Basic Research—Biology
Differentially Expressed Protein Profile of Human Dental
Pulp Cells in the Early Process of Odontoblast-like
Differentiation In Vitro
Xi Wei, MDS, PhD,* Liping Wu, MS,* Junqi Ling, PhD,* Lu Liu, MS,* Shaojun Liu, MS,†
Wei Liu, PhD,† Mingtao Li, PhD,† and Yin Xiao, PhD‡
Abstract
Dental pulp cells (DPCs) are capable of differentiating
into odontoblasts that secrete reparative dentin after
pulp injury. The molecular mechanisms governing re-
parative dentinogenesis are yet to be fully understood.
Here we investigated the differential protein profile of
human DPCs undergoing odontogenic induction for 7
days. Using two-dimensional differential gel electrophore-
sis coupled with matrix-assisted laser adsorption ioniza-
tion time of flight mass spectrometry, 23 protein spots
related to the early odontogenic differentiation were
identified. These proteins included cytoskeleton pro-
teins, nuclear proteins, cell membrane-bound mole-
cules, proteins involved in matrix synthesis, and met-
abolic enzymes. The expression of four identified
proteins, which were heteronuclear ribonuclear pro-
teins C, annexin VI, collagen type VI, and matrilin-2,
was confirmed by Western blot and real-time real-
time polymerase chain reaction analyses. This study
generated a proteome reference map during odon-
toblast-like differentiation of human DPCs, which
will be valuable to better understand the underlying
molecular mechanisms in odontoblast-like differenti-
ation. (J Endod 2008;34:1077–1084)
Key Words
Dental pulp cells, differentiation, odontoblasts, pro-
teome, two-dimensional differential gel electrophoresis
From the *Department of Operative Dentistry and End-
odontics, Guanghua School of Stomatology, Sun Yat-sen Uni-
versity, Guangzhou, Guangdong, China; †Proteomic Labora-
tory, Zhongshan Medical College, Sun Yat-sen University,
Guangzhou, Guangdong, China; and ‡Institute of Health and
Biomedical Innovation, Queensland University of Technology,
Brisbane, Queensland, Australia.
Supported by the National Nature Science Grant (no.
30672318) and Nature Science Grant of Guangdong province
(no. 06021264), China.
Address requests for reprints to Dr Junqi Ling, PhD, De-
partment of Operative Dentistry and Endodontics, Guanghua
School of Stomatology, Sun Yat-sen University, Guangzhou
510055, China. E-mail address: lingjq@mail.sysu.edu.cn.
0099-2399/$0 - see front matter
Copyright © 2008 American Association of Endodontists.
doi:10.1016/j.joen.2008.06.014
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A fter tissue injury, the dentin-pulp complex may need to undergo complete
regeneration, including the differentiation of various cell types and the induc-
tion of new proteins. Initially, during pulp wound healing, necrosis of pulp tissue
including odontoblasts is induced followed by reparative dentinogenesis, the result
of successive and interrelated processes, including chemotaxis, proliferation, an-
giogenesis, extracellular matrix remodeling, and cell differentiation. Dentin regen-
eration is mediated by the differentiation of a new generation of odontoblasts from
a precursor population of dental pulp stem cells during the process of reparative
dentinogenesis (1). With conventional molecular biological approaches, some
signaling pathways, gene expression, and protein activities have been identified in
the process of dentinogenesis (2, 3). The molecular mechanisms controlling odon-
toblast differentiation are yet to be fully understood. The identification of the
regulatory factors governing these processes will enhance our understanding and
open up potential treatment strategies for dental pulp regeneration.
Several new methods for detection of protein expression have been developed.
Proteomic approaches using two-dimensional gel electrophoresis (2-DE), coupled
with protein identification by mass spectrometry (MS) and bioinformatics, offer a
high-throughput technology to study the differential protein expression profiles
relating to particular pathophysiological conditions. The recently introduced two-
dimensional differential gel electrophoresis (2D-DIGE) technology is thought to be
a more reliable tool for exact quantitative analysis of differentially expressed pro-
teins. 2D-DIGE allows three independent samples labeled with different fluorescent
dyes to be analyzed on one gel simultaneously and viewed individually using the different
fluorescent properties of Cy2 (3-[4-carboxymethyl] phenylmethyl-3-ethyloxacarbocya-
nine halide N-hydroxysuccinimidyl ester), Cy3 (1-[5-carboxypentyl]-1-propylindocar-
bocyanine halide N-hydroxysuccinimidyl ester), and Cy5 (1-[5-carboxypentyl]-1-
methylindodicarbocyanine halide N-hydroxysuccinimidyl ester). Compared with other
staining methods, this method circumvents some of the reproducibility problems
associated with the gel-to-gel variation of 2-DE and provides more accurate quan-
titative information (4). Proteins of interest can subsequently be identified by
matrix-assisted laser adsorption ionization time of flight mass spectrometry
(MALDI-TOF-MS).
Proteomic techniques are now widely accepted as an important tool for find-
ing potential biomarkers and studying the mechanisms involved in cell differenti-
ation (5, 6). However, proteomic studies on odontoblast-like differentiation have
yet to be performed. In a previous study, we have isolated human dental pulp cells
(DPCs) with stem-cell characteristics and shown their odontoblast-like differenti-
ation (3). In this study, we used 2D-DIGE and MS-based proteomic approaches to
investigate the differential protein expression in the early process of odontoblast-
like differentiation of human DPCs. Twenty-three proteins were identified by
MALDI-TOF-MS, which were either up- or downregulated during odontogenic dif-
ferentiation. These data should provide some further insight into the molecular
mechanisms of odontoblast-like differentiation in reparative dentinogenesis.
Protein Profile of Human DPCs 1077
Basic Research—Biology
TABLE 1. Experimental Design of DIGE Gels
Gel No. 3-(4-carboxymethyl) Phenylmethyl-3-
Ethyloxacarbocyanine Halide
N-hydroxysuccinimidyl Ester (Cy2)
1 Internal standard
2 Internal standard
3 Internal standard
4 Internal standard
DIGE, differential gel electrophoresis.
Materials and Methods
Cell Culture and Induced Odontogenic Differentiation
Eight healthy human third molars were extracted from four pa-
tients (19 –25 years of age) undergoing orthodontic treatment in the
Department of Orthodontics, Sun Yat-sen University Dental Hospital,
after obtaining written informed consent from each subject, and the
protocols were approved by the University Ethic Committee.
DPCs were isolated and subjected to odontogenic induction as
previously reported (3). Surfaces of the extracted third molars were
cleaned with 70% alcohol. Teeth were split using tooth forceps after a
horizontal groove was cut around the cementoenamel junction. The
dental pulp tissue was removed from the crown and root using dental
probe and barbed broach. Minced pulp tissue was digested in a solution
of 3 mg/mL type I collagenase (Worthington Biochem, Freehold, NJ)
and 4 mg/mL dispase (GIBCO-BRL Life Technologies, Breda, The Neth-
erlands) at 37°C for 30 to 60 minutes. Once digested, the solution was
filtered onto a 70-␮m strainer (Becton Dickinson, Sunnyvale, CA). The
released cells were plated in 6-well plates containing alpha modified
Eagle’s medium (␣-MEM, GIBCO-BRL Life Technologies) supple-
mented with 20% fetal bovine serum (FBS, GIBCO-BRL), 100 U/mL
penicillin, and 100ug/mL streptomycin (Sigma, St Louis, MO) and then
incubated at 37°C in 5% CO2. DPCs at passage 3 were cultured in
␣-MEM with 15% FBS until they reached 70% to 80% confluence. Cells
were then induced in odontogenic medium consisting of 10 mmol/L
␤-glycerophosphate, 0.2 mmol/L ascorbate-2-phosphate, and 100
nmol/L dexamethasone (Sigma) in ␣-MEM with 15% FBS for 7 days.
Control samples were DPCs grown in ␣-MEM with 15% FBS and har-
vested at 80% confluence.
Protein Extraction
Both control and induced DPCs were incubated in serum-free
medium for 24 hours before harvesting after which the medium was
removed from the flasks and the cells washed three times with ice-cold
Tris-buffered sucrose (10 mmol/L Tris-base, 250 mmol/L sucrose, pH
7.4). The cells were carefully scraped off and incubated on ice for 10
minutes in 200 ␮L SDS lysis buffer containing 7 mol/L urea, 2 mol/L
thiourea, 4% CHAPS (GE Healthcare, Bucks, UK), and 30 mmol/L Tris.
The cellular lysates were sonicated on ice and the nuclear fraction
removed by centrifugation at 20,000g for 30 minutes at 4oC. The protein
samples were purified by using a 2-D Clean-Up Kit (GE Healthcare) and
protein concentration measured with a 2-D Quant kit (GE Healthcare)
according to the manufacturer’s instructions.
Cy Dye Labeling
Before running the 2-DE experiments, the protein extracts were
labeled with fluorescent cyanine dyes developed for 2D-DIGE technol-
ogy (GE Healthcare) following the manufacturer’s instructions. The
internal standard was prepared by combining equal amounts of each of
the eight test samples. A total of four DIGE gels were performed to
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Fluorescent CyDye Labeling
1-(5-carboxypentyl)-1- 1-(5-carboxypentyl)-l-
Propylindocarbocyanine Halide Methylindodicarbocyanine Halide
N-hydroxysuccinimidyl Ester (Cy3) N-hydroxysuccinimidyl Ester (Cy5)
Control culture 1 Induced culture1
Control culture 2 Induced culture2
Induced culture3 Control culture 3
Induced culture4 Control culture 4
analyze control or induced samples. Fifty milligrams of protein from
control or induced cultures were labeled with Cy3 or Cy5, and 50 mg of
the internal standard mixture was labeled with Cy2 (Table 1) followed
by incubation on ice for 30 minutes in darkness. The reactions were
quenched with the addition of 1 ␮L 10 mmol/L lysine for 10 minutes on
ice in darkness, after which the Cy3- and Cy5-labeled samples and
Cy2-labeled internal standard were all pooled before 2D-DIGE analysis.
2D-DIGE and 2-DE
Immobilized dry strips (pH 4-7 NL, 24 cm, GE Healthcare) were
rehydrated for 12 hours in 450 ␮L rehydration buffer (7 mol/L urea, 2
mol/L thiourea, 4% w/v CHAPS, 1% v/v IPG buffer [pH 4-7]) using an
Ettan IPGphor IEF system (GE Healthcare). Once rehydrated, samples
were focused at 500 V for 1 hour, 1,000 V for 1 hour, and at 8,000 V for
a total of 80,000 Vh at 20oC. The strips were incubated in equilibration
buffer I (6 mol/L urea, 30% glycerol, 2% SDS, 50 mmol/L Tris-HCl [pH
8.8], 1% DTT) for 15 minutes at room temperature and then soaked in
equilibration buffer II (6 mol/L urea, 30% glycerol, 2% SDS, 50 mmol/L
Tris-HCl [pH 8.8], 2.5% iodoacetamide) for an additional 15 minutes.
The equilibrated strips were then embedded in 0.5 w/v agarose on top
of 12.5% acrylamide slab gels. Second-dimension separations were
performed on an Ettan DALT six electrophoresis system (GE Health-
care). The proteins were initially separated at 15oC at 2 W/gel for 50
minutes, after which the power was increased to 17 W/gel until the dye
front reached the bottom of the gel. The gels were rinsed with Milli-Q
water before the Cy2, Cy3, and Cy5 components of each gel were indi-
vidually visualized by using a Typhoon Variable Mode Imager 9400 (GE
Healthcare).
Two preparative gels, each containing 500 mg of unlabeled inter-
nal standard mixture proteins extracted from eight samples (four con-
trol and four induced samples) were also analyzed. After 2-DE, two
preparative gels were stained with Deep Purple Total Protein Stain
(RPN6306, GE Healthcare) according to the manufacturer’s instruction
and scanned by the Typhoon Variable Mode Imager 9400. The emission
filters were Blue 488 nm (Cy2), Green 532 nm (Cy3), Red 633 nm
(Cy5), and 532 nm (Deep Purple) with the resolution of 100 ␮m.
Image Analysis
The 2-D image from each induced sample gel was compared with
that of the control sample gel by means of the internal standard sample
gel. Protein-expression analysis was performed for each of the four gels
in parallel with the differential in-gel analysis (DIA) module of the
DeCyder software 6.0 (GE Healthcare) using a value of 3,000 as the
initial estimate of the number of protein spots present. The intensities of
individual protein spots within the proteomes of control and induced
samples were compared. These DIA analyses were collated into a single
analysis using the DeCyder biological variation analysis (BVA) module,
and final values for the expression ratio of specific protein spots be-
tween control and induced samples were determined. A total of 2,459 to
JOE — Volume 34, Number 9, September 2008

2,775 protein spots were analyzed across all cell samples. A Student t
test was used to calculate significant differences in the relative abun-
dance of individual protein spot features between control and induced
samples. Protein spots with abundance ratios larger than ⫹1.5 or
smaller than ⫺1.5 were determined to be differentially expressed and
added to a list of proteins of interest.
In-gel Digestion
Spot map images generated from Deep Purple post-stained prepara-
tive gels were matched to the analytical 2D-DIGE gels. Picking selected
protein spots from the preparative gels, in-gel digestion, peptides extraction,
preparation of the samples for MS, and spotting on target slides were carried
out automatically with an Ettan spot handling workstation (GE Healthcare).
Samples were prepared for MS by reconstituting the dried peptides in 3 ␮L
of matrix solution (␣-cyano-4-hydroxycinnaminic acid [CHCA] saturated
solution in 50% acetonitrile [ACN] containing 0.3% trifluoroacetic acid
[TFA]). Briefly, each selected gel plug was rinsed by three wash cycles in
which 150 ␮L of 1:1 methanol/water containing 50 mmol/L ammonium
bicarbonate was applied, left for 15 minutes, and then removed. The plugs
were dehydrated in 75% ACN for 20 minutes; the solution was removed and
placed into a heated, air-circulating drying module and dried for 20 minutes
at 40oC. The digestion solution (10 ␮L, containing 20 ng/␮L sequencing
grade trypsin from Promega (San Luis Obispo, CA) in 20 mmol/L ammo-
nium bicarbonate) was then added to the dried plugs and maintained at
37oC for 2 hours; 80 ␮L of extraction solution (50% ACN containing 0.1%
TFA) was then added and left to stand for 20 minutes. The extract solutions
were transferred to a 96-well plate, and the extraction was repeated. The
plate containing the pooled extracts was then evaporated to dryness. Mixed
solution (0.3 ␮L, CHCA saturated solution in 50% ACN containing 0.3%
TFA) was then spotted on the target for MALDI-TOF-MS identification.
MALDI-TOF-MS
Protein identification by peptide mass fingerprint (PMF) was per-
formed on an Ettan MALDI-TOF mass spectrometer (GE Healthcare)
operating in the reflectron mode. Internal calibration was performed by
using the trypsin autodigestion peaks at m/z 842.509 and 2211.104.
Mass spectra were obtained over the m/z range of 700 to 3,000 Da, and
each spectrum was produced by accumulating data from 200 consecutive
laser shots. The results from PMF were used to search the human NCBI nr
protein database using the following Profound search engine: http://
www.matrixscience.com/cgi/search_form.pl?FORMVER⫽2&SEARCH⫽
PMF, (GE Healthcare), which compared the experimentally determined
tryptic peptide masses with theoretical peptide masses calculated for
proteins contained in these databases. Search parameters are shown as
follows: type of search, peptide mass fingerprint; enzyme, trypsin; fixed
modification, carbamidomethylation (Cys); variable modifications, ox-
idation (Met); mass values, monoisotopic; peptide charge state, 1⫹;
maximum missed cleavages, 1; and peptide mass tolerance, 100 ppm.
Basic Research—Biology
Western Blotting
Control and induced DPCs (days 7, 14, and 21) were harvested in
sodium dodecyl sulfate (SDS) lysis buffer, sonicated, and centrifuged at
12,000g for 5 minutes. The total protein in cell extract was measured by
using a Bio-Rad Coomassie Blue protein assay (Bio-Rad Laboratories,
Richmond, CA). Twenty micrograms of protein were separated by so-
dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to a nitrocellulose membrane using 100 mA for 3
hours. The membranes were blocked in 5% low-fat milk at room
temperature for 1 hour, rinsed, and incubated with individual pri-
mary antibodies of rabbit antihuman annexin VI (Lab Vision Corp,
Fremont, CA), mouse antihuman heteronuclear ribonuclear pro-
teins C (hnRNP C), rabbit antihuman collagen type VI, rabbit anti-
human matrilin-2, or mouse antihuman ␤-actin (Santa Cruz Biotech-
nology, Santa Cruz, CA), at a dilution of 1:1,000, at 4oC overnight. After
washing, the membrane was incubated with the HRP-conjugated sec-
ondary antibodies (Santa Cruz, 1:5000 dilution) at room temperature
for 1 hour. Immunoreactive proteins were then visualized by incubating
membranes with electrogenerated chemiluminescence plus detection
agents (GE Healthcare).
Quantitative Real-time Polymerase Chain Reaction
Total RNA was harvested from the control and induced DPCs (days
7, 14, and 21) using the RNeasy Mini Kit (Qiagen, Valencia, CA). Com-
plementary DNA was synthesized from 1 ␮g total RNA using a Omnis-
cript RT kit (Qiagen) and RNase-free DNase Set (Qiagen) in a total
volume of 20 ␮L according to manufacturer’s instructions; 5 ␮L of
reaction mixture was incubated with the PCR master mix containing
double-stranded DNA dye SYBR Green I (Qiagen) in a total volume of 50
␮L. Primers used for the quantitative PCR are listed in Table 2. All
reactions were run with a hot start preincubation step of 3 minutes at
93°C, followed by 40 cycles of 1 minute at 93°C, 1 minute at 55°C, and
1 minute at 72°C. Messenger RNA expression of genes of interest were
normalized against the expression of ␤-actin and designated as a ex-
pression coefficient in the present study. One-way analysis of variance
for repeated measures and Bonferroni test were performed to analyze
the messenger RNA (mRNA) expressions between induced and control
cultures; p ⬍ 0.05 was considered significant. The data were analyzed by
using the SPSS software package (version 13.0; SPSS Inc, Chicago, IL).
Results
2D-DIGE Analysis of Differential Protein Expression
2D-DIGE was used to compare the protein expression profiles on
day 7 between control and induced DPCs (Fig. 1). The data were ana-
lyzed on the DeCyder image analysis software. More than 2,100 protein
spots were detected across all four gels. Twenty-four protein spots more
than 1.5-fold upregulated and 22 protein spots more than 1.5-fold

TABLE 2. Primer Sequences Used in Quantitative Real-Time RT-PCR

Gene Primers Length
hnRNPC Forward: 5=-ATTGTGGGCTGCTCTGTTCAT-3= 67 bp
Reverse: 5=-CCCGGGCATTTCTCTCATTA-3=
Annexin VI Forward: 5=-AGCAAGCAGCATCTTCGGTT-3= 76 bp
Reverse: 5=-CTCGGATGCTGGCTTCAATC-3=
Collagen VI(␣1) Forward: 5=-CGTGGACATGATCAAAAATAACG-3= 51 bp
Reverse: 5=-CATTCGAAGGAGCAGCACACT-3=
Matrilin-2 Forward: 5=-TCCTTGTGGCCAATTTCAGC-3= 68 bp
Reverse: 5=-GGCCGTGCACAACTTCTTCT-3=
␤-actin Forward: 5=-GCGCGGCTACAGCTTCA-3= 59 bp
Reverse: 5=-TCTCCTTAATGTCACGCACGAT-3=
RT-PCR, reverse transcriptase polymerase chain reaction.

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Basic Research—Biology
Figure 1. Proteomic profiling of DPCs on day 7 of odontogenic induction as
revealed by 2D-DIGE analysis. Protein extracted from control and induced DPCs
were differentially labeled with Cy3 and Cy 5, respectively. An internal standard
comprising proteins from a combination of control and induced DPCs was
labeled with Cy2. The pooled protein samples were loaded on IPG strips and
subjected to isoelectrofocusing before second-dimension electrophoresis in
12.5% acrylamide gels. The pH range was 4 to 7, and the molecular mass
separation was from 14 to 220 kDa. Protein spots with abundance ratios larger
than ⫹1.5 or smaller than ⫺1.5 were determined to be differentially expressed
and subjected to MALDI-TOF-MS and database searching. The identified pro-
teins are indicated with arrows and the numbers corresponding to the spot ID
in Table 3 and Figure 2.
downregulated were identified in the induced DPC samples compared
with controls (p ⬍ 0.05) (Table 3). A partial view of four protein spots
of induced and control groups of the 2-D gel and three-dimensional
images are shown in Figure 2.
MALDI-TOF-MS Identification
In order to identify the 46 spots flagged by the image software,
separate preparative gels containing 500 mg of each extract were ran
and post stained with Deep Purple. The protein spots were matched to
the CyDye-labeled images by the DeCyder software and then excised
from the gel and trypsin digested. Peptide mass maps were obtained
from MALDI-TOF PMF analysis, and a total of 23 proteins were identified
(Table 3 and Fig. 1). The identified proteins were grouped into func-
tional categories including cytoskeleton proteins, nuclear proteins, cell
membrane-bound molecules, proteins involved in matrix synthesis,
protein synthesis and degradation, metabolic enzymes, and signal trans-
duction.
Western Blotting and Quantitative Real-time Polymerase
Chain Reaction Analyses
Western blotting and quantitative real-time polymerase chain re-
action (qRT-PCR) analyses were performed on four proteins of interest
(hnRNP C, annexin VI, collagen type VI, and matrilin-2) (Fig. 3) to
highlight protein expression changes in DPCs after odontogenic induc-
tion and to confirm the data from the DIGE gels. There was a significant
difference between control and induced groups in each transcript. The
mRNA levels of hnRNPC (p ⬍ 0.05), collagen type VI(␣1) (p ⬍ 0.05),
and matrilin-2 (p ⬍ 0.001) were significantly downregulated in the
induced cultures compared with controls at the same time points. By
contrast, the expression of annexin VI was significantly higher in the
induced DPCs compared with controls (p ⬍ 0.05). There was no sta-
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tistical difference in the expression of each transcript among induced
groups (p ⬎ 0.05), but there was a high correlation between the mRNA
expression and protein expression of these proteins. Data obtained on
day 7 of induction correlated well with the DIGE results for the analyzed
proteins.
Discussion
The mechanisms controlling differentiation of odontoblast-like
cells from their precursor cells is a major question in the field of re-
parative dentinogenesis. Proteomic profiling provides a complementary
and potentially more comprehensive approach to identify important
candidates in the process of cell differentiation (5, 6). In this study, we
have attempted to address the molecular basis of odontoblast-like dif-
ferentiation from human DPCs by means of proteomic approaches
based on highly sensitive 2D-DIGE and MALDI-TOF-MS technologies.
Twenty-three proteins were identified during the odontogenic differen-
tiation of DPCs.
Cytoskeleton Proteins
Cellular differentiation, generally associated with phenotypic mod-
ification of the cell, often involves reorganization of cytoskeletal pro-
teins. The expression of vimentin in neurons showed that vimentin is
essential for neurogenesis in culture (7). Kryszke et al. (8) reported the
downregulation of vimentin during skeletal muscle differentiation. By
contrast, vimentin expression was upregulated during osteoblast matu-
ration (9). In the present study, three isoforms of vimentin were up-
regulated at different levels during odontoblast-like differentiation; it is
therefore possible that a post-translational modification of vimentin may
accompany functional modifications.
Cardiac muscle alpha actin (␣-CAA) is predominantly expressed
in the adult heart and is crucial for maintenance of the contractility of
the heart muscle (10). ␣-CAA has also been detected in umbilical cord
vessels, human myometrium, and smooth muscle cells of various ath-
erosclerotic lesions. It is speculated that the “ectopic” expression of
␣-CAA may represent a shift away from a filamentous contractile char-
acter to a more cytoskeletal structural character (11). In our study, the
concomitant upregulation of ␣-CAA and vimentin may be caused by a
large net requirement for actin in DPCs at day 7 of induction, suggesting
that significant reorganization of the cytoskeleton accompanies the early
odontoblast-like differentiation of DPCs.
The Nuclear Proteins
hnRNP C proteins are localized to the nucleoplasm and excluded
from the nucleoli in interphase cells, becoming dispersed throughout
the cell during mitosis (12). It has been suggested that hnRNP C regu-
lates cell cycle by modulating the translation of c-myc mRNA (13).
Furthermore, the increased expression of hnRNP C induces the trans-
lation of p27 mRNA, which triggers the inhibition of cell growth via
p27-regulated cell-cycle arrest (14). Foster et al. (15) observed the
coregulation of five members of hnRNPs family, including hnRNP C, Q,
U, K, and G, as mesenchymal stem cells differentiated into osteoblasts by
proteomic approaches. Based on the findings from these studies, the
downregulation of the hnRNP C proteins observed in our study suggests
that they play a regulatory role in the odontoblast-like differentiation of
DPCs. To understand the molecular mechanism of hnRNP C function,
the detailed roles of hnRNP C in the cell cycle, cell growth, and differ-
entiation must be investigated further.
Calcium-binding Proteins
Annexins are a structurally related family of calcium-binding pro-
teins, several of which have been noted for their roles in osteogenesis
and mineralization. Annexins A2, V, and VI are major components of
JOE — Volume 34, Number 9, September 2008
 Basic Research—Biology
TABLE 3. List of Proteins with Altered Expression Identified by 2D-DIGE/MALDI-TOF-MS During Odontogenic Differentiation of DPCs
NCBI Coverage Average
Spot ID Protein Name MW (kDa) pI t Test
Accession No. (%) Ratio
Cytoskeleton proteins
1410 Vimentin gi|37852 41.6 53.72 5.1 ⫹2.25 0.0075
1431 Vimentin gi|37852 33.9 53.72 5.1 ⫹1.94 0.023
1310 Vimentin gi|37852 41 53.72 5.1 ⫹1.67 0.007
1516 Cardiac muscle alpha actin gi|4885049 17 42.34 5.2 ⫹4.25 0.01
Nuclear proteins
1540 Heterogeneous nuclear gi|13937888 18 33.64 5 ⫺2.56 6.4E-05
ribonucleoprotein C (hnRNPC
protein)
1580 Heterogeneous nuclear gi|14249959 22.5 32.43 5 ⫺3.56 0.0005
ribonucleoprotein C (hnRNPC
protein)
Cell membrane-bound
molecules
1730 Annexin VI gi|71773415 12.6 75.6 5.5 ⫹2.66 0.0023
Proteins involved in matrix
synthesis
370 Collagen, type VI, alpha 1 gi|15011913 17.2 109.64 5.3 ⫺1.77 0.019
2188 Matrilin-2 gi|62298084 8.1 110.7 5.8 ⫺2.28 0.02
Metabolic enzymes
1554 Ribonucleotide reductase gi|227541 13.4 34 5 ⫹3.25 0.011
1706 Pyruvate dehydrogenase E1-beta gi|387010 31.3 36.81 5.4 ⫹1.55 0.048
subunit precursor
1931 Peroxiredoxin IV gi|49456297 22.1 30.74 5.9 ⫹1.61 0.0032
1543 Chain A, Uroporphyrinogen gi|40889570 14.7 41.13 5.8 ⫹2.56 0.0036
decarboxylase single mutant
D86e in complex with
coproporphyrinogen-I
Protein synthesis and
degradation
1973 Chain D, Cathepsin B gi|999911 30.7 22.97 5.2 ⫹1.83 0.0056
1655 Eukaryotic translation gi|25453474 13.9 71.85 6 ⫺2.71 0.0042
elongation factor 1 delta
Signal transduction
1709 OSBP-related protein 6 gi|33187713 7.4 103.36 6.6 ⫺1.99 0.0075
Others
1542 SGT1B protein gi|33286853 14.5 41.32 5.1 ⫺2.97 0.0002
1996 Chain A, ternary complex of an gi|24987410 39.4 12.11 6.8 ⫹3.17 9.2E-05
Crk Sh2 domain, Crk-derived
phophopeptide, and Abl Sh3
domain by nmr spectroscopy
1187 Human-HERV-F(c)1_Xq21.33 gi|52000661 18.5 51.97 9.4 ⫹2.58 0.023
provirus ancestral Gag
polyprotein
1895 Ventricular zone expressed PH gi|75517805 6.9 95.54 6.3 ⫺1.92 0.0083
domain homolog 1
1933 Unnamed protein product gi|21757045 24.2 52.48 5 ⫺2.95 0.017
1956 Unnamed protein product gi|16552261 13.9 47.53 5 ⫺3.99 0.018
2042 Unnamed protein product gi|16552261 10.5 47.53 5 ⫺2.04 0.029
DPC, dental pulp cell.

matrix vesicles that enable influx of Ca2⫹ into the vesicles and initiate
the mineralizating process of the growth plate cartilage (16). Annexin VI
together with alkaline phosphatase is predominantly expressed by ter-
minally differentiated, mineralizing chondrocytes during endochondral
ossification and thus considered as late markers of chondrocyte differ-
entiation (17). The upregulation of annexin VI in our study fit with
previous reports, and it is believed to be involved in Ca2⫹ homeostasis
and mineralization of DPCs. In light of recent findings (18), annexin VI
has been shown to be involved in transforming growth factor (TGF)-␤1
binding to the acidic pH-binding site and capable of mediating internal-
ization and degradation of cell surface-bound TGF-␤1, suggesting its
regulatory functions in cell growth and differentiation. Our finding that
the expression of annexin VI was increased as DPCs differentiated into
odontoblast-like cells lends additional support to the regulatory role of
annexin VI in the mineralization process of DPCs and thus may serve as
the potential mineralization marker.
Proteins Involved in Matrix Synthesis
Collagen type VI is a minor but ubiquitous constituent of the collagens
found in the interstitial extracellular matrix (ECM) of dental tissues; it forms
microfibrils that most likely serve as a flexible anchor interconnecting col-
lagen fibers with cells and adjacent collagen fibers (19). Collagen type VI has
been found in the odontoblast layer in human dental pulp (20) and predentin,
whereasitisabsentindentin(21).Immunoelectron microscopy has revealed
the presence of collagen type VI in dentin with dentinogenesis imperfecta
(22). Our data have shown a downregulation of collagen type VI during the
mineralization of DPCs. Taken together, we postulate that collagen type VI
may be actively involved in odontoblast-like differentiation and dentinogen-
esis. It has been reported that collagen type VI probably mediates interleu-
kin-4 –induced mineralization and regulates collagen type I expression in
human osteoblast-like cells (23, 24). As for odontoblast-like differentiation
of DPCs, the exact function of collagen type VI needs further investigation.

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Basic Research—Biology

Figure 2. Comparative analysis of four protein spots intensities using the BVA module of the DeCyder software. The 2-D image from each induced sample gel was
compared with that of the control sample gel by means of the internal standard sample gel. Protein-expression analysis was performed for each of the four gels in
parallel with the DIA module of the DeCyder software. These DIA analyses were collated into a single analysis by using the DeCyder BVA module, and final values for
the expression ratio of specific protein spots between control and induced samples were determined. A Student t test was used to calculate significant differences in
the relative abundance of individual protein spots between control and induced samples. The four selected differentially expressed protein spots of the control and
induced DPCs are displayed as partial views of 2-D gel (top panels) and as three-dimensional images (bottom panels). The spot boundary of selected proteins is
displayed in pink. (A) HnRNPC, (C) collagen type VI, and (D) matrilin-2 were downregulated, whereas (B) annexin VI was upregulated.

Matrilins are typical modular proteins belonging to the superfam-
ily with von Willebrand factor type A (vWFA) modules and have been
used as markers for the characterization of chondrocyte differentiation
stages (25). In a recent study on antler development, matrilin-2 was
identified as a marker of cells with high differentiation potential and was
expressed predominantly by mesenchymal cells, prechondrocytes, and
preosteoblasts (26). Based on these reports, the downregulation of
matrilin-2 found in early odontoblast-like differentiation suggests that
matrilin-2 may be involved in the regulation of DPCs differentiation. It
has been shown that matrilin-2 interacts with collagens I, II, III, IV, and
V, which makes matrilin-2 a good candidate as an adapter or mediator
molecule for interactions between other matrix macromolecules during
the assembly of ECM (27). Our result showed a coregulation collagen
type VI and matrilin-2 during odontogenic differentiation of DPCs.
Therefore, some kind of interaction is likely to exist between these two
proteins, but at this stage this interaction needs to be confirmed by
further studies.
Metabolic Enzymes
Peroxiredoxins (PrdxsTHO) have been implicated in a number of
cellular functions such as cell proliferation and differentiation, protec-
tion of free-radical–sensitive proteins, hemoglobin metabolism, and
intracellular signaling (28, 29). Prdx4 plays a regulatory role in the
activation of the transcription factor NF-␬B by modulating I␬B-␣ phos-
phorylation in the cytoplasm (30) and has been shown to be associated
with the stem-cell differentiation by proteomic analysis (5, 6). The
upregulation of Prdx4 in our study indicates the involvement of Prdx4 in
the odontoblast-like differentiation of DPCs.
Cell Signaling
Oxysterol binding protein-related proteins (ORPs) are homologs
of the oxysterol-binding protein (OSBP). OSBP is a cholesterol-depen-
dent scaffolding protein that regulates the extracellular signal-regulated
kinase (ERK) signaling pathway (31) and whose activation plays a pos-
itive role in odontoblast-like differentiation in DPCs (2). The downregu-
lation of ORP6 in this study implicates its putative role in activating ERK
signaling in a similar fashion to OSBP, thus regulating the odontoblast-
like differentiation. Further investigation should be undertaken to de-
fine the regulation of ERK signal pathway by ORP6.
Others
Human Sgt1B is a splice variant of Sgt1A, and their respective
proteins are 91% identical (32). Sgt1 was originally identified as a
suppressor of the G2 allele of Skp1 and was found to be important for
both the G1/S and G2/M transitions in the cell cycle. Furthermore, Sgt1
was capable of interacting with several members of the S100 protein

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Figure 3. Western blotting and qRT-PCR analyses of the expression of hnRNPC,
annexin VI, collagen type VI, and matrilin-2. (A) Equal amounts of protein
extracted from control and induced DPCs (days 7, 14, and 21) were separated
by SDS-PAGE. The proteins were transferred to the nitrocellulose membranes;
then probed with antibodies against hnRNPC, annexin VI, collagen type VI, and
matrilin-2; and visualized by electrogenerated chemiluminescence. This is a
representative result from three independent experiments. (B) Blot quantifica-
tion of differential protein expression is shown in the histogram. (C) Total
cellular RNA was isolated from control and induced DPCs (days 7, 14, and 21),
and qRT-PCR analysis was performed. The mRNA levels of the genes of interest
were normalized against ␤-actin mRNA expression and designated as an ex-
pression coefficient. These data were calculated from three different cell sam-
ples with the same cell sample in triplicate.
family, functions of which include regulation of protein phosphoryla-
tion, cytoskeleton polymerization, and cell-cycle progression (33). The
downregulation of Sgt1B expression in the induced DPCs found in our
study indicates it has a role in cell differentiation and mineralization.
Conclusions
This study shows, for the first time, the differential protein
expression profile of DPCs on day 7 of odontogenic induction com-
pared with noninduced controls. Expression changes of the identi-
fied proteins reveal the involvement of various regulation mecha-
nisms in odontoblast-like differentiation including cell cycle,
protein synthesis and degradation, Ca2⫹ homeostasis, signal trans-
duction, translation, and cellular energy regulation, all of which are
interesting targets for further investigations. Our results show that
existing proteomics technologies and bioinformatics tools are ca-
pable of supporting comprehensive assessment of the proteome in
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Basic Research—Biology
the process of cell differentiation. However, it must be pointed out
that the success of proteomic analysis depends on the accuracy and
completeness of existing genomic and proteomic databases and on
the rapid and accurate interconvertibility of such information
across database platforms (34). Among the proteins identified in
the present study, some are of unknown function (eg, unnamed
protein products, which may have important roles in cell differen-
tiation). Further studies of these proteins and their function will
lead to a more in-depth understanding of the biological processes
related to odontoblast-like differentiation and reparative dentino-
genesis.
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