Heo et al. Arthritis Research & Therapy 2011, 13:R113 http://arthritis-research.com/content/13/4/R113 RESEARCHARTICLE Open Access The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1 Yu-Jung Heo1†, Hye-Jwa Oh1†, Young Ok Jung2*†, Mi-La Cho1,4*†, Seon-Yeong Lee1, Jun-Geol Yu1, Mi-Kyung Park1, Hae-Rim Kim3, Sang-Heon Lee3, Sung-Hwan Park1 and Ho-Youn Kim1 Abstract Introduction: The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-B (NF-B) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated. Methods: RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production. Results: RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1b (*P<0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-a in RA-FLS. The combined stimuli of both IL-17 and IL-1b significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1b alone (P<0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL- 17. Conclusions: RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL- 1b. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment. Introduction The receptor for advanced glycation end-products Rheumatoid arthritis (RA) is a systemic autoimmune (RAGE) is a novel receptor that binds products of none- disease characterized by chronic synovial inflammation, nzymatic glycation of proteins or advanced glycation which ultimately leads to the destruction of cartilage end-products (AGEs) [1]. AGEs are a heterogeneous and bone in the affected joints. Synovial hyperplasia is a group of irreversible products formed from the none- hallmark pathology of RA, and fibroblast-like synovio- nzymatic reaction of reducing sugars [2]. AGEs accumu- cytes (FLS) play a critical role in RA pathogenesis by late under a wide variety of biological conditions, such producing pro-inflammatory soluble factors or activating as diabetes, renal failure, aging, and inflammation [3]. other immune cells. The interaction of AGE and RAGE has been implicated in the activation of inflammatory signaling cascades and sequelae of AGE accumulation, such as diabetic compli- * Correspondence: [email protected]; [email protected] cations, amplification of inflammation, and tissue injury † Contributed equally 1The Rheumatism Research Center, Catholic Research Institute of Medical [3]. AGEs cannot be removed until the protein degrades, Science, The Catholic University of Korea, Seoul, 505 Banpo-dong, Seocho- and they alter tissue integrity and metabolism. Several gu, Seoul 137-040, South Korea receptors for the AGEs are known, and RAGE is a cen- 2Division of Rheumatology, Department of Internal Medicine, Hallym University Kang-Nam Sacred Heart Hospital, Seoul, 143-729, Korea tral signal transduction receptor for AGEs. RAGE is a Full list of author information is available at the end of the article © 2011 Heo et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Heo et al. Arthritis Research & Therapy 2011, 13:R113 Page 2 of 12 http://arthritis-research.com/content/13/4/R113 member of the superfamily of immunoglobulin type cell anti-rheumatic drugs) and the rheumatoid factor was surface receptors [4]. This receptor is strongly activated positive in five patients. ESR (erythrocyte segmentation by cross-linked AGE-modified proteins. The activation rate), and CRP (C-reactive protein) checked pre-opera- of RAGE results in activation of an inflammatory signal- tively were median 34 (range: 12 to 84) mm/hr and ing cascade, and up-regulation of RAGE is associated median 1.22 (range: 0.08 to 5.94) mg/dL respectively. with sustained cellular perturbation and tissue injury The diagnosis of RA was confirmed by the revised cri- [5]. Up-regulation of RAGE has also been reported teria of the American College of Rheumatology [18]. under various pathologic conditions, such as vascular Informed consent was provided according to the injury, diabetes, neurodegenerative disorders, and Declaration of Helsinki and obtained from all patients. inflammatory diseases [6]. Overexpression of RAGE is Approval by the ethical committee of the Seoul St. implicated in the pathogenesis of RA. RAGE is overex- Mary’s Hospital (Seoul, Korea) was obtained. pressed in synovial macrophages obtained from patients with RA, and synovial tissue cell culture supernatants Isolation and culture of FLS strongly induce cell surface RAGE [7]. The increased Synoviocytes were isolated by enzymatic digestion of level of RAGE pro-inflammatory ligands, such as high- synovial tissue specimens obtained from patients with mobility group box chromosomal protein 1 (HMBG-1) RA undergoing total joint replacement surgery. The tis- and S100/calgranulin in serum and synovial fluid in sue samples were minced into 2- to 3-mm pieces and patients with RA may contribute to RAGE up-regulation treated for four hours with 4 mg/ml type I collagenase [8,9]. (Worthington Biochemical Company, Freehold, NJ, Interleukin (IL)-17 and its major cell source, the type USA) in Dulbecco’s modified Eagle’s medium (DMEM) 17 T helper cells (Th17), have been implicated in the at 37°C in 5% CO2. Dissociated cells were then centri- pathogenesis of various inflammatory diseases [10,11]. fuged at 500 × g and resuspended in 10% fetal bovine IL-17 mediates inflammatory responses including angio- serum in DMEM. After an overnight culture, the non- genesis, recruitment of inflammatory cells, and induc- adherent cells were removed, and the adherent cells tion of pro-inflammatory mediators in endothelial and were cultured in DMEM supplemented with 20% fetal epithelial tissues [12]. An up-regulated Th17 response calf serum. Synoviocytes from passages 4 to 8 were used or increased IL-17 production is associated with the in each experiment. The RA-FLS were incubated with pathogenesis of autoimmune diseases and chronic IL-17, IL-1b,orTNF-a (R&D Systems, Minneapolis, inflammation, including RA [13,14]. IL-17 mediates cru- MN, USA) alone and in combination. To evaluate signal cial cross talk between the immune system and tissues. transduction, the RA-FLS were pretreated with 20 μM Signaling through IL-17 receptors on synoviocytes LY294002, 50 μM AG490, 10 μM SB203580, 20 μM induces immune cells to produce inflammatory factors PD98059, 10 μM parthenolide, or 10 μMcurcuminand such as IL-1 and IL-6 [15]. Many studies have been con- then treated with IL-17 for 12 h. The inhibitors were ducted regarding signaling molecules under IL-17 recep- purchased from Calbiochem (Schwalbach, Germany). tors, and nuclear factor-B (NF-B) activator 1 (Act1) is considered an essential protein for linking IL-17 recep- Immunohistochemistry of RA synovium and FLS tors and downstream signaling pathways. Act1 is a Immunohistochemical staining was performed on sec- recently identified 60-kD cytoplasmic adaptor protein tions of synovium. Briefly, the synovial samples were that activates IB kinase (IKK), liberating NF-Bfrom obtained from eight patients with RA and one patient its complex with IB [16]. with osteoarthritis (OA) and fixed in 4% paraformalde- We investigated whether pro-inflammatory cytokines, hyde solution overnight at 4°C, dehydrated with alcohol, including IL-1, tumor necrosis factor (TNF)-a,and washed, embedded in paraffin, and sectioned into 7-μm- especially IL-17, can induce RAGE expression and pro- thick slices. The sections were depleted of endogenous duction in RA-FLS. We also determined whether the sti- peroxidase activity by adding methanolic hydrogen per- mulatory effect of IL-17 on RAGE is mediated by Act-1. oxide (H2O2) and were blocked with normal serum for 30 minutes. After an overnight incubation at 4°C with Materials and methods goat anti-human RAGE, anti-Act1 antibody (Santa Cruz Patients Biotechnology, Santa Cruz, CA, USA) and antihuman IL- Human FLSs were isolated from synovial tissues from 17 antibody (R&D Systems), pS727-STAT3, p-AKT, and patients with RA (F/M 7/1, median age 56 (range 26 to p-C-Jun (Cell Signaling Technology, Danvers, MA, USA), 65)), and patients with OA (F/M 6/1, median age 64 the samples were incubated with the secondary antibo- (range 46 to 71)) at the time of knee-joint arthroscopic dies, biotinylated anti-goat IgG and anti-rabbit IgG for 20 synovectomy, as described previously [17]. The RA minutes. The sections were then incubated with strepta- patients were all taking DMARDs (disease modifying vidin-peroxidase complex (Vector Laboratories Ltd., Heo et al. Arthritis Research & Therapy 2011, 13:R113 Page 3 of 12 http://arthritis-research.com/content/13/4/R113 Peterborough, UK) for one hour followed by incubation GAT CAG GTC TTT G-3’ (antisense) in a LightCy- with 3, 3-diaminobenzidine (DAKO, Glostrup, Denmark). clerÔ (Roche Diagnostics, Mannheim, Germany). The The sections were counterstained with hematoxylin, and relative expression levels were calculated by normalizing the samples were photographed with a photomicroscope the targets to the endogenously expressed housekeeping (Olympus, Tokyo, Japan).