Inflammation ( # 2014) DOI: 10.1007/s10753-014-9837-x

SUMO-Conjugating Enzyme UBC9 Promotes Proliferation and Migration of Fibroblast-like Synoviocytes in Rheumatoid Arthritis Faxin Li,1,4 Xueyan Li,1 Liqing Kou,2 Yi Li,3 Fanhua Meng,1 and Fang Ma1

Abstract—Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Fibroblast-like synoviocytes (FLS) in the synovial tissues play critical roles in joint destruction. Recent studies implicate the sumoylation in the regulation of the inflammation and arthritis. Thus, we explored whether SUMO-conjugating enzyme UBC9 is involved in the progression of RA using a mouse collagen-induced arthritis (CIA) model. The effects of UBC9 siRNA on cell invasion and migration in human RA-FLS were also assessed in vitro. Treatment with siRNA against UBC9 for 3 weeks reduced the arthritis score and joint destruction. The expression of SUMO-1 and UBC9 protein in CIA joints was inhibited by UBC9 knockdown. Serum levels of anti-collagen (CII) antibodies, vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were also decreased in CIA mice. In vitro, UBC9 silencing inhibited the secretion of VEGF-A, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS. TNF-α-induced RA-FLS proliferation and migration were significantly attenuated by UBC9 knockdown. These findings indicate that SUMO-conjugating enzyme UBC9 promotes proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis. Inhibition of UBC9 activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating FLS proliferation, migration, and invasion. KEY WORDS: rheumatoid arthritis; collagen-induced arthritis; fibroblast-like synoviocytes; sumoylation, UBC9.

INTRODUCTION Rheumatoid arthritis (RA) is a common chronic inflammatory disorder characterized by excess synovial hyperplasia and progressive destruction of joint [1]. Multiple cell types, including macrophages, osteoclasts, and chondrocytes, are involved in the damaged joints of RA [2, 3]. However, accumulated evidence indicates that

Faxin Li and Xueyan Li contributed equally to this work. 1

Department of Rheumatology, Jinan Central Hospital Affiliated to Shandong University, No. 105 Jiefang RoadLixia DistrictJinan, 250013, China 2 Department of Radiology, Jinan Central Hospital Affiliated to Shandong University, Jinan, China 3 Department of Pharmacy, Jinan Central Hospital Affiliated to Shandong University, Jinan, China 4 To whom correspondence should be addressed at Department of Rheumatology, Jinan Central Hospital Affiliated to Shandong University, No. 105 Jiefang RoadLixia DistrictJinan, 250013, China. E-mail: [email protected]

activated fibroblast-like synoviocytes (FLS), present in great numbers in RA synovium, play a critical role in the development of pannus by migrating into cartilage and bone [4–7]. More importantly, FLS and T cells can interact with each other in antigen-dependent systems in vitro and in vivo, which is crucial for the progression of RA [8, 9]. Post-translational modifications of protein are important regulators of cell signaling pathways that regulate enzymatic activity and protein stability or subcellular localization [10]. Sumoylation is among these post-translational modifications. In contrast to other common protein modifications, such as phosphorylation, only a rather limited spectrum of substrates for sumoylation has been identified [11]. Sumoylation describes the covalent attachment of small ubiquitin-like modifiers (SUMO) to lysine residues of target proteins [12]. Besides modifying the activity of individual transcription factors, sumoylation of histones provides an additional epigenetic mechanism for SUMO proteins to modify cellular pathways [13]. In this study, we explored whether blockade of SUMO-conjugating enzyme UBC9 inhibits the development of collagen-induced

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Li, Kou, Li, Meng, Ma, and Li arthritis (CIA) model as well as the proliferation and migration of human RA-FLS.

MATERIALS AND METHODS Collagen-induced Arthritis Model Thirty-six 8-week-old male DBA/1 mice (Nanjing, China) were maintained under standard conditions. Mice were immunized with Freund’s adjuvant (Sigma Aldrich), Complete on day 21 and Incomplete on day 0, with 100 μg per mouse of bovine type II collagen (Chondrex) in a 1:1 mixture of adjuvant and collagen injected subcutaneously into the base of the tail. This protocol has been well characterized by Wooley et al. [14]. Animal experiment protocols were approved by the Animal Care and Use Committee of Jinan Central Hospital, Shandong University. In Vivo Treatments with siRNAs Against UBC9 in CIA Complexes of siRNA and atelocollagen (Boppard, China) were prepared as described before [15]. The siRNA/atelocollagen complex was formed by mixing siRNAs with atelocollagen, and these complexes were prepared in an injectable form. Duplexes of siRNAs against UBC9 (sense 5′-CAAUGAACCUGAUGAACUGUU-3′, antisense 5′-PCAGUUCAUCAGGUUC AUUGUU-3′) from Dharmacon Research (Lafayette, CO) were used. Non-targeting siRNA duplexes (Dharmacon Research) served as controls. Atelocollagen/siRNA complexes (0.5 mg/kg b.w.) were administered twice weekly for a total of 3 weeks, starting on day 25 after the first immunization, when clear onset of arthritis occurred. Arthritic Score Clinical arthritic score was performed every 3 days with the following scoring system [16]: 0=normal paw; 1= mild but definite swelling of either the ankle or digits; 2= moderate redness and swelling of an ankle±any number of digits; 3=maximal redness and swelling of the entire paw and digits with or without ankylosis. The maximum score per paw was 3 with a total score of 12 per mouse.

polyvinylidene difluoride membrane (Millipore). After blocking non-specific binding sites for 60 min with 5 % non-fat milk, the membranes were incubated with rabbit monoclonal antibody against SUMO-1 and UBC9 (1:1,000, Abcam), and GAPDH (1:1,000, Santa Cruz) at 4 °C over night, respectively. Then, membranes were washed with Tris buffer saline (TBS) containing 10 % Tween 20 (TBST) for three times, 15 min each time and then incubated with horseradish peroxidase (HRP)conjugated anti-rabbit secondary antibody (1:10,000, Santa Cruz) for 60 min at room temperature. The membrane was developed by an enhanced chemiluminescence system (ECL, Millipore) after washed with TBST for three times. Histological Analysis At the termination of experiment, hind limbs were fixed (4 % paraformaldehyde), decalcified, and embedded in paraffin. Serial 5-μm sections were cut and stained with hematoxylin and eosin (H&E) according to standard protocols for morphologic analysis. Sections were analyzed microscopically for the degree of inflammation and for cartilage and bone destruction according to the method reported previously [17]. The histological scores were determined as follows: 0, no signs of inflammation; 1, mild inflammation with hyperplasia of the synovial lining without cartilage destruction; and 2 through 4, increasing degrees of inflammatory cell infiltration and cartilage/bone destruction. Rheumatoid Arthritis (RA) Patients RA patients gave their written informed consent. All enrolled patients with active RA satisfied the 2010 American College of Rheumatology/European League Against Rheumatism criteria for RA [18]. Active RA was defined as ≥6 swollen joints (28-joint count), ≥6 tender joints (28-joint count), and at least 1 of the following: erythrocyte sedimentation rate greater than the upper limit of normal (ULN) for the local laboratory or C-reactive protein level greater than the ULN for the central reference laboratory (>8.0 mg/L). Cell Culture

Western Blot The frozen joint tissue samples were homogenized in a RIPA buffer (Qiagen, China). After centrifugation at 15,000 rpm, 4 °C for 10 min, 60 μg of protein samples was run on a 12.5 % SDS–PAGE gel and transferred to

Fibroblast-like synoviocytes (FLS) were obtained from the synovium of active anti-citrullinated protein antibodies-positive RA patients (n=15) during knee joint arthroscopy according to the approval of the Ethics Committee of Jinan Central Hospital, Shandong

SUMO/UBC9 in Rheumatoid Arthritis University. FLS were isolated from synovial tissues by enzymatic digestion as previously described [19]. FLS were grown in Dulbecco’s modified Eagle’s medium (GIBCO, USA) medium containing 10 % fetal bovine serum (FBS), supplemented with antibiotics (100 mg/ml streptomycin and 100 U/ml penicillin) in a humidified incubator at 37 °C under 5 % CO2. Cells used for experiments were at the third to sixth passage. Gene silencing In Vitro Repression of UBC9 expression was achieved using UBC9 siRNA and non-targeting control (Dharmacon Research). Cells were electroporated with siRNA using the Amaxa nucleofection system as previously described [20, 21]. Transfected synoviocyte cells were seeded in 6well plates at 2×106 cells/well. Cells were cultured for 48 h post-transfection. Verification of UBC9 protein ablation was achieved using Western blot analysis. ELISA Mice were killed at the end of experiment, and sera were analyzed for anti-collagen (CII) IgG, IgG1, and IgG2a antibody levels by ELISA (Chondrex, USA) according to standard protocols by the manufacturer. The serum levels of vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were determined using specific ELISA Kits (R&D Systems) as per the instructions. For the measurement of VEGF-A, MMP-3, and MMP-9 levels in the supernatants, RA-FLS cells were seeded at 2×106/ml and pretreated with UBC9 siRNA for 24 h followed by 48-h stimulation with human TNF-α (20 ng/ml, eBioscience). Cell supernatants were centrifuged to avoid any cell debris prior to ELISA analysis. In order to determine the levels of VEGF-A, MMP-3, and MMP-9, ELISA was performed per manufacturer’s instructions (Abnova). BrdU-Labeled Cell Proliferation Assay A BrdU kit (Amersham Biosciences, USA) was used to measure the incorporation of BrdU during DNA synthesis according to the manufacturer’s protocols. Briefly, after treatment with TNF-α for 72 h, BrdU (10 μM) was added to the culture medium for 2 h, the BrdU-labeled cells were fixed, and the DNA was denatured in fixative solution for 30 min at room temperature. The cells were then incubated with peroxidase-conjugated anti-BrdU antibody for 2 h at room temperature, followed by washing three times with

washing solution. The immune complex was detected using a 3,3′,5,5′-tetramethylbenzidine substrate reaction, and absorbance was measured at 490 nm.

Cell Migration and Invasion Assay FLS cells were seeded at a density of 4×103 cells/well in a 96-well plate. After 48 h of UBC9siRNA infection, the cells formed a fluent monolayer and were observed under a fluorescent scope. A linear scratch was formed using a 10 μl pipette tip at 120 h after infection. Wounded monolayers were washed with PBS to remove detached cells and debris. Transwell migration assays were performed using a 24-well Boyden chamber (6.5 mm diameter, 8.0 μm; BD) according to the manufacturer’s instructions. In brief, FLS cells were infected and cultured for 48 h. Cells were then harvested by trypsinization, washed, and resuspended in serum-free media at a density of 2×104 cells/well. A 100 μl cell suspension was placed onto the upper, and the lower chambers of the transwell were filled with 500 μl of media containing serum as an adhesive substrate. Cells were incubated at 37 °C under 5 % CO2 for 48 h, and then non-migrating cells on the upper side of the membrane were removed with a cotton swab. Migrating cells on the lower side of the membrane were fixed with 4 % methanol for 20 min and stained with 0.1 % crystal violet for 1 min. Photomicrographs of five random fields were obtained (original magnification, ×100), and cells were counted to calculate the average number of cells that had migrated. For the in vitro invasion assay, similar experiments were performed using inserts coated with a Matrigel basement membrane matrix. The Matrigel (Matrigel basement membrane matrix, Becton Dickinson, USA) was diluted in serum-free cold media and placed into upper chambers of a 24 well Transwell and incubated at 37 °C for 1 h. Cells were resuspended with serum-free media at a density of 5×104 cells/well and incubated for 48 h to evaluate cell migration. All experiments were performed in duplicate.

Data Analysis Statistical analysis of data was performed using SPSS16.0 and GraphPad Prism 6.0 software. Data points and the bars represent the mean±SEM of three independent determinations. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Significance is expressed as p