Calcineurin-binding protein 1 (CABIN-1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLS), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of CABIN-1 in FLS apoptosis is not clear. This study was undertaken to define the regulatory role of CABIN-1 in FLS from mice with collagen-induced arthritis (CIA).
Transgenic mice overexpressing human CABIN-1 in joint tissue under the control of a type II collagen promoter were generated. Expression of human CABIN-1 (hCABIN-1) in joints and FLS was determined by reverse transcription–polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLS was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were stained with hematoxylin and eosin and with tartrate-resistant acid phosphatase for histologic analysis.
Human CABIN-1–transgenic mice with CIA had less severe arthritis than wild-type mice with CIA, as assessed according to hind paw thickness and histologic features. The milder arthritis was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by a significantly greater number of TUNEL-positive cells in synovial tissue. Expression of inflammatory cytokines and MMPs in the transgenic mice with CIA was reduced, and they exhibited decreased Akt activation and increased expression of p53, caspase 3, caspase 9, and Bax.
Our findings demonstrate that hCABIN-1 plays a critical role in promoting apoptosis of FLS and in attenuating inflammation and cartilage and bone destruction in RA. These results help elucidate the pathogenic mechanisms of RA and suggest that CABIN-1 is a potential target for treatment of this disease.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of joint tissue, infiltration by activated immune cells, and synovial hyperplasia, leading to cartilage and bone destruction (1). In the synovium of patients with RA, fibroblast-like synoviocytes (FLS) actively participate in the chronic inflammatory responses as a major cell population in the invasive pannus (2). FLS from RA patients have the potential to produce matrix-degrading enzymes and cytokines such as interleukin-1β (IL-1β), IL-6, and IL-8 (3). Moreover, synovial fibroblasts proliferate abnormally, invade the local environment, and exhibit characteristics of tumor cells, including somatic mutation of p53 (4, 5); an example of this was the observation that RA FLS invaded normal cartilage that was coimplanted into SCID mice, whereas normal and osteoarthritis FLS did not (6). Hence, FLS are altered or transformed by their exposure to the inflammatory environment in RA, and these changes are major contributors to the destructive phase of the disease. The abnormal FLS biology in RA may result, in part, from the presence of somatic mutations of the p53 gene in human RA synovium and cultured FLS (5, 7, 8). These mutations can be dominant-negative, indicating a loss of p53 function in these cells (9).
The tumor suppressor p53 is a multifunctional transcription factor that maintains genome integrity (10) and regulates cell proliferation, DNA repair, and apoptosis (11). The molecular mechanisms behind p53-mediated growth inhibition involve modulation of growth arrest and of the expression of proapoptotic genes such as p21, cyclin G, Bax, growth arrest and DNA damage–inducible protein 45, and p45 up-regulated modulator of apoptosis (12–14). Expression of p53 protein normally occurs in response to DNA damage caused by cellular stressors, including ionizing and ultraviolet radiation, hypoxia, heat shock, and inflammation (10). Functional inactivation of p53 can result from somatic mutations and has been associated with neoplastic disease (15).
Fas is one of the best-characterized members of the death domain–containing receptor family, is constitutively expressed in cultured FLS. Anti-Fas antibodies lead to apoptosis of RA FLS (16, 17). In animal models of RA, induction of apoptosis in the synovium through anti-Fas antibodies or Fas ligand gene transfer improves arthritis by the elimination of both proliferating synoviocytes and infiltrating lymphocytes in the inflamed synovium (18–20). Engagement of cell surface death receptors leads to the formation of the death-inducing signaling complex that includes FADD, caspases 3 and 8, and cFLIP, a negative regulator of caspase 8.
Calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase (21, 22), plays a critical role in various biologic processes, including cell proliferation, cardiovascular and skeletal muscle development, and apoptosis (23–25). Calcineurin-binding protein 1 (CABIN-1) is a ubiquitously expressed 2,220–amino acid protein that acts as a natural antagonist to inhibit calcineurin phosphatase activity and also regulates the transcriptional activity of myocyte enhancer factor 2 (26, 27). CABIN-1 has been implicated in FLS activation, apoptosis, and inflammatory responses (28).
In the present study, transgenic mice overexpressing human CABIN-1 in joint tissue under the control of the type II collagen promoter were generated and used in an experimental arthritis model. We explored the possible role of CABIN-1 in FLS apoptosis. Our results indicate that the inflammatory response was affected by CABIN-1 and that the protective effects of CABIN-1 and p53 in collagen-induced arthritis (CIA) are likely related to FLS apoptosis. These findings have important implications for understanding the role of CABIN-1 in inflammatory responses in RA and suggest that CABIN-1 is a potential therapeutic target in this disease.
MATERIALS AND METHODS
Generation of human CABIN-1 (hCABIN-1)–overexpressing transgenic mice.
The gene for hCABIN-1 was amplified from rheumatoid synoviocyte complementary DNA (cDNA) by polymerase chain reaction (PCR) with 2 pairs of primers encompassing the calcineurin-binding domain sequence (5641–6614). The forward primer had a Not I site, and the reverse primer had a Not I site for cloning. The resulting PCR product was cloned to pEGFP-C1 vector by the type II collagen promoter. The DNA construct was microinjected into fertilized embryos from BDF1 hybrid mice, and the embryos were transferred to the oviducts of pseudopregnant recipients. Transgenic mice, identified by PCR and Western blot analysis, were backcrossed with normal DBA/1 mice for at least 6 generations.
Expression of hCABIN-1 messenger RNA (mRNA) in transgenic mice.
Total RNA from joint tissue and FLS of transgenic mice was extracted using TRIzol reagent according to the instructions of the manufacturer (MRC). Complementary DNA was synthesized using a reverse transcription system (Promega), and the hCABIN-1 transcript was detected by PCR amplification using hCABIN-1–specific primers.
Isolation of joint tissue and FLS.
FLS were prepared from synovial tissue of hCABIN-1–transgenic mice. Fore and hind paws were removed at the ankle joint, the skin was removed, and the remaining tissue was carefully recovered with a scalpel and placed into 1 ml of phosphate buffered saline (PBS). The specimens were minced into small pieces and incubated for 4 hours with 2.5% collagenase (Sigma-Aldrich) in Dulbecco's modified Eagle's medium (DMEM) at 37°C. The cells were washed extensively and cultured overnight in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2 incubator. Nonadherent cells were removed, and adherent cells were cultivated in the same medium. FLS from passages 4–6 were seeded at 3 × 104 cells per well in 24-well plates (Nunc) in DMEM supplemented with 10% FBS, and cultivated for 24 hours at 37°C.
Western blot analysis of hCABIN-1, and apoptosis-related protein expression.
To prepare samples for Western blot analysis, FLS and tissue were washed twice in PBS and then homogenized in nuclear extract lysis buffer (10 mM Tris HCl, 500 mM NaCl, 0.1% Nonidet P40, 5 mM EDTA [pH 8.0]). The lysates were clarified by centrifugation for 10 minutes at 13,000 revolutions per minute. All procedures were carried out at 0–4°C. Lysate samples (40 μg) were electrophoresed in 8% polyacrylamide gels, and the resolved proteins were transferred to nitrocellulose membranes. The membranes were incubated with each of the following primary antibodies: anti–hCABIN-1 (Abcam), anti-p53 (Abcam), anti–Bcl-2, anti–caspase 3, anti-Bax, anti–caspase 9, anti-Akt, and anti-pAkt (Ser473) (all from Cell Signaling Technology), followed by incubation with horseradish peroxidase–conjugated anti-rabbit IgG (Santa Cruz Biotechnology).
Cells in decalcified paraffin-embedded tissue sections from arthritic ankle joints were labeled using TUNEL technology and an In Situ Cell Death Detection Kit, according to the protocol of the manufacturer (Roche). Light microscopy was used to identify individual apoptotic cells, as the DNA strand breaks in cells undergoing apoptosis were labeled. Five high-power fields from each section were examined, and the average number of apoptotic cells was calculated.
Age-matched male DBA/1 mice (Charles River) and CABIN-1–overexpressing mice were immunized with bovine type II collagen (CII; Chondrex) at 8–12 weeks of age, as previously described (29). Briefly, CII was dissolved at a concentration of 0.25% in 0.01N acetic acid. The mice were immunized on day 0 with an intradermal injection of 100 μg CII emulsified in complete Freund's adjuvant. On day 14, the mice were boosted by injection of 100 μg CII in incomplete Freund's adjuvant into the left footpad. Beginning 3 weeks after the primary immunization, the mice were examined 2–3 times per week for onset and severity of arthritis. Arthritis severity was scored under blinded conditions, with each paw assigned a clinical score as follows: 0 = normal, 1 = erythema and mild swelling confined to the ankle joint and toes, 2 = erythema and mild swelling extending from the ankle to the midfoot, 3 = erythema and severe swelling extending from the ankle to the metatarsal joints, and 4 = ankylosing deformity with joint swelling. Hind paw thickness was measured with electronic calipers placed across the ankle joint at its widest point.
Measurement of cytokine, matrix metalloproteinase (MMP), and apoptosis-related gene expression.
The protein levels of cytokines, MMPs, and apoptosis-related genes in FLS culture supernatants and in joint tissue were measured by enzyme-linked immunosorbent assay (ELISA), and mRNA expression levels were measured by reverse transcription–PCR (RT-PCR). Tumor necrosis factor α (TNFα), IL-1β, and IL-6 protein levels in cell-free FLS supernatants were measured using commercially available ELISA kits (Quantikine Mouse ELISA kits) according to the instructions of the manufacturer (R&D Systems). Total RNA was extracted from FLS from synovial tissue of mice with CIA, and expression of mRNA for Bcl-2, p53, caspase 3, caspase 9, Bax, MMP-2, and MMP-9 was analyzed by RT-PCR using primers specific for each. The PCR products were separated in 1% agarose gels. Gene expression levels, normalized to GAPDH, were measured using Multi Gauge, version 3.0.
The right hind paw of each mouse was fixed in 10% formalin on day 46. The paws were decalcified in EDTA, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. A semiquantitative scoring system was used to assess synovial inflammation, extraarticular inflammation, erosion, and proteoglycan loss. The degree of synovial hyperplasia and inflammation and pannus formation in the joints was determined using a standard scoring protocol, in which the severity was scored on a scale of 0–3 where 0 = absent, 1 = weak, 2 = moderate, and 3 = severe. The maximum possible score per mouse was 9. To investigate osteoclastic bone resorption in mice with CIA, sections were stained with a tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich). The total number of TRAP-positive cells containing 3 or more nuclei was ascertained in 10 areas of each ankle.
MMP-9 and MMP-2 activity in culture supernatants was measured by substrate gel electrophoresis (zymography). The culture supernatants were mixed with sample buffer (0.125M Tris HCl, 4% sodium dodecyl sulfate [SDS], 20% glycerol, and 0.01% bromphenol blue) and electrophoresed in 10% polyacrylamide gels containing 0.1% gelatin, according to a previously reported method (30). After electrophoresis, the gels were washed in 2.5% Triton X-100 for 40 minutes to remove the SDS and then incubated in digestion buffer (50 mM Tris HCl [pH 7.6], 0.15M NaCl, 10 mM CaCl2, and 0.02% NaN3) for 13 hours at 37°C. The gels were stained with 0.1% Coomassie brilliant blue R250 and destained to visualize the clear bands.
Data are expressed as the mean ± SD from at least 3 independent experiments. The significance of differences between groups was evaluated by chi-square test. P values less than 0.05 were considered significant.
Expression of hCABIN-1 in transgenic mice.
To generate hCABIN-1–overexpressing transgenic mice, a CII promoter vector (mouse CII collagen–specific) containing hCABIN-1 cDNA was constructed and microinjected into mouse embryos. The progeny were tested for the potential to be founders of transgenic lines. To identify transgenic mice, total genomic DNA was extracted from tail biopsy specimens from candidate pups and screened by PCR using hCABIN-1–specific primers. One mouse line was confirmed as a germline-transgenic line (Figure 1A).
As demonstrated by RT-PCR, hCABIN-1 mRNA was expressed in the joint tissue and FLS of transgenic mice, but not wild-type mice (Figures 1B and C). Western blot analysis confirmed that hCABIN-1 protein was also expressed in transgenic mice, with a pattern similar to that of the mRNA (Figure 1D).
Effects of hCABIN-1 on the progression of inflammatory arthritis.
To examine the effects of hCABIN-1 overexpression on arthritic conditions in vivo, arthritis was induced in transgenic and wild-type mice. Clinical severity was monitored over time, beginning 21 days after the primary immunization. All animals exhibited detectable disease activity, but the clinical scores were significantly lower in the transgenic mice than in the wild-type mice (P < 0.05) (Figure 2A). Consistent with the clinical scores, the severity of inflammation was significantly reduced in the transgenic mice. Hind paw swelling also correlated with the clinical scores (P < 0.05) (Figure 2B).
Histologic examination on day 46 postimmunization revealed a lower degree of inflammation in the joints of hCABIN-1–transgenic mice compared with wild-type mice. Although destruction of cartilage and bone, infiltration of mononuclear cells, and proliferation of synovial cells were observed in both transgenic and wild-type mice (Figure 3A), histopathologic evaluation revealed marked reductions in synovial proliferation, cartilage damage, pannus formation, and bone erosion in the transgenic mice compared with the wild-type mice (P < 0.05) (Figure 3B).
Studies have indicated that bone-resorbing osteoclasts in the synovium play an important role in bone destruction in RA and that administration of hCABIN-1 may reduce the clinical and pathologic severity of arthritis (31). To examine the effect of hCABIN-1 overexpression on the RA process, we stained mouse joint tissue for TRAP, which is secreted into the circulation by osteoclasts during bone resorption (32). There were significantly fewer TRAP-positive cells in the joint tissue of transgenic mice with CIA compared to wild-type mice with CIA on day 46 postimmunization, and the TRAP levels in the joint tissue of the transgenic mice were significantly reduced (Figure 3C). There were also fewer TRAP-positive cells in the bone of transgenic mice compared to wild-type mice (P < 0.05) (Figure 3D). These results indicate that hCABIN-1 reduced the clinical and pathologic severity of arthritis.
Increased apoptosis in the synovium of hCABIN-1–overexpressing mice with CIA.
The reduced severity of CIA in the hCABIN-1–transgenic mice could be due to apoptosis in synovial tissue. We therefore evaluated apoptosis in the joint tissue of the mice with CIA. The number of apoptotic cells was generally low in the synovial tissue of wild-type mice with CIA, which showed only a small percentage of TUNEL-positive cells. In contrast, the percentage of TUNEL-positive cells in the synovial tissue of the transgenic mice was significantly higher (P < 0.05) (Figures 4A and B). These findings suggest that apoptosis may be positively regulated by hCABIN-1.
CABIN-1 induces apoptosis in FLS by modulating the expression of apoptosis-related genes.
To investigate the mechanism by which hCABIN-1 may regulate apoptosis, the expression of several apoptosis-related genes was examined in RA FLS from mice with CIA. Expression of Bcl-2 in RA FLS was reduced in transgenic mice, and expression of the proapoptotic proteins caspase 3, caspase 9, and Bax was increased (Figures 5A and B). Thus, hCABIN-1 may induce apoptosis in FLS by altering the expression of Bcl-2, caspase 3, caspase 9, and Bax.
Given that Akt is critical for the survival of RA FLS (33), we investigated the effect of hCABIN-1 on Akt phosphorylation and p53 activity in RA FLS. The phosphorylation of pAkt was decreased and the activity of p53 was increased in the hCABIN-1–transgenic mice with CIA (Figure 5C). Taken together with previously reported findings (33–37), these results suggest that hCABIN-1 induces FLS death through regulating Akt activity and the expression of Bcl-2, p53, caspases 3 and 9, and Bax.
Levels of cytokines and MMPs in FLS from hCABIN-1–transgenic mice.
In view of the antiarthritic effects of hCABIN-1, we next investigated changes in the levels of proinflammatory cytokines in transgenic mice. FLS from transgenic mice with CIA secreted less IL-6 and IL-1β than those from control mice with CIA (Figure 6A). The reduced IL-6 production may be directly related to the increased p53 expression in hCABIN-1–transgenic mice, because p53 is known to suppress IL-6 gene transcription (38). Alternatively, it may be related to the reduced IL-1β levels (35).
Altered MMP expression may contribute to the reduced joint damage in hCABIN-1–transgenic mice. RT-PCR analysis of mRNA extracted from FLS showed that MMP mRNA expression was significantly lower in transgenic compared to wild-type mice with CIA (P < 0.05) (Figures 6B–D). In addition, zymographic analysis revealed that the activity of MMP-2 and MMP-9 was reduced in RA FLS from transgenic mice (Figure 6E).
In this study of hCABIN-1–overexpressing transgenic mice with collagen-induced arthritis, we demonstrated that hCABIN-1 altered Akt activity and the subsequent expression of Bcl-2, p53, caspases 3 and 9, and Bax, leading to FLS apoptosis. Bcl-2 family members are critical for the regulation of survival via modulation of mitochondrial integrity, and the expression of antiapoptotic Bcl-2 family members, but not proapoptotic members such as Bad and Bax, has been implicated in the pathogenesis of RA (33). Phosphoactivation of Akt maintains mitochondrial integrity via up-regulation of Bcl-2 expression (34); however, it also reduces the expression and transcriptional activity of p53 (35). Furthermore, by increasing p53 expression, hCABIN-1 also suppressed the expression of proinflammatory cytokines and MMPs, thereby reducing the clinical and pathologic severity of arthritis. This mechanism functions in FLS, and caspase activation accompanies Fas- and p53-mediated cell death. The contribution of FLS to RA is particularly interesting as their sojourn through rheumatoid joints confers a uniquely aggressive phenotype that can perpetuate the disease and increase joint destruction.
The targeted inhibition of calcineurin by CABIN-1 has been studied in many diseases. The mouse homolog of human CABIN-1 has been shown to regulate synaptic endocytosis of neurotransmitter vesicles (39). Moreover, adenoviral gene transfer of CABIN-1 prevented agonist-induced cardiomyocyte hypertrophy (40). Notably, the overexpression of CABIN-1 in RA suppressed the production of IL-6 and MMP-2 in rheumatoid FLS (41), suggesting that calcineurin inhibition by CABIN-1 may be a good strategy for the treatment of RA.
The tumor suppressor p53 likely plays a protective role in RA by virtue of its ability to regulate synovial cell proliferation, apoptosis, and cytokine expression. Dominant-negative somatic mutations in the p53 gene have been identified in the joints of some patients with longstanding RA, suggesting that affected cells are no longer regulated by p53 (5, 7–9). Functional studies in p53-deficient mice have demonstrated increased expression of p53-regulated mediators such as IL-6 and MMP-13 (42). The reduced IL-6 production may be directly related to the increased p53 expression in hCABIN-1–transgenic mice, because p53 is known to suppress IL-6 gene transcription (38). In the present study, hCABIN-1–overexpressing mice with CIA exhibited increased p53 expression and decreased production of IL-6, MMP-2, and MMP-9.
CABIN-1 regulates both the local chromatin structure near the p53 response elements and the DNA binding of p53. Knockdown of CABIN-1 apparently increases the binding of p53 to chromatin, partially by regulating p53 acetylation. Thus, CABIN-1 may act as a reservoir for inactive p53, sequestering it close to regulatory genomic sequences so that when CABIN-1 is quickly degraded in response to DNA damage, the sequestered p53 is released and recruits coactivators to produce an immediate transcriptional response. This mechanism would facilitate rapid and specific regulation by p53, leading to numerous potential outcomes (43). In the present study, increased p53 expression in FLS from hCABIN-1–transgenic mice helped to protect against inflammation and joint damage.
In RA, FLS are key players in the propagation of both inflammation and joint destruction. In contrast to normal FLS, RA FLS are hyperplastic and invasive (44), and they help to perpetuate the RA inflammatory microenvironment by elaborating inflammatory mediators and stimulating localized inflammation (45). Increased proliferation and/or decreased apoptosis in RA FLS contribute to synovial hyperplasia (16). In the present study, hCABIN-1 overexpression increased the number of apoptotic FLS, as evidenced by a significantly increased number of TUNEL-positive synoviocytes in hCABIN-1–transgenic mice with CIA compared to wild-type littermates with CIA. In parallel with this finding, FLS from hCABIN-1–overexpressing transgenic mice also exhibited attenuated Akt phosphorylation, reduced expression of the antiapoptotic protein Bcl-2, and increased expression of the proapoptotic proteins p53, caspase 3, caspase 9, and Bax. Given the apoptotic pathway reported for RA FLS (33), our data suggest that hCABIN-1 promotes FLS apoptosis through the inhibition of Akt activation, which leads to a decrease in antiapoptotic signaling and an increase in proapoptotic signaling. In this way, hCABIN-1 may reduce inflammation in RA by limiting the abnormal proliferation of synoviocytes.
Increased cytokine production in RA enhances the expression of vascular adhesion proteins, which attract inflammatory cells to joints, and of MMPs, which degrade the extracellular matrix (46, 47). In the present study, hCABIN-1 effectively down-regulated the expression of IL-6, IL-1β, MMP-2, and MMP-9 in the FLS of transgenic mice with CIA. Histopathologic evaluation revealed that hCABIN-1 also reduced CIA-associated joint destruction.
The most widely used pharmacologic inhibitors of calcineurin are cyclosporine and tacrolimus (48). Both drugs exhibit potent immunosuppressive activity and have been used in the treatment of many chronic inflammatory diseases, including RA (29). However, systemic administration of these drugs can produce significant side effects. For example, prolonged treatment with tacrolimus is associated with hypertension, nephrotoxicity, psychiatric disturbances, and hyperlipidemia (49). Considering that CABIN-1 is a naturally occurring selective calcineurin inhibitor, its therapeutic use may minimize these side effects.
Previous studies have suggested that bone resorption in osteoclasts of the synovium plays an important role in bone and cartilage destruction in RA (31, 50). IL-6 is derived from activated synovial macrophages, and FLS stimulate local osteoclasts to resorb the bone matrix in affected joints (50). Consistent with these findings, the present study demonstrated that hCABIN-1 overexpression led to a decrease in IL-6 and TRAP-positive osteoclasts in the joint tissue. Thus, it is possible that hCABIN-1 minimizes joint destruction through reducing the number and activity of osteoclasts.
In summary, we generated transgenic mice that overexpress hCABIN-1 in joint tissue. The overexpression of hCABIN-1 reduced the severity of collagen-induced arthritis and the levels of various cytokines and MMPs, and promoted FLS apoptosis. These findings provide new knowledge regarding the pathogenic mechanisms of RA and demonstrate the usefulness of animal models in the study of chronic inflammatory conditions.
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. Ryoo 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. Yi, Hei-Jung Kim, Seo-Jin Park, Shin, Ji, Na-Ri Kim, Jae-Young Kim, Hyun-Shik Lee, Sang-Gyu Lee, Yoon, Ryoo.
Acquisition of data. Yi, Yu, Yuh, Si-Jun Park, Wan-Uk Kim, Ryoo.
Analysis and interpretation of data. Hei-Jung Kim, Shin, Yuh, Bae, Ji, Jae-Young Kim, Hyun-Shik Lee, Hyun, Wan-Uk Kim.