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MDM2 promotes rheumatoid arthritis via activation of MAPK and NF-κB
International Immunopharmacology 30 (2016) 69–73
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
MDM2 promotes rheumatoid arthritis via activation of MAPK and NF-κB Lin Zhang a, Jing Luo b, Hongyan Wen b, Tingting Zhang b, Xiaoxia Zuo a,⁎,1, Xiaofeng Li b,⁎⁎,1 a b
Department of Rheumatology, Xiangya School of Medicine, Central South University, Changsha 410008, Hunan, China Department of Rheumatology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China
a r t i c l e
i n f o
Article history: Received 20 August 2015 Received in revised form 15 November 2015 Accepted 23 November 2015 Available online 2 December 2015 Keywords: MDM2 Rheumatoid arthritis FLS MAPK NF-κB
a b s t r a c t Murine double minute-2 (MDM2) has pleiotropic roles in immune activation and regulation. However, the role of MDM2 in rheumatoid arthritis (RA) remains unknown. We undertook this study to investigate the role of MDM2 in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were isolated from 25 patients with active RA and 25 patients with osteoarthritis (OA). FLS were stimulated in the presence or absence of IL-1β in vitro. Mice with collagen-induced arthritis (CIA) were treated with Nutlin-3a (100 mg/kg) or vehicle twice daily for 2 weeks. MDM2 expression was determined by Western blot. MDM2 was down-regulated by specific gene silencing. The concentrations of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) were analyzed using enzyme-linked immunosorbent assay (ELISA). The pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were investigated by Western blot. Arthritis scoring and histological analysis were conducted. MDM2 expression was significantly higher in RA-FLS than in OA-FLS. MDM2 protein expression was positively correlated with disease activity of RA. MDM2 promoted the production of TNF-α, IL-6, MMP1 and MMP13 through MAPK and NF-κB pathways in RA-FLS. Nutlin-3a treatment decreased the arthritis severity and joint damage in CIA. Nutlin-3a also inhibited the activation of MAPK and NF-κB in arthritic joints. In conclusion, MDM2 inhibition exhibits anti-inflammatory activity and MDM2 might be a new therapeutic target for RA. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation and cartilage erosions [1]. Fibroblast-like synoviocytes (FLS) contribute greatly to RA progression and joint destruction by initiating and regulating a number of pathways [2]. Molecular insights into FLS biology and related signaling pathways are needed to identify therapeutic targets [3]. Murine double minute-2 (MDM2), an E3 ubiquitin ligase, encodes a negative regulator of the p53 tumor suppressor [4]. MDM2 is an intracellular molecule with diverse biological functions and amplification of MDM2 occurs in multiple malignancies [5]. Furthermore, MDM2 promotes tissue inflammation and MDM2 inhibition has potent antiinflammatory effects [6]. Nutlin-3a is a small-molecule antagonist that inhibits MDM2–p53 interactions and stabilizes the p53 protein, thereby inducing cell cycle arrest and apoptosis [7]. Inhibition of MDM2 attenuated neointimal hyperplasia via suppression of vascular proliferation and inflammation [8]. MDM2 links inflammation and tubular cell healing during acute kidney
injury in mice [9]. Here, we undertook this study to investigate the role of MDM2 in rheumatoid arthritis (RA). In this study, we found that inhibition of MDM2 attenuated collagen-induced arthritis (CIA), suggesting a role for MDM2 in the pathogenesis of RA. 2. Materials and methods 2.1. Patients Twenty-five patients who fulfilled the 2010 ACR/European League against Rheumatism (EULAR) classification criteria for RA [10] were enrolled from the Department of Rheumatology, Xiangya School of Medicine, Central South University. All patients had active disease with a 28-joint disease activity score (DAS28) more than 2.6. The patients with osteoarthritis (OA, n = 25) were included from the same Department as controls. This study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by Ethics Committee of our Hospital. Written informed consent was obtained from all patients. 2.2. Synovial tissue collection and FLS culture
⁎ Correspondence to: X. Zuo, Department of Rheumatology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha 410008, Hunan, China. ⁎⁎ Correspondence to: X. Li, Department of Rheumatology, The Second Hospital of Shanxi Medical University, No. 382 Wuyi Road, Taiyuan 030001, Shanxi, China. E-mail addresses: [email protected] (X. Zuo), [email protected] (X. Li). 1 Xiaoxia Zuo and Xiaofeng Li contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2015.11.030 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Closed needle was used to obtain the synovium samples from RA and OA patients [11]. All specimens were fixed in 10% neutral formalin and embedded in paraffin. Sections (5 μm) were cut serially and mounted on adhesive glass slides.
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FLS were isolated from the synovial tissues as previously described [12]. Fresh synovial tissues were digested in type I collagenase (Sigma-Aldrich, USA). The cells were cultured with DMEM-Ham's F-12 (Life Technologies, Shanghai, China), containing 20% fetal calf serum (Life Technologies, Australia) in a humidified 5% CO2 incubator. FLS from passages three to six were used in this study. 2.3. MDM2 inhibition in RA-FLS RA-FLS were pretreated with IL-1β (10 ng/ml, Minneapolis, USA) for 12 h. For MDM2 knockdown, RA-FLS were transfected with two different specific human MDM2 siRNAs (#1 and #2, Invitrogen, Life Technologies) using Lipofectamine 2000 reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. Scrambled control siRNAs were used as control. MDM2 siRNAs were added to RA-FLS in the presence of polybrene. In short, siRNAs and lipofectamine were diluted in Opti-MEM I Reduced Serum Medium (Invitrogen, Life Technologies) separately and incubated for 10 min at room temperature. The diluted solutions were then mixed and incubated for 20 min at room temperature. Subsequently, the mixtures were added to each well. The culture plates were then incubated for 6 h at 37 °C in a CO2 incubator. Then, the medium containing FBS was added. Cells or culture supernatants were subjected to the Western blot analysis or ELISA. In addition, we tested the effects of MDM2 inhibitor Nutlin-3a (0, 5, 10, 20, 100 μM) on the cytokine production using the system as described above.
using enhanced chemiluminescence (ECL) and exposed to hyperfilm– ECL film (GE Healthcare Bio-Sciences, Australia). 2.7. Measurements of cytokines Sera were obtained from anesthetized animals by retroorbital puncture at the end of the study. The levels of TNF-α, IL-6, MMP1 and MMP13 were measured by ELISA kits (R&D Systems) according to the manufacturer's instructions. 2.8. Histological assessment Mice were sacrificed and the hind paws were fixed in 10% neutral formalin, decalcified by immersing in 10% EDTA solution for 20 days and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E). The inflammation was evaluated as described previously [15]. Cartilage damage was examined using safranin-O staining and scored as follows [15]: 0, no destruction; 1, minimal erosion; 2, slight to moderate erosion in a limited area; 3, more extensive erosion; 4, general destruction. 2.9. Statistics Data are expressed as mean ± SEM. All data were processed using SPSS 16.0. Statistical comparisons were performed using one-way analysis of variance or the Mann–Whitney U test. The significance of differences between 2 groups was determined using Student's unpaired t-test. P b 0.05 was considered significant.
2.4. CIA induction and assessment 3. Results Male DBA/1 mice (8 weeks old) were purchased from the Shanghai Laboratory Animal Center and maintained under specific pathogen-free conditions. All experiments were approved by the Animal Use Committee of Xiangya School of Medicine, Central South University. CIA was induced according to the previous report [13]. Chicken type II collagen in an emulsion with complete Freund's adjuvant (CFA; Hooke Laboratories) was initially immunized, and at 3 weeks chicken type II collagen in an emulsion with incomplete Freund's adjuvant was used for boosted immunization. Mice were observed and scored from the boost. The scoring for the severity of arthritis was conducted on a scale of 0–4 for each paw as follows [13], 0 = no clinical disease; 1 = one toe inflamed and swollen; 2 = more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw; 3 = entire paw inflamed and swollen; and 4 = severely inflamed and swollen paw or ankylosed paw. 2.5. Administration of Nutlin-3a
3.1. MDM2 was up-regulated in RA-FLS The protein expression of MDM2 in RA-FLS was detected from 25 RA patients and 25 OA patients. Western blot analysis showed MDM2 was expressed in RA-FLS (Fig. 1A). The protein expression of MDM2 in RAFLS was significantly enhanced compared with OA-FLS (Fig. 1B). 3.2. MDM2 protein expression was positively correlated with disease activity Furthermore, we analyzed the correlations between MDM2 expression and clinical parameters including CRP, ESR, DAS28, RF titers, antiCCP, 28TJC, 28SJC, and pain VAS. We found that MDM2 protein expression in RA-FLS was positively correlated with DAS28 (r = 0.815, P = 0.005). There were no significant correlations between MDM2 expression and other clinical features (all P N 0.05).
The mice with CIA were orally treated with Nutlin-3a (100 mg/kg, n = 10 mice) twice daily [14], starting from day 22 (1 day after boosted immunization) for 2 weeks. The control animals (n = 10 mice) were treated with vehicle (1% Klucel, 0.1% Tween 80) alone. 2.6. Western blot The protein concentration was determined using the Bradford assay (BioRad). SDS sample buffer (5 ×) was added to the collected protein samples and boiled for 5 min. The proteins were then loaded onto a 10% SDS-PAGE gel and separated by electrophoresis. After transfer onto a nitrocellulose membrane, the membrane was blocked with 5% skim milk. The following primary antibodies were used: antip-ERK1/2, p-p38, p-JNK, NF-κB p65 and anti-GAPDH or Lamin B (all from Santa Cruz). The membrane was incubated with the primary antibodies overnight at 4 °C, followed by washing with PBST and incubation with peroxidase-labeled secondary antibody (goat anti-rabbit IgG-HRP, Santa Cruz Biotech). Protein visualization was achieved
Fig. 1. The expression of MDM2 protein in fibroblast-like synoviocytes (FLS). A. MDM2 protein expression was detected by Western blot in FLS samples from RA, OA patients or healthy volunteers. B. The relative level of MDM2 mRNA expression was corrected by GAPDH. Data represent fold change of relative MDM2 expression normalized to GAPDH levels. *P b 0.01 vs RA.
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3.3. MDM2 knockdown inhibited inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways To determine the role of MDM2 in RA-FLS, we evaluated the effects of MDM2 knockdown on the pro-inflammatory cytokines and MMPs in RA-FLS. We first confirmed the silencing efficacy of siMDM2 (#1 and #2) using Western blot analysis (Fig. 2A). MDM2 knockdown suppressed the production of TNF-α, IL-6, MMP1 and MMP13 in IL-1β-treated RA-FLS (Fig. 2B). To explore the potential signaling pathways involved in this process, MAPK and NF-κB activation was evaluated. We found that MDM2 knockdown suppressed the activation of MAPK pathway, including ERK1/2, p38 MAPK, and JNK in RA-FLS (Fig. 2C). MDM2 knockdown also suppressed the p-IκBα and the nuclear translocation of the NF-κB p65, indicating the inhibition of NF-κB pathway (Fig. 2C). 3.4. Nutlin-3a inhibited the production of cytokines in a dose-dependent manner In order to explore whether MDM2 inhibition acts in a dosedependent manner, RA-FLS were exposed to different doses of Nutlin3a (0, 5, 10, 20, 100 μM). As shown in Fig. 3, Nutlin-3a treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by RA-FLS in a dose-dependent manner. These results were consistent with those from the MDM2 silencing above. 3.5. Nutlin-3a treatment reduced arthritis score and joint inflammation in CIA mice Mice were treated from 22 days after first immunization. Clinical scores were evaluated every 2 days. Nutlin-3a treatment significantly attenuated the clinical scores of CIA (Fig. 4A). As depicted in Fig. 3B and C, vehicle-treated CIA mice showed marked infiltration of leukocytes in the joints, while Nutlin-3a-treated mice exhibited a lower degree of inflammation and cartilage damage
Fig. 2. MDM2 knockdown inhibited inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways. A. The silencing effect of MDM2 in RA-FLS transfected with MDM2siRNA #1 (siRNA #1) and MDM2siRNA #2(siRNA #2) was confirmed by Western blot. B. MDM2 knockdown suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by IL-1β-treated RA-FLS. *P b 0.01 vs siControl. Values represent the mean ± SEM of three independent experiments (n = 10 patients). C. MAPK signaling molecules, including p-ERK1/2, p-JNK and p-p36, and NF-κB signaling molecules, including p-IκB-α and p65 NF-κB, were measured by Western blot. D. The fold increase relative to respective control (their total form, GAPDH or Lamin B) was shown. *P b 0.01 compared with vehicle-treated group.
Fig. 3. Nutlin-3a treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 in a dose-dependent manner. Nutlin-3a (0, 5, 10, 20, 100 μM) treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by IL-1β-treated RA-FLS in a dose-dependent manner. *P b 0.01 compared with 0 μM-treated group.
on day 36 after immunization. Bone erosion in the CIA mice was significantly inhibited by Nutlin-3a treatment, as determined by safranin O staining (Fig. 4B and C).
3.6. Nutlin-3a treatment inhibited the production of pro-inflammatory cytokines and MMPs in CIA mice Pro-inflammatory cytokines and MMPs contributed significantly to the development of CIA. To ascertain whether Nutlin-3a inhibits the production of these mediators, the levels of TNF-α, IL-6, MMP1 and MMP13 from sera and joint homogenates were measured by ELISA. The Nutlin-3a-treated mice had significantly lower levels of TNF-α, IL-6, MMP1 and MMP13 than vehicle-treated mice (Fig. 5). These results suggest that Nutlin-3a may attenuate CIA by inhibiting the production of pro-inflammatory cytokines and MMPs.
Fig. 4. Effect of Nutlin-3a treatment on the arthritis and cartilage damage. A. Clinical scores were assessed every 2 days, starting at 22 days after first immunization. B. synovial inflammation (H&E, × 200) and cartilage damage (safranin-O, × 200). Bar graphs represent histological scores as described in Materials and methods. Data were analyzed using one-way ANOVA and results are mean ± SEM from 10 mice/group. *P b 0.01 compared with vehicle-treated group.
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Fig. 5. Nutlin-3a treatment inhibited the production of pro-inflammatory cytokines and MMPs in CIA. On day 36, sera were collected from mice. Cytokines and MMPs in sera were measured by ELISA. Values are the mean ± SEM (n = 10 mice per group). *P b 0.01 compared with vehicle-treated group.
3.7. Nutlin-3a treatment inhibited MAPK and NF-κB pathways in CIA mice NF-κB is a well-known factor in regulating inflammation. As shown in Fig. 6, we found that MDM2 inhibition suppressed the activation of the MAPK pathway, including ERK1/2, p38 MAPK, and JNK in arthritic joints. MDM2 inhibition also suppressed the p-IκBα and the nuclear translocation of the NF-κB p65, indicating the inhibition of NF-κB signaling. These findings suggest that Nutlin-3a suppressed MAPK and NF-κB pathways in CIA mice. 4. Discussion MDM2 was first described to limit p53-mediated cell cycle arrest and apoptosis, hence, gain of function mutations is associated with malignancies [16]. In recent years, MDM2 inhibitors have been successfully obtained and advanced into clinical trials for the treatment of human cancers [16]. Recent evidence suggests that MDM2 has p53-independent activity in inflammatory diseases [17]. Nutlin-3a is a selective inhibitor of MDM2. Our data here showed that inhibition of MDM2 by Nutlin-3a prevented the development of arthritis and cartilage destruction in CIA. Further investigation showed that the protective effects of Nutlin-3a were mediated by inhibition of the MAPK and NF-κB pathways. MDM2 blockade has anti-inflammatory and anti-mitotic effects in inflammatory and autoimmune disorders [6,9,18–20]. MDM2 was identified to act as a co-transcription factor for NF-κB [6]. MDM2 plays an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques [18]. Inhibition of MDM2 reduced systemic inflammation and abrogated immune complex-mediated disease by suppressing plasma cells and autoantibodies in lupus nephritis [19]. Nutlin-3a also prevented tubular necrosis by suppressing sterile inflammation via a pro-inflammatory, p53-independent mechanism in acute kidney injury [9]. TSLP induces mast cell development and aggravates
Fig. 6. Nutlin-3a treatment inhibited MAPK and NF-κB pathways in the arthritic joints in CIA. The MAPK signaling molecules, including p-ERK1/2, p-JNK and p-p36, and the NFκB signaling molecules, including p-IκB-α and p65 NF-κB, were measured by Western blot. The fold increase relative to respective control (their total form, GAPDH or Lamin B) was shown. *P b 0.01 compared with vehicle-treated group. Values are mean ± SEM (n = 10 mice per group).
allergic reactions through the activation of MDM2 [20]. In the present study, we demonstrated that Nutlin-3a treatment reduced the synovial inflammation and bone destruction in CIA. Targeting MDM2 has therapeutic potential for human RA. Pro-inflammatory cytokines and MMPs play important roles in RA. IL-1β-stimulated RA-FLS have been widely used in vitro to mimic synovial proliferation in patients with RA, as IL-1β is believed to play a major role in synovial inflammation in RA [21]. IL-1β promotes inflammation and destruction in synovial tissue, bone and cartilage in RA patients [21,22]. IL-1β is also a pivotal cytokine in inducing expression of other inflammatory cytokines, such as TNF-α and IL-6 [23]. Previous data have shown that increased level of IL-1β in synovial tissue was correlated with histological features of arthritis [24,25]. IL-1β enhances the expressions of pro-inflammatory mediators and MMPs, leading to subsequent joint destruction through the activation of transcriptional factors such as AP-1 and NF-κB [26]. NF-κB plays a central role in the regulation of many genes that express proteins functioning in inflammation, including MMPs and IL-6 [27]. Persistent NF-κB activation contributes to the pathogenesis of human RA [28]. In addition to NF-κB, MAPK has also been considered as a therapeutic target for RA [29]. In line with the previous findings, this study showed that Nutlin-3a treatment caused a marked decrease in the expression of TNF-α and IL-6 in CIA. In addition, our study showed that Nutlin-3a treatment inhibited the activation of MAPK and NF-κB in arthritic joints in CIA. To further support the clinical significances of our study, we examined the human RA-FLS. And we found that MDM2 inhibition suppressed inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways. Our findings suggest that inhibition of MDM2 ameliorated joint inflammation and cartilage destruction in CIA, and these effects were mediated via down-regulation of MAPK and NF-κB pathways. MDM2 blockade has beneficial effects in CIA. MDM2 blockade may be a feasible strategy to control human RA. Conflict of interest All authors declare no conflicts of interest in this study. References [1] S. Roser-Page, T. Vikulina, M. Zayzafoon, Weitzmann MN.CTLA-4Ig-induced T cell anergy promotes Wnt-10b production and bone formation in a mouse model, Arthritis Rheumatol. 66 (2014) 990–999. [2] C. Sohn, A. Lee, Y. Qiao, K. Loupasakis, L.B. Ivashkiv, G.D. Kalliolias, Prolonged tumor necrosis factor α primes fibroblast-like synoviocytes in a gene-specific manner by altering chromatin, Arthritis Rheumatol. 67 (2015) 86–95. [3] J. Li, H.C. Hsu, Y. Ding, H. Li, Q. Wu, P. Yang, B. Luo, A.L. Rowse, D.M. Spalding, S.L. Bridges Jr., J.D. Mountz, Inhibition of fucosylation reshapes inflammatory macrophages and suppresses type II collagen-induced arthritis, Arthritis Rheumatol. 66 (2014) 2368–2379. [4] J.D. Oliner, K.W. Kinzler, P.S. Meltzer, D.L. George, B. Vogelstein, Amplification of a gene encoding a p53-associated protein in human sarcomas, Nature 358 (1992) 80–83. [5] Y. Yuan, Y.M. Liao, C.T. Hsueh, H.R. Mirshahidi, Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP, J. Hematol. Oncol. 4 (2011) 16. [6] D. Thomasova, S.R. Mulay, H. Bruns, Anders HJ.p53-independent roles of MDM2 in NF-κB signaling: implications for cancer therapy, wound healing, and autoimmune diseases, Neoplasia 14 (2012) 1097–1101. [7] L.T. Vassilev, B.T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong, U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi, E.A. Liu, In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, Science 303 (2004) 844–848. [8] T. Hashimoto, T. Ichiki, J. Ikeda, E. Narabayashi, H. Matsuura, R. Miyazaki, K. Inanaga, K. Takeda, K. Sunagawa, Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation, Cardiovasc. Res. 91 (2011) 711–719. [9] S.R. Mulay, D. Thomasova, M. Ryu, H.J. Anders, MDM2 (murine double minute-2) links inflammation and tubular cell healing during acute kidney injury in mice, Kidney Int. 81 (2012) 1199–1211. [10] D. Aletaha, T. Neogi, A.J. Silman, J. Funovits, D.T. Felson, C.O. Bingham 3rd, et al., 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative, Arthritis Rheum. 62 (2010) 2569–2581. [11] J.A. Singh, T. Arayssi, P. Duray, H.R. Schumacher, Immunohistochemistry of normal human knee synovium: a quantitative study, Ann. Rheum. Dis. 63 (2004) 785–790.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
MDM2 promotes rheumatoid arthritis via activation of MAPK and NF-κB Lin Zhang a, Jing Luo b, Hongyan Wen b, Tingting Zhang b, Xiaoxia Zuo a,⁎,1, Xiaofeng Li b,⁎⁎,1 a b
Department of Rheumatology, Xiangya School of Medicine, Central South University, Changsha 410008, Hunan, China Department of Rheumatology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China
a r t i c l e
i n f o
Article history: Received 20 August 2015 Received in revised form 15 November 2015 Accepted 23 November 2015 Available online 2 December 2015 Keywords: MDM2 Rheumatoid arthritis FLS MAPK NF-κB
a b s t r a c t Murine double minute-2 (MDM2) has pleiotropic roles in immune activation and regulation. However, the role of MDM2 in rheumatoid arthritis (RA) remains unknown. We undertook this study to investigate the role of MDM2 in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were isolated from 25 patients with active RA and 25 patients with osteoarthritis (OA). FLS were stimulated in the presence or absence of IL-1β in vitro. Mice with collagen-induced arthritis (CIA) were treated with Nutlin-3a (100 mg/kg) or vehicle twice daily for 2 weeks. MDM2 expression was determined by Western blot. MDM2 was down-regulated by specific gene silencing. The concentrations of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) were analyzed using enzyme-linked immunosorbent assay (ELISA). The pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were investigated by Western blot. Arthritis scoring and histological analysis were conducted. MDM2 expression was significantly higher in RA-FLS than in OA-FLS. MDM2 protein expression was positively correlated with disease activity of RA. MDM2 promoted the production of TNF-α, IL-6, MMP1 and MMP13 through MAPK and NF-κB pathways in RA-FLS. Nutlin-3a treatment decreased the arthritis severity and joint damage in CIA. Nutlin-3a also inhibited the activation of MAPK and NF-κB in arthritic joints. In conclusion, MDM2 inhibition exhibits anti-inflammatory activity and MDM2 might be a new therapeutic target for RA. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation and cartilage erosions [1]. Fibroblast-like synoviocytes (FLS) contribute greatly to RA progression and joint destruction by initiating and regulating a number of pathways [2]. Molecular insights into FLS biology and related signaling pathways are needed to identify therapeutic targets [3]. Murine double minute-2 (MDM2), an E3 ubiquitin ligase, encodes a negative regulator of the p53 tumor suppressor [4]. MDM2 is an intracellular molecule with diverse biological functions and amplification of MDM2 occurs in multiple malignancies [5]. Furthermore, MDM2 promotes tissue inflammation and MDM2 inhibition has potent antiinflammatory effects [6]. Nutlin-3a is a small-molecule antagonist that inhibits MDM2–p53 interactions and stabilizes the p53 protein, thereby inducing cell cycle arrest and apoptosis [7]. Inhibition of MDM2 attenuated neointimal hyperplasia via suppression of vascular proliferation and inflammation [8]. MDM2 links inflammation and tubular cell healing during acute kidney
injury in mice [9]. Here, we undertook this study to investigate the role of MDM2 in rheumatoid arthritis (RA). In this study, we found that inhibition of MDM2 attenuated collagen-induced arthritis (CIA), suggesting a role for MDM2 in the pathogenesis of RA. 2. Materials and methods 2.1. Patients Twenty-five patients who fulfilled the 2010 ACR/European League against Rheumatism (EULAR) classification criteria for RA [10] were enrolled from the Department of Rheumatology, Xiangya School of Medicine, Central South University. All patients had active disease with a 28-joint disease activity score (DAS28) more than 2.6. The patients with osteoarthritis (OA, n = 25) were included from the same Department as controls. This study was performed in accordance with the Declaration of Helsinki, and the protocol was approved by Ethics Committee of our Hospital. Written informed consent was obtained from all patients. 2.2. Synovial tissue collection and FLS culture
⁎ Correspondence to: X. Zuo, Department of Rheumatology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha 410008, Hunan, China. ⁎⁎ Correspondence to: X. Li, Department of Rheumatology, The Second Hospital of Shanxi Medical University, No. 382 Wuyi Road, Taiyuan 030001, Shanxi, China. E-mail addresses: [email protected] (X. Zuo), [email protected] (X. Li). 1 Xiaoxia Zuo and Xiaofeng Li contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2015.11.030 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Closed needle was used to obtain the synovium samples from RA and OA patients [11]. All specimens were fixed in 10% neutral formalin and embedded in paraffin. Sections (5 μm) were cut serially and mounted on adhesive glass slides.
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FLS were isolated from the synovial tissues as previously described [12]. Fresh synovial tissues were digested in type I collagenase (Sigma-Aldrich, USA). The cells were cultured with DMEM-Ham's F-12 (Life Technologies, Shanghai, China), containing 20% fetal calf serum (Life Technologies, Australia) in a humidified 5% CO2 incubator. FLS from passages three to six were used in this study. 2.3. MDM2 inhibition in RA-FLS RA-FLS were pretreated with IL-1β (10 ng/ml, Minneapolis, USA) for 12 h. For MDM2 knockdown, RA-FLS were transfected with two different specific human MDM2 siRNAs (#1 and #2, Invitrogen, Life Technologies) using Lipofectamine 2000 reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. Scrambled control siRNAs were used as control. MDM2 siRNAs were added to RA-FLS in the presence of polybrene. In short, siRNAs and lipofectamine were diluted in Opti-MEM I Reduced Serum Medium (Invitrogen, Life Technologies) separately and incubated for 10 min at room temperature. The diluted solutions were then mixed and incubated for 20 min at room temperature. Subsequently, the mixtures were added to each well. The culture plates were then incubated for 6 h at 37 °C in a CO2 incubator. Then, the medium containing FBS was added. Cells or culture supernatants were subjected to the Western blot analysis or ELISA. In addition, we tested the effects of MDM2 inhibitor Nutlin-3a (0, 5, 10, 20, 100 μM) on the cytokine production using the system as described above.
using enhanced chemiluminescence (ECL) and exposed to hyperfilm– ECL film (GE Healthcare Bio-Sciences, Australia). 2.7. Measurements of cytokines Sera were obtained from anesthetized animals by retroorbital puncture at the end of the study. The levels of TNF-α, IL-6, MMP1 and MMP13 were measured by ELISA kits (R&D Systems) according to the manufacturer's instructions. 2.8. Histological assessment Mice were sacrificed and the hind paws were fixed in 10% neutral formalin, decalcified by immersing in 10% EDTA solution for 20 days and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E). The inflammation was evaluated as described previously [15]. Cartilage damage was examined using safranin-O staining and scored as follows [15]: 0, no destruction; 1, minimal erosion; 2, slight to moderate erosion in a limited area; 3, more extensive erosion; 4, general destruction. 2.9. Statistics Data are expressed as mean ± SEM. All data were processed using SPSS 16.0. Statistical comparisons were performed using one-way analysis of variance or the Mann–Whitney U test. The significance of differences between 2 groups was determined using Student's unpaired t-test. P b 0.05 was considered significant.
2.4. CIA induction and assessment 3. Results Male DBA/1 mice (8 weeks old) were purchased from the Shanghai Laboratory Animal Center and maintained under specific pathogen-free conditions. All experiments were approved by the Animal Use Committee of Xiangya School of Medicine, Central South University. CIA was induced according to the previous report [13]. Chicken type II collagen in an emulsion with complete Freund's adjuvant (CFA; Hooke Laboratories) was initially immunized, and at 3 weeks chicken type II collagen in an emulsion with incomplete Freund's adjuvant was used for boosted immunization. Mice were observed and scored from the boost. The scoring for the severity of arthritis was conducted on a scale of 0–4 for each paw as follows [13], 0 = no clinical disease; 1 = one toe inflamed and swollen; 2 = more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw; 3 = entire paw inflamed and swollen; and 4 = severely inflamed and swollen paw or ankylosed paw. 2.5. Administration of Nutlin-3a
3.1. MDM2 was up-regulated in RA-FLS The protein expression of MDM2 in RA-FLS was detected from 25 RA patients and 25 OA patients. Western blot analysis showed MDM2 was expressed in RA-FLS (Fig. 1A). The protein expression of MDM2 in RAFLS was significantly enhanced compared with OA-FLS (Fig. 1B). 3.2. MDM2 protein expression was positively correlated with disease activity Furthermore, we analyzed the correlations between MDM2 expression and clinical parameters including CRP, ESR, DAS28, RF titers, antiCCP, 28TJC, 28SJC, and pain VAS. We found that MDM2 protein expression in RA-FLS was positively correlated with DAS28 (r = 0.815, P = 0.005). There were no significant correlations between MDM2 expression and other clinical features (all P N 0.05).
The mice with CIA were orally treated with Nutlin-3a (100 mg/kg, n = 10 mice) twice daily [14], starting from day 22 (1 day after boosted immunization) for 2 weeks. The control animals (n = 10 mice) were treated with vehicle (1% Klucel, 0.1% Tween 80) alone. 2.6. Western blot The protein concentration was determined using the Bradford assay (BioRad). SDS sample buffer (5 ×) was added to the collected protein samples and boiled for 5 min. The proteins were then loaded onto a 10% SDS-PAGE gel and separated by electrophoresis. After transfer onto a nitrocellulose membrane, the membrane was blocked with 5% skim milk. The following primary antibodies were used: antip-ERK1/2, p-p38, p-JNK, NF-κB p65 and anti-GAPDH or Lamin B (all from Santa Cruz). The membrane was incubated with the primary antibodies overnight at 4 °C, followed by washing with PBST and incubation with peroxidase-labeled secondary antibody (goat anti-rabbit IgG-HRP, Santa Cruz Biotech). Protein visualization was achieved
Fig. 1. The expression of MDM2 protein in fibroblast-like synoviocytes (FLS). A. MDM2 protein expression was detected by Western blot in FLS samples from RA, OA patients or healthy volunteers. B. The relative level of MDM2 mRNA expression was corrected by GAPDH. Data represent fold change of relative MDM2 expression normalized to GAPDH levels. *P b 0.01 vs RA.
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3.3. MDM2 knockdown inhibited inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways To determine the role of MDM2 in RA-FLS, we evaluated the effects of MDM2 knockdown on the pro-inflammatory cytokines and MMPs in RA-FLS. We first confirmed the silencing efficacy of siMDM2 (#1 and #2) using Western blot analysis (Fig. 2A). MDM2 knockdown suppressed the production of TNF-α, IL-6, MMP1 and MMP13 in IL-1β-treated RA-FLS (Fig. 2B). To explore the potential signaling pathways involved in this process, MAPK and NF-κB activation was evaluated. We found that MDM2 knockdown suppressed the activation of MAPK pathway, including ERK1/2, p38 MAPK, and JNK in RA-FLS (Fig. 2C). MDM2 knockdown also suppressed the p-IκBα and the nuclear translocation of the NF-κB p65, indicating the inhibition of NF-κB pathway (Fig. 2C). 3.4. Nutlin-3a inhibited the production of cytokines in a dose-dependent manner In order to explore whether MDM2 inhibition acts in a dosedependent manner, RA-FLS were exposed to different doses of Nutlin3a (0, 5, 10, 20, 100 μM). As shown in Fig. 3, Nutlin-3a treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by RA-FLS in a dose-dependent manner. These results were consistent with those from the MDM2 silencing above. 3.5. Nutlin-3a treatment reduced arthritis score and joint inflammation in CIA mice Mice were treated from 22 days after first immunization. Clinical scores were evaluated every 2 days. Nutlin-3a treatment significantly attenuated the clinical scores of CIA (Fig. 4A). As depicted in Fig. 3B and C, vehicle-treated CIA mice showed marked infiltration of leukocytes in the joints, while Nutlin-3a-treated mice exhibited a lower degree of inflammation and cartilage damage
Fig. 2. MDM2 knockdown inhibited inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways. A. The silencing effect of MDM2 in RA-FLS transfected with MDM2siRNA #1 (siRNA #1) and MDM2siRNA #2(siRNA #2) was confirmed by Western blot. B. MDM2 knockdown suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by IL-1β-treated RA-FLS. *P b 0.01 vs siControl. Values represent the mean ± SEM of three independent experiments (n = 10 patients). C. MAPK signaling molecules, including p-ERK1/2, p-JNK and p-p36, and NF-κB signaling molecules, including p-IκB-α and p65 NF-κB, were measured by Western blot. D. The fold increase relative to respective control (their total form, GAPDH or Lamin B) was shown. *P b 0.01 compared with vehicle-treated group.
Fig. 3. Nutlin-3a treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 in a dose-dependent manner. Nutlin-3a (0, 5, 10, 20, 100 μM) treatment suppressed the production of TNF-α, IL-6, MMP1 and MMP13 by IL-1β-treated RA-FLS in a dose-dependent manner. *P b 0.01 compared with 0 μM-treated group.
on day 36 after immunization. Bone erosion in the CIA mice was significantly inhibited by Nutlin-3a treatment, as determined by safranin O staining (Fig. 4B and C).
3.6. Nutlin-3a treatment inhibited the production of pro-inflammatory cytokines and MMPs in CIA mice Pro-inflammatory cytokines and MMPs contributed significantly to the development of CIA. To ascertain whether Nutlin-3a inhibits the production of these mediators, the levels of TNF-α, IL-6, MMP1 and MMP13 from sera and joint homogenates were measured by ELISA. The Nutlin-3a-treated mice had significantly lower levels of TNF-α, IL-6, MMP1 and MMP13 than vehicle-treated mice (Fig. 5). These results suggest that Nutlin-3a may attenuate CIA by inhibiting the production of pro-inflammatory cytokines and MMPs.
Fig. 4. Effect of Nutlin-3a treatment on the arthritis and cartilage damage. A. Clinical scores were assessed every 2 days, starting at 22 days after first immunization. B. synovial inflammation (H&E, × 200) and cartilage damage (safranin-O, × 200). Bar graphs represent histological scores as described in Materials and methods. Data were analyzed using one-way ANOVA and results are mean ± SEM from 10 mice/group. *P b 0.01 compared with vehicle-treated group.
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Fig. 5. Nutlin-3a treatment inhibited the production of pro-inflammatory cytokines and MMPs in CIA. On day 36, sera were collected from mice. Cytokines and MMPs in sera were measured by ELISA. Values are the mean ± SEM (n = 10 mice per group). *P b 0.01 compared with vehicle-treated group.
3.7. Nutlin-3a treatment inhibited MAPK and NF-κB pathways in CIA mice NF-κB is a well-known factor in regulating inflammation. As shown in Fig. 6, we found that MDM2 inhibition suppressed the activation of the MAPK pathway, including ERK1/2, p38 MAPK, and JNK in arthritic joints. MDM2 inhibition also suppressed the p-IκBα and the nuclear translocation of the NF-κB p65, indicating the inhibition of NF-κB signaling. These findings suggest that Nutlin-3a suppressed MAPK and NF-κB pathways in CIA mice. 4. Discussion MDM2 was first described to limit p53-mediated cell cycle arrest and apoptosis, hence, gain of function mutations is associated with malignancies [16]. In recent years, MDM2 inhibitors have been successfully obtained and advanced into clinical trials for the treatment of human cancers [16]. Recent evidence suggests that MDM2 has p53-independent activity in inflammatory diseases [17]. Nutlin-3a is a selective inhibitor of MDM2. Our data here showed that inhibition of MDM2 by Nutlin-3a prevented the development of arthritis and cartilage destruction in CIA. Further investigation showed that the protective effects of Nutlin-3a were mediated by inhibition of the MAPK and NF-κB pathways. MDM2 blockade has anti-inflammatory and anti-mitotic effects in inflammatory and autoimmune disorders [6,9,18–20]. MDM2 was identified to act as a co-transcription factor for NF-κB [6]. MDM2 plays an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques [18]. Inhibition of MDM2 reduced systemic inflammation and abrogated immune complex-mediated disease by suppressing plasma cells and autoantibodies in lupus nephritis [19]. Nutlin-3a also prevented tubular necrosis by suppressing sterile inflammation via a pro-inflammatory, p53-independent mechanism in acute kidney injury [9]. TSLP induces mast cell development and aggravates
Fig. 6. Nutlin-3a treatment inhibited MAPK and NF-κB pathways in the arthritic joints in CIA. The MAPK signaling molecules, including p-ERK1/2, p-JNK and p-p36, and the NFκB signaling molecules, including p-IκB-α and p65 NF-κB, were measured by Western blot. The fold increase relative to respective control (their total form, GAPDH or Lamin B) was shown. *P b 0.01 compared with vehicle-treated group. Values are mean ± SEM (n = 10 mice per group).
allergic reactions through the activation of MDM2 [20]. In the present study, we demonstrated that Nutlin-3a treatment reduced the synovial inflammation and bone destruction in CIA. Targeting MDM2 has therapeutic potential for human RA. Pro-inflammatory cytokines and MMPs play important roles in RA. IL-1β-stimulated RA-FLS have been widely used in vitro to mimic synovial proliferation in patients with RA, as IL-1β is believed to play a major role in synovial inflammation in RA [21]. IL-1β promotes inflammation and destruction in synovial tissue, bone and cartilage in RA patients [21,22]. IL-1β is also a pivotal cytokine in inducing expression of other inflammatory cytokines, such as TNF-α and IL-6 [23]. Previous data have shown that increased level of IL-1β in synovial tissue was correlated with histological features of arthritis [24,25]. IL-1β enhances the expressions of pro-inflammatory mediators and MMPs, leading to subsequent joint destruction through the activation of transcriptional factors such as AP-1 and NF-κB [26]. NF-κB plays a central role in the regulation of many genes that express proteins functioning in inflammation, including MMPs and IL-6 [27]. Persistent NF-κB activation contributes to the pathogenesis of human RA [28]. In addition to NF-κB, MAPK has also been considered as a therapeutic target for RA [29]. In line with the previous findings, this study showed that Nutlin-3a treatment caused a marked decrease in the expression of TNF-α and IL-6 in CIA. In addition, our study showed that Nutlin-3a treatment inhibited the activation of MAPK and NF-κB in arthritic joints in CIA. To further support the clinical significances of our study, we examined the human RA-FLS. And we found that MDM2 inhibition suppressed inflammation in IL-1β-treated RA-FLS via MAPK and NF-κB pathways. Our findings suggest that inhibition of MDM2 ameliorated joint inflammation and cartilage destruction in CIA, and these effects were mediated via down-regulation of MAPK and NF-κB pathways. MDM2 blockade has beneficial effects in CIA. MDM2 blockade may be a feasible strategy to control human RA. Conflict of interest All authors declare no conflicts of interest in this study. References [1] S. Roser-Page, T. Vikulina, M. Zayzafoon, Weitzmann MN.CTLA-4Ig-induced T cell anergy promotes Wnt-10b production and bone formation in a mouse model, Arthritis Rheumatol. 66 (2014) 990–999. [2] C. Sohn, A. Lee, Y. Qiao, K. Loupasakis, L.B. Ivashkiv, G.D. Kalliolias, Prolonged tumor necrosis factor α primes fibroblast-like synoviocytes in a gene-specific manner by altering chromatin, Arthritis Rheumatol. 67 (2015) 86–95. [3] J. Li, H.C. Hsu, Y. Ding, H. Li, Q. Wu, P. Yang, B. Luo, A.L. Rowse, D.M. Spalding, S.L. Bridges Jr., J.D. Mountz, Inhibition of fucosylation reshapes inflammatory macrophages and suppresses type II collagen-induced arthritis, Arthritis Rheumatol. 66 (2014) 2368–2379. [4] J.D. Oliner, K.W. Kinzler, P.S. Meltzer, D.L. George, B. Vogelstein, Amplification of a gene encoding a p53-associated protein in human sarcomas, Nature 358 (1992) 80–83. [5] Y. Yuan, Y.M. Liao, C.T. Hsueh, H.R. Mirshahidi, Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP, J. Hematol. Oncol. 4 (2011) 16. [6] D. Thomasova, S.R. Mulay, H. Bruns, Anders HJ.p53-independent roles of MDM2 in NF-κB signaling: implications for cancer therapy, wound healing, and autoimmune diseases, Neoplasia 14 (2012) 1097–1101. [7] L.T. Vassilev, B.T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong, U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi, E.A. Liu, In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, Science 303 (2004) 844–848. [8] T. Hashimoto, T. Ichiki, J. Ikeda, E. Narabayashi, H. Matsuura, R. Miyazaki, K. Inanaga, K. Takeda, K. Sunagawa, Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation, Cardiovasc. Res. 91 (2011) 711–719. [9] S.R. Mulay, D. Thomasova, M. Ryu, H.J. Anders, MDM2 (murine double minute-2) links inflammation and tubular cell healing during acute kidney injury in mice, Kidney Int. 81 (2012) 1199–1211. [10] D. Aletaha, T. Neogi, A.J. Silman, J. Funovits, D.T. Felson, C.O. Bingham 3rd, et al., 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/ European League Against Rheumatism collaborative initiative, Arthritis Rheum. 62 (2010) 2569–2581. [11] J.A. Singh, T. Arayssi, P. Duray, H.R. Schumacher, Immunohistochemistry of normal human knee synovium: a quantitative study, Ann. Rheum. Dis. 63 (2004) 785–790.
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