Gadd45 deficiency in rheumatoid arthritisEnhanced synovitis through JNK signaling.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 11, November 2009, pp 3229–3240 DOI 10.1002/art.24887 © 2009, American College of Rheumatology Gadd45␤ Deficiency in Rheumatoid Arthritis Enhanced Synovitis Through JNK Signaling Camilla I. Svensson,1 Tomoyuki Inoue,1 Deepa Hammaker,1 Akihisa Fukushima,1 Salvatore Papa,2 Guido Franzoso,2 Georg Schett,3 Maripat Corr,1 David L. Boyle,1 and Gary S. Firestein1 Objective. JNK-mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45␤, which is an NF-B–regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK-7. This study was carried out to evaluate whether low Gadd45␤ expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45␤ deficiency increases arthritis severity in passive K/BxN murine arthritis. Methods. Activation of the NF-B and JNK pathways and Gadd45␤ expression were analyzed in human synovium and fibroblast-like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45␤ⴚ/ⴚ and wild-type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed. Results. Expression levels of the Gadd45␤ gene and protein were unexpectedly low in human RA synovium despite abundant NF- B activity. Forced Gadd45␤ expression in human FLS attenuated tumor necrosis factor–induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45␤ deficiency exacerbated K/BxN serum– induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts. Conclusion. Deficient Gadd45␤ expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45␤ expression or by inhibiting the activity of JNK or its upstream regulator, MKK-7. Dr. Svensson’s work was supported by NIH grant R21-DA021654, and by an Arthritis Foundation Postdoctoral Fellowship. Dr. Franzoso’s work was supported by NIH grants R01-CA-084040 and R01-CA-098583, and Cancer Research UK program grant C26587/ A8839. Dr. Schett’s work was supported by the Interdisciplinary Center for Clinic Research Erlangen (project C6). Dr. Firestein’s work was supported by NIH grant AR-047825. 1 Camilla I. Svensson, PhD (current address: Karolinska Institute, Stockholm, Sweden), Tomoyuki Inoue, PhD, Deepa Hammaker, PhD, Akihisa Fukushima, MS, Maripat Corr, MD, David L. Boyle, BA, Gary S. Firestein, MD: University of California San Diego School of Medicine, La Jolla; 2Salvatore Papa, PhD, Guido Franzoso, MD, PhD: Imperial College London, London, UK; 3Georg Schett, MD: University of Erlangen-Nuremberg, Erlangen, Germany. Address correspondence and reprint requests to Gary S. Firestein, MD, University of California San Diego School of Medicine, Division of Rheumatology, Allergy and Immunology, 9500 Gilman Drive, La Jolla, CA 92093. E-mail: email@example.com. Submitted for publication January 2, 2009; accepted in revised form July 11, 2009. Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by synovial lining hyperplasia, infiltration of the synovium with immune cells, and joint destruction (1). Matrix metalloproteinases (MMPs) are highly expressed in RA and are known to be involved in the joint degradation and remodeling of RA. JNK is thought to be especially important in extracellular matrix degradation, because JNK is a key regulator of MMP gene transcription. Furthermore, this signaling pathway, which includes its upstream kinases, MKK-4 and MKK-7, is activated in RA synovium, and JNK inhibition suppresses MMP gene expression and joint destruction in animal models of RA (1–3). Recently, growth arrest and DNA damage– 3229 3230 SVENSSON ET AL inducible gene 45␤ (Gadd45␤) was identified as a negative regulator of JNK. The Gadd45 genes, including Gadd45␣, Gadd45␤, and Gadd45␥, encode for evolutionarily conserved 18-kd proteins. Initially, Gadd45␤, also referred to as myeloid differentiation factor 118, was identified as a primary response gene that is activated in murine myeloid leukemia cells by interleukin-6 (IL-6) during terminal differentiation (4). Gadd45␤ is known to be involved in cellular stress responses, cell cycle control, and cell survival (5,6). Gadd45␤ is induced by NF-B, binds directly to the JNK-activating kinase MKK-7, and inhibits the catalytic function of MKK-7 by blocking access to ATP (6–8). Thus, Gadd45␤ serves as an endogenous inhibitor of MKK-7 that can blunt signaling through the JNK pathway (7,9). This is especially relevant in RA, since MKK-7 is expressed and activated in rheumatoid synovium (10), and MKK-7, rather than the other JNK-activating kinase, MKK-4, is required for JNK activation in cytokine-activated synoviocytes (11). Gadd45␤ deficiency has been linked to increased disease severity in murine experimental allergic encephalomyelitis (12), hepatocellular carcinoma (13,14), hepatic regeneration (15), and diabetes (16). Based on the MKK-7–inhibitory properties of Gadd45␤ and the loss of protection associated with Gadd45␤ deficiency in other models, we hypothesized that dysregulation of Gadd45␤ expression contributes to enhancement of MKK-7 and JNK responses and subsequent MMP production in RA synovium. To explore this hypothesis, we examined the expression of Gadd45␤ in human synovial tissue from patients with RA and patients with osteoarthritis (OA) and found no difference in Gadd45␤ gene or protein expression between the groups, despite markedly higher NF-B activation in patients with RA. The role of Gadd45␤ in the pathologic processes associated with inflammatory arthritis was also assessed using the K/BxN serum transfer model of arthritis in Gadd45␤⫺/⫺ mice. This experiment showed that deficient Gadd45␤ expression leads to enhanced JNK activity and exacerbation of the disease. These studies suggest that MKK-7 and JNK play a key role in synovial inflammation and joint damage. Therapeutic strategies designed to increase the expression of Gadd45␤, inhibit MKK-7, or block the activity of JNK might have potential utility in RA. MATERIALS AND METHODS Synovial tissue samples. Synovial tissue and fibroblastlike synoviocytes (FLS) were obtained from patients with OA or RA at the time of total joint replacement or synovectomy, as previously described (17). The protocol was approved by the Human Research Protection Program of the University of California, San Diego. The diagnosis of RA conformed to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (18). The samples were either processed for cell culture or snapfrozen and stored at ⫺80°C until processed for protein or gene expression analysis. Preparation of human FLS and Gadd45␤ⴚ/ⴚ mouse FLS. For preparation of human FLS, synovial tissue samples were minced and incubated for 1.5 hours at 37°C with 0.5 mg/ml of type VIII collagenase (Sigma, St. Louis, MO) in serum-free RPMI 1640 (Mediatech, Herndon, VA). Tissues were then filtered through a nylon strainer, washed, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gemini Biosciences, Calabasas, CA), L-glutamine, penicillin, streptomycin, and gentamicin in a humidified chamber containing an atmosphere of 5% CO2. After overnight culture, nonadherent cells were removed and adherent cells were trypsinized and split at a 1:3 ratio when the cells were 80–90% confluent. FLS from passages 5–9 were used, since these cells comprised a homogeneous population (⬍1% CD11b-positive, ⬍1% phagocytic, and ⬍1% Fc␥ receptor II–positive cells) (19). Mouse FLS were derived from Gadd45␤⫺/⫺ (15) and wild-type (WT) mouse knee and ankle joints by microdissecting the synovium and enzymatically dispersing the cells, as previously described (2). Mouse FLS from passages 4–5 and at 80–90% confluency were used. Antibodies and reagents. The antibodies and reagents used were as follows: rabbit antibodies against MKK-7, JNK1/2, phosphorylated MKK-4 (Ser257/Thr261), phosphorylated MKK-7 (Ser271/Thr275) (for Western blotting), phosphorylated JNK-1/2 (Thr183/Tyr185), and phosphorylated c-Jun (Ser63/ Ser73) (Cell Signaling Technology, Beverly, MA); mouse antibodies against p65 (Santa Cruz Biotechnology, Santa Cruz, CA), CD68 (Dako, Carpinteria, CA), ␤-actin (Sigma), and Gadd45␤ (8) (for immunohistochemistry); and goat antibodies against MKK-7 (for immunoprecipitation; Santa Cruz Biotechnology), Gadd45␤ (for Western blotting; Santa Cruz Biotechnology), recombinant human tumor necrosis factor (TNF) and IL-1␤ (R&D Systems, Minneapolis, MN), and anisomycin (Calbiochem, San Diego, CA). Glutathione S-transferase (GST)– c-Jun was a gift from Roche Bioscience (Palo Alto, CA). Immunohistochemistry. Serial cryosections (5 m) of synovial tissue from RA and OA patients were used for immunohistochemistry. The tissue was fixed with 4% formalin and endogenous peroxidase was depleted with 0.1% H2O2. Blocking was performed for 1 hour in phosphate buffered saline (PBS) containing 5% normal goat serum, 2.5% horse serum, and 1% human serum albumin followed by overnight incubation with antibodies against Gadd45␤, NF-B (p65), and CD68 in blocking buffer at 4°C. Mouse IgG served as a negative control. The primary antibodies were detected using biotinylated horse anti-mouse secondary antibodies (Vector, Burlingame, CA), followed by streptavidin–horseradish peroxidase (HRP) and aminoethylcarbazole substrate (Dako). Nuclei were counterstained with hematoxylin. Mouse K/BxN serum transfer arthritis model. All animal experiments were carried out according to protocols approved by the Institutional Animal Care Committee of the University of California, San Diego. To induce passive K/BxN arthritis (20,21), serum samples were pooled from arthritic ROLE OF GADD45␤ IN RHEUMATOID ARTHRITIS adult K/BxN mice, and the sera were injected intraperitoneally (IP) into Gadd45␤⫺/⫺ and WT mice. Preliminary studies were performed to determine a protocol for submaximal dosing that would permit detection of increased disease activity. In those studies, it was found that a low dose of 75 l, injected once on day 0 and then again on day 2, was sufficient to cause mild arthritis (results not shown), and therefore 150 l (75 l ⫻ 2) was selected as the submaximal dose. Arthritis severity was assessed using a semiquantitative clinical scoring system for each paw, where a score of 0 indicates normal, while a score of 1 indicates swelling of any of the following areas: the digits, mid–hind paw/mid–fore paw, and ankle/wrist joint. The maximum clinical score per leg was 4, and the maximum total score per mouse was 16. Histologic analysis. Paraffin sections of the hind paws were stained with hematoxylin and eosin (H&E) and tartrateresistant acid phosphatase (TRAP) using a leukocyte staining kit (Sigma). Areas of synovial inflammation and the number of osteoclasts were quantified by histomorphometry using an Axioskop 2 microscope (Zeiss, Oberkochen, Germany) and OsteoMeasure Analysis software (OsteoMetrics, Decatur, GA), as previously described (22). Forced Gadd45␤ expression in cultured human FLS. Using the Amaxa Human Dermal Fibroblast Nucleofector kit (NHDF-adult; Amaxa, Gaithersburg, MD) with program U-23, 1 ⫻ 106 cells were transfected with 5 g pcDNA3.1Gadd45␤ plasmid or control (empty, mock) pcDNA3.1 plasmid, in accordance with the manufacturer’s protocol (Amaxa). Following transfection, FLS were seeded in 6-well dishes and cultured in DMEM with 10% FCS at 37°C for 24 hours. The cells were incubated in fresh medium for 24 hours, and were subsequently synchronized (0.1% FCS/DMEM) for 48 hours and then stimulated with TNF (50 ng/ml) or anisomycin (1 g/ml) for 15 minutes for studies of phosphorylation of MKK-4, MKK-7, and JNK, or with TNF (50 ng/ml) for 60 minutes (for electrophoretic mobility shift assay [EMSA]), 7 hours (for luciferase reporter assays), or 24 hours (for gene expression analysis). Western blot analysis. Protein was extracted from human synovial tissue, human FLS, and mouse ankle joints using lysis buffer (150 mM NaCl, 50 mM Tris, 0.5% Triton X-100, 3% sodium dodecyl sulfate, 1 mM EDTA, protease inhibitor cocktail [Sigma], and phosphatase inhibitor cocktail I and II [Sigma]) followed by sonication. The homogenates were centrifuged at 14,000 revolutions per minute for 15 minutes, and the supernatant was fractionated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). After blocking nonspecific binding sites with 5% nonfat milk in 0.1% Tween 20/Tris buffered saline (TBS-T) for 1 hour at room temperature, the membranes were incubated with antibodies in 5% bovine serum albumin in TBS-T overnight at 4°C. After washing the membranes with TBS-T, the antibody–protein complexes were probed with appropriate HRP-conjugated secondary antibodies in 5% nonfat milk in TBS-T for 1 hour at room temperature. The immunoreactive proteins were detected with chemiluminescent reagents (Pico and Femto SuperSignal; Pierce, Rockford, IL). The nitrocellulose membranes were stripped with a Re-Blot Western blot recycling kit (Chemicon, Temecula, CA) and reblotted with different antibodies. Densitometry analysis was done using ImageQuant (Molecular Dynamics, Sunnyvale, CA) (23), and immunoposi- 3231 tive bands were normalized relative to the bands for ␤-actin or GAPDH. In vitro kinase assays. Kinase assays were performed according to previously described methods (11), with modifications. Following transfection, 3 ⫻ 106 FLS were seeded in 10-cm dishes and cultured in DMEM with 10% FCS at 37°C for 24 hours. The cells were incubated in fresh medium for 24 hours, and were subsequently synchronized (0.1% FCS/ DMEM) for 48 hours and then stimulated with TNF (50 ng/ml) with PBS or TNF alone (50 ng/ml) for 15 minutes. Cells were washed with cold PBS and scraped directly into lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 25 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 20 mM ␤ glycerophosphate, 10 mM sodium fluoride, 1 mM Na3VO4, protease inhibitor cocktail [Complete Mini; Roche Applied Sciences, Indianapolis, IN], 1 mM dithiothreitol, and 5 mM p-nitrophenyl phosphate). After 30 minutes of incubation on ice, the homogenate was centrifuged at 14,000g and the supernatant was retained for immunoprecipitation. Samples containing the same amount of protein were incubated with specific goat anti–MKK-4, anti–MKK-7, or control IgG for 3 hours at 4°C on a rotating wheel, after which protein A–Sepharose CL-4B (Oncogene Research Products, Cambridge, MA) was added and incubation was continued overnight. After centrifugation, the immune complexes were washed with lysis buffer and then with kinase buffer (25 mM HEPES, pH 7.4, 25 mM MgCl2, 20 mM ␤-glycerophosphate, 0.1 mM Na3VO4, protease inhibitor cocktail [Roche Applied Sciences], 2 mM dithiothreitol, 10 mM p-nitrophenyl phosphate, and 20 M ATP). The kinase reaction was started by adding 30 l of the kinase buffer, with GST–c-Jun as substrate at 8 g per reaction, and 2 Ci of 32P-ATP, and then incubating for 30 minutes at 37°C. Samples were heated for 5 minutes at 95°C, separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen), and visualized by autoradiography. EMSA. Following transfection, FLS were seeded in 10-cm dishes and cultured in DMEM with 10% FCS at 37°C for 24 hours. The cells were incubated in fresh medium for 24 hours and subsequently synchronized (0.1% FCS/DMEM) for 48 hours (for NF-B) or 72 hours (for activator protein 1 [AP-1]) and then stimulated with TNF (50 ng/ml) for 60 minutes. Nuclear extracts were isolated using a nuclear protein extraction kit (Chemicon), in accordance with the manufacturer’s instructions. Nuclear extracts (5 g) were incubated with ␥-32P–ATP–labeled or unlabeled AP-1 (5⬘-CGCTTGATGAGTCAGCCGGAA-3⬘) and NF-B (5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘) oligonucleotides (Promega, Madison, WI) for 30 minutes at room temperature, and resolved on 6% DNA retention gels (Invitrogen). The DNA binding was visualized by autoradiography, and bands were quantified using ImageQuant (Molecular Dynamics). Luciferase assays. The Amaxa program U-23 was also used for transfection of luciferase reporter constructs. FLS (5 ⫻ 105 cells) were transfected with 2.8 g of Gadd45␤ or control (empty, mock) pcDNA3.1 plasmid, 2 g AP-1– luciferase (Luc) or NF-B–Luc vector, and 0.2 g SV-40–Luc (Renilla) reporter control vector (generously provided by Dr. Michael David, San Diego, CA). Following transfection, FLS were seeded in 12-well dishes and cultured in DMEM with 10% FCS at 37°C for 24 hours. The cells were incubated in fresh medium for 24 hours and subsequently synchronized (0.1% FCS/DMEM) for 48 hours, and then stimulated with 3232 SVENSSON ET AL Figure 1. NF-B activity and Gadd45␤ gene and protein expression in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. A, Analysis of NF-B activity in 2 RA and 2 OA synovial samples by electrophoretic mobility shift assay (top), showing high NF-B nuclear binding in RA compared with OA synovium, and quantitative results determined by densitometry (bottom), expressed as the relative band density (optical density [OD]) of NF-B DNA binding. B and C, Gene expression of cyclooxygenase 2 (COX-2) (B) and Gadd45␤ (C) in RA and OA synovial tissue extracts, as determined by quantitative polymerase chain reaction. D, Gadd45␤ protein expression in RA and OA synovia, as assessed by Western blot analysis. These results indicate that NF-B DNA binding and products of NF-B activity are increased in RA synovium as compared with OA synovium, and yet there is no difference in Gadd45␤ gene or protein expression between the 2 groups. Bars in A–D show the mean and SEM results for 7 samples per group. E–H, Immunohistochemical analyses of RA and OA synovium, showing Gadd45␤ protein expression in RA synovial tissue (E) and OA synovial tissue (G), as well as staining for NF-B in consecutive RA and OA sections (F and H, respectively). Representative results are shown. I and J, Gadd45␤ protein expression in RA synovium (I), with a consecutive section stained for CD68, a macrophage marker (J). Original magnification ⫻ 400 in E, F, G, and H; ⫻ 200 in I and J. Bars ⫽ 50 m. TNF (50 ng/ml) for 7 hours. Luciferase activity was measured in cell lysates using a luminometer (MGM Instruments, Hamden, CT) and the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Relative AP-1 activity and NF-B activity were determined by normalization to the values for SV-40–Renilla Luc activity. Quantitative real-time polymerase chain reaction (PCR). Messenger RNA (mRNA) in frozen mouse and human tissues and cultured FLS was isolated using RNA Stat (TelTest, Friendswood, TX), as described previously (24). Complementary DNA (cDNA) was prepared, and quantitative real-time PCR was performed with TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA), both according to the manufacturer’s instructions, to determine the relative mRNA levels using the GeneAmp 7300 Sequence Detection system (Applied Biosystems). Predeveloped specific primers were used to detect Gadd45␤ (assay no. Hs01063373), MMP3 (assay no. Hs00233962, Mm00440295), MMP13 (assay no. Mm00439490), and cyclooxygenase 2 (COX-2) (assay no. Hs00153133) (all from Applied Biosystems). Threshold cycle values in each sample were used to calculate the number of cell equivalents in the test samples. The data were normalized to the values for GAPDH expression (for human samples) (cat- alog no. 402869; Applied Biosystems) and Hprt1 expression (for mouse samples) (assay no. Mm00446968; Applied Biosystems) to obtain the relative cell equivalents, with results expressed as relative expression units (REU). Statistical analysis. Results are expressed as the mean ⫾ SEM. Differences were assessed by one-way and two-way repeated-measures analysis of variance (ANOVA), followed by the Bonferroni post hoc test for comparison of multiple groups or a Student’s t-test for comparison of 2 groups. The criterion for significance was set at P values less than 0.05. RESULTS NF-B activity and Gadd45␤ expression in human RA and OA synovial tissue. Gadd45␤ is expressed at very low levels in most tissues, but its expression is induced by NF-B (4,6). Therefore, we expected Gadd45␤ levels to be high in RA synovial tissue relative to OA synovial tissue due to the marked activation of NK-B. To study the link between NF-B activation, ROLE OF GADD45␤ IN RHEUMATOID ARTHRITIS 3233 Figure 2. Gadd45␤ gene expression, as assessed by quantitative real-time polymerase chain reaction, in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HS68 human dermal fibroblasts at various time points following stimulation with tumor necrosis factor (TNF) (50 ng/ml) (A and D), interleukin-1␤ (IL-1␤) (2 ng/ml) (B), or anisomycin (1 g/ml) (C and E). Bars show the mean and SEM of 6–8 separate RA FLS lines in A–C and 3 HS68 dermal fibroblasts in D and E. ⴱ ⫽ P ⬍ 0.05 versus untreated control (C). Gadd45␤ expression, and JNK activity in human synovial tissue, we first assessed NF-B DNA binding by EMSA in synovia from RA and OA patients. In agreement with previous studies (3), we found higher NF-B DNA binding in rheumatoid synovium as compared with OA synovium (mean ⫾ SEM 4.7 ⫾ 0.6 REU versus 1.4 ⫾ 0.3 REU [n ⫽ 7 each]; P ⫽ 0.003) (Figure 1A). Moreover, expression of an NF-B–regulated gene, COX-2, was elevated in the RA group as compared with the OA group (17.5 ⫾ 6.3 REU versus 3.2 ⫾ 1.0 REU [n ⫽ 7 each]; P ⫽ 0.04) (Figure 1B). These data confirm that RA synovial tissue exhibits higher NF-B activity and increased expression of NF-B–regulated genes compared with OA synovial tissue. Surprisingly, when the relative expression levels of the Gadd45␤ gene and protein were assessed in the same synovial tissue samples, we found that Gadd45␤ mRNA and protein levels were similar between RA and OA (n ⫽ 7 each; P ⬎ 0.05) (Figures 1C and D). The cellular distribution of Gadd45␤ in synovial tissue was then determined by immunohistochemistry in serial sections from RA and OA synovia. Gadd45␤ was expressed predominantly in the intimal lining (Figures 1E, G, and I) and was mainly present in CD68⫹ macrophages rather than intimal lining FLS (Figures 1I and J). The Gadd45␤ detected in the synovial sublining was mainly present in macrophages rather than T cells. Gadd45␤ and NF-B (p65) immunoreactivity showed similar patterns of expression in RA and OA, with no apparent difference in Gadd45␤ immunoreactivity between the groups (Figures 1E and G), despite stronger immunoreactivity for NF-B (p65) in the RA group (Figures 1F and H). Gadd45␤ gene expression in human FLS. The discoordinate expression of Gadd45␤ and NF-B in RA synovium and the relatively low expression of Gadd45␤ in synovial lining fibroblast-like cells led us to explore the expression and regulation of Gadd45␤ in cultured FLS. To determine whether RA FLS express Gadd45␤ in response to cytokines, the cells were stimulated with TNF (50 ng/ml) or IL-1␤ (2 ng/ml), and Gadd45␤ gene expression was examined by quantitative PCR. Stimula- 3234 SVENSSON ET AL Figure 3. Effect of forced Gadd45␤ expression on the JNK signaling pathway in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). A and B, Quantitative results (top) and representative Western blots (bottom) for the extent of phosphorylation of MKK-7 (A) and MKK-4 (B) in tumor necrosis factor (TNF)–stimulated RA FLS transfected with Gadd45␤ cDNA plasmid or empty pcDNA3.1 (mock) plasmid. C, Representative autoradiographs of MKK-4 and MKK-7 activity, as determined from in vitro kinase assays in which control and Gadd45␤-overexpressing FLS were stimulated with TNF. These experiments were repeated 4 times, with similar results obtained each time. D and E, Quantitative results (top) and representative Western blots (bottom) for the extent of JNK phosphorylation in TNF- or anisomycin (Aniso)–stimulated RA FLS transfected with Gadd45␤ cDNA plasmid or empty pcDNA3.1 (mock) plasmid. Bars in A, B, D, and E show the mean and SEM percent change in expression in Gadd45␤-overexpressing RA FLS relative to the TNF- or anisomycin-stimulated mock control cultures (set at 100%). F–H, Activator protein 1 (AP-1) luciferase reporter activity (F), NF-B luciferase reporter activity (G), and matrix metalloproteinase 3 (MMP3) gene expression (H) in mock control and Gadd45␤-overexpressing RA FLS stimulated with TNF. Bars in F–H show the mean and SEM results in TNF-stimulated cultures relative to the phosphate buffered saline (PBS)–treated control cultures. MMP3 expression values were normalized to the values for GAPDH. In each experiment, PBS was used as the unstimulated control, and 5–7 separate RA FLS lines were studied. tion of RA FLS with TNF or IL-1␤ under conditions that optimized the activation of NF-B (see below and ref. 25) induced only low-level, transient expression of Gadd45␤ (Figures 2A and B). The cells were also stimulated with anisomycin, which is a potent and nonselective activator of kinases (including the MKK-4/ MKK-7/JNK pathway), and thus was used as a positive control for comparison with cytokine receptor–mediated JNK signaling. Stimulation of the cells with anisomycin (1 g/ml) evoked massive and persistent Gadd45␤ expression in human FLS (Figure 2C), indicating that synoviocytes are able to express abundant Gadd45␤ under some circumstances. Similar results were observed in OA FLS (results not shown) and in HS68 cells, a human dermal fibroblast cell line (Figures 2D and E), suggesting that this is a general property of cultured FLS as well as fibroblast-lineage cells. Gadd45␤ regulation of MKK-4 and MKK-7 activity in FLS. To assess the effect of Gadd45␤ expression on JNK-mediated signaling, we used human RA FLS transfected with Gadd45␤ cDNA containing pcDNA3.1 plasmid or empty pcDNA3.1 control plasmid. The effect of forced Gadd45␤ protein expression on the TNFinduced phosphorylation and activity of MKK-4 and MKK-7 was examined. Using Western blotting, phosphorylation of MKK-4 and MKK-7 was readily detected in RA FLS stimulated with TNF (50 ng/ml for 15 minutes), with no difference observed between Gadd45␤ pcDNA3.1–transfected cells and empty pcDNA3.1– transfected cells (n ⫽ 5 each; P ⬎ 0.05) (Figures 3A and B). Because Gadd45 ␤ is thought to inactivate MKK-7 through binding within the kinase ATP pocket, rather than via the prevention of phosphorylation (7), the functional activity of MKK-7 and MKK-4 was measured using an in vitro protein kinase activity assay, with GST–c-Jun as a substrate. JNK forms a tight complex with c-Jun, and therefore GST–c-Jun may serve as a substrate in MKK-4/MKK-7 kinase activity assays (26). As shown in Figure 3C, forced expression of Gadd45␤ in FLS blocked the TNF-induced function of MKK-7 without affecting the function of MKK-4. ROLE OF GADD45␤ IN RHEUMATOID ARTHRITIS Gadd45␤ regulation of JNK phosphorylation in FLS. After observing that forced Gadd45␤ expression in FLS attenuates MKK-7 activity, we assessed the effect of Gadd45␤ on JNK phosphorylation. RA FLS were transfected with the Gadd45␤ plasmid or control plasmid and then stimulated with TNF (50 ng/ml for 15 minutes). Forced Gadd45␤ overexpression significantly reduced JNK phosphorylation compared with that in the TNFstimulated mock control cultures (mean ⫾ SEM phosphorylation of JNK 0.31 ⫾ 0.05 relative to TNFstimulated mock control cultures [n ⫽ 7 each]; P ⬍ 0.0001) (Figure 3D). JNK phosphorylation was normal in Gadd45␤-transfected cells that had been stimulated with anisomycin (1.26 ⫾ 0.14–fold increase relative to TNF-stimulated mock control cultures [n ⫽ 5 each]; P ⬎ 0.05) (Figure 3E). This finding is consistent with the observations in previous studies showing that cytokineinduced JNK phosphorylation is strictly MKK-7 dependent, whereas anisomycin-induced JNK activation can utilize either MKK-4 or MKK-7 (11). Thus, Gadd45␤ modulation of MKK-7/JNK signaling suppresses cytokine-mediated FLS activation while leaving other stress responses through MKK-4 intact. Gadd45␤ regulation of JNK function in FLS. The consequences of increased Gadd45␤ expression for JNK function were then assessed by examining the transcriptional activity of AP-1 and NF-B. Cultured RA FLS were cotransfected with Gadd45␤ plasmid or mock pcDNA3.1 plasmid and with an AP-1 or NF-B promoter luciferase construct. Stimulation of the cells with TNF (50 ng/ml for 7 hours) increased the transcriptional activity of AP-1 and NF-B in the pcDNA3.1 mock–transfected cultured FLS. Forced Gadd45␤ overexpression blocked AP-1 promoter activity (2.8 ⫾ 0.3– fold change in TNF-stimulated mock cultures versus 0.9 ⫾ 3–fold change in TNF-stimulated Gadd45␤ cultures [n ⫽ 4 each, relative to PBS cultures]; P ⫽ 0.004), but had no effect on NF-B promoter activity (7.3 ⫾ 1.7–fold change in TNF-stimulated mock cultures versus 5.5 ⫾ 0.6–fold change in TNF-stimulated Gadd45␤ cultures [n ⫽ 4 each, relative to PBS cultures]; P ⫽ 0.34) (Figures 3F and G). In addition, cultured RA FLS transfected with Gadd45␤ plasmid or mock pcDNA3.1 plasmid and stimulated with TNF (50 ng/ml for 1 hour) were also assessed by EMSA for AP-1 and NF-B DNA binding. In agreement with the findings from the luciferase reporter construct assays described above, Gadd45␤ overexpression attenuated AP-1 DNA binding, but had no effect on NF-B DNA binding (results not shown). The MMP3 gene contains key AP-1 binding sites in its promoter. Thus, the presence of JNK-activated 3235 Figure 4. Gadd45␤ gene expression in the ankle joints of wild-type mice with K/BxN serum transfer arthritis. K/BxN serum was injected at the standard dose (150 l intraperitoneally on day 0), which caused severe arthritis, and Gadd45␤ gene expression in the knee joints was then assessed by quantitative reverse transcription–polymerase chain reaction. Gadd45␤ gene expression was modestly increased on day 4 and day 8, confirming that this protein is expressed in response to innate immune challenges in normal mice. Bars show the mean and SEM results in 7 samples. Gadd45␤ gene expression values were normalized to the values for Hprt1. ⴱ ⫽ P ⬍ 0.05 versus day 0. AP-1 is required for transcription of this MMP gene, and increased Gadd45␤ expression should suppress this pathway. As shown in Figure 3H, TNF-stimulated (50 ng/ml for 24 hours) expression of MMP3 mRNA was reduced in cells transfected with the Gadd45␤ plasmid compared with that in TNF-stimulated mock control– transfected FLS (n ⫽ 6 each; P ⬍ 0.0001). Previous studies have demonstrated that the expression levels of MMP3 mRNA regulated by MKK-7 and JNK in cultured FLS closely parallel the protein levels in culture supernatants (11). Gadd45␤ expression and function in K/BxN serum transfer arthritis. Initial studies were performed to determine the time course of Gadd45␤ expression in the passive K/BxN arthritis model (20,21). Serum (150 l) was injected IP on day 0, and the joints were harvested on days 0, 1, 4, 8, and 12. Quantitative PCR showed that articular Gadd45␤ gene expression could be detected in naive WT mice, similar to that seen in human RA and OA synovium. The levels of Gadd45␤ mRNA increased modestly after serum injection, and were similar in magnitude to the changes observed in cytokinestimulated human FLS (Figure 4). The regulatory role of Gadd45␤ in this murine model was then evaluated using Gadd45␤⫺/⫺ mice. To permit detection of disease exacerbation, a modified 3236 SVENSSON ET AL Figure 5. Development of K/BxN serum–induced arthritis, synovial inflammation, and bone destruction in Gadd45␤⫺/⫺ mice. A, To determine the arthritis severity in Gadd45␤⫺/⫺ and wild-type (WT) mice over time after the induction of K/BxN serum transfer arthritis, clinical scores were assessed showing that inflammatory arthritis was exacerbated in Gadd45␤⫺/⫺ mice compared with WT mice. Bars show the mean ⫾ SEM results in 8 mice per group, representative of 1 of 3 separate experiments. ⴱ ⫽ P ⬍ 0.01. B and C, The ankles from Gadd45␤⫺/⫺ and WT mice were harvested on day 6 and prepared for histologic evaluation by hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining, to assess synovial inflammation (B) and bone destruction (C). In B, results are expressed as the area of inflammation (in mm2) on H&E-stained sections, as determined using image analysis. ⴱ ⫽ P ⬍ 0.002. In C, results are expressed as the number of osteoclasts per bone perimeter, as assessed by TRAP staining. ⴱ ⫽ P ⬍ 0.05. Bars in B and C show the mean and SEM results in 10 mice per group. D and E, Representative photomicrographs of the WT mouse ankle joints (D) and Gadd45␤⫺/⫺ mouse ankle joints (E), as analyzed by TRAP staining 6 days after the induction of K/BxN passive transfer arthritis (original magnification ⫻ 20). The results show greater local bone erosions and osteoclast formation in the Gadd45␤⫺/⫺ mice (indicated by purple staining and arrows). protocol with a submaximal dose of K/BxN serum was used (see Materials and Methods). Mild arthritis was observed in the WT group, but disease severity was significantly greater in the Gadd45␤⫺/⫺ group (P ⬍ 0.01 by 2-way ANOVA) (representative results are shown in Figure 5A). Synovial inflammation and osteoclast generation in K/BxN serum transfer arthritis. Consistent with the increased clinical score of arthritis in the Gadd45␤⫺/⫺ group, histologic evaluation of the joints at the peak of K/BxN serum–induced arthritis (day 6) demonstrated significantly greater synovial inflammation in Gadd45␤⫺/⫺ mice compared with WT mice (area of H&E-stained sections, 0.47 ⫾ 0.09 mm2 in Gadd45␤⫺/⫺ mice versus 0.15 ⫾ 0.05 mm2 in WT mice [n ⫽ 10 each]; P ⬍ 0.002) (Figure 5B). Because NF-B and JNK participate in osteoclast differentiation and bone erosion (27,28), we assessed the number of osteoclasts in the mouse joints. Using TRAP staining, we found that the number of osteoclasts was higher in Gadd45␤⫺/⫺ mice compared with that in the WT group (5.00 ⫾ 1.09 per mm in Gadd45␤⫺/⫺ mice versus 2.10 ⫾ 0.46 per mm in WT mice [n ⫽ 10 each]; P ⬍ 0.05) (Figures 5C–E). JNK phosphorylation in Gadd45␤ⴚ/ⴚ mice. In separate experiments, the joints were collected at the peak of K/BxN serum–induced arthritis (day 6) and evaluated by Western blot analysis to determine the phosphorylation state of JNK. Whereas only a modest increase in the levels of phosphorylated JNK was observed in the WT group, a marked increase in the levels of phosphorylated JNK was observed in the Gadd45␤⫺/⫺ mice (16.7 ⫾ 0.4–fold increase in Gadd45␤⫺/⫺ mice versus 3.6 ⫾ 0.6–fold increase in WT mice relative to ROLE OF GADD45␤ IN RHEUMATOID ARTHRITIS untreated WT controls [n ⫽ 7 each]; P ⬍ 0.05 versus untreated WT controls) (Figures 6A and B). MMP3 and MMP13 gene expression in Gadd45␤ⴚ/ⴚ mice and cultured murine FLS. The relationship between elevated JNK activity in the mouse joints and changes in MMP gene expression was then evaluated by quantitative PCR. On day 6 after serum administration, the expression levels of the MMP3 and MMP13 genes were significantly greater in Gadd45␤⫺/⫺ mice compared with WT mice (for MMP3, mean ⫾ SEM 3.6 ⫾ 0.8 REU versus 1.3 ⫾ 0.3 REU [n ⫽ 7 each] [P ⫽ 0.02]; for MMP13, 2.2 ⫾ 0.3 REU versus 1.2 ⫾ 0.1 REU [n ⫽ 7 each] [P ⫽ 0.03]) (Figure 6C). These findings link Gadd45␤ not only to a protective role in the progression of inflammatory arthritis but also to critical regulatory mechanisms in relation to the activities and function of MKK-7 and JNK. FLS are thought to be a primary source of MMPs in inflammatory arthritis (29). Therefore, we determined whether MMP gene expression is altered in murine FLS from Gadd45␤-deficient mice. As shown in Figure 6D, Gadd45␤⫺/⫺ FLS expressed significantly more MMP3 and MMP13 mRNA than did WT mouse FLS after TNF stimulation (for TNF-stimulated MMP3, mean ⫾ SEM 179 ⫾ 11 REU versus 82 ⫾ 4 REU [n ⫽ 3 each] [P ⬍ 0.001]; for TNF-stimulated MMP13, 19.3 ⫾ 1.8 REU versus 9.7 ⫾ 2.4 REU [n ⫽ 3 each] [P ⬍ 0.05]). DISCUSSION RA is a chronic autoimmune disease that is marked by synovial inflammation and joint destruction (1). Degradation of articular extracellular matrix in the RA joint is, in part, mediated by MMPs (29,30). The production of these enzymes is regulated by cytokines, such as TNF and IL-1, most notably through activation of the MAPKs. Activation of a key MAPK, namely JNK, is especially important in this process, since it phosphorylates transcription factors, such as AP-1, that are required for MMP transcription (2,31). JNK activity is regulated by 2 upstream kinases, MKK-4 and MKK-7. Of these 2 kinases, MKK-7 is particularly important in RA, since only MKK-7 is required for JNK activation in FLS after cytokine stimulation (10,11). The kinases in the JNK pathway are highly activated in RA synovium and contribute to the production of cytokines and MMPs. Although this could be a response to proinflammatory cytokines, we considered whether Gadd45␤ deficiency might also be a contributor to overactivation of JNK in rheumatoid synovitis. Our initial studies showed that Gadd45␤ expression in RA synovium is similar to that in OA synovium, 3237 despite the activation of NF-B in the intimal lining. In contrast, other NF-B–driven genes are readily detected in higher amounts in RA than in OA samples, which indicates that the NF-B activity is sufficient for induction of other NF-B–regulated genes in the synovial environment. Similarly, cultured synoviocytes had only minimal and transient Gadd45␤ induction, despite stimulation with cytokines that maximally increase NF-B translocation. The mechanism for this deficient transcriptional response to NF-B activation has not yet been defined. However, it is stimulus specific, because anisomycin induced abundant Gadd45␤ expression. It appears to be a common feature of fibroblasts, rather than being disease specific, because OA FLS and HS68 dermal fibroblasts also had limited TNF-induced Gadd45␤ gene expression as compared with that after stimulation with anisomycin. Therefore, the relative lack of Gadd45␤ is not specifically associated with RA. Preprogrammed deficient Gadd45␤ responses in the intimal lining would not cause RA but could amplify the innate immune processes that lead to joint damage. Uncoupled NF-B activation and Gadd45␤ in the presence of proinflammatory cytokines is a potential mechanism that could explain why the JNK pathway is highly responsive in an inflammatory disease state such as RA. Thus, MKK-7 could be phosphorylated but would not be restrained by Gadd45␤. Because MKK-7 is the pivotal upstream kinase that regulates JNK in FLS, it could readily phosphorylate JNK and increase MMP production. The end result would be enhanced extracellular matrix destruction (31). This hypothesis is supported by our studies demonstrating that Gadd45␤ overexpression in cultured FLS reverses this process by suppressing AP-1 binding, AP-1–mediated transcription, and MMP expression. Other negative regulators of JNK activity could also be important and may contribute to the regulation of cytokines and MMPs. The relative contribution of one such factor, X-linked inhibitor of apoptosis protein, has not been studied in relation to Gadd45␤, but it has been associated with deficient synoviocyte apoptosis in RA (32). Injection of K/BxN serum increased synovial Gadd45␤ expression transiently, up to 2–3-fold, which was similar to that observed in TNF-stimulated cultured FLS. Direct comparisons between RA and normal tissue with respect to Gadd45␤ expression, and how this correlates with observations in the mouse model, are difficult because matched normal human samples are rarely available. Thus, the passive model needs to be interpreted with some caution. However, the experiments allowed us to evaluate the role of Gadd45␤ in inflammation and the consequences of a deficient 3238 SVENSSON ET AL Figure 6. JNK phosphorylation and matrix metalloproteinase (MMP) gene expression in the joints of Gadd45␤⫺/⫺ and wild-type (WT) mice. A, JNK phosphorylation was evaluated by Western blotting in the ankle joints of Gadd45␤⫺/⫺ and WT mice on day 6 after induction of K/BxN serum transfer arthritis as compared with naive WT controls. Representative results are shown. ␤-actin was used as the reference. tot ⫽ total. B, Phosphorylation of JNK was assessed quantitatively in Gadd45␤⫺/⫺ and WT mice with K/BxN serum–induced arthritis, expressed as the percent change relative to naive WT controls. C, Gene expression levels of MMP3 and MMP13 in the mouse joints were determined on day 6 by quantitative reverse transcription–polymerase chain reaction. Bars show the mean and SEM results in 7 mice per group. ⴱ ⫽ P ⬍ 0.05 versus naive WT; # ⫽ P ⬍ 0.05 versus WT group that received K/BxN serum. D, Fibroblast-like synoviocytes (FLS) were established from the knee joints of Gadd45␤⫺/⫺ and WT mice, and the relative gene expression of MMP3 and MMP13 in response to tumor necrosis factor (TNF) (50 ng/ml for 24 hours) was determined in both FLS groups. Bars show the mean and SEM results in 3 mice per group. Gene expression values were normalized to the values for Hprt1. ⴱ ⫽ P ⬍ 0.05 versus phosphate buffered saline (PBS)–treated WT; # ⫽ P ⬍ 0.05 versus TNF-treated WT. Gadd45␤ response. A submaximal dose of serum in Gadd45␤⫺/⫺ mice led to rapid onset of arthritis, higher clinical scores, increased inflammation, and increased numbers of osteoclasts in the joints compared with the findings in serum-treated WT mice. However, it is not known whether the differences in osteoclast differentiation in vivo are due to an inherent property of Gadd45␤⫺/⫺ precursors or are a reflection of an altered cytokine milieu. These findings demonstrate that the net effect of Gadd45␤ deficiency is disease exacerbation. Gadd45␤ deficiency was accompanied by increased JNK phosphorylation and elevated MMP3 and MMP13 gene expression in the joints of Gadd45␤⫺/⫺ mice, as com- pared with the arthritic controls. Of note, the K/BxN serum transfer model represents an inflammatory arthritis that is independent of adaptive immune responses (33), and thus our studies are the first demonstration that Gadd45␤ influences diseases that are strictly dependent on innate immunity. The expression and role of Gadd45␤ in arthritis were not clearly delineated in previous studies. For instance, a previous report suggested that Gadd45␤ expression in synovial fluid T cells in RA might suppress apoptosis (34). In that case, Gadd45␤ deficiency could enhance lymphocyte death and ameliorate disease. However, this distribution of Gadd45␤ was not observed ROLE OF GADD45␤ IN RHEUMATOID ARTHRITIS in our studies of synovial tissue, since we observed that Gadd45␤ expression was localized to the lining synoviocytes and sublining macrophages rather than T cells. In addition to focusing on the role of Gadd45␤ in human FLS in this study, we also assessed the role of Gadd45␤ in murine macrophages. Preliminary studies have shown that MMP13 gene expression in Gadd45␤-deficient peritoneal macrophages is higher than that in WT cells in response to TNF stimulation (results not shown). Therefore, Gadd45␤ might be an important factor in synovial FLS and macrophages and warrants further studies to examine how Gadd45␤ may regulate JNK activity in RA synovial macrophages in situ. In contrast to FLS, chondrocytes from normal individuals and patients with early OA (35) constitutively express Gadd45␤ mRNA. The role of Gadd45␤ in chondrocytes is complex and age dependent. At the embryonic stage, Gadd45␤ is critical for terminal differentiation of mouse chondrocytes in the growth plate. However, at this stage, Gadd45␤ activates, rather than inhibits, JNK through interactions with MTK-1/ MEKK-4, upstream activators of JNK (36). As a consequence, Gadd45␤⫺/⫺ mouse embryos display a decreased MMP13 gene expression, which leads to defective mineralization and decreased bone growth. In adult cartilage, chondrocytes are quiescent, and cell survival is essential for maintenance of the avascular tissue. In late-stage OA, a reduction in Gadd45␤ expression by chondrocytes is associated with increased TNFinduced cell death (35,36). Thus, endogenous Gadd45␤ might provide a protective function in adult chondrocytes by promoting cell survival. On the other hand, Gadd45␤ decreases collagen production by chondrocytes, which could have a negative effect on cartilage composition and integrity. Gadd45␤ might play an important role in many other diseases by mediating a delicate balance between different cell survival and cell death pathways. For example, inadequate expression of Gadd45␤ is thought to be a key feature in insulin-secreting beta cells undergoing apoptosis in response to IL-1␤ (16). In contrast, down-regulation of Gadd45␤ has been associated with cell survival, since it was strongly correlated with the degree of malignancy in hepatocellular carcinoma (13,14). Gadd45␤ is also thought to play an especially important role in adaptive immunity (12). Liu et al showed that Gadd45␤ limits the proliferation of CD4⫹ T cells in response to T cell receptor signaling and cytokines, and that T cells lacking Gadd45␤ proliferated faster than WT controls and were more resistant to activation-induced cell death (12). In addition, the study 3239 by Liu et al showed that deletion of Gadd45␤ exacerbates murine experimental allergic encephalomyelitis (12). While Gadd45␤ is a complex molecule that can potentially affect many cellular functions, its role in the JNK pathway is especially noteworthy in arthritis. Previous studies have implicated JNK activity in RA (34), and recent in vitro studies with cultured FLS suggested that MKK-7 is the dominant upstream kinase for cytokine-mediated processes (10,11). However, MKK-4 does play a role in some circumstances, such as in Toll-like receptor 3–mediated signaling or anisomycin stimulation (37). One potential therapeutic approach in RA is to block a subset of relevant JNK functions, which might be safer than inhibiting all JNK activity. This could be accomplished by suppressing MKK-7 while leaving MKK-4 intact. Gadd45␤ accomplishes this goal by binding to MKK-7 and preventing it from activating JNK (8). Whereas the role of JNK in RA has been well documented, our present findings in Gadd45␤⫺/⫺ mice suggest that MKK-7 is a pivotal kinase for synovial JNK regulation. The beneficial effects of Gadd45␤ provide support for a therapeutic strategy that targets the JNK pathway for diseases involving innate immunity, either by directly inhibiting JNK or MKK-7 or by enhancing Gadd45␤ expression. This approach could potentially suppress osteoclast development, expression of MMPs, and synovial inflammation in RA. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Firestein had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Svensson, Schett, Corr, Boyle, Firestein. Acquisition of data. Svensson, Inoue, Hammaker, Fukushima, Papa, Franzoso. Analysis and interpretation of data. Svensson, Boyle, Firestein. REFERENCES 1. Firestein GS. Evolving concepts of rheumatoid arthritis. 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