Accepted Article
Original Research Article Effect
of
CTGF/CCN2
on
osteo/cementoblastic
and
fibroblastic †
differentiation of a human periodontal ligament stem/progenitor cell line
Asuka Yuda 1, Hidefumi Maeda 2*, Shinsuke Fujii 3, Satoshi Monnouchi 1, Naohide Yamamoto 1, Naohisa Wada 2, Katsuaki Koori 2, Atsushi Tomokiyo 1, Sayuri Hamano 1, Daigaku Hasegawa 1, Akifumi Akamine 1, 2 1
Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
2
3
Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
*Correspondence to: Hidefumi Maeda, D.D.S., Ph.D Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Fukuoka 812-8582, Japan Phone: +81-92-642-6432 Fax: +81-92-642-6366 E-mail: [email protected] †
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please ite this article as doi: [10.1002/jcp.24693] Received 30 December 2013; Revised 29 May 2014; Accepted 30 May 2014 Journal of Cellular Physiology © 2014 Wiley Periodicals, Inc. DOI 10.1002/jcp.24693
Accepted Article
Running title: Effects of CTGF/CCN2 on immature PDL cells
Keywords:
Connective tissue growth factor Fibroblastic differentiation Human periodontal ligament stem/progenitor cell line Osteoblastic differentiation Transforming growth factor-β1
Contract grant sponsor: Ministry of Education, Culture, Sports, Science, and Technology, Japan; Contract grant numbers: 23689077, 24390426, 24659848, 24792028, 25293388, and 25670811.
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Abstract Appropriate mechanical loading during occlusion and mastication play an important role in maintaining the homeostasis of periodontal ligament (PDL) tissue. Connective tissue growth factor (CTGF/CCN2), a matricellular protein, is known to up-regulate extracellular matrix production, including collagen in PDL tissue. However, the underlying mechanisms of CTGF/CCN2 in regulation of PDL tissue integrity remain unclear. In this study, we investigated the effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of human PDL stem cells using the cell line 1-11. CTGF/CCN2 expression in rat PDL tissue and human PDL cells (HPDLCs) was confirmed immunohisto/cytochemically. Mechanical loading was found to increase gene expression and secretion of CTGF/CCN2 in HPDLCs. CTGF/CCN2 up-regulated the proliferation and migration of 1-11 cells. Furthermore, increased bone/cementum-related gene expression in this cell line led to mineralization. In addition, combined treatment of 1-11 cells with CTGF/CCN2 and transforming growth factor-1 (TGF-1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-1 treatment alone. Thus, these data suggest the underlying
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biphasic effects of CTGF/CCN2 in 1-11 cells, inducible osteo/cementoblastic and fibroblastic
differentiation
dependent
on
the
environmental
condition.
CTGF/CCN2 may contribute to preservation of the structural integrity of PDL tissue, implying its potential use as a therapeutic agent for PDL regeneration.
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Introduction Periodontal ligament (PDL) tissues originate from cranial neural crest-derived ectomesenchymal cells (Chai et al., 2000) and consist of connective tissue located between the alveolar bone and cementum. Tooth roots are anchored in the bone socket by collagen bundles with terminals inserted into the bone and cementum covering the root surface, termed as Sharpey's fiber. The PDL tissue therefore plays a key role in the lifespan of the tooth. Once the PDL tissue is destroyed by deep caries, severe periodontitis, and trauma, its reconstruction is very difficult, resulting in tooth loss.
In 2004, Seo et al. reported the isolation of human PDL stem cells (PDLSCs) that express STRO-1 and CD146, and differentiate into fibroblasts, osteoblasts, and cementoblasts in the periodontium of immunocompromised mice (Maeda et al., 2011; Seo et al., 2004). Furthermore, these cells have important roles in maintenance and regeneration of PDL tissue (Maeda et al., 2011; Maeda et al., 2013b). In this context, we have established immortalized human PDL fibroblast cell lines by transduction of both simian virus 40 T-antigen and human telomerase reverse transcriptase (Fujii et al., 2006), and isolated a clonal cell
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line (1-11) (Fujii et al., 2008). In addition to CD13, CD29, CD44, CD71, CD90, CD105, and CD166, 1-11 cells express STRO-1 and CD146, exhibiting a similarity to bone marrow mesenchymal stem cells (Maeda et al., 2013a; Maeda et al., 2011). This cell line exhibits the potential to differentiate into osteoblastic and adipocytic cells, and produces bone/cementum-like tissues involving Sharpey’s-like fibers when transplanted into immunodeficient mice (Fujii et al., 2008).
Mechanical loading is an essential stimulus for homeostasis of various tissues such as PDL, tendons, ligaments, bone, and cartilage during development and healing. Recent studies suggest that appropriate mechanical loading is required to maintain PDL tissues (Monnouchi et al., 2011; Shi et al., 2005). Mechanical shear stress enhances the functional properties of engineered PDL constructs (Kim et al., 2011). Moreovoer, Saminathan et al. (2012) have shown that mechanical loading up-regulates connective tissue growth factor (CTGF/CCN2) gene expression in human PDL cells (Saminathan et al., 2012).
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CTGF/CCN2 is a cysteine-rich secretory protein belonging to the CCN family whose members include cysteine-rich 61 (CYP61/CCN1), nephroblastoma overexpressed (NOV/CCN3), Wnt-induced secreted protein-1 (WISP-1/CCN4), WISP-2 (CCN5), and WISP-3 (CCN6) (Holbourn et al., 2008; Lau and Lam, 1999; Moussad and Brigstock, 2000). A prototypical CCN protein includes four functional domains; an insulin-like growth factor binding protein-like module, von Willebrand factor type C repeat module, thrombospondin type-1 repeat module, and C-terminal cysteine knot-containing module (CT), although CCN5 lacks a CT module (Holbourn et al., 2008; Lau and Lam, 1999; Moussad and Brigstock, 2000). CTGF/CCN2 has been implicated as a key regulatory factor in many biological and pathological events including cell adhesion (Babic et al., 1999; Kireeva et al., 1997), proliferation (Asano et al., 2005; Shimo et al., 1999), migration (Babic et al., 1999; Guo et al., 2009; Muromachi et al., 2012; Shimo et al., 1999), extracellular matrix (ECM) production (Frazier et al., 1996), wound healing (Shi et al., 2012), angiogenesis (Hall-Glenn et al., 2012; Shimo et al., 1999), and endochondral ossification (Ivkovic et al., 2003; Takigawa et al., 2003). Furthermore, CTGF/CCN2 induces osteogenesis of mouse adipose-derived stromal cells (Xu et al., 2010).
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In
this
study,
we
investigated
the
effects
of
CTGF/CCN2
on
osteo/cementoblastic differentiation of 1-11 cells and those of combined treatment with CTGF/CCN2 and TGF-1 on fibroblastic differentiation.
MATERIALS AND METHODS Materials Recombinant human CTGF/CCN2 was purchased from BioVendor (Candler, NC). Recombinant human TGF-1 was purchased from Millipore (Temecula, CA). Goat polyclonal anti-rat/human CTGF/CCN2 and rabbit polyclonal anti-rat/human TGF-1 antibodies, and normal goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Normal rabbit IgG was purchased from R&D Systems (Minneapolis, MN). Alexa Fluor 568-conjugated donkey anti-goat and Alexa Fluor 488-conjugated chicken anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA).
Immunohistochemistry
Three five-week-old male Sprague-Dawley rats were purchased from Kyudo (Saga, Japan). The rats were sacrificed by transcardial perfusion with 4%
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paraformaldehyde (Merck, Darmstadt, Germany) in phosphate buffered saline (PBS) under anesthesia [2 mg/kg midazolam (Sandoz, Tokyo, Japan), 0.15 mg/kg medetomidine (Kyoritsu Seiyaku, Tokyo, Japan), and 2.5 mg/kg butorphanol tartrate (Meiji Seika Pharma, Tokyo, Japan)]. Maxillae were removed and then immersed in the same fixative for a further 12 h. The tissues were washed with PBS and decalcified in 10% ethylenediaminetetraacetic acid (Wako, Osaka, Japan) at 4 C for 4 weeks. Then, the tissues were dehydrated and embedded in O.C.T. Compound (Sakura Finetek USA, CA), followed by preparation of 5 μm horizontal sections. After blocking with 2% bovine serum albumin (BSA) in PBS for 1 h, immunofluorescence staining was performed with goat polyclonal anti-rat/human CTGF/CCN2 or normal IgG (1:50), rabbit polyclonal anti-rat/human TGF-1 primary antibodies or normal IgG (1:50). After washing with PBS, the sections were reacted with Alexa Fluor 568-conjugated donkey anti-goat (1:100) and Alexa Fluor 488-conjugated chicken anti-rabbit (1:100) secondary antibodies for 1 h. The rat PDL tissue was then washed with PBS and counterstained with DAPI (Vector Laboratories, Burlingame, CA). The sections were observed under a Biozero digital microscope (Keyence, Osaka,
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Japan). All procedures were approved by the Animal Ethics Committee and conformed to the regulations of Kyushu University.
In addition, HPDLCs were fixed with 4% paraformaldehyde and 0.5% dimethyl sulfoxide (Wako) in PBS for 20 min. After blocking with 2 % BSA in PBS for 1 h, the cells were immunostained as described above.
Cell culture Human PDL cells (HPDLCs) were isolated from healthy premolars or third molars from three different patients, a 23-year-old male (HPDLC-3S), a 25-year-old female (HPDLC-3U), and a 21-year-old female (HPDLC-3Q) who visited Kyushu University Hospital for extraction as described previously. HPDLCs and 1-11 cells were maintained in alpha-minimum essential medium (α-MEM; Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; BioWest, Nuaille, France), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan) at 37 C in a humidified atmosphere with 5% CO2. All procedures were performed in compliance with the Research Ethics Committee of the Faculty of Dentistry, Kyushu University.
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Application of mechanical stress HPDLCs (2x105 cells/chamber) were pre-cultured in flexible-bottomed culture chambers coated with type I collagen (Cellmatrix I-P, Nitta Gelatin Inc, Osaka, Japan) until subconfluence according to our recent study (Monnouchi et al., 2011). Then, stretch loading was applied to HPDLC culture chambers in a CO2 incubator by a STB-140 (STREX, Osaka, Japan). HPDLCs were stretched at 60 cycles/min (0.5 sec stretch and 0.5 sec relaxation per cycle) with a tension force producing 8% elongation. After stretch loading, HPDLCs were subjected to RNA extraction and an enzyme-linked immunosorbent assay (ELISA).
Quantitative RT-PCR First-strand cDNA was synthesized with a Prime Script RT Reagent kit (Takara Bio, Shiga, Japan). RT-PCR was performed with a SYBR Green II RT-PCR kit (Takara Bio) using a Thermal Cycler Dice Real Time System (Takara Bio) as described previously (Maeda et al., 2010). Specific primer sequences, annealing temperatures, and product sizes for each gene are listed in Table 1. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal
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control. Expression levels of the target genes were calculated using ∆∆Ct values.
ELISA HPDLC-3S, -3U, and -3Q cells (1.5x105 cells/chamber) were subjected to stretch loading for 3 h or non-loading in 1% FBS/α-MEM. The concentration of CTGF/CCN2 in the culture supernatant was measured with a commercially available ELISA kit (Aviscera Bioscience, Santa Clara, CA) according to the manufacturer’s instructions.
Cell proliferation assay Cell line 1-11 (1.5x103 cells/well) was cultured on 48-well plates (Becton Dickinson Labware, Lincoln Park, NJ) with or without CTGF/CCN2 (1, 10, and 100 ng/ml) in 2% FBS/α-MEM for 24 or 48 h. The proliferation rate was measured at the indicated time points using a Premix WST-1 Cell Proliferation Assay System (Takara Bio) according to the manufacturer’s instructions.
Cell cycle analysis
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Cell line 1-11 (1.2x105 cells/dish) was cultured on 10-cm dishes (Becton Dickinson Labware) with or without CTGF/CCN2 (100 ng/ml) in 2% FBS/-MEM for 48 h. The cells were trypsinized and detached, washed with PBS, and then fixed in 70% ethanol overnight at 4 °C. Then, the cells were washed with PBS and incubated with RNase (Sigma-Aldrich, St. Louis, MO) and propidium iodine (Sigma-Aldrich) for 30 min. The percentage of cells in the three phases of the growth cycle (G0/G1, G2/M, and S phase) was then measured by flow cytometry (EC800 Cell Analyzer, Sony, Tokyo, Japan). Data were analyzed using Eclipse software (Sony).
Cell migration assay A scratch wound healing assay was performed to assess the effect of CTGF/CCN2 on 1-11 cell migration in response to injury according to our recent study (Yamamoto et al., 2012). Cell line 1-11 (5x104
cells/well) was seeded on
12-well plates (Becton Dickinson Labware) in 10% FBS/α-MEM and grown to subconfluence. Then, a scratch wound was made across the diameter of the well using the end of a 200 μl pipette tip. The scraped cells were removed by three washes with PBS. The cells were then treated with CTGF/CCN2 (1, 10,
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and 100 ng/ml) in 2% FBS/α-MEM. For each well, images were taken at 0 and 6 h after injury, and the number of cells that migrated into the wound space was manually counted in three wells.
CTGF/CCN2
treatment
of
cell
line
1-11
and
analysis
of
bone/cementum-related gene expression Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates (Becton Dickinson Labware) in 10% FBS/α-MEM as control medium (CM) or in osteogenic differentiation
medium
[DM;
10%
FBS/α-MEM
containing
2
mM
-glycerophosphate (Sigma-Aldrich), 50 μg/ml ascorbic acid (Nacalai Tesque), and 0.1 mM dexamethasone (Calbiochem, Billerica, MA)] with CTGF/CCN2 (1, 10, and 100 ng/ml) for 7 days. The cells were lysed in TRIzol Reagent (Invitrogen) according to our previous study (Wada et al., 2004), and then total RNA was extracted and analyzed by quantitative RT-PCR.
Staining and quantification of alkaline phosphatase activity Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates in 10% FBS/α-MEM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 14 days. The
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cells were then fixed in a solution of 25% citrate (Sigma-Aldrich), 8% formalin (Wako), and 66% acetone (Wako) for 10 seconds at room temperature. After rinsing with sterile water, alkaline phosphatase (ALP) in the cells was stained according to the manufacturer’s instructions (Vector Laboratories) in the dark for 30 min at room temperature. ALP activity of the differentiated cells was determined using an ALP assay kit (Takara Bio) according to the manufacturer’s instructions. 1-11 cells (7x102 cells/well) were cultured on 96-well plates in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 14 days. Then, the cells were exposed to extraction solution and buffer from the ALP assay kit for 30 min at 37 C. ALP activity in the cell lysate was assayed by measuring p-nitrophenol used as the substrate at an absorbance of 405 nm.
Alizarin red staining and quantification Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 16 days. The cells were fixed for 60 min in 10% formalin and then stained with an Alizarin red S solution (pH 4.1–4.3)
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(Sigma-Aldrich) for 60 min. Stained cells were washed extensively with sterile water to remove nonspecific precipitation.
CTGF/CCN2 and/or TGF-1 treatment of cell line 1-11 Cell line 1-11 (1x104 cells/dish) was cultured on 35-mm dishes (Becton Dickinson Labware) with CTGF/CCN2 (100 ng/ml) and/or TGF-1 (0.5 ng/ml) in 2% FBS/α-MEM for 48 h. The cells were lysed in TRIzol Reagent according to our previous study (Wada et al., 2004), and then total RNA was extracted and analyzed by quantitative RT-PCR.
Collagen assay Collagen synthesis in 1-11 cells (2x103 cells/well) cultured on 24-well plates with CTGF/CCN2 (100 ng/ml) and TGF-1 (0.5 ng/ml) in 2% FBS/α-MEM for 72 h was determined using a Sircol collagen assay kit (Biocolor, Northern Ireland, UK) according to the manufacturer’s instructions. Culture supernatants were collected and mixed with Sircol dye regent. The mixed solution was transferred to a microcentrifuge and spun at maximum speed for 10 min. The collagen pellet was washed once with acid-salt wash reagent, and then the alkaline reagent
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was added to the pellet. Each sample was transferred to an individual well of a 96-well plate to measure the absorbance at 570 nm.
Statistical analysis All values are expressed as means SD. Statistical analyses of the results were performed using one-way ANOVA followed by Dunnett’s test and Tukey’s test, or two-way ANOVA followed by Tukey’s test for multiple comparisons. Student’s unpaired t-test was also used for comparison of two means. A p-value of less than 0.05 was considered statistically significant.
Results Expression of CTGF/CCN2 in rat PDL tissue and HPDLCs Immunofluorescence analysis revealed positive staining for CTGF/CCN2 throughout rat PDL tissue (Fig. 1A). Immunocytochemical staining also exhibited positive staining for CTGF/CCN2 in HPDLC-3S, -3U, and -3Q (Fig. 1C, E, G). CTGF/CCN2 staining was intense in the cytoplasm of all HPDLCs. Control staining with normal goat IgG was negative in PDL tissue and HPDLCs (Fig. 1B, D, F, H).
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Stretch loading-induced CTGF/CCN2 gene expression and secretion in HPDLCs The effects of 8% stretch loading on CTGF/CCN2 expression in HPDLC-3S, -3U, and -3Q were examined by quantitative RT-PCR and ELISA (Fig. 2). Compared with non-loading, the expression level of CTGF/CCN2 mRNA was up-regulated by exposure to stretch loading (Fig. 2A, B, C). Next, we determined the concentration of CTGF/CCN2 secreted from stretch-loaded HPDLC-3S, -3U, and -3Q. Compared with non-loading, secretion of CTGF/CCN2 was significantly increased by exposure to stretch loading (Fig. 2D, E, F).
Effects of CTGF/CCN2 on the proliferation, cell cycle distribution, and migration of cell line 1-11
A WST-1 assay was performed to evaluate the proliferation of 1-11 cells treated with CTGF/CCN2. Compared with other concentrations, 1-11 cell proliferation was significantly increased by treatment with 100 ng/ml CTGF/CCN2 for 48 h (Fig. 3A). To furthermore confirm this result, the cell cycle distribution was
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analyzed by determining DNA content. Compared with untreated cells, the proportion of CTGF/CCN2-treated 1-11 cells was significantly increased in G2/M phase and decreased in G0/G1 phase, indicating increased cell growth (Fig. 3B-D). A scratch wound healing assay revealed an increase in the migration of 1-11 cells treated with CTGF/CCN2 for 6 h compared with that of untreated cells. The number of migrated cells was increased in a dose-dependent manner (1, 10, and 100 ng/ml CTGF/CCN2) (Fig. 3E, F).
Expression of bone/cementum-related genes in CTGF/CCN2-treated cell line 1-11 1-11 cells cultured in DM showed a significant increase in the expression of bone/cementum-related genes such as ALP, bone sialoprotein (BSP), osteopontin (OPN), and cementum-derived attachment protein (CAP) compared with that in CM-cultured cells (Fig. 4A-H). Moreover, addition of CTGF/CCN2 (100 ng/ml) to DM resulted in further augmentation of the expression of these genes (Fig. 4B, D, F, H). However, 1-11 cells cultured in CM with CTGF/CCN2 (1,
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10, and 100 ng/ml) showed no significant increase in these genes (Fig. 4A, C, E, G).
Promotion of osteo/cementoblastic differentiation of CTGF/CCN2-treated cell line 1-11 We examined ALP activity and mineralization in CTGF/CCN2-treated 1-11 cells. After 14 days of culture in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml), the ALP activity was significantly and dose-dependently increased in DM-cultured 1-11 cells (Fig. 5C) while CM-cultured cells with CTGF/CCN2 (1, 10, and 100 ng/ml) revealed no significant increase (Fig. 5A). In addition, ALP staining of 1-11 cells treated as described above revealed the same tendency (Fig. 5B, D). Next, 1-11 cells were treated in the same manner for 16 days and then subjected to Alizarin red staining. The results demonstrated a remarkable enhancement of reactive products in DM-cultured cells treated with 100 ng/ml CTGF/CCN2 (Fig. 5F, G) while CM-cultured cells with CTGF/CCN2 (1, 10, and 100 ng/ml) showed no positive reaction (Fig. 5E).
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Expression of CTGF/CCN2 and TGF-1 in rat PDL tissues and HPDLCs Immunofluorescence analysis revealed co-localized positive staining for CTGF/CCN2 and TGF-1 in rat PDL tissue (Fig. 6A-C). Immunocytochemical staining exhibited positive staining for CTGF/CCN2 and TGF-1 in HPDLC-3S (Fig. 6E-G), -3U (Fig. 6I-K), and -3Q (Fig. 6M-O), and demonstrated cytoplasmic co-localization of these proteins. Control staining with normal goat and rabbit IgGs was negative in PDL tissue and HPDLCs (Fig. 6D, H, L, P).
Expression of fibroblast-related genes and soluble collagen in cell line 1-11 treated with both CTGF/CCN2 and TGF-1 Recently we have reported that TGF-1 promotes fibroblastic differentiation of 1-11 cells (Fujii et al., 2010; Kono et al., 2013). Qi et al. (2005) furthermore reported that combined treatment of human renal cells with CTGF/CCN2 and TGF-1 promotes their fibroblastic differentiation (Qi et al., 2005). In this context, we investigated whether combined treatment with CTGF/CCN2 and TGF-1 up-regulated fibroblastic differentiation of 1-11 cells. The cells were treated with CTGF/CCN2 (100 ng/ml) and/or TGF-1 (0.5 ng/ml) for 48 h. As a result, the expression of fibroblast-related genes, type I collagen (COL I) and fibronectin,
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was clearly up-regulated by TGF-1 treatment. Additionally, combined treatment with CTGF/CCN2 and TGF-1 exhibited further enhancement of the expression of these genes (Fig. 7A, B). Secreted collagen products from 1-11 cells subjected to combined treatment with CTGF/CCN2 and TGF-1 was compared with that of TGF-1 treatment alone (Fig. 7C). Similar to the results shown in Fig. 7A, the combination of CTGF/CCN2 and TGF-1 stimuli significantly up-regulated collagen synthesis (Fig. 7C).
Discussion Although PDL tissue is continually subjected to mechanical loading because of bite force, this is very important to maintain the structural and physiological conditions, and heal PDL tissue (Fujihara et al., 2010). Our recent study indicated that mechanical loading induces the expression of ALP and TGF-1 in HPDLCs through the renin-angiotensin system (Monnouchi et al., 2011). These molecules are expressed in functional PDL cells in vivo. In addition, another study reported increased synthesis of ECM components, such as OPN, dentin matrix protein 1, and COL I, in PDL cells subjected to mechanical loading (Berendsen et al., 2009).
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CTGF/CCN2 reportedly has a role in the repair of many tissues, including bone regeneration and organ fibrosis (Kikuchi et al., 2008; Shi-Wen et al., 2008). It is also known to contribute to mouse tooth development as well as proliferation and differentiation of mouse PDL cells (Asano et al., 2005; Shimo et al., 2002; Yamaai et al., 2005). In addition, a recent study reported that CTGF/CCN2 gene expression in human PDL cells is promoted by stretch loading (Saminathan et al., 2012). Our current study clearly demonstrated increased production of CTGF/CCN2 protein in HPDLCs exposed to stretch loading. In this context, we focused on the physiological effects of CTGF/CCN2 on the cell line 1-11. Although the ELISA data detected low concentration of CTGF/CCN2 in culture media of HPDLCs, we consider that the concentration of CTGF secreted from HPDLC exposed to stretch-loading would get higher at the limited area around secreting cells, and therefore secreted CTGF might probably work in an autocrine or paracrine manner. First, we examined the proliferative and migratory effects of CTGF/CCN2 on 1-11 cells. The results indicated that CTGF/CCN2 treatment led to effective expansion of the cells and induction of cell mobility, suggesting a therapeutic effect on wounded PDL using PDL stem cells. Recent studies have also shown
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that CTGF/CCN2 promotes the proliferation of mouse PDL cells (Asano et al., 2005), and induces migration of human dental pulp cells (Muromachi et al., 2012) and mouse mesenchymal cells (Song et al., 2007), which supports our data. Previous studies have reported that CTGF/CCN2 promotes mineral nodule formation of human PDL progenitor cells (Dangaria et al., 2009) and up-regulates osteogenic differentiation of human adipose-derived stromal cells (Xu et al., 2010). However, the underlying mechanism of CTGF/CCN2 in osteo/cementoblastic and fibroblastic differentiation of immature PDL cells has not been examined in detail. Asano et al. (2005) reported that CTGF/CCN2 promotes the synthesis of PDL-related molecules, such as Alp and Col I, but not bone-related markers, Opn and osteocalcin in mouse PDL cells, suggesting a certain role in development and regeneration of PDL tissue. We also found that 1-11 cells cultured in CM did not show an increase of bone/cementum-related molecule expression independently of the increasing concentration of CTGF/CCN2. However, 1-11 cells cultured in DM showed up-regulation of ALP, OPN, BSP, and even CAP, subsequently resulting in mineralization, suggesting their osteo/cementoblastic differentiation because CAP is a specialized protein
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of the cementoblast although this cell shares most of the characteristics with the osteoblast, such as the expression of ALP, OPN, and BSP. These results suggest that CTGF/CCN2 has an intrinsic effect on the differentiation of immature PDL cells, which is dependent on the environment. As PDL tissue does not mineralize per se, we examined how CTGF/CCN2 contributed to these events, viz fibroblastic and osteo/cementoblastic differentiation of immature PDL cells.
Our recent studies indicate that TGF-1 is abundantly expressed in the entire PDL tissue and involved in fibroblastic differentiation of immature PDL cells, suggesting its role in maintaining the homeostasis of PDL tissue (Fujii et al., 2010; Kono et al., 2013). In addition, CTGF/CCN2 acts downstream of TGF-β1 signaling as a co-operative mediator (Lee et al., 2010) although it could be also induced by BMP9 and Wnt3a (Luo et al., 2004). Our present results revealed co-localization of CTGF/CCN2 and TGF-β1 in PDL tissue. Qi et al reported that combined treatment with these proteins induces fibroblastic differentiation of human renal cells, indicating that CTGF/CCN2 facilitates the effects of TGF-1 to induce their fibroblastic differentiation (Qi et al., 2005). Therefore, we also examined the combined effects of CTGF/CCN2 with
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TGF-1 on the differentiation of 1-11 cells to clarify the role of CTGF/CCN2 in PDL tissue, because PDL tissue is a fibrous tissue that does not exhibit mineralization. We first confirmed that both proteins were co-expressed in HPDLCs and throughout PDL tissue, which suggested a co-operative relationship between these factors in the biological function of PDL tissue. We found increased expression of COL1 gene and protein, and fibronectin in co-treated cells compared with that in cells treated with TGF-1 alone. In support of our results, a recent study showed that simultaneous knockdown of TGF-β1, TGFβR2, and CTGF reduces Col I expression in rabbit corneal fibroblasts (Sriram et al., 2013). Li et al. (2013) reported expression of CTGF/CCN2 and TGF-1 in the developing periodontium, suggesting that regulation of CTGF/CCN2 by TGF-1 might be cell-specific in the periodontium. Taken together, CTGF/CCN2 might have biphasic effects on the differentiation of immature PDL cells, which is probably dependent on the surrounding
cell
populations
via
the
influence
of
osteoblasts
and
cementoblasts on the surface of bone and cementum or PDL fibroblasts in ligamentous tissue. As the periodontium is composed of hard tissue such as bone and cementum, and soft tissue such as the ligamentous tissue and
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gingiva, these features of CTGF/CCN2 imply that it could be efficient as an optimal therapeutic agent for repair of the damaged periodontium.
Acknowledgements This study was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant numbers: 23689077, 24390426, 24659848, 24792028, 25293388, and 25670811). We thank Dr. Hiroko Tsuda for useful suggestion on statistical analysis, and Drs. Hideki Sugii, Shinichirou Yoshida, Suguru Serita, Yumi Mitarai, and Hiroyuki Mizumachi for their great support in preparation of this work.
Literature Cited Asano M, Kubota S, Nakanishi T, Nishida T, Yamaai T, Yosimichi G, Ohyama K, Sugimoto T, Murayama Y, Takigawa M. 2005. Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells. Cell Commun Signal 3:11. Babic AM, Chen CC, Lau LF. 1999. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19(4):2958-2966.
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Berendsen AD, Smit TH, Walboomers XF, Everts V, Jansen JA, Bronckers AL. 2009. Three-dimensional loading model for periodontal ligament regeneration in vitro. Tissue Eng Part C Methods 15(4):561-570. Chai Y, Jiang X, Ito Y, Bringas P, Jr., Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM. 2000. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127(8):1671-1679. Dangaria SJ, Ito Y, Walker C, Druzinsky R, Luan X, Diekwisch TG. 2009. Extracellular matrix-mediated differentiation of periodontal progenitor cells. Differentiation 78(2-3):79-90. Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR. 1996. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107(3):404-411. Fujihara C, Yamada S, Ozaki N, Takeshita N, Kawaki H, Takano-Yamamoto T, Murakami S. 2010. Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells. J Biol Chem 285(36):28286-28297. Fujii S, Maeda H, Tomokiyo A, Monnouchi S, Hori K, Wada N, Akamine A. 2010. Effects of TGF-beta1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line. Cell Tissue Res 342(2):233-242. Fujii S, Maeda H, Wada N, Kano Y, Akamine A. 2006. Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer. Cell Tissue Res 324(1):117-125. Fujii S, Maeda H, Wada N, Tomokiyo A, Saito M, Akamine A. 2008. Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo. J Cell Physiol 215(3):743-749.
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Guo CM, Wang YS, Hu D, Han QH, Wang JB, Hou X, Hui YN. 2009. Modulation of migration and Ca2+ signaling in retinal pigment epithelium cells by recombinant human CTGF. Curr Eye Res 34(10):852-862. Hall-Glenn F, De Young RA, Huang BL, van Handel B, Hofmann JJ, Chen TT, Choi A, Ong JR, Benya PD, Mikkola H, Iruela-Arispe ML, Lyons KM. 2012. CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis. PLoS One 7(2):e30562. Holbourn KP, Acharya KR, Perbal B. 2008. The CCN family of proteins: structure-function relationships. Trends Biochem Sci 33(10):461-473. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM. 2003. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130(12):2779-2791. Kikuchi T, Kubota S, Asaumi K, Kawaki H, Nishida T, Kawata K, Mitani S, Tabata Y, Ozaki T, Takigawa M. 2008. Promotion of bone regeneration by CCN2 incorporated into gelatin hydrogel. Tissue Eng Part A 14(6):1089-1098. Kim SG, Viechnicki B, Kim S, Nah HD. 2011. Engineering of a periodontal ligament construct: cell and fibre alignment induced by shear stress. J Clin Periodontol 38(12):1130-1136. Kireeva ML, Latinkic BV, Kolesnikova TV, Chen CC, Yang GP, Abler AS, Lau LF. 1997. Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development. Exp Cell Res 233(1):63-77. Kono K, Maeda H, Fujii S, Tomokiyo A, Yamamoto N, Wada N, Monnouchi S, Teramatsu Y, Hamano S, Koori K, Akamine A. 2013. Exposure to transforming growth factor-beta1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines. Cell Tissue Res 352(2):249-263.
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Lau LF, Lam SC. 1999. The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res 248(1):44-57. Lee CH, Shah B, Moioli EK, Mao JJ. 2010. CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J Clin Invest 120(9):3340-3349. Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG, Haydon RC, He TC. 2004. Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279(53):55958-55968. Maeda H, Fujii S, Tomokiyo A, Wada N, Akamine A. 2013a. Periodontal tissue engineering: defining the triad. Int J Oral Maxillofac Implants 28(6):e461-471. Maeda H, Nakano T, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Hori K, Akamine A. 2010. Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells. J Endod 36(4):647-652. Maeda H, Tomokiyo A, Fujii S, Wada N, Akamine A. 2011. Promise of periodontal ligament stem cells in regeneration of periodontium. Stem Cell Res Ther 2(4):33. Maeda H, Wada N, Tomokiyo A, Monnouchi S, Akamine A. 2013b. Prospective potency of TGF-beta1 on maintenance and regeneration of periodontal tissue. Int Rev Cell Mol Biol 304:283-367. Monnouchi S, Maeda H, Fujii S, Tomokiyo A, Kono K, Akamine A. 2011. The roles of angiotensin II in stretched periodontal ligament cells. J Dent Res 90(2):181-185. Moussad EE, Brigstock DR. 2000. Connective tissue growth factor: what's in a name? Mol Genet Metab 71(1-2):276-292.
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Muromachi K, Kamio N, Narita T, Annen-Kamio M, Sugiya H, Matsushima K. 2012. MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration. J Cell Biochem 113(4):1348-1358. Qi W, Twigg S, Chen X, Polhill TS, Poronnik P, Gilbert RE, Pollock CA. 2005. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol 288(4):F800-809. Saminathan A, Vinoth KJ, Wescott DC, Pinkerton MN, Milne TJ, Cao T, Meikle MC. 2012. The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture. J Periodontal Res 47(2):212-221. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. 2004. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364(9429):149-155. Shi-Wen X, Leask A, Abraham D. 2008. Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 19(2):133-144. Shi L, Chang Y, Yang Y, Zhang Y, Yu FS, Wu X. 2012. Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing. PLoS One 7(2):e32128. Shi L, Kodama Y, Atsumi Y, Honma S, Wakisaka S. 2005. Requirement of occlusal force for maintenance of the terminal morphology of the periodontal Ruffini endings. Arch Histol Cytol 68(4):289-299. Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T, Takigawa M. 1999. Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126(1):137-145.
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Shimo T, Wu C, Billings PC, Piddington R, Rosenbloom J, Pacifici M, Koyama E. 2002. Expression, gene regulation, and roles of Fisp12/CTGF in developing tooth germs. Dev Dyn 224(3):267-278. Song JJ, Aswad R, Kanaan RA, Rico MC, Owen TA, Barbe MF, Safadi FF, Popoff SN. 2007. Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation. J Cell Physiol 210(2):398-410. Sriram S, Robinson PM, Pi L, Lewin AS, Schultz GS. 2013. Triple Combination of siRNAs Targeting TGFbeta1, TGFbetaR2 and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts. Invest Ophthalmol Vis Sci 54(13):8214-8223. Takigawa M, Nakanishi T, Kubota S, Nishida T. 2003. Role of CTGF/HCS24/ecogenin in skeletal growth control. J Cell Physiol 194(3):256-266. Wada N, Maeda H, Yoshimine Y, Akamine A. 2004. Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha. Bone 35(3):629-635. Xu Y, Wagner DR, Bekerman E, Chiou M, James AW, Carter D, Longaker MT. 2010. Connective tissue growth factor in regulation of RhoA mediated cytoskeletal tension associated osteogenesis of mouse adipose-derived stromal cells. PLoS One 5(6):e11279. Yamaai T, Nakanishi T, Asano M, Nawachi K, Yoshimichi G, Ohyama K, Komori T, Sugimoto T, Takigawa M. 2005. Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development. J Bone Miner Metab 23(4):280-288. Yamamoto N, Maeda H, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Kono K, Koori K, Teramatsu Y, Akamine A. 2012. Expression and effects of glial
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cell line-derived neurotrophic factor on periodontal ligament cells. J Clin Periodontol 39(6):556-564.
Figure Legends Figure. 1. Immunofluorescence analysis of CTGF/CCN2 in rat PDL tissue and HPDLCs.
Immunofluorescence staining for CTGF/CCN2 (red; A, C, E, G) was performed using rat PDL tissue (A), and HPDLC-3S (C, D), -3U (E, F), and -3Q (G, H). No staining was observed in samples incubated with normal control IgG (negative control; B, D, F, H). Cells were counterstained with DAPI (blue). AB, alveolar bone; PDL, periodontal ligament; D, dentin. Scale bars = 100 μm (A) and 50 μm (C, E, G).
Figure. 2. Effects of stretch loading on gene and protein expression of CTGF/CCN2 in HPDLCs.
HPDLC-3S (A, D), -3U (B, E), and -3Q (C, F) were exposed to stretch loading of 0 and 8% elongation for 1 h. Gene expression of CTGF/CCN2 in HPDLC-3S (A),
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-3U (B), and -3Q (C) was examined by quantitative RT-PCR. Human GAPDH was used as an internal standard. CTGF/CCN2 secreted into the culture medium from HPDLC-3S (D), -3U (E), and -3Q (F) exposed to 8% stretch loading or non-loading for 3 h was measured with an ELISA. Y-axis values in A, B, C represent the expression ratio relative to the control. Values are the means ± SD from three independent experiments. All data were analyzed by Student’s unpaired t-test; **p
Original Research Article Effect
of
CTGF/CCN2
on
osteo/cementoblastic
and
fibroblastic †
differentiation of a human periodontal ligament stem/progenitor cell line
Asuka Yuda 1, Hidefumi Maeda 2*, Shinsuke Fujii 3, Satoshi Monnouchi 1, Naohide Yamamoto 1, Naohisa Wada 2, Katsuaki Koori 2, Atsushi Tomokiyo 1, Sayuri Hamano 1, Daigaku Hasegawa 1, Akifumi Akamine 1, 2 1
Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation, Faculty of Dental Science Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
2
3
Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
*Correspondence to: Hidefumi Maeda, D.D.S., Ph.D Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Fukuoka 812-8582, Japan Phone: +81-92-642-6432 Fax: +81-92-642-6366 E-mail: [email protected] †
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please ite this article as doi: [10.1002/jcp.24693] Received 30 December 2013; Revised 29 May 2014; Accepted 30 May 2014 Journal of Cellular Physiology © 2014 Wiley Periodicals, Inc. DOI 10.1002/jcp.24693
Accepted Article
Running title: Effects of CTGF/CCN2 on immature PDL cells
Keywords:
Connective tissue growth factor Fibroblastic differentiation Human periodontal ligament stem/progenitor cell line Osteoblastic differentiation Transforming growth factor-β1
Contract grant sponsor: Ministry of Education, Culture, Sports, Science, and Technology, Japan; Contract grant numbers: 23689077, 24390426, 24659848, 24792028, 25293388, and 25670811.
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Abstract Appropriate mechanical loading during occlusion and mastication play an important role in maintaining the homeostasis of periodontal ligament (PDL) tissue. Connective tissue growth factor (CTGF/CCN2), a matricellular protein, is known to up-regulate extracellular matrix production, including collagen in PDL tissue. However, the underlying mechanisms of CTGF/CCN2 in regulation of PDL tissue integrity remain unclear. In this study, we investigated the effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of human PDL stem cells using the cell line 1-11. CTGF/CCN2 expression in rat PDL tissue and human PDL cells (HPDLCs) was confirmed immunohisto/cytochemically. Mechanical loading was found to increase gene expression and secretion of CTGF/CCN2 in HPDLCs. CTGF/CCN2 up-regulated the proliferation and migration of 1-11 cells. Furthermore, increased bone/cementum-related gene expression in this cell line led to mineralization. In addition, combined treatment of 1-11 cells with CTGF/CCN2 and transforming growth factor-1 (TGF-1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-1 treatment alone. Thus, these data suggest the underlying
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biphasic effects of CTGF/CCN2 in 1-11 cells, inducible osteo/cementoblastic and fibroblastic
differentiation
dependent
on
the
environmental
condition.
CTGF/CCN2 may contribute to preservation of the structural integrity of PDL tissue, implying its potential use as a therapeutic agent for PDL regeneration.
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Introduction Periodontal ligament (PDL) tissues originate from cranial neural crest-derived ectomesenchymal cells (Chai et al., 2000) and consist of connective tissue located between the alveolar bone and cementum. Tooth roots are anchored in the bone socket by collagen bundles with terminals inserted into the bone and cementum covering the root surface, termed as Sharpey's fiber. The PDL tissue therefore plays a key role in the lifespan of the tooth. Once the PDL tissue is destroyed by deep caries, severe periodontitis, and trauma, its reconstruction is very difficult, resulting in tooth loss.
In 2004, Seo et al. reported the isolation of human PDL stem cells (PDLSCs) that express STRO-1 and CD146, and differentiate into fibroblasts, osteoblasts, and cementoblasts in the periodontium of immunocompromised mice (Maeda et al., 2011; Seo et al., 2004). Furthermore, these cells have important roles in maintenance and regeneration of PDL tissue (Maeda et al., 2011; Maeda et al., 2013b). In this context, we have established immortalized human PDL fibroblast cell lines by transduction of both simian virus 40 T-antigen and human telomerase reverse transcriptase (Fujii et al., 2006), and isolated a clonal cell
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line (1-11) (Fujii et al., 2008). In addition to CD13, CD29, CD44, CD71, CD90, CD105, and CD166, 1-11 cells express STRO-1 and CD146, exhibiting a similarity to bone marrow mesenchymal stem cells (Maeda et al., 2013a; Maeda et al., 2011). This cell line exhibits the potential to differentiate into osteoblastic and adipocytic cells, and produces bone/cementum-like tissues involving Sharpey’s-like fibers when transplanted into immunodeficient mice (Fujii et al., 2008).
Mechanical loading is an essential stimulus for homeostasis of various tissues such as PDL, tendons, ligaments, bone, and cartilage during development and healing. Recent studies suggest that appropriate mechanical loading is required to maintain PDL tissues (Monnouchi et al., 2011; Shi et al., 2005). Mechanical shear stress enhances the functional properties of engineered PDL constructs (Kim et al., 2011). Moreovoer, Saminathan et al. (2012) have shown that mechanical loading up-regulates connective tissue growth factor (CTGF/CCN2) gene expression in human PDL cells (Saminathan et al., 2012).
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CTGF/CCN2 is a cysteine-rich secretory protein belonging to the CCN family whose members include cysteine-rich 61 (CYP61/CCN1), nephroblastoma overexpressed (NOV/CCN3), Wnt-induced secreted protein-1 (WISP-1/CCN4), WISP-2 (CCN5), and WISP-3 (CCN6) (Holbourn et al., 2008; Lau and Lam, 1999; Moussad and Brigstock, 2000). A prototypical CCN protein includes four functional domains; an insulin-like growth factor binding protein-like module, von Willebrand factor type C repeat module, thrombospondin type-1 repeat module, and C-terminal cysteine knot-containing module (CT), although CCN5 lacks a CT module (Holbourn et al., 2008; Lau and Lam, 1999; Moussad and Brigstock, 2000). CTGF/CCN2 has been implicated as a key regulatory factor in many biological and pathological events including cell adhesion (Babic et al., 1999; Kireeva et al., 1997), proliferation (Asano et al., 2005; Shimo et al., 1999), migration (Babic et al., 1999; Guo et al., 2009; Muromachi et al., 2012; Shimo et al., 1999), extracellular matrix (ECM) production (Frazier et al., 1996), wound healing (Shi et al., 2012), angiogenesis (Hall-Glenn et al., 2012; Shimo et al., 1999), and endochondral ossification (Ivkovic et al., 2003; Takigawa et al., 2003). Furthermore, CTGF/CCN2 induces osteogenesis of mouse adipose-derived stromal cells (Xu et al., 2010).
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In
this
study,
we
investigated
the
effects
of
CTGF/CCN2
on
osteo/cementoblastic differentiation of 1-11 cells and those of combined treatment with CTGF/CCN2 and TGF-1 on fibroblastic differentiation.
MATERIALS AND METHODS Materials Recombinant human CTGF/CCN2 was purchased from BioVendor (Candler, NC). Recombinant human TGF-1 was purchased from Millipore (Temecula, CA). Goat polyclonal anti-rat/human CTGF/CCN2 and rabbit polyclonal anti-rat/human TGF-1 antibodies, and normal goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Normal rabbit IgG was purchased from R&D Systems (Minneapolis, MN). Alexa Fluor 568-conjugated donkey anti-goat and Alexa Fluor 488-conjugated chicken anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA).
Immunohistochemistry
Three five-week-old male Sprague-Dawley rats were purchased from Kyudo (Saga, Japan). The rats were sacrificed by transcardial perfusion with 4%
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paraformaldehyde (Merck, Darmstadt, Germany) in phosphate buffered saline (PBS) under anesthesia [2 mg/kg midazolam (Sandoz, Tokyo, Japan), 0.15 mg/kg medetomidine (Kyoritsu Seiyaku, Tokyo, Japan), and 2.5 mg/kg butorphanol tartrate (Meiji Seika Pharma, Tokyo, Japan)]. Maxillae were removed and then immersed in the same fixative for a further 12 h. The tissues were washed with PBS and decalcified in 10% ethylenediaminetetraacetic acid (Wako, Osaka, Japan) at 4 C for 4 weeks. Then, the tissues were dehydrated and embedded in O.C.T. Compound (Sakura Finetek USA, CA), followed by preparation of 5 μm horizontal sections. After blocking with 2% bovine serum albumin (BSA) in PBS for 1 h, immunofluorescence staining was performed with goat polyclonal anti-rat/human CTGF/CCN2 or normal IgG (1:50), rabbit polyclonal anti-rat/human TGF-1 primary antibodies or normal IgG (1:50). After washing with PBS, the sections were reacted with Alexa Fluor 568-conjugated donkey anti-goat (1:100) and Alexa Fluor 488-conjugated chicken anti-rabbit (1:100) secondary antibodies for 1 h. The rat PDL tissue was then washed with PBS and counterstained with DAPI (Vector Laboratories, Burlingame, CA). The sections were observed under a Biozero digital microscope (Keyence, Osaka,
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Japan). All procedures were approved by the Animal Ethics Committee and conformed to the regulations of Kyushu University.
In addition, HPDLCs were fixed with 4% paraformaldehyde and 0.5% dimethyl sulfoxide (Wako) in PBS for 20 min. After blocking with 2 % BSA in PBS for 1 h, the cells were immunostained as described above.
Cell culture Human PDL cells (HPDLCs) were isolated from healthy premolars or third molars from three different patients, a 23-year-old male (HPDLC-3S), a 25-year-old female (HPDLC-3U), and a 21-year-old female (HPDLC-3Q) who visited Kyushu University Hospital for extraction as described previously. HPDLCs and 1-11 cells were maintained in alpha-minimum essential medium (α-MEM; Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; BioWest, Nuaille, France), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan) at 37 C in a humidified atmosphere with 5% CO2. All procedures were performed in compliance with the Research Ethics Committee of the Faculty of Dentistry, Kyushu University.
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Application of mechanical stress HPDLCs (2x105 cells/chamber) were pre-cultured in flexible-bottomed culture chambers coated with type I collagen (Cellmatrix I-P, Nitta Gelatin Inc, Osaka, Japan) until subconfluence according to our recent study (Monnouchi et al., 2011). Then, stretch loading was applied to HPDLC culture chambers in a CO2 incubator by a STB-140 (STREX, Osaka, Japan). HPDLCs were stretched at 60 cycles/min (0.5 sec stretch and 0.5 sec relaxation per cycle) with a tension force producing 8% elongation. After stretch loading, HPDLCs were subjected to RNA extraction and an enzyme-linked immunosorbent assay (ELISA).
Quantitative RT-PCR First-strand cDNA was synthesized with a Prime Script RT Reagent kit (Takara Bio, Shiga, Japan). RT-PCR was performed with a SYBR Green II RT-PCR kit (Takara Bio) using a Thermal Cycler Dice Real Time System (Takara Bio) as described previously (Maeda et al., 2010). Specific primer sequences, annealing temperatures, and product sizes for each gene are listed in Table 1. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal
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control. Expression levels of the target genes were calculated using ∆∆Ct values.
ELISA HPDLC-3S, -3U, and -3Q cells (1.5x105 cells/chamber) were subjected to stretch loading for 3 h or non-loading in 1% FBS/α-MEM. The concentration of CTGF/CCN2 in the culture supernatant was measured with a commercially available ELISA kit (Aviscera Bioscience, Santa Clara, CA) according to the manufacturer’s instructions.
Cell proliferation assay Cell line 1-11 (1.5x103 cells/well) was cultured on 48-well plates (Becton Dickinson Labware, Lincoln Park, NJ) with or without CTGF/CCN2 (1, 10, and 100 ng/ml) in 2% FBS/α-MEM for 24 or 48 h. The proliferation rate was measured at the indicated time points using a Premix WST-1 Cell Proliferation Assay System (Takara Bio) according to the manufacturer’s instructions.
Cell cycle analysis
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Cell line 1-11 (1.2x105 cells/dish) was cultured on 10-cm dishes (Becton Dickinson Labware) with or without CTGF/CCN2 (100 ng/ml) in 2% FBS/-MEM for 48 h. The cells were trypsinized and detached, washed with PBS, and then fixed in 70% ethanol overnight at 4 °C. Then, the cells were washed with PBS and incubated with RNase (Sigma-Aldrich, St. Louis, MO) and propidium iodine (Sigma-Aldrich) for 30 min. The percentage of cells in the three phases of the growth cycle (G0/G1, G2/M, and S phase) was then measured by flow cytometry (EC800 Cell Analyzer, Sony, Tokyo, Japan). Data were analyzed using Eclipse software (Sony).
Cell migration assay A scratch wound healing assay was performed to assess the effect of CTGF/CCN2 on 1-11 cell migration in response to injury according to our recent study (Yamamoto et al., 2012). Cell line 1-11 (5x104
cells/well) was seeded on
12-well plates (Becton Dickinson Labware) in 10% FBS/α-MEM and grown to subconfluence. Then, a scratch wound was made across the diameter of the well using the end of a 200 μl pipette tip. The scraped cells were removed by three washes with PBS. The cells were then treated with CTGF/CCN2 (1, 10,
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and 100 ng/ml) in 2% FBS/α-MEM. For each well, images were taken at 0 and 6 h after injury, and the number of cells that migrated into the wound space was manually counted in three wells.
CTGF/CCN2
treatment
of
cell
line
1-11
and
analysis
of
bone/cementum-related gene expression Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates (Becton Dickinson Labware) in 10% FBS/α-MEM as control medium (CM) or in osteogenic differentiation
medium
[DM;
10%
FBS/α-MEM
containing
2
mM
-glycerophosphate (Sigma-Aldrich), 50 μg/ml ascorbic acid (Nacalai Tesque), and 0.1 mM dexamethasone (Calbiochem, Billerica, MA)] with CTGF/CCN2 (1, 10, and 100 ng/ml) for 7 days. The cells were lysed in TRIzol Reagent (Invitrogen) according to our previous study (Wada et al., 2004), and then total RNA was extracted and analyzed by quantitative RT-PCR.
Staining and quantification of alkaline phosphatase activity Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates in 10% FBS/α-MEM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 14 days. The
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cells were then fixed in a solution of 25% citrate (Sigma-Aldrich), 8% formalin (Wako), and 66% acetone (Wako) for 10 seconds at room temperature. After rinsing with sterile water, alkaline phosphatase (ALP) in the cells was stained according to the manufacturer’s instructions (Vector Laboratories) in the dark for 30 min at room temperature. ALP activity of the differentiated cells was determined using an ALP assay kit (Takara Bio) according to the manufacturer’s instructions. 1-11 cells (7x102 cells/well) were cultured on 96-well plates in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 14 days. Then, the cells were exposed to extraction solution and buffer from the ALP assay kit for 30 min at 37 C. ALP activity in the cell lysate was assayed by measuring p-nitrophenol used as the substrate at an absorbance of 405 nm.
Alizarin red staining and quantification Cell line 1-11 (4x103 cells/well) was cultured on 24-well plates in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml) for 16 days. The cells were fixed for 60 min in 10% formalin and then stained with an Alizarin red S solution (pH 4.1–4.3)
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(Sigma-Aldrich) for 60 min. Stained cells were washed extensively with sterile water to remove nonspecific precipitation.
CTGF/CCN2 and/or TGF-1 treatment of cell line 1-11 Cell line 1-11 (1x104 cells/dish) was cultured on 35-mm dishes (Becton Dickinson Labware) with CTGF/CCN2 (100 ng/ml) and/or TGF-1 (0.5 ng/ml) in 2% FBS/α-MEM for 48 h. The cells were lysed in TRIzol Reagent according to our previous study (Wada et al., 2004), and then total RNA was extracted and analyzed by quantitative RT-PCR.
Collagen assay Collagen synthesis in 1-11 cells (2x103 cells/well) cultured on 24-well plates with CTGF/CCN2 (100 ng/ml) and TGF-1 (0.5 ng/ml) in 2% FBS/α-MEM for 72 h was determined using a Sircol collagen assay kit (Biocolor, Northern Ireland, UK) according to the manufacturer’s instructions. Culture supernatants were collected and mixed with Sircol dye regent. The mixed solution was transferred to a microcentrifuge and spun at maximum speed for 10 min. The collagen pellet was washed once with acid-salt wash reagent, and then the alkaline reagent
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was added to the pellet. Each sample was transferred to an individual well of a 96-well plate to measure the absorbance at 570 nm.
Statistical analysis All values are expressed as means SD. Statistical analyses of the results were performed using one-way ANOVA followed by Dunnett’s test and Tukey’s test, or two-way ANOVA followed by Tukey’s test for multiple comparisons. Student’s unpaired t-test was also used for comparison of two means. A p-value of less than 0.05 was considered statistically significant.
Results Expression of CTGF/CCN2 in rat PDL tissue and HPDLCs Immunofluorescence analysis revealed positive staining for CTGF/CCN2 throughout rat PDL tissue (Fig. 1A). Immunocytochemical staining also exhibited positive staining for CTGF/CCN2 in HPDLC-3S, -3U, and -3Q (Fig. 1C, E, G). CTGF/CCN2 staining was intense in the cytoplasm of all HPDLCs. Control staining with normal goat IgG was negative in PDL tissue and HPDLCs (Fig. 1B, D, F, H).
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Stretch loading-induced CTGF/CCN2 gene expression and secretion in HPDLCs The effects of 8% stretch loading on CTGF/CCN2 expression in HPDLC-3S, -3U, and -3Q were examined by quantitative RT-PCR and ELISA (Fig. 2). Compared with non-loading, the expression level of CTGF/CCN2 mRNA was up-regulated by exposure to stretch loading (Fig. 2A, B, C). Next, we determined the concentration of CTGF/CCN2 secreted from stretch-loaded HPDLC-3S, -3U, and -3Q. Compared with non-loading, secretion of CTGF/CCN2 was significantly increased by exposure to stretch loading (Fig. 2D, E, F).
Effects of CTGF/CCN2 on the proliferation, cell cycle distribution, and migration of cell line 1-11
A WST-1 assay was performed to evaluate the proliferation of 1-11 cells treated with CTGF/CCN2. Compared with other concentrations, 1-11 cell proliferation was significantly increased by treatment with 100 ng/ml CTGF/CCN2 for 48 h (Fig. 3A). To furthermore confirm this result, the cell cycle distribution was
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analyzed by determining DNA content. Compared with untreated cells, the proportion of CTGF/CCN2-treated 1-11 cells was significantly increased in G2/M phase and decreased in G0/G1 phase, indicating increased cell growth (Fig. 3B-D). A scratch wound healing assay revealed an increase in the migration of 1-11 cells treated with CTGF/CCN2 for 6 h compared with that of untreated cells. The number of migrated cells was increased in a dose-dependent manner (1, 10, and 100 ng/ml CTGF/CCN2) (Fig. 3E, F).
Expression of bone/cementum-related genes in CTGF/CCN2-treated cell line 1-11 1-11 cells cultured in DM showed a significant increase in the expression of bone/cementum-related genes such as ALP, bone sialoprotein (BSP), osteopontin (OPN), and cementum-derived attachment protein (CAP) compared with that in CM-cultured cells (Fig. 4A-H). Moreover, addition of CTGF/CCN2 (100 ng/ml) to DM resulted in further augmentation of the expression of these genes (Fig. 4B, D, F, H). However, 1-11 cells cultured in CM with CTGF/CCN2 (1,
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10, and 100 ng/ml) showed no significant increase in these genes (Fig. 4A, C, E, G).
Promotion of osteo/cementoblastic differentiation of CTGF/CCN2-treated cell line 1-11 We examined ALP activity and mineralization in CTGF/CCN2-treated 1-11 cells. After 14 days of culture in CM or DM with CTGF/CCN2 (1, 10, and 100 ng/ml), the ALP activity was significantly and dose-dependently increased in DM-cultured 1-11 cells (Fig. 5C) while CM-cultured cells with CTGF/CCN2 (1, 10, and 100 ng/ml) revealed no significant increase (Fig. 5A). In addition, ALP staining of 1-11 cells treated as described above revealed the same tendency (Fig. 5B, D). Next, 1-11 cells were treated in the same manner for 16 days and then subjected to Alizarin red staining. The results demonstrated a remarkable enhancement of reactive products in DM-cultured cells treated with 100 ng/ml CTGF/CCN2 (Fig. 5F, G) while CM-cultured cells with CTGF/CCN2 (1, 10, and 100 ng/ml) showed no positive reaction (Fig. 5E).
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Expression of CTGF/CCN2 and TGF-1 in rat PDL tissues and HPDLCs Immunofluorescence analysis revealed co-localized positive staining for CTGF/CCN2 and TGF-1 in rat PDL tissue (Fig. 6A-C). Immunocytochemical staining exhibited positive staining for CTGF/CCN2 and TGF-1 in HPDLC-3S (Fig. 6E-G), -3U (Fig. 6I-K), and -3Q (Fig. 6M-O), and demonstrated cytoplasmic co-localization of these proteins. Control staining with normal goat and rabbit IgGs was negative in PDL tissue and HPDLCs (Fig. 6D, H, L, P).
Expression of fibroblast-related genes and soluble collagen in cell line 1-11 treated with both CTGF/CCN2 and TGF-1 Recently we have reported that TGF-1 promotes fibroblastic differentiation of 1-11 cells (Fujii et al., 2010; Kono et al., 2013). Qi et al. (2005) furthermore reported that combined treatment of human renal cells with CTGF/CCN2 and TGF-1 promotes their fibroblastic differentiation (Qi et al., 2005). In this context, we investigated whether combined treatment with CTGF/CCN2 and TGF-1 up-regulated fibroblastic differentiation of 1-11 cells. The cells were treated with CTGF/CCN2 (100 ng/ml) and/or TGF-1 (0.5 ng/ml) for 48 h. As a result, the expression of fibroblast-related genes, type I collagen (COL I) and fibronectin,
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was clearly up-regulated by TGF-1 treatment. Additionally, combined treatment with CTGF/CCN2 and TGF-1 exhibited further enhancement of the expression of these genes (Fig. 7A, B). Secreted collagen products from 1-11 cells subjected to combined treatment with CTGF/CCN2 and TGF-1 was compared with that of TGF-1 treatment alone (Fig. 7C). Similar to the results shown in Fig. 7A, the combination of CTGF/CCN2 and TGF-1 stimuli significantly up-regulated collagen synthesis (Fig. 7C).
Discussion Although PDL tissue is continually subjected to mechanical loading because of bite force, this is very important to maintain the structural and physiological conditions, and heal PDL tissue (Fujihara et al., 2010). Our recent study indicated that mechanical loading induces the expression of ALP and TGF-1 in HPDLCs through the renin-angiotensin system (Monnouchi et al., 2011). These molecules are expressed in functional PDL cells in vivo. In addition, another study reported increased synthesis of ECM components, such as OPN, dentin matrix protein 1, and COL I, in PDL cells subjected to mechanical loading (Berendsen et al., 2009).
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CTGF/CCN2 reportedly has a role in the repair of many tissues, including bone regeneration and organ fibrosis (Kikuchi et al., 2008; Shi-Wen et al., 2008). It is also known to contribute to mouse tooth development as well as proliferation and differentiation of mouse PDL cells (Asano et al., 2005; Shimo et al., 2002; Yamaai et al., 2005). In addition, a recent study reported that CTGF/CCN2 gene expression in human PDL cells is promoted by stretch loading (Saminathan et al., 2012). Our current study clearly demonstrated increased production of CTGF/CCN2 protein in HPDLCs exposed to stretch loading. In this context, we focused on the physiological effects of CTGF/CCN2 on the cell line 1-11. Although the ELISA data detected low concentration of CTGF/CCN2 in culture media of HPDLCs, we consider that the concentration of CTGF secreted from HPDLC exposed to stretch-loading would get higher at the limited area around secreting cells, and therefore secreted CTGF might probably work in an autocrine or paracrine manner. First, we examined the proliferative and migratory effects of CTGF/CCN2 on 1-11 cells. The results indicated that CTGF/CCN2 treatment led to effective expansion of the cells and induction of cell mobility, suggesting a therapeutic effect on wounded PDL using PDL stem cells. Recent studies have also shown
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that CTGF/CCN2 promotes the proliferation of mouse PDL cells (Asano et al., 2005), and induces migration of human dental pulp cells (Muromachi et al., 2012) and mouse mesenchymal cells (Song et al., 2007), which supports our data. Previous studies have reported that CTGF/CCN2 promotes mineral nodule formation of human PDL progenitor cells (Dangaria et al., 2009) and up-regulates osteogenic differentiation of human adipose-derived stromal cells (Xu et al., 2010). However, the underlying mechanism of CTGF/CCN2 in osteo/cementoblastic and fibroblastic differentiation of immature PDL cells has not been examined in detail. Asano et al. (2005) reported that CTGF/CCN2 promotes the synthesis of PDL-related molecules, such as Alp and Col I, but not bone-related markers, Opn and osteocalcin in mouse PDL cells, suggesting a certain role in development and regeneration of PDL tissue. We also found that 1-11 cells cultured in CM did not show an increase of bone/cementum-related molecule expression independently of the increasing concentration of CTGF/CCN2. However, 1-11 cells cultured in DM showed up-regulation of ALP, OPN, BSP, and even CAP, subsequently resulting in mineralization, suggesting their osteo/cementoblastic differentiation because CAP is a specialized protein
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of the cementoblast although this cell shares most of the characteristics with the osteoblast, such as the expression of ALP, OPN, and BSP. These results suggest that CTGF/CCN2 has an intrinsic effect on the differentiation of immature PDL cells, which is dependent on the environment. As PDL tissue does not mineralize per se, we examined how CTGF/CCN2 contributed to these events, viz fibroblastic and osteo/cementoblastic differentiation of immature PDL cells.
Our recent studies indicate that TGF-1 is abundantly expressed in the entire PDL tissue and involved in fibroblastic differentiation of immature PDL cells, suggesting its role in maintaining the homeostasis of PDL tissue (Fujii et al., 2010; Kono et al., 2013). In addition, CTGF/CCN2 acts downstream of TGF-β1 signaling as a co-operative mediator (Lee et al., 2010) although it could be also induced by BMP9 and Wnt3a (Luo et al., 2004). Our present results revealed co-localization of CTGF/CCN2 and TGF-β1 in PDL tissue. Qi et al reported that combined treatment with these proteins induces fibroblastic differentiation of human renal cells, indicating that CTGF/CCN2 facilitates the effects of TGF-1 to induce their fibroblastic differentiation (Qi et al., 2005). Therefore, we also examined the combined effects of CTGF/CCN2 with
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TGF-1 on the differentiation of 1-11 cells to clarify the role of CTGF/CCN2 in PDL tissue, because PDL tissue is a fibrous tissue that does not exhibit mineralization. We first confirmed that both proteins were co-expressed in HPDLCs and throughout PDL tissue, which suggested a co-operative relationship between these factors in the biological function of PDL tissue. We found increased expression of COL1 gene and protein, and fibronectin in co-treated cells compared with that in cells treated with TGF-1 alone. In support of our results, a recent study showed that simultaneous knockdown of TGF-β1, TGFβR2, and CTGF reduces Col I expression in rabbit corneal fibroblasts (Sriram et al., 2013). Li et al. (2013) reported expression of CTGF/CCN2 and TGF-1 in the developing periodontium, suggesting that regulation of CTGF/CCN2 by TGF-1 might be cell-specific in the periodontium. Taken together, CTGF/CCN2 might have biphasic effects on the differentiation of immature PDL cells, which is probably dependent on the surrounding
cell
populations
via
the
influence
of
osteoblasts
and
cementoblasts on the surface of bone and cementum or PDL fibroblasts in ligamentous tissue. As the periodontium is composed of hard tissue such as bone and cementum, and soft tissue such as the ligamentous tissue and
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gingiva, these features of CTGF/CCN2 imply that it could be efficient as an optimal therapeutic agent for repair of the damaged periodontium.
Acknowledgements This study was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant numbers: 23689077, 24390426, 24659848, 24792028, 25293388, and 25670811). We thank Dr. Hiroko Tsuda for useful suggestion on statistical analysis, and Drs. Hideki Sugii, Shinichirou Yoshida, Suguru Serita, Yumi Mitarai, and Hiroyuki Mizumachi for their great support in preparation of this work.
Literature Cited Asano M, Kubota S, Nakanishi T, Nishida T, Yamaai T, Yosimichi G, Ohyama K, Sugimoto T, Murayama Y, Takigawa M. 2005. Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells. Cell Commun Signal 3:11. Babic AM, Chen CC, Lau LF. 1999. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19(4):2958-2966.
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Berendsen AD, Smit TH, Walboomers XF, Everts V, Jansen JA, Bronckers AL. 2009. Three-dimensional loading model for periodontal ligament regeneration in vitro. Tissue Eng Part C Methods 15(4):561-570. Chai Y, Jiang X, Ito Y, Bringas P, Jr., Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM. 2000. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127(8):1671-1679. Dangaria SJ, Ito Y, Walker C, Druzinsky R, Luan X, Diekwisch TG. 2009. Extracellular matrix-mediated differentiation of periodontal progenitor cells. Differentiation 78(2-3):79-90. Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR. 1996. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107(3):404-411. Fujihara C, Yamada S, Ozaki N, Takeshita N, Kawaki H, Takano-Yamamoto T, Murakami S. 2010. Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells. J Biol Chem 285(36):28286-28297. Fujii S, Maeda H, Tomokiyo A, Monnouchi S, Hori K, Wada N, Akamine A. 2010. Effects of TGF-beta1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line. Cell Tissue Res 342(2):233-242. Fujii S, Maeda H, Wada N, Kano Y, Akamine A. 2006. Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer. Cell Tissue Res 324(1):117-125. Fujii S, Maeda H, Wada N, Tomokiyo A, Saito M, Akamine A. 2008. Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo. J Cell Physiol 215(3):743-749.
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Guo CM, Wang YS, Hu D, Han QH, Wang JB, Hou X, Hui YN. 2009. Modulation of migration and Ca2+ signaling in retinal pigment epithelium cells by recombinant human CTGF. Curr Eye Res 34(10):852-862. Hall-Glenn F, De Young RA, Huang BL, van Handel B, Hofmann JJ, Chen TT, Choi A, Ong JR, Benya PD, Mikkola H, Iruela-Arispe ML, Lyons KM. 2012. CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis. PLoS One 7(2):e30562. Holbourn KP, Acharya KR, Perbal B. 2008. The CCN family of proteins: structure-function relationships. Trends Biochem Sci 33(10):461-473. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM. 2003. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130(12):2779-2791. Kikuchi T, Kubota S, Asaumi K, Kawaki H, Nishida T, Kawata K, Mitani S, Tabata Y, Ozaki T, Takigawa M. 2008. Promotion of bone regeneration by CCN2 incorporated into gelatin hydrogel. Tissue Eng Part A 14(6):1089-1098. Kim SG, Viechnicki B, Kim S, Nah HD. 2011. Engineering of a periodontal ligament construct: cell and fibre alignment induced by shear stress. J Clin Periodontol 38(12):1130-1136. Kireeva ML, Latinkic BV, Kolesnikova TV, Chen CC, Yang GP, Abler AS, Lau LF. 1997. Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development. Exp Cell Res 233(1):63-77. Kono K, Maeda H, Fujii S, Tomokiyo A, Yamamoto N, Wada N, Monnouchi S, Teramatsu Y, Hamano S, Koori K, Akamine A. 2013. Exposure to transforming growth factor-beta1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines. Cell Tissue Res 352(2):249-263.
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Lau LF, Lam SC. 1999. The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res 248(1):44-57. Lee CH, Shah B, Moioli EK, Mao JJ. 2010. CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J Clin Invest 120(9):3340-3349. Luo Q, Kang Q, Si W, Jiang W, Park JK, Peng Y, Li X, Luu HH, Luo J, Montag AG, Haydon RC, He TC. 2004. Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279(53):55958-55968. Maeda H, Fujii S, Tomokiyo A, Wada N, Akamine A. 2013a. Periodontal tissue engineering: defining the triad. Int J Oral Maxillofac Implants 28(6):e461-471. Maeda H, Nakano T, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Hori K, Akamine A. 2010. Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells. J Endod 36(4):647-652. Maeda H, Tomokiyo A, Fujii S, Wada N, Akamine A. 2011. Promise of periodontal ligament stem cells in regeneration of periodontium. Stem Cell Res Ther 2(4):33. Maeda H, Wada N, Tomokiyo A, Monnouchi S, Akamine A. 2013b. Prospective potency of TGF-beta1 on maintenance and regeneration of periodontal tissue. Int Rev Cell Mol Biol 304:283-367. Monnouchi S, Maeda H, Fujii S, Tomokiyo A, Kono K, Akamine A. 2011. The roles of angiotensin II in stretched periodontal ligament cells. J Dent Res 90(2):181-185. Moussad EE, Brigstock DR. 2000. Connective tissue growth factor: what's in a name? Mol Genet Metab 71(1-2):276-292.
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Muromachi K, Kamio N, Narita T, Annen-Kamio M, Sugiya H, Matsushima K. 2012. MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration. J Cell Biochem 113(4):1348-1358. Qi W, Twigg S, Chen X, Polhill TS, Poronnik P, Gilbert RE, Pollock CA. 2005. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol 288(4):F800-809. Saminathan A, Vinoth KJ, Wescott DC, Pinkerton MN, Milne TJ, Cao T, Meikle MC. 2012. The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture. J Periodontal Res 47(2):212-221. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. 2004. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364(9429):149-155. Shi-Wen X, Leask A, Abraham D. 2008. Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 19(2):133-144. Shi L, Chang Y, Yang Y, Zhang Y, Yu FS, Wu X. 2012. Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing. PLoS One 7(2):e32128. Shi L, Kodama Y, Atsumi Y, Honma S, Wakisaka S. 2005. Requirement of occlusal force for maintenance of the terminal morphology of the periodontal Ruffini endings. Arch Histol Cytol 68(4):289-299. Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T, Takigawa M. 1999. Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126(1):137-145.
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Shimo T, Wu C, Billings PC, Piddington R, Rosenbloom J, Pacifici M, Koyama E. 2002. Expression, gene regulation, and roles of Fisp12/CTGF in developing tooth germs. Dev Dyn 224(3):267-278. Song JJ, Aswad R, Kanaan RA, Rico MC, Owen TA, Barbe MF, Safadi FF, Popoff SN. 2007. Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation. J Cell Physiol 210(2):398-410. Sriram S, Robinson PM, Pi L, Lewin AS, Schultz GS. 2013. Triple Combination of siRNAs Targeting TGFbeta1, TGFbetaR2 and CTGF Enhances Reduction of Collagen I and Smooth Muscle Actin in Corneal Fibroblasts. Invest Ophthalmol Vis Sci 54(13):8214-8223. Takigawa M, Nakanishi T, Kubota S, Nishida T. 2003. Role of CTGF/HCS24/ecogenin in skeletal growth control. J Cell Physiol 194(3):256-266. Wada N, Maeda H, Yoshimine Y, Akamine A. 2004. Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha. Bone 35(3):629-635. Xu Y, Wagner DR, Bekerman E, Chiou M, James AW, Carter D, Longaker MT. 2010. Connective tissue growth factor in regulation of RhoA mediated cytoskeletal tension associated osteogenesis of mouse adipose-derived stromal cells. PLoS One 5(6):e11279. Yamaai T, Nakanishi T, Asano M, Nawachi K, Yoshimichi G, Ohyama K, Komori T, Sugimoto T, Takigawa M. 2005. Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development. J Bone Miner Metab 23(4):280-288. Yamamoto N, Maeda H, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Kono K, Koori K, Teramatsu Y, Akamine A. 2012. Expression and effects of glial
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cell line-derived neurotrophic factor on periodontal ligament cells. J Clin Periodontol 39(6):556-564.
Figure Legends Figure. 1. Immunofluorescence analysis of CTGF/CCN2 in rat PDL tissue and HPDLCs.
Immunofluorescence staining for CTGF/CCN2 (red; A, C, E, G) was performed using rat PDL tissue (A), and HPDLC-3S (C, D), -3U (E, F), and -3Q (G, H). No staining was observed in samples incubated with normal control IgG (negative control; B, D, F, H). Cells were counterstained with DAPI (blue). AB, alveolar bone; PDL, periodontal ligament; D, dentin. Scale bars = 100 μm (A) and 50 μm (C, E, G).
Figure. 2. Effects of stretch loading on gene and protein expression of CTGF/CCN2 in HPDLCs.
HPDLC-3S (A, D), -3U (B, E), and -3Q (C, F) were exposed to stretch loading of 0 and 8% elongation for 1 h. Gene expression of CTGF/CCN2 in HPDLC-3S (A),
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-3U (B), and -3Q (C) was examined by quantitative RT-PCR. Human GAPDH was used as an internal standard. CTGF/CCN2 secreted into the culture medium from HPDLC-3S (D), -3U (E), and -3Q (F) exposed to 8% stretch loading or non-loading for 3 h was measured with an ELISA. Y-axis values in A, B, C represent the expression ratio relative to the control. Values are the means ± SD from three independent experiments. All data were analyzed by Student’s unpaired t-test; **p
