Differentiation 88 (2014) 33–41

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Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells Tadashi Yamamoto, Yuki Ugawa, Keisuke Yamashiro, Masayuki Shimoe, Kazuya Tomikawa 1, Shoichi Hongo, Shinsuke Kochi, Hidetaka Ideguchi, Hiroshi Maeda, Shogo Takashiba n Department of Pathophysiology—Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 10 October 2013 Received in revised form 26 August 2014 Accepted 3 September 2014 Available online 29 September 2014

The periodontal ligament is a multifunctional soft connective tissue, which functions not only as a cushion supporting the teeth against occlusal force, but is also a source of osteogenic cells that can regenerate neighboring hard tissues. Periodontal ligament cells (PDL cells) contain heterogeneous cell populations, including osteogenic cell progenitors. However, the precise mechanism underlying the differentiation process remains elusive. Cell differentiation is regulated by the local biochemical and mechanical microenvironment that can modulate gene expression and cell morphology by altering actin cytoskeletal organization mediated by Rho-associated, coiled-coil containing protein kinase (ROCK). To determine its role in PDL cell differentiation, we examined the effects of ROCK on cytoskeletal changes and kinetics of gene expression during osteogenic differentiation. PDL cells were isolated from human periodontal ligament on extracted teeth and cultured in osteogenic medium for 14 days. Y-27632 was used for ROCK inhibition assay. Osteogenic phenotype was determined by monitoring alkaline phosphatase (ALP) activity and calcium deposition by Alizarin Red staining. ROCK-induced cytoskeletal changes were examined by immunofluorescence analysis of F-actin and myosin light chain 2 (MLC2) expression. Real-time PCR was performed to examine the kinetics of osteogenic gene expression. F-actin and phospho-MLC2 were markedly induced during osteogenic differentiation, which coincided with upregulation of ALP activity and mineralization. Subsequent inhibition assay indicated that Y-27632 significantly inhibited F-actin and phospho-MLC2 expression in a dose-dependent manner with concomitant partial reversal of the PDL cell osteogenic phenotype. PCR array analysis of osteogenic gene expression indicated that extracellular matrix genes, such as fibronectin 1, collagen type I and III, and biglycan, were significantly downregulated by Y27632. These findings indicated crucial effects of ROCK in cytoskeletal reorganization and differentiation of PDL cells toward osteogenic cells. ROCK contributes to induction of osteogenic differentiation by synergistic increases in extracellular matrix gene expression in PDL cells. & 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Keywords: Periodontal ligament cells Osteogenic differentiation Rho-associated coiled-coil containing protein kinase (ROCK) Y-27632 Actin cytoskeleton

1. Introduction Periodontitis involves the loss of supporting alveolar bone around the teeth, and is one of the most prevalent infectious diseases worldwide (Pihlstrom et al., 2005). The periodontal ligament is a multifunctional soft connective tissue that plays a crucial role in homeostasis of periodontal tissue, generates fibrous attachment between the tooth root and bone, and functions as a sensor and n

Corresponding author. Tel.: þ 81 86 235 6677; fax: þ 81 86 235 6679. E-mail address: [email protected] (S. Takashiba). 1 Present address: Division of General Oral Care, Kyushu University Hospital, 31-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

cushion supporting teeth against occlusal force. Moreover, the periodontal ligament acts a source of osteogenic cells that can regenerate neighboring hard tissues although it maintains its unmineralized state (Beertsen et al., 2000). Periodontal ligament cells (PDL cells) are composed of a heterogeneous fibroblast population with different functional characteristics, including osteogenic progenitor cells (McCulloch et al., 2000). The progenitor cells may contribute to reconstruction of the lost periodontium, but no defined origin or markers that specify the individual populations are available and these cells are too rare to isolate for clinical use. In response to a variety of extracellular stimuli, PDL cells can ultimately produce more differentiated cells that can synthesize adjacent bone, cementum, and extracellular matrix (ECM) of the periodontal ligament

http://dx.doi.org/10.1016/j.diff.2014.09.002 Join the International Society for Differentiation (www.isdifferentiation.org) 0301-4681/& 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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(Seo et al., 2004; Iwata et al., 2010). PDL cells show several osteogenic properties, such as alkaline phosphatase (ALP), osteocalcin (bone gamma-carboxyglutamate (gla) protein: BGLAP), osteopontin (secreted phosphoprotein 1: SPP1), runt-related transcription factor 2 (RUNX2), and osterix (SP7) gene expression, and the capacity to form mineralized nodules (Nishida et al., 2007; Fujii et al., 2008). Meanwhile, PDL cells express negative regulators of mineralization, such as twist basic helix-loop-helix transcription factor 1 (TWIST1) and periodontal ligament-associated protein-1 (PLAP-1), to maintain their fibroblastic properties (Komaki et al., 2007; Yamada et al., 2007). The intricate combinatorial intercellular mechanisms of osteogenic differentiation remain poorly understood. The extracellular environment may principally determine the lineage commitment of mixed cell populations of PDL cells in a specific spatiotemporal manner. Cell differentiation is induced by the sensing of changes in the local biochemical and mechanical microenvironment, defined by coordinated interactions with soluble factors, other cells, and ECM with complex and dynamic regulation (Engler et al., 2006; Discher et al., 2009). Progenitor cells are highly sensitive to the intrinsic properties of their ECM (Reilly and Engler, 2010), and the cells tune their internal stiffness to match the compliance of the ECM by modulating actin polymerization and crosslinking, which may be responsible for altering cell morphology and gene expression during tissue remodeling (Mammoto and Ingber, 2009). Recent studies have provided evidence that the cytoskeleton is one of the crucial factors contributing to the overall control of cell differentiation (Cooley et al., 2011; Mammoto et al., 2012). The mammalian Rho guanosine triphosphatases (Rho GTPases) are a family of 20 intracellular signaling molecules within the superfamily of Ras-related small GTP-binding proteins, which act as molecular switches cycling between an active GTP-bound state and an inactive GDP-bound state. They are key regulators of cytoskeletal dynamics and affect many cellular processes, including morphogenesis, polarity, migration, and cell division (Jaffe and Hall, 2005). RhoA, one of most extensively characterized Rho GTPases, plays critical roles in regulating actin cytoskeleton organization in response to various stimuli (Etienne-Manneville and Hall, 2002), mediated by one of its downstream effectors, Rhoassociated, coiled-coil containing protein kinase (ROCK) (Ishizaki et al., 1996). ROCK directly phosphorylates myosin light chain 2 (MLC2) (Amano et al., 1996), which leads to increased contractility of actin–myosin bundles (stress fibers) due to an increase in myosin ATPase activity (Katoh et al., 2001), and both are prevented by exposure of the cells to Y-27632, a pharmacological inhibitor of ROCK (Uehata et al., 1997). Recent studies suggested that RhoA/ ROCK-dependent generation of actin cytoskeleton appears to be critical for switching of cell fate from growth to differentiation (Mammoto and Ingber, 2009). Changes in cell shape regulate osteogenic or adipogenic differentiation of human mesenchymal stem cells (hMSC) by modulating RhoA/ROCK signaling (McBeath et al., 2004; Kilian et al., 2010), suggesting that the cytoskeleton affects mesenchymal lineage commitment. Moreover, RhoA/ROCK serves as a mechanotransducer of matrix stiffness, and can mediate cytoskeletal reorganization and osteogenic differentiation of hMSC (Shih et al., 2011). These studies suggested that RhoA/ ROCK signaling may be essential for direct cell differentiation in response to both mechanical and soluble biochemical factors. It has been reported that the homeostasis and regeneration of PDL cells are closely related to mechanical changes due to occlusal forces (Beertsen et al., 2000; Pavlin and Gluhak-Heinrich, 2001), and it has been suggested that mechanical stress-mediated RhoA-ROCK signaling is linked to intracellular signaling in PDL cells (Yamashiro et al., 2007). We hypothesized that RhoA/ROCK signaling may have an intercellular interaction with osteogenic gene expression, and contribute to regulation of differentiation in PDL cells. This study

demonstrated that ROCK activity and the actin cytoskeleton are enhanced during osteogenic differentiation of PDL cells, and that ROCK-mediated cytoskeletal alterations have significant effects on osteogenic gene expression and differentiation of PDL cells.

2. Materials and methods 2.1. Cell culture and reagents Periodontal ligament (PDL) samples were obtained from periodontally healthy third molars or premolars extracted from donors with informed consent. Seven donors were included in this study: three for cell staining analysis and Western blotting analysis, cells from one of whom were also used for MTS assay, another three for verification of PCR array analysis, and one for Western blotting analysis (Fig. 1C) and temporal mRNA expression analysis. Prior to the experiment, the protocol (no. 975) was approved by the Research Ethics Committee for Human Genome/Gene Analysis Research in Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. Fibroblastic PDL cells were obtained as described previously with slight modifications (Seo et al., 2004; Iwata et al., 2010). Briefly, PDL was recovered from the middle of the root surface, and digested in a solution of 2 mg/mL dispase-II (Sanko Junyaku, Tokyo, Japan) and 4 mg/mL collagenase Type-I (Worthington, Lakewood, NJ) for 1 h at 37 1C. A single-cell suspension (1  104 cells in 10-cm culture dishes: Corning, Corning, NY) obtained from each donor was maintained and expanded in control medium consisting of α-modified minimum essential medium (α-MEM; Sigma-Aldrich, St. Louis, MO) supplemented with 20% fetal bovine serum (Biowest, Logan, UT), 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin). For osteogenic differentiation, subconfluent PDL cells were maintained in osteogenic medium (control medium supplemented with 50 μM ascorbic acid2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone) for 14 days. Culture medium was changed every 3 days. All cell types were used for the experiments between passages 2 and 5. All experiments were confirmed in three independent experiments, each of which was performed in triplicate. To inhibit ROCK activity, a specific inhibitor of ROCK, Y-27632 (Nacalai Tesque, Kyoto, Japan), was dissolved to a final concentration of either 1 mM or 10 mM in sterile water (recommended solvent for Y-27632), and used at different concentrations up to 100 μM diluted with culture medium with daily application. Control samples without the inhibitor were supplemented with an equivalent volume of sterile water. 2.2. Cell staining For immunofluorescence staining, PDL cells were fixed in 3.7% formaldehyde/phosphate-buffered saline, incubated with 10% goat serum (Life Technologies, Gaithersburg, MD), and then incubated with 1:40 dilution of Alexa Fluor 594 phalloidin (Life Technologies) for filamentous actin (F-actin). The samples were subsequently incubated with 1:50 dilution of anti-phospho-myosin light chain 2 (Thr18/Ser19) antibody (pMLC2; Cell Signaling, Beverly, MA) followed by addition of 1:500 dilution of Alexa Fluor 488labeled secondary antibody (Life Technologies). Nuclear staining was performed using 40 ,6-diamidino-2-phenylindole (DAPI). Staining signals were visualized using a fluorescence microscope (BX50, DP70; Olympus, Tokyo, Japan). For ALP staining, PDL cells were fixed in citrate/acetone/ formaldehyde solution, and stained using alkaline phosphatase kit #86 (Sigma-Aldrich) in accordance with the manufacturer’s instructions. For Alizarin Red staining, PDL cells were fixed in formaldehyde/methanol solution (30/70 [v/v]: 37% formaldehyde and absolute methanol), and accumulation of calcium was detected by staining with 1% Alizarin Red S (Sigma-Aldrich) in

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Fig. 1. Induction of ROCK signaling during osteogenesis. Subconfluent PDL cells were cultured for 14 days in either osteogenic or normal medium, and then subjected to the following assays. Cells from three donors were used for staining assays, and typical images are shown. (A) ALP ((a), (b)) and Arizarin red ((c), (d)) staining were performed on osteogenic ((a), (c)) and normal ((b), (d)) PDL cells. Osteogenic PDL cells were more densely stained for the calcified markers compared to normal cells. Scale bar: 5 mm. (B) Immunofluorescence analysis was performed on osteogenic ((a)–(d)) and normal ((e)–(h)) PDL cells for F-actin (red: (b), (f)), pMLC (green: (c), (g)), and DAPI (Blue: (a), (e)) on the same sections. Overlays of the two images are shown (merge: (d), (h)) with colocalization shown in yellow. The signals of both F-actin and pMLC were intense in osteogenic cells compared to normal cells. Scale bar: 100 μm. (C) Western blotting was performed on protein extracts (30 μg in each lane) from PDL cells cultured for 14 days in control medium (normal) or osteogenic medium (osteogenic). The immunoblots present F-actin and GAPDH as an internal control. F-actin signals were quantified by densitometric analysis. The levels of expressions relative to GAPDH are shown on the y-axis. Results represent the means7SD; n¼ 3 (three independent experiments using PDL cells from a single donor). *Po0.05, Student’s t test.

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28% ammonium hydroxide (pH 5.0). Stained cells were photographed using a commercial digital camera, and light microscopic analysis was performed (Eclipse TS100, DS-2Mv; Nikon, Tokyo, Japan).

statistical significance. For temporal mRNA analysis, PDL cell cultures were harvested at 3, 6, 9, and 12 days.

2.3. Cell viability assay

To examine the histochemical localization in periodontal tissue, 12-week-old C57BL/6J mice (CLEA Japan, Tokyo, Japan) were used for the experiments. The upper jaw including the teeth and periodontal tissue was obtained from the mice under diethyl ether inhalation anesthesia. The samples were fixed immediately in 4% paraformaldehyde-phosphate buffered saline for 1 h, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 5 days, and embedded in paraffin. Coronal cross-sections 2 μm thick were taken from the second molar region. After routine deparaffinization and rehydration, the sections were heated in 1 mM EDTA for epitope retrieval. The sections were then blocked, and incubated with an antibody against pMLC2 (Thr18/ Ser19) Cell Signaling, Beverly) at 1:25 dilution followed by incubation with Alexa Fluor 594-conjugated secondary antibodies (Life Technologies) at 1:200 dilution. Nuclear staining was performed using DAPI. Staining signals were visualized using a fluorescence microscope. Staining was performed in triplicate on three separate samples. The primary antibody was excluded from negative controls. This experiment was conducted in accordance with the Guidelines for the Treatment of Experimental Animals and approved by the Animal Research Control Committee of Okayama University (#OKU-2013230).

Cell viability was assessed using a reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt; MTS] (CellTiter96s AQueous One Solution Reagent Cell Proliferation Assay; Promega, Madison, WI). PDL cells were seeded into 96well plates (Corning) in osteogenic medium at 5  103 cells per well, and incubated for 24 h. Thereafter, PDL cells were treated with Y-27632 at concentrations up to 100 μM. After 48 h or 14 days of incubation, color development was performed according to the manufacturer’s technical bulletin. The absorbance at 490 nm (A490) was measured using a microplate reader (Model-680; BioRad, Hercules, CA). The data are presented as means 7 standard deviation (SD) from three independent experiments using PDL cells from a single donor, and analyzed for statistical significance by Student’s t test. P o0.05 was taken to indicate statistical significance. 2.4. Western blotting analysis PDL cells (1  105 cells/well) were seeded into 6-well plates (Corning). After 14 days in culture, aliquots of 30 μg of total protein from each sample were subjected to Western blotting as described previously (Shimoe et al., 2014) using antibodies specific to F-actin (Abcam, Cambridge, MA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (Cell Signaling) at a dilution of 1:500 and 1:3000. The signal intensities were quantified by densitometric analysis using ImageJ (http://rsb.info.nih.gov/ij/). The data are presented as means 7 SD and were analyzed for statistical significance by Student’s t test. Po0.05 was taken to indicate statistical significance. 2.5. Real-time RT-PCR PDL cell cultures were harvested at 14 days. Aliquots of 1 μg of total RNA were recovered from the cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol, including Proteinase K and DNase I treatment. PCR array analysis of the expression of 84 spotted osteogenic genes was performed using Human osteogenesis RT² Profiler™ PCR Array (PAHS-026Z) (Qiagen) according to the manufacturer’s protocol. Data were analyzed and a heat map was generated by PCR Array Data Analysis Web Portal (version 3.5) using the default set format (Supplementary Fig. 1). Five endogenous control genes, β-actin, β2microglobulin, GAPDH, hypoxanthine phosphoribosyltransferase 1, and ribosomal protein, large, P0 were used for data normalization. Quantitative RT-PCR analyses were performed using cDNA transcribed by SuperScript III (Life Technologies), an ABI 7300 RealTime PCR system, and Power SYBRR Green PCR Master Mix (both from Applied Biosystems, Foster City, CA). Gene-specific primers were designed using Primer3 (http://bioinfo.ut.ee/primer3–0.4.0/ primer3/), and are described in Supplementary Table 1. The amplification conditions consisted of an initial 10 min of denaturation at 95 1C, followed by 40 cycles of denaturation at 95 1C for 10 s, annealing at 60 1C for 15 s, and elongation at 72 1C for 20 s. Relative expression is shown after normalization relative to the expression of GAPDH mRNA. The data were quantified using the ΔCt method (Shimoe et al., 2014). The data are presented as means from three different donors, and were analyzed for statistical significance by Student’s t test. P o0.05 was taken to indicate

2.6. Immunofluorescence analysis of murine periodontal tissue

3. Results 3.1. Induction of F-actin and ROCK signaling during osteogenic differentiation To examine the effects of ROCK signaling during osteogenic differentiation, immunofluorescence analysis was performed using differentiated PDL cells in osteogenic medium and normal medium after 14 days in culture. The osteogenic properties of each cell type were confirmed by ALP and Alizarin Red staining. Differentiated PDL cells showed intense staining with both ALP and Alizarin Red, whereas normal PDL cells showed only sparse ALP staining and no visible staining with Alizarin Red (Fig. 1A). Immunofluorescence analysis demonstrated that differentiated PDL cells showed abundant actin filaments accompanied by increased MLC2 phosphorylation (pMLC2), a major effector of ROCK, indicating that ROCK signaling was activated during osteogenic differentiation of PDL cells. Normal PDL cells showed thinner actin filaments and only weak pMLC2 staining (Fig. 1B). Western blotting analysis indicated that F-actin expression was increased in differentiated PDL cells compared with normal PDL cells (Fig. 1C). 3.2. ROCK-dependent expression of F-actin and pMLC2 Although Y-27632 has been widely used as a ROCK inhibitor, the biologically optimal concentration of Y-27632 in PDL cells has not yet been determined. Therefore, MTS-based viability assay was performed to determine cell toxicity of Y27632 (Fig. 2A and B), and the inhibitory effect of Y27632 on ROCK signaling was assessed based on the levels of F-actin and pMLC2 (Fig. 2C). After 48 h of culture with Y-27632, PDL cell viability was stable or slightly increased by Y27632 at concentrations up to 10 μM. PDL cell viability was reduced slightly in the presence of 20 μM Y27632, and showed a significant decrease above this concentration (Fig. 2A). After 14 days of culture with Y-27632 at concentrations up to 20 μM, PDL cell viability was stable (Fig. 2B), indicating that long-term treatment with Y-27632 at concentrations up to 20 μM does not reduce cell numbers. Moreover, pMLC2 was decreased in a dose-dependent manner, and there was no detectable staining for pMLC2 in the presence of 20 μM Y27632.

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F-actin bundles appeared significantly thinner and less dense in the presence of 10 μM Y27632. Even in the presence of 20 μM Y-27632, disassembled and dot-like aggregated F-actin was abundant, but no significant morphogenic changes were observed in the nuclei (Fig. 2C). Densitometric analysis indicated that dose-dependent Y27632 inhibited F-actin expression (Fig. 2D). These results indicated that 10 μM Y27632 has no toxic effect on PDL cell viability and is the minimum requirement for inactivation of ROCK signaling in PDL cells.

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3.3. Inhibition of osteogenic differentiation by Y27632 ROCK inhibition assay was performed using several doses of Y27632 to elucidate the correlation between ROCK signaling and osteogenic differentiation in PDL cells. After cultivation in osteogenic medium with or without Y27632 for 14 days, the effects of Y27632 on mineralization markers were assessed (Fig. 3). Macroscopic analysis of ALP and Alizarin Red staining showed that Y27632 decreased the intensity of staining for each marker in a dose-dependent manner. Microscopic analysis also indicated the dose-dependent inhibition of the intracellular ALP activity and extracellular formation of

Fig. 3. Inhibitory effects of Y27632 on osteogenesis. Subconfluent PDL cells were cultured for 14 days in osteogenic medium supplemented with Y27632 up to 20 μM. After routine fixation, ALP and Alizarin Red staining were performed. Cells from three donors were used for the assays, and typical images are shown. Lower panels show higher magnification images of ALP staining of the identical samples supplemented with several doses of Y27632 (0 μM: (a), (e); 1 μM: (b), (f); 10 μM: (c), (g); 20 μM: (d), (h)) and alizarin red staining (0 μM: (i), (m); 1 μM: (j), (n); 10 μM: (k), (o); 20 μM: (l), (p)). Scale bars: 5 mm for lower magnification; 100 μm for higher magnification.

Fig. 2. Effects of Y27632 on cell viability and ROCK activity. Subconfluent PDL cells were cultured for 48 h (A) or 14 days (B) in osteogenic medium with Y27632. Cell viability was quantified by MTS analysis. The fold increase relative to control (without Y27632) absorbance at 490 nm is shown on the y-axis while the x-axis indicates the concentrations of Y27632 up to 100 μM used in these analysis. Results represent the means 7 SD; n ¼ 3 (three independent experiments using PDL cells from a single donor). Cells from different donors were used for the assays in (A) and (B). *P o 0.05, Student’s t test. The cell viability was maintained in the presence of Y27632 at concentrations up to 20 μM. (C) Subconfluent PDL cells were cultured for 14 days in osteogenic medium supplemented with several dose of Y27632 (0 μM: (a), (e), (i), (m); 1 μM: (b), (f), (j), (n); 10 μM: (c), (g), (k), (o); 20 μM: (d), (h), (l), (p)). Immunofluorescence analysis was performed for F-actin (Red: (e), (f), (g), (h)), pMLC (green: (i), (j), (k), (l)), and DAPI (blue: (a), (b), (c), (d)). Overlays of the two images are shown (merge: (m), (n), (o), (p)) with colocalization shown in yellow. Cells from three donors were used for the assays, and typical images are shown. Y27632 at 10 μM and 20 μM markedly inhibited F-actin and pMLC, an effector of ROCK. Scale bar: 100 μm. (D) Western blotting was performed on protein extracts (30 μg in each lane) from PDL cells cultured for 14 days in osteogenic medium in the absence (0) or presence of Y27632 at several concentrations up to 20 μM (1, 10, 20). The immunoblots present F-actin and GAPDH. F-actin signals were quantified by densitometric analysis. The levels of expression relative to GAPDH are shown on the y-axis, while the doses of Y27632 (μM) are shown on the x-axis. Cells from three donors were used for the assays. The results represent the means 7 SD; n ¼ 3, *P o 0.05, **P o 0.01, Student’s t test.

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Table 1 Differentially expressed genes regulated by Y-27632 in PDL cells. Gene symbol

Gene description

Fold change

P-value

FN1 COL1A1 COL3A1 BGN SERPINH1 ANXA5 CTSK Proliferation genes

Fibronectin 1 Collagen, type I, alpha 1 Collagen, type III, alpha 1 Biglycan Serpin peptidase inhibitor member 1 Annexin A5 Cathepsin K

 3.72  3.85  2.68  2.83  1.17 1.95 1.82

0.0002nn 0.0071nn 0.0058nn 0.0323n 0.5291 0.3267 0.2888

TGFB1 TGFB2 SMAD3 PCNA Differentiation genes

Transforming growth factor beta 1 Transforming growth factor beta 2 SMAD family member 3 Proliferating cell nuclear antigen

1.20 1.96 10.60 7.12

0.0348n 0.3053 0.3949 0.3449

ALPL SPP1 BMP2 SMAD1 BGLAP TWIST1 FGFR2 NOG

Alkaline phosphatase, liver/bone/kidney Secreted phosphoprotein 1 Bone morphogenetic protein 2 SMAD family member 1 Bone gamma-carboxyglutamate (gla) protein Twist basic helix-loop-helix transcription factor 1 Fibroblast growth factor receptor 2 Noggin

 2.68 11.25 7.71 1.73 4.31 3.32 1.61 1.42

0.0272n 0.1416 0.2906 0.2322 0.0944 0.4104 0.0513 0.6554

ECM-related genes

Expression analysis of 84 spotted osteogenic genes in PDL cells w/o Y27632 using human osteogenesis PCR array. Differential expression was verified by real-time RT-PCR analysis, and the fold changes in gene expression levels with Y27632 (þ , upregulation;  , downregulation) are presented as means (n¼3 from three different donors). Student’s t test. The asterisks indicate statistical significance for mRNA expression with Y27632 versus control. nnP o 0.01, nP o 0.05. Nineteen selected genes were classified into 3 categories. The gene symbol and designation of each gene are shown.

mineralized nodules. Although PDL cells supplemented with all doses of Y27632 examined grew to overconfluency, the ratio of ALP-positive cells to total cells and the frequency of Alizarin-positive nodules were decreased significantly in the presence of 10 μM Y27632 compared with lower doses (0 or 1 μM). However, 20 μM Y27632 was not sufficient to completely inhibit expression of these osteogenic markers. 3.4. Effects of Y27632 on mRNA expression during osteogenic differentiation To analyze the gene expression profile in PDL cells altered by 10 μM Y27632, we performed PCR array analysis and compared the mRNA levels in PDL cells with vs. without inhibitor treatment. After cultivation in osteogenic medium for 14 days, 19 of the 84 spotted genes were differentially expressed by more than 2-fold in PDL cells treated with Y27632 compared to control cells (Supplementary Fig. 1). The expression profile was verified by real-time PCR in PDL cells from three different donors, and the statistical significance of differences in expression of 6 genes was also confirmed (Table 1). ECM genes, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), collagen, type III alpha 1 (COL3A1), and biglycan (BGN) were significantly downregulated, accompanied with ALP gene downregulation in PDL cells treated with Y27632. Moreover, the cell proliferation gene, transforming growth factor beta 1 (TGFB1), was upregulated. As there were no significant differences in expression of representative osteoblastic genes at 14 days, such as bone morphogenetic protein 2 (BMP2), BGLAP, SP7, and RUNX2, another set of PDL cell cultures was prepared and harvested at 3, 6, 9, and 12 days. Temporal mRNA analysis was performed to further examine the mechanism underlying the inhibition of osteogenic differentiation by Y27632 (Supplementary Fig. 2). The level of BMP2 and BGLAP expression increased over time, and showed elevated expression in PDL cells treated with Y27632 compared to control cells. The levels of SP7

and RUNX2 expression showed a similar pattern, with a peak at 3 days followed by a decrease during culture with no significant differences in their expression between inhibitor-treated cells and control cells. 3.5. Localization of pMLC2 in periodontal tissue To examine the localization of ROCK activation and elucidate how RhoA/ROCK signaling contributes to the maintenance of periodontal tissue, immunofluorescence analysis was performed using normal murine periodontal tissue specimens in vivo (Fig. 4). In the periodontal tissue, pMLC2, a direct effector of ROCK, was expressed strongly in dental pulp cells adjacent to the dentin, stratum spinosum in gingival epithelium, and PDL cells. Interestingly, dispersed central PDL cells showed only weak pMLC2 expression, while pMLC2 signal was significantly higher at the boundary adjacent to the bone and cementum where fibers were abundantly inserted.

4. Discussion Several studies have indicated that soluble and ECM molecules regulate the differentiation of PDL cells (Cochran et al., 2000; Tour et al., 2012). Combining the effects of cytoskeletal changes in cellular signaling appears to result in synergistic expression of markers of differentiated cells, and its effects may extend the influence of soluble factors and ECM in the differentiating stage. In this study, we demonstrated a pivotal role of ROCK signaling in lineage commitment of PDL cells in response to cytoskeletal alterations; specific inactivation of ROCK in PDL cells was highly effective in inhibiting osteogenic differentiation, at least in part, via downregulation of the expression of ECM components. The actin cytoskeleton plays roles in many cellular functions, including motility, proliferation, and differentiation. In this study,

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Fig. 4. Histological localization of pMLC2 in vivo. Immunofluorescence analysis of murine periodontal tissue for DAPI (Blue: (a), (d)), pMLC2 (red: (b), (e)), and overlay images ((c), (f)) are shown. An enlarged image of the dotted rectangular area is shown ((d), (e), (f)). The pMLC2 signal was strong in the boundary adjacent to the bone and the cementum. DP, dental pulp; De, dentin; G, gingiva; PDL, periodontal ligament; AB, alveolar bone; Ce, cementum. Scale bar: 20 μm for lower magnification ((a), (b), (c)); 5 μm for higher magnification ((d), (e), (f)).

we demonstrated the production of a high-density actin–myosin complex in differentiated PDL cells, indicating that ROCK activation and actin stress fiber formation were enhanced during osteogenic differentiation (Fig. 1). These results were consistent with the earliest reports of osteogenic differentiation of MSC; thick actin filament bundles were located in differentiated MSC, while thin and parallel filaments were seen in undifferentiated MSC, suggesting that changes in the kinetics of ROCK activation and actin stress fiber formation may be critical in supporting osteogenic differentiation (Rodriguez et al., 2004; Arnsdorf et al., 2009). To address whether ROCK activation is required for osteogenic differentiation in PDL cells more directly, we performed inhibition assays using Y27632 to eliminate ROCK activity with only minimal alterations of other kinase pathways (Uehata et al., 1997). MTS assay indicated that Y27632 treatment maintained PDL cell proliferation although Y27632 decreased MLC2 phosphorylation accompanied by marked reduction of F-actin formation in a dose-dependent manner (Fig. 2). Moreover, Y27632 clearly inhibited indicators of osteogenic differentiation, ALP and calcium deposition (Fig. 3). These results indicated that the inhibition of ROCK suppressed stabilization of the actin cytoskeleton, and the

initiation and progression of osteogenesis in PDL cells. Y27632 is now commonly used to increase the cloning efficiency of pluripotent human embryonic stem cells (Watanabe et al., 2007). Thus, Y27632 may be effective to maintain proliferation and the undifferentiated state of PDL cells. However, Y27632 alone was not sufficient to inhibit differentiation into osteogenic cells, as shown in Fig. 3, suggesting that there may be alternative signaling in addition to the cytoskeletal network directing differentiation in response to either mechanical or soluble biochemical factors in PDL cells, resulting in the presence of a PDL cell subpopulation that retains osteogenic lineage commitment. PCR array analysis of mRNA expression altered by Y27632 indicated that osteogenic differentiation of PDL cells involves alterations in gene expression of ECM components (Supplementary Fig. 1, Table 1). The ECM has many effects beyond providing structural support. The ECM comprises an essential part of the cellular microenvironment, which together with various extracellular stimuli, such as growth factors, hormones, and mechanical stress, plays a crucial role in regulating differentiation (Allori et al., 2008). It has been reported that cell adhesion to the ECM via integrins activates intercellular signaling pathways directing osteoblast differentiation

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(Garcia and Reyes, 2005). However, the molecular details of structural links between the cytoskeleton and ECM of PDL cells remain unclear. The present study first demonstrated that ROCK plays a central role in regulating the gene expression of ECM components, such as FN1, COL1A1, COL3A1, and BGN, which may promote ALP expression and osteogenic differentiation of PDL cells (Table 1). It has already been demonstrated that ECM and cytoskeletal structures contribute to the regulation of RhoA activity (Ren et al., 1999). Based on these observations, we propose a model of autocrine regulation by which periodontal ECM regulates osteogenesis of PDL cells through a mechanism involving upregulation of ECM expression and production downstream of RhoA/ROCK signaling; ROCK may play a central role in defining the ECM microenvironment of PDL cells. Local increases in ECM turnover result in localized stretching due to contraction of the microenvironment, which pre-stresses the ECM (Ingber, 2006). These micromechanical forces are transmitted to the actin–myosin cytoskeleton via integrins and modulate intracellular signaling of PDL cells. Further studies are required to investigate the kinetics of mRNA and protein expression and phenotypes of RhoA- or ROCK-overexpressing PDL cells to clarify the mechanisms involved in the regulation of osteogenic differentiation of PDL cells by signaling interactions. The physiological roles of TGF-β show tissue specificity; the major role of TGF-β can be represented by inhibition of cell proliferation in epithelia but by induction in mesenchymal cells, such as fibroblasts (Wrighton et al., 2009). In this study, inhibition of ROCK activity by Y27632 demonstrated that TGFB1 was upregulated in PDL cells. Moreover, the downstream effectors, SMAD family member 3 (SMAD3) and proliferating cell nuclear antigen (PCNA), also tended to be upregulated (Table 1). The growthpromoting and anti-apoptotic effects of Y27632 have been applied for stem cell culture (Watanabe et al., 2007); these effects may be partially attributed to induction of TGFB1 activity by Y27632. The mechanism underlying TGFB1 upregulation by ROCK remains ambiguous; however, ECM-integrin signaling may be an effective activator of ROCK-dependent TGFB1 expression (Margadant and Sonnenberg, 2010). Although the differences were not significant, the expression levels of some osteoblastic genes, such as BMP2, SPP1, and BGLAP, tended to increase, while TWIST1, a negative regulator of osteoblast differentiation, also tended to increase with Y27632 treatment (Table 1). BMP-2 is one of the most powerful cytokines to induce ectopic bone formation and is a potent inducer of osteogenesis (Reddi, 1998). Moreover, SPP1 and BGLAP are expressed during the phases of matrix maturation and mineralization (Khanna-Jain et al., 2010). Regardless of the upregulated expression of these osteogenic genes, an early stage marker of osteogenesis, ALP expression, was downregulated and eventually osteogenic differentiation of PDL cells was inhibited by application of Y27632 (Table 1, Fig. 3). In this study, we used a common osteogenic medium containing ascorbic acid, β-glycerophosphate, and dexamethasone. Although both BMPs and the osteogenic medium are capable of inducing osteogenic differentiation in vitro, they seem to act through different signaling mechanisms and induce different transcriptional activities. As expression of RUNX2 in Y27632-treated PDL cells tended to decrease in the early phase (3 days) (Supplementary Fig. 2), it is also possible that RhoROCK signaling inhibits the early stage of osteogenesis. The whole picture of intercellular signaling cross-talk, including an estimated positive-feedback loop that regulates BMP signaling, is still unknown, and it remains unclear whether the observed transcriptional changes result in parallel changes in expression at the protein level. Therefore, further analyses of protein expression are required. The periodontal ligament is composed of different cell populations in various differentiation stages. PDL cells isolated by

sequential enzymatic digestion have been reported to exhibit different proliferation, ALP, and mineralization activities (Kaneda et al., 2006). Moreover, ALP activity and collagen type I expression were shown to be strongest adjacent to the bone, while cell proliferation was not significant in the PDL adjacent to the bone in vivo (Rooker et al., 2010). The present study suggested that the cytoskeletal and ECM stiffness regulated by Rho-ROCK signaling may define the graded distribution of differentiated PDL cells. In fact, immunofluorescence analysis in vivo indicated that pMLC2 was localized in the border region adjacent to the bone and the cementum (Fig. 4), where differentiated PDL cells are present. These observations strongly suggested that increased ROCK activity and ECM stiffness play critical roles during osteogenic differentiation of PDL cells. In summary, this study demonstrated that ROCK is essential for osteogenic differentiation of PDL cells. Our results highlighted that the PDL differentiation process was accompanied by F-actin polymerization and ROCK activation, and the activity has crucial roles for actin-myosin reorganization and osteogenic gene expression in PDL cells. Inhibition of ROCK activation by Y27632 abrogated ALP activity and mineralization, and altered osteogenic gene expression, particularly ECM gene expression, in the early stages of osteogenesis. RhoA-ROCK signaling is an important regulator that could transduce stimulation from ECM into the cytoskeletal component to link osteogenic differentiation of PDL cells. A better understanding of the mechanobiology in PDL cells is required to manipulate osteogenic differentiation for in vitro and future clinical applications.

Acknowledgments We would like to thank our colleagues at Okayama University, Drs. Wataru Sonoyama and Junji Mineshiba, for their helpful suggestions, and Drs. Ayaka Goto, Chiaki Yoshihara, and Mari Kawamura for excellent technical support. This work was supported by a Grant-in-Aid for Scientific Research (C) (20592429) from the JSPS and partially supported by 2010 Research Award from the Ryobi Teien Foundation.

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