Promotes Oncostatin M Production in Human Osteoblasts: Implication of Rheumatoid Arthritis Therapy

This information is current as Chen-Ming Su, Yi-Chun Chiang, Chun-Yin Huang, of October 2, 2021. Chin-Jung Hsu, Yi-Chin Fong and Chih-Hsin Tang J Immunol 2015; 195:3355-3364; Prepublished online 24 August 2015; doi: 10.4049/jimmunol.1403191 http://www.jimmunol.org/content/195/7/3355 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Osteopontin Promotes Oncostatin M Production in Human Osteoblasts: Implication of Rheumatoid Arthritis Therapy

Chen-Ming Su,*,† Yi-Chun Chiang,† Chun-Yin Huang,‡,x Chin-Jung Hsu,{,‖ Yi-Chin Fong,{,‖ and Chih-Hsin Tang†,#,**

Accumulating evidence indicates that subchondral bone might play an essential role in rheumatoid arthritis (RA). Osteopontin (OPN) induces the production of an important proinflammatory involved in the pathogenesis of RA. This study evaluated the activation of oncostatin M (OSM) by OPN in human primary osteoblasts to understand RA pathogenesis and characterized the intracellular signaling pathways involved in this activation. Quantitative PCR, ELISA, and Western blot results indicated that stimulation of human primary osteoblasts with OPN induces OSM expression through avb3 integrin/c-Src/platelet-derived receptor transactivation/MEK/ERK. Treatment of osteoblasts with OPN also increased c-Jun phosphorylation, AP-1 luciferase activity, and c-Jun binding to the AP-1 element on the OSM promoter, as demonstrated using chromatin immunopre- Downloaded from cipitation assay. Moreover, inhibition of OPN expression using lentiviral‐OPN short hairpin RNA resulted in the amelioration of articular swelling, cartilage erosion, and OSM expression in the ankle joint of mice with collagen-induced arthritis as shown using microcomputed tomography and immunohistochemistry staining. Our results imply that OSM expression in osteoblasts increases in response to OPN-induced inflammation in vitro. Finally, lentiviral‐OPN short hairpin RNA ameliorates the inflammatory response and bone destruction in mice with collagen-induced arthritis. Therefore, OPN may be a potential therapeutic target for RA. The Journal of Immunology, 2015, 195: 3355–3364. http://www.jimmunol.org/

heumatoid arthritis (RA) is an autoimmune disease, which of the IL-6 family, is capable of activating osteoblasts and stim- results in progressive articular cartilage erosion and sy- ulating mesenchymal progenitor differentiation toward the osteo- R novial hyperplasia. Although the pathogenesis of RA blastic cell lineage (3). OSM stimulates bone formation and is have not been completely elucidated, it is clear that both structural produced by osteocytes and macrophages (4). OSM secretion has damage and infiltration of proinflammatory occur at the been associated with the modulation of other inflammatory dis- articular capsule microenvironment (1). In fact, it is known that eases, including RA, osteoarthritis, and hepatic cellular carcinoma excessive production of proinflammatory mediators is a determining (5). by guest on October 2, 2021 factor for RA pathogenesis. Activated immune cells infiltrating the Osteopontin (OPN) is an extracellular matrix glycoprotein that synovial tissue secrete large quantities of cytokines such as IL-6, is present in the extracellular fluid surrounding the sites of min- which is a potent effector in RA and generates enzymes to de- eralized tissues and bone remodeling (6). OPN is an important grade cartilage and bone (1, 2). Oncostatin M (OSM), a member mediator of the immune response as it is involved in cell migration and Th1-mediated immunity (7, 8). OPN was found to selectively induce expression of proinflammatory and acted as *Department of Biomedical Sciences Laboratory, Affiliated Dongyang Hospital of Wenzhou Medical University, Dongyang, Zhejiang, China 322100; †Graduate In- a crucial mediator in the rheumatoid synovium (9, 10). Accu- stitute of Basic Medical Science, China Medical University, Taichung, Taiwan mulating evidence suggests that higher OPN levels could be ob- 40466; ‡Graduate Institute of Clinical Medical Science, China Medical University, x served in the synovial fluid and synovial membrane in RA patients Taichung, Taiwan 40466; Department of Orthopedic Surgery, China Medical Uni- versity Beigang Hospital, Yun-Lin County, Taiwan 65142; {School of Chinese (11, 12). OPN-deficient mice were found to attenuate articular Medicine, College of Chinese Medicine, China Medical University, Taichung, Tai- cartilage erosion and reduce arthritic scores in collagen-induced wan 40466; ‖Department of Orthopedic Surgery, China Medical University Hospi- tal, Taichung, Taiwan 40466; #Department of Pharmacology, School of Medicine, arthritis (CIA) (13). In addition, the arginine–glycine–aspartate China Medical University, Taichung, Taiwan 40466; and **Department of Biotech- sequence (RGD) motif in OPN acts as a multifunctional cytokine nology, College of Health Science, Asia University, Taichung, Taiwan 40466 that interacts with multiple ubiquitously expressed cell surface Received for publication December 22, 2014. Accepted for publication July 23, 2015. receptors, including various integrins (14). Thereby, OPN-induced This work was supported by Ministry of Science and Technology of Taiwan Grants effects on osteoblasts might be implicated in RA. 103-2628-B-039-002-MY3 and 101-2314-B-039-002-MY3 and China Medical Uni- During the development of RA, subchondral bone plays an es- versity Grant CMU102-BC. sential role in the mechanisms of bone remodeling that involves OSM Address correspondence and reprint requests to Dr. Chih-Hsin Tang, Graduate Insti- tute of Basic Medical Science, China Medical University, Number 91, Hsueh-Shih (3). A recent study has reported OSM to be constitutively expressed Road, Taichung, Taiwan 40466. E-mail address: [email protected] in the subchondral bone and possibly associated with osteoblasts (15). The online version of this article contains supplemental material. Nevertheless, the mechanism of interaction between OPN and OSM Abbreviations used in this article: BMD, bone mineral density; BS, bone surface; BV, during osteoblast inflammation has not yet been elucidated. bone volume; ChIP, chromatin immunoprecipitation; CIA, collagen-induced arthritis; Our results showed that OPN induces upregulation of OSM CII, collagen type II; DN, dominant-negative; IHC, immunohistochemistry; micro- CT, microcomputed tomography; OPN, osteopontin; OSM, oncostatin M; PDGFR, expression in primary osteoblasts. Similarly, in vivo results showed platelet-derived growth factor receptor; qPCR, quantitative real-time PCR; RA, rheu- that lentiviral knockdown of OPN significantly abolished bone matoid arthritis; RASF, RA synovial fibroblast; sh, short hairpin; siRNA, small erosion and inflammation in mice with CIA. These results provide interfering RNA. new insights into the mechanisms of OPN action that may have Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 therapeutic value for RA. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403191 3356 OPN INDUCES OSM EXPRESSION

Materials and Methods with 4% BSA for 1 h at room temperature and then probed with rabbit Abs Materials against human phospho-ERK, ERK, phospho-MEK, MEK, phospho–c-Jun or c-Jun (1:1000) for 1 h at room temperature. After three washes, the blots were Anti-mouse and anti-rabbit IgG-conjugated HRP, rabbit polyclonal Abs incubated with a donkey anti-rabbit peroxidase–conjugated secondary Ab specific for b-actin, phospho–c-Src, c-Src, platelet-derived growth factor (1:1000) for 1 h at room temperature. The blots with HRP-labeled substrate receptor (PDGFR)-b, phospho-MEK, MEK, phospho-ERK, ERK, phospho- were detected by ECL and visualized by using a Fujifilm LAS-3000 chemi- c-Jun, c-Jun, IgG, and OSM were purchased from Santa Cruz Biotech- luminescence detection system (Fujifilm, Tokyo, Japan). nology (Santa Cruz, CA). Rabbit polyclonal Ab specific for phospho– PDGFR-b was purchased from Technology (Danvers, Transfection and reporter assay MA). Mouse monoclonal Abs specific for OSM or avb3 integrin were Control ON-TARGETplus small interfering RNA (siRNA) and ON- purchased from R&D Systems (Minneapolis, MN). An OSM enzyme TARGETplus siRNAs against c-Jun and PDGFR-b were purchased from immunoassay was purchased from R&D Systems. Tanshinone IIA was Dharmacon Research (Lafayette, CO). Transient transfection of siRNA purchased from Biomol (Butler Pike, PA). The AP-1 luciferase plasmid (0.5 nM) was carried out using DharmaFECT 1 transfection reagent was purchased from Stratagene (La Jolla, CA). The c-Src dominant- (Thermo, Waltham, MA) for 24 h, according to the manufacturer’s instruc- negative (DN) mutant was a gift from Dr. S. Parsons (University of Vir- tions. For the reporter assay, cells were cotransfected with AP-1 luciferase ginia Health System, Charlottesville, VA). MEK-1 DN mutant was a gift (0.5 mg) and b-galactosidase expression vector (0.5 mg)for24husing from Dr. W. M. Fu (National Taiwan University, Taipei, Taiwan). ERK-2 Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. DN mutant was a gift from Dr. M. Cobb (Southwestern Medical Center, b Dallas, TX). pSV- -galactosidase vector and the luciferase kit were pur- Chromatin immunoprecipitation assay chased from Promega (Madison, WI). All other chemicals or inhibitors were obtained from Sigma-Aldrich (St. Louis, MO). Chromatin immunoprecipitation (ChIP) analysis was performed as de- scribed previously (18). DNA, immunoprecipitated with an anti–c-Jun Ab, Cell cultures was purified and extracted with phenol-chloroform and then amplified Downloaded from Human primary osteoblasts were isolated from bone chips obtained from using PCR. PCR products were resolved by electrophoresis in a 1.5% 9 20 donors (ages 40–60 y) who were generally healthy and had no bone agarose gel and visualized under UV . Primers 5 -AACCTCCTC- 9 9 9 disorder other than hip dysplasia, for which they had undergone hip CTTTCTGGCTG-3 and 5 -CTGGTCAGCACATCTCCCAG-3 were used arthroplasty at China Medical University Hospital. Informed consent was to amplify across the OSM promoter region (19). obtained from each donor. The protocol for this study was approved by the Institutional Review Board at China Medical University Hospital (16). Lentiviral production m The osteoblasts were cultured in DMEM containing 100 g/ml ascorbic Recombinant lentiviruses were produced by transient cotransfection of http://www.jimmunol.org/ acid, nonessential amino acids, penicillin/streptomycin, and 10% FBS 293T cells with short hairpin (sh)RNA-expressing plasmid (TRCN0000004877) (Invitrogen, Carlsbad, CA). Cultures were maintained in a humidified along with the packaging plasmid pCMVDR8.91 and the VSV-G enve- atmosphere of 5% CO2 at 37˚C. The human osteoblast-like cell line MG- lope glycoprotein expression plasmid pMD.G, all of which were ob- 63 was purchased from the American Type Culture Collection (Manassas, tained from the National RNAi Core Facility at the Academia Sinica VA). MG-63 cells were cultured in DMEM supplemented with 10% FBS in Taiwan. After 48 h, lentiviral particles carrying OPN shRNA (Lenti- m and antibiotics (100 U/ml penicillin G and 100 g/ml streptomycin). shOPN) were isolated form the supernatant of 293T cells. A plaque assay We obtained approval for this study from the local ethics committee and using serial dilution was performed in human osteoblasts to determine all subjects gave written informed consent to participate. Abnormal RA virus titers (in plaque forming units) of Lenti-shOPN. The viral titer of synovial fibroblasts (RASFs) were obtained from six patients (ages 40–60 y) Lenti-shOPN was determined to be ∼7.1 3 106 PFU. during total knee arthroplasty for RA. Human primary synovial fibroblasts were isolated from RA patients and incubated in a solution of type II In vivo collagen-induced arthritis model by guest on October 2, 2021 collagenase for 18 h and then filtered through 70-mm nylon filters. Cells were cultured in RPMI 1640 medium supplemented with 10% heat- Male C57BL/6J mice (ages 8–10 wk) were purchased from the National inactivated FCS (Life Technologies) and were seeded at a concentration Laboratory Animal Center in Taiwan. Mice were maintained under con- of 1 3 106 cells/T75 flask. After 1 wk, nonadherent cells were removed, ditions complying with the Guidelines of the Animal Care Committee of and the adherent RASFs were expanded in vitro. RASFs from passages 3 China Medical University. The CIA model was established according to to 9 were used for the experiments. published protocols (20). Briefly, 0.1 ml of an emulsion containing 100 mg chicken collagen type II (CII) (Sigma-Aldrich), dissolved at a concentra- OSM quantification tion of 2 mg/ml in 0.1 M acetic acid, and CFA was injected intradermally into the base of the tail on day 0. Two weeks after the primary immuni- Human primary osteoblasts were cultured in 24-well culture plates. Cells zation, the second booster injection of 100 mg CII dissolved and emulsified were treated with OPN and then incubated at 37˚C for 24 h. To examine 1:1 in IFA was administered into the hind paws. Paw swelling was mea- the downstream signaling pathways responsive to OPN treatment, cells sured in a blinded manner with a plethysmometer (Marsap, Mumbai, India) were pretreated with various inhibitors for 30 min before the addition of once weekly for 4 wk to determine the clinical severity of arthritis. In a OPN (100 ng/ml). After incubation, the supernatant medium was collected separate experiment, groups of 10 mice with CIA or control were injected and stored at 280˚C until the assay was performed. OSM in the medium with 7.1 3 106 PFU Lenti-shOPN or control-shRNA in the hind paw was assayed using an OSM ELISA kit, according to the manufacturer’s weekly for 4 wk, respectively, after the second CII immunization. Upon instructions. sacrifice on day 42, the mice phalanges and ankle joints were removed Quantitative real-time PCR immediately and fixed in 4% paraformaldehyde for microcomputed to- mography (micro-CT) analysis. The micro-CT scan of the paws was per- Total RNA was extracted from osteoblasts using a TRIzol kit (MDBio, formed using an in vivo micro-CT scanner (Skyscan 1176; Bruker, Kontich, Taipei, Taiwan). RNA quality and yield were determined by absorbance Belgium) at 9 mm resolution and with a rotation step of 0.30 degree per at 260-nm measurements performed with a Nanovue Spectrophotometer image. Scanning was done at 50 keV X-ray voltage, 500 mA current, and (GE Healthcare, Madison, WI). cDNA was synthetized from 1 mgtotal 885 ms of exposure time/image with a 0.5-mm aluminum filter. Recon- RNA using an Moloney Murine Leukemia Virus Reverse Transcription struction of sections was carried out with GPU-based scanner software kit (Invitrogen) following the manufacturer’s recommendations. Quantitative (NRecon; Bruker). The grayscale was based on Hounsfield units, and vali- real-time PCR (qPCR) analysis was carried out with TaqMan One-Step RT- dated calcium standards were scanned to use as density references. Three- PCR Master Mix (Applied Biosystems, Foster City, CA). All target gene dimensional microstructural volumes from micro-CT scans were analyzed primers and probes were purchased from Applied Biosystems. b-Actin was using Skyscan software (CTAn; Bruker). To evaluate bone focal erosion in used as an internal control. qPCR assays were carried out in triplicate using the ankle joint, quantification of volumetric bone mineral density (BMD), a StepOnePlus sequence detection system (Applied Biosystems), according bone surface (BS), and bone volume (BV) were performed in a defined bone to the manufacturer’s instructions. area by manually drawn regions of interest (913 slices from the proximal calcaneus to metatarsals, 8 mm each). Western blot analysis Immunohistochemistry Cellular lysates were prepared as described previously (17). were resolved using NaDodSO4-PAGE and transferred to Immobilon polyvinyldi- Immunohistochemistry (IHC) staining was performed on serial sections of fluoride membranes (Millipore, Billerica, MA). The membranes were blocked the mice ankle joints. After ankle joints from the mice were fixed in 1% The Journal of Immunology 3357 formaldehyde, the specimens were decalcified in 10% EDTA and dehy- dependent manner. These results demonstrate that OPN induces up- drated in ethanol/xylene as described previously (21). The sections were regulation of proinflammatory OSM expression in osteoblasts. stained with primary rabbit anti-mouse OSM (2 mg/ml), IL-6 (0.125 mg/ml), IL-1b (5 mg/ml), TNF-a (2 mg/ml), or control IgG (5 ng/ml) (Santa Cruz OPN induces OSM expression through avb3 integrin receptor Biotechnology). Biotin-conjugated goat anti-rabbit IgG was used as the sec- and c-Src signaling in human osteoblasts ondary Ab and 3,3ʹ-diaminobenzidine tetrahydrochloride as the substrate for color development. Some specimens were also stained with Safranin O-fast Although the interaction between the RGD motif of OPN and Green and H&E (21). integrins within osteoclastic bone resorption has been generally Statistical analysis accepted (26), it has not yet been elucidated whether OPN inter- acts with integrins in osteoblasts during RA inflammation. Therefore, Data are expressed as the mean 6 SEM. Parametric testing between two matched groups was performed by the paired t test. The Mann–Whitney to determine the role of integrin-dependent signaling in osteoblasts, sum test was used between two groups, and Kruskal–Wallis one-way cells were treated with OPN and the mRNA expression of different ANOVA on ranks among was used for four groups. The p value ,0.05 was integrins quantified. It was found that OPN-treatment induces mRNA considered significant. expression of av and b3 integrins, whereas expression of a2, a5, b1, and b integrins mRNA was not induced (Fig. 2A). Next, we Results 5 specifically inhibited avb3 to examine its involvement in the OPN- OPN increases OSM production in human osteoblasts mediated increase of OSM expression. OSM mRNA (Fig. 2B) Although much information regarding RA focuses on the effects and levels (Fig. 2C, 2D) in response to OPN-treatment of inflammation on osteoclast differentiation and function, recent selectively decreased in the presence of 2 mg/ml anti-avb3 mAb studies have begun to elucidate the impact of proinflammatory compared with control, supporting the role of avb3 integrin in this cytokines, such as OPN, on the osteoblast (22, 23). Studies regarding process. Downloaded from the inflammatory development of RA show that OSM potentially Recently, c-Src signaling downstream of integrin has been contributes to monocyte migration and macrophage activation in reported (27). As such, we investigated the role of c-Src in synovial tissue (24, 25). These findings suggest that the high con- mediating OPN-induced OSM expression. OPN-induced OSM centrations of OPN involved in bone remodeling within osteoblasts expression was markedly attenuated by pretreatment of cells for might play a dynamic role promoting the secretion of inflammatory 30 min with the specific c-Src inhibitor PP2 (10 mM) (Fig. 2E), as cytokines into the inflamed articular microenvironments observed in well as in cells expressing the c-Src DN mutant (Fig. 2F). C-Src http://www.jimmunol.org/ RA development. Therefore, we used human osteoblasts to investi- autophosphorylation at Tyr416 results in the recruitment of other gate the signaling pathways of OPN in the production of OSM. signal transduction molecules (28). Therefore, we measured the Treatment of osteoblasts (human primary osteoblasts and MG-63 level of c-Src phosphorylation at Tyr416 in response to OPN. As cells) with different OPN dosages (25–200 ng/ml) or performed at shown in Fig. 2G, treatment of osteoblasts with OPN resulted in different time intervals induced OSM mRNA expression in concen- time-dependent phosphorylation of c-Src at Tyr416, which was tration- and time-dependent manners (Fig. 1A, 1B, respectively). reduced by anti-avb3 Ab (Fig. 2H). On the basis of these results, Western blot (Fig. 1C, 1D) and ELISA (Fig. 1E, 1F) analysis dem- we concluded that OPN acts through a signaling pathway in- onstrated that OPN stimulation results in increased OSM expression volving avb3 integrin and c-Src to enhance OSM expression in by guest on October 2, 2021 in the supernatant and cellular lysates in a concentration- and time- human osteoblasts.

FIGURE 1. Concentration- and time-dependent increases in OSM production by OPN. (A and B) Human primary osteoblasts and osteoblast cell line MG63 were incubated with OPN for different concentration or time intervals, and OSM mRNA expression was determined by qPCR. Human primary osteoblasts were incubated with OPN for different concentration or time intervals, and OSM protein expression was determined by Western blotting (C and D) as well as the supernatant medium was collected to analyze by ELISA (E and F). Bars show the mean 6 SEM relative expression from three individual experiments. *p , 0.05 versus control (A, B, E, and F). 3358 OPN INDUCES OSM EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Involvement of avb3 integrin and c-Src signaling in OPN-mediated OSM production in human osteoblasts. (A) Osteoblasts were incubated with OPN for 24 h, and mRNA expression of various integrins was examined by qPCR. (B and C) Osteoblasts were pretreated with OPN or OPN plus control IgG (1 mg/ml) or plus anti-avb3mAb(2mg/ml) for 24 h, and the expression of OSM mRNA or protein was analyzed by qPCR or Western blotting, respectively. (D) Osteoblasts were pretreated with anti-avb3 mAb for 30 min, followed by stimulation with OPN for 24 h. The supernatant medium was analyzed by ELISA. (E and F) Osteoblasts were pretreated for 30 min with avb3 mAb and PP2 (10 mM) or transfected for 24 h with c-Src DN mutant, followed by stimulation with OPN for 24 h. The expression of OSM mRNA (E) and protein (F) was analyzed by qPCR and ELISA, re- spectively. (G) Osteoblasts were incubated with OPN for the indicated time intervals, and c-Src phosphorylation was examined by Western blotting. (H) Osteoblasts were pretreated with avb3 mAb for 30 min, followed by stimulation with OPN. Bars show the mean 6 SEM. *p , 0.05 versus control (A, E,andF) or IgG (B and D), #p , 0.05 versus OPN alone (B, D, E,andF). The Journal of Immunology 3359

Regulation of OPN-induced OSM expression through cells were pretreated with anti-avb3 Ab, PP2, or AG1296 (Fig. 4D). transactivation of PDGFR results in MEK/ERK activation In contrast, OPN-mediated PDGFR-b phosphorylation was sup- a b Studies have implicated transactivation of PDGFR in the mediation pressed by pretreatment of cells with anti- v 3 Ab or PP2 but not of cellular inflammation (29) and in the development of min- with PD98059 or U0126 (Fig. 4E). These results suggested that eralized nodules regulated by osteoblasts (30). To test whether OPN-induced OSM expression may be mediated through a c-Src/ b c-Src–dependent transactivation of PDGFR-b wasinvolvedinOPN- PDGFR- transactivation/MEK/ERK pathway in human osteo- induced OSM expression, cells were pretreated with a PDGFR-b blasts. tyrosine kinase inhibitor AG1296. OPN-induced OSM production Involvement of AP-1 in OPN-induced OSM expression was reduced upon pretreatment with 1 mM AG1296, a general inhibitor of PDGFR (Fig. 3A) or in the absence of PDGFR-b To examine whether AP-1 binding site is involved in OPN-induced expression (Fig. 3B), suggesting that PDGFR-b may play a role in OSM expression, cells were pretreated with the AP-1 inhibitors OPN-mediated OSM expression of osteoblasts. In addition, upon curcumin (20 mM) and tanshinone IIA (10 mM), leading to the treatment of osteoblasts with OPN, increased phosphorylation of reduction of OPN-mediated OSM expression (Fig. 5A, 5B). This PDGFR-b at Tyr1021 was observed (Fig. 3C). This OPN-mediated involvement was confirmed upon measuring the levels of OSM activation of PDGFR-b was abolished upon pretreatment of expression after siRNA suppression of c-Jun (Fig. 5A, 5B), a osteoblasts with the Ab against avb3 integrin or with the inhibitor component of the transcription factor complex AP-1 that binds to PP2 (Fig. 3D). These results were consistent with our hypothesis a specific DNA sequence (32). In fact, OPN treatment of cells that OPN-induced OSM production was mediated through the resulted in marked phosphorylation of c-Jun (Fig. 5C), which was b a b transactivation of PDGFR- . inhibited by pretreatment with anti- v 3 Ab, PP2, AG1296, or Downloaded from It has been reported that the activation of PDGFR-b is related U0126 (Fig. 5D). to the MEK/ERK signaling pathway (31). To determine whether It has been reported that c-Jun is recruited to the OSM promoter, OPN-dependent OSM expression depends on MEK/ERK signal- which contains an AP-1 binding site (19, 33). The binding of c-Jun ing downstream of PDGFR-b, osteoblasts were treated with the to the AP-1 element on the OSM promoter after OPN stimulation MEK inhibitors PD98059 (15 mM) and U0126 (10 mM) (Fig. 4A, was confirmed through ChIP (Fig. 5E). OPN-induced binding of 4B). It was observed that OPN-induced OSM expression on osteo- c-Jun to the AP-1 element was attenuated by PD98059, PP2, http://www.jimmunol.org/ blasts was greatly reduced upon treatment, as well as in osteoblasts AG1296, and anti-avb3 Ab (Fig. 5E). To further verify that the expressing the MEK-1 or ERK-2 DN mutants (Fig. 4A, 4B). In ad- AP-1 element itself is involved in OPN-induced OSM expression, dition, stimulation of osteoblasts with OPN increased MEK and we performed transient transfections with AP-1 promoter–lucif- ERK phosphorylation (Fig. 4C), which was markedly inhibited when erase constructs, and it was found that osteoblasts incubated with by guest on October 2, 2021

FIGURE 3. OPN-induced PDGFR-b transactivation in osteoblasts. (A and B) Primary osteoblasts were pretreated for 30 min with AG1296 (1 mM) or transfected with control or PDGFR-b siRNA, followed by stimulation with OPN for 24 h. Total RNA and media were collected, and the expression of OSM mRNA (A) and protein (B) was assessed by qPCR and ELISA, respectively. (C) Primary osteoblasts were incubated with OPN for the indicated time intervals, and PDGFR-b phosphorylation was examined by Western blotting. (D) OPN-induced PDGFR-b phosphorylation on osteoblasts was decreased by pretreatment with avb3 mAb and PP2 (10 mM) for 30 min. Bars show the mean 6 SEM. *p , 0.05 versus control, #p , 0.05 versus OPN treatment. 3360 OPN INDUCES OSM EXPRESSION Downloaded from http://www.jimmunol.org/

FIGURE 4. OPN induced MEK and ERK activation in osteoblasts. (A and B) Osteoblasts were pretreated for 30 min with PD98059 (15 mM) and U0126 (10 mM) or were transfected for 24 h with DN-MEK-1, DN-ERK-2, or control vector, followed by stimulation with OPN for 24 h. Media and total RNA by guest on October 2, 2021 were collected, and the expression of OSM mRNA (A) zand protein (B) was analyzed by qPCR and ELISA, respectively. (C) Primary osteoblasts were incubated with OPN for the indicated time intervals, and MEK and ERK phosphorylation was examined by Western blotting. (D) Primary osteoblasts were pretreated for 30 min with avb3 mAb, PP2, or AG1296, followed by stimulation with OPN for 60 min, and MEK phosphorylation was reduced by using Western blotting. (E) Primary osteoblasts were pretreated for 30 min with avb3 mAb, PP2, PD98059, or U0126, followed by stimulation with OPN for 30 min, and phosphorylated PDGFR-b expression was determined by Western blotting. Bars in (A) and (B) show the mean 6 SEM. *p , 0.05 versus control, #p , 0.05 versus OPN treatment.

OPN exhibited a 3.6-fold increase in AP-1 promoter activity. The volume data (Fig. 6B). In addition, micro-CT imaging of hind paws increase in AP-1 activity by OPN was antagonized by the inhib- showed that mice with CIA had significant reduction of BMD at the itors of upstream signals, including anti-avb3 Ab, PP2, AG1296, ankle joint (Fig. 6C, 6D), as well as a statistically significant in- PD98059, U0126, curcumin, and tanshinone IIA, as well as the crease in the ratio between BS and BV, parameter quantifying sur- by siRNA-mediated inhibition or the expression of DN mutants face density (Fig. 6E). In contrast, the Lenti-shOPN infected CIA (Fig. 5F, 5G). Taken together, these data suggest that activation of mice showed higher BMD and had a lower BS:BV ratio at the ankle the avb3 integrin/c-Src/PDGFR transactivation/MEK/ERK/c-Jun/ joints, as compared with the control group (Fig. 6D, 6E). Con- AP-1 pathway is required for the OPN-induced increase in OSM comitantly, the level of intra-articular cartilage erosion was assayed expression in human osteoblasts. by staining with H&E and Safranin O. IHC staining of the ankle joints revealed that the numbers of OSM-immunopositive cells were Knockdown of OPN attenuates CIA-induced OSM expression significantly increased in mice with CIA but markedly diminished in and cartilage erosion in vivo CIA mice treated with Lenti-shOPN (Fig. 6F). Taken together, our To further confirm the role of OPN in vivo, we first assessed the results confirmed that Lenti-shOPN prevents cartilage erosion and effect of shRNA‐mediated OPN knockdown in the amelioration of OSM expression in vivo, suggesting OPN as a potential therapeutic mice with CIA. For local delivery of Lenti-shOPN or control- target for the treatment of RA. shRNA into the joint cavity, hind paws of mice with CIA were injected with 7.1 3 106 PFU Lenti-shOPN or control-shRNA once Discussion weekly for 4 wk after second immunization. The incidence of Recent studies have focused on the impact of inflammation in arthritis in mice with CIA was very high within 4 wk, and 95% of osteoblasts, which exhibit important functions within the arthritic the mice developed severe arthritis. The CIA group injected with bone microenvironment and thus in RA pathogenesis (16, 34); Lenti-shOPN had significantly attenuation in paw swelling com- whereas, during the past decade, RA studies had focused on the pared with the control group (Fig. 6A) as reflected by the hind paw effects of inflammation on osteoclast-mediated bone resorption The Journal of Immunology 3361 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Involvement of AP-1 in the potentiation of OSM production by OPN. (A and B) Primary osteoblasts were pretreated for 30 min with curcumin (20 mM) or tanshinone IIA (10 mM) or were transfected for 24 h with control or c-Jun siRNA, followed by stimulation with OPN for 24 h. The expression of OSM mRNA (A) and protein (B) was analyzed by qPCR and ELISA, respectively. (C and D) Primary osteoblasts were incubated with OPN for the indicated time intervals (C) or were pretreated for 30 min with avb3 mAb, PP2, AG1296, or U0126 followed by stimulation with OPN (D). The expression of phospho–c-Jun was determined by Western blotting. (E) The recruitment of c-Jun to the OSM promoter was analyzed using ChIP. (F and G) Osteoblasts were pretreated with avb3 mAb, PP2, AG1296, PD98059, U0126, curcumin, or tanshinone IIA for 30 min (F) or were transfected with DN- c-Src, DN-MEK, or DN-ERK, or PDGFR-b or c-Jun siRNAs for 24 h (G), followed by stimulation with OPN for 24 h. Transient transfection with AP-1 promoter-luciferase constructs was performed, and the results were normalized to the b-galactosidase activity. Bars in (A), (B), (F), and (G) show the mean 6 SEM. *p , 0.05 versus control, #p , 0.05 versus OPN treatment.

(35, 36). These studies highlight that the influence of inflammation involving osteoclasts, monocytes, lymphocytes, and neutrophils on bone is specific to the site of inflammation and dependent on (37), and in fact, cumulative evidence suggests that OPN is involved the cytokines present within the local bone microenvironment. It in the pathogenesis of RA because it is important in both joint in- has also been shown that OPN has implications for inflammation flammation and destruction (38). The physiologic concentration of 3362 OPN INDUCES OSM EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. Administration of Lenti-shOPN attenuates bone erosion and OSM expression in a collagen-induced arthritis (CIA) model. (A) Group of 10 mice with CIA were intra-articularly injected with 7.1 3 106 PFU Lenti-shOPN or control-shRNA on day 14 and were sacrificed on day 42. The swelling of hind paws was photographed. Original magnification 1 cm. (B) A digital plethysmometer was used to measure hind paw volumes of mice once weekly until sacrifice. (C, upper panels) Representative micro-CT images of the hind paws on day 42 (original magnification 1 mm). Bottom panels, The severest area of hind paws in the indicated group. (D and E) Quantification of bone mineral density (D) and ratio between bone surface and bone volume (E). Values were analyzed using micro-CT Skyscan Software. (F, upper panels) Histologic sections of ankle joints were stained with H&E or Safranin O in indicated groups. Lower panels, Histologic sections of ankle joints were immunostained with IgG (5 ng/ml) or OSM (2 mg/ml). Results are repre- sentative of 10 mice per experimental group. Bars in (B), (D), and (E)showthemean6 SEM. *p , 0.05 versus corresponding day 0 value (B) or control group (D and E), #p , 0.05 versus mice with CIA.

OPN is 20–35 ng/ml (39, 40); however, high concentrations of OPN OSM being expressed in osteoblasts, we also found that other types have been detected in RASFs and have even been known to induce of proinflammatory cytokines, such as IL-6, IL-1b,andTNF-a,as chondrosarcoma cell migration (41, 42). Therefore, in our current well as other proteases like matrix metalloproteinases, were induced study, we treated osteoblasts with 25–200 ng/ml OPN. In addition to by OPN (Supplemental Figs. 1, 2). To further investigate OSM is not The Journal of Immunology 3363 only a molecular signaling protein but also a significant proin- luciferase as an indicator of AP-1 activity, we also observed that flammatory indicator of RA, we demonstrated OSM mAb reduced OPN induced an increase in AP-1 activity. Similarly, those in- OPN-increased proinflammatory cytokines expression, including IL- hibitors including anti-avb3 Ab, PP2, AG1296, PD98059, and 6, IL-1b,andTNF-a, in osteoblasts and RASF (Supplemental Figs. U0126, mutant including DN-c-Src, DN-MEK, and DN-ERK, and 1, 3), resulting that OSM is a functional effector in vitro. Otherwise, siRNA against PDGFR and c-Jun also reduced OPN-induced AP-1 a previous in vivo study also reported the significance of OSM in promoter activity. arthritis pathology using an OSM neutralizing Ab in CIA model (43). In mice with CIA are possible to observe the most histopatho- Above in vitro and in vivo results both displayed OSM as a functional logical characteristics of RA, such as articular cartilage destruction mediator of RA pathogenesis. Consequently, because OSM expres- and infiltration of inflammatory cells in subchondral bone (51). IHC sion is dependent on OPN, OPN could be developed as a useful target staining of the ankle joints showed articular cartilage erosion in mice in osteoblasts for RA treatment; however, the function and the with CIA, which was ameliorated in Lenti-shOPN mice. In addition, mechanism of action of OPN in RASF remain to be elucidated. the IHC staining demonstrated the presence of other proinflammatory Extensive research has characterized the central role of the immunopositive cytokines in the subchondral bone of the CIA mice, interaction between OPN and the integrins expressed in inflam- which were reduced by Lenti-shOPN (Supplemental Fig. 4), sug- matory cells derived from activated lymphocytes and macrophages gesting the effect of OPN in the CIA model might be a response to (44, 45). In particular, the interaction of OPN and avb3 integrin the expression of other proinflammatory mediators. A recent study has been implicated in the pathogenesis of RA by a preclinical demonstrated that OSM is constitutively expressed in the subchondral study (46). During the process of osteoclastic bone resorption, the bone of patients with RA and might be secreted by osteoblasts (15). interaction between the thrombin cleaved NH2-terminal fragment Of all proinflammatory cytokines, however, OSM-immunopositive of OPN containing the RGD sequence and integrin has been well cells were significantly suppressed in Lenti-shOPN mice (Fig. 6F, Downloaded from characterized (26, 47); however, in the context of RA osteoblasts, Supplemental Fig. 4), suggesting that OSM expression in subchon- the interaction between OPN and integrin remains to be elucidated. dral bone is a functional consequence of RA through expression of We scanned a variety of cell surface integrins for their involvement Lenti-shOPN. in OPN-mediated OSM expression. Only av and b3 integrins were This study, performed on a murine model of RA pathogenesis, found to play a role; in fact, treatment with Ab against avb3 integrin provides a direct demonstration that reduced inflammation within abolishes the up-regulation of OSM production. Therefore, the the intra-articular microenvironment entails the involvement of http://www.jimmunol.org/ axis of OPN/avb3 integrin mediated inflammation of human pri- subchondral bone and repairs of focal cartilage erosions. Further- mary osteoblasts is suggested as a functional target in the patho- more, it demonstrates that PDGFR-b transactivation in osteoblasts genesis of RA. results in OPN-induced OSM expression, which significantly con- Cumulative evidence suggests that PDGFR signaling plays an tributes to a signaling pathway that ultimately influences inflam- important role in the regulation of osteoblasts; in fact, the most mation in the articular microenvironment. We also observed that recent study has revealed that enhancement of bone formation OPN-induced OSM expression requires an interaction with avb3 responds to osteoblasts through transactivation of PDGFRs, in integrin to trigger c-Src and PDGFR transactivation of MEK and particular PDGFR-a and PDGFR-b (48). In agreement with these ERK signals, which enhanced binding of c-Jun to the AP-1 site in by guest on October 2, 2021 findings, our data showed that transactivation of PDGFR-b is in- human osteoblasts. Furthermore, in agreement with our in vitro volved in OPN-induced OSM expression. In addition, we dem- studies, our in vivo results show that silencing of OPN via Lenti- onstrated that OPN stimulation of osteoblasts result in PDGFR-b shOPN infection reduces inflammation and cartilage destruction in phosphorylation at the Tyr1021 residue in a time-dependent man- mice with CIA. In conclusion, our in vitro and in vivo findings ner, and that AG1296 or PDGFR-b siRNA inhibited OPN- provide a better understanding of the mechanisms involving OPN in mediated OSM expression. Furthermore, our results provide evi- RA pathogenesis, thus making it a better therapeutic target against dence that MEK/ERK signaling transduction participates in OPN- the inflammation of osteoblasts in RA. induced OSM activation in human primary osteoblasts. The fact that the MEK/ERK inhibitors PD98059 and U0126 did not affect Acknowledgments OPN-increased PDGFR-b phosphorylation suggests that PDGFR- We thank hyTec!mage Solution Core in micro-CT experiment. We also b transactivation is an upstream event of the MEK/ERK pathway thank Dr. S. Parsons for providing the c-Src DN mutant, Dr. W. M. Fu in response to OPN stimulation. for providing the MEK-1 DN mutant, and Dr. M. Cobb for providing As mentioned earlier, AP-1 is one of the most important bind- the ERK-2 DN mutant. ing sites in the 59 region of the OSM gene (49). AP-1 binds to members of the Jun and Fos families of transcription factors that Disclosures interact with the AP-1 site to form Jun homodimers or Jun-Fos The authors have no financial conflicts of interest. heterodimers by protein dimerization through their leucine zipper motifs (50). In support of this notion, we showed that OPN- induced OSM production involves the translocation of one of References 1. Bartok, B., and G. S. Firestein. 2010. Fibroblast-like synoviocytes: key effector the AP-1 binding complex subunits, c-Fos or c-Jun, into the nu- cells in rheumatoid arthritis. Immunol. Rev. 233: 233–255. cleus of osteoblasts. We also demonstrated that OPN induced 2. Grabiec, A. M., O. Korchynskyi, P. P. Tak, and K. A. Reedquist. 2012. 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