Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Comparative Gene Expression Analysis of the Human Periodontal Ligament in Deciduous and Permanent Teeth

  • Je Seon Song ,

    Contributed equally to this work with: Je Seon Song, Dong Hwan Hwang

    Affiliations Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea, Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea

  • Dong Hwan Hwang ,

    Contributed equally to this work with: Je Seon Song, Dong Hwan Hwang

    Affiliation Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea

  • Seong-Oh Kim,

    Affiliations Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea, Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea

  • Mijeong Jeon,

    Affiliation Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea

  • Byung-Jai Choi,

    Affiliations Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea, Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea

  • Han-Sung Jung,

    Affiliation Division in Anatomy & Developmental Biology, Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Korea

  • Seok Jun Moon,

    Affiliation Division in Pharmacology, Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Korea

  • Wonse Park,

    Affiliation Department of General Dentistry, College of Dentistry, Yonsei University, Seoul, Korea

  • Hyung-Jun Choi

    CHOIHJ88@yuhs.ac

    Affiliations Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea, Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea

Comparative Gene Expression Analysis of the Human Periodontal Ligament in Deciduous and Permanent Teeth

  • Je Seon Song, 
  • Dong Hwan Hwang, 
  • Seong-Oh Kim, 
  • Mijeong Jeon, 
  • Byung-Jai Choi, 
  • Han-Sung Jung, 
  • Seok Jun Moon, 
  • Wonse Park, 
  • Hyung-Jun Choi
PLOS
x

Abstract

There are histological and functional differences between human deciduous and permanent periodontal ligament (PDL) tissues. The aim of this study was to determine the differences between these two types of tissue at the molecular level by comparing their gene expression patterns. PDL samples were obtained from permanent premolars (n = 38) and anterior deciduous teeth (n = 31) extracted from 40 healthy persons. Comparative cDNA microarray analysis revealed several differences in gene expression between the deciduous and permanent PDL tissues. These findings were verified by qRT-PCR (quantitative reverse-transcription–polymerase chain reaction) analysis, and the areas where genes are expressed were revealed by immunohistochemical staining. The expressions of 21 genes were up-regulated in deciduous relative to PDL tissues, and those of 30 genes were up-regulated in permanent relative to deciduous PDL tissues. The genes that were up-regulated in deciduous PDL tissues were those involved in the formation of the extracellular matrix (LAMC2, LAMB3, and COMP), tissue development (IGF2BP, MAB21L2, and PAX3), and inflammatory or immune reactions leading to tissue degradation (IL1A, CCL21, and CCL18). The up-regulated genes in permanent PDL tissues were related to tissue degradation (IL6 and ADAMTS18), myocontraction (PDE3B, CASQ2, and MYH10), and neurological responses (FOS, NCAM2, SYT1, SLC22A3, DOCK3, LRRTM1, LRRTM3, PRSS12, and ARPP21). The analysis of differential gene expressions between deciduous and permanent PDL tissues aids our understanding of histological and functional differences between them at the molecular level.

Introduction

The periodontal ligament (PDL) tissues are components of the dental apparatus that connect the tooth to the alveolar jaw bone in the area surrounding the root surfaces. Similar to tendons, collagen type I is a predominant component of PDL tissue, but collagen types III, V, VI, and XII, and proteoglycans, which are known to regulate collagen fibril formation, are also found in this tissue [1], [2]. The PDL tissues comprise various cells such as PDL fibroblasts, epithelial cell rests of Malassez, osteoblasts, cementoblasts, vascular cells, and sensory nerve cells.

There are many anatomical, embryological, functional, and structural differences between human deciduous and permanent teeth. For example, deciduous and permanent teeth exhibit different responses to external stimuli, and the sensory nerve endings are fewer and looser in permanent than in deciduous teeth [3], [4]. Because of the different responses of dental pulp tissues, the modalities of pulp therapy should differ between deciduous and permanent teeth [5]. Besides, in vitro and in vivo experiments have shown that cells originating from deciduous teeth behave differently to those from permanent teeth [6], [7].

Since deciduous teeth are resorbed and exfoliated in association with the eruption of successive permanent teeth, the periodontal tissues of the former are more easily resorbed than those of the latter [8], [9]. To explain this at the molecular level, some investigators reported that the periodontal tissues of deciduous teeth contain more bone sialoprotein and osteopontin with the Arg-Gly-Asp (RGD) sequence, to which odontoclasts bind [10], [11]. Others reported that PDL cells obtained from areas being resorbed in deciduous teeth express more of the receptor activator of the nuclear factor κ-light-chain-enhancer of activated B cells ligand (RANKL) gene, which is known to be associated with osteoclastogenesis [12], [13]. In addition, extracellular-matrix-degrading enzymes such as collagenase [14], metalloproteinases [15], [16], and mucopolysaccharidase [17] were found to be up-regulated upon the resorption of deciduous periodontium. However, these findings are not sufficient to explain the differences in the normal-functioning periodontium of deciduous and permanent teeth.

The recent development of microarray analysis allows evaluation of the expressions of large numbers of genes simultaneously, and has been used to investigate periodontal tissues [18] and periodontal cell cultures [19], [20]. Given the anatomical and functional differences between the periodontal tissues of deciduous and permanent teeth, it is reasonable to assume that there are also differences in the gene expression patterns of the cells within those tissues. Therefore, the aims of the present study were to identify and compare the gene expression patterns of human deciduous and permanent periodontal tissues in order to enhance our understanding of the molecular basis of the observed functional differences between these two tissue types.

Materials and Methods

PDL samples

The experimental protocol was approved by the Institutional Review Board of the Yonsei University Dental Hospital, and informed consent to participate was obtained from all of the subjects and their parents (#2-2011-0009). PDL samples were obtained from healthy permanent premolars (n = 38; from 4 males and 10 females, aged 10–19 years) extracted for orthodontic reasons and from anterior deciduous teeth (n = 31; from 14 males and 12 females aged 5–13 years) extracted for space management in 40 healthy persons. For RNA isolation, each of the extracted teeth (36 permanent teeth and 29 deciduous teeth) was immediately frozen and stored in liquid nitrogen. All of the teeth were subsequently thawed at room temperature, and the PDL tissues were obtained carefully using sterile curettes from the middle-third in permanent teeth or from the nonresorbed root surface in deciduous teeth. We mixed the PDL tissues and divided them into three equal groups, and then immediately submerged them in a RNA stabilizing reagent (RNAlater, Qiagen, Valencia, CA, USA). The remaining four teeth (two permanent teeth and two deciduous teeth) were used for immunohistochemical (IHC) staining.

RNA isolation

The PDL tissues were homogenized using a homogenizer (Bullet Blender, Next Advance, NY, USA). The RNeasy Fibrous Mini kit (Qiagen) was then used to extract total RNA from the PDL tissues, according to the manufacturer's instructions. The extracted RNA was eluted in 25 µl of RNase-free sterile water (provided with the kit). The quality and concentration of RNA were determined by measuring the absorbance at a wavelength of 260 nm with the aid of a spectrophotometer (NanoDrop ND-1000, Thermo Scientific Inc, Rockford, IL, USA). The RNA samples used in this study had 260/280 nm ratios of ≥1.8. Half of isolated RNA was used for complementary DNA (cDNA) microarray experiments, and the other half was used for gene expression analysis with the quantitative reverse-transcription–polymerase chain reaction (qRT-PCR).

cDNA microarray

Global gene expression analyses were conducted using oligonucleotide arrays (GeneChip Human Gene 1.0 ST, Affymetrix, Santa Clara, CA, USA). The samples were prepared according to the instructions and recommendations provided by the manufacturer. The quality and quantity of RNA were assessed using a bioanalyzer (model 2100 with RNA 6000 Nano Chips, Agilent Technologies, Amstelveen, The Netherlands).

As recommended by the manufacturer's protocol, 300-ng samples were used. In brief, 300 ng of total RNA from each sample was converted to double-stranded cDNA. Amplified RNA (cRNA) was generated from the double-stranded cDNA template using a random hexamer incorporating a T7 promoter, via in-vitro transcription, and purified with the Affymetrix sample cleanup module. cDNA was regenerated through a random-primed reverse transcription (RT) using a dNTP mix containing dUTP. The cDNA was then fragmented by UDG and APE1 restriction endonucleases and end-labeled by a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. The fragmented end-labeled cDNA was hybridized to the GeneChip Human Gene 1.0 ST arrays for 16 hours at 45°C and 60 rpm, as per the GeneChip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). The chips were then stained and washed in a GeneChip Fluidics Station 450 (Affymetrix) and scanned using a GeneChip Array scanner (3000 G7, Affymetrix). The image data were extracted using Affymetrix Command Console software (version 1.1, Affymetrix). The raw file generated using this procedure provided the expression intensity data that was used for the next step.

Analysis of microarray data

The generated expression data were normalized using the Robust MultiAverage (RMA) algorithm in the Affymetrix Expression Console software. Whether genes were differentially expressed between the three groups was determined by subjecting the RMA expression data to one-way ANOVA. Multiple testing correction was applied to the p values of the F-statistics to adjust for the false discovery rate. Genes with adjusted F-statistic p values of <0.05 were extracted. Strongly expressed genes in each test group that were up-regulated by over twofold compared to the signal value were selected for further study. The coexpression gene group, with similar expression patterns, was classified using hierarchical clustering and k-means clustering with Multi Experiment Viewer software (version 4.4, www.tm4.org, Dana-Farber Cancer Institute, Boston, MA, USA). The Web-based tool Database for Annotation, Visualization, and Integrated Discovery was used to interpret the biological implications of the differentially expressed genes. These genes were then classified based on the gene function information provided in the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes Pathway database (http://david.abcc.ncifcrf.gov/home.jsp).

qRT-PCR

The single-stranded cDNA required in the polymerase chain reaction (PCR) analysis was produced using 500 ng of extracted total RNA as a templates for RT (Superscript III Reverse Transcriptase and random primer, Invitrogen, Renfrew, UK). The RT reaction was performed at 65°C for 5 minutes, followed by 25°C for 5 minutes, 50°C for 1 hour, and 70°C for 15 minutes to inactivate the activity of the reverse transcriptase. The synthesized cDNA was diluted 10∶1 in distilled water and used as a template for qRT-PCR, which was performed using the ABI 7300 RT-PCR system (Applied Biosystems, Warrington, UK). Samples of 25 µl containing 1× Universal TaqMan Master Mix (4369016, Applied Biosystems), PCR primers at a concentration of 0.9 µM, and the diluted cDNA were prepared in triplicate. The amplification conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The following TaqMan gene expression assay primers (Applied Biosystems) were used: chemokine (C-C motif) ligand 21 (CCL21), laminin subunit γ-2 (LAMC2), insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), interleukin (IL)-6 (IL6), ADAM metalloproteinase with thrombospondin motifs-like 3 (ADAMTSL3), and leucine-rich repeat transmembrane neuronal protein 1 (LRRTM1). 18S rRNA which is known to be a reliable housekeeping gene, was used as an internal control [21][23]. ABI 7300 SDS 1.3.1 software (Applied Biosystems) recorded the fluorescence intensity of the reporter and quencher dyes; the results are plotted versus time, quantified as the cycle number. A precise quantification of the initial target was obtained by examining the amplification plots during the early log phase of product accumulation above background [the threshold cycle (Ct) number]. Ct values were subsequently used to determine ΔCt values (ΔCt = Ct of the gene minus Ct of the 18S rRNA control), and differences in Ct values were used to quantify the relative amount of PCR product, expressed as the relative change by applying the equation 2−ΔCt. The specific primer assay ID and product sizes for each gene are listed in Table 1.

IHC staining

IHC staining was performed in order to determine where the genes were expressed. In preparation for IHC staining, deciduous and permanent teeth were fixed in 10% buffered formalin (Sigma, St. Louis, MO, USA) for 1 day, decalcified with 10% EDTA (pH 7.4; Fisher Scientific, Houston, TX, USA) for 8 weeks, embedded in paraffin, and then sectioned at a thickness of 3 µm. The sections were subjected to IHC staining with antihuman IGF2BP1 (rabbit polyclonal, diluted 1∶100; Ab82968, Abcam, Cambridge, UK), antihuman IL6 (rabbit polyclonal, diluted 1∶1600; Ab6672, Abcam), antihuman LRRTM1 (rabbit polyclonal, diluted 1∶50; Ab102968, Abcam), and antihuman ADAMTSL3 (rabbit polyclonal, diluted 1∶50; NBP1-81426, Novus Biologicals, CO, USA). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. The sections were then incubated in 5% bovine serum albumin (Sigma) to block nonspecific binding and then incubated overnight with primary antibodies, which had been diluted to give optimal staining. After incubation, EnVision+ System-HRP-labeled polymer anti-rabbit (K4003, Dako North America, Carpinteria, CA, USA; ready to use) was applied for 20 minutes or Vectastain Elite ABC Kit (PK-6105, Vector Laboratories, Burlingame, CA, USA; goat IgG, diluted 1∶200) was applied for 30 minutes. Labeled streptavidin biotin kits (Dako) were used for color development according to the manufacturer's instructions. The sections were counterstained with Gill's hematoxylin (Sigma). Control sections were treated in the same manner but without primary antibodies.

Results

Gene expression profiles of deciduous and permanent PDL tissues

The distribution and frequency of all of the data were first confirmed using density and box plots. The cDNA microarray results, presented as plots of the standardized log intensity ratio (M) to the average intensity (A) in three deciduous PDL tissues and three permanent PDL tissues (i.e., “M-A” plots), are shown in Figure S1. M-A plots are used to visualize intensity-dependent ratios of raw microarray data, and provide an easy-to-understand overview of the data distribution. These plots confirmed the normalization and standardization of the distributions of the data yielded in this study.

The results demonstrate that the expressions of 51 genes were up-regulated by twofold or more in one tissue type relative to the other. Seven genes (five in deciduous PDL tissue and two in permanent PDL tissue) were not identified well. Ultimately, 44 genes were analyzed further. In deciduous PDL tissues, the expressions of 16 genes were up-regulated by twofold or more compared to permanent PDL tissue, especially LAMC2, cartilage oligomeric matrix protein (COMP), and CCL21 were expressed more than threefold (Table 2). Whereas, those of 28 genes were up-regulated by twofold in permanent compared to deciduous PDL tissue, especially IL6, SH3 and cysteine-rich domain (STAC), and FBJ murine osteosarcoma viral oncogene homolog B (FOSB) were found to be up-regulated by more than fourfold (Table 3).

thumbnail
Table 2. Up-regulated genes in deciduous PDL tissue (compared to permanent PDL tissue).

https://doi.org/10.1371/journal.pone.0061231.t002

thumbnail
Table 3. Up-regulated genes in permanent PDL tissue (compared to deciduous PDL tissue).

https://doi.org/10.1371/journal.pone.0061231.t003

GO analysis

GO classes with an F-statistic p value of <0.05 following analysis on the basis of their biological processes are shown in Figure 1. Those GO-class processes found more frequently in the PDL tissues of permanent teeth include neurological system processes, cognition, and response to organic substances. In contrast, the GO-class processes found more frequently in the PDL tissues of deciduous teeth included positive regulation of cellular biosynthetic processes and response to wounding.

thumbnail
Figure 1. Main categories of genes expressed specifically in deciduous PDL tissues (de-PDL) and permanent PDL tissues (pe-PDL) on the basis of their biological processes.

Numbers of involved genes are listed in x-axis, F-statistic p<0.05.

https://doi.org/10.1371/journal.pone.0061231.g001

The GO classes with an F-statistic p value of <0.05 following analysis on the basis of their molecular functions are shown in Figure 2. These classes did not differ noticeably between the PDL tissues of deciduous teeth and those of permanent teeth.

thumbnail
Figure 2. Main categories of genes expressed specifically in de-PDL and pe-PDL on the basis of their molecular functions.

Numbers of involved genes are listed in the pie charts, F-statistic p<0.05.

https://doi.org/10.1371/journal.pone.0061231.g002

qRT-PCR

qRT-PCR analysis was performed to verify the differential expression levels determined via cDNA microarray analysis. Among interesting genes, we selected three genes that are expressed more in the deciduous PDL tissues and three genes that are expressed more in the permanent PDL tissues. The six genes (i.e., LAMC2, CCL21, IGF2BP1, IL6, LRRTM1, and ADAMTSL3) that were selected for this verification procedure exhibited an increase of at least fourfold in the gene expression level compared to the other tissue types (Table 4). The results are consistent with those of the cDNA microarray analysis.

thumbnail
Table 4. The relative gene expressions in the deciduous and permanent PDL tissues.

https://doi.org/10.1371/journal.pone.0061231.t004

IHC staining

The following four proteins were the targets of the IHC study: IGF2BP1, IL6, ADAMTLS3, and LRRTM1 (Figure 3). IGF2BP1 was broadly stained in the deciduous PDL tissues, but only weakly in permanent PDL tissues, with the exception of the epithelial cell rests of Malassez. ADAMTLS3 and LRRTM1 were stained more strongly across the board in permanent compared to deciduous PDL tissues. IL6 was located mainly around blood vessels, and its staining was more prominent in the permanent PDL tissues.

thumbnail
Figure 3. Immunohistochemical (IHC) staining of permanent and deciduous PDL tissues (A–P).

(A, E) IHC staining for IGF2BP1 in permanent and (I, M) deciduous PDL tissues. (B, F) IHC staining for IL6 in permanent and (J, N) deciduous PDL tissues. (C, G) IHC staining for LRRTM1 in permanent and (K, O) deciduous PDL tissues. (D, H) IHC staining for ADAMTSL3 in permanent PDL tissues and (L, P) deciduous PDL tissues. Abbreviations: pe-PDL, permanent PDL tissues; de-PDL, deciduous PDL tissues. Scale bars: 100 µm in A–D and M–P; 20 µm in E–H and I–L.

https://doi.org/10.1371/journal.pone.0061231.g003

Discussion

The periodontal tissues used in this study were located at the surface of the extracted teeth. PDLs usually tear across their middle during tooth extraction, and they contain not only PDL fibroblasts, epithelial cell rests of Malassez, cementoblasts, vascular cells, and sensory nerve cells, but also osteoblasts, which make and maintain the alveolar bone. However, since there are no reports about differences in cell populations between the PDLs of permanent and deciduous teeth, the obtained RNAs must have originated from these cell populations.

Just 51 of the known >20,000 human genes [24] were differentially expressed by more than twofold between the two PDL tissue types. This indicates that while differences do exist between the tissues, their compositions and functions are very similar. There were similar quantities of genes predominantly or specific expressed in PDLs (periostin, scleraxis, and collagen XII) [25][27] and cementoblasts [cementum protein-23 (CP-23) and cementum attachment protein (CAP)] [28], [29]. In addition, inhibitors of PDL mineralization (aspirin) [30], matrix metalloproteinases (MMPs) [31], which are involved with turnover of the PDL matrix, and factors associated with osteoclast differentiation [RANKL and osteoprotegerin (OPG)] [32] were expressed to similar degrees in the two tissue types. This similarity probably explains why histological differences between permanent PDLs and deciduous PDLs have seldom been reported previously.

The changes that the PDL undergoes during the root resorption of deciduous teeth have been investigated previously. Fukushima et al. [12] insisted that cells obtained from the root-resorbing area of deciduous PDLs expressed more RANKL (encoding a factor that enhances osteoclastogenesis) and less OPG (encoding a factor that suppresses osteoclastogenesis), and that cells isolated from either nonresorbing deciduous teeth or permanent teeth expressed equal ratios of RANKL and OPG, in line with our present findings. Furthermore, one group reported that the mucopolysaccharidase and collagenolytic activities of deciduous PDL were higher in a resorbing root than in nonresorbing roots and permanent PDLs [17], [33]. Another study showed that the IHC signals of bone sialoprotein and osteopontin, which contain the osteoclast-binding RGD sequence, were higher in the root-resorbing area than in normal deciduous PDL and permanent PDL areas [10]. Our finding of no differences in those genes between the two types of PDL tissues is probably due to the PDL in the root-resorbing area not being included in the analysis. However, although several of the molecular and cellular events that occur during the resorption of deciduous roots have been revealed, further investigation is required to determine which factor awakens silent deciduous PDLs to become involved in the root-resorbing process.

Several of the genes that were up-regulated in deciduous PDL tissues were associated with the formation of the extracellular matrix and organ development. For example, LAMC2 and laminin beta 3 (LAMB3) encode the laminin family proteins, which are components of the basement membrane. Laminin is known to be involved in a wide variety of biological processes such as cell adhesion, differentiation, migration, signaling, neurite outgrowth, and metastasis [34], while cartilage oligomeric matrix protein (COMP) is known to be a type of noncollagenous extracellular matrix protein, a marker of cartilage turnover [35], and an abundant component of tendon [36]. IGF2BP1 plays an important role in control dental development [37], and regulates the growth factor IGF2. Phosphate-regulating endopeptidase homolog, X-linked (PHEX) is involved in bone and dentin mineralization and renal phosphate reabsorption [38]. It is thought that Mab-21-like 2 (C. elegans) (MAB21L2) is involved in neural development [39]. Paired box 3 (PAX3) has been identified in association with fetal development [40]. The present study is the first to demonstrate that these genes are expressed more strongly in deciduous PDL than permanent PDL, but their precise functions in periodontal tissues remain to be elucidated.

Interestingly, in spite of their origin in the nonresorbed area, the deciduous PDL tissues expressed more genes associated with inflammation or immune reaction than did the permanent PDL tissues. The biological function of CCL21 and chemokine (C-C motif) ligand 18 (CCL18) is chemotaxis for lymphocytes, the inflammatory response, and the immune response [41], [42]. In addition, these chemoattractants exhibit a slight chemotactic activity for monocytes [43], [44], which are precursors of osteoclasts or odontoclasts. It is therefore suggested that these chemokines are involved in the degradation of deciduous PDL tissues and roots. Interleukin 1, alpha (IL1A) is involved in the immune response, inflammatory processes, and apoptosis [45], [46], and can enhance the biosynthesis of prostaglandin [47] and some kinds of MMPs [48]. In addition, it up-regulates RANKL and down-regulates OPG in PDL cells [49], and positively affects the survival and differentiation of osteoclasts or odontoclasts and consecutive bone or tooth resorption [50][52]. Therefore, deciduous PDL tissues appear to be prone to resorption because of the relatively strong expressions of genes associated with tissue destruction even in the normal functioning state.

Several of the up-regulated genes in permanent PDL tissues seem to be associated with degradation of the extracellular matrix. IL6 functions as a proinflammatory cytokine and plays a role in orthodontic movement and lipopolysaccharide-induced inflammatory reactions [53][55]. ADAMTS18 belongs to the ADAMTS family of extracellular proteases, which are zinc-dependent metalloproteinases that play an important role in both normal and pathological events, and especially in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis, and cell migration [56]. It has been reported that PDL cells and cementoblasts regulate the extracellular accumulation of a large extracellular matrix proteoglycan using the degrading enzymes ADAMTS1, -4, and -5, which are members of ADAMTS family [57]. Interestingly, stimulation of IL6 up-regulates the expressions of ADAMTS4 and ADAMTS-5 [58]. Although it is unknown whether IL6 and ADAMTS18 are related, genes that were shown in the present study to be up-regulated in permanent PDL tissues are likely to play a part in the turnover of the extracellular matrix in permanent PDL tissues.

On the other hand, several of the genes that are relatively strongly expressed in permanent PDL tissues are associated with myocontraction. In the present study, IL6 was found mainly around the blood vessels, and it is known that IL6 induces the expression of vascular endothelial growth factor and is associated with angiogenesis [59]. Phosphodiesterase 3B, cGMP-inhibited (PDE3B) is expressed in vascular smooth muscle cells and is thought to modulate contraction [60], [61]. Calsequestrin 2 (CASQ2) is a calcium-binding protein of the sarcoplasmic reticulum in cardiac and slow skeletal muscle; the release of calsequestrin-bound calcium triggers muscle contraction [62]. Myosin, heavy chain 10, non-muscle (MYH10) regulates cell adhesion and migration [63]. While it is not certain that these aforementioned genes are related to the presence of abundant blood vessels or abundant myofibroblasts, which are generally present in organs with a high remodeling capacity such as the PDL [64], unfortunately there are no reports of quantitative differences in blood vessels or myofibroblasts between permanent and deciduous PDL tissues.

Some of the genes that were relatively strongly expressed in permanent PDL tissues are associated with neural function. According to the GO groupings identified for biological processes and molecular functions, the genes involved in neurological system processes were expressed much more strongly in permanent than in deciduous PDL tissues. Only PAX3 plays a role in this function in deciduous PDL tissues, but in permanent PDL tissues, eight genes were identified: FOS, V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), neural cell adhesion molecule 2 (NCAM2), MYH10, synaptotagmin I (SYT1), solute carrier family 22, member 3 (SLC22A3), cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), and jun proto-oncogene (JUN). Among these genes, FOS is known to be an indirect marker of neuronal activity because it is often expressed when neurons fire action potentials [65], NCAM2 is a type I membrane protein and is thought to induce neurite outgrowth [66], [67], SYT1 functions as a calcium regulator of neurotransmitter release [68], and SLC22A3 is an extraneuronal monoamine transporter and is involved in neurotransmission [69]. Furthermore, among the genes that are relatively strongly expressed in permanent PDL tissues, dedicator of cytokinesis 3 (DOCK3), LRRTM1, leucine-rich repeat transmembrane neuronal 3 (LRRTM3), protease, serine 12 (PRSS12), and cAMP-regulated phosphoprotein, 21 kDa (ARPP21) are genes known to be associated with neuronal signaling systems. DOCK3 is specifically expressed in neurons, especially in the central nervous system, and induces axonal outgrowth [70]. LRRTM1 and LRRTM3 are transmembrane proteins that contain many leucine-rich repeats; they are found in neurons and are associated with synaptic function [71], [72]. PRSS12 is a mosaic serine protease that is expressed preferentially in motor neurons, and defects of this gene are correlated with mental retardation [73]. ARPP21 is enriched in the caudate nucleus and cerebellar cortex, and regulates the effects of dopamine [74]. Although we were unable to determine the precise function of these genes, the up-regulation of genes related to neurological system processes in permanent PDL tissues is consistent with the degree of innervation being lower for deciduous incisors and canines than for permanent premolars [3], [75], [76].

In conclusion, the genes in deciduous PDL tissues that are up-regulated relative to their permanent counterparts are involved in the formation of the extracellular matrix and inflammation or immune reactions, leading to PDL degradation and root resorption. Those that are up-regulated in permanent compared to deciduous PDL tissues are related to the regulation of extracellular components, myocontraction, and neurological responses. Although only the RNA from the entire periodontal tissue of permanent and deciduous teeth was investigated in this study, and not from the individual diverse types of cells that constitute these tissues, our results provide clues as to their respective functions and support the histologic findings for permanent and deciduous PDL tissues.

Supporting Information

Figure S1.

M-A plot comparing three deciduous PDL tissue samples and three permanent PDL tissue samples. In each plot the x-axis (A) is 0.5×(log2(case)+log2(control)) and the y-axis (M) is log2(case/control). M and A correspond to the difference between the log intensities and the average log intensity, respectively. The data of all plots were normally distributed.

https://doi.org/10.1371/journal.pone.0061231.s001

(TIF)

Author Contributions

Conceived and designed the experiments: JSS SOK. Performed the experiments: DHH JSS SOK MJ. Analyzed the data: DHH JSS SOK MJ HSJ SJM BJC HJC. Contributed reagents/materials/analysis tools: DHH JSS SOK HJC WP. Wrote the paper: JSS DHH. Approved the paper finally: HJC.

References

  1. 1. Lukinmaa PL Waltimo WJ (1992) Immunohistochemical localization of types I, V, and VI collagen in human permanent teeth and periodontal ligament. J Dent Res 71: 391–397.
  2. 2. MacNeil RL, Berry JE, Strayhorn CL, Shigeyama Y, Somerman MJ (1998) Expression of type I and XII collagen during development of the periodontal ligament in the mouse. Arch Oral Biol 43: 779–787.
  3. 3. Itoh K (1976) The distribution of nerves in human deciduous and permanent teeth. Arch Histol Jpn 39: 379–399.
  4. 4. Johnsen D, Johns S (1978) Quantitation of nerve fibres in the primary and permanent canine and incisor teeth in man. Arch Oral Biol 23: 825–829.
  5. 5. Ranly D, Garcia-Godoy F (2000) Current and potential pulp therapies for primary and young permanent teeth. J Dent 28: 153–161.
  6. 6. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, et al. (2003) SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100: 5807–5812.
  7. 7. Song JS, Kim SO, Kim SH, Choi HJ, Son HK, et al. (2012) In vitro and in vivo characteristics of stem cells derived from the periodontal ligament of human deciduous and permanent teeth. Tissue Eng Part A 18: 2040–2051.
  8. 8. Harokopakis-Hajishengallis E (2007) Physiologic root resorption in primary teeth: molecular and histological events. J Oral Sci 49: 1–12.
  9. 9. Davies KR, Schneider GB, Southard TE, Hillis SL, Wertz PW, et al. (2001) Deciduous canine and permanent lateral incisor differential root resorption. Am J Orthod Dentofacial Orthop 120: 339–347.
  10. 10. Lee A, Schneider G, Finkelstein M, Southard T (2004) Root resorption: the possible role of extracellular matrix proteins. Am J Orthod Dentofacial Orthop 126: 173–177.
  11. 11. Bosshardt DD, Degen T, Lang NP (2005) Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth. Cell Tissue Res 320: 399–407.
  12. 12. Fukushima H, Kajiya H, Takada K, Okamoto F, Okabe K (2003) Expression and role of RANKL in periodontal ligament cells during physiological root-resorption in human deciduous teeth. Eur J Oral Sci 111: 346–352.
  13. 13. Lossdorfer S, Gotz W, Jager A (2002) Immunohistochemical localization of receptor activator of nuclear factor kappaB (RANK) and its ligand (RANKL) in human deciduous teeth. Calcif Tissue Int 71: 45–52.
  14. 14. Alexander SA, Swerdloff M (1980) Collagenolytic activity in traumatized human primary teeth undergoing accelerated resorption. Pediatr Dent 2: 287–290.
  15. 15. Wu YM, Richards DW, Rowe DJ (1999) Production of matrix-degrading enzymes and inhibition of osteoclast-like cell differentiation by fibroblast-like cells from the periodontal ligament of human primary teeth. J Dent Res 78: 681–689.
  16. 16. Linsuwanont B, Takagi Y, Ohya K, Shimokawa H (2002) Expression of matrix metalloproteinase-9 mRNA and protein during deciduous tooth resorption in bovine odontoclasts. Bone 31: 472–478.
  17. 17. Alexander SA, Swerdloff M (1979) Mucopolysaccharidase activity during human deciduous root resorption. Arch Oral Biol 24: 735–738.
  18. 18. Suda N (2008) Comprehensive gene expression analysis in human periodontal ligaments of the mandibular third molars performing vertical movement and the maxillary second premolars with occlusal contact. Orthod Craniofac Res 11: 1–7.
  19. 19. Lee YH, Nahm DS, Jung YK, Choi JY, Kim SG, et al. (2007) Differential gene expression of periodontal ligament cells after loading of static compressive force. J Periodontol 78: 446–452.
  20. 20. Kim SH, Kim YS, Lee SY, Kim KH, Lee YM, et al. (2011) Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow. J Periodontal Implant Sci 41: 192–200.
  21. 21. Zhang Y, Kim SO, Opsahl-Vital S, Ho SP, Souron JB, et al. (2011) Multiple effects of the cellular prion protein on tooth development. Int J Dev Biol 55: 953–960.
  22. 22. Goidin D, Mamessier A, Staquet M-J, Schmitt D, Berthier-Vergnes O (2001) Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and β-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 295: 17–21.
  23. 23. Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46: 69–81.
  24. 24. Pertea M, Salzberg SL (2010) Between a chicken and a grape: estimating the number of human genes. Genome Biol 11: 206.
  25. 25. Seo BM, Miura M, Gronthos S, Mark Bartold P, Batouli S, et al. (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364: 149–155.
  26. 26. Horiuchi K, Amizuka N, Takeshita S, Takamatsu H, Katsuura M, et al. (1999) Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor β. J Bone Miner Res 14: 1239–1249.
  27. 27. Karimbux N, Nishimura I (1995) Temporal and spatial expressions of type XII collagen in the remodeling periodontal ligament during experimental tooth movement. J Dent Res 74: 313–318.
  28. 28. Alvarez-Pérez MA, Narayanan S, Zeichner-David M, Rodríguez Carmona B, Arzate H (2006) Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23). Bone 38: 409–419.
  29. 29. Wu D, Ikezawa K, Parker T, Saito M, Narayanan AS (1996) Characterization of a collagenous cementum-derived attachment protein. J Bone Miner Res 11: 686–692.
  30. 30. Yamada S, Tomoeda M, Ozawa Y, Yoneda S, Terashima Y, et al. (2007) PLAP-1/asporin, a novel negative regulator of periodontal ligament mineralization. J Biol Chem 282: 23070–23080.
  31. 31. Takahashi I, Nishimura M, Onodera K, Bae JW, Mitani H, et al. (2003) Expression of MMP-8 and MMP-13 genes in the periodontal ligament during tooth movement in rats. J Dent Res 82: 646–651.
  32. 32. Kanzaki H, Chiba M, Shimizu Y, Mitani H (2001) Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition. J Dent Res 80: 887–891.
  33. 33. Alexander SA, Swerdloff M (1980) Mucopolysaccharidase activity and glycosaminoglycan content in traumatized resorbing deciduous teeth. J Dent Res 59: 766–770.
  34. 34. Kallunki P, Sainio K, Eddy R, Byers M, Kallunki T, et al. (1992) A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J Cell Biol 119: 679–693.
  35. 35. Paulsson M, Heinegard D (1981) Purification and structural characterization of a cartilage matrix protein. Biochem J 197: 367–375.
  36. 36. DiCesare P, Hauser N, Lehman D, Pasumarti S, Paulsson M (1994) Cartilage oligomeric matrix protein (COMP) is an abundant component of tendon. FEBS Lett 354: 237–240.
  37. 37. Kim KM, Lim J, Choi Y, Kim JY, Shin HI, et al. (2012) Gene expression profiling of oral epithelium during tooth development. Arch Oral Biol 57: 1100–1107.
  38. 38. Quarles LD (2003) FGF23, PHEX, and MEPE regulation of phosphate homeostasis and skeletal mineralization. Am J Physiol Endocrinol Metab 285: E1–E9.
  39. 39. Mariani M, Baldessari D, Francisconi S, Viggiano L, Rocchi M, et al. (1999) Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development. Hum Mol Genet 8: 2397–2406.
  40. 40. Lang D, Powell SK, Plummer RS, Young KP, Ruggeri BA (2007) PAX genes: roles in development, pathophysiology, and cancer. Biochem Pharmacol 73: 1–14.
  41. 41. Schutyser E, Richmond A, Van Damme J (2005) Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes. J Leukoc Biol 78: 14–26.
  42. 42. Marsland BJ, Bättig P, Bauer M, Ruedl C, Lässing U, et al. (2005) CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells. Immunity 22: 493–505.
  43. 43. Cravens PD, Hayashida K, Davis LS, Nanki T, Lipsky PE (2007) Human peripheral blood dendritic cells and monocyte subsets display similar chemokine receptor expression profiles with differential migratory responses. Scand J Immunol 65: 514–524.
  44. 44. Schraufstatter I, Takamori H, Sikora L, Sriramarao P, DiScipio RG (2004) Eosinophils and monocytes produce pulmonary and activation-regulated chemokine, which activates cultured monocytes/macrophages. Am J Physiol Lung Cell Mol Physiol 286: L494–L501.
  45. 45. Nesic O, Xu GY, McAdoo D, Westlund High K, Hulsebosch C, et al. (2001) IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury. J Neurotrauma 18: 947–956.
  46. 46. Parker KP, Benjamin WR, Kaffka KL, Kilian PL (1989) Presence of IL-1 receptors on human and murine neutrophils. Relevance to IL-1-mediated effects in inflammation. J Immunol 142: 537–542.
  47. 47. Romero R, Durum S, Dinarello C, Oyarzun E, Hobbins J, et al. (1989) Interlukin-1 stimulates prostaglandin biosynthesis by human amnion. Prostaglandins 37: 13–22.
  48. 48. Shi J, Schmitt-Talbot E, DiMattia D, Dullea R (2004) The differential effects of IL-1 and TNF-a on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells. Inflamm Res 53: 377–389.
  49. 49. Fukushima H, Jimi E, Okamoto F, Motokawa W, Okabe K (2005) IL-1-induced receptor activator of NF-κB ligand in human periodontal ligament cells involves ERK-dependent PGE 2 production. Bone 36: 267–275.
  50. 50. Tani-Ishii N, Tsunoda A, Teranaka T, Umemoto T (1999) Autocrine regulation of osteoclast formation and bone resorption by IL-1α and TNFα. J Dent Res 78: 1617–1623.
  51. 51. Lee ZH, Lee SE, Kim CW, Lee SH, Kim SW, et al. (2002) IL-1α stimulation of osteoclast survival through the PI 3-kinase/Akt and ERK pathways. J Biochem 131: 161–166.
  52. 52. Zhang D, Goetz W, Braumann B, Bourauel C, Jaeger A (2003) Effect of soluble receptors to interleukin-1 and tumor necrosis factor alpha on experimentally induced root resorption in rats. J Periodontal Res 38: 324–332.
  53. 53. Kiecolt-Glaser JK, Preacher KJ, MacCallum RC, Atkinson C, Malarkey WB, et al. (2003) Chronic stress and age-related increases in the proinflammatory cytokine IL-6. Proc Natl Acad Sci U S A 100: 9090–9095.
  54. 54. Yamamoto T, Kita M, Kimura I, Oseko F, Terauchi R, et al. (2006) Mechanical stress induces expression of cytokines in human periodontal ligament cells. Oral Dis 12: 171–175.
  55. 55. Yamaji Y, Kubota T, Sasaguri K, Sato S, Suzuki Y, et al. (1995) Inflammatory cytokine gene expression in human periodontal ligament fibroblasts stimulated with bacterial lipopolysaccharides. Infect Immun 63: 3576–3581.
  56. 56. Tang BL (2001) ADAMTS: a novel family of extracellular matrix proteases. Int J Biochem Cell Biol 33: 33–44.
  57. 57. Sone S, Nakamura M, Maruya Y, Takahashi I, Mizoguchi I, et al. (2005) Expression of versican and ADAMTS during rat tooth eruption. J Mol Histol 36: 281–288.
  58. 58. Mimata Y KA, Oikawa S, Murakami K, Uzuki M, Shimamura T, et al. (2012) Interleukin-6 upregulates expression of ADAMTS-4 in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Int J Rheum Dis 15: 36–44.
  59. 59. Motro B, Itin A, Sachs L, Keshet E (1990) Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis. Proc Natl Acad Sci U S A 87: 3092–3096.
  60. 60. Rybalkin SD, Yan C, Bornfeldt KE, Beavo JA (2003) Cyclic GMP phosphodiesterases and regulation of smooth muscle function. Circ Res 93: 280–291.
  61. 61. Polson JB, Strada SJ (1996) Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annu Rev Pharmacol Toxicol 36: 403–427.
  62. 62. Terentyev D, Kubalova Z, Valle G, Nori A, Vedamoorthyrao S, et al. (2008) Modulation of SR Ca release by luminal Ca and calsequestrin in cardiac myocytes: effects of CASQ2 mutations linked to sudden cardiac death. Biophys J 95: 2037–2048.
  63. 63. Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR (2009) Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10: 778–790.
  64. 64. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3: 349–363.
  65. 65. VanElzakker M, Fevurly RD, Breindel T, Spencer RL (2008) Environmental novelty is associated with a selective increase in Fos expression in the output elements of the hippocampal formation and the perirhinal cortex. Learn Mem 15: 899–908.
  66. 66. Kulahin N, Walmod PS (2010) The neural cell adhesion molecule NCAM2/OCAM/RNCAM, a close relative to NCAM. Adv Exp Med Biol 663: 403–420.
  67. 67. Winther M, Berezin V, Walmod PS (2012) NCAM2/OCAM/RNCAM: Cell adhesion molecule with a role in neuronal compartmentalization. Int J Biochem Cell Biol 44: 441–446.
  68. 68. Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF, et al. (2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature 410: 41–49.
  69. 69. Mooney JJ, Samson JA, Hennen J, Pappalardo K, McHale N, et al. (2008) Enhanced norepinephrine output during long-term desipramine treatment: a possible role for the extraneuronal monoamine transporter (SLC22A3). J Psychiatr Res 42: 605–611.
  70. 70. Namekata K, Harada C, Taya C, Guo X, Kimura H, et al. (2010) Dock3 induces axonal outgrowth by stimulating membrane recruitment of the WAVE complex. Proc Natl Acad Sci U S A 107: 7586–7591.
  71. 71. Laurén JAM, Saarma M, Timmusk T (2003) A novel gene family encoding leucine-rich repeat transmembrane proteins differentially expressed in the nervous system. Genomics 81: 411–421.
  72. 72. Linhoff MW, Laurén J, Cassidy RM, Dobie FA, Takahashi H, et al. (2009) An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers. Neuron 61: 734–749.
  73. 73. Mitsui S, Yamaguchi N, Osako Y, Yuri K (2007) Enzymatic properties and localization of motopsin (PRSS12), a protease whose absence causes mental retardation. Brain Res 1136: 1–12.
  74. 74. Ouimet C, Hemmings H Jr, Greengard P (1989) ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Immunocytochemical localization in rat brain. J Neurosci 9: 865–875.
  75. 75. Johnsen DC (1985) Innervation of teeth: qualitative, quantitative, and developmental assessment. J Dent Res 64 Spec No: 555–563.
  76. 76. Rapp R, Avery JK, Strachan DS (1967) The distribution of nerves in human primary teeth. Anat Rec 159: 89–103.