QUT Digital Repository: http://eprints.qut.edu.au/

This is the accepted version of this journal article. Published as: 

Wu, Liping and Wei, Xi and Ling, Junqi and Liu, Lu and Liu, Shaojun and Li, Mintao and Xiao, Yin (2009) Early osteogenic differential protein profile detected by proteomic analysis in human periodontal ligament cells. Journal of Periodontal Research, 44(5). pp. 645‐654. 

© Copyright 2008 Blackwell Publishing The definitive version is available at www.blackwell‐synergy.com 

1

Early Osteogenic Differential Protein Profile Detected by Proteomic Analysis in

Human Periodontal Ligament Cells

Liping Wu1*, Xi Wei1*, Junqi Ling1†, Lu Liu1, Shaojun Liu2, Mintao Li2, and Yin Xiao3

1Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong,

China

2Proteomics Laboratory, Zhongshan Medical College, Sun Yat-Sen University,

Guangzhou, China

3Institute of Health and Biomedical Innovation, Queensland University of Technology,

Brisbane, Queensland, Australia;

*Contributed equally to this study

†Corresponding author

A running title: Differential Protein Profile in PDLCs

Correspondance to:

Dr. Junqi Ling

Professor and Dean

Guanghua School of Stomatology

Sun Yat-sen University

Guangzhou 510055, P. R. China

2

Tel: +86-20-8386-2621

Fax: +86-20-8382-2807

E-mail: lingjq@ mail.sysu.edu.cn

Keywords: Human periodontal ligament cells; Osteogenic differentiation; Proteomics；

2D-DIGE

Number of words in the abstract: 200

Number of words in the abstract and the text (excluding tables, figure legends,

acknowledgments, and cited references): 3890

Number of figures: 6

Number of cited references: 31

There is no conflict of interest in this study and no author has financial affiliation or

involvement with any commercial organization with direct financial interest in the subject

or materials discussed in this manuscript.

3

Early Osteogenic Differential Protein Profile Detected by Proteomic Analysis in

Human Periodontal Ligament Cells

Abstract

Background and Objective: Human periodontal ligament cells (PDLCs) play a pivotal

role in maintaining periodontal ligament space, have the ability to differentiate into

cementoblasts/osteoblasts-like cells, and a tremendous potential to regenerate periodontal

tissue. The exact molecular mechanisms governing the differentiation status of PDLCs

remains largely unknown. This study was to investigate the differentially expressed

proteins involved in the osteogenic differentiation of PDLCs.

Material and Methods: Using two-dimensional gel electrophoresis (2D-DIGE), mass

spectrometry (MS), and peptide mass fingerprinting (PMF), we have analyzed the

differential protein profiles of PDLCs undergoing mineralization condition.

Results: Compared to undifferentiated PDLCs, 61 proteins with at least a 1.5-fold change

were detected, of which 29 differentially expressed proteins were successfully identified

by matrix assisted laser desorption ionization time-of-flight mass spectrometry

(MALDI-TOF MS). The expression of some of the identified proteins was further

confirmed by western blot and RT-PCR analysis. The identified proteins were found to

belong to cytoskeleton proteins and cytoskeleton-associated proteins, nuclear proteins

and cell membrane-bound molecules.

Conclusion: Our results suggest that these proteins may be associated with the unique

function of PDLCs in maintaining periodontal tissue homeostasis, providing a

comprehensive reference to understand and further study the molecular mechanisms of

4

PDLCs involved in periodontal regeneration.

Keywords: Human periodontal ligament cells; Osteogenic differentiation; Proteomics;

2D- DIGE.

Introduction

The periodontal ligament (PDL) is a narrow fibrous connective tissue that connects the

alveolar bone to teeth and plays an important role in providing anchorage and supporting

tooth function, maintaining homeostasis, and repairing damaged tissue in response to

periodontal disease or mechanical trauma. PDL contains a heterogeneous cell population

at various stages of differentiation and lineage commitment. Previous studies indicate that

periodontal ligament cells (PDLCs) can form mineralized nodules in vitro and express

bone-related proteins, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and

osteocalcin (OCN) (1-3). Recently Seo et al. reported that PDLCs contain stem cells with

high proliferative capacity, self-renewal propertie, multilineage differentiation potential,

and capable of forming cementum/PDL-like tissue in vivo (4). Thus, PDLCs play pivotal

role in maintaining periodontal ligament space with the ability to differentiate into

cementoblasts/osteoblasts-like cells, and have tremendous potential to regenerate

periodontal tissue. However, a comprehensive understanding of molecular mechanisms

controlling PDLCs differentiation into functional cells remains poor.

Proteomic techniques such as two-dimensional gel electrophoresis (2-DE), coupled with

protein identification by mass spectrometry (MS) and bioinformatics, has led to a

5

revolution in the ability of researchers to tease out the temporal and spatial changes of the

entire protein complement and their functions in a given cell type (5). The task of

detecting changes in protein expression has recently been made possible by the

introduction of difference in gel electrophoresis (DIGE) (6), This method increases

statistical confidence when combining experimental repetition with internal standards and

facilitates the identification of quantitative and qualitative protein alterations during cell

differentiation. Proteomics techniques are now widely accepted as an important tool for

finding potential biomarkers or studying the mechanisms of cell differentiation (7). In

order to identify early differential proteins in PDLCs osteogenic differentiation, DIGE

and MS based proteomics approaches were utilized to investigate PDLCs cultures for 7

days in osteogenic culture conditions. Twenty-nine proteins were identified by matrix

assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS),

of which 12 were up-regulated and 17 were down-regulation during early osteogenic

differentiation.

Material and Methods

PDLCs isolation and cell culture

Healthy periodontal ligament tissues were collected from the middle third part of the root

of premolars obtained from patients (12-18 years of age) undergoing orthodontic

treatment in the Department of Orthodontics, Sun Yat-sen University Dental Hospital.

Informed consent was obtained from each subject and the research protocols were

approved by the University Ethic Committee. PDL tissues were minced and digested in a

solution containing 3mg/mL type I collagenase (Worthington Biochem, Freehold, NJ,

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USA) and 4mg/mL dispase (GIBCO-BRL Life Technologies, Breda, Netherlands) at

370C for 30 to 60 minutes as previously described (4). The digested solution was filtered

through a 70-μm strainer (Becton Dickinson, Sunnyvale, CA, USA) and the released cells

plated in 6-well plates containing alpha modified Eagle’s medium (α-MEM,

GIBCO-BRL) supplemented with 20% fetal bovine serum (FBS, GIBCO-BRL), 100

units/mL penicillin and 100µg/mL streptomycin (Sigma, St. Louis, MO, USA), and

incubated at 370C in 5% CO2. Cells from passages 2 to 4 were used in the following

experiments.

PDLCs surface characterization using flow cytometry analysis

1×105 PDLCs at passage 2 were harvested in phosphate buffered solution (PBS)

containing 1mM EDTA, washed in PBS, and then incubated with 0.5µg/100µL of mouse

antihuman STRO-1 antibodies (R&D systems, Minneapolis, MN, USA) and CD146

(Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at 4°C. Cells were washed

twice with cold PBS containing 2% FBS and incubated with 1µg/100µL of fluorescein

isothiocyanate (FITC onjugated goat antimouse IgM or IgG antibodies (Santa Cruz) for

30 minutes at 4°C. Mouse antihuman antibodies to CD29-FITC, CD44-FITC, CD106-PE,

CD34-FITC (BD Bioscience, Farmington, CT) were also used for phenotype

characterization. Mouse isotype antibodies (BD Bioscience) served as a control. Labeled

cells were analyzed by flow cytometer (Becton -Dickinson, Franklin Lakes, NJ, USA).

The experiments were repeated three times on all three patient samples.

Immunocytochemical staining and alizarin red staining

Comment [QSOE1]: 0.5ug per mL??

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Cells at passage two were subcultured into 22×22mm slides (2×104cells/mL) for 24 hours

and fixed in 4% paraformaldehyde for 15 minutes, then blocked and incubated with

monoclonal antibody of anti-vimentin or anti-pancytokeratin (Boster Biotechnology, INC,

Wuhan, China) at 1:100 dilution, and rabbit anti-mouse antibody was as a secondary

antibody.

Osteogenic differentiation of PDLCs was verified by the bone nodule formation after

PDLCs had been cultured for 21 days in osteogenic media (α-MEM supplemented with

10mM β-glycerophosphate, 0.2mM ascorbic acid, 100nM dexamethasone [Sigma] with

15% FBS); controls were cultured in α-MEM with 15% FBS, Mineral deposits were

detected at day 21 by 2% alizarin red staining, and osteocalcin expression was monitored

at days 21 by immunocytochemical staining using polyclonal mouse antihuman

osteocalcin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:100 dilution).

Protein preparation

PDLCs were cultured in osteogenic and control medium for 7 days. Prior to the harvest

both the differentiated cells and controls were incubated with serum-free medium for 24

hours after which the medium was removed and the cells washed three times with

ice-cold Tris-buffered sucrose (10mM Tris-base, 250mM sucrose, pH 7.4). The cells were

lysed with sodium dodecyl sulfate (SDS) lysis buffer containing 7M urea, 2M thiourea,

4% CHAPS, and 30mM Tris (GE healthcare), the lysates sonicated on ice and then

centrifuged at 20000g for 30 minutes at 40C. The protein samples were purified using the

2-D Clean-Up Kit (GE healthcare) and protein content determined by 2D-Quant kit (GE

healthcare) according to the manufacturer’s instructions.

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CyDye labeling of extracted proteins and two-dimensional electrophoresis (2-DE)

Prior to 2-DE the protein extracts were labeled with fluorescent cyanine dyes developed

for 2-D DIGE technology (GE Healthcare) following the manufacturer’s

recommendations. 50mg of differentiated and control proteins were labeled with Cy3 or

Cy5, and 50mg of the internal standard mixture was labeled with Cy2 (Table 1), followed

by incubation on ice for 30 minutes in the dark. The reactions were quenched by the

addition of 1μL of 10mM lysine for 10 minutes on ice in the dark. The quenched Cy3-

and Cy5-labeled samples and the Cy2-labeled internal standard were pooled prior to

analysis by 2-DE. Two preparative gels, each containing 500mg of unlabeled mixture

proteins, were analyzed, and four DIGE gels were run to analyze differentiated and

control samples.

2-DE was performed according to the manufacturer’s instructions (GE Healthcare).

Briefly, immobilized dry strips (pH4-7, 24 cm, GE Healthcare) were rehydrated for 12 h

in the 450μL rehydration buffer (7M urea, 2M thiourea, 4% w/v CHAPS, 1% v/v IPG

buffer (pH 4–7) ,40mM DTT and 0.002% (w/v) Bromophenol blue ) at 30 V for 12 hours

using an Ettan IPGphor II IEF system (GE Healthcare). After rehydration the samples

were focused at 500 V for 1 hour, 1000 V for 1 hour, and at 8000 V for a total of 80000

Vh at 200C. The strips were incubated in Equilibration Buffer I containing 6 M urea, 30%

glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), 1% DTT for 15 minutes at room

temperature in the dark and then soaked in Equilibration Buffer II for an additional 15

minutes. Buffer II was identical to Buffer I with the exception of 2.5% iodoacetamide

instead of 40mM DTT. The equilibrated strips were embedded in 0.5 w/v agarose on top

9

of 12.5% acrylamide slab gels. Second dimension separations were performed on an

Ettan DALT six electrophoresis system (GE Healthcare). The proteins were separated

initially at 150C, 2 W/gel for 50 minutes, followed by 17 W/gel until the dye front

reached the bottom of the gel. The two preparative gels were stained with Deep purple

total protein stain (RPN6306, GE Healthcare) following the manufacturer’s instructions.

Gel scanning and image analysis

Both sample and preparative gels were imaged using a Typhoon Variable Mode Imager

9400 (GE Healthcare) using the excitation filters Blue 488nm (Cy2), Green 532nm (Cy3),

Red 633nm (Cy5), and 532nm (Deep Purple) with the resolution of 100μm. The 2-D

image of the gel from each treated sample was compared to that of the control sample via

the internal standard sample. Protein expression analysis was performed for each of the

four gels in parallel using the differential in-gel analysis (DIA) module of DeCyder

software 6.0 (GE Healthcare) using a value of 3000 as the initial estimation of the

number of protein spots present. The intensities of individual protein spots of the

proteomes of the differentiated samples were compared with the control. The DIA

analysis data was collated into a single analysis using the DeCyder biological variation

analysis (BVA) module, and final values for the expression ratio of specific protein spots

between differentiated samples and the control were determined. A total of 2787~3039

protein spots were analyzed across all cell samples and Student’s t-test and one-way

ANOVA were used to calculate significant differences in the relative abundance of

individual protein spot features between differentiated samples and the control. Protein

spots an abundance ratio greater than +1.5 or less than -1.5 were determined to be

10

differentially expressed.

In-gel digestion and MALDI-TOF MS

Selecting protein spots from the 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). Identification of proteins by peptide

mass fingerprinting (PMF) was carried out on an Ettan MALDI-TOF mass spectrometer

(GE Healthcare) operating in reflectron mode. Internal calibration was performed using

the trypsin autodigestion peaks at m/z 842.509 and 2211.104. Mass spectra were obtained

over the m/z range of 700–3000 Da. PMF data was used to search the human NCBI nr

protein database using the Profound search engine (http://www.

matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF, GE Healthcare).

Search parameters were as follows: type of search, peptide mass fingerprint; enzyme,

trypsin; fixed modification, carbamidomethylation (Cys); variable modifications,

oxidation (Met); mass values, monoisotopic; peptide charge state, 1+; maximum missed

cleavages, 1; and peptide mass tolerance, 100ppm.

Western blot

PDLCs on days 0 (at 80% confluence, before the addition of osteogenic induction

medium) and 7 of osteogenic induction were harvested and lysed in SDS lysis buffer,

sonicated, and centrifuged at 12000g for 5 minutes. Equal amounts of protein (50μg)

were separated by electrophoresis on a 12.5% polyacrylamide gel and transferred to

nitrocellulose membranes by 100mA for 3 hours. The membranes were blocked in 5%

11

nonfat dry milk at room temperature for 1 hour, rinsed, and incubated with primary

antibodies (1:1000 dilution) of mouse anti-human heteronuclear ribonuclear proteins

C1/C2 (hnRNPC, Santa Cruz), mouse anti-human caldesmon (CaD), mouse anti-human

tropomyosin (Tm), rabbit anti-human polyclonal antibody AnnexinA4 (Cell Signaling,

LAB VISION Corporation NeoMarkers), or mouse anti-human β-actin (Santa Cruz) at

40C overnight. The membranes were washed, then incubated with HRP-conjugated

secondary antibodies (Santa Cruz, 1:5000 dilutions) at room temperature for 1 hour, and

the proteins visualized using enhanced chemiluminescence (Amersham Pharmacia

Biotech, Piscataway, NJ, USA).

Quantitative real-time reverse-transcription polymerase chain reaction

Prior to osteogenic induction and after 7 days of induction, cells were lysed to extract

total RNA by using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The primers

used for detection were listed in Table 2. Quantifications of mRNAs were performed with

an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA).

cDNA was synthesized from 1�g of each RNA sample using the omniscript RT kit

(Qiagen) and RNase-free DNase Set (Qiagen) following the manufacturer’s instructions,

5µL of the reaction mixture was incubated with PCR master mix with double-stranded

DNA dye SYBR Green I (Qiagen) in a total volume of 50µL. 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. The amount of template was quantified by

using the comparative cycle threshold method according to the manufacturer’s protocol.

Measured mRNA levels were normalized against β-actin mRNA expression. Experiments

12

were performed in triplicates on three different cell samples.

Statistical analysis

All experiments have been repeated three to four times, and the representative results are

presented. The quantitative data is presented as mean ± standard deviation. One-way

analysis of variance (ANOVA) and Bonferroni test was performed to analyze mRNA

expressions between induced samples and controls. Differences were considered

significant at p<0.05. The SPSS software package (version 13.0, SPSS Inc., Chicago, IL,

USA) was used for the statistical analysis.

Results

Cell characterization

PDLCs expressed cell surface epitopes associated with mesenchymal progenitors such as

STRO-1, CD-146, CD29, CD44, CD106, but did not express CD34, a hematopoietic

origin marker as shown by flow cytometry. The expression of STRO-1, CD-146, CD29,

CD44, CD106 was 15.3%, 47.6%, 99.9%, 99.8% and 76.8% respectively (Fig. 1A-F,

Table 3). The positive immunocytochemical staining for vimentin antibody, the most

widely distributed intermediate filament in fibroblasts, and negative staining for

pancytokeratin, a marker for tissue of epithelial origin, indicated that the PDLCs islolated

from PDL tissue in this study were of mesenchymal origin (Fig. 2A-B).

Alizarin red positive nodules were seen in PDLC cultures on day 21 of osteogenic

induction, however, no staining was detected in the controls (Fig. 2C). Furthermore,

PDLCs showed positive expression of OCN after 21 days of osteogenic induction (Fig.

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2D). Quantitative real-time RT-PCR showed an upregulation of ALP, OCN and matrix

extracellular phosphoglycoprotein (MEPE) mRNA expressions in osteogenic culture

conditions (Fig 3). The difference between the induced cells versus controls at the same

time points was statistically significant (on days 14 and 21 for ALP and OCN, on day 21

for MEPE, ANOVA, p﹤0.05 or p﹤0.001) for each transcript. These results suggest that

PDLCs are able to differentiate into cementoblasts/osteoblasts in vitro.

Osteogenic differential protein expression in PDLCs identified by 2D-DIGE and

MALDI-TOF MS

Protein profiles were compared between the osteogenic induced cells and controls. The

DeCyder software detected more than 2,787 protein spots across all four gels. 61 protein

spots showed at least 1.5-fold changes in expression of which 14 proteins were

up-regulated and 47 proteins were down-regulated (p<0.05). The protein profile of

2D-DIGE is shown in Figure 4. MALDI-TOF MS identified 29 proteins in PDLCs by

their PMF using the Swiss-Prot database. These proteins were classified into seven

functional categories: cytoskeleton proteins, nuclear proteins, cell membrane-bound

molecules, proteins involved in matrix synthesis, protein synthesis, metabolic enzymes

and signal transduction (Table 4).

Western blot and RT-PCR analysis of up- or down-regulated proteins

To confirm the results obtained in 2D-DIGE gels, several proteins, such as the

cytoskeleton associated proteins caldesmon (CaD) and tropomyosin (Tm), heterogeneous

nuclear ribonucleoprotein C (hnRNPC) and calcium-binding protein annexin A4, were

14

selected for the further confirmation by western blot and quantitative PCR (qPCR). The

proteins and mRNA were extracted from PDLCs at days 0 and 7 of induction. Western

blot analysis confirmed the down-regulation of CaD, Tm, hnRNPC and up-regulation of

annexinA4 after osteogenic induction of PDLCs on days 7. Furthermore, qPCR analysis

showed similar changes in mRNA expressions of CaD, Tm, hnRNPC and annexin A4 in

osteogenic culture (Fig. 6).

Discussion

In this study, PDLCs isolated from periodontal ligament showed a similar profile of cell

surface epitopes compared with the studies reported by others (4, 8, 9). Seo et al. showed

that periodontal ligament stem cells (PDLSCs) are immunocytochemically positive for

STRO-1 and CD146, markers which have been used to isolate and purify human bone

marrow mesenchymal stem cells (MSCs) (4). By flow cytometry analysis, we found that

the percentage of STRO-1 positive cells at passage 2 was higher than previously reported

(8). Our findings that PDLCs expressed the mesenchymal stem cell-associated antigens

CD44 and CD106 and an absence of hematopoietic marker CD34 expression agreed with

that reported by Gronthos et al (9). These results indicate that PDLCs contain a

population of mesenchymal stem cell. We also demonstrated that PDLCs formed a

mineralized matrix and expressed the bone-related markers ALP, OCN and MEPE when

grown in osteogenic media. These data suggest that cultured PDLCs may represent a

heterogeneous stem-cell enriched population and have the potential to differentiate into

cementoblasts or osteoblasts in vitro.

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Although some growth factors, genes and signaling molecular have been identified in the

process of cementogenesis/osteogenesis with conventional molecular biological

approaches (10-12), the differentiation mechanisms of stem/progenitor cells in

periodontal regeneration need to be further understood. Proteomic profiling analysis

provides a complementary and potentially more comprehensive approach to tease out

important protein candidates in the process of differentiation. In this study, PDLCs were

induced along cementoblasts/osteoblasts lineages. Changes in protein expression levels

during PDLCs differentiation on day 7 was determined by proteomics based on the

highly sensitive 2D-DIGE and MALDI-TOF MS technologies. 29 differentially

expressed proteins were identified in PDLCs during the cementoblast/osteoblast

differentiation. These proteins mainly effect the cytoskeleton, nuclear regulation, cell

membrane-bounding, matrix synthesis, metabolic enzymes and signal transduction.

We found that cytoskeleton proteins, including vimentin, CaD, Tm and Clathrin heavy

chain polypeptide (CHC), were down-regulated during cementoblastic/osteoblastic

differentiation of PDLCs. Significant down-regulation of vimentin has been demonstrated

in vitro in MSCs differentiating into cardiomyocytes (13), suggesting differentiation

stage-specific reorganization of the cytoskeleton. In addition, vimentin exhibits a

complex pattern of developmental and tissue-specific expression regulated by

transforming growth factor beta 1 (TGFβ-1), which plays an important role in cell

differentiation (14). CaD contains actin-, myosin-, tropomyosin-, and Ca2+-calmodulin

(CaM)-binding domains (15), and is often regarded as differentiation marker of smooth

muscle cells (16). CaD was down-regulated in MSCs during the process of neuron-like

16

differentiation (17), providing further evidence of its involvement in cell differentiation.

CaD can be phosphorylated by p21-activated kinases (PAKs), a dynamic process required

to regulate actin dynamics and membrane protrusions in wound-induced cell migration

(18). As a natural partner of CaD, tropomyosin regulate actomyosin-based contractility,

mediate the inhibitory effects of CaD on actomyosin and regulate cytokinesis speed and

membrane stability during cell division (19). CHC, another actin filament binding protein

identified in this study, has, together with Tm and annexin 4, been indentified to be

involved in the osteogenic differentiation of MSCs (20). An earlier proteomics study of

periodontal ligament fibroblasts found that the expression of actin, vimentin and Tms

were related to mechanical loading and remodeling of PDL (21). In our study, we found a

down-regulation of vimentin, CaD, beta Tm, skeletal muscle Tm, CHC and related kinase

PAK65 at day 7 of PDLCs osteogenic differentiation. Co-regulation of the functional

class of proteins during PDLCs differentiation suggests that these cytoskeleton proteins

are intimately involved in the differentiation process and that their regulation proceeds

via similar regulatory mechanisms. It is possible that these proteins may be involved in

regulating cell migration during the osteogenic differentiation of PDLCs, but this

hypothesis needs to be confirmed by further experiments.

The hnRNPC proteins (C1 and C2) belong to a class of proteins that bind to pre-mRNA

in the cell nucleus. Increased expression of hnRNPC induced the translation of p27

mRNA and triggered the inhibition of cell growth via p27 regulated cell cycle arrest (22).

Forster and colleagues, applying a proteomic approach, observed the co-regulation of five

members of hnRNPs family (hnRNP C1/C2, Q, U, K and G) during MSC osteogenic

17

differentiation; altered expression of hnRNPs was regarded to be related with cell

adhesion (20). The down-regulation of hnRNP C1/C2 found in our study further supports

this protein’s critical role in cell adhesion in PDLC osteogenic differentiation. In addition

to hnRNP C1/C2 proteins, the two nuclear proteins nucleophosmin 1 (NPM1) and lamins

A/C, were also down-regulated during PDLCs differentiation. NPM1 protein serves as a

shuttle protein for the nuclear transport of ribosomal components and presumably

collaborates with other nucleolar proteins in ribosome assembly (23). Nuclear lamins A/C

are intermediate filament proteins which form the nuclear lamina underlying the inner

nuclear membrane (24). The absence of lamin A/C has been identified as a novel marker

for undifferentiated embryoic stem (ES) cells and lamin A/C expression as an early

indicator of ES cell differentiation, indicating the functional role of lamin A/C in the

maintenance of the differentiated state (25). The concomitant down-regulation of hnRNP

C1/C2, NPM1 and lamin A/C in our study suggest that these nuclear proteins may work

together to allow osteogenic differentiation of PDLCs to be proceed.

Annexins belong to a family of structurally related of calcium-binding proteins (26),

several of which have already been found to play roles in osteogenesis and mineralization.

Ye et al found, by 2-DE and MS, that three isoforms of annexin A1 are down-regulated to

various degrees during rat MSC differentiation (13). Other proteomics studies have

demonstrated the differential expression of anexins A1, A2, A4 and V in hMSC

osteogenic differentiation (27, 28). Annexin A4 is reported to regulate a

calmodulin-dependent kinase II-activated (Ca2+/CaMKII) chloride conductance, and has

been proposed to play a role in exocytosis, vesicle aggregation and trafficking in a

18

Ca2+-dependent manner (29-31). In our study both the protein and mRNA expression of

annexins A4 were up-regulated in the mineralization process of PDLCs. Although some

possible biological functions of annexin A4 have been proposed, the exact role of this

molecule in osteogenic differentiation of PDLCs requires further investigation.

It is worth noting that typical bone-related markers, such as ALP, OCN and MEPE, were

not seen to be up-regulated in the proteomic map during early differentiation, although

these markers were significantly up-regulated during the late stages of osteogenic

differentiation of PDLCs, confirmed by qPCR. ALP is reported to be detectable in early

osteogenic differentiation, but in our study neither mRNA nor protein levels showed any

significant difference at day 7 of differentiation. One possible reason for this apparent

variation may be the source of cells and differentiation protocols utilized in the various

research laboratories. However, the early regulatory proteins identified in our study may

provide some insights into the potential molecular interactions between early and late

stage differential proteins.

Conclusion

In this study we present for the first time the differential protein expression profiles of

PDLCs at day 7 of osteogenic induction. 29 proteins were identified by MS analysis

showing differential expression during osteogenesis. These proteins mainly included

cytoskeleton proteins and cytoskeleton-associated proteins, nuclear proteins, cell

membrane-bound molecules, and may be associated with the unique function of PDLCs

in maintaining periodontal tissue homeostasis, especially in the mineralization nature of

PDL. Our results provide a comprehensive reference to understand and further study

19

molecular mechanisms of PDLCs in periodontal regeneration.

Acknowledgements

This investigation was supported by the National Nature Science Research Grant

(30672318) of China.

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