J Dent Res 77(9): 1694-1699, September, 1998
cDNA Cloning of S100 Calcium-binding
Proteins from Bovine Periodontal Ligament
and Their Expression in Oral Tissues
W.R. Duartel,2*, S. Kasugai2, T. Iimura3, S. Oida4, K. Takenaga5, K. Ohya2, and I. Ishikawal
Departments of Periodontologyl*, Pharmacology2, Developmental Biology3, and Biochemistry4, Faculty of Dentistry, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan; and Division of Chemotherapy5, Chiba Cancer Center Research Institute, 6-
66-2, Nitona, Chuoh-ku, Chiba 260, Japan; *corresponding author
Abstract. The periodontal ligament (PDL) is a unique tissue
that is crucial for tooth function. However, little is known of
the molecular mechanisms controlling PDL function. To
characterize PDL cells at the molecular level, we constructed
a cDNA library from bovine PDL tissue. We then focused
on the isolation of S100 calcium-binding proteins (CaBPs),
because they mediate Ca2+ signaling and control important
cellular processes such as differentiation and metabolism.
We screened the PDL cDNA library with a mouse S100A4
cDNA, and cloned the bovine cDNAs of two S100 CaBPs
(S1OOA4 and S10OA2). In Northern blotting analysis, the
highest expression of S10OA4 was detected in PDL from
erupted teeth (PDLE). PDL from teeth under eruption
(PDLU) showed a lower expression of S10OA4, and its
expression in gingiva was faintly detectable. S100A4
expression was also high in the pulp tissue followed by the
dental papilla of the tooth germ. S10OA2 expression was
high in PDLE and gingiva. Interestingly, only PDLE
exhibited a high expression of both S100A4 and S100A2.
PDLE also expressed the highest level of r-actin, a target
cytoskeletal protein for S100A4. It is conceivable that the
high expression of S100A4 in PDLE is a result of the
maturation of the PDL and/or a response to mechanical
stress generated by mastication. Since there was a marked
difference of S100A4 expression between PDL and gingiva,
we propose that 5100A4 could be a useful marker for
distinguishing cells from these two tissues.
Key words: S100, calcium-binding proteins, periodontal
ligament, oral tissues, cDNA cloning.
Received March 12, 1997; Last Revision November 24, 1997;
Accepted December 17,1997
1694
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Introduction
The periodontal ligament tissue (PDL) anchors the tooth to
the alveolar bone and acts as a cushion that distributes
multidirectional forces generated by mastication. The PDL
is unique since it can maintain its normal function under
such strong occlusal forces, and is able to maintain its thin
space free of mineralization. Obviously, cells that reside
within the PDL contribute to such uniqueness. Therefore,
the characterization of PDL cells at cellular and molecular
levels could explain such peculiar features of the PDL.
These cells have been extensively characterized in cell
culture, and previous studies have demonstrated that most
PDL cells are morphologically fibroblast-like cells but
express osteoblastic phenotypes, such as high alkaline phos-
phatase activity (Arceo et al., 1991), parathyroid hormone
responsiveness (Nohutcu et al., 1995), production of bone-
like matrix proteins, and formation of mineralized nodules
(Mukai et al., 1993; Ramakrishnan et al., 1995). However, a
specific phenotype marker which distinguishes PDL cells
from other cells has not been reported, and molecular
mechanisms controlling PDL cell function remain unclear.
On the other hand, S100 calcium-binding proteins are
small, acidic proteins which, after binding to Ca2+, interact
with target molecules and control various cellular processes
such as cell cycle progression, differentiation, and cell
metabolism (Schafer and Heizmann, 1996). Interestingly,
Strutz et al. (1995) have isolated one S100 calcium-binding
protein known as S10OA4 as a fibroblast-specific protein and
demonstrated its high gene expression in the periodontal
mesenchyme during early stages of tooth morphogenesis in
an in situ hybridization study. In the present study, to
characterize PDL cells at the molecular level, we constructed
a cDNA library from bovine PDL. This study is the first
report of a cDNA library construction from PDL. We then
cloned the bovine S10OA4 and S10OA2 (another S100 CaBP)
cDNAs, and we observed high expression levels of these
two CaBPs in PDL as compared with other oral tissues.
J Dent Res 77(9) 1998 S100 Calcium-binding Proteins in Oral Tissues 1695

Since there was a marked dif- MM x1 illB 60

ference of S100A4 expression M A Y P
between PDL and gingiva, we
propose that S100A4 could be a 61 120
useful marker for distin-
guishing cells from these two
L E K A L D V M V S T F H KY ns G K G
tissues. 121 - -L-a 180
E K F TiL N K S [ L K E L L T R E L P S
Materials and methods 181 -MC 240
'P T. r- K' T?r1I LIn P.
J T £A 'P n K T.i*1J.M .q L"d
M* T.U Li 'XJ '-
Isolation of oral tissues,
total RNA extraction, 241 300
and mRNA purification Y

F Q mY C V F L S C I A M
Fresh bovine oral tissues were
K UD N E V

isolated from 10 mandibles of 301 M 360

1.5-year-old cattle obtained in a M C N E F F E G F P D K Q P R K K
local slaughterhouse. The animal
experimental protocol used in 361 CICAx?JXoG 420
this study is in agreement with 421 CA1CXrIACX'TUGGC TYIMAAGAA ItCCAGAO~IGIGCACCATCIGAACAAGI'ICA
AGTTCI 480 GCACATIU
the standards of the Tokyo 481 TGAAAGCrAAAAAAJ ,AAAAAAAA 509
Medical and Dental University.
The following tissues were used
Figure 1. Nucleotide and deduced amino acid sequence of the bovine S10OA4 CaBP cDNA. The 303-
in this study: periodontal nucleotide coding region, which encodes a 101-amino-acid protein, is underlined. Upper bars indicate
ligaments from erupted teeth the ATG starting codon and the TGA termination codon. The polyadenylation signal (AATAAA) is
(PDLE), periodontal ligaments double-underlined. Plain and bold boxes represent the first and second Ca2+ binding loop-structures,
from teeth under eruption respectively.
(PDLU), gingivae, dental pulps,
dental papillae, and dental
follicles. Fully erupted permane nt central incisors were the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA, USA)
extracted, and PDLE was scraped fr4om the middle third of the protocol. A bovine PDL X ZAP cDNA library was constructed
root with surgical blades; contamiination with surrounding by means of the instructions in the ZAP-cDNA Gigapack II
tissues was carefully avoided. PDLU was also scraped from the Gold cloning kit (Stratagene, La Jolla, CA, USA). For the
middle third of the root of each pern manent central incisor that screening procedure, approximately 5 x i05 independent clones
had not visibly reached the occlusal level when compared with were transferred to Colony/Plaque Screen® nylon membranes
adjacent teeth, and that displayed afpproximately two-thirds of (Du Pont, Boston, MA, USA), following the manufacturer's
the roots formed. Gingivae were lremoved from the buccal instructions. One-hour pre-hybridization was carried out at
anterior regions of mandibles with suirgical blades. Dental pulps 65°C in a buffer containing 6 x SSC, 1 x Denhardt's, and 0.5%
were removed from erupted central incisors through the apical SDS. A 0.5-kb S100A4 mouse cDNA (Goto et al., 1987; kindly
foramen. Tooth germs of permanLent lateral incisors were supplied by Dr. H. Endo, Department of Biochemistry, Medical
removed from mandibles, and then dental papillae and dental Institute of Bioregulation, Kyushu University, Fukuoka, Japan)
follicles were carefully separated. Tc obtain sufficient amounts was labeled with [(X 32p] dCTP (Du Pont, Boston, MA, USA) by
of total RNA for the subsequent sttudies, we pooled corres- random priming (Ready to Go Kit, Pharmacia Biotech, Uppsala,
ponding tissues and extracted and jpurified total RNA by the Sweden) and used as a probe. Overnight hybridization was
guanidinium thiocyanate-phenol -chloroform method, as carried out at 65°C in a buffer containing 1 M NaCl, 1 x
described by Chomczynski and Saccihi (1987). Immediately after Denhardt's, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% SDS,
removal, all tissues were placed in se?parate tubes containing an 50 pg/mL yeast tRNA, 50 iig/mL denatured salmon sperm
excess volume of guanidinium thio cyanate solution (solution DNA, and 106 cpm/mL of the [(x-32p] dCTP-labeled mouse
D). We observed that the use of a L arge amount of solution D S10OA4 cDNA probe. The membranes were washed twice at
ar
yielded a higher-quality RNA. Aft( the spectrophotometric 65°C in 0.2 x SSC, 0.1% SDS for 30 min, and exposed to x-ray
measurement of the RNA, RNA qual ity was checked by electro- films (X-OMAT, Kodak, Rochester, NY, USA) at -80°C with two
phoresis in denaturing agarose gel. ]PDLE mRNA was purified intens'ifying screens. Positively hybridized recombinant phage
with an oligo(dT)-latex beads system (Oligotex® dT3O<SUPER>, clones were selected for further analyses.
Takara, Tokyo, Japan), accordinig to the manufacturer's
instructions.
cDNA sequencing and homology search
cDNA synthesis, cDNA library co:nstruction, Positive cDNA clones were sequenced by the dideoxynucleo-
and screening tide chain termination method (Sanger et al., 1977). GENETYX-
First- and second-strand cDNAs werre synthesized according to SV-DB 34.1 software was used for the sequence analysis.
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1696 Dutarte et a]. j Dciff Rcs 77(9) 1998
containing de-ionized formamide and formaldehyde. The
,

;4$.'
,
samples were immediately cooled on ice for 5 min and
electrophoresed on a 1.2%" (w/v) agarose gel containing 17"`
formalin, stained with ethidium bromide, and photographed.
Total RNAs were transferred onto nylon membranes (Zeta-
Probe"", BIO-RAD, Hercules, CA, USA) by capillary blotting
with 10 x SSC and fixed by being baked at 80°C for 30 min. The
membranes were then pre-hybridized overnight at 42°C in
hybridization buffer containing 50%, formamide, 0.12 M
Na2HP04, pH 4; 0.25 M NaCL; 1 mM EDTA; and 7'0,o SDS.
Hybridizations proceeded overnight at 42°C in fresh
S10OA4-"- S; hybridization buffer containing a radiolabeled probe. Clones 371
and 381-corresponding to the full sequence of bovine S1OOA4
and SI OOA2 cDNAs, respectively-were used as probes in two
distinct hybridizations. In addition, a BamHI fragment of the
2.1-kb human ]-actin cDNA clone pHFjA-1 (Gunning ct al.,
1983) was used. All probes were labeled with [a-32P] dCTP by
random priming (Ready to Go Kit, Pharmacia Biotech, Uppsala,
Sweden). The membranes were briefly rinsed in 2 x SSC and
sequentially washed for 15 min at room temperatuLre in 2 x
SSC/0.100, SDS, and 0.5 x SSC/O.I',, SDS. A final washing at
65°C in 0.1 x SSC/0.1°/, SDS was performed for 15 min for
removal of non-specifically-bound probe. The membranes were
exposed to x-ray films (X-OMAT, Kodak, Rochester, NY, USA)
S1OOA2-'o 0 at -80°C with two intensifying screens. Autoradiographs were
scanned with an imaging densitometer (BIO-RAD GS-670,
Richmond, CA), and the signal intensities were evaluated and
compared by means of Molecular Analyst 2.0.1 Software on a
Macintosh computer. All signals were normalized to the
ethidium bromide staining of the 28S ribosomal RNA.

Results
f-actin-. Isolation of S100A4 and S100A2 CaBPs
from a bovine PDL cDNA library
We screened 5 x 105 independent clones in a bovine PDL
cDNA library with a mouse S100A4 cDNA probe. We
picked up 25 positive clones and selected 12 for further
analyses. A search in the database revealed that the deduced
Ribosomal amino acid sequences of two clones, named 361 and 371,
RNA were 100% homologous to the bovine amino acid sequLence
of a member of the S100 CaBPs family known as CAPL-a
synonym for S1OOA4 (Polans et al., 1994). The bovine S10OA4
nucleotide sequence has not been reported. The other three
Figure 2. Northern blotting analysis of the expression level and clones, named 381, 391, and 431, were 100'S, homologous to a
distribution of S10OA4, SIOOA2, and f-actin mRNA in bovine oral bovine member of the S100 CaBPs family known as S1OOL-
tissues. Total RNA was extracted from bovine oral tissues; 10-pjg a synonym for S100A2 (Glenney et al., 1989)-at both amino
aliquots of total RNA were fractionated on 1.2% agarose gels, acid and cDNA levels. The bovine S100A4 cDNA contains
transferred to nylon membranes, and hybridized with cDNA 509 nucleotides, a 21-nucleotide poly (A) tail, and a single
probes for bovine S1OOA4, S100A2, and human f-actin. The
ethidium-bromide staining of the 28S/18S ribosomal RNAs is long open reading frame that extends from nucleotide
shown in the lower panel. PDLE, PDL from erupted teeth; PDLU, position 1 to position 351. With the first ATG nucleotide
PDL from teeth under eruption; GT, gingiva; PT, pulp tissue; DP, sequence at position 49 as the starting codon for translation,
dental papilla; and DF, dental follicle. the bovine S1OOA4 cDNA contains a 48-nucleotide 5'-
untranslated region, a 303-nucleotide sequence encoding a
101-amino acid protein, and a 3-untranslated region of 137
Northern blotting nucleotides. The AATAAA polyadenylation signal in the 3'-
Aliquots of PDLE, PDLU, gingiva, dental pulp, dental papilla, untranslated region is located 20 nucleotides upstream from
and dental follicle containing 10 pg of total RNA were the first adenine of the poly (A) tail (Fig. 1). Amino acids of
659C from
denatured by being heated toDownloaded 5 min inatloading
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j Dcnt Res 77(9) 1998 SIO Calciunb-bindinzg Proteins in Oral TissnIcs 1 697

double-boxed in Fig. 1. The bovine S10OA4 and S10OA2

(Glenney et al., 1989) cDNAs exhibited 70.7(X1 homology in 1(
their nucleotide sequences. The bovine S100A4 cDNA was
80.7'% homologous to the corresponding mouse S10OA4
cDNA (Goto et al., 1987), whereas the bovine S10OA2 was
62.6%0, homologous to the mouse SI0OA4 cDNA.

Expression of S10OA4, S100A2, and 3-actin mRNAs

in oral tissues
We conducted Northern blot analysis to determine the
patterns of expression of S10OA4 and S10OA2 mRNAs as
well as the expression of the cytoskeletal protein 3-actin
mRNA in various oral tissues. Similar results were obtained 1
from two sets of separate experiments. Hybridizations with
bovine S10OA4 and S10OA2 probes yielded bands of
approximately 0.5 kb. The highest expression of S10OA4
mRNA was detected in PDLE. S10OA4 mRNA was also
detected in dental pulp, followed by tissues from early
stages of tooth development, such as PDLU, dental papilla,
and dental follicle (Fig. 2). The expression of S10OA4 mRNA
in gingiva was slightly detectable (Fig. 2). Interestingly, the
expression of S1OOA4 mRNA in PDLE was much higher
than that in PDLU. On the other hand, S10OA2 mRNA
expression exhibited a pattern different from that of S10OA4 3-actin
(Fig. 2). S10OA2 mRNA was highly expressed in both PDLE 100-
and gingiva; however, in other tissues, the expression was
either very low or absent. The only tissue expressing high
levels of both S10OA4 and S10OA2 mRNAs was PDLE.
Hybridization with the 3-actin probe showed that the 50
highest expression of 3-actin mRNA was observed in PDLE
as compared with other tissues. The relative values for
signal intensity of all mRNAs after normalization to the
ethidium bromide staining of the 28S ribosomal RNA are
shown in Fig. 3. 0-
DF
I DP PT
PDLE PDLU
I GT
Figure 3. Relative values for signal intensity of S100A4, S100A2,
Discussion and 3-actin mRNAs after normalization to the ethidium bromide
To date, little is known of the gene expression of S100 staining of the 28S ribosomal RNA. Scanning densitometry of two
sets of separate experiments was performed, and values are
calcium-binding proteins and their role in oral tissues. Strutz represented by the black and grey bars. All values are shown as a
et al. (1995) have briefly mentioned the high expression of percentage of the maximal level reached.
S10OA4 mRNA, one of the S100 CaBPs, in "the periodontal
mesenchyme" during early stages of mouse tooth
morphogenesis, indicating the implication of S10OA4 in
tooth development. In the present study, we de-monstrated also increase intracellular Ca2+ and exhibit changes in actin
that the expression of S10OA4 mRNA was higher in PDLE filament polymerization as the most rapid response to
than in PDLU. PDLE represents a PDL at a later mechanical stress in vitra (Pender and McCulloch, 1991;
developmental stage which is more exposed to occlusal Nakago-Matsuo et al., 1996). Thus, it is likely that PDL cells
forces as compared with the PDLU. The maturation of the possess a mechanism coupling Ca2+ and cytoskeletal
PDL is achieved after tooth eruption and establishment of components. S10OA4 could participate in such a mechanism
full occlusal function (Moxham and Grant, 1995). Therefore, because in vitro experiments have demonstrated the inter-
the difference of S10OA4 mRNA expression between PDLE action of S10OA4 with several cytoskeletal or cytoskeletal-
and PDLU could be a result of the maturation of the PDL related proteins, such as actin (Watanabe et al., 1993), non-
and/or a response of PDL cells to mechanical stress. muscle myosin (Kriejevska et al., 1994), and non-muscle
Mechanical stress activates mechanosensitive ion tropomyosin (Takenaga et al., 1994), in a calcium-dependent
channels located on the cellular membrane (Davidson et al., manner in other cell types. Furthermore, PDL cells are
1990; Davidson, 1993), which is followed by an increase in constantly under strain, which requires a highly active and
intracellular Ca2" levels (Glogauer et al., 1995). Actin organized cytoskeleton for their normal function. This is
filaments are also involved in mechanotransduction (Wang supported by the fact that PDL fibroblasts express x-
et al., 1993; Glogauer et al., 1997). Cells of the periodontium smooth-muscle actin and smooth-muscle myosin
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1698 Duarte et al.
(Giannapoulou and Cimasoni, 1996), and that PDL cells
contain a three-fold-higher amount of actin than gingival
fibroblasts (Pender and McCulloch, 1991). We also showed
that 3-actin mRNA was highly expressed in PDLE, coinci-
ding with the high expression of S100A4 mRNA. The
variation of I-actin mRNA expression observed in our
results is supported by other reports in which the expression
level of 1-actin mRNA is inconstant and depends on the
stage of cell differentiation, stage of tissue development, and
origins of the tissues (Spanakis, 1993; Biragyn et al., 1994;
Carlyle et al., 1996; Nagahara and Matsuda, 1996).
A relatively high expression of S100A4 mRNA was also
seen in the pulp tissue (PT). Unlike PDL, PT is not exposed
to forces derived from mastication. Therefore, we cannot
speculate that the high expression of S100A4 mRNA is a
response of pulp cells to mechanical stress. However,
similarly to PDLU and PDLE, S100A4 mRNA was expressed
in dental papilla (DP) and PT in the same pattem, i.e., higher
expression at a later developmental stage (PT) and lower
expression in a younger tissue (DP). Thus, it is likely that
S100A4 is involved in the developmental process of the pulp
tissue.
Another member of the S100 CaBPs family, presently
known as S100A2, was also isolated. S100A2 is localized
both in the cytoplasm and in the nucleus (Glenney et al.,
1989), but its role is still unknown. It has been reported that
the S100A2 gene expression pattern is opposite that of
S100A4 (Pedrocchi et al., 1994). In our experiments, the
pattern of expression of S100A2 mRNA also differed from
that of S100A4. However, only PDLE highly expressed both
S100A4 and S100A2 mRNAs.
Notably, the highest expression of S100A4 mRNA was
detected in PDLE, while the lowest was expressed in
gingiva. Several markers to distinguish PDL cells from
gingival cells have been suggested: mineralized nodule
formation in culture (Mukai et al., 1993), high alkaline
phosphatase activity (Arceo et al., 1991), PTH hormone
responsiveness (Nohutcu et al., 1995), and production of
bone-like matrix proteins (Ramakrishnan et al., 1995), which
are highly expressed in PDL cells but not in gingival cells.
Most of these data, however, derive from cultured cells and
should be interpreted with caution, because significant
differences were observed when freshly extracted PDL was
compared with cultured samples (Nohutcu et al., 1996).
Similarly, in the present study, we used freshly extracted
tissues and showed a clear difference in the expression of
S10OA4 mRNA between PDL and gingiva. Conclusively, we
propose that S100A4 could be a useful marker to distinguish
between cells from these two tissues, although the exact
function of S100A4 in oral tissues is not clear.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific
Research (No. 08457486) from the Ministry of Education,
Japan, and a Research Grant for the Future (No. 96100205)
from the Japanese Society for the Promotion of Science.
The bovine S10OA4 cDNA nucleotide sequence is
registered in the DDBJ/EMBL/GenBank databases.
Accession number: D89056.
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f Dent Res 77(9) 1998
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