archives of oral biology 59 (2014) 187–192
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress Rui Mauricio Santos de Araujo a, Yasuo Oba a, Shingo Kuroda a, Eiji Tanaka a,*, Keiji Moriyama b a
Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan b Department of Maxillofacial Orthognathics, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
article info
abstract
Article history:
Objectives: RhoE and regulator of G-proteins signalling (RGS) 2 were identified as the up-
Accepted 18 November 2013
regulated genes in human periodontal ligament (PDL) cells under compression. RhoE belongs to the Rho GTPase family, and RGS2, a novel family of GTPase-activating proteins,
Keywords:
turns off the G-protein signalling. Rho family proteins have recently been known to regulate
RhoE
actin cytoskeleton dynamics in various cell types. In this study, we investigated the
Periodontal ligament cells
involvement of RhoE and RGS2 in the regulation of actin filament organization in the
Mechanical stress
PDL cells under mechanical stress. Methods: Human PDL cells were cultured and subjected to a static compressive force (3.0 g/ cm2) for 48 h. To observe changes in the actin cytoskeleton and the expression of RhoE and RGS2 in response to mechanical stress, immunofluorescence analysis was performed. To examine the role of RhoE and RGS2 in actin filament organization, cells were transfected with antisense S-oligonucleotides (ODNs) to RhoE and RGS2. Results: Compressive force caused a loss and disassembly of actin stress fibres leading to cell spreading. Immunocytochemical study revealed that RhoE and RGS2 expressions were induced by mechanical stress and localized in the perinuclear and in the cell membrane, respectively. The impaired formation of stress fibres caused by compressive forces was recovered by treatment with antisense S-ODN to RhoE to the control levels. However, addition of antisense S-ODN to RGS2 did not affect the stress fibre formation. Conclusions: These results indicate that the loss and disassembly of stress fibres due to mechanical stress are mediating RhoE signalling, without the exertion of RGS2. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The periodontal ligament (PDL) is the connective tissue situated between the cementum of dental root and the
alveolar bone and receives mechanical stress such as occlusal pressure and orthodontic force.1,2 The biological response of the PDL to mechanical stress may have an effect on the homeostasis of the PDL itself as well as other components of periodontal tissues. Because the rate and extent of bone
* Corresponding author. Tel.: +81 88 633 7356; fax: +81 88 633 9138. E-mail address: [email protected] (E. Tanaka). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.11.010
188
archives of oral biology 59 (2014) 187–192
remodelling are very limited when forces are applied to teeth devoid of PDL, the PDL is thought to play a critical role in resorption and formation of bone matrix during the physiological and orthodontic tooth movement.3 Mechanical stress has been suggested to regulate the production of biological mediators in PDL cells such as interleukin-1b (IL-1b),4,5 interleukin 6 (IL-6),4 tumour necrosis factor-a (TNF-a),6 cyclooxygenase-2 (COX-2),7,8 type I collagen,9 osteocalcin,10 cAMP,11 and receptor activator of nuclear factor k ligand (RANKL).12 Thus, PDL cells are known as a transducer of mechanical stress into biological signalling in which a set of genes starts to be expressed under the regulation of their own mechanisms. To identify genes differentially expressed in human PDL cells due to mechanical stress, we previously conducted a microarray analysis and detected RGS2 and RhoE as up-regulating genes by mechanical stress.13 RhoE is a member of the Ras superfamily of small GTP-binding proteins, which regulate multiple aspects of cell behaviour.14–23 Like other small G-proteins, Rho family members generally function as molecular switches that cycle between an active (GTP-bound) and inactive (GDP-bound) state. RhoA appears to be the major regulator of stress fibre formation under physiological conditions. The regulation of stress fibres comes from the downstream effects of RhoA, in particular the ROCK protein kinase and the activity of mDia1.24 Meanwhile, RGS2 is a member of the regulators of G-protein signalling (RGS) superfamily.25–27 RGS proteins act as GTPase activating proteins (GAPs), thereby the hydrolysis of GTP to GDP is catalysed by the RGS protein. RhoE can inhibit cell proliferation and transformation in addition to its known effects on the actin cytoskeleton and can contribute to balanced coordination of cell proliferation and migration. RGS proteins are thought to play a critical role in shutting off G-proteinmediated cell responses in all eukaryotes. However, the role of RhoE and RGS2 in PDL cell has not been elucidated yet. The present study examined the role of RhoE and RGS2 in the PDL cells under mechanical stress.
gels were prepared according to the manufacturer’s protocol (Cellmatrix I-A from Nitta Gelatin Inc., Osaka, Japan). The collagen gel matrix containing 1 106 cells (800 ml) was placed onto a 24-well plate and allowed to polymerize for 30 min at 37 8C. After polymerization, the gels were transferred to a 6-well plate to promote nutrient diffusion from their surroundings in 2 ml of fresh media (a-MEM with 10% FBS). Three gels in each well were cultured and allowed to set for 24–36 h prior to force loading. For the mechanical stress application, a polystyrene plate was placed over the collagen gels. The weight used to apply the compressive force consisted of a plastic cylinder filled with lead that was placed on top of the polystyrene plate (Fig. 1). The amount of force used was 6.0 g/cm2 for every three gels, which was empirically determined to yield a 50% deformation of the gels. Prior to force application to the cells, the weight was calibrated by adding lead granules to the plastic cylinder using a laboratory balance. For the control groups, the polystyrene plate alone was placed over the gels containing cells. For immunofluorescence microscopic analysis, PDL cells were cultured on coverslips in 6-well plate and the cells were subjected to 3.0 g/ cm2 of compressive force.
2.
Materials and methods
2.3. Isolation of F-actin (triton X-100-insoluble) fractions of PDL cells
2.1.
Cell culture
Human PDL cells were derived from the ligament tissues of periodontally healthy non-carious human premolar teeth extracted from donors for orthodontic reasons with informed consent, and this study was approved by the Ethical Committee of the University of Tokushima. The cells were isolated and maintained in alpha-minimum essential medium (a-MEM) (Sigma–Aldrich, St Louis, MO) supplemented with 10% foetal bovine serum (FBS) and 100 U/ml penicillin at 37 8C in a humidified atmosphere of 5% CO2 at 37 8C as previously described with slight modification.28 In this study, we used PDL cells that have been cultured through nine passages.
2.2.
Mechanical stress
PDL cells were embedded in collagen gel to mimic in vivo conditions and submitted to compressive force. The collagen
Fig. 1 – Schematic illustration of the apparatus for compressive force application. A polystyrene plate was lowered onto the gels and a plastic container filled with lead granules was used to compress the gels.
To obtain the whole-cell extracts, cells were lysed in a lysis buffer (50 mM Hepes, pH 6.4, 1 mM MgCl2, 10 mM EDTA, 1% Triton X-100) containing the following protease inhibitors: 1 mg/ml aproptin, 1 mg/ml leupeptin, 1 mg/ml pepstatin and 1 mM PMSF (phenylmethanesulfonylfluoride). Triton X-100-soluble and Triton X-100-insoluble cytoskeleton fractions were obtained by centrifuging cell lysates at 12,000 g for 10 min at 25 8C, recovering the supernatants (Triton X-100-soluble fraction, containing Gactin), and suspending the pellets (Triton X-100-insoluble fraction, containing F-actin) in the same lysis buffer under vigorous agitation.
2.4.
Western blot analysis
The PDL cells embedded in collagen gel were isolated by dissolving the collagen with 1 mg/ml of collagenase according to the method of Kagami et al.,29 and the cells were then lysed in a lysis buffer. Samples were resolved by SDS-PAGE and
archives of oral biology 59 (2014) 187–192
transferred to PVDF membranes (Millipore, Bedford, MA). After blocking, the membranes were incubated with monoclonal mouse anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal mouse anti-RhoE (Upstate Biotechnology, Lake Placid, NY), polyclonal rabbit anti-RGS2 (Santa Cruz Biotechnology), monoclonal mouse anti-actin (Santa Cruz Biotechnology) at 1:1000 dilution at 1:1000 dilution, or monoclonal mouse anti-b-actin (Sigma–Aldrich) at 1:10,000 dilution for 2 h at room temperature. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Santa Cruz Biotechnology) at 1:2000 dilution or HRP-conjugated goat anti-mouse IgG (BioSource, Camarillo, CA) at 1:10,000 dilution were used as secondary antibodies for 1 h at room temperature. The blots were developed with an enhanced chemiluminescent (ECL) system (Amersham Biosciences, Buckinghamshire, UK) in X-ray films (Amersham Biosciences).
2.5.
Immunofluorescence microscopy
After applying compressive force, cells on coverslips were fixed with 10% formalin in phosphate-buffered saline (PBS), pH 7.3 for 10 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. After blocking the cells with 1% skim milk for 1 h at room temperature, the cells were incubated with polyclonal mouse anti-RhoE (Upstate Biotechnology), monoclonal mouse anti-RhoA (Santa Cruz Biotechnology), or polyclonal rabbit antiRGS2 (Santa Cruz Biotechnology) antibodies at a 1:100 dilution for overnight at 4 8C. Rhodamine-anti-mouse IgG (Chemicon International, Temecula, CA), FITC-anti-mouse IgG (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) or FITC-anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies at a 1:200 dilution for 1 h at room temperature. F-actin filaments were localized by incubating cells for 45 min with 0.1 mg/ml of tetramethyl rhodamine isothiocyanate (TRITC)-labelled phalloidin (Sigma–Aldrich). Images were generated with a fluorescent microscope (BX 60, Olympus Co, Tokyo, Japan) operating a digital camera (DP 70, Olympus).
2.6.
Results
3.1. Effects of compressive force on stress fibres formation and F-actin level in the PDL cells To evaluate the stress fibre formation under compressive force, immunocytochemical study was performed for F-actin filaments. The F-actin levels were examined by immunoblotting. Compressive force (3.0 g/cm2) for 48 h inhibited stress fibre formation leading to cell spreading compared with the control (Fig. 2A, c and f). At 24 h after removing compressive loading the stress fibres formation returned to the control levels (Fig. 2A, d and g). The decrease in stress fibres caused by mechanical stress was further confirmed by examining the F-actin expression by western blot analysis. Fig. 1B shows that the F-actin level was reduced after 48 h of mechanical loading.
3.2. Effects of compressive force on RhoE, RhoA and RGS2 expression in the PDL cells To determine if PDL cells express RhoE, RhoA, and RGS2 protein in response to compressive force, immunocytochemical and Western blot analysis were performed using specific antibodies for RhoE, RhoA, and RGS2. RhoE expression was induced after compressive force (3.0 g/cm2) application for 48 h in the perinuclear region as well as in the plasma membrane (Fig. 3A, a and d). RhoA was localized in the plasma membrane of the PDL cells (Fig. 3A, b and e), but RhoA expression did not alter. The expression of RGS2 was also enhanced by compressive force in the plasma membrane of the PDL cells (Fig. 3A, c and f).
Antisense S-oligonucleotide treatment
To determine the involvement of native RhoE and RGS2 in cellular response to mechanical stress, antisense and sense Soligonucleotides (S-ODNs) that included the ATG and ribosome binding site of each gene were designed. The cells on coverslips were transfected with 10 nM antisense and sense SODNs to RGS2 or RhoE using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The S-ODN sequences used in this study were as follows: RGS2 antisense 50 -CATAGCACTTTGCATTATCG-30 , and sense 50 RhoE antisense 50 CGATAATGCAAAAGTGCTATG-30 ; 0 0 TCTTCTCTCCTTCATTGATG-3 , and sense 5 -CATCAATGAAGGAGAGAAGA-30 .
2.7.
3.
189
Statistical analysis
Results are reported as the mean SD for a typical experiment and compared by Student t-test. Results were considered significantly different for p values less than 0.05.
Fig. 2 – Actin cytoskeleton dynamics and F-actin levels in PDL cells under compressive force. Actin filaments were stained with TRITC-phalloidin in the cells subject to compressive force (3.0 g/cm2) at the indicated time (A, e, f and g) and controls (A, a, b, c and d). F-actin fractions were separated from soluble actin (G-actin) by centrifugation as described in Section 2 and were subjected to SDS-PAGE followed by immunoblotting with anti-actin antibody (B). b-actin was used as a control for equal loading. Similar results were seen in three independent experiments.
190
archives of oral biology 59 (2014) 187–192
Fig. 3 – Immunofluorescence analysis was performed for the cells subjected to compressive force (3.0 g/cm2) for 48 h (A, d, e and f) and controls (A, a, b and c). RhoE (A, a and d), RhoA (A, b and e) and RGS2 (A, c and f) expression were localized using each specific antibody. Lysates from PDL cells stimulated by compressive force for 12 and 24 h were examined by Western blot analysis for RhoE, RhoA and RGS2 as described in Section 2 (B). b-actin was used as a control for equal loading. Arrows indicate the localization of RhoE, RGS2 and RhoA. Similar results were seen in three independent experiments.
As shown in Fig. 2B, the RhoE protein level were increased after 12 h of compressive force loading and the RGS2 protein levels were markedly enhanced at 24 h in response to compressive force, but RhoA expression level did not alter under this force loading condition.
3.3. Effects of antisense S-oligonucleotide to RGS2 or RhoE on stress fibre formation in the PDL cells under compressive force To determine if endogenous RhoE and RGS2 was involved in regulating actin cytoskeleton organization in PDL cells under mechanical stress, the effects of an antisense S-ODN to RhoE or RGS2 on stress fibre assembly were examined. As shown in Fig. 4, the attenuation of RhoE and RGS2 expressions induced by compressive force was observed in PDL cells treated with the antisense S-ODN. Blockage of RhoE promoted the formation of stress fibre under mechanical stress compared with the controls (Fig. 4c). On the other hand, inhibition of RGS2 expression did not affect stress fibre formation (Fig. 4f).
4.
Discussion
The activation of signalling molecules in response to mechanical stimuli appears to be mediated through changes in
Fig. 4 – Effects of antisense S-ODN to RhoE or RGS2 on the stress fibre formation in the PDL cells under compressive force. Cells were transfected with antisense S-ODN to RhoE (c) and RGS2 (f) 24 h prior to mechanical stress application. After 48 h of compression, the cells were stained with anti-RhoE (a, b and c) and anti-RGS2 (d, e and f) antibodies. To visualize stress fibres, cells were incubated with TRITC-phalloidin. Cells transfected with sense S-ODN to RhoE (a and b) and RGS2 (d and e) were used as controls. Arrows indicate the localization of RhoE and RGS2. Similar results were observed in three different experiments.
the cytoskeleton. The cytoskeleton is a complex network of protein filaments extending throughout the cell, and is formed by three major types of filaments: microtubules, actin filaments and intermediate filaments. Small GTPases of Rho family – Rho, Rac, and Cdc42 – are known to regulate actin cytoskeleton dynamics, thereby controlling cytoskeleton organization and cell motility response.14–19 RhoE binds only weakly to GDP and intrinsic GTPase activity is very low. Consequently, RhoE exists predominantly in a GTP-bound state. RhoE protein activity is presumably regulated differently from GTP hydrolysis, for example, by altering protein expression levels. Indeed, RhoE expression has been shown to be upregulated upon platelet-derived growth factor (PDGF)19 or hepatocyte growth factor20 stimulation of Swiss 3T3 or MDCK cells. Recent studies have elucidated that RhoE expression in fibroblasts and epithelial cells induces loss of actin stress fibres, and therefore, they seem to function antagonistically to RhoA in actin cytoskeleton regulation. RhoA is well known to induce the actin cytoskeleton organization. In order to investigate the involvement of small G-protein signalling in PDL cells under mechanical stress, the changes in the actin cytoskeleton organization and the expression of RhoE and RGS2 due to compressive force were examined. By applying compressive force to the PDL cells, a disruption of stress fibre formation leading to cell spreading was observed. It is likely that this modification in the actin cytoskeleton represents a self-protection mechanism in the cells to adapt to the mechanical stress. The recovery of stress fibres to control levels 24 h after removing the compressive loading indicates that the loss of stress fibres was caused only by the mechanical stress application. It has been reported that RhoE is widely expressed, although its expression levels vary significantly between different cell types.30 RhoE has been found in both membrane and cytoplasmic fractions of cells, and localized at
archives of oral biology 59 (2014) 187–192
least partly to the Golgi complex as well as to the plasma membrane.30 The results of fluorescent immunocytochemical examination for RhoE expression are consistent with the previous reports. However, in this immunostaining condition with the polyclonal anti-RhoE antibody, the positive signals in the nuclear may be due to a cross-reactivity of the antibody with a nuclear protein. RGS2 has been found to be predominantly a nuclear protein, but is recently reported to localize in the cytoplasm and plasma membrane.30 RGS2 is recruited to the plasma membrane by G-proteins.31,32 The immunocytochemical examination for RGS2 revealed that its expression was induced in the plasma membrane in mechanically stressed PDL cells. These data suggest the interactions of RGS2 with membrane-bound G-proteins under mechanical stress. RhoA is known to translocate to the cell membrane upon activation.15 It is not clear why RhoA has not been induced by compressive force in this immunocytochemistry. RhoA expression is well respondent to various stimuli including serum stimulation. Since these culture media for PDL cells contained FBS, RhoA induction due to the serum stimulation might occur in the cells without compression, thereby in which RhoA expression levels were similar to compressed cells. With regard to the decrease in stress fibre disassembly under compressive force, two regulatory mechanisms are hypothesized, which the up-regulated RhoE and the inactivation of other Rho GTPases such as RhoA by RGS2 induce the disassembly. In order to determine the role of RhoE and RGS2 in the stress fibre disruption, stress fibre formation due to compressive force was examined in inhibition of native RhoE and RGS2 expressions by antisense S-ODN. By treatment with antisense S-ODN to RhoE, the impaired formation of stress fibres caused by compressive force was recovered to the control levels, but the addition of antisense S-ODN to RGS2 did not affect the stress fibre formation. These results indicate that the loss of stress fibres due to mechanical stress is mediating RhoE signalling without the exertion of RGS2. Recently, RhoE has been described as ROCK I inhibitor. ROCK I, also known to be a target of RhoA, stimulates actin organization by phosphorylating a number of actin associated proteins such as MLC phosphatase.33 RhoE binds only to the amino-terminal part of ROCK I and might therefore physically interfere with its kinase activity. RhoE does not bind to other RhoA targets such as ROCK II. The regulatory mechanisms of ROCK I downstream of RhoE or RhoA in PDL cells have yet to be further clarified. This study imply that RhoE and RGS2 are the stress-related molecules that are highly expressed in human PDL cells under compressive force and play a role in the signalling of mechanotransduction events in human PDL cells. The enhanced RhoE by compressive force is responsible for the stress fibre disassembly in human PDL cells. Further studies on their physiological roles are necessary and may contribute to clarify the molecular function of periodontal ligament tissue and the remodelling mechanisms of periodontal tissues in response to mechanical stress.
Author contributions Conceived and designed the experiments: R.D., Y.O., K.M., Performed the analysis: R.D., Y.O., S.K., Analysed the data: R.D., Y.O., K.M., Wrote the paper: R.D., S.K., E.T.
191
Funding This research was supported, in part, by a Grant (No. 23659966) for Science Research from the Ministry of Education, Science and Culture, Japan.
Competing interests None declared.
Ethical approval This work was approved by the Ethical Committee of the University of Tokushima.
references
1. Tencate AR. Oral histology: development, structure and function. St. Louis: Mosby; 1998: 256. 2. Roberts WE, Goodwin Jr WC, Heiner SR. Cellular response to orthodontic force. Dent Clin North Am 1981;25:3–17. 3. Mitchell DL, West JD. Attempted orthodontic movement in the presence of suspected ankylosis. Am J Orthod Dentofacial Orthop 1975;68:404–11. 4. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop 2001;119:307–12. 5. Saito M, Saito S, Ngan PW, Shanfeld J, Davidovitch Z. Interleukin 1 beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. Am J Orthod Dentofacial Orthop 1991;99:226–40. 6. Lowney JJ, Norton LA, Shafer DM, Rossomando EF. Orthodontic forces increase tumor necrosis factor alpha in the human gingival sulcus. Am J Orthod Dentofacial Orthop 1995;108:519–24. 7. Shimizu N, Ozawa Y, Yamaguchi M, Goseki T, Ohzeki K, Abiko Y. Induction of COX-2 expression by mechanical tension force in human periodontal ligament cells. J Periodontol 1998;69:670–7. 8. Ohzeki K, Yamaguchi M, Shimizu N, Abiko Y. Effect of cellular aging on the induction of cyclooxygenase-2 by mechanical stress in human periodontal ligament cells. Mech Ageing Dev 1999;108:151–63. 9. Nakagawa M, Kukita T, Nakasima A, Kurisu K. Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization. Arch Oral Biol 1994;39:289–94. 10. Matsuda N, Yokoyama K, Takeshita S, Watanabe M. Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro. Arch Oral Biol 1998;43:987–97. 11. Yousefian J, Firouzian F, Shanfeld J, Ngan P, Lanese R, Davidovitch Z. A new experimental model for studying the response of periodontal ligament cells to hydrostatic pressure. Am J Orthod Dentofacial Orthop 1995;108: 402–9. 12. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–20.
192
archives of oral biology 59 (2014) 187–192
13. Araujo RMS, Oba Y, Moriyama K. Identification of genes related to mechanical stress in human periodontal ligament cells using microarray analysis. J Periodontal Res 2007;42:15–22. 14. Downes GB, Gautam N. The G protein subunit gene families. Genomics 1999;62:544–52. 15. Van Aelst L, D’Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 1997;11:2295–322. 16. Foster R, Hu KQ, Lu Y, Nolan KM, Thissen J, Settleman J. Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation. Mol Cell Biol 1996;16:2689–99. 17. Guasch RM, Scambler P, Jones GE, Ridley AJ. RhoE regulates actin cytoskeleton organization and cell migration. Mol Cell Biol 1998;18:4761–71. 18. Nobes CD, Lauritzen I, Mattei MG, Paris S, Hall A, Chardin P. A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion. J Cell Biol 1998;141:187–97. 19. Riento K, Guasch RM, Garg R, Jin B, Ridley AJ. RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol 2003;23:4219–29. 20. Tanimura S, Nomura K, Ozaki K, Tsujimoto M, Kondo T, Kohno M. Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility. J Biol Chem 2002;277:28256–64. 21. Hansen SH, Zegers MM, Woodrow M, Rodriguez-Viciana P, Chardin P, Mostov KE, et al. Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway. Mol Cell Biol 2000;24:9364–75. 22. Riento K, Totty N, Villalonga P, Garg R, Guasch R, Ridley AJ. RhoE function is regulated by ROCK I-mediated phosphorylation. EMBO J 2005;24:1170–80.
23. Villalonga P, Guasch RM, Riento K, Ridley AJ. RhoE inhibits cell cycle progression and Ras-induced transformation. Mol Cell Biol 2004;24:7829–40. 24. Pellegrin S, Mellor H. Actin stress fibres. J Cell Sci 2007;120:3491–9. 25. Ishii M, Kurachi Y. Physiological actions of regulators of Gprotein signaling (RGS) proteins. Life Sci 2003;74: 163–71. 26. De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 2000;40:235–71. 27. Song L, Zmijewski JW, Jope RS. RGS2: regulation of expression and nuclear localization. Biochem Biophys Res Commun 2001;283:102–6. 28. Han X, Amar S. Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis. J Dent Res 2002;81: 399–405. 29. Kagami S, Urushihara M, Kondo S, Loster K, Reutter W, Tamaki T, et al. Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells. Exp Cell Res 2001;268:274–83. 30. Riento K, Villalonga P, Garg R, Ridley A. Function and regulation of RhoE. Biochem Soc Trans 2005;33:649–51. 31. Kehrl JH, Sinnarajah S. RGS2: a multifunctional regulator of G-protein signaling. Int J Biochem Cell Biol 2002;34: 432–8. 32. Zmijewski JW, Song L, Harkins L, Cobbs CS, Jope RS. Second messengers regulate RGS2 expression which is targeted to the nucleus. Biochim Biophys Acta 2001;1541:201–11. 33. Riento K, Ridley AJ. ROCKs: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003;4:446–56.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress Rui Mauricio Santos de Araujo a, Yasuo Oba a, Shingo Kuroda a, Eiji Tanaka a,*, Keiji Moriyama b a
Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan b Department of Maxillofacial Orthognathics, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
article info
abstract
Article history:
Objectives: RhoE and regulator of G-proteins signalling (RGS) 2 were identified as the up-
Accepted 18 November 2013
regulated genes in human periodontal ligament (PDL) cells under compression. RhoE belongs to the Rho GTPase family, and RGS2, a novel family of GTPase-activating proteins,
Keywords:
turns off the G-protein signalling. Rho family proteins have recently been known to regulate
RhoE
actin cytoskeleton dynamics in various cell types. In this study, we investigated the
Periodontal ligament cells
involvement of RhoE and RGS2 in the regulation of actin filament organization in the
Mechanical stress
PDL cells under mechanical stress. Methods: Human PDL cells were cultured and subjected to a static compressive force (3.0 g/ cm2) for 48 h. To observe changes in the actin cytoskeleton and the expression of RhoE and RGS2 in response to mechanical stress, immunofluorescence analysis was performed. To examine the role of RhoE and RGS2 in actin filament organization, cells were transfected with antisense S-oligonucleotides (ODNs) to RhoE and RGS2. Results: Compressive force caused a loss and disassembly of actin stress fibres leading to cell spreading. Immunocytochemical study revealed that RhoE and RGS2 expressions were induced by mechanical stress and localized in the perinuclear and in the cell membrane, respectively. The impaired formation of stress fibres caused by compressive forces was recovered by treatment with antisense S-ODN to RhoE to the control levels. However, addition of antisense S-ODN to RGS2 did not affect the stress fibre formation. Conclusions: These results indicate that the loss and disassembly of stress fibres due to mechanical stress are mediating RhoE signalling, without the exertion of RGS2. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The periodontal ligament (PDL) is the connective tissue situated between the cementum of dental root and the
alveolar bone and receives mechanical stress such as occlusal pressure and orthodontic force.1,2 The biological response of the PDL to mechanical stress may have an effect on the homeostasis of the PDL itself as well as other components of periodontal tissues. Because the rate and extent of bone
* Corresponding author. Tel.: +81 88 633 7356; fax: +81 88 633 9138. E-mail address: [email protected] (E. Tanaka). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.11.010
188
archives of oral biology 59 (2014) 187–192
remodelling are very limited when forces are applied to teeth devoid of PDL, the PDL is thought to play a critical role in resorption and formation of bone matrix during the physiological and orthodontic tooth movement.3 Mechanical stress has been suggested to regulate the production of biological mediators in PDL cells such as interleukin-1b (IL-1b),4,5 interleukin 6 (IL-6),4 tumour necrosis factor-a (TNF-a),6 cyclooxygenase-2 (COX-2),7,8 type I collagen,9 osteocalcin,10 cAMP,11 and receptor activator of nuclear factor k ligand (RANKL).12 Thus, PDL cells are known as a transducer of mechanical stress into biological signalling in which a set of genes starts to be expressed under the regulation of their own mechanisms. To identify genes differentially expressed in human PDL cells due to mechanical stress, we previously conducted a microarray analysis and detected RGS2 and RhoE as up-regulating genes by mechanical stress.13 RhoE is a member of the Ras superfamily of small GTP-binding proteins, which regulate multiple aspects of cell behaviour.14–23 Like other small G-proteins, Rho family members generally function as molecular switches that cycle between an active (GTP-bound) and inactive (GDP-bound) state. RhoA appears to be the major regulator of stress fibre formation under physiological conditions. The regulation of stress fibres comes from the downstream effects of RhoA, in particular the ROCK protein kinase and the activity of mDia1.24 Meanwhile, RGS2 is a member of the regulators of G-protein signalling (RGS) superfamily.25–27 RGS proteins act as GTPase activating proteins (GAPs), thereby the hydrolysis of GTP to GDP is catalysed by the RGS protein. RhoE can inhibit cell proliferation and transformation in addition to its known effects on the actin cytoskeleton and can contribute to balanced coordination of cell proliferation and migration. RGS proteins are thought to play a critical role in shutting off G-proteinmediated cell responses in all eukaryotes. However, the role of RhoE and RGS2 in PDL cell has not been elucidated yet. The present study examined the role of RhoE and RGS2 in the PDL cells under mechanical stress.
gels were prepared according to the manufacturer’s protocol (Cellmatrix I-A from Nitta Gelatin Inc., Osaka, Japan). The collagen gel matrix containing 1 106 cells (800 ml) was placed onto a 24-well plate and allowed to polymerize for 30 min at 37 8C. After polymerization, the gels were transferred to a 6-well plate to promote nutrient diffusion from their surroundings in 2 ml of fresh media (a-MEM with 10% FBS). Three gels in each well were cultured and allowed to set for 24–36 h prior to force loading. For the mechanical stress application, a polystyrene plate was placed over the collagen gels. The weight used to apply the compressive force consisted of a plastic cylinder filled with lead that was placed on top of the polystyrene plate (Fig. 1). The amount of force used was 6.0 g/cm2 for every three gels, which was empirically determined to yield a 50% deformation of the gels. Prior to force application to the cells, the weight was calibrated by adding lead granules to the plastic cylinder using a laboratory balance. For the control groups, the polystyrene plate alone was placed over the gels containing cells. For immunofluorescence microscopic analysis, PDL cells were cultured on coverslips in 6-well plate and the cells were subjected to 3.0 g/ cm2 of compressive force.
2.
Materials and methods
2.3. Isolation of F-actin (triton X-100-insoluble) fractions of PDL cells
2.1.
Cell culture
Human PDL cells were derived from the ligament tissues of periodontally healthy non-carious human premolar teeth extracted from donors for orthodontic reasons with informed consent, and this study was approved by the Ethical Committee of the University of Tokushima. The cells were isolated and maintained in alpha-minimum essential medium (a-MEM) (Sigma–Aldrich, St Louis, MO) supplemented with 10% foetal bovine serum (FBS) and 100 U/ml penicillin at 37 8C in a humidified atmosphere of 5% CO2 at 37 8C as previously described with slight modification.28 In this study, we used PDL cells that have been cultured through nine passages.
2.2.
Mechanical stress
PDL cells were embedded in collagen gel to mimic in vivo conditions and submitted to compressive force. The collagen
Fig. 1 – Schematic illustration of the apparatus for compressive force application. A polystyrene plate was lowered onto the gels and a plastic container filled with lead granules was used to compress the gels.
To obtain the whole-cell extracts, cells were lysed in a lysis buffer (50 mM Hepes, pH 6.4, 1 mM MgCl2, 10 mM EDTA, 1% Triton X-100) containing the following protease inhibitors: 1 mg/ml aproptin, 1 mg/ml leupeptin, 1 mg/ml pepstatin and 1 mM PMSF (phenylmethanesulfonylfluoride). Triton X-100-soluble and Triton X-100-insoluble cytoskeleton fractions were obtained by centrifuging cell lysates at 12,000 g for 10 min at 25 8C, recovering the supernatants (Triton X-100-soluble fraction, containing Gactin), and suspending the pellets (Triton X-100-insoluble fraction, containing F-actin) in the same lysis buffer under vigorous agitation.
2.4.
Western blot analysis
The PDL cells embedded in collagen gel were isolated by dissolving the collagen with 1 mg/ml of collagenase according to the method of Kagami et al.,29 and the cells were then lysed in a lysis buffer. Samples were resolved by SDS-PAGE and
archives of oral biology 59 (2014) 187–192
transferred to PVDF membranes (Millipore, Bedford, MA). After blocking, the membranes were incubated with monoclonal mouse anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal mouse anti-RhoE (Upstate Biotechnology, Lake Placid, NY), polyclonal rabbit anti-RGS2 (Santa Cruz Biotechnology), monoclonal mouse anti-actin (Santa Cruz Biotechnology) at 1:1000 dilution at 1:1000 dilution, or monoclonal mouse anti-b-actin (Sigma–Aldrich) at 1:10,000 dilution for 2 h at room temperature. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Santa Cruz Biotechnology) at 1:2000 dilution or HRP-conjugated goat anti-mouse IgG (BioSource, Camarillo, CA) at 1:10,000 dilution were used as secondary antibodies for 1 h at room temperature. The blots were developed with an enhanced chemiluminescent (ECL) system (Amersham Biosciences, Buckinghamshire, UK) in X-ray films (Amersham Biosciences).
2.5.
Immunofluorescence microscopy
After applying compressive force, cells on coverslips were fixed with 10% formalin in phosphate-buffered saline (PBS), pH 7.3 for 10 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. After blocking the cells with 1% skim milk for 1 h at room temperature, the cells were incubated with polyclonal mouse anti-RhoE (Upstate Biotechnology), monoclonal mouse anti-RhoA (Santa Cruz Biotechnology), or polyclonal rabbit antiRGS2 (Santa Cruz Biotechnology) antibodies at a 1:100 dilution for overnight at 4 8C. Rhodamine-anti-mouse IgG (Chemicon International, Temecula, CA), FITC-anti-mouse IgG (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) or FITC-anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies at a 1:200 dilution for 1 h at room temperature. F-actin filaments were localized by incubating cells for 45 min with 0.1 mg/ml of tetramethyl rhodamine isothiocyanate (TRITC)-labelled phalloidin (Sigma–Aldrich). Images were generated with a fluorescent microscope (BX 60, Olympus Co, Tokyo, Japan) operating a digital camera (DP 70, Olympus).
2.6.
Results
3.1. Effects of compressive force on stress fibres formation and F-actin level in the PDL cells To evaluate the stress fibre formation under compressive force, immunocytochemical study was performed for F-actin filaments. The F-actin levels were examined by immunoblotting. Compressive force (3.0 g/cm2) for 48 h inhibited stress fibre formation leading to cell spreading compared with the control (Fig. 2A, c and f). At 24 h after removing compressive loading the stress fibres formation returned to the control levels (Fig. 2A, d and g). The decrease in stress fibres caused by mechanical stress was further confirmed by examining the F-actin expression by western blot analysis. Fig. 1B shows that the F-actin level was reduced after 48 h of mechanical loading.
3.2. Effects of compressive force on RhoE, RhoA and RGS2 expression in the PDL cells To determine if PDL cells express RhoE, RhoA, and RGS2 protein in response to compressive force, immunocytochemical and Western blot analysis were performed using specific antibodies for RhoE, RhoA, and RGS2. RhoE expression was induced after compressive force (3.0 g/cm2) application for 48 h in the perinuclear region as well as in the plasma membrane (Fig. 3A, a and d). RhoA was localized in the plasma membrane of the PDL cells (Fig. 3A, b and e), but RhoA expression did not alter. The expression of RGS2 was also enhanced by compressive force in the plasma membrane of the PDL cells (Fig. 3A, c and f).
Antisense S-oligonucleotide treatment
To determine the involvement of native RhoE and RGS2 in cellular response to mechanical stress, antisense and sense Soligonucleotides (S-ODNs) that included the ATG and ribosome binding site of each gene were designed. The cells on coverslips were transfected with 10 nM antisense and sense SODNs to RGS2 or RhoE using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The S-ODN sequences used in this study were as follows: RGS2 antisense 50 -CATAGCACTTTGCATTATCG-30 , and sense 50 RhoE antisense 50 CGATAATGCAAAAGTGCTATG-30 ; 0 0 TCTTCTCTCCTTCATTGATG-3 , and sense 5 -CATCAATGAAGGAGAGAAGA-30 .
2.7.
3.
189
Statistical analysis
Results are reported as the mean SD for a typical experiment and compared by Student t-test. Results were considered significantly different for p values less than 0.05.
Fig. 2 – Actin cytoskeleton dynamics and F-actin levels in PDL cells under compressive force. Actin filaments were stained with TRITC-phalloidin in the cells subject to compressive force (3.0 g/cm2) at the indicated time (A, e, f and g) and controls (A, a, b, c and d). F-actin fractions were separated from soluble actin (G-actin) by centrifugation as described in Section 2 and were subjected to SDS-PAGE followed by immunoblotting with anti-actin antibody (B). b-actin was used as a control for equal loading. Similar results were seen in three independent experiments.
190
archives of oral biology 59 (2014) 187–192
Fig. 3 – Immunofluorescence analysis was performed for the cells subjected to compressive force (3.0 g/cm2) for 48 h (A, d, e and f) and controls (A, a, b and c). RhoE (A, a and d), RhoA (A, b and e) and RGS2 (A, c and f) expression were localized using each specific antibody. Lysates from PDL cells stimulated by compressive force for 12 and 24 h were examined by Western blot analysis for RhoE, RhoA and RGS2 as described in Section 2 (B). b-actin was used as a control for equal loading. Arrows indicate the localization of RhoE, RGS2 and RhoA. Similar results were seen in three independent experiments.
As shown in Fig. 2B, the RhoE protein level were increased after 12 h of compressive force loading and the RGS2 protein levels were markedly enhanced at 24 h in response to compressive force, but RhoA expression level did not alter under this force loading condition.
3.3. Effects of antisense S-oligonucleotide to RGS2 or RhoE on stress fibre formation in the PDL cells under compressive force To determine if endogenous RhoE and RGS2 was involved in regulating actin cytoskeleton organization in PDL cells under mechanical stress, the effects of an antisense S-ODN to RhoE or RGS2 on stress fibre assembly were examined. As shown in Fig. 4, the attenuation of RhoE and RGS2 expressions induced by compressive force was observed in PDL cells treated with the antisense S-ODN. Blockage of RhoE promoted the formation of stress fibre under mechanical stress compared with the controls (Fig. 4c). On the other hand, inhibition of RGS2 expression did not affect stress fibre formation (Fig. 4f).
4.
Discussion
The activation of signalling molecules in response to mechanical stimuli appears to be mediated through changes in
Fig. 4 – Effects of antisense S-ODN to RhoE or RGS2 on the stress fibre formation in the PDL cells under compressive force. Cells were transfected with antisense S-ODN to RhoE (c) and RGS2 (f) 24 h prior to mechanical stress application. After 48 h of compression, the cells were stained with anti-RhoE (a, b and c) and anti-RGS2 (d, e and f) antibodies. To visualize stress fibres, cells were incubated with TRITC-phalloidin. Cells transfected with sense S-ODN to RhoE (a and b) and RGS2 (d and e) were used as controls. Arrows indicate the localization of RhoE and RGS2. Similar results were observed in three different experiments.
the cytoskeleton. The cytoskeleton is a complex network of protein filaments extending throughout the cell, and is formed by three major types of filaments: microtubules, actin filaments and intermediate filaments. Small GTPases of Rho family – Rho, Rac, and Cdc42 – are known to regulate actin cytoskeleton dynamics, thereby controlling cytoskeleton organization and cell motility response.14–19 RhoE binds only weakly to GDP and intrinsic GTPase activity is very low. Consequently, RhoE exists predominantly in a GTP-bound state. RhoE protein activity is presumably regulated differently from GTP hydrolysis, for example, by altering protein expression levels. Indeed, RhoE expression has been shown to be upregulated upon platelet-derived growth factor (PDGF)19 or hepatocyte growth factor20 stimulation of Swiss 3T3 or MDCK cells. Recent studies have elucidated that RhoE expression in fibroblasts and epithelial cells induces loss of actin stress fibres, and therefore, they seem to function antagonistically to RhoA in actin cytoskeleton regulation. RhoA is well known to induce the actin cytoskeleton organization. In order to investigate the involvement of small G-protein signalling in PDL cells under mechanical stress, the changes in the actin cytoskeleton organization and the expression of RhoE and RGS2 due to compressive force were examined. By applying compressive force to the PDL cells, a disruption of stress fibre formation leading to cell spreading was observed. It is likely that this modification in the actin cytoskeleton represents a self-protection mechanism in the cells to adapt to the mechanical stress. The recovery of stress fibres to control levels 24 h after removing the compressive loading indicates that the loss of stress fibres was caused only by the mechanical stress application. It has been reported that RhoE is widely expressed, although its expression levels vary significantly between different cell types.30 RhoE has been found in both membrane and cytoplasmic fractions of cells, and localized at
archives of oral biology 59 (2014) 187–192
least partly to the Golgi complex as well as to the plasma membrane.30 The results of fluorescent immunocytochemical examination for RhoE expression are consistent with the previous reports. However, in this immunostaining condition with the polyclonal anti-RhoE antibody, the positive signals in the nuclear may be due to a cross-reactivity of the antibody with a nuclear protein. RGS2 has been found to be predominantly a nuclear protein, but is recently reported to localize in the cytoplasm and plasma membrane.30 RGS2 is recruited to the plasma membrane by G-proteins.31,32 The immunocytochemical examination for RGS2 revealed that its expression was induced in the plasma membrane in mechanically stressed PDL cells. These data suggest the interactions of RGS2 with membrane-bound G-proteins under mechanical stress. RhoA is known to translocate to the cell membrane upon activation.15 It is not clear why RhoA has not been induced by compressive force in this immunocytochemistry. RhoA expression is well respondent to various stimuli including serum stimulation. Since these culture media for PDL cells contained FBS, RhoA induction due to the serum stimulation might occur in the cells without compression, thereby in which RhoA expression levels were similar to compressed cells. With regard to the decrease in stress fibre disassembly under compressive force, two regulatory mechanisms are hypothesized, which the up-regulated RhoE and the inactivation of other Rho GTPases such as RhoA by RGS2 induce the disassembly. In order to determine the role of RhoE and RGS2 in the stress fibre disruption, stress fibre formation due to compressive force was examined in inhibition of native RhoE and RGS2 expressions by antisense S-ODN. By treatment with antisense S-ODN to RhoE, the impaired formation of stress fibres caused by compressive force was recovered to the control levels, but the addition of antisense S-ODN to RGS2 did not affect the stress fibre formation. These results indicate that the loss of stress fibres due to mechanical stress is mediating RhoE signalling without the exertion of RGS2. Recently, RhoE has been described as ROCK I inhibitor. ROCK I, also known to be a target of RhoA, stimulates actin organization by phosphorylating a number of actin associated proteins such as MLC phosphatase.33 RhoE binds only to the amino-terminal part of ROCK I and might therefore physically interfere with its kinase activity. RhoE does not bind to other RhoA targets such as ROCK II. The regulatory mechanisms of ROCK I downstream of RhoE or RhoA in PDL cells have yet to be further clarified. This study imply that RhoE and RGS2 are the stress-related molecules that are highly expressed in human PDL cells under compressive force and play a role in the signalling of mechanotransduction events in human PDL cells. The enhanced RhoE by compressive force is responsible for the stress fibre disassembly in human PDL cells. Further studies on their physiological roles are necessary and may contribute to clarify the molecular function of periodontal ligament tissue and the remodelling mechanisms of periodontal tissues in response to mechanical stress.
Author contributions Conceived and designed the experiments: R.D., Y.O., K.M., Performed the analysis: R.D., Y.O., S.K., Analysed the data: R.D., Y.O., K.M., Wrote the paper: R.D., S.K., E.T.
191
Funding This research was supported, in part, by a Grant (No. 23659966) for Science Research from the Ministry of Education, Science and Culture, Japan.
Competing interests None declared.
Ethical approval This work was approved by the Ethical Committee of the University of Tokushima.
references
1. Tencate AR. Oral histology: development, structure and function. St. Louis: Mosby; 1998: 256. 2. Roberts WE, Goodwin Jr WC, Heiner SR. Cellular response to orthodontic force. Dent Clin North Am 1981;25:3–17. 3. Mitchell DL, West JD. Attempted orthodontic movement in the presence of suspected ankylosis. Am J Orthod Dentofacial Orthop 1975;68:404–11. 4. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop 2001;119:307–12. 5. Saito M, Saito S, Ngan PW, Shanfeld J, Davidovitch Z. Interleukin 1 beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. Am J Orthod Dentofacial Orthop 1991;99:226–40. 6. Lowney JJ, Norton LA, Shafer DM, Rossomando EF. Orthodontic forces increase tumor necrosis factor alpha in the human gingival sulcus. Am J Orthod Dentofacial Orthop 1995;108:519–24. 7. Shimizu N, Ozawa Y, Yamaguchi M, Goseki T, Ohzeki K, Abiko Y. Induction of COX-2 expression by mechanical tension force in human periodontal ligament cells. J Periodontol 1998;69:670–7. 8. Ohzeki K, Yamaguchi M, Shimizu N, Abiko Y. Effect of cellular aging on the induction of cyclooxygenase-2 by mechanical stress in human periodontal ligament cells. Mech Ageing Dev 1999;108:151–63. 9. Nakagawa M, Kukita T, Nakasima A, Kurisu K. Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization. Arch Oral Biol 1994;39:289–94. 10. Matsuda N, Yokoyama K, Takeshita S, Watanabe M. Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro. Arch Oral Biol 1998;43:987–97. 11. Yousefian J, Firouzian F, Shanfeld J, Ngan P, Lanese R, Davidovitch Z. A new experimental model for studying the response of periodontal ligament cells to hydrostatic pressure. Am J Orthod Dentofacial Orthop 1995;108: 402–9. 12. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–20.
192
archives of oral biology 59 (2014) 187–192
13. Araujo RMS, Oba Y, Moriyama K. Identification of genes related to mechanical stress in human periodontal ligament cells using microarray analysis. J Periodontal Res 2007;42:15–22. 14. Downes GB, Gautam N. The G protein subunit gene families. Genomics 1999;62:544–52. 15. Van Aelst L, D’Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 1997;11:2295–322. 16. Foster R, Hu KQ, Lu Y, Nolan KM, Thissen J, Settleman J. Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation. Mol Cell Biol 1996;16:2689–99. 17. Guasch RM, Scambler P, Jones GE, Ridley AJ. RhoE regulates actin cytoskeleton organization and cell migration. Mol Cell Biol 1998;18:4761–71. 18. Nobes CD, Lauritzen I, Mattei MG, Paris S, Hall A, Chardin P. A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion. J Cell Biol 1998;141:187–97. 19. Riento K, Guasch RM, Garg R, Jin B, Ridley AJ. RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol 2003;23:4219–29. 20. Tanimura S, Nomura K, Ozaki K, Tsujimoto M, Kondo T, Kohno M. Prolonged nuclear retention of activated extracellular signal-regulated kinase 1/2 is required for hepatocyte growth factor-induced cell motility. J Biol Chem 2002;277:28256–64. 21. Hansen SH, Zegers MM, Woodrow M, Rodriguez-Viciana P, Chardin P, Mostov KE, et al. Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway. Mol Cell Biol 2000;24:9364–75. 22. Riento K, Totty N, Villalonga P, Garg R, Guasch R, Ridley AJ. RhoE function is regulated by ROCK I-mediated phosphorylation. EMBO J 2005;24:1170–80.
23. Villalonga P, Guasch RM, Riento K, Ridley AJ. RhoE inhibits cell cycle progression and Ras-induced transformation. Mol Cell Biol 2004;24:7829–40. 24. Pellegrin S, Mellor H. Actin stress fibres. J Cell Sci 2007;120:3491–9. 25. Ishii M, Kurachi Y. Physiological actions of regulators of Gprotein signaling (RGS) proteins. Life Sci 2003;74: 163–71. 26. De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 2000;40:235–71. 27. Song L, Zmijewski JW, Jope RS. RGS2: regulation of expression and nuclear localization. Biochem Biophys Res Commun 2001;283:102–6. 28. Han X, Amar S. Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis. J Dent Res 2002;81: 399–405. 29. Kagami S, Urushihara M, Kondo S, Loster K, Reutter W, Tamaki T, et al. Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells. Exp Cell Res 2001;268:274–83. 30. Riento K, Villalonga P, Garg R, Ridley A. Function and regulation of RhoE. Biochem Soc Trans 2005;33:649–51. 31. Kehrl JH, Sinnarajah S. RGS2: a multifunctional regulator of G-protein signaling. Int J Biochem Cell Biol 2002;34: 432–8. 32. Zmijewski JW, Song L, Harkins L, Cobbs CS, Jope RS. Second messengers regulate RGS2 expression which is targeted to the nucleus. Biochim Biophys Acta 2001;1541:201–11. 33. Riento K, Ridley AJ. ROCKs: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003;4:446–56.
