Mechanisms of Ageing and Development
110 (1999) 37 – 48
www.elsevier.com/locate/mechagedev

Age-dependent alterations in mRNA level and
promoter methylation of collagen a1(I) gene in
human periodontal ligament
Masaki Takatsu a, Shinji Uyeno b,1, Jun-ichiro Komura b,
Makoto Watanabe a, Tetsuya Ono b,*
a

Department of Geriatric Dentistry, Tohoku Uni6ersity School of Dentistry, Seiryo-machi 4 -1,
Aoba-ku, Sendai 980 -8575, Japan
b
Department of Cell Biology, Graduate School of Medicine, Tohoku Uni6ersity, Seiryo-machi 2 -1,
Aoba-ku, Sendai 980 -8575, Japan
Received 6 April 1999; received in revised form 28 May 1999; accepted 31 May 1999

Abstract
In an attempt to understand the molecular mechanisms of age-dependent degenerative
alteration in human periodontal tissues, we examined mRNA level and DNA methylation of
collagen a1(I) gene. Using healthy periodontal ligament tissues from humans aged 9 – 76
years, we found that the collagen a1(I) mRNA level decreased almost linearly with age. It
was observed in both Northern blot and dot blot hybridization. Examination of DNA
methylation in the collagen a1(I) gene promoter region by its susceptibility to methylationsensitive restriction enzyme followed by Southern blot analysis showed age-dependent
increase of DNA methylation at − 1705 and −80 positions located upstream of the gene.
The data suggest the possible importance of alterations in collagen a1(I) gene expression and
its DNA methylation in promoter region in age-dependent degeneration of periodontal
ligament. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Aging; Periodontal ligament; Collagen a1(I); mRNA; DNA methylation

* Corresponding author. Tel.: +81-22-717-8131; fax: +81-22-717-8136.
E-mail address: tono@mail.cc.tohoku.ac.jp (T. Ono)
1
Present address: Department of Neurosurgery, Jichi Medical School, Minamikawachi-machi,
Kawachi-gun 329-0498, Japan.
0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 4 1 - X

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M. Takatsu et al. / Mechanisms of Ageing and De6elopment 110 (1999) 37–48

1. Introduction
Periodontal ligament is a membrane-like connective tissue surrounding the root
of a tooth. It plays important roles in supporting the tooth in the bone socket of
the jaw and also in maintaining homeostasis of the surrounding tissues, such as
alveolar bone and cementum (Bhaskar, 1990). Many histological studies, using
light- and electron-microscopes, have demonstrated that the integrity of the tissue
is lost as a function of age in human as well as in experimental animals (Grant and
Bernick, 1972; Severson et al., 1978; van der Velden, 1984; Berglundh et al., 1990;
Moxham and Evans, 1995). The change is believed to lead to edentulousness, often
observed in old individuals. Two kinds of factors are proposed as the cause of this
age-dependent degenerative change of periodontal ligament: extrinsic and intrinsic
(van der Velden, 1984). Major extrinsic factor is dental plaque caused by microbes
in the oral cavity followed by inﬂammation of the periodontium. Its detailed
processes are under investigation (van der Velden, 1984; Havemose-Poulsen and
Holmstrup, 1997). On the other hand, few data are available for intrinsic factors.
One approach to this problem would be to ﬁnd out genes whose expression alters
in the aging process. In other tissues like the liver, skin, colon etc. several genes are
now known to show age-dependent alteration in the expression levels (Finch, 1990;
Takeda et al., 1992; Heydari et al., 1993; Issa et al., 1994; Pahlavani and Richardson, 1996; Fujita et al., 1996). Among them, estrogen receptor gene expression in
the human colon has been found to decline with age in parallel to an increase in
DNA methylation in the 5% region of the gene (Issa et al., 1994). Since DNA
methylation is shown to be one of the factors involved in the regulation of gene
expression (Graessmann and Graessmann, 1993; Komura et al., 1995; Baylin et al.,
1998), the age-dependent hypermethylation in the estrogen receptor gene has been
thought to play a causative role in the decline in gene expression (Issa et al., 1994).
Furthermore, these age-dependent alterations are accelerated in colon cancers in
old individuals (Issa et al., 1994). A further search of such genes in each tissue
would eventually elucidate molecular mechanisms of age-dependent degeneration of
tissues. For periodontal ligament, histological studies have suggested that collagen
could be one of the molecules whose expression declines in the aging process
(Moxham and Evans, 1995). Here, we found that the mRNA level of collagen a1(I),
one of the major components of collagen ﬁbers in periodontal ligament, decreased
with age. By extending the analyses to DNA methylation in collagen a1(I) gene
promoter, we found that the degree of methylation at two sites increased with age.

2. Materials and methods

2.1. Tissue preparation
Periodontal ligament tissues were obtained from teeth which were extracted
because of dental caries, orthodontal reasons or third molar. The teeth extracted
from periodontitis were not used. Immediately after extraction, the tooth was

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39

frozen in liquid nitrogen and kept at − 70°C until use. After thawing of the tooth
at 4°C, the tissues at cervical and apical portions were removed ﬁrst by a scalpel
to avoid contamination of gingiva and pulp tissues. Then, the remaining periodontal ligament at the middle part of the root was removed by scraping with a
scalpel.

2.2. RNA analysis
Total RNA was extracted from the tissue by acid guanidinium thiocyanate-phenol-chloroform method (Uyeno et al., 1996). For Northern blotting, : 2 mg of total
RNA was denatured at 65°C for 10 min in 1 M glyoxal, 10 mM Na2HPO4, 50%
DMSO, electrophoresed in 1% agarose gel, transferred to nylon membrane ﬁlter
(Magnagraph, Micron Separation, Westborough, MA) and ﬁxed on the ﬁlter by
UV irradiation. For dot blotting, 0.1 and 0.02 mg of total RNA was denatured at
68°C for 15 min in 50% formamide, 7% formaldehyde, 1×SSC, trapped on nylon
membrane and ﬁxed by UV. The blotted RNA was hybridized to 32P-labeled
collagen a1(I) cDNA (clone Hf677), which was obtained from ATCC (Rockville,
MD). The 32P-labeling of DNA was performed using a random-primer labeling kit
(Boehringer – Mannheim). The hybridization was done for 16 h at 65°C in hybridization buffer (1× Denhardt’s, 1% SDS, 100 mg/ml salmon testis DNA, 6×
SSC). After hybridization, the ﬁlter was washed with 0.2× SSC, 0.1% SDS for 1 h
at 65°C. Autoradiography and determination of quantity of radioactivity were done
using Bio-Image Analyzer BAS 2000 (Fuji, Tokyo). In order to estimate accurately
the amount of total RNA ﬁxed on the ﬁlter, it was rehybridized to human
28S ribosomal DNA after removal of the labeled collagen gene probe by heating
the ﬁlter at 100°C in water. The removal of the probe was checked by autoradiography. The 28S ribosomal probe was 1.4 kbp BamHI fragment of pHr14E3
which was provided by the Japanese Cancer Research Resources Bank (Mishima,
Japan).

2.3. DNA analysis
The tissue was suspended in 10 ml of TNE (10 mM Tris–HCl, pH 8.0, 100 mM
NaCl, 1 mM EDTA) and lysed with SDS (ﬁnal 0.5%). DNA was extracted with
phenol and dissolved in TE (10 mM Tris, pH 8.0, 1 mM EDTA), digested with
restriction enzymes BamHI, EcoRV and NaeI, as suggested by the manufacturer
(NEB, Beverly, MA), run through 1% agarose gel, denatured, transferred to nylon
membrane ﬁlter and ﬁxed by UV light (Ono et al., 1985). Hybridization to collagen
a1(I) gene was done at 65°C for 16 h in the hybridization buffer mentioned above.
The probe was obtained as follows. The genomic collagen a1(I) gene was ampliﬁed
by PCR using two primers located upstream of exon 1 (− 1974 to − 1950) and
downstream of it ( +1270 to + 1294), 5%-AGGGGGCAGGACTTTGGTGGGTTCA (upper primer) and 5%-ACAGGAGGAGGGCGAGGGAGGAGAG
(lower primer), respectively (Jimenez et al., 1994). PCR ampliﬁcation condition was
94°C× 1
min+(98°C ×30
s+68°C× 5
min)× 30
cycles+ 72°C× 10

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M. Takatsu et al. / Mechanisms of Ageing and De6elopment 110 (1999) 37–48

min. Polymerase used was ex-Taq (Takara, Kyoto). The PCR product (3.3 kbp)
was digested with EcoRV (see Fig. 3) and the fragments were separated by gel
electrophoresis. Hybridization, washing of the ﬁlter and analysis of data were
similar to those used in RNA analysis.

Fig. 1. Northern blot analysis of collagen a1(I) mRNA level in human periodontal ligament tissues.
RNAs extracted from the tissues obtained from people at 17 – 61 years of age were run through agarose
gel, transferred to a ﬁlter and hybridized to 32P-labeled collagen a1(I) cDNA. Two bands were observed
at 5.8 and 4.8 kb (A). The ﬁlter was rehybridized to 28S ribosomal gene clone (the bottom part of A).
The total amount of collagen a1(I) mRNA was expressed as the sum of radioactivities in 4.8 and 5.8 kb
bands. The summed activity was further divided by the activity in 28S band and plotted as a relative
mRNA level against age (B). Almost linear decrease was observed. The straight line indicates a
regression line.

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41

3. Results

3.1. mRNA le6el
Total RNAs from 12 periodontal ligaments obtained from people aged 17–61
years were analysed by Northern blotting (Fig. 1A). Two bands were observed as
collagen a1(I) mRNA at 5.8 and 4.8 kb. They corresponded to those observed
before in cultured human ﬁbroblast cells (Chu et al., 1985). The ratio of radioactivity in 5.8 kb band to that in 4.8 kb was :0.25 and did not show any age-dependent
change. The total amount of collagen a1(I) mRNA was estimated as the sum of the
radioactivities in the two bands, and its relative amount to rRNA was calculated by
dividing the summed activity by the radioactivity of ribosomal 28S band. When the
relative amount of collagen a1(I) mRNA was plotted as a function of the donor’s
age, almost linear decline was observed (Fig. 1B). Regression analysis showed the
relationship between age in years (A) and relative mRNA level (R) as R= 1.87−
0.030 ×A (r =0.74). The ﬁtting of the data to an exponential curve gave less
likelihood (r = 0.69).
Similar analysis was done by dot blot hybridization (Fig. 2A). In this experiment,
the total RNA was ﬁxed on the ﬁlter and hybridized to collagen a1(I) cDNA. To
evaluate variations caused by technical problems, a different amount of RNA was
plotted for each sample in duplicates. The radioactivity in each dot was measured
and its ratio to the radioactivity of 28S ribosomal RNA was calculated as a relative
level of collagen a1(I) mRNA. Four values were obtained for each sample. The
variations among them were within reasonable ranges. The averages of them are
plotted as a function of age in Fig. 2B. Age-dependent decline was observed again.
The linear regression analysis showed R= 1.20− 0.016× A (r= 0.76). The slopes of
decline were different between Fig. 1A and Fig. 2B. It could be reﬂecting the fact
that Northern blot analysis monitors only intact length mRNA, while dot blot
analysis detects both intact length mRNA and degraded mRNA as far as its size is
enough to be trapped on the ﬁlter paper. If it is the case, the less steep slope
observed in dot blotting may represent higher fractions of degraded mRNA in aged
than in young tissues. This, of course, needs more direct evidence.

3.2. DNA methylation
Since DNA methylation, especially in the promoter area of each gene, is one of
the factors which regulate gene expression (Baylin et al., 1998), we examined
methylation status of collagen a1(I) gene in the upstream region. The region has
been sequenced before (Jimenez et al., 1994) and has two NaeI sites. NaeI cuts
DNA at a GCCGGC site but not when the cytosine in the middle of recognizing
sequence is methylated (McClelland et al., 1994). Fig. 3 shows the map of collagen
a1(I) promoter region. The locations of two NaeI sites are at − 1705 and − 80.
The status of methylation at these sites can be studied by digestibility of the sites by
NaeI. DNAs of periodontal ligaments collected from 15 people aged 9–76 years
were digested with BamHI, EcoRV and NaeI and analysed by Southern blotting.

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M. Takatsu et al. / Mechanisms of Ageing and De6elopment 110 (1999) 37–48

Fig. 2. Dot blot analysis of collagen a1(I) mRNA. The total RNAs used in Fig. 1 were blotted directly
on a ﬁlter paper and hybridized to 32P-labeled collagen a1(I) cDNA (A). The amount of RNA was 0.02
and 0.1 mg for each sample. The spotting was done in duplicate. After hybridization to collagen a1(I)
cDNA, the same ﬁlter was rehybridized to 28S ribosomal DNA probe. The ratios of radioactivities in
collagen a1(I) signals to those in 28S ribosomal signals for each sample were determined and their
averages were plotted as a function of age (B). Almost linear decline was observed. The straight line
indicates a regression line.

Representative data are shown in Fig. 4A and Fig. 5A. Fig. 4A demonstrates
age-dependent increase in resistance at − 82 position to NaeI digestion indicating
hypermethylation. The degree of DNA methylation was expressed by a ratio of
radioactivity in NaeI resistant band (3.2 kbp) to the sum of radioactivities in
NaeI-sensitive (2.9 kbp) and NaeI-resistant (3.2 kbp) bands. The values (% methylation) were plotted in Fig. 4B as a function of age. The increase was almost linear
and the regression analysis revealed M= −7.40+ 1.10× A (r= 0.89), where M is

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43

a degree of methylation in percentage and A is age in years. The ﬁtting of the data
to an exponential curve gave a slightly less likelihood (r=0.83).
Results of similar analysis on the other NaeI site at − 1705 are shown in Fig. 5.
At this site, the degree of methylation at a young age was higher than that at − 80
site. An age-dependent increase was found and the regression analysis showed a
relationship of M = 36.7 + 0.89 × A (r= 0.89). When similar analyses were applied
to c-MYC and GST-y genes, we observed no age-dependent alteration in DNA
methylation, suggesting that the change in DNA methylation is not ubiquitous
among different genes.

4. Discussion
The present study demonstrates that mRNA level and promoter methylation of
collagen a1(I) gene alter in the aging process of human periodontal ligament. The
change in DNA methylation was found at two loci, − 80 and − 1705. These sites
are located in the regions where regulatory sequences for collagen a1(I) gene
expression have been shown to exist (Ritzenthaler et al., 1991; Jimenez et al., 1994;
McClelland et al., 1994). Since many lines of evidence suggest that methylation of
DNA at regulatory region of genes could suppress gene expression (Graessmann
and Graessmann, 1993; Komura et al., 1995; Baylin et al., 1998), the age-dependent
increase of methylation at these sites could be involved in the decrease of mRNA
level of the gene. In fact, DNA methylation at − 87 of murine collagen a1(I) gene
has been shown to repress gene expression in mouse ﬁbroblasts and embryonic
carcinoma cells in vitro (Rhodes et al., 1994). Lately, deacetylation of histones
located at the region where DNA is methylated has been shown to be involved in
repression of gene transcription by making the chromatin structure inaccessible to
factors needed for transcription (Jones et al., 1998; Nan et al., 1998). It should be
remembered, however, that the age-dependent decrease of mRNA level observed in

Fig. 3. Map of collagen a1(I) gene 5% region and its 5% ﬂanking area. E1 represents exon 1. B, N, E
represent the BamHI, NaeI and EcoRV site, respectively. The probes used for analyses of DNA
methylation are shown by thick horizontal lines. The sizes of DNA bands observed in Southern blot
analyses are shown at the bottom.

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M. Takatsu et al. / Mechanisms of Ageing and De6elopment 110 (1999) 37–48

Fig. 4. Southern blot analysis of DNA methylation at the −80 NaeI site of collagen a1(I) gene.
Genomic DNAs derived from periodontal ligaments of 9 – 64 year-olds were digested with BamHI, NaeI
and EcoRV, run through gel, transferred to a ﬁlter and hybridized to a probe covering exon 1, shown
in Fig. 3. Two bands were observed at 2.9 and 3.2 kbp (A). The upper band represent DNA fragments
which were resistant to NaeI digestion at −80 position. The radioactivities in the two bands were
measured and the ratio of radioactivity in the 3.2 kbp band to the sum of those in the 3.2 and 2.9 kbp
were calculated as the degree of methylation. A similar study was done with 15 samples from people
aged 9–76 years and plotted in (B). An almost linear age-dependent increase was observed. The straight
line shows linear regression.

the present study could result from either a decrease in mRNA synthesis or an
increase in its degradation. Evidence showing a direct correlation between the
increase of DNA methylation and the decrease of mRNA level is still missing.
When the age-related changes in mRNA level and DNA methylation are
compared from a quantitative point of view, the decrease in mRNA level between
20 and 60 years of age was 1/2 (dot blot) or 1/10 (Northern blot), whereas the

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45

decreases in the fraction of unmethylated DNA during the same period was 2/3 at
− 80 and 1/5 at − 1705. The magnitudes of the changes in the two indices look
comparable. But, since the degree of suppressive effect on gene transcription caused
by DNA methylation is not yet known, it is difﬁcult to discuss in detail.
In order to ﬁnd out if the protein level of collagen a1(I) might change with age,
we tried Western blot analysis and immunohistochemical study. But we could not
obtain speciﬁc signals, probably because of the low speciﬁcities of antibodies used.
In the literature, however, collagen protein level and its synthetic rate in periodontal ligament have been suggested to decline with age (Stahl and Tonna 1977;

Fig. 5. Southern blot analysis of DNA methylation at −1705 position NaeI site. Similar experiment as
in Fig. 4 was done using a different probe, which monitors upstream region of collagen a1(I) gene shown
in Fig. 3. Again, age-dependent increase of NaeI-resistant DNA fragments was observed (A). Quantitative expression is shown in (B). The method of calculation was the same as described in Fig. 4. The
degree of methylation increased with age. The straight line shows the result of regression analysis.

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M. Takatsu et al. / Mechanisms of Ageing and De6elopment 110 (1999) 37–48

Moxham and Evans 1995). Since type I collagen is a major component of collagen
ﬁbers in periodontal tissues (Karimbux et al., 1992; Lukinmaa and Waltimo 1992),
it is likely that collagen a1(I) protein level declines with age. But, it should be
proved.
It is well known that each tooth root are established at different ages. Since the
age is similar among individuals (Schour and Massler, 1940), we tried to plot the
changes of mRNA level and the degree of DNA methylation as a function of
period after establishment of each tooth root instead of donor’s age. The results
indicated that the age-dependent decline of mRNA level and the increase of DNA
methylation were similar to those obtained using the ages of individuals (data not
shown).
An age-dependent decline of collagen a1(I) mRNA has been observed in human
skin ﬁbroblast cells (Takeda et al., 1992), but DNA methylation has not yet been
examined. Age-dependent alteration of DNA methylation has been found on many
genes in different tissues (Ono et al., 1993; Choi et al., 1996; Issa et al., 1996; Ahuja
et al., 1998; Baylin et al., 1998). However, it is only the estrogen receptor gene
whose age-dependent increase in DNA methylation has been suggested to be related
to age-related decrease in mRNA level in colon (Issa et al., 1994). Collagen a1(I) in
periodontal ligament is the second example which directly suggests the possible
importance of simultaneous alterations in promoter methylation and gene expression in the aging process. Although reasons for these age-dependent alterations are
only speculative at present (Jones, 1999), further accumulation of this kind of
information would eventually be helpful in understanding the intrinsic causes of
age-dependent degenerative alteration of periodontal ligament. We are now trying
to extend the study to different kinds of genes.

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