Effect of follicular dendritic cell secreted protein on gene expression of human periodontal ligament cells

Archives of Oral Biology 81 (2017) 151–159

Contents lists available at ScienceDirect

Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio

Effect of follicular dendritic cell secreted protein on gene expression of human periodontal ligament cells ⁎

Lin Xianga,b, Na Xina,b, Ying Yuana,b, Xiaogang Houc, Junwei Chend, Na Weia,b, , Ping Gonga,b,

MARK ⁎

a State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China b Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China c College of Hydraulic and Hydroelectric Engineering, Sichuan University, Chengdu 610041, China d Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100000, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Follicular dendritic cell secreted protein (FDCSP) Lentiviral vector Human periodontal ligament cells (hPDLCs) Microarray analysis Differential gene expression

Objective: The objective of this study was to investigate the specific roles of follicular dendritic cell secreted protein (FDC-SP), a protein exists in saliva, in the inhibition of calcium precipitation during periodontal regeneration, as well as affect phenotype expression of human periodontal ligament cells (hPDLCs) during the differentiation process. Design: To investigate this, we applied microarray technology to identify gene expression changes in hPDLCs transfected with FDC-SP and then clustered them according to their biological functions. Results: One hundred seventy-one genes were found differentially expressed by at least two-fold between FDC-SP -transfected and empty vector-transfected cells. Besides, genes encoding cell-cycle proteins, blood-related and cell differentiation-related proteins tended to be up-regulated after FDC-SP transfection, whereas cytokine/ growth factors, signal transduction and metabolism-related genes tended to be down-regulated in hPDLCs overexpression FDC-SP. Conclusions: The present study investigated FDC-SP’s roles in hPDLCs’ phenotype expression, via comparing the gene expression profiles between FDC-SP -transfected hPDLCs and empty vector-transfected cells upon microarray analysis. hPDLCs overexpression FDC-SP appear to display different gene expression patterns. In all, these observations showed a potential of FDC-SP in the maintenance of PDL homeostasis and its ultimate contribution to periodontal would-healing processes.

1. Introduction Periodontal ligament (PDL) plays quite important roles in proprioception, tooth support and acts as a receptor of biting forces and shock absorber against the mastication impact (Lv et al., 2009; Ralph 1982; Poiate, de Vasconcellos, de Santana, & Poiate, 2009). It is an unmineralized connective tissue that connects cementum and alveolar bone, which located between the alveolar bone and the tooth. Periodontal ligament cells (PDLCs) are considered as an ideal cell type for periodontal regeneration including the restoration of periodontal ligament, cementum and alveolar bone, due to their mesenchymal stem cell-like properties (Gjertsen, Stothz, Neiva, & Pileggi, 2011; Ivanovski S, Haase, & Bartold, 2001; Ishikawa et al., 2009). Interestingly, the PDL always maintains its width unmineralized despite the mechanical stress

in physiological conditions. Therefore, it has been hypothesized that PDLCs may have regulatory mechanisms to inhibit their osteogenesis (Kato et al., 2005). Recently, follicular dendritic cell secreted protein (FDC-SP), a novel small protein, has been originally identified in primary follicular dendritic cells isolated from human tonsils (Al-Alwan et al., 2007; Marshall et al., 2002). Then, Nakamura et al. identified this novel protein in human PDL tissue (Nakamura et al., 2005). FDC-SP has been proved to prevent calcium precipitation in PDL tissue (Xiang, Ma, He, Wei, & Gong, 2014). However, the molecular mechanism of how FDCSP regulates osteogenic activity of PDLCs has been less studied. Nowadays, lentiviral vectors have been broadly used in functional genomics (Li et al., 2010; Logan, Lutzko, & Kohn, 2002). A series of studies have shown application of lentiviral vectors containing specific

⁎ Corresponding authors at: State Key Laboratory of Oral Diseases, Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, No 14th, 3rd section, Renmin South Road, Chengdu, Sichuan 610041, China. E-mail addresses: [email protected] (L. Xiang), [email protected] (N. Xin), [email protected] (Y. Yuan), [email protected] (X. Hou), [email protected] (J. Chen), [email protected] (N. Wei), [email protected] (P. Gong).

http://dx.doi.org/10.1016/j.archoralbio.2017.05.005 Received 22 August 2016; Received in revised form 27 April 2017; Accepted 14 May 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.

Archives of Oral Biology 81 (2017) 151–159

L. Xiang et al.

US) in Hybridization Oven (Agilent technologies, Santa Clara, CA, US). After 17 h hybridization, slides were washed in staining dishes (Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US). Slides were scanned by Agilent Microarray Scanner (Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution = 3 μm, 20bit. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). The entire microarray experiments were performed at the National Engineering Center for Biochip at Shanghai, China. The differentiated genes were selected based on the criteria of P < 0.05 and a fold change of ≥ 2 of their expression values between the two groups. As for gene enrichment, GO analysis was applied to analyze the main function of the differential expression genes according to the Gene Ontology. Generally, Fisher’s exact test and χ2 test were used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P-value. The FDR was defined as N FDR = 1 − Tk , where Nk refers to the number of Fisher’s test P-values less than χ2 test P-values. We computed P-values for the GOs of all the differential genes. Enrichment provides a measure of the significance of the function: as the enrichment increases, the corresponding function is more specific, which helps us to find those GOs with more concrete function description in the experiment. Within the significant category, the enrichment Re was given by: Re = (nf/n)/(Nf/N), where nf is the number of differential genes within the particular category, n is the total number of genes within the same category, Nf is the number of differential genes in the entire microarray, and N is the total number of genes in the microarray. The data were submitted to the GEO database (http://www.ncbi. nlm.nih.gov/geo/) with a series accession number GSE61384. All data are compliant with MIAME standards.

genes to obtain efficient and stable transgene expression in bone marrow and other tissue cells (Bu, Xin, Toneff, Li, & Li, 2009; Pawelczyk et al., 2009). In the present study, we firstly established a recombinant lentiviral vector containing FDC-SP and obtained safe and efficient FDC-SP overexpression in human periodontal ligament cells (hPDLCs). After that, we applied microarray technology to identify differentially expressed genes between empty vector-transfected hPDLCs and FDC-SP -transfected ones and then clustered them according to their biological functions. It is hypothesized that this information will provide insight into changes in cell phenotype expression during periodontal restoration, as well as the molecular process that FDC-SP regulates hPDLCs’ differentiation. 2. Materials and methods 2.1. Cell culture hPDLCs were isolated and cultured via standard techniques as previously described (Xiang et al., 2014). Briefly stated, premolars extracted from healthy voluntary donors with matching ages (12–14 years old) were used. Informed consent had been obtained from patients, and the study protocol was approved by the Ethics Committee of Sichuan University. Periodontal ligament were separated from only the middle third of the roots and were cut into small pieces. After a 30min enzymatic digestion (0.05% trypsin and 0.15% collagenase; Sigma, St Louis, MO, USA) and centrifugation, single cells in suspension were obtained and then cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA), containing 20% fetal bovine serum (FBS; Gibco, Grand Island, NY), 200 mM L-glutamine (Invitrogen, Life Technologies Co.), 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate, at 37 °C with 5% CO2. Cell culture was continued with medium changes every three days until hPDLCs reached subconfluency. Cells at passage 2–4 were used in the following tests.

2.4. Real-time PCR 2.2. FDC-SP genes transfection For real-time PCR, cDNA was synthesized using PrimeScript Reverse Transcriptase (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. Real-time PCR was carried out in triplicate and performed using an ABI PRISM 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA), the cycling conditions were chosen according to the manufacturer’s instructions. Expression levels of formin2 (FMN2), SRY (sex determining region Y)-box 12 (SOX12), early growth response 1 (EGR1) and insulin-like growth factor 1 (somatomedin C) (IGF1) were normalized to glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) house-keeping gene expression and performed according to the 2−ΔΔCt method, then presented as fold increase relative to the control group.

hPDLCs’ transfection with FDC-SP was conducted as previously described (Xiang et al., 2014). Briefly, 2 × 105 hPDLCs were incubated in the mixture containing growth medium and viral supernatant (1:1) with 5 ug/ml polybrene (Sigma) for 10 h. The transfected cells were then washed twice with PBS, and cultured in normal DMEM medium containing 20% FBS for 96 h. Two different groups were designed for further research experiments, including the negative control group (transfection with empty lentiviral vector), and the experimental group (transfection with lentiviral vector containing FDC-SP). As for the transfection efficiency, we applied an inverted fluorescent microscope (OLYMPUS IX70, Japan) to analyze the expression of green fluorescence protein (GFP) 2 days later, and the transfection efficiency turned to be approximately 80%.

2.5. Statistical analysis

2.3. RNA extraction and microarray analysis

All assays were performed in triplicate and each experiment was repeated at least three times. The results were presented as mean standard deviation (SD). The microarray bioinformatic analysis enrolled in this study applied Student’s t-test to analyze the differential expression genes. In addition, P-value < 0.05 was considered to indicate a statistically significant difference.

Agilent Whole Human Genome Oligo Microarray (4 × 44 K) (Agilent Technologies, Palo Alto, CA) was applied in the following experiments. Firstly, total RNA was extracted using TRIZOL Reagent (Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (QIAGEN, GmBH, Germany) and RNase-Free DNase Set (QIAGEN, GmBH, Germany). Then, total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (QIAGEN, GmBH, Germany). Each Slide was hybridized with 1.65 μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Agilent technologies, Santa Clara, CA,

3. Results 3.1. Identification of robust and consistent differences in gene expression patterns Differences in gene expression were analyzed via the SAS analysis system (Shanghai Biotechnology Co, Ltd). We compared all experimental samples to all control ones. This comparison of FDC-SP -transfected hPDLCs to empty vector-transfected cells identified 171 152

Archives of Oral Biology 81 (2017) 151–159

L. Xiang et al.

genes (21 vs. 13) were expressed at higher level in the control group than in the experimental one. 3.3. Verification of microarray results by real-time PCR analysis To confirm the microarray data, we selected 4 genes (FMN2, SOX12, EGR1 and IGF1) with various levels of differential expression, and representing different functional groups, for analysis by Real-time PCR (Fig. 2). The relative amount of FMN2 mRNA and SOX12 mRNA to GAPDH were 2.2-fold and 2.3-fold higher in FDC-SP -transfected hPDLCs than in empty vector-transfected cells, while the relative amount of EGR1 and IGF1 were 4.1-fold and 2.4-fold higher in the control group than in the experimental one, concordant with their 2.09fold, 2.22-fold, 4.17-fold and 2.38-fold differential expression, respectively, observed in microarray results. 4. Discussion Up to now, several studies have suggested that PDLCs which consist of heterogeneous cell populations have the capacity to differentiate into various cell types due to their mesenchymal stem cell-like properties with multilineage differentiation potential (Nagatomo et al., 2006; Seo et al., 2004; Silvério et al., 2010). However, the characteristics and intrinsic functions of PDLCs are not yet fully understood. In our previous studies, we successfully transfected FDC-SP into hPDLCs (confirmed by western blot) and found that transfection with FDC-SP had negligible adverse effect on proliferation of hPDLCs and implied the biological function of FDC-SP as a fibroblastic phenotype stabilizer by inhibiting hPDLCs differentiation into mineralized tissue-forming cells (Xiang et al., 2014). To further provide insight into molecular process of hPDLCs phenotype expression regulated by FDC-SP, we applied microarray technology to investigate the pattern of gene differential expression after FDC-SP transfection. It was first observed that cell-cycle regulation proteins were up-regulated in FDC-SP −transfected hPDLCs compared with the empty vector control. FMN2 was observed up-regulated in hPDLCs overexpressing FDC-SP, with a differential expression ratio of 2.09. FMN2 belongs to a family of ubiquitous, conserved multidomain proteins called formins (Faix & Grosse, 2006). Formins are defined by the presence of a formin homology (FH) domain, which confers an actin-nucleating activity to these proteins. Besides, formin domains are required for nucleation of actin filaments in the cytoskeleton. Yamada et al. concluded in their previous study that FMN2 enhances expression of the cell-cycle inhibitor p21 by preventing its degradation. FMN2 is also induced by activation of other oncogenes, hypoxia, and DNA damage. Their results identified FMN2 as a crucial component in the regulation of p21 and consequent oncogene/stress-induced cell-cycle arrest in human cells (Yamada, Ono, Perkins, Rocha, & Lamond, 2013). In addition, Yamada et al. have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, which have in common activation of cell cycle arrest (Yamada, Ono, Bensaddek, Lamond, & Rocha, 2013). Therefore, the preferential expression of cell-cycle regulation genes in FDC-SP -transfected hPDLCs provides information of FDC-SP’s role in the regulation of cell proliferation status. This could also help us understand the reaction of PDLCs in the response to the early periodontal wound-healing processes (Oates, Mumford, Carnes, & Cochran, 2001). Another interesting observation in this study was the up-regulation of cell differentiation genes in FDC-SP -transfected group compared with the control one (3 vs. 1). SOX12 is a protein that in humans is encoded by the SOX12 gene (Hoser et al., 2008; Jay et al., 1997). Members of the SOX family of transcription factors are characterized by the presence of a DNA-binding high mobility group (HMG) domain, homologous to the HMG box of sex-determining region Y (SRY). Forming a subgroup of the HMG domain superfamily, SOX proteins have been implicated in cell fate decisions in a diverse range of

Fig. 1. Hierarchical clustering analysis of differential expression genes in hPDLCs with and without FDC-SP transfection. Red color represents the up-regulated genes, and green or blue color represents the down-regulated genes in hPDLCs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

genes (73 increases and 98 decreases) which were differentially expressed. To determine the relationships between the individual samples, hierarchical clustering was carried out (Fig. 1). The hierarchical clustering demonstrated that the 3 experimental samples and the 3 control samples clustered together. This result showed that the two cell populations had distinct transcriptional profiles. 3.2. Functional analysis of differential gene expression To analyze the changes in gene expression pattern more thoroughly, we sorted all 171 of these genes according to their biological functions (Table 1). We found that there were significant differences between FDC-SP -transfected hPDLCs and empty vector- transfected hPDLCs on several functional groups. First, 14 genes regulating cell-cycle were more abundantly expressed in FDC-SP -transfected hPDLCs compared with the control, with the most significant one, NIMA (never in mitosis gene A)-related kinase 2 (NEK2), having a 3.17-fold differential expression. However, only 1 gene in the same functional group was up-regulated in empty vector-transfected hPDLCs, with a comparatively low differential expression ratio (2.27). Furthermore, 6 blood-related and cell differentiation related genes were overexpressed in the experimental group, whereas only 2 genes showed higher expression in the control group. On the other hand, many more cytokine/growth factors (8 vs. 3), signal transduction (38 vs. 17), and metabolism-related 153

154

CEBPA

2.18

LIPH

ZNF229 CLUL1

HSPC072

C6orf223

TDRD12

DNAJC3-AS1

MYPN

PREDICTED: Homo sapiens DNAJC3 antisense RNA 1 (non-protein coding) (DNAJC3-AS1), miscRNA [XR_109147] Homo sapiens tudor domain containing 12 (TDRD12), mRNA [NM_001110822] Homo sapiens chromosome 6 open reading frame 223 (C6orf223), transcript variant 1, mRNA [NM_153246] Homo sapiens uncharacterized LOC29075 (HSPC072), transcript variant 1, non-coding RNA [NR_026883] Homo sapiens zinc finger protein 229 (ZNF229), mRNA [NM_014518] Homo sapiens clusterin-like 1 (retinal) (CLUL1), transcript variant 1, mRNA [NM_014410] lipase, member H [Source:HGNC Symbol;Acc:18483] [ENST00000296252] 2.1

2.12 2.11

2.19

2.42

2.43

2.69

2.82

3.6

LOC100505633

Metabolism-related: CTAG1A

RBP5

RBP1

APC2

FAM107A

FAM197Y2P

CARHSP1

ATF7

3.72

RGS6

FSIP2

NR4A3

Homo sapiens regulator of G-protein signaling 6 (RGS6), transcript variant 10, mRNA [NM_001204424] Homo sapiens fibrous sheath interacting protein 2 (FSIP2), mRNA [NM_173651] Homo sapiens myopalladin (MYPN), mRNA [NM_032578]

NR4A1

2.01

NFIB

ZBTB24

MIAT

FOXA3

POLB

2.05

2.09

2.12

2.14

EFCAB6

PRG3

2.18

2.14

KLF6

PRUNE2

2.3 2.25

FOS

Transcription and protein processing:

2.74 2.32

Cell-cycle regulation PER1

3.17

2.03

Homo sapiens forkhead box G1 (FOXG1), mRNA [NM_005249]

Homo sapiens SPC25, NDC80 kinetochore complex component, homolog (S. cerevisiae) (SPC25), mRNA [NM_020675] Homo sapiens ubiquitin-conjugating enzyme E2C (UBE2C), transcript variant 6, mRNA [NM_181803] Homo sapiens asp (abnormal spindle) homolog, microcephaly associated (Drosophila) (ASPM), transcript variant 1, mRNA [NM_018136] Homo sapiens sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A (SEMA6A), mRNA [NM_020796] Homo sapiens discs, large (Drosophila) homolog-associated protein 5 (DLGAP5), transcript variant 1, mRNA [NM_014750] Homo sapiens kinesin family member C1 (KIFC1), mRNA [NM_002263] Homo sapiens neuromedin U receptor 2 (NMUR2), mRNA [NM_020167] Homo sapiens formin 2 (FMN2), mRNA [NM_020066]

Homo sapiens NIMA (never in mitosis gene a)-related kinase 2 (NEK2), transcript variant 1, mRNA [NM_002497] Homo sapiens TTK protein kinase (TTK), transcript variant 1, mRNA [NM_003318] Homo sapiens E2F transcription factor 2 (E2F2), mRNA [NM_004091]

Homo sapiens testis-specific transcript, Y-linked 5 (non-protein coding) (TTTY5), non-coding RNA [NR_001541] HJURP Homo sapiens Holliday junction recognition protein (HJURP), mRNA [NM_018410] Transcription and protein processing：

TTTY5

FOXG1

FMN2

NMUR2

KIFC1

DLGAP5

SEMA6A

ASPM

UBE2C

SPC25

E2F2

TTK

Cell-cycle regulation NEK2

4.17

2.04

2.04

2.04

2.08

2.22

2.22

2.22

2.27

2.33

2.38

2.38

2.50

2.56

2.63

2.78

2.86

2.94

3.13

3.23

5.00

2.27

Ratio

Homo sapiens uncharacterized LOC100505633 (LOC100505633), non3.03 coding RNA [NR_038849] (continued on next page)

Homo sapiens cancer/testis antigen 1A (CTAG1A), mRNA [NM_139250]

Homo sapiens myocardial infarction associated transcript (non-protein coding) (MIAT), transcript variant 1, non-coding RNA [NR_003491] Homo sapiens zinc finger and BTB domain containing 24 (ZBTB24), transcript variant 1, mRNA [NM_014797] Homo sapiens nuclear factor I/B (NFIB), transcript variant 3, mRNA [NM_005596] polymerase (DNA directed), beta [Source:HGNC Symbol;Acc:9174] [ENST00000522610] Homo sapiens nuclear receptor subfamily 4, group A, member 1 (NR4A1), transcript variant 1, mRNA [NM_002135] Homo sapiens nuclear receptor subfamily 4, group A, member 3 (NR4A3), transcript variant 4, mRNA [NM_173199] Homo sapiens activating transcription factor 7 (ATF7), transcript variant 2, mRNA [NM_006856] Homo sapiens calcium regulated heat stable protein 1, 24kDa (CARHSP1), transcript variant 2, mRNA [NM_001042476] Homo sapiens family with sequence similarity 197, Y-linked, member 2, pseudogene (FAM197Y2P), non-coding RNA [NR_001553] Homo sapiens family with sequence similarity 107, member A (FAM107A), transcript variant 1, mRNA [NM_007177] Homo sapiens adenomatosis polyposis coli 2 (APC2), mRNA [NM_005883] Homo sapiens retinol binding protein 1, cellular (RBP1), transcript variant 1, mRNA [NM_002899] Homo sapiens retinol binding protein 5, cellular (RBP5), mRNA [NM_031491]

Homo sapiens EF-hand calcium binding domain 6 (EFCAB6), transcript variant 1, mRNA [NM_022785] Homo sapiens forkhead box A3 (FOXA3), mRNA [NM_004497]

Homo sapiens CCAAT/enhancer binding protein (C/EBP), alpha (CEBPA), mRNA [NM_004364]

Homo sapiens FBJ murine osteosarcoma viral oncogene homolog (FOS), mRNA [NM_005252] prune homolog 2 (Drosophila) [Source:HGNC Symbol;Acc:25209] [ENST00000492157] Homo sapiens Kruppel-like factor 6 (KLF6), transcript variant A, mRNA [NM_001300] Homo sapiens proteoglycan 3 (PRG3), mRNA [NM_006093]

period homolog 1 (Drosophila) [Source:HGNC Symbol;Acc:8845] [ENST00000354903]

Description

Name

Ratio

Name

Description

Up-regulated in Empty Vector transfected hPDLCs

Up-regulated in FDC-SP transfected hPDLCs

Table 1 Differentially Expressed Genes between FDC-SP and Empty Vector Transfected hPDLCs by Microarray Analysis.a

L. Xiang et al.

Archives of Oral Biology 81 (2017) 151–159

155

EFR3B

PF4V1 CXCL12 Signal transduction：

Cytokine/growth factor： P2RY14

MAMDC2

Extracellular matrix：

FIGN ABCC8

NCF2

ATP6V0A4

SLFNL1

LBP

SERPINA12

MUCL1 POLQ

LOC100505683

CAMK2B

Homo sapiens mRNA for KIAA0953 protein, partial cds. [AB023170]

Homo sapiens purinergic receptor P2Y, G-protein coupled, 14 (P2RY14), transcript variant 2, mRNA [NM_014879] Homo sapiens platelet factor 4 variant 1 (PF4V1), mRNA [NM_002620] Homo sapiens mRNA for FLJ00404 protein. [AK090482]

Homo sapiens MAM domain containing 2 (MAMDC2), mRNA [NM_153267]

6.06

2.45 2.17

2.7

2.35

2.06 2.02

2.1

2.19

2.2

EGR3

Cytokine/growth factor： EGR1 EGR2

MEPE

SPON1

RTP1

ACAN

Extracellular matrix： HAS2

LOC100128098

MLXIPL

LOC100652951

LOC100130428

DTWD2

2.38

2.22

APOA4 DDX43

LOC100287616

SOAT2

2.52 2.49

2.63

2.69

MMP14

3.21

SLFN13

LOC728763

3.98

PREDICTED: Homo sapiens hypothetical LOC100506262 (LOC100506262), miscRNA [XR_109925] Homo sapiens schlafen family member 13 (SLFN13), mRNA [NM_144682] Homo sapiens calcium/calmodulin-dependent protein kinase II beta (CAMK2B), transcript variant 6, mRNA [NM_172082] PREDICTED: Homo sapiens hypothetical LOC100505683 (LOC100505683), miscRNA [XR_133258] Homo sapiens mucin-like 1 (MUCL1), mRNA [NM_058173] Homo sapiens polymerase (DNA directed), theta (POLQ), mRNA [NM_199420] Homo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 12 (SERPINA12), mRNA [NM_173850] Homo sapiens lipopolysaccharide binding protein (LBP), mRNA [NM_004139] Homo sapiens schlafen-like 1 (SLFNL1), transcript variant 1, mRNA [NM_144990] Homo sapiens ATPase, H+ transporting, lysosomal V0 subunit a4 (ATP6V0A4), transcript variant 1, mRNA [NM_020632] Homo sapiens neutrophil cytosolic factor 2 (NCF2), transcript variant 1, mRNA [NM_000433] Homo sapiens fidgetin (FIGN), mRNA [NM_018086] Homo sapiens ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (ABCC8), mRNA [NM_000352]

LOC100506262

JUN L1TD1 LOC283588

2.02 2.01

OXR1

LOC100506795

2.05 2.02

LOC285141

2.05

Metabolism-related：

FLCN IL5RA

C9orf84

LINC00320

C9orf153

PTGS2

2.07

IQCA1

2.07

Homo sapiens coiled-coil domain containing 158 (CCDC158), mRNA [NM_001042784] Homo sapiens cell division cycle associated 7 (CDCA7), transcript variant 1, mRNA [NM_031942] chromosome 9 open reading frame 153 [Source:HGNC Symbol;Acc:31456] [ENST00000469914] Homo sapiens long intergenic non-protein coding RNA 320 (LINC00320), non-coding RNA [NR_024090] Homo sapiens chromosome 9 open reading frame 84 (C9orf84), transcript variant 1, mRNA [NM_173521] folliculin [Source:HGNC Symbol;Acc:27310] [ENST00000466317] Homo sapiens interleukin 5 receptor, alpha (IL5RA), transcript variant 3, mRNA [NM_175725]

CCDC158

CDCA7

Name

Ratio

Description

Name

2.08

2.22

2.27

2.27

3.70

2.04

2.08

2.13

2.13

2.22

2.22 2.22

2.33

2.38

2.38

2.38

2.38

2.44 2.38

2.56

2.56

2.70

2.94

2.94

Ratio

Homo sapiens early growth response 1 (EGR1), mRNA [NM_001964] 4.17 2.86 Homo sapiens early growth response 2 (EGR2), transcript variant 1, mRNA [NM_000399] Homo sapiens early growth response 3 (EGR3), transcript variant 1, 2.78 (continued on next page)

Homo sapiens aggrecan (ACAN), transcript variant 2, mRNA [NM_013227] Homo sapiens receptor (chemosensory) transporter protein 1 (RTP1), mRNA [NM_153708] Homo sapiens spondin 1, extracellular matrix protein (SPON1), mRNA [NM_006108] Homo sapiens matrix extracellular phosphoglycoprotein (MEPE), transcript variant 2, mRNA [NM_020203]

Homo sapiens hyaluronan synthase 2 (HAS2), mRNA [NM_005328]

PREDICTED: Homo sapiens IGYY565 (LOC100130428), miscRNA [XR_110533] PREDICTED: Homo sapiens hypothetical LOC100652951 (LOC100652951), miscRNA [XR_132888] Homo sapiens MLX interacting protein-like (MLXIPL), transcript variant 1, mRNA [NM_032951] Homo sapiens uncharacterized LOC100128098 (LOC100128098), noncoding RNA [NR_034129]

Homo sapiens uncharacterized LOC100287616 (LOC100287616), transcript variant 1, non-coding RNA [NR_040066] Homo sapiens apolipoprotein A-IV (APOA4), mRNA [NM_000482] Homo sapiens DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 (DDX43), mRNA [NM_018665] Homo sapiens DTW domain containing 2 (DTWD2), mRNA [NM_173666]

Homo sapiens matrix metallopeptidase 14 (membrane-inserted) (MMP14), mRNA [NM_004995] Homo sapiens sterol O-acyltransferase 2 (SOAT2), mRNA [NM_003578]

Homo sapiens IQ motif containing with AAA domain 1 (IQCA1), mRNA [NM_024726] Homo sapiens prostaglandin-endoperoxide synthase 2 (prostaglandin G/ H synthase and cyclooxygenase) (PTGS2), mRNA [NM_000963] PREDICTED: Homo sapiens hypothetical protein LOC285141 (LOC285141), mRNA [XM_001714892] Homo sapiens uncharacterized LOC100506795 (LOC100506795), noncoding RNA [NR_038426] oxidation resistance 1 [Source:HGNC Symbol;Acc:15822] [ENST00000497705] Homo sapiens jun proto-oncogene (JUN), mRNA [NM_002228] Homo sapiens LINE-1 type transposase domain containing 1 (L1TD1), transcript variant 2, mRNA [NM_019079] PREDICTED: Homo sapiens hypothetical LOC283588 (LOC283588), miscRNA [XR_110237] –

Description

Up-regulated in Empty Vector transfected hPDLCs

Up-regulated in FDC-SP transfected hPDLCs

Table 1 (continued)

L. Xiang et al.

Archives of Oral Biology 81 (2017) 151–159

156 3.11

Homo sapiens hemoglobin, alpha 2 (HBA2), mRNA [NM_000517]

Homo sapiens hemoglobin, epsilon 1 (HBE1), mRNA [NM_005330]

Homo sapiens plasminogen (PLG), transcript variant 1, mRNA [NM_000301]

HBA2

HBE1

PLG

NETO1

Homo sapiens SRY (sex determining region Y)-box 12 (SOX12), mRNA [NM_006943]

SOX12

2.22

LAIR1

Homo sapiens one cut homeobox 2 (ONECUT2), mRNA [NM_004852]

ONECUT2

2.71

DIO1

CKMT1A

EMCN

Homo sapiens carbonic anhydrase XII (CA12), transcript variant 1, mRNA [NM_001218] KIAA1324L

ARHGAP28

Cell differentiation：

2.04

2.42

CA12

2.17

Blood-related：

KIF18B

2.21

CNTN3

Homo sapiens kinesin family member 20A (KIF20A), mRNA [NM_005733] Homo sapiens hypothetical protein LOC146909, mRNA (cDNA clone IMAGE:4418755), partial cds. [BC048263]

KIF20A

C18orf1 SAMD3

2

C21orf90

C11orf87

2.08

2.01

C11orf21

TCHH

C1orf115

NPFFR2

GRID1

OPALIN

Signal transduction： MC2R

CRLF1

IGF1

FGF13

2.09

2.1

2.13

2.18

2.21

2.24

2.3 2.27

2.32

2.39

2.47

TNFAIP8L3

Cytoskeleton-related：

FAM84A

HMMR

KCNN1

CLK1

FAM163A

ACP1

BCOR

TPH1

RRM2

EPHB1 RAD51AP1

DTL

SHCBP1

BCL6 corepressor [Source:HGNC Symbol;Acc:20893] [ENST00000501455] Homo sapiens acid phosphatase 1, soluble (ACP1), transcript variant 4, mRNA [NM_001040649] Homo sapiens family with sequence similarity 163, member A (FAM163A), mRNA [NM_173509] Homo sapiens CDC-like kinase 1 (CLK1), transcript variant 1, mRNA [NM_004071] Homo sapiens potassium intermediate/small conductance calciumactivated channel, subfamily N, member 1 (KCNN1), mRNA [NM_002248] Homo sapiens hyaluronan-mediated motility receptor (RHAMM) (HMMR), transcript variant 2, mRNA [NM_012484] Homo sapiens family with sequence similarity 84, member A (FAM84A), mRNA [NM_145175]

Homo sapiens minichromosome maintenance complex component 10 (MCM10), transcript variant 1, mRNA [NM_182751] Homo sapiens SHC SH2-domain binding protein 1 (SHCBP1), mRNA [NM_024745] Homo sapiens denticleless homolog (Drosophila) (DTL), mRNA [NM_016448] Homo sapiens EPH receptor B1 (EPHB1), mRNA [NM_004441] Homo sapiens RAD51 associated protein 1 (RAD51AP1), transcript variant 2, mRNA [NM_006479] Homo sapiens ribonucleotide reductase M2 (RRM2), transcript variant 2, mRNA [NM_001034] Homo sapiens tryptophan hydroxylase 1 (TPH1), mRNA [NM_004179]

2.58

CXCL14

MCM10

STBD1

2.93

glutamate-rich 1 [Source:HGNC Symbol;Acc:27234] [ENST00000523415] Homo sapiens cDNA FLJ90154 fis, clone HEMBB1002162. [AK074635]

ERICH1

2.63

2.78

2.86

2.86

2.94

2.94

2.94

3.03

3.13

3.70

3.70

4.17

4.17

2.04

2.38

2.38

2.56

2.78

Ratio

Homo sapiens KIAA1324-like (KIAA1324L), transcript variant 2, mRNA 2.56 [NM_152748] Homo sapiens endomucin (EMCN), transcript variant 1, mRNA 2.44 [NM_016242] Homo sapiens creatine kinase, mitochondrial 1A (CKMT1A), nuclear 2.33 gene encoding mitochondrial protein, mRNA [NM_001015001] Homo sapiens deiodinase, iodothyronine, type I (DIO1), transcript 2.27 variant 1, mRNA [NM_000792] Homo sapiens leukocyte-associated immunoglobulin-like receptor 1 2.27 (LAIR1), transcript variant b, mRNA [NM_021706] Homo sapiens neuropilin (NRP) and tolloid (TLL)-like 1 (NETO1), 2.27 transcript variant 3, mRNA [NM_138966] (continued on next page)

Homo sapiens chromosome 21 open reading frame 90 (C21orf90), transcript variant 1, non-coding RNA [NR_026547] Homo sapiens chromosome 18 open reading frame 1 (C18orf1), transcript variant a2, mRNA [NM_181482] Homo sapiens sterile alpha motif domain containing 3 (SAMD3), transcript variant 1, mRNA [NM_001017373] Homo sapiens contactin 3 (plasmacytoma associated) (CNTN3), mRNA [NM_020872] Homo sapiens Rho GTPase activating protein 28 (ARHGAP28), mRNA [NM_001010000] 2.56

chromosome 11 open reading frame 21 [Source:HGNC Symbol;Acc:13231] [ENST00000381153] Homo sapiens chromosome 11 open reading frame 87 (C11orf87), mRNA [NM_207645]

Homo sapiens melanocortin 2 receptor (adrenocorticotropic hormone) (MC2R), mRNA [NM_000529] Homo sapiens oligodendrocytic myelin paranodal and inner loop protein (OPALIN), transcript variant 3, mRNA [NM_001040103] Homo sapiens glutamate receptor, ionotropic, delta 1 (GRID1), mRNA [NM_017551] Homo sapiens neuropeptide FF receptor 2 (NPFFR2), transcript variant 2, mRNA [NM_053036] Homo sapiens chromosome 1 open reading frame 115 (C1orf115), mRNA [NM_024709] Homo sapiens trichohyalin (TCHH), mRNA [NM_007113]

mRNA [NM_004430] Homo sapiens chemokine (C-X-C motif) ligand 14 (CXCL14), mRNA [NM_004887] Homo sapiens tumor necrosis factor, alpha-induced protein 8-like 3 (TNFAIP8L3), mRNA [NM_207381] Homo sapiens fibroblast growth factor 13 (FGF13), transcript variant 1, mRNA [NM_004114] Homo sapiens insulin-like growth factor 1 (somatomedin C) (IGF1), transcript variant 4, mRNA [NM_000618] Homo sapiens cytokine receptor-like factor 1 (CRLF1), mRNA [NM_004750]

Description

Name

Description

Name

Ratio

Up-regulated in Empty Vector transfected hPDLCs

Up-regulated in FDC-SP transfected hPDLCs

Table 1 (continued)

L. Xiang et al.

Archives of Oral Biology 81 (2017) 151–159

a

157 Cell differentiation： IBSP

Blood-related： PRG4

TPTE2

CYP19A1

Cytoskeleton-related： HSD11B1

PHACTR1

GDF15

ARHGAP25

ADCY4

PTX3 TMED6

CHRNB3

LRRC32

ISLR

HLA-DRB5

FRAS1

SLC7A14

RGMA

KIAA1644 PRRG2

CNTNAP2

C1orf94

C15orf50

Homo sapiens integrin-binding sialoprotein (IBSP), mRNA [NM_004967]

Homo sapiens proteoglycan 4 (PRG4), transcript variant A, mRNA [NM_005807]

Homo sapiens hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), transcript variant 2, mRNA [NM_181755] Homo sapiens cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), transcript variant 2, mRNA [NM_031226] Homo sapiens transmembrane phosphoinositide 3-phosphatase and tensin homolog 2 (TPTE2), transcript variant 3, mRNA [NM_199254]

Homo sapiens chromosome 15 open reading frame 50 (C15orf50), noncoding RNA [NR_026764] Homo sapiens chromosome 1 open reading frame 94 (C1orf94), transcript variant 2, mRNA [NM_032884] Homo sapiens contactin associated protein-like 2 (CNTNAP2), mRNA [NM_014141] Homo sapiens KIAA1644 (KIAA1644), mRNA [NM_001099294] Homo sapiens proline rich Gla (G-carboxyglutamic acid) 2 (PRRG2), mRNA [NM_000951] Homo sapiens RGM domain family, member A (RGMA), transcript variant 4, mRNA [NM_020211] Homo sapiens solute carrier family 7 (orphan transporter), member 14 (SLC7A14), mRNA [NM_020949] Homo sapiens cDNA FLJ14927 fis, clone PLACE1009094, weakly similar to FURIN-LIKE PROTEASE 2 PRECURSOR (EC 3.4.21.75). [AK027833] Homo sapiens major histocompatibility complex, class II, DR beta 5 (HLA-DRB5), mRNA [NM_002125] Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR), transcript variant 1, mRNA Homo sapiens leucine rich repeat containing 32 (LRRC32), transcript variant 1, mRNA [NM_005512] Homo sapiens cholinergic receptor, nicotinic, beta 3 (CHRNB3), mRNA [NM_000749] Homo sapiens pentraxin 3, long (PTX3), mRNA [NM_002852] Homo sapiens transmembrane emp24 protein transport domain containing 6 (TMED6), mRNA [NM_144676] Homo sapiens adenylate cyclase 4 (ADCY4), transcript variant 2, mRNA [NM_139247] Homo sapiens Rho GTPase activating protein 25 (ARHGAP25), transcript variant 1, mRNA [NM_001007231] Homo sapiens growth differentiation factor 15 (GDF15), mRNA [NM_004864] phosphatase and actin regulator 1 [Source:HGNC Symbol;Acc:20990] [ENST00000379350]

Genes with defined threshold of two-fold differential expression for 9 functional groups are listed. Each gene is represented with a name, a brief description and the differential expression ratio.

2. 1

Homo sapiens homeobox B3 (HOXB3), mRNA [NM_002146]

HOXB3

Description

Name

Description

Name

Ratio

Up-regulated in Empty Vector transfected hPDLCs

Up-regulated in FDC-SP transfected hPDLCs

Table 1 (continued)

3.57

2.08

2.04

2.44

3.45

2.04

2.04

2.04

2.04

2.08 2.08

2.08

2.13

2.13

2.13

2.13

2.17

2.17

2.17 2.17

2.17

2.17

2.17

Ratio

L. Xiang et al.

Archives of Oral Biology 81 (2017) 151–159

Archives of Oral Biology 81 (2017) 151–159

L. Xiang et al.

structural protein of the bone matrix. It constitutes approximately 12% of the noncollagenous proteins in human bone and is synthesized by skeletal-associated cell types, the only extraskeletal site of its synthesis is the trophoblast. This protein binds to calcium and hydroxyapatite via its acidic amino acid clusters, and mediates cell attachment (Ayari & Bricca, 2012). Taken together, based on the relatively downexpression of IBSP gene in FDC-SP -transfected hPDLCs, we speculated that FDC-SP might prevent calcium precipitation in PDL tissue, thus maintaining PDL width unmineralized despite the mechanical stress in tooth movement. However, due to the sample selection of this study, it actually has not represented the overall populations in regard to gender, age, as well as genetic background. Cautions must be conducted in extrapolating the current data to the general population. Meanwhile, due to limitations intrinsic to the microarray technology, currently available chips include only a subset of human genes. Therefore, it seems that some significant genes may be missing from the present array results. In summary, the present study investigated FDC-SP’s role in hPDLCs’ phenotype expression, via comparing the gene expression profile between FDC-SP -transfected hPDLCs to empty vector-transfected cells upon microarray analysis. hPDLCs overexpression FDC-SP appear to display different gene expression patterns that may reflect intrinsic functions of FDC-SP in the maintenance of PDL homeostasis and its ultimate contribution to periodontal would-healing processes. The authors declare that they have no conflict of interest.

Fig. 2. Confirmation of differentially expressed genes observed in microarray results. Four genes including FMN2, SOX12, EGR1 and IGF1 selected from array results were analyzed by Real-time PCR. The expression levels of these four genes tested by Real-time PCR were concordant with their differential expression observed in microarray results.

developmental processes. SOX transcription factors have diverse tissuespecific expression patterns during early development and have been proposed to act as target-specific transcription factors and/or as chromatin structure regulatory elements. Expression of SOX12 in various tissues suggests a role in both differentiation and maintenance of several cell types (Dy et al., 2008; Hoser et al., 2008; Jay et al., 1997). Taken together, the presence of these specific genes suggests a significant role of FDC-SP in phenotype expression of hPDLCs during the differentiation process. On the other hand, we noticed that genes encoding cytokine/growth factors tended to be more abundantly expressed in empty vectortransfected cells than in FDC-SP −transfected hPDLCs (8 vs. 3). Members of the early growth response (EGR) gene family, has been found to play a critical role in hindbrain development and myelination of the peripheral nervous system (O’Donovan, Tourtellotte, Millbrandt & Baraban, 1999). Transcription factors of this gene family have nonredundant biological functions. EGR1, a zinc finger transcription factor, regulates the transcription of a number of genes involved in immune response, differentiation, development and plays a central role in the induction and maintenance of various vascular pathologies (Anderson et al., 2006; Harris et al., 2004; Knapska & Kaczmare, 2004; Saffor et al., 2005). Fang et al. provided the evidence that EGR with potent effects on inflammation and immunity, is necessary and sufficient for profibrotic responses, suggesting important and distinct roles in the pathogenesis of fibrosis (Fang et al., 2013). Furthermore, we found IGF1 mRNA to be highly expressed in empty vector control group (2.38-fold). IGF1, a protein that in humans is encoded by the IGF1 gene (Höppener et al., 1985; Jansen et al., 1983). IGF1 has also been referred to as a “sulfation factor” and its effects were termed “nonsuppressible insulin-like activity” (NSILA) in the 1970s (Salmon & Daughaday, 1957). Its primary action is mediated by binding to its specific receptor, the insulin-like growth factor 1 receptor (IGF1R), which is present on many cell types in various tissues. Binding to the IGF1R, a receptor tyrosine kinase, initiates intracellular signaling; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. Therefore, regulation of these cytokine/growth factors suggests a key role of PDLCs in the maintenance of PDL homeostasis during periodontal restoration process. Interestingly, integrin-binding sialoprotein (IBSP) was found to be highly expressed in the control group compared with FDC-SP -transfected hPDLCs, with a differential expression of 3.57-fold. Previous studies have shown that the protein encoded by this gene is a major

Author contribution All authors had substantial contributions to the design of the work or the acquisition, analysis, or interpretation of data for the work. All authors were involved in drafting the work or revising it critically for important intellectual content. All authors have approved the final version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Source of funding This work was supported by grants from the National Natural Science Foundation of China (No. 81000443, 81400522) and the Youth Science Foundation of West China Hospital of Stomatology (No. 2016-11). References Al-Alwan, M., Du, Q., Hou, S., Nashed, B., Fan, Y., Yang, X., et al. (2007). Follicular dendritic cell secreted protein (FDC-SP) regulates germinal center and antibody responses. The Journal of Immunology, 178(12), 7859–7867. Anderson, P. O., Manzo, B. A., Sundstedt, A., Minaee, S., Symonds, A., Khalid, S., et al. (2006). Persistent antigenic stimulation alters the transcription program in T cells, resulting in antigen-specific tolerance. European Journal of Immunology, 36(6), 1374–1385. Ayari, H., & Bricca, G. (2012). Microarray analysis reveals overexpression of IBSP in human carotid plaques. Advances in Medical Sciences, 57(2), 334–340. Bu, W., Xin, L., Toneff, M., Li, L., & Li, Y. (2009). Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo. Journal of Mammary Gland Biology and Neoplasia, 14(4), 401–404. Dy, P., Penzo-Méndez, A., Wang, H., Pedraza, C. E., Macklin, W. B., & Lefebvre, V. (2008). The three SoxC proteins-Sox4, Sox11 and Sox12-exhibit overlapping expression patterns and molecular properties. Nucleic Acids Research, 36(9), 3101–3117. Faix, J., & Grosse, R. (2006). Staying in shape with formins. Developmental Cell, 10(6), 693–706. Fang, F., Shangguan, A. J., Kelly, K., Wei, J., Gruner, K., Ye, B., et al. (2013). Early growth response 3 (Egr-3) is induced by transforming growth factor-β and regulates fibrogenic responses. American Journal of Pathology, 183(4), 1197–1208. Gjertsen, A. W., Stothz, K. A., Neiva, K. G., & Pileggi, R. (2011). Effect of propolis on proliferation and apoptosis of periodontal ligament fibroblasts. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics, 112(6), 843–848. Höppener, J. W., de Pagter-Holthuizen, P., Geurts van Kessel, A. H., Jansen, M., Kittur, S. D., Antonarakis, S. E., et al. (1985). The human gene encoding insulin-like growth factor I is located on chromosome 12. Human Genetics, 69(2), 157–160. Harris, J. E., Bishop, K. D., Phillips, N. E., Mordes, J. P., Greiner, D. L., Rossini, A. A., et al.

158

Archives of Oral Biology 81 (2017) 151–159

L. Xiang et al.

Research, 41(4), 303–310. Nakamura, S., Terashima, T., Yoshida, T., Iseki, S., Takano, Y., Ishikawa, I., et al. (2005). Identification of genes preferentially expressed in periodontal ligament: Specific expression of a novel secreted protein, FDC-SP. Biochemical and Biophysical Research Communications, 338(2), 1197–1203. O’Donovan, K. J., Tourtellotte, W. G., Millbrandt, J., & Baraban, J. M. (1999). The EGR family of transcription-regulatory factors: Progress at the interface of molecular and systems neuroscience. Trends in Neurosciences, 22(4), 167–173. Oates, T. W., Mumford, J. H., Carnes, D. L., & Cochran, D. L. (2001). Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model. Journal of Periodontology, 72(3), 324–330. Pawelczyk, E., Jordan, E. K., Balakumaran, A., Chaudhry, A., Gormley, N., Smith, M., et al. (2009). In vivo transfer of intracellular labels from locally implanted bone marrow stromal cells to resident tissue macrophages. PUBLIC LIBRARY OF SCIENCE, 4(8), e6712. Poiate, I. A., de Vasconcellos, A. B., de Santana, R. B., & Poiate, E. (2009). Threedimensional stress distribution in the human periodontal ligament in masticatory, parafunctional, and trauma loads: Finite element analysis. Journal of Periodontology, 80(11), 1859–1867. Ralph, W. J. (1982). Tensile behaviour of the periodontal ligament. Journal of Periodontal Research, 17(4), 423–426. Salmon, W., & Daughaday, W. (1957). A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. Journal of Laboratory and Clinical Medicine, 49(6), 825–836. Seo, B. M., Miura, M., Gronthos, S., Bartold, P. M., Batouli, S., Brahim, J., et al. (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 364(9429), 149–155. Silvério, K. G., Rodrigues, T. L., Coletta, R. D., Benevides, L., Da Silva, J. S., Casati, M. Z., et al. (2010). Mesenchymal stem cell properties of periodontal ligament cells from deciduous and permanent teeth. Journal of Periodontology, 81(8), 1207–1215. Xiang, L., Ma, L., He, Y., Wei, N., & Gong, P. (2014). Transfection with follicular dendritic cell secreted protein to affect phenotype expression of human periodontal ligament cells. Journal of Cellular Biochemistry, 115(5), 940–948. Yamada, K., Ono, M., Bensaddek, D., Lamond, A. I., & Rocha, S. (2013). FMN2 is a novel regulator of the cyclin-dependent kinase inhibitor p21. Cell Cycle, 12(15), 2348–2354. Yamada, K., Ono, M., Perkins, N. D., Rocha, S., & Lamond, A. I. (2013). Identification and functional characterization of FMN2, a regulator of the cyclin-Dependent kinase inhibitor p21. Molecular Cell, 49(5), 922–933.

(2004). Early growth response gene-2, a zinc-finger transcription factor, is required for full induction of clonal anergy in CD4+ T cells. The Journal of Immunology, 173(12), 7331–7338. Hoser, M., Potzner, M. R., Koch, J. M., Bösl, M. R., Wegner, M., & Sock, E. (2008). Sox12 deletion in the mouse reveals nonreciprocal redundancy with the related sox4 and sox11 transcription factors. Molecular and Cellular Biology, 28(15), 4675–4687. Ishikawa, I., Iwata, T., Washio, K., Okano, T., Nagasawa, T., & Ando, T. (2009). Cell sheet engineering and other novel cell-based approaches to periodontal regeneration. Periodontol, 2000(51), 220–238. Ivanovski, S., Haase, H. R., & Bartold, P. M. (2001). Isolation and characterization of fibroblasts derived from regenerating human periodontal defects. Archives of Oral Biology, 46(8), 679–688. Jansen, M., van Schaik, F. M., Ricker, A. T., Bullock, B., Woods, D. E., Gabbay, K. H., et al. (1983). Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature, 306(5943), 609–611. Jay, P., Sahly, I., Goze, C., Taviaux, S., Poulat, F., Couly, G., et al. (1997). SOX22 is a new member of the SOX gene family, mainly expressed in human nervous tissue. Human Molecular Genetics, 6(7), 1069–1077. Kato, C., Kojima, T., Komaki, M., Mimori, K., Duarte, W. R., Takenaga, K., et al. (2005). S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells. Biochemical and Biophysical Research Communications, 326(1), 147–153. Knapska, E., & Kaczmare, K. L. (2004). A gene for neuronal plasticity in the mammalian brain: Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Progress in Neurobiology, 74(4), 183–211. Li, M., Husic, N., Lin, Y., Christensen, H., Malik, I., & McIver, S. (2010). Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells. Journal of Neuroscience Methods, 189(1), 56–64. Logan, A. C., Lutzko, C., & Kohn, D. B. (2002). Advances in lentiviral vector design for gene-modification of hematopoietic stem cells. Current Opinion in Biotechnology, 13(5), 429–436. Lv, T., Kang, N., Wang, C., Han, X., Chen, Y., & Bai, D. (2009). Biologic response of rapid tooth movement with periodontal ligament distraction. American Journal of Orthodontics and Dentofacial Orthopedics, 136(3), 401–411. Marshall, A. J., Du, Q., Draves, K. E., Shikishima, Y., HayGlass, K. T., & Clark, E. A. (2002). FDC-SP, a novel secreted protein expressed by follicular dendritic cells. The Journal of Immunology, 169(5), 2381–2389. Nagatomo, K., Komaki, M., Sekiya, I., Sakaguchi, Y., Noguchi, K., Oda, S., et al. (2006). Stem cell properties of human periodontal ligament cells. Journal of Periodontal

159