (1991). Role of fibroblast subpopulations in periodontal physiology and pathology. J. Periodontal Res. 26,144 -154.[Medline]
McCulloch, C. A. G. and Melcher, A. H. (1983). Cell density and cell generation in the periodontal ligament of mice. Am. J. Anat. 167,43 -58.[Medline]
McCulloch, C. A. G., Nemeth, E., Lowenberg, B. and Melcher, A. H. (1987). Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations. Anat. Rec. 219,2233 -2242.
Melcher, A. H., Cheong, T., Cox, J., Nemeth, E. and Shiga, A. (1986). Synthesis of cement-like tissue in vitro by cells cultured from bone: a light and electron microscopic study. J. Periodont. Res. 21,592 -619.[Medline]
Nojima, N., Kobayashi, M., Shionome, M., Takahashi, N., Suda, T. and Hasegawa, K. (1990). Fibroblastic cells derived from bovine periodontal ligaments have the phenotypes of osteoblasts. J. Periodont. Res. 25,179 -185.[Medline]
Rajishankar, D., McCulloch, C. A. G., Tenenbaum, H. C. and Lekic, P. C. (1998). Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenetic protein-7 activity in vivo. Cell Tissue Res. 294,475 -483.[Medline]
Roberts, W. E. and Chamberlain, J. G. (1978). Scanning electron microscopy of the cellular elements of rat periodontal ligament. Arch. Oral. Biol. 23,587 -589.[Medline]
Roberts, W. E., Mozsary, P. G. and Klingler, E. (1982). Nuclear size as a cell-kinetic marker for osteoblast differentiation. Am. J. Anat. 165,373 -384.[Medline]
Rose, G. G., Yamasaki, A., Pinero, G. J. and Mahan, C. J. (1987). Human periodontal ligament cells in vitro. J. Periodont. Res. 22,20 -28.[Medline]
Sasaki-Iwaoka, H., Maruyama, K., Endoh, H., Komori, T., Kato, S. and Kawashima, H. (1999). A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts. J. Bone Miner. Res. 14,248 -255.[Medline]
Stanford, C. M., Jacobson, P. A., Eanes, E. D., Lembke, L. A.
and Midura, R. J. (1995). Rapidly forming apatitic mineral in
an osteoblastic cell line (UMR 106-01 BSP). J. Biol.
Chem. 270,9420
-9428.
Takeshita, S., Kikuno, R., Tezuka, K. and Amann, E. (1993). Osteoblast-specific factor 2: Cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem. J. 294,271 -278.[Medline]
Xiao, Z. S., Thomas, R., Hison, T. K. and Quarles, L. D. (1998). Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor. Gene 14,187 -197.
Yamashita, Y., Sato, M. and Noguchi, T. (1987). Alkaline phosphatase in the periodontal ligament of the rabbit and macaque monkey. Arch. Oral. Biol. 32,677 -678.[Medline]
Yanai, N., Suzuki, M. and Obinata, M. (1991). Hepatocyte cell lines established from transgenic mice harbouring temperature-sensitive Simian Virus 40 large T-antigen gene. Exp. Cell Res. 197,50 -56.[Medline]