Synovial tissues were obtained from patients with rheumatoid arthritis and osteoarthritis at joint replacement surgery. The diagnosis of RA conformed to the American College of Rheumatology 1987 revised criteria 15. The protocol was approved by the University of California at San Diego Human Subjects Research Protection Program. FLS were isolated from individual tissues with 1 mg/ml collagenase and cultured in DMEM supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glutamine as described previously. Cell lines were used from the third to ninth passage, when they are a homogeneous population of fibroblast-like cells 16. Although the origin of these cells cannot be certain, they probably derive from the intimal lining, on the basis of vascular cell adhesion molecule (VCAM)-1 and CD55 expression. In addition to RA FLS, we also examined FLS derived from osteoarthritis FLS in most experiments. No differences were observed between RA and osteoarthritis FLS in these assays. p53^+/+ and p53^-/- murine synoviocytes were obtained as described previously from DBA/1J wild-type mice (Jackson Laboratory, Bar Harbor, ME, USA) and DBA/1J p53^-/- mice 17.

Affinity-purified rabbit polyclonal anti-p53 (for immunohistochemistry), mouse monoclonal anti-p53 (for Western blotting), and rabbit polyclonal antibodies against p21 and hemagglutinin (HA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Rabbit anti-PUMA polyclonal antibody was purchased from ProSci, Inc. (Poway, CA, USA).

Scrambled RNA and p53 siRNA were purchased from Dharmacon Research, Inc. (Lafayette, CO, USA). Plasmids encoding HA-tagged full-length PUMA (HA-PUMA) and PUMA with a deletion of the BH3 domain (HA-PUMA-dBH3) were kindly provided by Dr B Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA) 5. R213* encoding mutant p53 was isolated from a patient with RA and has previously been characterized as dominant-negative 14. Bax-luc (BF72-2 PGL3) is a reporter construct containing the p53-responsive promoter for bax with the luciferase cDNA 14. The control construct contains the β-Gal cDNA and the cytomegalovirus (CMV) promoter in pCI. Cells were transfected with the use of the Amaxa Human Dermal Fibroblast Nucleofactor kit (NHDF-adult) with program U-23 for human FLS. Murine FLS were transfected with the use of the Mouse Embryonic Fibroblasts kit (MEF1) with program T-20. Cells (2 × 10⁵ to 10⁶) were transfected with siRNAs, cDNAs, or control plasmids in each reaction.

Cultured FLS were washed with phosphate-buffered saline, and protein was extracted with lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl₂, 1.5 mM EDTA, 20 mM β-glycerophosphate, 50 mM NaF, 1 mM Na₃VO₄, 10 μg/ml aprotonin, 1 μM pepstatin A, 1 mM phenylmethylsulphonyl fluoride). The protein concentrations were determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Whole cell lysates containing 50 μg of protein were fractionated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% non-fat milk for 1 hour at room temperature followed by incubation overnight with the appropriate antibody at 4°C. The membrane was washed three times and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. Immunoreactive protein was detected by chemiluminescence with Kodak X-AR film (Eastman Kodak, Rochester, NY, USA).

siRNA-transfected cells for immunostaining were cultured in four-well chamber slides at 4.0 × 10⁴ cells per well. They were then fixed with methanol, permeabilized with 0.05% Triton X-100 and blocked with 10% human serum. The fixed cells were incubated overnight with anti-p53 antibody or matched control antibody at 4°C. Endogenous peroxidase was then depleted with 0.1% H₂O₂ and 0.1% NaN₃. The cells were then washed and stained with biotinylated secondary antibody anti-mouse or anti-rabbit IgG and Vectastain ABC and developed with diaminobenzidine (Vector, Burlingame, CA, USA).

FLS were harvested and suspended in 0.2% trypan blue and counted with a hemocytometer. Cells that excluded dye were considered viable. Apoptosis was determined with a Cell Death Detection ELISA^PLUS kit (Roche Applied Science, Mannheim, Germany). FLS (4 × 10³) were seeded into each well of a 96-well plate after transfection. Nine hours later, samples were collected and ELISA was performed in accordance with the manufacturer's instructions. Results are presented as the fold induction compared with control. To confirm the role of apoptosis, caspase-3 activation was also determined in transfected cells with the use of the human active caspase-3 ELISA (R&D Systems, Minneapolis, MN, USA). PUMA or PUMA-dBH3 plasmids were transduced into p53 siRNA-transfected or scrambled siRNA-transfected cells. The cells were then cultured at 4.5 × 10⁵ cells per well in six-well plates. Eight hours later, the cells were lysed and assayed as described by the manufacturer.

Alamar Blue assays incorporate a fluorimetric/colorimetric growth indicator based on the detection of metabolic activity. FLS (3 × 10³) were plated into 96-well plate after siRNA transfection. At various time points, medium was replaced by DMEM without phenol red supplemented with 10% Alamar Blue. After incubation for 4 hours at 37°C, fluorescence was measured with a microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The number of cells is expressed as relative fluorescence units.

Data are expressed as means ± SEM. Statistics were performed with Student's t test, one-way analysis of variance and repeated-measures analysis of variance. A comparison was considered significant at p < 0.05.

Initial studies were performed to determine whether siRNA could knock down p53 expression in cultured FLS. A representative time course and dose response are shown in Figure 1a. The residual expression of p53 protein was about 8 to 10%, 5%, and 1 to 2% for 1, 2.5, and 5 μg of siRNA, respectively, as determined by Western blot analysis. Immunohistochemistry also demonstrated a marked decrease in the percentage of p53-positive cells after siRNA transduction (Figure 1b). The percentage of cells with detectable p53 protein was 75.1 ± 2.9% for scrambled siRNA, 2.8 ± 0.6% for 1 μg of p53 siRNA, and 0.9 ± 0.5% for 5 μg of p53 siRNA.

To confirm that p53 knockdown with siRNA was functionally relevant, the effect on cell proliferation and p21 expression was examined 18. As shown in Figure 2a, growth of FLS was increased by p53 siRNA. Figure 2b shows that siRNA also blocked the expression of p21, which is normally induced by p53. These data indicate that p53 knockdown leads to functional alterations consistent with p53 deficiency.

To determine whether p53 is required for PUMA-induced apoptosis, FLS were transduced with p53 siRNA. After p53 expression reached its minimum 3 days later, cells were transfected with the PUMA cDNA, PUMA-dBH3, or empty plasmid pCEP4. As shown in Figure 3a,b, PUMA-induced apoptosis in scrambled siRNA-transfected cells, as measured by either histone release or cell viability, was similar to that in p53 siRNA-transfected cells. PUMA-dBH3, which lacks the BH3 domain and does not induce apoptosis, had no effect on cell viability. As further evidence of PUMA-mediated apoptosis, caspase-3 activation was also evaluated in p53-deficient FLS. Figure 3c shows that PUMA does not require p53 to engage this pathway.

Small amounts of residual p53 might contribute to PUMA apoptosis in the siRNA studies, so we repeated the experiments in p53^+/+ and p53^-/- murine synoviocytes. Figure 4a shows that cell viability overall was decreased in the transfected cells. However, the effect of PUMA compared with PUMA-dBH3 or empty vector was the same in p53^+/+ and p53^-/- synoviocytes (Figure 4).

Because some RA synoviocytes could potentially express dominant-negative p53 protein, we determined whether PUMA could induce cell death in the presence of mutant p53. A known dominant-negative gene that was isolated from a patient with RA (R213*) was used for these experiments 14. Sequential transfection of cultured human FLS with R213* and followed with PUMA 2 days later was performed. Figure 5 shows that PUMA effectively induced apoptosis in FLS even in the presence of a dominant-negative p53.