Generation of homozygous p21-/-mice
Homozygous B6.129S6 (Cg)-Cdkn1atm1 Led/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). We backcrossed these mice for ten generations against a C57BL/6 background, obtained from CREA Japan, Inc (Tokyo, Japan), and studied 10-week-old male mice (n=16). p21+/+ littermates were used as WT controls (n=16). Genotyping was performed using PCR-based amplification of mouse-tail DNA with allele-specific probes. Both the p21+/+ and p21-/- groups contained four mice. All animals were bred in a mouse house with automatically controlled lighting (12 h light/dark cycle) and a stable temperature of 23 °C and were allowed ad libitum access to food and water throughout the study. This study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA). All procedures were approved by the Animal Studies Committee of Kobe University, Japan (permit number: P180404).
Establishment of a CAIA mouse model
A cocktail of five monoclonal antibodies recognizing the conserved epitopes on various species of type II collagen (Chondrex Inc., Redmond, WA, USA) was prepared as described previously (2) and used according to the manufacturer’s instructions. Mice were injected with the cocktail of antibodies intraperitoneally (i.p.; 5 mg). Three days later, they were injected with 50 µg lipopolysaccharide (LPS) from Escherichia coli 0111: B4 (Chondrex Inc.) i.p. to induce arthritis. On days 7, 14, and 28 (counting from day 0 as the day of cocktail injection), at least four mice each from the p21-/- and WT groups were euthanized using CO2. We defined the mice without injection of monoclonal antibodies as the control mice.
Evaluation of arthritis
The mice were blindly evaluated for disease progression on days 0, 3, 7, 10, 14, and 28. The severity of arthritis in each paw was graded on a scale of 0–4, as follows: 0, normal; 1, mild swelling; 2, moderate swelling; 3, severe swelling; 4, pronounced edema of the entire paw. The cumulative score from all four paws (maximum score of 16 per mouse) was used as the overall disease score (32).
Histological evaluation for cartilage degeneration and synovitis
Mouse knee joints were fixed using 4% paraformaldehyde (163-20415, WAKO, Osaka, Japan) for 24 h, decalcified with 14% ethylenediaminetetraacetic acid (EDTA; 345-01865, Dojindo, Kumamoto, Japan) for 7 d, and embedded in paraffin. Histological coronal sections were obtained from the joint at 80-µm intervals and stained with Safranin-O (S0145, Tokyo Chemical Industry, Tokyo, Japan) and Fast Green (10720, Chroma-Gesellschaft, Thermo Fisher Scientific, Inc). RA histopathology was evaluated using the Osteoarthritis Research Society International (OARSI) cartilage OA-histopathology scoring system. Histological scores were measured in the four quadrants (i.e., medial femoral condyle, medial tibial plateau, lateral femoral condyle, and lateral tibial plateau) of the knee joints at all sectioned levels (eight sections per knee) to obtain summed scores. The summed scores were calculated from all four quadrants of all sections that represented whole-joint changes (33). Synovitis was also evaluated using the OARSI-recommended scoring system of hematoxylin-eosin-stained sections (34). Two specimens from each compartment were evaluated and the highest score was recorded. The average of each compartment score was considered as the whole-knee score.
Deparaffinized sections were digested with proteinase (Dako, Glostrup, Denmark) for 10 min and treated with 3% hydrogen peroxide (Wako Pure Chemical Industries, Osaka, Japan) to block endogenous peroxidase activity. We assessed F4/80 expression—using a previously reported scoring system for immunohistochemistry—as an immune and inflammatory cell marker because it is a well-known macrophage marker (35).
The sections were probed with the following antibodies (1:50 dilution) at 4 °C overnight: anti-F4/80 (MCA497P647T, AbDSerotec, Kidlington, UK), anti-IL-1β (ab9722, Abcam), phosphor-IκB kinase complex (IKK) α/β (#2697S, Cell Signaling Technology, Danvers, MA, USA), anti-MMP-3 (SC-21732, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MMP-13 (ab39012, Abcam), or anti-MMP-9 (10375-2-AP, Proteintech Group, Chicago, IL, USA). Sections were subsequently probed with peroxidase-labeled anti-rabbit or rat IgG (Histofine Simple Stain MAX PO; Nichirei Bioscience, Tokyo, Japan) antibody at 23–27 °C for 1 h. The brown reaction product was generated as a signal upon addition of the peroxidase substrate 3,3'-diaminobenzidine (Histofine Simple Stain DAB solution; Nichirei Bioscience), and the sections were examined under an optical microscope. Hematoxylin was used as a counterstain.
One coronal section from the center of the most severe lesion in each tibial plateau was scored. The numbers of stained cells were counted in three areas of high-magnification fields at both superficial and deep zones of the cartilage tissue by triple-blinded observers. The average percentages of MMP-3-, MMP-13-, p-IKKα/β-and IL-1β-positive cells/total cells were calculated. Each coronal section from each of the four mice was evaluated for each group. The positive cells were included superior of the tidemark.
Preparation of human synovium
Synovial tissues were obtained during a total knee joint replacement surgery from five patients with RA. All RA patients fulfilled the American College of Rheumatology 1987 revised criteria for RA (36). OA synovial tissues were also obtained during total knee joint replacement surgery from five patients, as controls. Diagnoses of OA were based on clinical, laboratory, and radiographic evaluations. All samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. The study protocol was approved by the Kobe University Graduate School of Medicine Ethics Committee, and all participants provided informed consent.
Preparation of cell culture
Primary synoviocytes were isolated and cultured from the RA and OA synovial tissues. The tissues were minced and incubated with trypsin (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C, after which the synovium was treated with Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Life Technologies, Grand Island, NY, USA) containing 0.2% collagenase (Sigma-Aldrich) at 37 °C for 15 h. Dissociated cells were cultured overnight in DMEM supplemented with 10% fetal bovine serum (BioWhittaker FBS; Lonza, Walkersville, MD, USA) and 100 U/ml penicillin-streptomycin. The non-adherent cells were removed, and the adherent cells were further incubated on a 6-well plate with fresh medium (3 × 105 cells/well). All experiments were conducted using 3–5 passage cells.
Transfection of small-interfering RNA
LipofectamineTM RNAiMax transfection reagent (Invitrogen) was used to transfect p21 small-interfering RNA (siRNA) and nonspecific siRNA control into the RA and OA human knee synoviocytes, respectively, according to the manufacturer’s recommendations. Briefly, a day before transfection, the cells (3 × 105 cells/well) were seeded in a 6-well plate with growth medium without antibiotics to achieve 30–50 % confluence at the time of transfection. Subsequently, 5 pmol of siRNA and LipofectamineTM RNAiMax complexes were prepared and added to each well. After transfection for 24 h, the complexes were removed and fresh medium containing 10% FBS was added.
Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
Cultured RA and OA synoviocytes were transfected with the p21 siRNA or nonspecific siRNA control. FLSs without siRNA transfection were used as controls. After transfection for 24 h, the cells were incubated for another 24 h with or without stimulation with 10 ng/ml recombinant human IL-1β(R&D systems, McKinnley, MN, USA), followed by RNA extraction using a QIA shredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Briefly, 1 μg of total RNA was reverse-transcribed to first-strand cDNA using 1.25 μM oligo-dT primer in 40 μl PCR buffer II containing 2.5 mM MgC12, 0.5 mM dNTP mix, 0.5 U of RNase inhibitor, and 1.25 U of murine leukemia virus reverse transcriptase (PerkinElmer/Applied Biosystems, Foster City, CA, USA), at 42 °C for 1 h. The relative expression levels of mRNA encoding human p21, MMP-3, and MMP-9 were analyzed using SYBR Green RT-PCR on an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Relative gene expression was normalized against the GAPDH housekeeping gene using the comparative cycle threshold (Ct) method. The difference between the mean Ct values of the gene of interest and those of the housekeeping gene is denoted as ΔCt, whereas the difference between the ΔCt and the Ct value of the calibrator sample is denoted as ΔΔCt. The log2 (ΔΔCt) value gives the relative level of gene expression. The primer sequences used for the detection of human p21, MMP-3, and MMP-9 are listed in Supplementary Table 1.
Western blot analysis
First, the cultured RA synoviocytes were treated with or without 10 ng/ml recombinant human IL-1β (R&D Systems) for 5, 10,15, 30, and 60 min; stimulation time for IL-1β was determined as previously reported (37). The synoviocytes were washed with Tris-buffered saline with Tween-20 (TBST) and lysed in a buffer containing 25 mM Tris, 1% Nonidet P-40, 150 mM NaCl, 1.5 mM ethylene glycol tetraacetic acid, and a protease/phosphatase inhibitor mix (Roche Diagnostics, Basel, Switzerland). The lysates were centrifuged at 4 °C at 15,000 × g for 10 min to remove cellular debris. Next, the cellular debris-free lysates were collected and mixed with 4× electrophoresis sample buffer; 15 μl of cell lysates (1.0 × 107 cells/ml) were electrophoresed on a 7.5–15% SDS‑polyacrylamide gradient gel (Biocraft, Tokyo, Japan) and electrically transferred onto a polyvinylidene difluoride blotting membrane (GE Healthcare Life Sciences, Little Chalfont, UK). The membrane was blocked with 5% skimmed milk in TBST at 25 °C for 30 min, incubated with antibodies against anti-p-IKKα/β (Cell Signaling Technology) at 4 °C for 12 h, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody at 25 °C for 1 h. The proteins were subsequently visualized using ECL Plus reagent (GE Healthcare Life Sciences) in a chemilumino analyzer (LAS-3000 mini; Fujifilm, Tokyo, Japan).
The cultured RA and OA synoviocytes were then transfected with p21 siRNA or nonspecific siRNA control. After 24 h of transfection, the cells were incubated with or without IL-1β stimulation for the period with the highest level of p-IKKα/β in the western blot. Western blots of the synoviocytes were subsequently subjected to the same procedure as described above. Expression of the alpha-tubulin protein was detected using rabbit anti-alpha-tubulin polyclonal antibody (catalog no. ab4074; Abcam) as a primary antibody. Protein expression was determined semi-quantitatively with the National Institutes of Health ImageJ software (http://imagej.nih.gov/ij/) using digitally captured images. Five different samples were analyzed for each experiment.
Statistical analysis was performed using one-way (Figures 5B and 6A) or two-way (Figures 1A, 1C, 1E, 2B, 3B, 3D 4B, 4E, 5C, 5D, and 6B) analysis of variance and Tukey’s post hoc test for multiple comparisons of paired samples. The Mann-Whitney U test was used for comparisons between two groups (Figure 5A). Results are presented as means with 95% confidence intervals and were considered statistically significant at P < 0.05.