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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Objective

Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor–inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA.

Methods

Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy.

Results

Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-κB signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17–stimulated FLS proliferation.

Conclusion

Our findings indicate that Cyr61 plays a critical role in IL-17–mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and variable degrees of bone and cartilage erosion. Recent evidence suggests that fibroblast-like synoviocytes (FLS) are potent players in all aspects of the pathogenesis of RA. FLS in the rheumatoid synovium are aggressive and proliferative and are known to attack cartilage, characteristics that are similar to those of transformed cells (1, 2). Cyr61 is a heparin-binding protein encoded by an immediate early gene (IEG) and is part of the CCN family (including CCN1–CCN6) (3). The expression of Cyr61 is rapidly induced by growth factors in various types of cells. Associated with the extracellular matrix (ECM), Cyr61 mediates cell adhesion, migration, proliferation, and neovascularization in a cell type–specific and function-specific manner (4–7). Using complementary DNA (cDNA) microarray analysis, it was recently found that Cyr61 was overexpressed in B cells infiltrating RA synovial tissue (ST) (8). However, the expression of Cyr61 in RA synovial fluid (SF) and its role in FLS proliferation have not been investigated.

Interleukin-17 (IL-17) is an important proinflammatory cytokine involved in RA pathogenesis. IL-17–knockout mice develop significantly less severe arthritis than do wild-type mice, and treatment with neutralizing IL-17 antibodies or the soluble IL-17 receptor alleviates joint inflammation (9–11). In vitro, IL-17 can up-regulate and/or synergize with local mediators of inflammation, such as tumor necrosis factor α (TNFα) and IL-1β (12, 13), and can stimulate FLS to generate matrix metalloproteinases (MMPs), which then aggravate the ECM injury (14). Nevertheless, whether IL-17 is involved in the proliferation of FLS and in the expression of Cyr61 remains unknown.

In the present study, we explored the possible role of Cyr61 in FLS proliferation and its functional relationship to IL-17. We found that Cyr61 was overexpressed in ST and FLS derived from RA patients. Furthermore, we observed that RA SF contained high levels of Cyr61, which could effectively stimulate FLS proliferation, and that the effect was abrogated by neutralizing antibody against human Cyr61. Additionally, expression of Cyr61 in RA FLS was up-regulated by IL-17 via the NF-κB and p38 MAPK signaling pathways. Importantly, knockdown of the expression of Cyr61 by RNA interference (RNAi) techniques significantly inhibited IL-17–stimulated FLS proliferation. To our knowledge, our study is the first to reveal that Cyr61 plays a critical role in IL-17–dependent proliferation of FLS derived from patients with RA. Our findings suggest that this molecule likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in RA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Patients and specimens.

A total of 79 patients (7 men and 72 women) with a diagnosis of RA according to the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) 1987 revised criteria (15) were included in the study. The mean ± SD age of the patients was 55 ± 10 years (range 35–75 years), and the mean ± SD disease duration was 14 ± 10 years. A total of 35 patients with a diagnosis of osteoarthritis (OA) according to the ACR criteria (16) and 2 trauma patients with no history of acute or chronic arthritis served as controls. ST samples were obtained from patients during total knee replacement surgery or arthroscopy. Peripheral blood mononuclear cells (PBMCs), synovial tissue mononuclear cells (STMCs), and synovial fluid mononuclear cells (SFMCs) from RA patients were isolated and purified by Ficoll-Hypaque separation (Amersham Biosciences, Piscataway, NJ). PBMCs from healthy individuals were used as controls. SF specimens were centrifuged at 500g for 10 minutes, and supernatants were collected and immediately stored at −80°C until used. All study protocols and consent forms were approved by the Institutional Medical Ethics Review Board of the Shanghai Jiao Tong University School of Medicine.

RNA extraction and real-time quantitative polymerase chain reaction (PCR).

Real-time PCR was performed as previously described (17). Briefly, total RNA was extracted from specimens using an RNeasy Mini kit (Qiagen, Chatsworth, CA). Messenger RNA (mRNA) was converted to cDNA using a Sensiscript RT kit (Qiagen). Two-step real-time PCR was performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Primers were developed using Primer Express 2.0 software (Applied Biosystems) (see Supplementary Table 1, available on the Arthritis & Rheumatism Website at http://www3.interscience.wiley.com/journal/76509746/home). Thermocycler conditions were as follows: initial holding at 50°C for 2 minutes, then 95°C for 10 minutes, followed by a 2-step PCR process that consisted of 95°C for 15 seconds and 60°C for 60 seconds for 40 cycles. All primers were validated according to the protocol. Data were collected, and quantitative analysis was performed using an ABI Prism 7900 sequence detection system (Applied Biosystems). The GAPDH gene was used as the endogenous control. Gene expression was then calculated as the difference in cycle threshold (ΔCt) between the target gene and GAPDH; ΔΔCt was the difference between the ΔCt values of the test sample and that of the control. Relative expression of target genes was calculated as 2math image.

Culture and identification of FLS.

ST specimens were minced into small pieces and incubated for 2 hours with 1 mg/ml type I collagenase (Sigma-Aldrich, Bornem, Belgium) in Dulbecco's modified Eagle's medium (DMEM) at 37°C. Cells were collected by filtering the suspension through nylon mesh (70 μm). Cells were extensively washed and cultured in complete high-glucose DMEM (Hyclone, Logan, UT) supplemented with 10% fetal calf serum (FCS; Gibco, Grand Island, NY), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2 incubator as previously described (18). FLS at passages 4–6 were used in our study and were overwhelmingly negative (>99%) for CD14, CD11b, CD3, and CD19 surface markers, as identified using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA).

Immunohistochemical analysis of Cyr61 expression in ST.

ST specimens were fixed in 4% paraformaldehyde. Using immunocytochemistry, samples were stained with rabbit anti-Cyr61 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions and as previously described (8). Rabbit and goat IgG were used as negative controls. The stained samples were examined by microscopy, and representative sections were photographed.

Expression of Cyr61 regulated by cytokines and SF.

FLS were seeded into 12-well plates (density 1 × 105/well). Confluent cells were starved by progressively decreasing serum from 10% to 1% in medium for 12 hours. The starved FLS were stimulated for 2 hours with either dilutions of SF (1:32, 1:16, 1:8, 1:4, 1:2) or inflammatory cytokines (50 ng/ml IL-17 alone or in combination with 10 ng/ml of TNFα, 10 ng/ml of interferon-γ [IFNγ], or 10 ng/ml of IL-12) (PeproTech, Rocky Hill, NJ). Expression of Cyr61 was detected by real-time PCR and Western blotting.

Preparation of anti-human Cyr61 monoclonal antibody (mAb).

The cDNA fragment encoding human Cyr61 residues 26–381 (GenBank accession no. NM_001554) was amplified by PCR using an appropriate pair of primers (5′-TTGGATCCTGCCCCGCTGCCTGCCACT-3′ and 5′-TTAAGCTTCTAGTCCCTAAATTTGTGAATGTCATT-3′) and cloned into the pET-28(a) vector (Novagen, Madison, WI) via the Bam HI and Hind III sites. BL21 (DE3) cells from Escherichia coli were transformed by the resulting plasmid. The Cyr61 fusion protein used to immunize BALB/c mice was isolated from bacterial lysate and purified using a nickel–nitrilotriacetic acid column (Qiagen) according to the manufacturer's instructions, and mAb were prepared and identified using previously described methods (19). The specificity of the prepared anti-human Cyr61 mAb was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunocytochemistry. Commercial recombinant human Cyr61 (rHuCyr61; PeproTech) and anti-Cyr61 polyclonal antibody (Santa Cruz Biotechnology) were used as a standard and a positive control, respectively. Isotypes of prepared mAb were determined using a Mouse Monoclonal Antibody Isotyping kit (Roche, Indianapolis, IN).

Measurement of Cyr61 in SF and serum by ELISA.

Concentrations of Cyr61 were measured quantitatively using a sandwich ELISA. Briefly, microtiter plates were coated with rabbit anti-human Cyr61 (Santa Cruz Biotechnology) and stored overnight at 4°C. SF or serum along with the standard (rHuCyr61; PeproTech) were diluted with phosphate buffered saline (PBS) and added to duplicate wells. Plates were incubated at 37°C for 2 hours and subsequently washed with PBS–Tween 20. An IgG1 mouse anti-human Cyr61 mAb (produced in our laboratory) was added and incubated at 37°C for 2 additional hours. After washing, horseradish peroxidase–conjugated goat anti-mouse IgG antibodies and, later, tetramethylbenzidine were used for color development. Concentrations of Cyr61 were evaluated according to absorbance at 450 nm using Microplate software (Bio-Rad, Hercules, CA).

FLS proliferation and antibody neutralization assays.

RA FLS were cultured for 24 hours at a density of 5 × 103/well in 96-well plates in complete DMEM in the presence of diluted RA SF (1:2, 1:4, 1:8, 1:16, 1:32) or serial concentrations of IL-17 (25–100 ng/ml) or Cyr61 (5–20 μg/ml). FLS were pulsed with 1 μCi thymidine (Amersham Biosciences) for an additional 16 hours, and incorporated radioactivity was measured as counts per minute using a beta-plate counter. For the antibody blocking assay, mouse anti-Cyr61 mAb (1 mg/ml, diluted 1:25–1:200) was preincubated for 1 hour with RA SF (diluted 1:4) alone or in combination with IL-17 (50 ng/ml) before stimulation of FLS proliferation. An isotype-matched antibody was used as a control.

RNAi knockdown of gene expression.

Cyr61 small interfering RNA (siRNA) was designed and synthesized at Shanghai GenePharma (Shanghai, China). FLS from RA patients were cultured in 12-well plates. A transfection mixture of siRNA oligonucleotides and GenePorter reagent (Gene Therapy Systems, San Diego, CA) in serum-free medium was added to medium-aspirated FLS for 4 hours. Then, the medium was replaced with complete DMEM containing 10% FCS for an additional 24 hours and incubation was continued. At the end of culture, expression of Cyr61 in FLS was analyzed using real-time PCR and Western blotting; expression levels of Bcl-2 family members were detected using real-time PCR. Intracellular staining of the Bcl-2 protein was examined by fluorescence-activated cell sorting. Cell proliferation in FLS in which expression of Cyr61 had been knocked down was measured as described above. Small interfering RNA for human p38 (catalog no. 29433) and control (catalog no. 37007) were purchased from Santa Cruz Biotechnology. FLS were transfected with siRNA according to the manufacturer's instructions. Expression levels of Cyr61 and total p38 were detected using Western blotting as previously described (20, 21).

FLS division and apoptosis assays.

FLS were labeled with carboxyfluorescein succinimidyl ester (CFSE), and cell division was detected using flow cytometry after knockdown of expression of the Cyr61 gene. For apoptosis assay, apoptotic FLS were induced by serum deprivation in the presence or absence of rHuCyr61 (5 μg/ml) for 24 hours and were detected using an annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. To further confirm the role of Cyr61 in protecting FLS from apoptosis, the apoptosis of FLS in which expression of Cyr61 had been knocked down was also determined using the apoptosis detection kit.

Probing of signaling pathways involved in Cyr61 induction.

Special inhibitors of the NF-κB, MAPK, and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways were purchased from Sigma-Aldrich and used to analyze IL-17–dependent expression of Cyr61. Briefly, 4 μM pyrrolidine dithiocarbamate (PDTC; an inhibitor of NF-κB activation), 20 μM LY294002 (an inhibitor of PI 3-kinase activation), 10 μM SB203580 (an inhibitor of p38 MAPK), or 1 μM PD98059 (an inhibitor of ERK-1/2) was added into cell culture in the presence of 50 ng/ml of IL-17 for 2 hours, then expression of Cyr61 was determined using real-time PCR. The activations of p38 MAPK and ERK were analyzed using Western blotting, with specific antibodies to ERK-1/2, phosphorylated ERK-1/2, p38 MAPK, and phosphorylated p38 MAPK (Cell Signaling Technology, Beverly, MA) serving as primary antibodies.

Confocal laser scanning fluorescence microscopy for NF-κB nuclear translocation.

FLS grown on glass coverslips were stimulated with 50 ng/ml IL-17 for 0, 15, and 30 minutes and fixed with acetone. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS, stained overnight with anti–NF-κB p65 antibody (Thermo Scientific, Waltham, MA), and incubated for 1 additional hour with an FITC-labeled secondary antibody (Santa Cruz Biotechnology). After washing, cells were incubated for 3 minutes with 0.25 mg/ml of 4′,6-diamidino-2-phenylindole (Roche) and examined using an LSM 510 confocal fluorescence microscope (Zeiss, Jena, Germany).

Statistical analysis.

Except where indicated otherwise, data are expressed as the mean ± SEM. Student's t-test was used to analyze the differences between groups. Statistically significant changes were first determined by one-way analysis of variance and then by Student's paired or unpaired 2-tailed t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Overexpression of Cyr61 in ST and FLS from RA patients.

In a preliminary microarray analysis, we observed that Cyr61 was overexpressed in RA ST (data not shown). To confirm this result, expression of Cyr61 in ST obtained from RA patients (n = 10), OA patients (n = 10), and trauma patients with no history of arthritis (n = 2) was measured using real-time PCR. Expression of Cyr61 mRNA was higher in RA ST than in OA ST or in normal samples (Figure 1A). Consistent with these findings, immunohistochemical analysis revealed that expression of the Cyr61 protein (dark brown staining) was prominent in RA ST and mostly localized in the synovial lining and sublining cells, whereas expression was much less prominent in normal samples and in OA ST (Figure 1A). These findings revealed that Cyr61 was overexpressed in RA ST.

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Figure 1. Expression of Cyr61 in synovial tissue (ST) and in fibroblast-like synoviocytes (FLS). A, Expression of Cyr61 in ST from patients with rheumatoid arthritis (RA; n = 10), patients with osteoarthritis (OA; n = 10), and normal individuals (Nor; n = 2). Cyr61 expression was determined by real-time polymerase chain reaction (PCR) analysis (left) and immunohistochemical analysis (right) (original magnification × 40). B, Expression of Cyr61 mRNA in peripheral blood mononuclear cells (PBMCs) from normal individuals (n = 10) and in PBMCs, synovial fluid MCs (SFMCs), STMCs, and ST from patients with RA (n = 10). C, Expression of Cyr61 in FLS isolated from the ST of normal individuals and patients with OA and RA. Cyr61 mRNA expression was determined by real-time PCR and calculated as 2math image, with the GAPDH gene used as an endogenous control (left), and expression of the Cyr61 protein was evaluated by Western blot analysis of FLS (passage 4), with the GAPDH protein used as a loading control (right). Values are the mean and SEM of at least 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001.

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Since ST consists of different types of cells, we also examined levels of Cyr61 mRNA in isolated STMCs, SFMCs, and PBMCs, as well as in total RA ST (n = 10). Expression of Cyr61 was higher in RA ST than in STMCs, SFMCs, and PBMCs from RA patients (Figure 1B). These results suggest that FLS likely contributed to the prominent expression of Cyr61 in RA ST. To further test this, we examined the expression of Cyr61 in isolated and cultured FLS from RA and OA patients and from normal individuals. Expression levels of both Cyr61 mRNA and the Cyr61 protein were significantly higher in RA FLS than in OA FLS or in FLS from normal patients (Figure 1C).

FLS proliferation stimulated by Cyr61 in RA SF.

Because Cyr61 is a secreted protein, we examined whether Cyr61 was present in RA SF. Levels of Cyr61 were higher in RA SF than in OA SF or in RA serum (P < 0.0001) (Figure 2A). Cyr61 has previously been reported to be involved in skin fibroblast proliferation (5). Thus, we explored whether Cyr61 in RA SF could regulate FLS proliferation. In order to measure FLS proliferation, we incubated FLS with diluted RA SF. As shown in Figure 2B, FLS proliferation was markedly increased in a dose-dependent manner in samples with diluted RA SF as compared with proliferation in samples with the control medium. Using a specific anti-human Cyr61 mAb that we have developed (see Materials and Methods), we found that preincubation of SF with the anti-Cyr61 antibody inhibited SF-mediated FLS proliferation (Figure 2B). To further confirm the stimulatory effect of Cyr61 on FLS proliferation, rHuCyr61 was used to stimulate FLS proliferation. Cyr61 indeed enhanced FLS proliferation in a dose-dependent manner (Figure 2C).

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Figure 2. Enhancement of FLS proliferation by Cyr61. A, Concentrations of Cyr61 in RA serum (n = 30), OA SF (n = 25), and RA SF (n = 30). Each symbol represents an individual patient. Bars show the mean. B, Decrease in SF-stimulated FLS proliferation by treatment with an anti-Cyr61 neutralizing antibody. FLS proliferation was stimulated with dilutions of RA SF (1:32, 1:16, 1:8, 1:4, 1:2) (left), but SF-stimulated FLS proliferation was inhibited in a dose-dependent manner by the addition of an anti-human Cyr61 monoclonal antibody (anti-Cyr61 mAb) (right). Murine isotype-matched antibody (con-IgG) and medium (M) were used as controls. C, Increase in FLS proliferation by treatment with exogenous recombinant human Cyr61. D, Inhibition of FLS proliferation by knockdown of Cyr61 expression. Expression of Cyr61 mRNA (top left) and the Cyr61 protein (bottom left) was inhibited in FLS treated with specific Cyr61 small interfering RNA (siRNA [siCyr61]). FLS treated with scrambled siRNA (siNC) were used as controls. Knockdown of Cyr61 expression decreased SF-stimulated proliferation, as measured using 3H-thymidine incorporation (right). Values in BD are the mean ± SEM of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001. See Figure 1 for other definitions.

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Since Cyr61 is overexpressed in RA FLS (Figure 1), we tested whether this expression is required for optimal FLS proliferation. Our initial screening identified specific siRNA that could reduce expression levels of both Cyr61 mRNA and the Cyr61 protein by >60% (Figure 2D). Interestingly, knockdown of Cyr61 expression inhibited even SF-stimulated FLS proliferation (P < 0.001) (Figure 2D), suggesting that optimal FLS proliferation requires Cyr61 that is produced autonomously from FLS. Previous studies have identified several integrins as the cell surface receptors for Cyr61 in different cell types (4, 5, 22, 23). We therefore measured mRNA profiles of the α and β integrin families in RA FLS and found high levels of αv, α6, β1, and β5 integrins. However, only the anti-αvβ5 antibody blocked Cyr61-mediated stimulation of FLS proliferation (data not shown). Collectively, these data demonstrate that Cyr61 plays a pivotal role in FLS proliferation.

Increased division and decreased apoptosis of FLS by Cyr61.

FLS proliferation up-regulated by Cyr61 could be due to enhanced cell division and/or reduced cell apoptosis. The CFSE assay showed that cell division was reduced in FLS in which expression of Cyr61 had been knocked down, suggesting that Cyr61 plays a positive role in FLS division (Figure 3A). We also investigated whether Cyr61 could protect FLS from apoptosis. Exogenous Cyr61 was added to cultures of starved FLS, and FLS apoptosis was assayed using annexin V–FITC and propidium iodide staining. Apoptosis of FLS induced by serum deprivation was significantly decreased in the presence of exogenous Cyr61 (Figure 3B). To further confirm that Cyr61 enhances FLS survival, expression of the Cyr61 gene was knocked down in FLS, and in these FLS, apoptosis was significantly increased (Figure 3C). Because Bcl-2 family molecules are important regulators of cell survival and cell death, we used real-time PCR to examine the expression profiles of Bcl-2, Bcl-xL, Bax, Bim, and Bad in FLS in which the expression of Cyr61 had been knocked down. Expression levels of Bcl-2 mRNA were decreased significantly, while the expression levels of the others did not change (Figure 3D). Using intracellular staining, we confirmed that Bcl-2 expression levels were also significantly decreased in FLS in which the expression of Cyr61 had been knocked down (Figure 3D). These results suggest that FLS survival is positively regulated by Cyr61, likely through sustaining the expression of Bcl-2.

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Figure 3. Effects of Cyr61 on FLS division and apoptosis. A, Assay of FLS division. FLS were treated with either scrambled small interfering RNA (siRNA [siNC]) or specific Cyr61 siRNA (siCyr61). Different color peaks represent FLS from different passages labeled with carboxyfluorescein succinimidyl ester (CFSE). B, FLS apoptosis analyzed by flow cytometry. Apoptotic FLS stained with annexin V–fluorescein isothiocyanate were induced by serum deprivation in the presence of exogenous Cyr61 or phosphate buffered saline (PBS) (control) for 24 hours. Values in each compartment are the percentage of positive cells. C, Comparison of the percentage of apoptotic cells in FLS in which Cyr61 expression had been knocked down and in FLS controls. Values are the mean and SEM. D, Expression levels of Bcl-2 family molecules (Bcl-2, Bcl-xL, Bax, Bim, and Bad) in FLS in which Cyr61 expression had been knocked down and in controls. Results were analyzed using real-time PCR (n = 10) (left) and intracellular staining (right). Values are the mean and SEM. ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001. FL1-H = fluorescence channel 1; PI = propidium iodide (see Figure 1 for other definitions).

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Up-regulation of Cyr61 expression in FLS by IL-17.

Because the expression of Cyr61 is elevated in RA SF (Figure 2A) and because SF is known to contain proinflammatory cytokines (17, 24), we tested whether the expression of Cyr61 is stimulated by these cytokines. Using ELISA, we determined that there were high levels of TNFα, IFNγ, IL-12, and IL-17 in RA SF (data not shown). In a dose-response study, we also observed that SF from RA patients significantly induced expression of Cyr61 (Figure 4A). We then investigated which proinflammatory cytokines enhanced the expression of Cyr61 and found that only IL-17 consistently and significantly stimulated the expression of Cyr61. Moreover, we observed that IL-17 had no synergistic effect when combined with other proinflammatory cytokines (Figure 4B). To examine whether IL-17 indeed plays a critical role in RA SF–stimulated expression of Cyr61, RA SF was preincubated with the anti–IL-17 mAb. The anti–IL-17 antibody inhibited SF-stimulated expression of Cyr61 in a dose-dependent manner (Figure 4B).

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Figure 4. Expression of Cyr61 and its role in interleukin-17 (IL-17)–dependent FLS proliferation. A, Expression of Cyr61 in RA FLS, as determined by real-time PCR and Western blotting after cells were incubated with either diluted RA SF (1:32, 1:16, 1:8, 1:4, 1:2) or control medium (M). B, Effects of cytokines on expression of Cyr61 in RA FLS. Expression of Cyr61 after stimulation with IL-17 and with tumor necrosis factor α (TNFα), interferon-γ (IFNγ), and IL-12 individually and in combination with IL-17 was determined using real-time PCR. SF and medium were used as controls (left). Inhibition of SF-stimulated expression of Cyr61 by anti-human IL-17 monoclonal antibody (mAb) was assessed by real-time PCR and Western blotting (right). Murine control IgG1 (con-IgG) and medium were used as controls. C, IL-17–stimulated FLS proliferation as measured by 3H-thymidine incorporation. FLS proliferation induced by IL-17 was neutralized by anti–IL-17 mAb. D, Role of Cyr61 in IL-17–dependent FLS proliferation. IL-17–induced FLS proliferation was decreased in FLS in which Cyr61 expression had been knocked down (siCyr61) as compared with proliferation in controls (siNC) (left). IL-17–induced FLS proliferation was decreased by addition of serial dilutions of the anti-human Cyr61 mAb (right). Values are the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. See Figure 1 for other definitions.

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Critical role of Cyr61 in IL-17–mediated FLS proliferation.

Since IL-17 could stimulate FLS to produce Cyr61, we explored whether enhanced expression of Cyr61 might be involved in IL-17–mediated FLS proliferation. As expected, we observed that FLS proliferation was significantly increased by stimulation with IL-17 (5–100 ng/ml) and that the proliferation reached a maximum level with 50 ng/ml IL-17 (data not shown). Consistent with these findings, the stimulatory effect of IL-17 was neutralized by the anti–IL-17 antibody in a dose-dependent manner (Figure 4C). Interestingly, IL-17–stimulated cell proliferation was dramatically reduced in FLS in which the expression of Cyr61 had been knocked down (Figure 4D). Moreover, adding an anti-Cyr61 neutralizing mAb to the FLS culture also inhibited IL-17–mediated stimulation of FLS proliferation in a dose-dependent manner (Figure 4D). These data support our hypothesis that IL-17 in RA SF positively regulates the expression of Cyr61 in FLS, which in turn, plays a critical role in mediating the stimulatory effect of this proinflammatory cytokine on FLS proliferation.

Induction of Cyr61 expression by IL-17 through the p38 MAPK and NF-κB pathways.

To investigate which signaling pathways are responsible for IL-17–induced expression of Cyr61 in FLS, we used known inhibitors of several pathways, including PDTC (an inhibitor of NF-κB activation), LY294002 (an inhibitor of PI 3-kinase activation), SB203580 (an inhibitor of p38 MAPK), and PD98059 (an inhibitor of ERK-1/2), and we documented that these chemical inhibitors exhibited no cytotoxicity at the concentrations used in our experiments (data not show). IL-17–stimulated expression of Cyr61 mRNA in FLS was markedly decreased in the presence of the NF-κB and p38 MAPK inhibitors. In contrast, the PI 3-kinase and ERK-1/2 inhibitors had little effect (Figure 5A). These results suggest that IL-17 might regulate the expression of Cyr61 in RA FLS via the NF-κB and p38 MAPK pathways. IL-17–mediated expression of Cyr61 via the p38 MAPK pathway was further confirmed by the results of p38 siRNA transfection analysis (Figure 5B). Additionally, IL-17 activated the phosphorylation of p38 MAPK in FLS, while the phosphorylation of ERK was not affected (Figure 5C). Furthermore, confocal laser scanning immunofluorescence microscopy revealed that IL-17 could induce NF-κB translocation into the nucleus of FLS (Figure 5D). All of these results indicated that the NF-κB and p38 MAPK signaling pathways might be involved in IL-17–stimulated expression of Cyr61.

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Figure 5. Signaling pathways involved in interleukin-17 (IL-17)–regulated expression of Cyr61 in RA FLS. A, Effect of inhibitors of signaling pathways on IL-17–induced expression of Cyr61. RA FLS were treated for 2 hours with IL-17 or with pyrrolidine dithiocarbamate (PDTC), SB203580, PD98028, or LY294002 in combination with IL-17 (50 ng/ml). Expression of Cyr61 in cell cultures and in control medium (M) was evaluated by real-time PCR. Values are the mean and SEM of 4 individual experiments. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus FLS treated with IL-17 alone. B, Expression of Cyr61 in FLS in which the p38 MAPK signaling pathway has been knocked down. FLS were treated with either small interfering p38 (sip38; to knock down the p38 MAPK signaling pathway) or siNC (control), and results were analyzed using real-time PCR (left) or Western blotting (right). Values are the mean and SEM. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus FLS treated with control. C, Phosphorylation of p38 MAPK and ERK detected using Western blotting. FLS were left unstimulated or were stimulated with recombinant human IL-17 (50 ng/ml) for 10 minutes or 20 minutes. D, Nuclear translocation of NF-κB observed using confocal fluorescence microscopy. NF-κB was detected using fluorescein isothiocyanate anti-p65 staining (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The merged images show NF-κB translocation into the nucleus (arrow). Results represent 1 of 3 independent experiments. See Figure 1 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Cyr61 was first identified as a cysteine-rich protein of ∼40 kd particularly encoded by a growth factor–inducible IEG with functions in regulating the proliferation of cells, particularly endothelial cells and human skin fibroblasts (5, 6, 25). In a previous study, mutations in the Wnt-induced signaling protein 3, another CCN family member, were found to cause progressive pseudorheumatoid dysplasia (26). In a more recent study, cDNA microarray analysis revealed that Cyr61 was overexpressed in ST obtained from 11 pairs of monozygotic twins discordant for RA, and immunostaining revealed that significant overexpression of Cyr61 was located in RA synovial macrophages, lining cells, and infiltrated B cells (8). Nevertheless, no investigation of the expression of Cyr61 in FLS and its potential function in FLS proliferation has previously been reported.

We initially observed that Cyr61 was overexpressed in RA ST, based on a preliminary microarray analysis of ST from RA patients and normal individuals. Further analyses using real-time PCR and immunohistochemistry confirmed the overexpression of Cyr61 in RA FLS (Figure 1). Since Cyr61 has been shown to promote the proliferation of endothelial cells and human skin fibroblasts (5, 6, 25), we hypothesized that overexpression of Cyr61 might also regulate FLS proliferation. Using rHuCyr61 to stimulate FLS and RNAi to knockdown the expression of Cyr61 in FLS, we observed that the addition of exogenous Cyr61 enhanced FLS proliferation, whereas knockdown of the expression of Cyr61 markedly inhibited proliferation. Consistent with the fact that Cyr61 is a known secreted protein (3), we found that RA SF contained higher levels of Cyr61 than did OA SF. While diluted RA SF promoted FLS proliferation in a dose-dependent manner, an anti-Cyr61 neutralizing antibody added to SF significantly decreased its effect. We have confirmed that Cyr61 in RA SF is a key factor in promoting FLS proliferation, and the conclusion can be drawn that Cyr61 plays a pivotal role in FLS proliferation.

In RA, increased FLS proliferation and/or decreased FLS apoptosis contributes to synovial hyperplasia (27). It has been suggested that enhanced expression of Bcl-2 in RA FLS may inhibit FLS apoptosis (28, 29). In the current study, we demonstrated that knockdown of Cyr61 expression by siRNA resulted in a significant increase in FLS apoptosis induced by serum deprivation and a decrease in expression of both Bcl-2 mRNA and protein, leading to the conclusion that Cyr61 prevented RA FLS apoptosis and that the protective effect of Cyr61 is likely due to its role in sustaining the expression of Bcl-2.

IL-17 is an important proinflammatory cytokine involved in the pathogenesis of RA (30, 31). Previous studies have shown that IL-17 is present in RA SF and can up-regulate several mediators of inflammation, such as TNFα, IL-1β, IL-6, IL-8, and MMPs, in FLS (13, 32–34). Our observation that Cyr61 plays an important role in FLS proliferation led us to explore the possibility that Cyr61 might be functionally linked to IL-17. Indeed, we found that IL-17 represents an essential cytokine in RA SF and is responsible for promoting the expression of Cyr61 in FLS. Moreover, in experiments with actinomycin D, Cyr61 mRNA stability was not obviously increased in IL-17–stimulated FLS (data not shown). Importantly, the knockdown of Cyr61 expression in FLS dramatically reduced IL-17–stimulated FLS proliferation, thus establishing a novel functional link between IL-17 and Cyr61.

TNFα is another important proinflammatory cytokine that contributes to RA pathogenesis. Previous studies have shown that TNFα significantly induces FLS proliferation and that IL-17 exhibits a synergistic effect on TNFα in inducing cytokine expression in FLS (13). In human skin fibroblasts, Cyr61 was found to modify the activities of TNFα, converting it from a proliferation-enhancing factor into a potent apoptotic agent (35). In the current study, we found somewhat unexpectedly that TNFα did not exhibit a synergistic effect on IL-17–dependent expression of Cyr61. The fact that TNFα exhibited different characteristics in RA SF is interesting, and the reasons for this need to be investigated further.

Previous studies have shown that IL-17 induces cytokine production in FLS via the NF-κB, ERK, p38 MAPK, and PI 3-kinase pathways (21, 33, 36). To investigate which pathway is used by IL-17 to up-regulate the expression of Cyr61 in FLS, we used specific inhibitors to evaluate possible involvement of these pathways. IL-17–induced expression of Cyr61 in RA FLS was significantly hampered by PDTC (an inhibitor of NF-κB activation) and by SB203580 (an inhibitor of p38 MAPK), while inhibition of PI 3-kinase and ERK did not affect Cyr61 expression. The activation of NF-κB and p38 by IL-17 in FLS was also further confirmed by confocal microscopy and Western blotting, respectively.

Moreover, αvβ3, αvβ5, α6β1, αIIβ3, and αMβ2 integrins were identified as 5 receptors for Cyr61 in a certain cell type (5, 22, 23, 37, 38). Among these receptors, α6β1, αvβ3, and αvβ5 mediate cell adhesion (23), cell proliferation, and migration of human skin fibroblasts (5). However, in the current study, we observed that only αvβ5 mediated Cyr61-dependent FLS proliferation (data not shown), suggesting that Cyr61 regulates diverse cellular processes in a cell type–specific and a function-specific manner. The interaction between Cyr61 and its receptors on RA FLS warrants further investigation.

In summary, our study is the first to reveal that Cyr61 plays a critical role in IL-17–dependent proliferation of FLS in RA and, thus, might contribute to hyperplasia of cells of the synovial lining and eventually to joint destruction in RA (Figure 6). Based on our findings and the findings of others (30, 39), we propose that IL-17 secreted by Th17 cells binds to its receptor on FLS and induces the activation of the NF-κB and p38 MAPK signaling pathways. The activation of these 2 pathways is necessary for the induction of Cyr61 expression. The induced Cyr61 is then released by FLS into the SF, which in turn, acts on FLS in a paracrine or autocrine manner via binding to its surface receptors, such as αvβ5. As a result, FLS proliferation is stimulated, due to both inhibition of apoptosis and enhancement of cell division.

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Figure 6. Schematic model of Cyr61-stimulated FLS proliferation and its regulation by interleukin-17 (IL-17) signaling. IL-17 secreted by Th17 cells enhances the expression of Cyr61 via the p38 and NF-κB signaling pathways, leading to activation of Cyr61 transcription. Cyr61, in turn, stimulates FLS division and protects FLS against apoptosis via up-regulation of Bcl-2. In addition to IL-17, other mechanisms (such as growth factors [GF]) might be involved in up-regulating expression of Cyr61. The process by which Cyr61 regulates FLS division is currently unclear. IL-17Ra = IL-17 receptor antagonist; PDTC = pyrrolidine dithiocarbamate; AP-1 = activator protein 1.

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Our findings suggest that the critical role of Cyr61 in FLS proliferation likely contributes to hyperplasia of cells of the synovial lining and eventually to joint destruction in RA. Thus, Cyr61 might be a potential target for use in the development of new therapeutic approaches for RA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. N. Li had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Q. Zhang, Wu, N. Li.

Acquisition of data. Wu, Cao, Xiao, He, Ouyang, Lin, Shen, Shi.

Analysis and interpretation of data. Wang, Y. Zhang, D. Li, N. Li.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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ART_24999_sm_SupplementaryTable1.doc75KSupplementary table 1: Specific Primers Used in Real-time PCR Analysis

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