RIAA was supplied by Hopsteiner (New York, NY); the chemical composition of RIAA was described in . Phospho-ERK1/2, phospho-p38, phospho-JNK, phospho-β-catenin anti-bodies were purchased from Cell Signaling Technology (Danvers, MA). SB216763 was purchased from Biomol (Plymouth Meeting, PA). LPS (from E. coli), anti-actin antibody, parthenolide and other analytical grade chemicals were purchased from Sigma (St. Louis, MO). Electrophoresis gels and reagents were purchased from Bio-Rad (Hercules, CA).
RAW 264.7 macrophages were purchased from ATCC (Manassas, VA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) in the presence of 10% fetal bovine serum (FBS), 100 U penicillin/ml and 100 μg streptomycin/ml, according to manufacturer instructions. All test compounds were dissolved in DMSO, then diluted in serum-free media and used at a final concentration of 0.1% DMSO.
Electrophoretic mobility shift assays (EMSA)
RAW 264.7 cells were sub-cultured and grown overnight in 6-well plates at a density of 2 × 106 cells per well, and incubated in the absence or presence of RIAA for 1 h followed by LPS (1 μg/ml) stimulation for 2 h. Nuclear extract was prepared as previously described  with modifications. Briefly, cells were lysed with lysis buffer containing 10 mM Hepes-KOH (pH 7.9), 0.1% NP-40, 10 mM KCl, 1.5 mM MgCl2, and protease inhibitor cocktail (Amersham Biosciences, Piscataway, NJ) for 15 min on ice and centrifuged at 10,000× for 10 min. Cell pellet was washed with the lysis buffer, resuspended in nuclear extract buffer containing 20 mM Hepes-KOH, 25% glycerol (v/v), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA and protease inhibitor cocktail, and sonicated (2 × 10 sec at 60% power level). The samples were centrifuged at 10,000× for 10 min and the nuclear extract was stored at -80°C until analysis. For DNA binding activity, 5 μg of the nuclear extract was incubated with ~3 × 104 cpm of [32P]ATP -labeled NF-κB consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGGC) at room temperature for 20 min. This EMSA probe has been previously shown to be specific for NF-κB . Following electrophoresis at on 5% nondenaturing acrylamide gel, the gel was dried and exposed to X-ray film and developed by autoradiography.
Western blot analysis
RAW 264.7 cells were grown overnight in 12-well plates at a density of 106 cells per well, serum starved for 5 h, and incubated with various concentrations of RIAA for 1 h. Cells were washed with PBS and lysed in lysis buffer containing 0.1% Triton X-100, 20 mM Tris (pH 8.0), 100 mM KCl, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail. Total cell lysates were electrophoresed, and incubated overnight at 4°C with primary antibodies of phospho β-catenin (Ser33/37), phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182) and phosphor-JNK (Thr183/Tyr185). These antibodies have been shown previously to be specific . Secondary antibody linked to horseradish peroxidase (Amersham Biosciences) was incubated for 1 h at room temperature, after which proteins were visualized using the enhanced chemiluminescence (ECL) system from Pierce (Rockford, IL). For the loading control, the membranes were stripped and the blot analyzed by using anti-actin antibody.
NF-κB driven luciferase activity
RAW 264.7 cells were sub-cultured in 96-well plates at a density of 7 × 104 cells per well and transiently transfected using SuperFect transfection reagent with an NF-κB or cAMP-responsive-element (CRE) firefly luciferase construct (SuperArray, Frederick, MD). After 2 days, cells were pre-incubated with various concentrations of RIAA or the NF-κB inhibitor parthenolide (10 μM) for 1 h in serum-free media, followed by 8 h LPS (1 μg/ml) stimulation. Luciferase activity was measured using Dual-Luciferase® Reporter Assay System (Promega, Madison, WI) per the manufacturer's instructions. Transfection was normalized with constitutively expressing Renilla luciferase.
Kinase assays were performed at the Upstate Biotechnology (Dundee, UK). Briefly, GSK-3α/β activity was measured in the absence or presence of RIAA. In a final reaction volume of 25 μl the kinase of interest (5–10 mU) was incubated with 20 μM peptide substrate (YRRAAVPPSPSLSRHSSPHQS(p)EDEEE), 10 mM MgAcetate and [γ-33P-ATP] (specific activity approximately 500 cpm/mM) in the presence of 8 mM MOPS (pH 7.0) and 0.2 mM EDTA. The reaction was initiated by the addition of the 10 μM MgATP mix. After 40 min of incubation at room temperature, the reaction was stopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 μl of the reaction was then spotted onto a P30 filtermat and washed 3 times for 5 min in 50 mM phosphoric acid and once in methanol prior to drying and scintillation counting. Detailed protocols are available online (at http://www.millipore.com/drugdiscovery/dd3/assayprotocols).
RAW 264.7 cells were sub-cultured overnight in 96-well plates at a density of 7 × 104 cells per well and incubated with various concentrations of RIAA for 1 h in serum-free media, followed by overnight LPS (1 μg/ml) stimulation. Nitrate/nitrite levels in the medium were measured using the Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI) per the manufacturer's instructions.
Osteoclastogenisis and tartrate-resistant acid phosphatase (TRAP) activity
RAW 264.7 cells were sub-cultured in 48-well plates at a density of 7 × 104 cells per well and incubated with various concentrations of RIAA in medium at a final concentration of 0.1% DMSO. Following overnight incubation, 50 ng/ml of soluble receptor activator of NF-κB-ligand (sRANKL) was added. After 2 days, medium and all reagents were replaced and incubation was continued for 3 more days. Cells were washed with ice cold PBS, lysed in 150 μl of 0.2% Triton X-100 in PBS, and TRAP activity was determined using a TRAP Kit from Sigma (Cat. #387A1). Briefly, 100 μl lysate was added to 100 μl of TRAP solution and incubated at 37°C for 1 h followed by measurement of absorbance at 555 nm. Protein concentration was estimated using BCA reagent (Bio-Rad) and final activity was normalized for equal protein.
Chondrocytes and matrix metalloproteinase (MMP)-13 expression
The human chondrosarcoma cell line SW1353 was purchased from ATCC and maintained in L-15 medium in the presence of 10% FBS, according to manufacturer instructions. Cells were sub-cultured overnight in 96-well plates at a density of 8 × 104 cells per well. Following 1 h of incubation with various concentrations of RIAA, TNF-α or IL-1β (10 ng/ml) was added for 20–24 h and MMP-13 levels measured in medium using an ELISA kit (Amersham Biosciences), according to the manufacturer's instructions.
The study was performed at the Washington Biotechnology, Inc. (Simpsonville, MD) laboratories with approved standard protocol (CIA-MI). (i) Induction of collagen-induced arthritis: 6.5 ml of bovine type II collagen was emulsified with an equal volume of Complete Freund's Adjuvant (CFA, Chondrex, Redmond, WA, 4 mg/ml). Female DBA/1J mice (6–7 weeks)  were injected subcutaneously at the base of the tail with 50 μl of the emulsion (containing 100 μg type II collagen), with a booster injection after 21 days. After 7 days, mice that developed arthritis were used for the study. (ii) Experimental groups: Each group consists of 10 animals for (1) vehicle treatment – 50 μl distilled water/CFA emulsion, (2) celecoxib – 20 mg/kg, (3–5) RIAA – 250, 50 and 10 mg/kg, respectively, and (6) the "non-diseased" control. Test agents in 2% Tweeen-80 were administered daily by gavage (10 ml/kg). Treatment continued for 14 days and the arthritic index determined every other day. (iii) Determination of the arthritic index: Each paw was evaluated on the basis of a 4-point ordinal scale: 0 – no visible signs, 1 – edema/erythema of a single joint or digit; 2 – edema/erythema of 2 joints; 3 – edema/erythema of > 2 joints; 4 – severe arthritis of the entire paw and digits accompanied by ankylosis of the ankle and limb deformity. The index was calculated by summing from all four paws with a maximum score of 16. (iv) Histological evaluation: At day 42, mice were euthanized. One limb from each mouse was removed and preserved in 10% buffered formalin, decalcified, and subsequently trimmed so as to render a longitudinal section through the limb and digits. The specimens were processed, blocked, sectioned, stained with Haematoxylin and Eosin for microscopic examination. Soft tissue, bone and joint changes were evaluated using a standardized severity score whereby 0 = not present, 1 = minimal, 2 = mild, 3 = moderate and 4 = severe.
SAS 9.0 (Cary, NC) was used for the statistical analyses. The in vitro data (NO inhibition, TRAP activity and MMP-13 inhibition) and the in vivo data (arthritis index and histological index) were analyzed using one-way ANOVA with Dunnett's post-hoc multiple comparisons in which the treatment groups were compared to the positive control; data are expressed as mean ± SEM. The significance level was at 0.05. No statistical tests were performed on data from a single experiment.