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BxN serum-induced arthritis
International Immunopharmacology 10 (2010) 1170–1176
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Critical role for apoptosis signal-regulating kinase 1 in the development of inflammatory K/BxN serum-induced arthritis Stephen J. Mnich, Patrick M. Blanner, Liangbiao G. Hu, Alexander F. Shaffer, Fernando A. Happa, Shawn O'Neil, Okechukwu Ukairo, Dave Weiss, Eric Welsh, Chad Storer, Gabriel Mbalaviele, Hidenori Ichijo 1, Joseph B. Monahan, Medora M. Hardy, Hiroyuki Eda ⁎ Discovery Biology, Global Research and Development, St. Louis Laboratories Pfizer Inc., 700 Chesterfield Parkway West, mail zone AA3C, Chesterfield, MO 63017, USA
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Article history: Received 31 May 2010 Accepted 22 June 2010 Keywords: Apoptosis signal-regulating kinase 1 (ASK1) Rheumatoid arthritis Mitogen-activated protein kinase (MAPK) p38 MAPK c-jun N-terminal kinase (JNK) K/BxN serum transfer model
a b s t r a c t In this report, we show that apoptosis signal-regulating kinase 1−/− (ASK1 KO) mice were resistant to inflammatory arthritis induced in the K/BxN serum transfer model of rheumatoid arthritis (RA). The p38 inhibitor, SD-0006 was administered to wild type (WT) mice as a comparator. Both ASK1 KO and p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses. Transcriptional profiling of mRNA prepared from paw tissue demonstrated that the production of many proinflammatory genes including cytokines, chemokines, and extracellular matrix degradative enzymes were maintained at basal levels by either ASK1 KO or prophylactic p38 MAPK inhibition. In the mouse whole blood (MWB) assay, tumor necrosis factor-α (TNF-α)-induced KC and CCL2 levels and also LPS-induced interleukin-6 (IL-6), CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Furthermore, both p38 and JNK were activated by TNF-α in human synovial fibroblasts isolated from RA patients (RASF). SD-0006 or SP600125, a JNK inhibitor, partially blocked the elevation of IL-6 production in RASF following stimulation with TNF-α. In contrast, dual inhibition with both p38/JNK inhibitors almost completely abolished TNF-α-induced IL-6 production from these cells. Ablation of ASK1 expression in RASF using siRNA for ASK1 resulted in inhibition of TNF-α-induced IL-6 and PGE2 production. This study is the first to suggest that ASK1 is critical for the development of RA and that ASK1 may be involved in the production of proinflammatory mediators in response to TNF-α stimulation in the RA joint. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Mitogen-activated protein kinases (MAPKs) have critical roles in the regulation of key cellular process including cell survival and apoptosis, proliferation, and inflammatory responses. Especially, activation of these MAPK pathways leads to the production of inflammatory mediators, such as cytokines and chemokines which are involved in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis [1]. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase kinase kinase (MAP3K) family that activates p38 MAPK and c-jun N-terminal kinase (JNK) via activating the MAP kinase kinase (MAP2K), MKK3/MKK6 and MKK4/MKK7, respectively [2,3] ASK1 is activated in response to various stresses, such as calcium influx, endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and oxidative
⁎ Corresponding author. Exploratory Research Laboratories, Research Center, AJINOMOTO PHARMACEUTICALS CO., LTD., 1-1, Suzuki, Kawasaki, Kanagawa, 2108681, Japan. Tel.: +81 44 210 5824; fax: +81 44 210 5875. E-mail address: [email protected] (H. Eda). 1 Laboratory of Cell Signaling, The University of Tokyo, Japan. 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.06.023
stress [4–7], and therefore ASK1 is involved in a variety of cellular functions, such as immune response. ASK1 is involved in LPS-induced cytokine production; LPS-induced production of IL-6, TNF-α, IL-1β, and IL-12 was impaired in ASK1−/− dendritic cells and splenocytes as well as macrophages [7,8]. Recent evidence has shown that ASK1 is closely linked to the pathogenesis of a variety of apoptosis-, immune-, and stress-related diseases, such as neurodegenerative diseases and autoimmune diseases [9–12]. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial cytokine production, infiltration of the synovium with immune cells, and joint destruction [13,14]. Cytokines including IL-1, IL-17, and TNF-α and downstream inflammatory mediators such as prostaglandins were produced from infiltrated cells [13,14]. Recent evidence reveals that not only infiltrated immune cells, but also RA synovial fibroblasts (RASFs), in the inflamed synovium are engaged in the initiation and perpetuation of RA and produce a variety of cytokines and other inflammatory mediators that mediate the interaction with infiltrated immune cells and other cells [15]. The production of these cytokines and inflammatory mediators from infiltrated immune cells and RASF are regulated by MAPKs [16– 18]. Therefore, MAPKs, especially p38 MAPK, is considered to be
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potential therapeutic targets for RA because p38 MAPK plays a crucial role in the regulation of IL-1 and TNF-α synthesis as well as IL-1stimulated IL-6 and IL-8 production and induction of other mediators such as matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) [1]. Indeed, a number of p38 MAPK inhibitors have been developed for the treatment of RA; however, none has yet made it to the market. The failure of p38 MAPK inhibitors in the clinic suggests the importance of other downstream pathways playing critical roles in the pathogenesis of RA and it has not yet been fully elucidated. In the current study, we investigated the role of ASK-1 in the K/ BxN serum transfer inflammatory arthritis model in mice and found that ASK1−/− mice were resistant to the edema and the infiltrations of various types of inflammatory cells into the ankle joints induced in this model. Furthermore, we have demonstrated that ASK1 plays a crucial role in the proinflammatory response to TNF-α in human RASF.
histological examination was scored as follows; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Scoring examination was conducted in a double blinded manner.
2. Materials and methods
2.5. Isolation of total RNA from the joint and amplification of RNA
2.1. Animals
Right-sided hind paws, sectioned just proximal to the ankle joint, were individually flash-frozen in liquid nitrogen and stored at −80 °C. Frozen tissues were powdered using SPEX Certiprep 6750 Freezer mill (SPEX CertiPrep, Middlesex, UK) and 6753 microvials (Wolf Laboratories, York UK). Pulverized tissues were lysed in TriZol (Invitrogen, Carlsbad, CA) and extracted with chloroform and then phenol: chloroform:isoamylalcohol (Applied Biosystems/Ambion, Austin, TX). Four volumes of Zymo RNA binding buffer (Zymo Research, Orange, CA) was added into an aqueous phase of sample and applied to Zymo RNA spin column to purify RNA. Total RNA integrity (size and quality) was assessed with an Agilent HP2100 Electrophoresis Bioanalyzer (Agilent Technologies, Santa Clara CA), and quantified using a NanoDrop 1000 UV spectrophotometer by measuring the absorbance at A260 and A280 nm (Thermo Fisher Scientific, Waltham MA). Typical yields ranged from 200 to 800 ng/μl. 10 ng of total RNA from each paw was amplified for two rounds using a modified amplification protocol (Message Amp II aRNA Amplification Kit, Ambion, Austin TX).
C57BL/6 ASK1−/− (ASK1 KO) mice were provided by Dr. Hidenori Ichijo (Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, the University of Tokyo) and bred in Charles River Laboratories International (Wilmington, MA) [19]. Age-matched female wild-type (WT) mice on the same genetic background (C57BL/6) were purchased from Charles River Laboratories International. Animals were acclimatized to the laboratory conditions for several days before the start of the experiments. The St. Louis Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. 2.2. K/BxN serum transfer arthritis model and treatment Development of K/BxN serum transfer induced arthritis model was developed by injection of K/BxN serum to recipient mice. Briefly, K/ BxN serum (200 μl/mouse, Charles River Laboratories) was injected intraperitoneally into both female ASK1 KO (n = 24) and WT mice (n = 24) on day 0. Progression of disease was assessed by measuring the joint swelling on day 4, 5, 7, 10, and 12 after K/BxN serum transfer. Orally available specific p38 MAPK inhibitor [20,21] at a dose of 30 mg/kg was administered orally to WT mice as a comparator from day 0 to 12 (qd). Hind paws were collected for histological examination and microarray analysis on days 3, 4, 7, and 12, or on days 3, 4, and 7, respectively. All experiments were conducted in a double-blinded manner. 2.3. Histology Immediately following the necropsy, the left hind paw (including ankle) was fixed in 10% Neutral Buffered Formalin (NBF) for 24 h at 20 °C, followed by decalcification in Immunocal™ (Decal Chemical Corporation, Tallman NY) for 7 days at 20 °C. Decalcified joints were then processed and paraffin-embedded with dorsal aspect down for frontal view, sectioned twice (4 μm each), and stained with hematoxylin and eosin (H&E) for general evaluation or toluidine blue for specific assessment of cartilage changes. Cumulative histological score was determined according to a sum of three major parameters; 1) general inflammation including synovial epithelial changes, edema, inflammatory cell infiltrations, and inflammation; 2) cartilage damage including cartilage matrix changes, degeneration and/or necrosis of chondrocytes, ulceration and loss of cartilage; 3) bone resorption including reactions of osteoclasts, cortical bone loss and necrosis of osteocytes, etc. Each
2.4. Measurement of cytokine production from mouse whole blood Whole blood from WT and ASK1 KO mice was collected into sodium heparin tubes and used immediately. Whole blood was incubated with mouse TNF-α (10 ng/ml; R&D systems, Minneapolis, MN) or LPS (10 μg/ml; Sigma Aldrich, St. Louis, MO) for 20 h. Then, plasma was collected by centrifugation and used for further evaluation. Cytokine and chemokine levels in plasma were determined using MSD MULTI-ARRAY and MULTI-SPOT kits (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer's directions.
2.6. Preparation and analysis of Agilent whole mouse genome arrays Amplified RNA (aRNA) was labeled using a modified protocol with the Kreatech Universal Linkage System (ULS) aRNA Fluorescent Cy5 labeling kit (Kreatech Diagnostics, Amsterdam, The Netherlands); specifically, 2 μl of labeling buffer and 4 μl of ULS were added to 5 μg of aRNA in a total volume of 20 μl. Once combined, samples were incubated for 15 min at 85 °C and after incubation samples were returned to room temperature and spun down to collect condensate. Labeled samples were purified using Zymo Research RNA Clean and Concentrate spin columns according to manufacturer's protocol (Zymo Research Corporation, Orange CA). Samples were eluted in 100 μl of RNase/DNase-free water. Labeled samples were quantified on the Nanodrop ND-1000 spectrophotometer. Each labeled aRNA (750 ng) from a comparison was added to a total volume of 230 μl water and 9 μl of fragmentation buffer (Agilent Technologies, Santa Clara, CA). Hybridization mixtures were then incubated for 30 min at 60 °C. Hybridization mixtures were cooled to room temperature and spun down to collect condensate. 2× Hi-RPM hybridization buffer (240 μl; Agilent Technologies) was added to each mixture. These probes were then hybridized to 4 × 44 K mouse Whole Genome Microarrays for 18 h at 65 °C in Sure-Hyb Hybridization chambers (Agilent Technologies). Chambers were disassembled in a 0.6× SSC 0.005% Triton X-102 wash bath at room temperature and washed for 10 min in wash bath 1. Microarrays were transferred to a 0.01× SSC 0.005% Triton X-102 wash for 5 min at 40 °C. Microarrays were then dried with HEPA filtered compressed nitrogen and scanned on the Agilent Technologies DNA Microarray Scanner at 5 μm resolution.
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Data were extracted from the scanned image using Agilent Technologies Feature Extraction Software version 9.5. 2.7. Data normalization Signal normalization against a common sample was performed using a smoothed piecewise linear curve, trained on the log intensities that differed by b10% rank order between two samples. Local background subtraction was performed on each array prior to normalization. A computational normal pool was created from the averaged normalized signals of the individual normal animal arrays. Within the normal arrays, a median array was selected as least distant from the other normal arrays. All normal arrays were normalized against the median array prior to calculating the averaged normalized
signals of the pool. Technical replicates for individual samples were treated in a similar fashion, choosing a median array within each group of replicates, and flagging spots with a coefficient of variation ≥0.7 as bad data. A final normalization was performed on all averaged signals vs. the computational normal pool. Fold changes between samples were calculated, raising weak signals to a minimum value of 10 prior to comparison. 2.8. In vitro cellular assay Human rheumatoid arthritis synovial fibroblast cultures (RASF) were established from the synovial membrane of a female RA patient and cultured in Dulbecco's Modified Eagle Medium (Invitrogen) containing 15 % FBS. Cells were preincubated with SD0006 (3 μM)
Fig. 1. Ablation of ASK1 is efficacious in the K/BxN serum transfer arthritis model. ASK1 KO and WT mice were injected with K/BxN serum to induce arthritis. Progression of disease was assessed by joint swelling (A), cumulative inflammation score (B), and H&E stain on day 12 (C). Data in graph A and B were indicated by mean ± S.E.M (WT: n = 6, WT + p38i: n = 6, ASK1 KO: n = 10). p38i: p38 MAPK inhibitor (SD-0006: 30 mg/kg, qd, po). Cumulative histology score is indicated by the sum of three parameters; general inflammation (H&E stain), cartilage damage (trichrome stain), and bone resorption. 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. *: p b 0.001 vs. WT (one-way ANOVA on Turkey– Friedman Square Root, one tailed analysis).
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and/or JNK inhibitor (SP600125, 3 μM: TOCRIS, Ellisville, MO) for 1 h, followed by stimulation with 10 ng/ml human TNF-α (R&D systems) for 18 h. IL-6 production from the cells was determined using MSD MULTI-ARRAY-MULTI-SPOT kit (Meso Scale Discovery) and prostaglandin E2 (PGE2) production was determined using PGE2 EIA (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions or PGE2 ELIA established in house [22]. 2.9. Western blot analyses Western Blot analysis for evaluation of MAPK signaling pathways was performed using cells lysates prepared after stimulation with TNF-α for 30 min. Cells were lysed using RIPA buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, phosphatase inhibitor cocktails I and II, 100 μg/ml PMSF (Sigma Aldrich) and Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Whole cell protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to PVDF membrane (Invitrogen). Membranes were incubated with primary antibodies for overnight at 4 °C. The following primary antibodies were used for detection of protein of interests: antibody to human ASK1 (Epitomics, Burlingame, CA), human total JNK and p38 (Santa Cruz Biotechnology, Santa Cruz, CA), human phosphorylated MKK3/6, human phosphorylated HSP-27, human phosphorylated MKK4, human phosphorylated c-Jun human total and phosphorylated Erk (Cell Signaling Technology, Danvers, MA). After incubation for 1 h with the appropriate fluorescent-labeled secondary antibodies, proteins of interest were detected using Odyssey infrared imaging System (LiCor, Lincoln, NE). 2.10. Small interfering RNA (siRNA) study and quantitative real-time polymerase chain reaction (qRT-PCR) assay Validated Stealth ASK1 and ASK2 siRNA duplexes were obtained from Invitrogen. RASF were transfected with siRNA at a concentration of 15 nM using DharmaFect 1 transfection reagent (Thermo Scientific, Waltham, MA). At 72 h post transfection, cells were stimulated with 10 ng/ml TNF-α (R&D) for 8 h. The effect of ASK siRNA was determined using qRT-PCR. In qRT-PCR assay, cells were lysed with Nucleic Acid Purification Lysis Solution (Applied Biosystems, Wamington, UK). Total RNA was isolated using 6100 Nucleic Acid PrerStation according to the manufacturer's directions (Applied Biosystems). qRT-PCR was conducted using qSCriptTM One-Step qRT-PCR kit (Quanta BIOSCIENCES, Gaithersburg, MD). Amplified reactions were quantified on an ABI PRISM 7900 Sequence detection system (Applied Biosystems). Relative gene quantities were obtained using the comparative Ct method after normalization to appropriate control genes (cyclophilin A: NM_021130.3). TaqMan probes were also purchased from Applied Biosystems. 3. Results 3.1. ASK-1 deficiency attenuates arthritis, cartilage destruction, and bone erosion To determine whether ASK-1 has a critical role in the synovial inflammation and joint destruction, ASK-1 KO and WT were investigated in the K/BxN serum transfer arthritis model. As a comparator, a specific p38 MPK inhibitor was administered orally to WT. Fig. 1A and B shows the ankle thickness and cumulative histological score of mice injected with K/BxN serum. ASK1 deficiency significantly decreased joint swelling and cumulative histological score compared to those of WT from day 4 to the end of the study (day 12) (p b 0.001) and the decrease in these arthritis severity scores of ASK1 KO was comparable to those of mice treated with the p38 inhibitor. Ankle histopathology of WT showed significantly severe
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inflammation including indicated by synovial epithelial changes, edema, inflammatory cell infiltrations compared to those of WT without K/BxN serum injection. Also, cartilage degradation and bone erosion was observed (Fig. 1C). In contrast, ankle histology from ASK1 KO demonstrated markedly less inflammation and no cartilage degradation and bone erosion. 3.2. Comparison of transcriptional gene profiling of mouse paw from WT with those from ASK1 KO Transcriptional profiling of mRNA prepared from pulverized paw tissue was investigated to determine the proinflammatory gene expression in WT, ASK1 KO, and WT treated with p38 inhibitor (Table 1). Transcriptional profiling demonstrated that the production of many proinflammatory genes including cytokines (IL-6 and IL-1β), chemokines (CXCL3, CXCL5, KC, CCL2, CCL3, CCL7, and CCL8), matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase (MMP-3 and TIMP-1) and cell surface marker (FcγRI, FcγRIII, and F4/ 80) were dramatically increased in K/BxN serum-injected WT compared to those of WT without K/BxN serum injection. On the other hand, expression of these inflammatory cytokines/chemokines, MMPs and cell surface marker was decreased in K/BxN seruminjected ASK1 KO as well as K/BxN serum-injected WT treated with p38 inhibitor. 3.3. Induction of cytokine level by inflammatory mediators in mouse whole blood Induction of cytokine or chemokine by TNF-α and LPS was investigated using whole blood collected from WT and ASK1 KO mice (Fig. 2). IL-1β, IL-6, TNF-α, CCL2, and KC levels in MWB from both WT and ASK1 KO were significantly elevated by LPS while IL-1β was elevated by TNF-α. TNF-α-induced KC and CCL2 levels and also LPS-induced IL-6, CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Interestingly, IL-1β, IL-6, and KC levels in naïve MWB from ASK1 KO were significantly lower than those from WT. 3.4. The role of ASK1 in the production of IL-6 and PGE2 from human RASF stimulated with TNF-α To understand the role of ASK1 in human RASF, the role of p38 MAPK and JNK in the production of IL-6 and PGE2 in RASF stimulated with TNF-α. TNF-α-stimulated RASF produced IL-6 and PGE2 and the production was inhibited by both p38 MAPK inhibitor and JNK Table 1 Comparison of transcriptional gene profiling of mouse paw from WT with those from ASK1 KO on day 7. Gene
WT
ASK1 KO
WT+SD-0006
CXCL5 CXCL3 CCL7 CCL2 CCL8 CCL3 KC IL-6 IL-1β TNF-α TIMP-1 MMP3 FcγRI FcγRIII F4/80
40.9 15.0 12.6 7.6 6.0 5.8 5.5 19.0 13.9 1.6 11.9 10.2 9.1 6.0 3.6
2.1 2.2 2.7 2.4 1.8 1.2 1.3 2.9 1.5 0.9 1.2 1.3 1.6 1.2 1.2
3.5 4.2 3.5 2.6 1.4 1.6 1.9 4.6 2.5 0.9 1.5 1.8 2.0 1.3 1.2
Data (n = 2: WT and WT + SD-0006, n = 6: ASK1 KO) are indicated by fold change of gene expression from that of treatment naïve (no K/BxN serum injection). SD-0006: specific p38 MAPK inhibitor (30 mg/kg, qd, po).
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Fig. 2. Induction of cytokine level by TNF-α or LPS in mouse whole blood. Mouse whole blood (MWB) collected from ASK1 KO and WT was treated with mouse TNF-α (10 ng/ml) or LPS (10 μg/ml) for 20 h. Data were indicated by mean ± S.E.M (n = 3). *: p b 0.05 vs. CTRL; **: p b 0.01 vs. CTRL; ***: p b 0.001 vs. CTRL (one-way ANOVA with Dunnett). #: p b 0.05 vs. WT; ##: p b 0.01 vs. WT (two-way ANOVA with Bonferroni).
inhibitor (Fig. 3A and B). Dual inhibition of p38 MAPK and JNK resulted in the enhanced inhibition of IL-6 and PGE2 production in RASF, indicating that ASK1 may have a role in TNF-α-induced IL-6 and PGE2 production in human RASF. Inhibition of IL-6 and PGE2 production by these inhibitors was accompanied by inhibition of p38 and JNK, phosphorylation as well as downstream activation of heat shock protein 27 (HSP27) and c-jun (Fig. 4). To confirm the role of ASK1 in RASF activation, we investigated whether TNF-α-induced IL-6 and PGE2 production was decreased by ablation of ASK1 gene using ASK1 siRNA in RASF. ASK1 mRNA level was decreased by up to 75% by ASK1 siRNA (Fig. 5A). Decreased ASK1
protein expression in ASK1 siRNA-treated cells was confirmed by Western blot analysis (data not shown). Importantly, ASK1 gene ablation in RASF resulted in a significant decrease in TNF-α-induced production of IL-6 and PGE2 compared to those treated with scramble siRNA (Fig. 5B). 4. Discussion In this study, we have demonstrated that ASK1 is critical for the progression of disease in the mouse K/BxN serum transfer model of RA. These data suggest that ASK1 is involved in the production if
Fig. 3. Dual inhibition of p38 MAPK and JNK results in the enhanced inhibition of IL-6 (A) PGE2 (B) production in human RASF. Human RASF were preincubated with 3 μM p38 MAPK inhibitor (p38i: SD-0006), SP600125 (JNKi), and combination of these two compound (Combo) for 1 h, followed by stimulation with 10 ng/ml human TNF-α for 18 h. Data were indicated by mean ± S.D. of 2 independent assay (n = 3). *: p b 0.05 vs. Combo (one-way ANOVA with Dunnett).
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Fig. 4. Phosphorylation status of downstream signaling of p38 MAPK and JNK. Human RASF were preincubated with 3 μM p38 MAPK inhibitor (p38i: SD-0006) or SP600125 (JNKi) for 1 h, followed by stimulation with 10 ng/ml human TNF-α for 30 min. pMKK3/6: phosphorylated MKK3/6; p-HSP27: phosphorylated heat shock protein 27 (HSP27); p-MKK4: phosphorylated MKK4; JNK Total (green); p-c-jun: phosphorylated c-jun (red); p-ERK: phosphorylated extracellular signal-regulated kinase (ERK; red). ERK Total (green).
proinflammatory mediators in response to TNF-α stimulation in the RA joint. Terauchi et al. reported that arthritis was significantly promoted by transduction of the constitutively active form of ASK1 in rat collagen induced arthritis (CIA) model [23]. In that study, introduction of constitutively active ASK1 into the joint promoted ankle swelling, as well as enhanced infiltration of inflammatory cells into the synovial membrane. Our results using ASK1 KO mice are consistent with this report and provide strong evidence that ASK1 plays a role in the pathogenesis of arthritis.. In our study, ASK1 KO mice showed marked attenuation of joint edema, cartilage damage, bone absorption, and inflammatory cell infiltration in the K/BxN serum transfer model of RA. Furthermore, in the mouse CIA model of arthritis, we observed that the incidence and severity of arthritis was significantly lower in ASK1 KO mice than those in WT (data not shown). These data indicate that ASK1 has a critical role in the progression of collagen- or K/BxN serum-induced RA in rodents. Transcriptional gene profiling of paw from ASK1 KO showed that expression of inflammatory cytokines and chemokines, cellular surface markers, and extracellular degradative enzymes were decreased in ASK1 KO compared to those in WT. Down-regulation of the Fcγ receptors (FcγRs) which have a critical role in the development of
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paw swelling in this model [24], may be one factor that is responsible for reduction of these key pro-inflammatory mediators in the ASK1 KO. FcγRs are expressed on immune cells such as macrophages, mast cells, and neutrophils as well as fibroblast-like synoviocytes [25–28]. FcγR activation, especially predominantly FcγRIII, leads to the production of inflammatory cytokines, chemokines as well as degradative enzymes from macrophages, neutrophils, and mast cells, resulting in inflammation and arthritis in K/BxN serum transfer model in mice [27,28]. In fact, FcγRIII−/− mice exhibited a slower progression of arthritis in K/BxN serum transfer model [29]. However, we cannot exclude the possibility that an increase in signal intensity can be observed due to cellularity changes in tissues tested. Immune cells basally expressing several relatively selective transcripts that infiltrate into a tissue will cause an increase in the signal intensity for those selective transcripts, independent of any upregulation of those mRNAs. Therefore, by profiling whole paw, gene changes as well as cellular infiltration events are both represented. Further investigation is needed to elucidate whether the infiltration of immune cells expressing FcγRs are decreased in K/BxN study using ASK1 KO. In this model, inflammatory cells, especially neutrophils and mast cells, are required as effecter cells to mediate joint destruction [30,31]. Infiltration of these inflammatory cells into the joint may be suppressed in ASK1 KO mice since the expression level of CC and CXC chemokines was much lower than those in WT. Indeed, histological examination showed that few infiltrated inflammatory cells were seen in the joint of ASK1 KO compared to WT. Furthermore, inflammatory mediator-induced CCL2 and KC production from mouse whole blood in ASK1 KO were significantly lower than those in WT in this study. We also observed that the induction level of chemokines (KC and MCP-1) by LPS in granulocytes from ASK1 KO mice was significantly lower than those from WT in mice (Eda H, in preparation). It has been reported that chemokine production from splenocytes, dendritic cells, or endothelial cells in ASK1 KO was lower than that in WT [7,32]. Osaka et al. reported that gene expression of chemokines in wounded skin samples from ASK1 KO was significantly lower than those in WT [33]. Collectively, these results indicate that ASK1 may be involved in the chemotaxis of the inflammatory cells via regulating the chemokine production. However, there is no report describing the association of ASK1 with chemotaxis at present time. Whether ASK1 has a major role in the production of chemokines and regulates chemotaxis remains to be investigated. Consistent with a role for ASK1, dual inhibition of p38 MAPK and JNK resulted in enhanced inhibition of IL-6 and PGE2 production in TNF-α-stimulated cultured RASF. MAPKs have been implicated in inflammatory diseases. In particular p38 MAPK is thought to play a critical role in RA because it is activated in the rheumatoid synovial intimal lining and p38 MPK inhibitors show effects on RA models [34–
Fig. 5. ASK1 gene ablation inhibits IL-6 and PGE2 production in human RASF stimulated with TNF-α. A: Relative quantity of ASK1 mRNA expression. mRNA was collected at 72 h post transfection and relative gene quantities were obtained using the comparative Ct method after normalization to human control gene (cyclophilin A: NM_021130.3). Data [mean ± S.D. (n= 3)] were indicated by % of control (non-treatment). Scramble: non-targeted scramble siRNA (Negative CTRL). B: At 72 h post transfection of siRNA, RASF were stimulated with 10 ng/ ml TNF-α for 8 h. Data were indicated by mean ± S.D. of 2 independent assay (n= 3). *: p b 0.05 vs. Scramble siRNA treatment (one-way ANOVA with Dunnett).
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36]. In fact, in this study, p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses that are indistinguishable from those observed in ASK1 KO. On the other hand, JNK also appears to play a role in RA. JNK is thought to be important in extracellular matrix degradation. JNK is a critical MAPK pathway for IL-1-induced collagenase gene expression and regulation of MMP secretion in RASF [37–39]. Furthermore, JNK-1deficient osteoclast progenitors were unable to mature into bone-resorbing osteoclasts following exposure to receptor activator of nuclear factor-κB ligand (RANKL) [40]. Although JNK inhibitors have been reported to have marginal effect on the joint inflammation and destruction [41,42], this study showed that either JNK inhibition alone or combination with the p38 MAPK inhibition resulted in decreased TNF-α-stimulated production of IL-6 and PGE2 in RASF. These data indicate that JNK signaling contributes to the production of cytokines and inflammatory mediators. In conclusion, we demonstrated that ASK1 is critical for the progression of disease in the mouse K/BxN serum transfer model of RA. Furthermore, our findings in ASK1 KO mice and human RASF suggest that ASK1 is involved in the production of proinflammatory mediators in RA. Thus, inhibition of ASK1 upstream signaling of p38 MAPK and JNK, may provide a novel therapeutic intervention for RA.
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Arterioscler Thromb Vasc Biol 2005;25:1877–83. [33] Osaka N, Takahashi T, Murakami S, Matsuzawa A, Noguchi T, Fujiwara T, et al. ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds. J Cell Biol 2007;176:903–9. [34] Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12. [35] Mclay LM, Halley F, Souness JE, McKenna J, Benning V, Birrell M, et al. The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral antiarthritic efficacy. Bioorg Med Chem 2001;9:537–54. [36] Hope HR, Anderson GD, Burnette BL, Compton RP, Devraj RV, Hirsch JL, et al. Antiinflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogenactivated protein kinase: preclinical-to-clinical translation. J Pharmacol Exp Ther 2009;331:882–95. [37] Han Z, Boyle DL, Aupperle KR, Bennett B, Manning AM, Firestein GS. Jun Nterminal kinase in rheumatoid arthritis. J Pharmacol Exp Ther 1999;291:124–30. [38] Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:73–81. [39] Inoue T, Hammaker D, Boyle DL, Firestein GS. Regulation of JNK by MKK-7 in fibroblast-like synoviocytes. Arthritis Rheum 2006;54:2127–35. [40] David JP, Sabapathy K, Hoffmann O, Idarraga MH, Wagner EF. JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms. J Cell Sci 2002;115:4317–25. [41] Han Z, Chang L, Yamanishi Y, Karin M, Firestein GS. 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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Critical role for apoptosis signal-regulating kinase 1 in the development of inflammatory K/BxN serum-induced arthritis Stephen J. Mnich, Patrick M. Blanner, Liangbiao G. Hu, Alexander F. Shaffer, Fernando A. Happa, Shawn O'Neil, Okechukwu Ukairo, Dave Weiss, Eric Welsh, Chad Storer, Gabriel Mbalaviele, Hidenori Ichijo 1, Joseph B. Monahan, Medora M. Hardy, Hiroyuki Eda ⁎ Discovery Biology, Global Research and Development, St. Louis Laboratories Pfizer Inc., 700 Chesterfield Parkway West, mail zone AA3C, Chesterfield, MO 63017, USA
a r t i c l e
i n f o
Article history: Received 31 May 2010 Accepted 22 June 2010 Keywords: Apoptosis signal-regulating kinase 1 (ASK1) Rheumatoid arthritis Mitogen-activated protein kinase (MAPK) p38 MAPK c-jun N-terminal kinase (JNK) K/BxN serum transfer model
a b s t r a c t In this report, we show that apoptosis signal-regulating kinase 1−/− (ASK1 KO) mice were resistant to inflammatory arthritis induced in the K/BxN serum transfer model of rheumatoid arthritis (RA). The p38 inhibitor, SD-0006 was administered to wild type (WT) mice as a comparator. Both ASK1 KO and p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses. Transcriptional profiling of mRNA prepared from paw tissue demonstrated that the production of many proinflammatory genes including cytokines, chemokines, and extracellular matrix degradative enzymes were maintained at basal levels by either ASK1 KO or prophylactic p38 MAPK inhibition. In the mouse whole blood (MWB) assay, tumor necrosis factor-α (TNF-α)-induced KC and CCL2 levels and also LPS-induced interleukin-6 (IL-6), CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Furthermore, both p38 and JNK were activated by TNF-α in human synovial fibroblasts isolated from RA patients (RASF). SD-0006 or SP600125, a JNK inhibitor, partially blocked the elevation of IL-6 production in RASF following stimulation with TNF-α. In contrast, dual inhibition with both p38/JNK inhibitors almost completely abolished TNF-α-induced IL-6 production from these cells. Ablation of ASK1 expression in RASF using siRNA for ASK1 resulted in inhibition of TNF-α-induced IL-6 and PGE2 production. This study is the first to suggest that ASK1 is critical for the development of RA and that ASK1 may be involved in the production of proinflammatory mediators in response to TNF-α stimulation in the RA joint. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Mitogen-activated protein kinases (MAPKs) have critical roles in the regulation of key cellular process including cell survival and apoptosis, proliferation, and inflammatory responses. Especially, activation of these MAPK pathways leads to the production of inflammatory mediators, such as cytokines and chemokines which are involved in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis [1]. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase kinase kinase (MAP3K) family that activates p38 MAPK and c-jun N-terminal kinase (JNK) via activating the MAP kinase kinase (MAP2K), MKK3/MKK6 and MKK4/MKK7, respectively [2,3] ASK1 is activated in response to various stresses, such as calcium influx, endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and oxidative
⁎ Corresponding author. Exploratory Research Laboratories, Research Center, AJINOMOTO PHARMACEUTICALS CO., LTD., 1-1, Suzuki, Kawasaki, Kanagawa, 2108681, Japan. Tel.: +81 44 210 5824; fax: +81 44 210 5875. E-mail address: [email protected] (H. Eda). 1 Laboratory of Cell Signaling, The University of Tokyo, Japan. 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.06.023
stress [4–7], and therefore ASK1 is involved in a variety of cellular functions, such as immune response. ASK1 is involved in LPS-induced cytokine production; LPS-induced production of IL-6, TNF-α, IL-1β, and IL-12 was impaired in ASK1−/− dendritic cells and splenocytes as well as macrophages [7,8]. Recent evidence has shown that ASK1 is closely linked to the pathogenesis of a variety of apoptosis-, immune-, and stress-related diseases, such as neurodegenerative diseases and autoimmune diseases [9–12]. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial cytokine production, infiltration of the synovium with immune cells, and joint destruction [13,14]. Cytokines including IL-1, IL-17, and TNF-α and downstream inflammatory mediators such as prostaglandins were produced from infiltrated cells [13,14]. Recent evidence reveals that not only infiltrated immune cells, but also RA synovial fibroblasts (RASFs), in the inflamed synovium are engaged in the initiation and perpetuation of RA and produce a variety of cytokines and other inflammatory mediators that mediate the interaction with infiltrated immune cells and other cells [15]. The production of these cytokines and inflammatory mediators from infiltrated immune cells and RASF are regulated by MAPKs [16– 18]. Therefore, MAPKs, especially p38 MAPK, is considered to be
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potential therapeutic targets for RA because p38 MAPK plays a crucial role in the regulation of IL-1 and TNF-α synthesis as well as IL-1stimulated IL-6 and IL-8 production and induction of other mediators such as matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) [1]. Indeed, a number of p38 MAPK inhibitors have been developed for the treatment of RA; however, none has yet made it to the market. The failure of p38 MAPK inhibitors in the clinic suggests the importance of other downstream pathways playing critical roles in the pathogenesis of RA and it has not yet been fully elucidated. In the current study, we investigated the role of ASK-1 in the K/ BxN serum transfer inflammatory arthritis model in mice and found that ASK1−/− mice were resistant to the edema and the infiltrations of various types of inflammatory cells into the ankle joints induced in this model. Furthermore, we have demonstrated that ASK1 plays a crucial role in the proinflammatory response to TNF-α in human RASF.
histological examination was scored as follows; 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Scoring examination was conducted in a double blinded manner.
2. Materials and methods
2.5. Isolation of total RNA from the joint and amplification of RNA
2.1. Animals
Right-sided hind paws, sectioned just proximal to the ankle joint, were individually flash-frozen in liquid nitrogen and stored at −80 °C. Frozen tissues were powdered using SPEX Certiprep 6750 Freezer mill (SPEX CertiPrep, Middlesex, UK) and 6753 microvials (Wolf Laboratories, York UK). Pulverized tissues were lysed in TriZol (Invitrogen, Carlsbad, CA) and extracted with chloroform and then phenol: chloroform:isoamylalcohol (Applied Biosystems/Ambion, Austin, TX). Four volumes of Zymo RNA binding buffer (Zymo Research, Orange, CA) was added into an aqueous phase of sample and applied to Zymo RNA spin column to purify RNA. Total RNA integrity (size and quality) was assessed with an Agilent HP2100 Electrophoresis Bioanalyzer (Agilent Technologies, Santa Clara CA), and quantified using a NanoDrop 1000 UV spectrophotometer by measuring the absorbance at A260 and A280 nm (Thermo Fisher Scientific, Waltham MA). Typical yields ranged from 200 to 800 ng/μl. 10 ng of total RNA from each paw was amplified for two rounds using a modified amplification protocol (Message Amp II aRNA Amplification Kit, Ambion, Austin TX).
C57BL/6 ASK1−/− (ASK1 KO) mice were provided by Dr. Hidenori Ichijo (Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, the University of Tokyo) and bred in Charles River Laboratories International (Wilmington, MA) [19]. Age-matched female wild-type (WT) mice on the same genetic background (C57BL/6) were purchased from Charles River Laboratories International. Animals were acclimatized to the laboratory conditions for several days before the start of the experiments. The St. Louis Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. 2.2. K/BxN serum transfer arthritis model and treatment Development of K/BxN serum transfer induced arthritis model was developed by injection of K/BxN serum to recipient mice. Briefly, K/ BxN serum (200 μl/mouse, Charles River Laboratories) was injected intraperitoneally into both female ASK1 KO (n = 24) and WT mice (n = 24) on day 0. Progression of disease was assessed by measuring the joint swelling on day 4, 5, 7, 10, and 12 after K/BxN serum transfer. Orally available specific p38 MAPK inhibitor [20,21] at a dose of 30 mg/kg was administered orally to WT mice as a comparator from day 0 to 12 (qd). Hind paws were collected for histological examination and microarray analysis on days 3, 4, 7, and 12, or on days 3, 4, and 7, respectively. All experiments were conducted in a double-blinded manner. 2.3. Histology Immediately following the necropsy, the left hind paw (including ankle) was fixed in 10% Neutral Buffered Formalin (NBF) for 24 h at 20 °C, followed by decalcification in Immunocal™ (Decal Chemical Corporation, Tallman NY) for 7 days at 20 °C. Decalcified joints were then processed and paraffin-embedded with dorsal aspect down for frontal view, sectioned twice (4 μm each), and stained with hematoxylin and eosin (H&E) for general evaluation or toluidine blue for specific assessment of cartilage changes. Cumulative histological score was determined according to a sum of three major parameters; 1) general inflammation including synovial epithelial changes, edema, inflammatory cell infiltrations, and inflammation; 2) cartilage damage including cartilage matrix changes, degeneration and/or necrosis of chondrocytes, ulceration and loss of cartilage; 3) bone resorption including reactions of osteoclasts, cortical bone loss and necrosis of osteocytes, etc. Each
2.4. Measurement of cytokine production from mouse whole blood Whole blood from WT and ASK1 KO mice was collected into sodium heparin tubes and used immediately. Whole blood was incubated with mouse TNF-α (10 ng/ml; R&D systems, Minneapolis, MN) or LPS (10 μg/ml; Sigma Aldrich, St. Louis, MO) for 20 h. Then, plasma was collected by centrifugation and used for further evaluation. Cytokine and chemokine levels in plasma were determined using MSD MULTI-ARRAY and MULTI-SPOT kits (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer's directions.
2.6. Preparation and analysis of Agilent whole mouse genome arrays Amplified RNA (aRNA) was labeled using a modified protocol with the Kreatech Universal Linkage System (ULS) aRNA Fluorescent Cy5 labeling kit (Kreatech Diagnostics, Amsterdam, The Netherlands); specifically, 2 μl of labeling buffer and 4 μl of ULS were added to 5 μg of aRNA in a total volume of 20 μl. Once combined, samples were incubated for 15 min at 85 °C and after incubation samples were returned to room temperature and spun down to collect condensate. Labeled samples were purified using Zymo Research RNA Clean and Concentrate spin columns according to manufacturer's protocol (Zymo Research Corporation, Orange CA). Samples were eluted in 100 μl of RNase/DNase-free water. Labeled samples were quantified on the Nanodrop ND-1000 spectrophotometer. Each labeled aRNA (750 ng) from a comparison was added to a total volume of 230 μl water and 9 μl of fragmentation buffer (Agilent Technologies, Santa Clara, CA). Hybridization mixtures were then incubated for 30 min at 60 °C. Hybridization mixtures were cooled to room temperature and spun down to collect condensate. 2× Hi-RPM hybridization buffer (240 μl; Agilent Technologies) was added to each mixture. These probes were then hybridized to 4 × 44 K mouse Whole Genome Microarrays for 18 h at 65 °C in Sure-Hyb Hybridization chambers (Agilent Technologies). Chambers were disassembled in a 0.6× SSC 0.005% Triton X-102 wash bath at room temperature and washed for 10 min in wash bath 1. Microarrays were transferred to a 0.01× SSC 0.005% Triton X-102 wash for 5 min at 40 °C. Microarrays were then dried with HEPA filtered compressed nitrogen and scanned on the Agilent Technologies DNA Microarray Scanner at 5 μm resolution.
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Data were extracted from the scanned image using Agilent Technologies Feature Extraction Software version 9.5. 2.7. Data normalization Signal normalization against a common sample was performed using a smoothed piecewise linear curve, trained on the log intensities that differed by b10% rank order between two samples. Local background subtraction was performed on each array prior to normalization. A computational normal pool was created from the averaged normalized signals of the individual normal animal arrays. Within the normal arrays, a median array was selected as least distant from the other normal arrays. All normal arrays were normalized against the median array prior to calculating the averaged normalized
signals of the pool. Technical replicates for individual samples were treated in a similar fashion, choosing a median array within each group of replicates, and flagging spots with a coefficient of variation ≥0.7 as bad data. A final normalization was performed on all averaged signals vs. the computational normal pool. Fold changes between samples were calculated, raising weak signals to a minimum value of 10 prior to comparison. 2.8. In vitro cellular assay Human rheumatoid arthritis synovial fibroblast cultures (RASF) were established from the synovial membrane of a female RA patient and cultured in Dulbecco's Modified Eagle Medium (Invitrogen) containing 15 % FBS. Cells were preincubated with SD0006 (3 μM)
Fig. 1. Ablation of ASK1 is efficacious in the K/BxN serum transfer arthritis model. ASK1 KO and WT mice were injected with K/BxN serum to induce arthritis. Progression of disease was assessed by joint swelling (A), cumulative inflammation score (B), and H&E stain on day 12 (C). Data in graph A and B were indicated by mean ± S.E.M (WT: n = 6, WT + p38i: n = 6, ASK1 KO: n = 10). p38i: p38 MAPK inhibitor (SD-0006: 30 mg/kg, qd, po). Cumulative histology score is indicated by the sum of three parameters; general inflammation (H&E stain), cartilage damage (trichrome stain), and bone resorption. 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. *: p b 0.001 vs. WT (one-way ANOVA on Turkey– Friedman Square Root, one tailed analysis).
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and/or JNK inhibitor (SP600125, 3 μM: TOCRIS, Ellisville, MO) for 1 h, followed by stimulation with 10 ng/ml human TNF-α (R&D systems) for 18 h. IL-6 production from the cells was determined using MSD MULTI-ARRAY-MULTI-SPOT kit (Meso Scale Discovery) and prostaglandin E2 (PGE2) production was determined using PGE2 EIA (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions or PGE2 ELIA established in house [22]. 2.9. Western blot analyses Western Blot analysis for evaluation of MAPK signaling pathways was performed using cells lysates prepared after stimulation with TNF-α for 30 min. Cells were lysed using RIPA buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, phosphatase inhibitor cocktails I and II, 100 μg/ml PMSF (Sigma Aldrich) and Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Whole cell protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to PVDF membrane (Invitrogen). Membranes were incubated with primary antibodies for overnight at 4 °C. The following primary antibodies were used for detection of protein of interests: antibody to human ASK1 (Epitomics, Burlingame, CA), human total JNK and p38 (Santa Cruz Biotechnology, Santa Cruz, CA), human phosphorylated MKK3/6, human phosphorylated HSP-27, human phosphorylated MKK4, human phosphorylated c-Jun human total and phosphorylated Erk (Cell Signaling Technology, Danvers, MA). After incubation for 1 h with the appropriate fluorescent-labeled secondary antibodies, proteins of interest were detected using Odyssey infrared imaging System (LiCor, Lincoln, NE). 2.10. Small interfering RNA (siRNA) study and quantitative real-time polymerase chain reaction (qRT-PCR) assay Validated Stealth ASK1 and ASK2 siRNA duplexes were obtained from Invitrogen. RASF were transfected with siRNA at a concentration of 15 nM using DharmaFect 1 transfection reagent (Thermo Scientific, Waltham, MA). At 72 h post transfection, cells were stimulated with 10 ng/ml TNF-α (R&D) for 8 h. The effect of ASK siRNA was determined using qRT-PCR. In qRT-PCR assay, cells were lysed with Nucleic Acid Purification Lysis Solution (Applied Biosystems, Wamington, UK). Total RNA was isolated using 6100 Nucleic Acid PrerStation according to the manufacturer's directions (Applied Biosystems). qRT-PCR was conducted using qSCriptTM One-Step qRT-PCR kit (Quanta BIOSCIENCES, Gaithersburg, MD). Amplified reactions were quantified on an ABI PRISM 7900 Sequence detection system (Applied Biosystems). Relative gene quantities were obtained using the comparative Ct method after normalization to appropriate control genes (cyclophilin A: NM_021130.3). TaqMan probes were also purchased from Applied Biosystems. 3. Results 3.1. ASK-1 deficiency attenuates arthritis, cartilage destruction, and bone erosion To determine whether ASK-1 has a critical role in the synovial inflammation and joint destruction, ASK-1 KO and WT were investigated in the K/BxN serum transfer arthritis model. As a comparator, a specific p38 MPK inhibitor was administered orally to WT. Fig. 1A and B shows the ankle thickness and cumulative histological score of mice injected with K/BxN serum. ASK1 deficiency significantly decreased joint swelling and cumulative histological score compared to those of WT from day 4 to the end of the study (day 12) (p b 0.001) and the decrease in these arthritis severity scores of ASK1 KO was comparable to those of mice treated with the p38 inhibitor. Ankle histopathology of WT showed significantly severe
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inflammation including indicated by synovial epithelial changes, edema, inflammatory cell infiltrations compared to those of WT without K/BxN serum injection. Also, cartilage degradation and bone erosion was observed (Fig. 1C). In contrast, ankle histology from ASK1 KO demonstrated markedly less inflammation and no cartilage degradation and bone erosion. 3.2. Comparison of transcriptional gene profiling of mouse paw from WT with those from ASK1 KO Transcriptional profiling of mRNA prepared from pulverized paw tissue was investigated to determine the proinflammatory gene expression in WT, ASK1 KO, and WT treated with p38 inhibitor (Table 1). Transcriptional profiling demonstrated that the production of many proinflammatory genes including cytokines (IL-6 and IL-1β), chemokines (CXCL3, CXCL5, KC, CCL2, CCL3, CCL7, and CCL8), matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase (MMP-3 and TIMP-1) and cell surface marker (FcγRI, FcγRIII, and F4/ 80) were dramatically increased in K/BxN serum-injected WT compared to those of WT without K/BxN serum injection. On the other hand, expression of these inflammatory cytokines/chemokines, MMPs and cell surface marker was decreased in K/BxN seruminjected ASK1 KO as well as K/BxN serum-injected WT treated with p38 inhibitor. 3.3. Induction of cytokine level by inflammatory mediators in mouse whole blood Induction of cytokine or chemokine by TNF-α and LPS was investigated using whole blood collected from WT and ASK1 KO mice (Fig. 2). IL-1β, IL-6, TNF-α, CCL2, and KC levels in MWB from both WT and ASK1 KO were significantly elevated by LPS while IL-1β was elevated by TNF-α. TNF-α-induced KC and CCL2 levels and also LPS-induced IL-6, CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Interestingly, IL-1β, IL-6, and KC levels in naïve MWB from ASK1 KO were significantly lower than those from WT. 3.4. The role of ASK1 in the production of IL-6 and PGE2 from human RASF stimulated with TNF-α To understand the role of ASK1 in human RASF, the role of p38 MAPK and JNK in the production of IL-6 and PGE2 in RASF stimulated with TNF-α. TNF-α-stimulated RASF produced IL-6 and PGE2 and the production was inhibited by both p38 MAPK inhibitor and JNK Table 1 Comparison of transcriptional gene profiling of mouse paw from WT with those from ASK1 KO on day 7. Gene
WT
ASK1 KO
WT+SD-0006
CXCL5 CXCL3 CCL7 CCL2 CCL8 CCL3 KC IL-6 IL-1β TNF-α TIMP-1 MMP3 FcγRI FcγRIII F4/80
40.9 15.0 12.6 7.6 6.0 5.8 5.5 19.0 13.9 1.6 11.9 10.2 9.1 6.0 3.6
2.1 2.2 2.7 2.4 1.8 1.2 1.3 2.9 1.5 0.9 1.2 1.3 1.6 1.2 1.2
3.5 4.2 3.5 2.6 1.4 1.6 1.9 4.6 2.5 0.9 1.5 1.8 2.0 1.3 1.2
Data (n = 2: WT and WT + SD-0006, n = 6: ASK1 KO) are indicated by fold change of gene expression from that of treatment naïve (no K/BxN serum injection). SD-0006: specific p38 MAPK inhibitor (30 mg/kg, qd, po).
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Fig. 2. Induction of cytokine level by TNF-α or LPS in mouse whole blood. Mouse whole blood (MWB) collected from ASK1 KO and WT was treated with mouse TNF-α (10 ng/ml) or LPS (10 μg/ml) for 20 h. Data were indicated by mean ± S.E.M (n = 3). *: p b 0.05 vs. CTRL; **: p b 0.01 vs. CTRL; ***: p b 0.001 vs. CTRL (one-way ANOVA with Dunnett). #: p b 0.05 vs. WT; ##: p b 0.01 vs. WT (two-way ANOVA with Bonferroni).
inhibitor (Fig. 3A and B). Dual inhibition of p38 MAPK and JNK resulted in the enhanced inhibition of IL-6 and PGE2 production in RASF, indicating that ASK1 may have a role in TNF-α-induced IL-6 and PGE2 production in human RASF. Inhibition of IL-6 and PGE2 production by these inhibitors was accompanied by inhibition of p38 and JNK, phosphorylation as well as downstream activation of heat shock protein 27 (HSP27) and c-jun (Fig. 4). To confirm the role of ASK1 in RASF activation, we investigated whether TNF-α-induced IL-6 and PGE2 production was decreased by ablation of ASK1 gene using ASK1 siRNA in RASF. ASK1 mRNA level was decreased by up to 75% by ASK1 siRNA (Fig. 5A). Decreased ASK1
protein expression in ASK1 siRNA-treated cells was confirmed by Western blot analysis (data not shown). Importantly, ASK1 gene ablation in RASF resulted in a significant decrease in TNF-α-induced production of IL-6 and PGE2 compared to those treated with scramble siRNA (Fig. 5B). 4. Discussion In this study, we have demonstrated that ASK1 is critical for the progression of disease in the mouse K/BxN serum transfer model of RA. These data suggest that ASK1 is involved in the production if
Fig. 3. Dual inhibition of p38 MAPK and JNK results in the enhanced inhibition of IL-6 (A) PGE2 (B) production in human RASF. Human RASF were preincubated with 3 μM p38 MAPK inhibitor (p38i: SD-0006), SP600125 (JNKi), and combination of these two compound (Combo) for 1 h, followed by stimulation with 10 ng/ml human TNF-α for 18 h. Data were indicated by mean ± S.D. of 2 independent assay (n = 3). *: p b 0.05 vs. Combo (one-way ANOVA with Dunnett).
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Fig. 4. Phosphorylation status of downstream signaling of p38 MAPK and JNK. Human RASF were preincubated with 3 μM p38 MAPK inhibitor (p38i: SD-0006) or SP600125 (JNKi) for 1 h, followed by stimulation with 10 ng/ml human TNF-α for 30 min. pMKK3/6: phosphorylated MKK3/6; p-HSP27: phosphorylated heat shock protein 27 (HSP27); p-MKK4: phosphorylated MKK4; JNK Total (green); p-c-jun: phosphorylated c-jun (red); p-ERK: phosphorylated extracellular signal-regulated kinase (ERK; red). ERK Total (green).
proinflammatory mediators in response to TNF-α stimulation in the RA joint. Terauchi et al. reported that arthritis was significantly promoted by transduction of the constitutively active form of ASK1 in rat collagen induced arthritis (CIA) model [23]. In that study, introduction of constitutively active ASK1 into the joint promoted ankle swelling, as well as enhanced infiltration of inflammatory cells into the synovial membrane. Our results using ASK1 KO mice are consistent with this report and provide strong evidence that ASK1 plays a role in the pathogenesis of arthritis.. In our study, ASK1 KO mice showed marked attenuation of joint edema, cartilage damage, bone absorption, and inflammatory cell infiltration in the K/BxN serum transfer model of RA. Furthermore, in the mouse CIA model of arthritis, we observed that the incidence and severity of arthritis was significantly lower in ASK1 KO mice than those in WT (data not shown). These data indicate that ASK1 has a critical role in the progression of collagen- or K/BxN serum-induced RA in rodents. Transcriptional gene profiling of paw from ASK1 KO showed that expression of inflammatory cytokines and chemokines, cellular surface markers, and extracellular degradative enzymes were decreased in ASK1 KO compared to those in WT. Down-regulation of the Fcγ receptors (FcγRs) which have a critical role in the development of
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paw swelling in this model [24], may be one factor that is responsible for reduction of these key pro-inflammatory mediators in the ASK1 KO. FcγRs are expressed on immune cells such as macrophages, mast cells, and neutrophils as well as fibroblast-like synoviocytes [25–28]. FcγR activation, especially predominantly FcγRIII, leads to the production of inflammatory cytokines, chemokines as well as degradative enzymes from macrophages, neutrophils, and mast cells, resulting in inflammation and arthritis in K/BxN serum transfer model in mice [27,28]. In fact, FcγRIII−/− mice exhibited a slower progression of arthritis in K/BxN serum transfer model [29]. However, we cannot exclude the possibility that an increase in signal intensity can be observed due to cellularity changes in tissues tested. Immune cells basally expressing several relatively selective transcripts that infiltrate into a tissue will cause an increase in the signal intensity for those selective transcripts, independent of any upregulation of those mRNAs. Therefore, by profiling whole paw, gene changes as well as cellular infiltration events are both represented. Further investigation is needed to elucidate whether the infiltration of immune cells expressing FcγRs are decreased in K/BxN study using ASK1 KO. In this model, inflammatory cells, especially neutrophils and mast cells, are required as effecter cells to mediate joint destruction [30,31]. Infiltration of these inflammatory cells into the joint may be suppressed in ASK1 KO mice since the expression level of CC and CXC chemokines was much lower than those in WT. Indeed, histological examination showed that few infiltrated inflammatory cells were seen in the joint of ASK1 KO compared to WT. Furthermore, inflammatory mediator-induced CCL2 and KC production from mouse whole blood in ASK1 KO were significantly lower than those in WT in this study. We also observed that the induction level of chemokines (KC and MCP-1) by LPS in granulocytes from ASK1 KO mice was significantly lower than those from WT in mice (Eda H, in preparation). It has been reported that chemokine production from splenocytes, dendritic cells, or endothelial cells in ASK1 KO was lower than that in WT [7,32]. Osaka et al. reported that gene expression of chemokines in wounded skin samples from ASK1 KO was significantly lower than those in WT [33]. Collectively, these results indicate that ASK1 may be involved in the chemotaxis of the inflammatory cells via regulating the chemokine production. However, there is no report describing the association of ASK1 with chemotaxis at present time. Whether ASK1 has a major role in the production of chemokines and regulates chemotaxis remains to be investigated. Consistent with a role for ASK1, dual inhibition of p38 MAPK and JNK resulted in enhanced inhibition of IL-6 and PGE2 production in TNF-α-stimulated cultured RASF. MAPKs have been implicated in inflammatory diseases. In particular p38 MAPK is thought to play a critical role in RA because it is activated in the rheumatoid synovial intimal lining and p38 MPK inhibitors show effects on RA models [34–
Fig. 5. ASK1 gene ablation inhibits IL-6 and PGE2 production in human RASF stimulated with TNF-α. A: Relative quantity of ASK1 mRNA expression. mRNA was collected at 72 h post transfection and relative gene quantities were obtained using the comparative Ct method after normalization to human control gene (cyclophilin A: NM_021130.3). Data [mean ± S.D. (n= 3)] were indicated by % of control (non-treatment). Scramble: non-targeted scramble siRNA (Negative CTRL). B: At 72 h post transfection of siRNA, RASF were stimulated with 10 ng/ ml TNF-α for 8 h. Data were indicated by mean ± S.D. of 2 independent assay (n= 3). *: p b 0.05 vs. Scramble siRNA treatment (one-way ANOVA with Dunnett).
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36]. In fact, in this study, p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses that are indistinguishable from those observed in ASK1 KO. On the other hand, JNK also appears to play a role in RA. JNK is thought to be important in extracellular matrix degradation. JNK is a critical MAPK pathway for IL-1-induced collagenase gene expression and regulation of MMP secretion in RASF [37–39]. Furthermore, JNK-1deficient osteoclast progenitors were unable to mature into bone-resorbing osteoclasts following exposure to receptor activator of nuclear factor-κB ligand (RANKL) [40]. Although JNK inhibitors have been reported to have marginal effect on the joint inflammation and destruction [41,42], this study showed that either JNK inhibition alone or combination with the p38 MAPK inhibition resulted in decreased TNF-α-stimulated production of IL-6 and PGE2 in RASF. These data indicate that JNK signaling contributes to the production of cytokines and inflammatory mediators. In conclusion, we demonstrated that ASK1 is critical for the progression of disease in the mouse K/BxN serum transfer model of RA. Furthermore, our findings in ASK1 KO mice and human RASF suggest that ASK1 is involved in the production of proinflammatory mediators in RA. Thus, inhibition of ASK1 upstream signaling of p38 MAPK and JNK, may provide a novel therapeutic intervention for RA.
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