Susceptibility of Cyclin-dependent Kinase Inhibitor-1–decient Mice to Rheumatoid Arthritis From IL-1β–induced Inammation

Background: Rheumatoid arthritis (RA) is a chronic and systemic inammatory disorder whose progression is modulated by broblast-like synoviocytes (FLSs). Cyclin-dependent kinase (CDK) inhibitor 1 (p21) regulates the activation of other CDKs, and we recently reported that p21 deciency induces susceptibility to osteoarthritis. Here, we focused on joint inammation to determine the mechanisms associated with p21 function in synovial and cartilage tissues in RA. Methods: p21-knockout (p21 -/- ) mice and wild-type C57BL/6 (WT p21 +/+ ) mice were used to establish a collagen antibody-induced arthritis (CAIA) model. The severity of arthritis was evaluated visually, and histological and immunohistological analyses performed 7, 14, and 28 days after injection with a cocktail of ve monoclonal antibodies that recognize conserved epitopes on various species of type II collagen. The response of p21 siRNA-treated human RA FLSs to IL-1β stimulation was also determined. Results: Arthritis scores were higher in p21 -/- mice than those in p21 +/+ mice. More severe and prolonged synovitis of the knee joints and earlier loss of staining and cartilage destruction were observed in p21 -/-mice than in p21 +/+ mice. p21 -/- mice expressed higher levels of IL-1β, F4/80, p-IKKα/β, and MMPs in cartilage and synovial tissues at each time point, except for before injection of the monoclonal antibodies, via IL-1β-induced NF-kB signaling. IL-1β stimulation signicantly increased MMP expression and enhanced IKKα/β phosphorylation in human FLSs. Conclusion: p21-decient CAIA mice are susceptible to alterations in the RA phenotype, including joint cartilage destruction and severe synovitis, via IL-1β-induced inammation. Therefore, p21 regulation may constitute a possible strategy for RA treatment.


Background
Rheumatoid arthritis (RA) is characterized by chronic synovial in ammation of multiple joints (1,2). The affected synovial tissues contain activated macrophages, broblasts, and T and B lymphocytes that are induced by pro-in ammatory cytokines, such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6. The cytokines provoke and perpetuate the synovial membrane in ammation in the joints, which leads to articular cartilage damage and bone erosion (3,4). Especially, activated RA broblast-like synoviocytes (FLSs) contribute to the in ammatory and destructive potential of the aggressive pannus tissue in patients with RA by producing pro-in ammatory mediators and matrix metalloproteinases (MMPs), such as MMP-1, MMP-3, and MMP-9 (5)(6)(7)(8)(9)(10)(11). Moreover, IL-1β, which is produced by chondrocytes, osteoblasts, mononuclear cells, and cells forming synovial membranes, is secreted into the hip and knee joints during an in ammatory response (12). IL-1β affects the synthesis of MMPs, speci cally MMP- 13, in the chondrocytes, resulting in cartilage destruction (13). Patients with RA display elevated IL-1β levels in FLSs, the synovial uid, the synovial membrane, cartilage, and the subchondral bone layer (6), suggesting that IL-1β may be involved in the pathogenesis and progression of RA.
Animal models of RA, including collagen rat and mouse models of type II-induced arthritis, rat model of adjuvant-induced arthritis, and models of antigen-induced arthritis in several species, have proven to be highly predictive of therapeutic e cacy in humans (14). In particular, the collagen antibody-induced arthritis (CAIA) model mouse is ideal for rapid screening of novel arthritis therapeutics and elucidating the mechanisms involved in the development of arthritis (15). In this model, arthritis is induced through systemic administration of antibody mixtures that target various epitopes of type II collagen. Moreover, this method can induce arthritis in various mouse strains, not just CAIA-susceptible mice, making it ideal for studying the pathological role of individual gene products, such as cytokines, without the in uence of complete or incomplete Freund's adjuvant that may strongly affect the host immune system.
Cyclin-dependent kinase inhibitor 1 (p21) was initially identi ed as a potent inhibitor of cell-cycle progression (16)(17)(18)(19). Its knockout induces a regenerative response in an appendage of an otherwise nonregenerating mouse strain (20). It has been shown to regulate cell proliferation and in ammation after arterial injury in local vascular cells (21). Subsequent studies have also identi ed the importance of p21 in controlling in ammation, cytostasis, and cell death (22). Transcription of p21, which is activated by the tumor suppressor protein p53, is part of a negative-feedback mechanism that controls p53 activity during apoptosis (23). p21 -/mice have been shown to lack any overt skeletal phenotype and are unlikely to develop spontaneous malignancies (24)(25)(26).
We recently reported that p21 de ciency induces susceptibility to osteoarthritis (OA) through signal transducer and activator of transcription 3 (STAT3)-and IL-1β-induced activation of nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) signaling (27,28). Recent studies have also shown that p21 plays a key role in suppressing activated macrophages (29), and that expression of p21 in rheumatoid synovial broblasts resulted in downregulation of in ammatory mediators and tissuedegrading proteinases in RA (30).While the expression of p21 is decreased in RA FLSs (31), it is unclear how p21 affects the joint synovial tissues and cartilage in RA.
In this study, we hypothesized that p21 de ciency enhances joint in ammation and cartilage destruction in RA. We evaluated the degree of joint in ammation to determine the mechanisms associated with p21 function in vitro as well as in vivo using RAFLS and the systemic arthritis model, respectively.

Methods
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 ampli cation of mouse-tail DNA with allele-speci c 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 ve 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 CO 2 . We de ned 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 xed using 4% paraformaldehyde (163-20415, WAKO, Osaka, Japan) for 24 h, decalci ed with 14% ethylenediaminetetraacetic acid (EDTA; 345-01865, Dojindo, Kumamoto, Japan) for 7 d, and embedded in para n. 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 Scienti c, 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.

Immunohistochemistry
Depara nized 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 in ammatory cell marker because it is a well-known macrophage marker (35).

Preparation of human synovium
Synovial tissues were obtained during a total knee joint replacement surgery from ve patients with RA.
All RA patients ful lled the American College of Rheumatology 1987 revised criteria for RA (36). OA synovial tissues were also obtained during total knee joint replacement surgery from ve 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 modi ed 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 × 10 5 cells/well). All experiments were conducted using 3-5 passage cells.
Transfection of small-interfering RNA Lipofectamine TM RNAiMax transfection reagent (Invitrogen) was used to transfect p21 small-interfering RNA (siRNA) and nonspeci c siRNA control into the RA and OA human knee synoviocytes, respectively, according to the manufacturer's recommendations. Brie y, a day before transfection, the cells (3 × 10 5 cells/well) were seeded in a 6-well plate with growth medium without antibiotics to achieve 30-50 % con uence at the time of transfection. Subsequently, 5 pmol of siRNA and Lipofectamine TM 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 nonspeci c 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. Brie y, 1 μg of total RNA was reversetranscribed to rst-strand cDNA using 1.25 μM oligo-dT primer in 40 μl PCR buffer II containing 2.5 mM MgC1 2 , 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 log 2 (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 × 10 7 cells/ml) were electrophoresed on a 7.5-15% SDS-polyacrylamide gradient gel (Biocraft, Tokyo, Japan) and electrically transferred onto a polyvinylidene di uoride 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; Fuji lm, Tokyo, Japan).
The cultured RA and OA synoviocytes were then transfected with p21 siRNA or nonspeci c 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
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% con dence intervals and were considered statistically signi cant at P < 0.05. Results p21-de cient mice are susceptible to joint destruction in vivo Severe arthritis was observed in p21 -/mice after injection of the monoclonal antibody cocktail. The arthritis scores on days 7, 10, 14, and 28 of p21 -/mice were signi cantly higher than those of p 21 +/+ mice at each time point ( Figure 1A). Based on Safranin-O and Fast Green staining, p21 -/mice showed an earlier loss of Safranin-O staining than both the control and p21 +/+ mice on day 7 ( Figure 1B). Both p21 -/and p21 +/+ groups showed loss of Safranin-O staining, but the articular surface layer remained intact on day 14 ( Figure 1B). While p21 -/mice showed mid-zone excavation of cartilage tissue, the articular surface of p21 +/+ mice remained intact on day 28 ( Figure 1B). According to the OARSI cartilage OAhistopathology scoring system, the average sum score of p21 -/mice increased signi cantly compared with that of p21 +/+ mice on day 28 ( Figure 1C). p21-de cient mice are susceptible to severe synovitis Histological analysis using hematoxylin-eosin staining showed that synovitis of the knee joints was more severe and more prolonged in p21 -/mice than in p21 +/+ mice ( Figure 1D). On days 7 and 14, p21 -/mice showed marked cellular in ltration mixed with lymphoid follicles, multiple-layered synovial lining cells, and villous hyperplasia compared with the control ( Figure 1D). Moreover, on day 28, p21 -/mice displayed prolonged synovitis, whereas synovitis was attenuated in p21 +/+ mice at this time point ( Figure 1D).
According to the OARSI synovitis-severity scoring system, more severe synovitis was observed in p21 -/mice than in p21 +/+ mice at all time points (Figure 1D, E). There was no signi cant difference in synovitis severity of p21 -/mice between days 7 and 14 ( Figure 1D, E). p21-de cient mice exhibited enhanced in ammatory cytokine expression p21 -/mice showed higher IL-1β expression in cartilage tissues compared with p21 +/+ mice at each time point except for the control (Figure 2A). IL-1β expression in synovial tissues was also increased in p21 -/mice compared with the p21 +/+ mice at each time point except for the control ( Figure 2C). The IL-1βpositive-cell ratio in the cartilage was signi cantly higher in p21 -/mice than in p21 +/+ mice at each time point except for the control ( Figure 2B), and those of IL-1β on day 7 and thereafter were signi cantly higher than those of the control ( Figure 2B). There was no signi cant difference in the positive-cell ratio of p21 -/mice between days 7 and 14 (Figure 2A, B). Moreover, the expression of IL-1β in p21 -/peaked on days 7 and 14 and was delayed compared with the peak IL-1β in p21 +/+ mice (Figure 2A, B).  Figure 3A). The F4/80 score was signi cantly higher in p21 -/mice than in p21 +/+ mice at each time point except for the control ( Figure 3A, B). On day 7 and thereafter, the F4/80 score was signi cantly higher in p21 -/mice than in controls ( Figure 3B).
Moreover, F4/80 expression in p21 -/mice peaked on day 14, which was delayed compared with that in p21 +/+ mice ( Figure 3A, B). p21 -/mice showed higher p-IKKα/β expression in the cartilage and synovial tissues compared with p21 +/+ mice at each time point except for the control (Figure 3C, E). The positivecell ratio of p-IKKα/β in the cartilage was signi cantly higher in p21 -/mice than in p21 +/+ mice at each time point except for control ( Figure 3C, D), and the ratios of p-IKKα/β in p21 -/mice on day 7 and thereafter were signi cantly higher than those in the control ( Figure 3D). There was no signi cant difference in the positive-cell ratio of p21 -/mice between day 7 and day 14 ( Figure 3B-D). Moreover, the expression of p-IKKα/β in p21 -/peaked on day 14, which was delayed compared with that in p21 +/+ mice ( Figure 3D). p21-de cient mice exhibited joint destruction through elevated MMPs expression MMP-3 and MMP-9 expression levels were elevated in the synovial tissues of p21 -/mice at each time point except for the control ( Figure 4C and Supplementary Figure 1A). The positive-cell ratios of MMP-3 and MMP-13 in the cartilage were signi cantly higher in p21 -/mice than in p21 +/+ mice at each time point except for the control ( Figure 4A, D), and positive-cell ratios of MMP-3 and MMP-13 on day 7 and thereafter were signi cantly higher than those of the control (Figure 4B, E).
Downregulation of p21 gene expression enhanced MMPs expression through IL-β stimulation in RA synovial tissues RT-PCR analysis showed that expression levels of p21 were lower in human RAFLS than OA FLS and that MMP3 and MMP9 were highly expressed in RA FLS ( Figure 5A). Expression of the p21 gene was inhibited by 14.7% and 18.6% in RA FLS and OA FLS, respectively, after transfection with p21-speci c siRNA ( Figure 5B). MMP3 expression was increased by 157-fold and 26-fold in p21 knockdown RA FLS and OA FLS, respectively, through IL-β stimulation ( Figure 5C), whereas MMP9 expression was increased by 3.7fold and 3.4-fold in p21 knockdown RA FLS and OA FLS, respectively ( Figure 5D).

Downregulation of p21 gene expression enhanced phosphorylation of IKKα/β in RA synovial tissues
Western blot demonstrated that the expression of phosphorylated IKKα/β (p-IKKα/β) markedly increased after a 15 min treatment with 10 ng/ml IL-1β ( Figure 6A). Therefore, the RA FLS and OA FLS were treated with IL-1β for 15 min in subsequent experiments. The downregulation of p21 gene expression enhanced the phosphorylation of IKKα/β in RA FLS compared with that in OA FLS ( Figure 6B).

Discussion
In this study, we demonstrated that p21-de cient mice presented enhanced cartilage degradation and more severe synovitis in response to systemic in ammation through NF-κB signaling in cartilage and synovial tissues.
Similar to our study, previous reports have shown increased arthritis scores and observed histological changes, including a marked increase in cellular in ltration, in the knee synovial membrane of p21 +/+ CAIA mice (38). Moreover, we found severe arthritis in p21 -/mice than in p21 +/+ mice on day 7 and thereafter, suggesting that downregulation of p21 may exacerbate CAIA and enhance macrophage in ltration in mice. IL-1β expression in p21 -/mice reportedly increased 2.4-fold compared to that in p21 +/+ mice in an experimental endotoxic shock model in vivo, and that in p21 -/bone marrow-derived macrophages with LPS stimulation increased 3.4-fold compared to that in p21 +/+ cells in vitro (39). We have previously reported that p21 knockout in a murine model of destabilization of the medial meniscus increased IL-1β serum levels and local IL-1β expression in knee joints on day 1 and day 56 post-surgery (28). Macrophages play important roles in in ammation, ranging from antigen presentation, phagocytosis, and immunomodulation via the production of various in ammatory cytokines, including IL-1β and TNF-α (39,40). The importance of macrophages in RA has been shown in several studies, including the study by Trakala et. al that reported increased IL-1β and TNF-α expression in p21 -/macrophages in an in vitro study (41), and by Mavers et al. who found remarkably increased macrophage in ltration in the ankles of a p21 -/arthritis mouse model on days 7, 14, and 25 and signi cantly elevated IL-1α serum levels on day 7 (29). Our current study also found that p21 -/-CAIA model mice exhibited prolonged joint arthritis with macrophage in ltration and elevated local IL-1β expression in vivo. These ndings suggest that macrophages have a critical role in joint in ammation in this mouse model.
Previous reports have shown that IL-1β stimulates NF-κB signaling and induces the expression of MMP-3, MMP-9, and MMP-13 (13,42), and it is known that the IKK complex plays a central role in the regulation of NF-κB activity (43). In this study, we con rmed that the expression levels of p-IKKα/β, MMP-3, and MMP-13 in chondrocytes and of p-IKKα/β, MMP-3, and MMP-9 in synovial tissues were found to be elevated in p21 -/mice compared with those in p21 +/+ mice. These ndings suggest that rapid joint destruction was caused by elevated levels of in ammation induced via p-IKKα/β signaling. Perlman et al. reported that synovial broblasts from p21-de cient mice enhances IL-6 and MMP-3 mRNA levels and cause a 100-fold increase in IL-6 protein levels (31). Hence, alterations in p21 expression may enhance pro-in ammatory cytokine and MMP production, thereby promoting development of autoimmune diseases (31). These earlier studies support our results that p21-de cient mice exhibit enhanced MMP-3, MMP-9, and MMP-13 expression through IL-1β-induced NF-κB signaling.
Consistent with our results, RA FLSs have been shown to exhibit decreased p21 expression (31) and higher IL-1β levels (6, 44) compared with OA FLSs. Moreover, previous studies have demonstrated increased IL-6 and MMP levels in RA FLSs through TNF-α stimulation (45,46). Consequently, the decline in p21 expression in RA FLSs might cause severe in ammation through IL-1β-induced NF-κB signaling.
Furthermore, we have demonstrated that downregulation of p21 in FLSs alters the cellular response to IL-1β stimulation. IL-1β stimulation increased MMP-3 and MMP-9 expression and enhanced p-IKKα/β activation in both RA FLSs and OA FLSs. However, the responses to IL-1β in RA FLSs and OA FLSs were different, indicating that decreased expression of p21 in RA joints may account for the observed joint destruction.
The applicability of the results from this study to human arthritis is limited by the use of an experimental animal model of antibody to collagen-induced arthritis. Clearly, the human scenario is much more complex than can be recreated in the CAIA model. In this study, we showed only the relationship between p21 and IL-1β, but there are many in ammatory pathways associated with RA pathogenesis, for instance, TNF-α and IL-6, among others. Hence, further investigation of these other pathways is needed.

Conclusions
Overall, we demonstrated that p21-de cient CAIA mice were susceptible to joint cartilage destruction and severe synovitis via IL-1β-induced in ammation in vivo. We have also shown that downregulation of p21 led to enhanced susceptibility to in ammation through IL-1β stimulation in RA FLSs compared with OA FLSs. Therefore, p21 regulation may constitute a possible therapeutic strategy for RA treatment. However, given that p21 is an oncogene involved in cell-cycle regulation, further research is required to verify the viability and safety of p21-based therapies for RA treatment. Availability of data and materials

List Of Abbreviations
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
All the authors state that they have no con icts of interest.

Funding
This study was supported in part by the Japan Society for the Promotion of Science (grant-in-aid for scienti c research 20K17998).

Authors' contributions
Page 13/22 YT, KF, and SH made substantial contributions to the design of the study, acquisition, analysis, interpretation of data, and writing of the manuscript. MT, TK, KK and MF participated in the design of the study, acquiring data. TM critically contributed to revising the manuscript enhancing its intellectual content. TM and RK participated in approving the nal content of the manuscript. All authors read and approved the nal manuscript. average severity of synovitis scores with 95% CI from four quadrants (i.e., medial femoral condyle, medial tibial plateau, lateral femoral condyle, and lateral tibial plateau) and eight sections per knee. Four mice were analyzed from each group. CI: con dence interval; CAIA: collagen antibody-induced arthritis; p21: cyclin-dependent kinase inhibitor 1.   Four mice were analyzed from each group. CI: con dence interval; CAIA: collagen antibody-induced arthritis; p21: cyclin-dependent kinase inhibitor 1.

Figure 5
Downregulation of the p21 gene affects MMP expression in rheumatoid arthritis and osteoarthritis synovial tissues. (A) The relative expression of p21, MMP-3, and MMP-9 mRNA was determined in broblast-like synoviocytes (FLSs). (B) FLSs were transfected with p21 siRNA or nonspeci c control siRNA for 24 h, and the relative expression of p21 mRNA was determined. The relative expression of (C) MMP-3 and (D) MMP-9 mRNA was determined after transfection with p21 siRNA or nonspeci c control siRNA and treatment with or without 10 ng/ml recombinant human IL-1β for 24 h. The relative expression of MMP-3 and MMP-9 mRNA with respect to the control is shown with 95% CI. Five human synovial tissues were analyzed for each group.CI: con dence interval; FLSs: broblast like synoviocytes; p21: cyclin-dependent kinase inhibitor 1.