Rheumatology Advance Access published March 4, 2014
RHEUMATOLOGY
Original article
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doi:10.1093/rheumatology/keu013
Increased phosphorylation of ezrin is associated with the migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis
Objective. Increasing evidence indicates that the cytoskeletal protein ezrin may play a critical role in cell motility. This study aims to investigate the role of ezrin in regulating the migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with RA. Methods. Synovial tissues were obtained from 12 patients with RA and 6 with OA, and then FLSs were separated from synovial tissues. The expression of ezrin and phosphorylated ezrin (p-ezrin) was examined by Western blotting or IF staining. A specific inhibitor of ezrin phosphorylation and small interference RNAmediated ezrin knockdown were used to inhibit the phosphorylation of ezrin. Migration and invasion of FLSs in vitro were measured by the Boyden chamber assay. Results. Increased expression of p-ezrin protein was found in synovial tissue and FLSs in patients with RA compared with patients with OA. Stimulation with TNF-a and IL-1b increased ezrin phosphorylation in RA FLSs. Inhibition of p-ezrin protein by a specific inhibitor of phosphorylation of ezrin and small interfering RNAmediated knockdown reduced in vitro migration and invasion, as well as actin stress fibre formation in RA FLS. Furthermore, rho kinase and p38 mitogen-activated protein kinase (MAPK) signal pathways were involved in the phosphorylation of ezrin and invasion of RA FLSs. Conclusion. Increased expression of p-ezrin may contribute to aberrant aggressive behaviours of RA FLSs, which are mediated by rho kinase and the p38 MAPK pathway. This suggests a novel strategy targeting phosphorylation of ezrin to prevent synovial invasiveness and joint destruction in RA. Key words: rheumatoid arthritis, ezrin, phosphorylation, invasion, fibroblast-like synoviocytes, signal transduction.
Introduction RA is a chronic inflammatory disease of the joints characterized by marked hyperplasia of the lining layer of synovium and erosion of bone and cartilage. Increasing evidence indicates that, in addition to macrophages and 1 Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Submitted 22 September 2013; revised version accepted 14 January 2014. Correspondence to: Hanshi Xu, Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, P.R. China. E-mail: [email protected] *Youjun Xiao, Mengying Sun and Zhongping Zhan contributed equally to this study.
T cells, activated fibroblast-like synoviocytes (FLSs) are key players in the pathophysiological process of RA [1]. Aberrant activated FLSs not only produce a number of proinflammatory cytokines and chemokines, but also secrete matrix metalloproteinases and cathepsins [2, 3]. More importantly, it has been shown that RA FLSs invade human cartilage in the absence of an inflammatory environment in severe combined immunodeficient mice [46]. These studies indicate that modulating behaviours of activated FLSs, such as migration and invasion, may be a novel therapeutic strategy for destructive progress of RA. Stable activated RA FLSs manifest the characteristics of tumour cells, including anchorage-independent proliferation, resistance to apoptotic stimuli and invasive behaviour [79]. However, the signal mechanisms regulating the aberrant activation of RA FLSs remain to be defined.
! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
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BASIC SCIENCE
Abstract
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Youjun Xiao1,*, Mengying Sun1,*, Zhongping Zhan1,*, Yujin Ye1, Mingcheng Huang1, Yaoyao Zou1, Liuqin Liang1, Xiuyan Yang1 and Hanshi Xu1
Youjun Xiao et al.
Materials and methods Reagents and antibodies Recombinant human TNF-a and IL-1b were obtained from R&D Systems (Minneapolis, MN, USA). DMEM/F12, fetal bovine serum (FBS), antibiotics, trypsin-EDTA, PBS and other products for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). Anti-tubulin antibody was obtained from Sigma Chemicals (St Louis, MO, USA). Rabbit polyclonal antibody to anti-ezrin and rabbit polyclonal antibody to p-ezrin (T567) were purchased from Cell Signaling Technologies (Beverly, MA, USA) and Abcam (Cambridge, UK), respectively. NSC668394, a specific inhibitor of ezrin phosphorylation, was obtained from Calbiochem (La Jolla, CA, USA) and dissolved in a solution containing 1% dimethyl sulphoxide (DMSO) when used.
Patients and controls Twelve patients with RA (10 women and 2 men, aged 4162 years) and 6 patients with OA (4 women and 2 men, aged 5568 years) who have undergone synovectomy were enrolled in this study. The demographics of the RA patients are shown in Table 1. RA was diagnosed according to the 1987 ACR revised criteria [17] and OA was diagnosed according to the ACR criteria [18]. The study was approved by the Medical Ethical Committee of the First Affiliated Hospital, Sun Yat-sen University, and was performed according to the recommendations of the Declaration of Helsinki. All patients provided written informed consent before taking part in the study.
Cell culture ST obtained from the patients was cut into small pieces and digested with collagenase in DMEM/F12 medium for 2 h at 37 C to isolate synoviocytes. The synoviocytes were grown in DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified incubator at 37 C under 5% CO2. At confluence, the
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cells were trypsinized and passaged and were used for three to five passages.
Western blot analysis For each experiment, a total of 5 105 cells were seeded, and at subconfluence (70%), cells were made quiescent for 24 h in DMEM/F12 medium containing 0.5% FBS and then treated with various agents. In order to detect the expression of signal proteins, we rinsed the cells twice with ice-cold PBS and added 0.5 ml of the ice-cold lysis buffer [50 mM TrisHCl, pH 7.5, 150 mM NaCl, 100 mg/ml phenylmethylsulphonyl fluoride (PMSF), 0.1% SDS, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 10 mg/ml aprotinin, 2 mg/ml leupeptin and 10 mM EDTA]. The cells were incubated for 20 min on ice and then scraped and centrifuged. As for the protein extracts of STs, the tissues were rapidly frozen in liquid nitrogen and homogenized in ice-cold lysis buffer containing 20 mM hydroxyethyl piperazineethanesulphonic acid (HEPES)NaOH, 10 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol and Complete (Roche Molecular Biochemicals, 1 tablet/50 ml) and then were centrifuged and the supernatant was stored at 80 C until use. Protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were solubilized in Laemmli buffer (62.5 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 5% b-mercaptoethanol and 0.00625% bromophenol blue), boiled for 5 min and then separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies diluted 1:1000 for ezrin and 1:500 for p-ezrin in TBS-T containing 5% nonfat milk at 4 C overnight. The membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Each blot is a representative of at least three similar independent experiments. The film was scanned by a high-resolution scanner, densitometry was performed using AlphaEaseFc software on a Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA) and the expression of p-ezrin and ezrin was calculated as the ratio of the optical density of p-ezrin and ezrin to that of tubulin bands (relative intensity).
Treatment with ezrin siRNA Ezrin small interfering RNA (siRNA) (50 -ATGTTCCTGATTT CACTCC-30 ) or non-silencing control siRNA was obtained from Shanghai GenePharma (Shanghai, China). RA FLSs were cultured in 12-well plates. A transfection mixture of 100 nM siRNA and 10 mg/ml lipofectin in serum-free medium was added to medium-aspirated FLSs for 4 h. The FLSs were then incubated with complete DMEM/ F12 containing 10% FBS for 48 h before experiments. At the end of culture, the effect of the siRNA on expression of ezrin and p-ezrin protein was analysed using western blot.
IF staining The samples were fixed in 4% paraformaldehyde to evaluate the expression of p-ezrin in STs. First, the samples
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Ezrin, a member of the ezrin/radixin/moesin proteins, is linker between membrane and cytoskeleton. Phosphorylation of ezrin is a critical step in modulating the structure and function of plasma membranes [10, 11]. Phosphorylated ezrin (p-ezrin) plays an important role in regulating the various cellular functions, such as migration, adhesion, stress fibre formation and signal transduction [1214]. Previous studies have shown that cytoskeletal ezrin plays an important role in tumour growth and metastasis [15, 16]. We therefore hypothesized that ezrin contributes to the progression of RA by modulating abnormal behaviour of activated RA FLS, including enhanced migration and invasion. In the present study we show that p-ezrin was increased in synovial tissues (STs) and FLSs from RA patients and may contribute to abnormal migration and invasion of RA FLSs. These results indicate that an increase in ezrin phosphorylation may be associated with the aggressive behaviour of RA FLSs.
Role of p-ezrin in regulating migration and invasion of RA FLS
TABLE 1 Demographics of patients with RA
Patient no.
DAS28
Prednisone used (410 mg/day)
NSAIDs used
DMARDs used
48 51 61 53 60 56 41 58 52 62 43 61
5.2 5.7 4.6 6.3 6.4 6.3 4.2 4.9 6.9 7.3 5.4 6.7
+ + + + +
+ + + + + + + + + + +
MTX + HCQ HCQ MTX SSZ SSZ MTX + HCQ
RA1 RA2 RA3 RA4 RA5 RA6 RA7 RA8 RA9 RA10 RA11 RA12 +: used; : not used.
were stained with rabbit anti-p-ezrin antibody and FITCconjugated secondary antibody according to the manufacturer’s instructions. Then the stained samples were examined by confocal fluorescence microscopy (Zeiss LSM510; Carl Zeiss MicroImaging, Thornwood, NY, USA) and representative sections were photographed. Cells were grown on glass coverslips to detect the expression of p-ezrin and cytoskeletal filamentous actin (F-actin) in RA FLSs. First, the cells were fixed with acetone and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated with anti-p-ezrin antibody (diluted 1:200) for 1 h at room temperature and then incubated with FITC-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing in PBS, cells were incubated for 3 min with 0.25 mg/ml 40 -6-diamidono-2-phenylindole dihydrochloride (DAPI). For detecting cytoskeletal F-actin, the cells were incubated with AlexaFluor-546 phalloidin (molecular probes; Invitrogen, Eugene, OR, USA). The coverslips were mounted on glass slides with anti-fade mounting media and examined using confocal fluorescence microscopy (Zeiss LSM510).
In vitro migration and invasion assay of FLSs Chemotaxis assay of FLSs was performed by the Boyden chamber method using a filter of 6.5 mm diameter and 8.0 mm pore size (Transwell; Corning, Corning, NY, USA). Briefly, DMEM containing 10% FBS as a chemoattractant was placed in the lower wells. FLSs (at a final concentration of 6 104 cells/ml) were suspended in serum-free DMEM in the upper wells. The compound was applied to the upper chamber in a serum-free media containing 1% DMSO and the lower chamber was loaded with 10% FBS media containing 1% DMSO to evaluate the effect of NSC668394 on migration. The chamber was incubated at 37 C in 5% CO2 for 10 h. After the incubation, the nonmigrating cells were removed from the upper surface of the filter using a cotton swab. The filters were fixed in methanol for 15 min and stained with 0.1% crystal violet for 15 min. Chemotaxis was quantified by counting the
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stained cells that migrated to the lower side of the filter using an optical microscope. The stained cells were counted as the mean number of cells per 10 random fields for each assay. For the in vitro invasion assay, similar experiments were performed using inserts coated with a Matrigel basement membrane matrix (BD Biosciences).
Wounding migration RA FLSs, plated to confluence on 60 mm culture dishes, were wounded with pipette tips and then treated with or without 10% FBS. After 24 h of incubation, migration was quantified by counting the cells that had moved beyond a reference line.
MTT test for FLS viability RA FLSs were pretreated for 48 h with NSC668394 at different concentrations (1, 5 and 10 mM). The culture supernatants were removed and the adherent cells were incubated for 30 min at 37 C with a solution of the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) salt (1 mg/ml in PBS). The dark blue crystals of formazan were dissolved in acidified isopropanol and formazan quantification was performed at a test wavelength of 570 nm and a reference wavelength of 620 nm.
Statistical analysis Data are expressed as mean (S.E.M.). Student’s t-test or one-way analysis of variance (ANOVA) was used to analyse the differences between groups. A P-value 40.05 was considered statistically significant.
Results Increased expression of p-ezrin protein in STs and FLSs from RA patients The expression of p-ezrin protein in STs and FLSs was determined by western blot and immunohistochemical analysis. As shown in Fig. 1A, the expression of p-ezrin
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Youjun Xiao et al.
FIG. 1 Expression of p-ezrin protein in joint STs and FLSs from RA and OA patients
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(A and B) The expression of p-ezrin in STs from RA and OA patients. The expression of p-ezrin was detected by (A) western blot analysis and (B) IF staining. Representative pictures of immune blot (upper panel of A) and the densitometric quantification [mean (S.E.M.), lower panel of A] of the expression of p-ezrin and ezrin were from patients with RA (n = 12) and OA (n = 6). vs OA: *P < 0.05. For IF staining, p-ezrin (green) and nuclei (blue) were evaluated using confocal microscopy and the pictures shown are representative of 10 RA and 5 OA patients. Original magnification 400. (C) Expression of p-ezrin (green) detected by IF staining in FLSs isolated from the STs of patients with RA and OA. The pictures shown are representative of nine RA and five OA patients. Original magnification 400. (D) Confluent FLSs were treated with TNF-a (10 ng/ml) or IL-1b (10 ng/ml) for 6 h. Data are expressed as mean (S.E.M.) of densitometric quantification (right panel) from three independent experiments on three patients. vs basal: *P < 0.05. p-ezrin: phosphorylated ezrin; STs: synovial tissues; FLSs: fibroblast-like synoviocytes.
(threonine 567 phosphorylation) was higher in STs from RA patients than from OA patients. However, the expression of ezrin protein in STs was similar between RA and OA patients. By using IF staining analysis, we further demonstrated that expression of p-ezrin protein was prominent in STs from RA patients and mostly
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localized in the synovial lining and sublining cells, whereas the expression was much less prominent in STs from OA patients (Fig. 1B). We also detected the p-ezrin expression in isolated and cultured FLSs and found increased expression of p-ezrin protein in RA FLSs as compared with OA FLSs (Fig. 1C). These results
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Expression of p-ezrin
tubulin
Role of p-ezrin in regulating migration and invasion of RA FLS
indicate that the expression of p-ezrin increased in RA synovial membrane. Since proinflammatory cytokines, especially TNF-a and IL-1b, play an important role in transforming RA FLSs to a more invasive phenotype, we examined the effect of the cytokines on the expression and activation of ezrin in confluent RA FLSs. As shown in Fig. 1D, treatment with TNF-a (10 ng/ml) or IL-1b (10 ng/ml) increased the phosphorylation of ezrin, but not the expression of total ezrin protein.
Inhibitory effect of ezrin phosphorylation on regulating migration of RA FLSs
FIG. 2 Inhibitory effect of ezrin phosphorylation on regulating RA FLS migration in vitro
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(A and B) RA FLSs were (A) pretreated with 1% dimethyl sulphoxide (as control) or various concentrations of NSC668394 (NSC), a specific inhibitor of ezrin phosphorylation for 2 h or (B) transfected with specific ezrin siRNA (SiE, 100 nM) or control siRNA (SiC, 100 nM) and then stimulated with or without IL-1b (10 ng/ml) for 6 h. The expression of p-ezrin and ezrin protein was assessed by western blot analysis. (C) Migration was performed in a Boyden chamber and chemotaxis was quantified by counting the migrated cells. The RA FLSs were seeded in a Boyden chamber and allowed to migrate for an additional 10 h. Fetal bovine serum (FBS, 10%) was used as a chemoattractant. The results show the mean (S.E.M.) in samples from five different RA patients. Arrows indicate migrated FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs DMSO or SiC: *P < 0.05. (D) Effect of p-ezrin inhibition on the wounding migration of RA FLSs (original magnification 100). Cells migrating beyond the reference line were photographed and counted. Data shown are representative of experiments from three different RA patients. FLSs: fibroblast-like synoviocytes; DMSO: dimethyl sulphoxide; siRNA: small interfering RNA; p-ezrin: phosphorylated ezrin.
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Ezrin has been implicated in the modulation of migration of several other cell lines. To examine the role of ezrin phosphorylation in RA FLS migration, the chemotaxis migration
of FLSs was evaluated using a Transwell chamber assay. As shown in Fig. 2A, p-ezrin expression was significantly inhibited in the cells treated with NSC668394 (110 mM), a compound that inhibits ezrin phosphorylation at T567. Since maximal inhibitory effect was observed at 5 mM, the optimal concentration of NSC668394 was defined as 5 mM for further experiments. Treatment with NSC668394 decreased the chemotaxis migration of RA FLSs (Fig. 2C). The viability of RA FLSs was measured by the MTT test to evaluate the toxic effect of NSC668394 on RA FLSs. Up to 10 mM, this compound did not reduce cell viability (data not shown), which indicates that the inhibitory effect observed was not due to cytotoxic effects. We further found that treatment with ezrin siRNA efficiently suppressed the expression of p-ezrin protein and
Youjun Xiao et al.
Inhibitory effect of ezrin phosphorylation on regulating in vitro invasion of RA FLSs
Inhibitory effect of ezrin phosphorylation on regulating actin organization of RA FLSs As a linker between membrane and cytoskeleton, ezrin plays an important role in regulating organization of the actin cytoskeleton, which is thought to have a critical role in cell migration. To determine the role of ezrin in the organization of the actin cytoskeleton in RA FLSs, fluorescent phalloidin staining was used to visualize polymerized actin in cells. As shown in Fig. 4, treatment with NSC668394 (5 mM) inhibited formation of actin stress fibres, as well as the expression of p-ezrin protein. Transfection with specific ezrin siRNA also inhibited IL-1b-induced condensation of actin stress fibre formation and p-ezrin protein expression compared with that treated with control siRNA. Thus the function of ezrin in cell migration and actin dynamics is likely to contribute to its role in invasion of RA FLSs.
The signalling mechanisms of phosphorylation of ezrin in RA FLSs To investigate the signalling mechanisms leading to phosphorylation of ezrin in RA FLSs, we used known inhibitors of several signalling pathways, including a special inhibitor of p38 MAPK, SB203580, and an inhibitor of rhoassociated protein kinase (ROCK), Y27632. As shown in Fig. 5, IL-1b stimulation induced increased expression of p-ezrin, and pre-treatment with SB203580 (20 mM) or Y27632 (10 mM) inhibited the expression of p-ezrin. Furthermore, we found that treatment with SB203580 or Y27632 inhibited in vitro invasion of RA FLSs. These results indicate that p38 MAPK and ROCK pathways might be involved in IL-1b-induced ezrin phosphorylation and therefore result in modification of the invasiveness of RA FLSs.
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For the in vitro invasion assay, the experiments were performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. FBS (10%) was used as a chemoattractant. The cells were allowed to migrate for 10 h. RA FLSs were treated with 1% DMSO (as control) or NSC668394 (NSC, 5 mM) or transfected with specific ezrin siRNA (SiE, 100 nM) or control siRNA (SiC, 100 nM). The results show the mean (S.E.M.) in samples from seven patients with RA. Arrows indicate migrated of FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs DMSO or SiC: *P < 0.05. FLSs: fibroblast-like synoviocytes; DMSO: dimethyl sulphoxide; siRNA: small RNA; FBS: fetal bovine serum.
Discussion In the present study we showed that expression of p-ezrin protein is increased in STs and FLSs derived from RA patients. It was also found that special siRNA for ezrin and a specific inhibitor of ezrin phosphorylation suppressed migration and invasion of RA FLSs, which indicates that the increased phosphorylation of ezrin plays an important role in the maintenance of the activated phenotype of FLSs from patients with RA. The migration of FLSs to cartilage and bone has been considered a critical step in the erosion of joints in RA. While they arrive in the cartilage or bone, FLSs destroy cartilage or activate osteoclasts to enhance bone erosion and destruction [2022]. Identifying the critical factors that control migration and invasion of RA FLSs through cartilage and into bone is an important step towards understanding what directs the disease process. Ezrin was considered not only as a cross-linker between the actin cytoskeleton and plasma membrane, but also as an important signal transducer involved in a wide variety of cellular functions. As a linker between membrane and cytoskeletal binding sites, ezrin needs to be conformationally activated/phosphorylated to fulfil its biological functions. Phosphorylation of ezrin is critical for regulating cell shape, motility, survival and signal transduction in several cell lines. For instance, it has been implicated in the regulation of tumour invasion and
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The in vitro invasion potential of RA FLSs is well associated with the rate of joint destruction in RA patients [19]. To investigate the role of ezrin in the invasive behaviour of RA FLSs in vitro, a thin layer of reconstituted extracellular matrix (Matrigel) was used to measure in vitro invasion of RA FLSs. As shown in Fig. 3, NSC668394 (5 mM) treatment suppressed Matrigel invasion of RA FLSs. Treatment with ezrin siRNA also inhibited invasion as compared with treatment with control siRNA in RA FLSs. These results indicate that ezrin plays an important role in regulating the invasive behaviour of RA FLSs.
FIG. 3 Inhibitory effect of ezrin phosphorylation on regulation of RA FLS invasion in vitro
Number of Martrige linv ading cells
resulted in a reduction in the migration of RA FLSs as compared with the non-silencing control vector transfection (Fig. 2B and C). Furthermore, we used a monolayer wound healing assay to detect the role of p-ezrin in cell migration. Wound closing was significantly slowed in RA FLSs treated with NSC668394 and specific ezrin siRNA, respectively (Fig. 2D).
Role of p-ezrin in regulating migration and invasion of RA FLS
FIG. 4 Inhibitory effect of ezrin phosphorylation on regulation of actin organization of RA FLSs
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RA FLSs were transfected with siRNA targeting ezrin (SiE) or control siRNA (SiC) or treated with 1% DMSO (as control) or NSC668394 (NSC, 5 mM). The cells were grown on glass coverslips and then were stimulated with IL-1b (10 ng/ml) for 6 h and p-ezrin (green), F-actin (red) and nuclei (blue) were evaluated by IF staining. F-actin and nuclei were stained with phalloidin and 40 -6-diamidono-2-phenylindole dihydrochloride (DAPI), respectively. Representative laser confocal microscopy images show F-actin and p-ezrin staining from five different patients. Original magnification 400. FLSs: fibroblast-like synoviocytes; siRNA: small interfering RNA; DMSO: dimethyl sulphoxide; FBS: fetal bovine serum; F-actin: filamentous actin; p-ezrin: phosphorylated ezrin.
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FIG. 5 The signals involved in IL-1b-induced phosphorylation of ezrin in RA FLSs
metastasis, such as in breast carcinoma cells [23], intraductal papillary mucinous neoplasms and pancreatic intraepithelial neoplasia [24] and gastrointestinal stromal tumours [25]. Dysregulation of ezrin phosphorylation at amino acid T567 inhibits metastasis and alters cellular metabolism in osteosarcoma [26]. A previous report shows that during Salmonella typhimurium infection,
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Rheumatology key messages Ezrin phosphorylation is increased in RA fibroblastlike synoviocytes (FLSs). . Elevated ezrin phosphorylation contributes to migration and invasion of RA FLSs. . Inhibition of ezrin phosphorylation may be a new therapeutic approach for RA. .
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(A) RA FLSs were pretreated for 1 h with or without 20 mM SB203580 (SB), a specific p38 inhibitor, or 10 mM Y27632 (Y), a ROCK inhibitor, and then stimulated with buffer or IL-1b (10 ng/ml) for 6 h. Data represent the mean (S.E.M.) of densitometric quantification (lower panel) from at least three independent experiments. vs basal: *P < 0.05; vs IL1b: #P < 0.05. (B) Inhibitory effect of p38 MAPK and ROCK pathway on in vitro invasion of RA FLSs. The experiments for in vitro invasion were performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. Ten per cent FBS was used as a chemoattractant. RA FLSs were treated with SB203580 (SB) or 10 mM Y27632 (Y). The results show the mean (S.E.M.) in samples from five patients with RA. Arrows indicate migrated FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs control: *P < 0.05. FLSs: fibroblast-like synoviocytes; ROCK: rho-associated protein kinase; MAPK: mitogenactivated protein kinase; FBS: fetal bovine serum.
ezrin activation also regulates neutrophil transmigration by modulating the apical localization of MRP2 in response to the SipA effector protein [27]. Phosphorylation of ezrin also participates in B cell antigen receptor signal transduction [28]. All these studies indicate that phosphorylation of ezrin plays an important role in many cellular biological functions. In this study we demonstrated for the first time that increased expression of p-ezrin protein in STs and FLSs from RA patients participates in the regulation of migration and invasion by RA FLSs. Since abnormal invasiveness of FLSs is considered a critical factor in joint destruction, our results also indicate that increased phosphorylation of ezrin in FLSs may be associated with the pathological progression of RA. Indeed, it has been reported that the mammalian target of rapamycin (mTOR) signal, a pathway that is associated with ezrin, is involved in invasion of RA FLSs [29]. Stress fibre formation is a critical step that controls cell motility [32]. Ezrin regulates stress fibre formation in other cell lines [30]. It has also been implied that modulation of cytoskeletal rearrangements may play a role in the aggravation of synovial pathophysiology in FLSs from a mice model with RA [31, 32]. Herein we showed that inhibition of ezrin with siRNA or a specific inhibitor of ezrin phosphorylation strongly prevents stress fibre formation, which indicates that p-ezrin controls RA FLS migration, at least in part, by modulating actin polymerization. These studies indicate that some actin-binding proteins such as ezrin may play an important role in the progression of RA through regulating stress fibre formation. Previous reports have shown that some signalling pathways, such as rho and p38 MAPK, are required for regulating phosphorylation of ezrin in several cell lines. For example, it has been demonstrated that the p38 MAPK signal mediates phosphorylation of ezrin in the activation of Na+H+ antiporter isoform 3 following Na+-glucose cotransport [33]. Another study also shows the modulation of the ROCK signal on phosphorylation of ezrin in epithelial cells [34] and nasopharyngeal carcinoma 6-10B cells [35]. Consistent with previous studies, we demonstrated that activation of rho kinases and p38 is required for phosphorylation of ezrin and migration and invasion of RA FLSs, as evaluated by the inhibitory effect of p38 inhibitor SB203580 and ROCK inhibitor Y27632, which suggests that ezrin may be the downstream target of p38 and ROCK for migration and invasion in RA FLSs. In summary, the present study reveals the role of phosphorylation of ezrin in modulating migration and invasion of RA FLSs, providing a novel therapeutic target that could be exploited to control pathological synovial invasion in RA.
Role of p-ezrin in regulating migration and invasion of RA FLS
Acknowledgements We would like to thank Jinjin Fan for her technical assistance.
Disclosure statement: The authors have declared no conflicts of interest.
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15 Yu Y, Khan J, Khanna C et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 2004;10:17581. 16 Khanna C, Wan X, Bose S et al. The membranecytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med 2004;10:1826. 17 Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31:31524. 18 Altman R, Asch E, Bloch D et al. Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheum 1986;29:103949. 19 Tolboom TC, van der Helm-Van Mil AH, Nelissen RG et al. Invasiveness of fibroblast-like synoviocytes is an individual patient characterize associated with the rate of joint destruction in patients with rheumatoid arthritis. Arthritis Rheum 2005;52:19992002. 20 Mu¨ller-Ladner U, Pap T, Gay RE, Neidhart M, Gay S. Mechanisms of disease: the molecular and cellular basis of joint destruction in rheumatoid arthritis. Nat Clin Pract Rheumatol 2005;1:10210. 21 Volin MV, Huynh N, Klosowska K, Chong KK, Woods JM. Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast-like synoviocyte signaling through MAP kinases and Akt. Arthritis Rheum 2007;56: 251222. 22 Gravallese EM, Manning C, Tsay A et al. Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum 2000;43:2508. 23 Mak H, Naba A, Varma S et al. Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells. BMC Cancer 2012;12:82. 24 Oda Y, Aishima S, Morimatsu K et al. Differential ezrin and phosphorylated ezrin expression profiles between pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and invasive ductal carcinoma of the pancreas. Hum Pathol 2013;44: 148798.
9 Roivainen A, Jalava J, Pirila L et al. H-ras oncogene point mutation in arthritic synovium. Arthritis Rheum 1997;40: 163643.
25 Weng WH, Yeh CN, Cheng YF, Lenka G, Liaw YW. Phosphorylated T567 ezrin is associated with merlin expression in KIT-mutant gastrointestinal stromal tumors. Mol Med Rep 2012;5:1721.
10 Bretscher A, Reczek D, Berryman M. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J Cell Sci 1997;110:301118.
26 Ren L, Hong SH, Chen QR et al. Dysregulation of ezrin phosphorylation prevents metastasis and alters cellular metabolism in osteosarcoma. Cancer Res 2012;72: 10012.
11 Louver-Vallee S. ERM proteins: from cellular architecture to cell signaling. Biol Cell 2000;92:30516.
27 Agbor TA, Demma ZC, Mumy KL, Bien JD, McCormick BA. The ERM protein, ezrin, regulates neutrophil transmigration by modulating the apical localization of MRP2 in response to the SipA effector protein during salmonella typhimurium infection. Cell Microbiol 2011;13: 200721.
12 Saotome I, Curto M, McClatchey AI. Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev Cell 2004;6:85564. 13 Faure S, Salazar-Fontana LI, Semichon M et al. ERM proteins regulate cytoskeleton relaxation promoting T cellAPC conjugation. Nat Immunol 2004;5:27279.
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28 Parameswaran N, Enyindah-Asonye G, Bagheri N, Shah NB, Gupta N. Spatial coupling of JNK activation to
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Funding: This work is supported by grants from the National Natural Science Foundation of China (81072484 and 81373182), China Ministry of Education Foundation (20100171110058), Guangdong Natural Science Foundation (S2011020002358 and S2013010015363), Guangdong Project of Science and Technology (2011B080701011 and 2011B080701098) and Guangdong Provincial Department of Education Foundation (2012KJCX0003).
14 Bretscher A, Edwards K, Fehon RG. ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 2002;3:58699.
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the B cell antigen receptor by tyrosine-phosphorylated ezrin. J Immunol 2013;190:201726. 29 Laragione T, Gulko PS. mTOR regulates the invasive properties of synovial fibroblasts in rheumatoid arthritis. Mol Med 2010;16:3528. 30 Yasui Y, Amano M, Nagata K et al. Roles of Rhoassociated kinase in cytokinesis; mutations in Rhoassociated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. J Cell Biol 1998;143: 124958.
32 Vasilopoulos Y, Gkretsi V, Armaka M, Aidinis V, Kollias G. Actin cytoskeleton dynamics linked to synovial fibroblast
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33 Hu Z, Wang Y, Graham WV et al. MAPKAPK-2 is a critical signaling intermediate in NHE3 activation following Na+-glucose cotransport. J Biol Chem 2006;281: 2424753. 34 Wakayama Y, Miura K, Sabe H, Mochizuki N. EphrinA1EphA2 signal induces compaction and polarization of Madin-Darby canine kidney cells by inactivating ezrin through negative regulation of RhoA. J Biol Chem 2011; 286:4424353. 35 Tang F, Zou F, Peng Z et al. N,N0 -dinitrosopiperazinemediated ezrin phosphorylation via activating Rho kinase and protein kinase C involves in metastasis of nasopharyngeal carcinoma 6-10B cells. J Biol Chem 2011;286: 3695667.
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31 Aidinis V, Carninci P, Armaka M et al. Cytoskeletal rearrangements in synovial fibroblasts as a novel determinant of modeled rheumatoid arthritis. PLoS Genet 2005;1:e48.
activation as a novel pathogenic principle in TNF-driven arthritis. Ann Rheum Dis 2007;66(Suppl III):iii238.
RHEUMATOLOGY
Original article
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doi:10.1093/rheumatology/keu013
Increased phosphorylation of ezrin is associated with the migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis
Objective. Increasing evidence indicates that the cytoskeletal protein ezrin may play a critical role in cell motility. This study aims to investigate the role of ezrin in regulating the migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with RA. Methods. Synovial tissues were obtained from 12 patients with RA and 6 with OA, and then FLSs were separated from synovial tissues. The expression of ezrin and phosphorylated ezrin (p-ezrin) was examined by Western blotting or IF staining. A specific inhibitor of ezrin phosphorylation and small interference RNAmediated ezrin knockdown were used to inhibit the phosphorylation of ezrin. Migration and invasion of FLSs in vitro were measured by the Boyden chamber assay. Results. Increased expression of p-ezrin protein was found in synovial tissue and FLSs in patients with RA compared with patients with OA. Stimulation with TNF-a and IL-1b increased ezrin phosphorylation in RA FLSs. Inhibition of p-ezrin protein by a specific inhibitor of phosphorylation of ezrin and small interfering RNAmediated knockdown reduced in vitro migration and invasion, as well as actin stress fibre formation in RA FLS. Furthermore, rho kinase and p38 mitogen-activated protein kinase (MAPK) signal pathways were involved in the phosphorylation of ezrin and invasion of RA FLSs. Conclusion. Increased expression of p-ezrin may contribute to aberrant aggressive behaviours of RA FLSs, which are mediated by rho kinase and the p38 MAPK pathway. This suggests a novel strategy targeting phosphorylation of ezrin to prevent synovial invasiveness and joint destruction in RA. Key words: rheumatoid arthritis, ezrin, phosphorylation, invasion, fibroblast-like synoviocytes, signal transduction.
Introduction RA is a chronic inflammatory disease of the joints characterized by marked hyperplasia of the lining layer of synovium and erosion of bone and cartilage. Increasing evidence indicates that, in addition to macrophages and 1 Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Submitted 22 September 2013; revised version accepted 14 January 2014. Correspondence to: Hanshi Xu, Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, P.R. China. E-mail: [email protected] *Youjun Xiao, Mengying Sun and Zhongping Zhan contributed equally to this study.
T cells, activated fibroblast-like synoviocytes (FLSs) are key players in the pathophysiological process of RA [1]. Aberrant activated FLSs not only produce a number of proinflammatory cytokines and chemokines, but also secrete matrix metalloproteinases and cathepsins [2, 3]. More importantly, it has been shown that RA FLSs invade human cartilage in the absence of an inflammatory environment in severe combined immunodeficient mice [46]. These studies indicate that modulating behaviours of activated FLSs, such as migration and invasion, may be a novel therapeutic strategy for destructive progress of RA. Stable activated RA FLSs manifest the characteristics of tumour cells, including anchorage-independent proliferation, resistance to apoptotic stimuli and invasive behaviour [79]. However, the signal mechanisms regulating the aberrant activation of RA FLSs remain to be defined.
! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
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BASIC SCIENCE
Abstract
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Youjun Xiao1,*, Mengying Sun1,*, Zhongping Zhan1,*, Yujin Ye1, Mingcheng Huang1, Yaoyao Zou1, Liuqin Liang1, Xiuyan Yang1 and Hanshi Xu1
Youjun Xiao et al.
Materials and methods Reagents and antibodies Recombinant human TNF-a and IL-1b were obtained from R&D Systems (Minneapolis, MN, USA). DMEM/F12, fetal bovine serum (FBS), antibiotics, trypsin-EDTA, PBS and other products for cell culture were purchased from Invitrogen (Carlsbad, CA, USA). Anti-tubulin antibody was obtained from Sigma Chemicals (St Louis, MO, USA). Rabbit polyclonal antibody to anti-ezrin and rabbit polyclonal antibody to p-ezrin (T567) were purchased from Cell Signaling Technologies (Beverly, MA, USA) and Abcam (Cambridge, UK), respectively. NSC668394, a specific inhibitor of ezrin phosphorylation, was obtained from Calbiochem (La Jolla, CA, USA) and dissolved in a solution containing 1% dimethyl sulphoxide (DMSO) when used.
Patients and controls Twelve patients with RA (10 women and 2 men, aged 4162 years) and 6 patients with OA (4 women and 2 men, aged 5568 years) who have undergone synovectomy were enrolled in this study. The demographics of the RA patients are shown in Table 1. RA was diagnosed according to the 1987 ACR revised criteria [17] and OA was diagnosed according to the ACR criteria [18]. The study was approved by the Medical Ethical Committee of the First Affiliated Hospital, Sun Yat-sen University, and was performed according to the recommendations of the Declaration of Helsinki. All patients provided written informed consent before taking part in the study.
Cell culture ST obtained from the patients was cut into small pieces and digested with collagenase in DMEM/F12 medium for 2 h at 37 C to isolate synoviocytes. The synoviocytes were grown in DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified incubator at 37 C under 5% CO2. At confluence, the
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cells were trypsinized and passaged and were used for three to five passages.
Western blot analysis For each experiment, a total of 5 105 cells were seeded, and at subconfluence (70%), cells were made quiescent for 24 h in DMEM/F12 medium containing 0.5% FBS and then treated with various agents. In order to detect the expression of signal proteins, we rinsed the cells twice with ice-cold PBS and added 0.5 ml of the ice-cold lysis buffer [50 mM TrisHCl, pH 7.5, 150 mM NaCl, 100 mg/ml phenylmethylsulphonyl fluoride (PMSF), 0.1% SDS, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 10 mg/ml aprotinin, 2 mg/ml leupeptin and 10 mM EDTA]. The cells were incubated for 20 min on ice and then scraped and centrifuged. As for the protein extracts of STs, the tissues were rapidly frozen in liquid nitrogen and homogenized in ice-cold lysis buffer containing 20 mM hydroxyethyl piperazineethanesulphonic acid (HEPES)NaOH, 10 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol and Complete (Roche Molecular Biochemicals, 1 tablet/50 ml) and then were centrifuged and the supernatant was stored at 80 C until use. Protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were solubilized in Laemmli buffer (62.5 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 5% b-mercaptoethanol and 0.00625% bromophenol blue), boiled for 5 min and then separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies diluted 1:1000 for ezrin and 1:500 for p-ezrin in TBS-T containing 5% nonfat milk at 4 C overnight. The membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Each blot is a representative of at least three similar independent experiments. The film was scanned by a high-resolution scanner, densitometry was performed using AlphaEaseFc software on a Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA) and the expression of p-ezrin and ezrin was calculated as the ratio of the optical density of p-ezrin and ezrin to that of tubulin bands (relative intensity).
Treatment with ezrin siRNA Ezrin small interfering RNA (siRNA) (50 -ATGTTCCTGATTT CACTCC-30 ) or non-silencing control siRNA was obtained from Shanghai GenePharma (Shanghai, China). RA FLSs were cultured in 12-well plates. A transfection mixture of 100 nM siRNA and 10 mg/ml lipofectin in serum-free medium was added to medium-aspirated FLSs for 4 h. The FLSs were then incubated with complete DMEM/ F12 containing 10% FBS for 48 h before experiments. At the end of culture, the effect of the siRNA on expression of ezrin and p-ezrin protein was analysed using western blot.
IF staining The samples were fixed in 4% paraformaldehyde to evaluate the expression of p-ezrin in STs. First, the samples
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Ezrin, a member of the ezrin/radixin/moesin proteins, is linker between membrane and cytoskeleton. Phosphorylation of ezrin is a critical step in modulating the structure and function of plasma membranes [10, 11]. Phosphorylated ezrin (p-ezrin) plays an important role in regulating the various cellular functions, such as migration, adhesion, stress fibre formation and signal transduction [1214]. Previous studies have shown that cytoskeletal ezrin plays an important role in tumour growth and metastasis [15, 16]. We therefore hypothesized that ezrin contributes to the progression of RA by modulating abnormal behaviour of activated RA FLS, including enhanced migration and invasion. In the present study we show that p-ezrin was increased in synovial tissues (STs) and FLSs from RA patients and may contribute to abnormal migration and invasion of RA FLSs. These results indicate that an increase in ezrin phosphorylation may be associated with the aggressive behaviour of RA FLSs.
Role of p-ezrin in regulating migration and invasion of RA FLS
TABLE 1 Demographics of patients with RA
Patient no.
DAS28
Prednisone used (410 mg/day)
NSAIDs used
DMARDs used
48 51 61 53 60 56 41 58 52 62 43 61
5.2 5.7 4.6 6.3 6.4 6.3 4.2 4.9 6.9 7.3 5.4 6.7
+ + + + +
+ + + + + + + + + + +
MTX + HCQ HCQ MTX SSZ SSZ MTX + HCQ
RA1 RA2 RA3 RA4 RA5 RA6 RA7 RA8 RA9 RA10 RA11 RA12 +: used; : not used.
were stained with rabbit anti-p-ezrin antibody and FITCconjugated secondary antibody according to the manufacturer’s instructions. Then the stained samples were examined by confocal fluorescence microscopy (Zeiss LSM510; Carl Zeiss MicroImaging, Thornwood, NY, USA) and representative sections were photographed. Cells were grown on glass coverslips to detect the expression of p-ezrin and cytoskeletal filamentous actin (F-actin) in RA FLSs. First, the cells were fixed with acetone and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated with anti-p-ezrin antibody (diluted 1:200) for 1 h at room temperature and then incubated with FITC-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing in PBS, cells were incubated for 3 min with 0.25 mg/ml 40 -6-diamidono-2-phenylindole dihydrochloride (DAPI). For detecting cytoskeletal F-actin, the cells were incubated with AlexaFluor-546 phalloidin (molecular probes; Invitrogen, Eugene, OR, USA). The coverslips were mounted on glass slides with anti-fade mounting media and examined using confocal fluorescence microscopy (Zeiss LSM510).
In vitro migration and invasion assay of FLSs Chemotaxis assay of FLSs was performed by the Boyden chamber method using a filter of 6.5 mm diameter and 8.0 mm pore size (Transwell; Corning, Corning, NY, USA). Briefly, DMEM containing 10% FBS as a chemoattractant was placed in the lower wells. FLSs (at a final concentration of 6 104 cells/ml) were suspended in serum-free DMEM in the upper wells. The compound was applied to the upper chamber in a serum-free media containing 1% DMSO and the lower chamber was loaded with 10% FBS media containing 1% DMSO to evaluate the effect of NSC668394 on migration. The chamber was incubated at 37 C in 5% CO2 for 10 h. After the incubation, the nonmigrating cells were removed from the upper surface of the filter using a cotton swab. The filters were fixed in methanol for 15 min and stained with 0.1% crystal violet for 15 min. Chemotaxis was quantified by counting the
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stained cells that migrated to the lower side of the filter using an optical microscope. The stained cells were counted as the mean number of cells per 10 random fields for each assay. For the in vitro invasion assay, similar experiments were performed using inserts coated with a Matrigel basement membrane matrix (BD Biosciences).
Wounding migration RA FLSs, plated to confluence on 60 mm culture dishes, were wounded with pipette tips and then treated with or without 10% FBS. After 24 h of incubation, migration was quantified by counting the cells that had moved beyond a reference line.
MTT test for FLS viability RA FLSs were pretreated for 48 h with NSC668394 at different concentrations (1, 5 and 10 mM). The culture supernatants were removed and the adherent cells were incubated for 30 min at 37 C with a solution of the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) salt (1 mg/ml in PBS). The dark blue crystals of formazan were dissolved in acidified isopropanol and formazan quantification was performed at a test wavelength of 570 nm and a reference wavelength of 620 nm.
Statistical analysis Data are expressed as mean (S.E.M.). Student’s t-test or one-way analysis of variance (ANOVA) was used to analyse the differences between groups. A P-value 40.05 was considered statistically significant.
Results Increased expression of p-ezrin protein in STs and FLSs from RA patients The expression of p-ezrin protein in STs and FLSs was determined by western blot and immunohistochemical analysis. As shown in Fig. 1A, the expression of p-ezrin
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Youjun Xiao et al.
FIG. 1 Expression of p-ezrin protein in joint STs and FLSs from RA and OA patients
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(A and B) The expression of p-ezrin in STs from RA and OA patients. The expression of p-ezrin was detected by (A) western blot analysis and (B) IF staining. Representative pictures of immune blot (upper panel of A) and the densitometric quantification [mean (S.E.M.), lower panel of A] of the expression of p-ezrin and ezrin were from patients with RA (n = 12) and OA (n = 6). vs OA: *P < 0.05. For IF staining, p-ezrin (green) and nuclei (blue) were evaluated using confocal microscopy and the pictures shown are representative of 10 RA and 5 OA patients. Original magnification 400. (C) Expression of p-ezrin (green) detected by IF staining in FLSs isolated from the STs of patients with RA and OA. The pictures shown are representative of nine RA and five OA patients. Original magnification 400. (D) Confluent FLSs were treated with TNF-a (10 ng/ml) or IL-1b (10 ng/ml) for 6 h. Data are expressed as mean (S.E.M.) of densitometric quantification (right panel) from three independent experiments on three patients. vs basal: *P < 0.05. p-ezrin: phosphorylated ezrin; STs: synovial tissues; FLSs: fibroblast-like synoviocytes.
(threonine 567 phosphorylation) was higher in STs from RA patients than from OA patients. However, the expression of ezrin protein in STs was similar between RA and OA patients. By using IF staining analysis, we further demonstrated that expression of p-ezrin protein was prominent in STs from RA patients and mostly
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localized in the synovial lining and sublining cells, whereas the expression was much less prominent in STs from OA patients (Fig. 1B). We also detected the p-ezrin expression in isolated and cultured FLSs and found increased expression of p-ezrin protein in RA FLSs as compared with OA FLSs (Fig. 1C). These results
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Expression of p-ezrin
tubulin
Role of p-ezrin in regulating migration and invasion of RA FLS
indicate that the expression of p-ezrin increased in RA synovial membrane. Since proinflammatory cytokines, especially TNF-a and IL-1b, play an important role in transforming RA FLSs to a more invasive phenotype, we examined the effect of the cytokines on the expression and activation of ezrin in confluent RA FLSs. As shown in Fig. 1D, treatment with TNF-a (10 ng/ml) or IL-1b (10 ng/ml) increased the phosphorylation of ezrin, but not the expression of total ezrin protein.
Inhibitory effect of ezrin phosphorylation on regulating migration of RA FLSs
FIG. 2 Inhibitory effect of ezrin phosphorylation on regulating RA FLS migration in vitro
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(A and B) RA FLSs were (A) pretreated with 1% dimethyl sulphoxide (as control) or various concentrations of NSC668394 (NSC), a specific inhibitor of ezrin phosphorylation for 2 h or (B) transfected with specific ezrin siRNA (SiE, 100 nM) or control siRNA (SiC, 100 nM) and then stimulated with or without IL-1b (10 ng/ml) for 6 h. The expression of p-ezrin and ezrin protein was assessed by western blot analysis. (C) Migration was performed in a Boyden chamber and chemotaxis was quantified by counting the migrated cells. The RA FLSs were seeded in a Boyden chamber and allowed to migrate for an additional 10 h. Fetal bovine serum (FBS, 10%) was used as a chemoattractant. The results show the mean (S.E.M.) in samples from five different RA patients. Arrows indicate migrated FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs DMSO or SiC: *P < 0.05. (D) Effect of p-ezrin inhibition on the wounding migration of RA FLSs (original magnification 100). Cells migrating beyond the reference line were photographed and counted. Data shown are representative of experiments from three different RA patients. FLSs: fibroblast-like synoviocytes; DMSO: dimethyl sulphoxide; siRNA: small interfering RNA; p-ezrin: phosphorylated ezrin.
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Ezrin has been implicated in the modulation of migration of several other cell lines. To examine the role of ezrin phosphorylation in RA FLS migration, the chemotaxis migration
of FLSs was evaluated using a Transwell chamber assay. As shown in Fig. 2A, p-ezrin expression was significantly inhibited in the cells treated with NSC668394 (110 mM), a compound that inhibits ezrin phosphorylation at T567. Since maximal inhibitory effect was observed at 5 mM, the optimal concentration of NSC668394 was defined as 5 mM for further experiments. Treatment with NSC668394 decreased the chemotaxis migration of RA FLSs (Fig. 2C). The viability of RA FLSs was measured by the MTT test to evaluate the toxic effect of NSC668394 on RA FLSs. Up to 10 mM, this compound did not reduce cell viability (data not shown), which indicates that the inhibitory effect observed was not due to cytotoxic effects. We further found that treatment with ezrin siRNA efficiently suppressed the expression of p-ezrin protein and
Youjun Xiao et al.
Inhibitory effect of ezrin phosphorylation on regulating in vitro invasion of RA FLSs
Inhibitory effect of ezrin phosphorylation on regulating actin organization of RA FLSs As a linker between membrane and cytoskeleton, ezrin plays an important role in regulating organization of the actin cytoskeleton, which is thought to have a critical role in cell migration. To determine the role of ezrin in the organization of the actin cytoskeleton in RA FLSs, fluorescent phalloidin staining was used to visualize polymerized actin in cells. As shown in Fig. 4, treatment with NSC668394 (5 mM) inhibited formation of actin stress fibres, as well as the expression of p-ezrin protein. Transfection with specific ezrin siRNA also inhibited IL-1b-induced condensation of actin stress fibre formation and p-ezrin protein expression compared with that treated with control siRNA. Thus the function of ezrin in cell migration and actin dynamics is likely to contribute to its role in invasion of RA FLSs.
The signalling mechanisms of phosphorylation of ezrin in RA FLSs To investigate the signalling mechanisms leading to phosphorylation of ezrin in RA FLSs, we used known inhibitors of several signalling pathways, including a special inhibitor of p38 MAPK, SB203580, and an inhibitor of rhoassociated protein kinase (ROCK), Y27632. As shown in Fig. 5, IL-1b stimulation induced increased expression of p-ezrin, and pre-treatment with SB203580 (20 mM) or Y27632 (10 mM) inhibited the expression of p-ezrin. Furthermore, we found that treatment with SB203580 or Y27632 inhibited in vitro invasion of RA FLSs. These results indicate that p38 MAPK and ROCK pathways might be involved in IL-1b-induced ezrin phosphorylation and therefore result in modification of the invasiveness of RA FLSs.
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For the in vitro invasion assay, the experiments were performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. FBS (10%) was used as a chemoattractant. The cells were allowed to migrate for 10 h. RA FLSs were treated with 1% DMSO (as control) or NSC668394 (NSC, 5 mM) or transfected with specific ezrin siRNA (SiE, 100 nM) or control siRNA (SiC, 100 nM). The results show the mean (S.E.M.) in samples from seven patients with RA. Arrows indicate migrated of FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs DMSO or SiC: *P < 0.05. FLSs: fibroblast-like synoviocytes; DMSO: dimethyl sulphoxide; siRNA: small RNA; FBS: fetal bovine serum.
Discussion In the present study we showed that expression of p-ezrin protein is increased in STs and FLSs derived from RA patients. It was also found that special siRNA for ezrin and a specific inhibitor of ezrin phosphorylation suppressed migration and invasion of RA FLSs, which indicates that the increased phosphorylation of ezrin plays an important role in the maintenance of the activated phenotype of FLSs from patients with RA. The migration of FLSs to cartilage and bone has been considered a critical step in the erosion of joints in RA. While they arrive in the cartilage or bone, FLSs destroy cartilage or activate osteoclasts to enhance bone erosion and destruction [2022]. Identifying the critical factors that control migration and invasion of RA FLSs through cartilage and into bone is an important step towards understanding what directs the disease process. Ezrin was considered not only as a cross-linker between the actin cytoskeleton and plasma membrane, but also as an important signal transducer involved in a wide variety of cellular functions. As a linker between membrane and cytoskeletal binding sites, ezrin needs to be conformationally activated/phosphorylated to fulfil its biological functions. Phosphorylation of ezrin is critical for regulating cell shape, motility, survival and signal transduction in several cell lines. For instance, it has been implicated in the regulation of tumour invasion and
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The in vitro invasion potential of RA FLSs is well associated with the rate of joint destruction in RA patients [19]. To investigate the role of ezrin in the invasive behaviour of RA FLSs in vitro, a thin layer of reconstituted extracellular matrix (Matrigel) was used to measure in vitro invasion of RA FLSs. As shown in Fig. 3, NSC668394 (5 mM) treatment suppressed Matrigel invasion of RA FLSs. Treatment with ezrin siRNA also inhibited invasion as compared with treatment with control siRNA in RA FLSs. These results indicate that ezrin plays an important role in regulating the invasive behaviour of RA FLSs.
FIG. 3 Inhibitory effect of ezrin phosphorylation on regulation of RA FLS invasion in vitro
Number of Martrige linv ading cells
resulted in a reduction in the migration of RA FLSs as compared with the non-silencing control vector transfection (Fig. 2B and C). Furthermore, we used a monolayer wound healing assay to detect the role of p-ezrin in cell migration. Wound closing was significantly slowed in RA FLSs treated with NSC668394 and specific ezrin siRNA, respectively (Fig. 2D).
Role of p-ezrin in regulating migration and invasion of RA FLS
FIG. 4 Inhibitory effect of ezrin phosphorylation on regulation of actin organization of RA FLSs
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RA FLSs were transfected with siRNA targeting ezrin (SiE) or control siRNA (SiC) or treated with 1% DMSO (as control) or NSC668394 (NSC, 5 mM). The cells were grown on glass coverslips and then were stimulated with IL-1b (10 ng/ml) for 6 h and p-ezrin (green), F-actin (red) and nuclei (blue) were evaluated by IF staining. F-actin and nuclei were stained with phalloidin and 40 -6-diamidono-2-phenylindole dihydrochloride (DAPI), respectively. Representative laser confocal microscopy images show F-actin and p-ezrin staining from five different patients. Original magnification 400. FLSs: fibroblast-like synoviocytes; siRNA: small interfering RNA; DMSO: dimethyl sulphoxide; FBS: fetal bovine serum; F-actin: filamentous actin; p-ezrin: phosphorylated ezrin.
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FIG. 5 The signals involved in IL-1b-induced phosphorylation of ezrin in RA FLSs
metastasis, such as in breast carcinoma cells [23], intraductal papillary mucinous neoplasms and pancreatic intraepithelial neoplasia [24] and gastrointestinal stromal tumours [25]. Dysregulation of ezrin phosphorylation at amino acid T567 inhibits metastasis and alters cellular metabolism in osteosarcoma [26]. A previous report shows that during Salmonella typhimurium infection,
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Rheumatology key messages Ezrin phosphorylation is increased in RA fibroblastlike synoviocytes (FLSs). . Elevated ezrin phosphorylation contributes to migration and invasion of RA FLSs. . Inhibition of ezrin phosphorylation may be a new therapeutic approach for RA. .
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(A) RA FLSs were pretreated for 1 h with or without 20 mM SB203580 (SB), a specific p38 inhibitor, or 10 mM Y27632 (Y), a ROCK inhibitor, and then stimulated with buffer or IL-1b (10 ng/ml) for 6 h. Data represent the mean (S.E.M.) of densitometric quantification (lower panel) from at least three independent experiments. vs basal: *P < 0.05; vs IL1b: #P < 0.05. (B) Inhibitory effect of p38 MAPK and ROCK pathway on in vitro invasion of RA FLSs. The experiments for in vitro invasion were performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. Ten per cent FBS was used as a chemoattractant. RA FLSs were treated with SB203580 (SB) or 10 mM Y27632 (Y). The results show the mean (S.E.M.) in samples from five patients with RA. Arrows indicate migrated FLSs, which were stained violet using a Diff-Quick kit (upper panel, original magnification 200). vs control: *P < 0.05. FLSs: fibroblast-like synoviocytes; ROCK: rho-associated protein kinase; MAPK: mitogenactivated protein kinase; FBS: fetal bovine serum.
ezrin activation also regulates neutrophil transmigration by modulating the apical localization of MRP2 in response to the SipA effector protein [27]. Phosphorylation of ezrin also participates in B cell antigen receptor signal transduction [28]. All these studies indicate that phosphorylation of ezrin plays an important role in many cellular biological functions. In this study we demonstrated for the first time that increased expression of p-ezrin protein in STs and FLSs from RA patients participates in the regulation of migration and invasion by RA FLSs. Since abnormal invasiveness of FLSs is considered a critical factor in joint destruction, our results also indicate that increased phosphorylation of ezrin in FLSs may be associated with the pathological progression of RA. Indeed, it has been reported that the mammalian target of rapamycin (mTOR) signal, a pathway that is associated with ezrin, is involved in invasion of RA FLSs [29]. Stress fibre formation is a critical step that controls cell motility [32]. Ezrin regulates stress fibre formation in other cell lines [30]. It has also been implied that modulation of cytoskeletal rearrangements may play a role in the aggravation of synovial pathophysiology in FLSs from a mice model with RA [31, 32]. Herein we showed that inhibition of ezrin with siRNA or a specific inhibitor of ezrin phosphorylation strongly prevents stress fibre formation, which indicates that p-ezrin controls RA FLS migration, at least in part, by modulating actin polymerization. These studies indicate that some actin-binding proteins such as ezrin may play an important role in the progression of RA through regulating stress fibre formation. Previous reports have shown that some signalling pathways, such as rho and p38 MAPK, are required for regulating phosphorylation of ezrin in several cell lines. For example, it has been demonstrated that the p38 MAPK signal mediates phosphorylation of ezrin in the activation of Na+H+ antiporter isoform 3 following Na+-glucose cotransport [33]. Another study also shows the modulation of the ROCK signal on phosphorylation of ezrin in epithelial cells [34] and nasopharyngeal carcinoma 6-10B cells [35]. Consistent with previous studies, we demonstrated that activation of rho kinases and p38 is required for phosphorylation of ezrin and migration and invasion of RA FLSs, as evaluated by the inhibitory effect of p38 inhibitor SB203580 and ROCK inhibitor Y27632, which suggests that ezrin may be the downstream target of p38 and ROCK for migration and invasion in RA FLSs. In summary, the present study reveals the role of phosphorylation of ezrin in modulating migration and invasion of RA FLSs, providing a novel therapeutic target that could be exploited to control pathological synovial invasion in RA.
Role of p-ezrin in regulating migration and invasion of RA FLS
Acknowledgements We would like to thank Jinjin Fan for her technical assistance.
Disclosure statement: The authors have declared no conflicts of interest.
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Funding: This work is supported by grants from the National Natural Science Foundation of China (81072484 and 81373182), China Ministry of Education Foundation (20100171110058), Guangdong Natural Science Foundation (S2011020002358 and S2013010015363), Guangdong Project of Science and Technology (2011B080701011 and 2011B080701098) and Guangdong Provincial Department of Education Foundation (2012KJCX0003).
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