Spontaneous development of synovitis and cartilage degeneration in transgenic mice overexpressing cathepsin K.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 12, December 2005, pp 3713–3717 DOI 10.1002/art.21423 © 2005, American College of Rheumatology Spontaneous Development of Synovitis and Cartilage Degeneration in Transgenic Mice Overexpressing Cathepsin K Jukka Morko, Riku Kiviranta, Kirsi Joronen, Anna-Marja Säämänen, Eero Vuorio, and Heli Salminen-Mankonen hypertrophic synovia were positive for cathepsin K. At 12 months, synovia of control mice revealed only a few isolated cathepsin K–positive cells and mild changes in articular cartilage. Conclusion. Our findings demonstrate that overexpression of the cathepsin K gene under its own promoter in transgenic mice makes them susceptible to progressive synovitis, which, upon aging, results in synovial hyperplasia and fibrosis and subsequent destruction of articular cartilage and bone. Objective. Several recent studies have demonstrated that cathepsin K, a proteolytic enzyme capable of degrading native fibrillar collagen, is overexpressed in osteoarthritic cartilage and inflamed synovial tissue. However, it is not known whether increased cathepsin K production is a primary or a secondary event in these diseases. The availability of transgenic UTU17 mice, which exhibit constitutive overexpression of the cathepsin K gene, prompted us to study possible arthritic changes in their knee joints. Methods. Progression of synovitis and articular cartilage degeneration in the knee joints of UTU17 mice and their nontransgenic littermates was monitored by histologic analyses at 7 and 12 months of age. Distribution of cathepsin K in the knee joints was studied by immunohistochemistry. Results. At the age of 7 months, UTU17 mice exhibited clear signs of synovitis, with strong immunostaining for cathepsin K in the synovial lining and the stroma, while control knee joints appeared normal. At 12 months, marked synovial thickening and fibrosis and severe degradation of cartilage and subchondral bone were observed in UTU17 mouse knee joints. In areas of cartilage degeneration, both chondrocytes and cells of Cysteine cathepsins B, H, K, L, and S form a group of proteolytic enzymes that are capable of degrading native collagens and other components of the extracellular matrix (1). Recent data have suggested that cathepsin K is the major cysteine cathepsin expressed in both osteoarthritic (OA) cartilage (2,3) and inflamed synovial tissue (4,5). Using a transgenic Del1 mouse model for OA (6), we have demonstrated up-regulation of cathepsin K expression in articular chondrocytes near sites of matrix destruction, particularly in clusters of proliferating cells, and in calcified cartilaginous matrix (3). Because cathepsin K is one of the few enzymes capable of degrading the networks of type II and I collagen fibrils (7) (which provide articular cartilage and bone, respectively, with structural strength), it is an obvious candidate for a key enzyme involved in the degradation of these tissues. In cultures of synovial fibroblasts, inflammation mediators interleukin-1␤ and tumor necrosis factor ␣ have been shown to activate cathepsin K gene expression (8), but the sequence of events leading to activation of cathepsin K production in arthritic joints remains poorly understood. We recently produced 3 lines of transgenic UTU17 mice containing variable numbers of extra copies of the murine gene for cathepsin K (Ctsk) under its Supported by the Academy of Finland (project 205346), the Maire Lisko Foundation, the Paulo Foundation, and the Research and Science Foundation of Farmos. Mr. Morko is recipient of a training grant from the Finnish Graduate School in Musculoskeletal Problems. Dr. Kiviranta is recipient of a training grant from Turku Biomedical Graduate School. Jukka Morko, MSc, Riku Kiviranta, MD, PhD, Kirsi Joronen, MD, PhD, Anna-Marja Säämänen, PhD, Eero Vuorio, MD, PhD, Heli Salminen-Mankonen, PhD: University of Turku, Turku, Finland. Address correspondence and reprint requests to Eero Vuorio, MD, PhD, Department of Medical Biochemistry and Molecular Biology, University of Turku, FI-20520 Turku, Finland. E-mail: firstname.lastname@example.org. Submitted for publication March 8, 2005; accepted in revised form August 18, 2005. 3713 3714 MORKO ET AL endogenous promoter (9). These mice exhibit increased production of cathepsin K and progressively develop high-turnover osteopenia of trabecular bone (9) and increased porosity of cortical bone (10). Considering the recent studies of increased cathepsin K production during (osteo)arthritic degradation of cartilage and bone, the availability of these mice provided us with an obvious possibility to study whether constitutive overexpression of cathepsin K could function as the primary cause of joint destruction. Our findings clearly demonstrate that overexpression of Ctsk in transgenic mice makes them susceptible to progressive synovitis, which, upon aging, results in destruction of articular cartilage and subchondral bone. MATERIALS AND METHODS Experimental animals. This study was conducted on samples of knee joints collected from 15 transgenic UTU17 male mice that overexpressed Ctsk and from their 19 nontransgenic male littermates, which served as controls. Transgenic UTU17 mice were produced at the Transgenic Core Facility at the University of Turku, as previously described (9). The UTU17 mice harbor several copies of an engineered 14-kb cathepsin K transgene with a silent mutation in an FVB/N genetic background. The UTU17 mice studied were homozygous for 1 of the 2 transgene alleles, UTU17.2 or UTU17.3. The litters were genotyped by Southern blot analysis, as previously described (9). Mice were killed at the ages of 7 and 12 months by cervical dislocation, and the hind limbs were collected for histology and immunohistochemistry studies. The study protocol was approved by the Institutional Committee for Animal Welfare of the University of Turku. Histology. For routine histology, dissected hind limbs were fixed overnight in fresh 4% paraformaldehyde prepared in phosphate buffered saline (PBS). Samples were then decalcified in 10% EDTA in PBS (pH 7.4) for 5–20 days, dehydrated, embedded in paraffin, and cut sagittally into 5-m– thick sections. Sections of the knee joints were either stained with hematoxylin and eosin (Merck, Darmstadt, Germany) and used for subsequent histologic grading or processed for immunohistochemistry. Histologic grading. Degenerative changes of articular cartilage were analyzed in sagittal sections of knee joints and classified into 5 grades based on the depth of erosion, as previously described (6). Hyperplasia and fibrosis were used as criteria in the scoring of synovial changes, as previously described (11). The score of each knee was calculated as the sum of the highest scores for fibrosis and hyperplasia observed in the serial sections. Immunohistochemistry. Tissue distribution of cathepsin K was studied using polyclonal anti-mouse cathepsin K antibodies (12), which have previously been tested for specificity by Western blotting (10). Sections of knee joints (5 m) were deparaffinized and rehydrated in a series of descending ethanol concentrations and digested for 1 hour with bovine testicular hyaluronidase (2,000 units/ml) in PBS (pH 5). Primary antibodies were detected using streptavidin–biotin and horseradish peroxidase complex (Histostain-Plus kit; Zymed, Figure 1. Histologic analysis of knee joints of male UTU17 mice. Hematoxylin and eosin–stained sagittal sections of knee joints of control mice at the ages of 7 months (A–C) and 12 months (G–I), and of UTU17 mice at 7 months (D–F) and 12 months (J–L). Bar in A ⫽ 25 m (A, D, G, and J); bar ⫽ 50 m (B, C, E, F, H, I, K, and L). Color figure can be viewed in the online issue, which is available at http: //www.arthritisrheum.org. South San Francisco, CA) and diaminobenzidine (Zymed) for color development. Tissue sections were counterstained with hematoxylin. The specificity of the reactions was controlled by replacing the primary antibodies with normal rabbit serum. Multinucleated cells in synovium and bone were studied in paraffin-embedded decalcified sections by enzyme histochemistry for tartrate-resistant acid phosphatase (TRAP) according to the protocol recommended by the supplier of the kit (Leukocyte Acid Phosphatase kit; Sigma-Aldrich, St. Louis, MO). The sections were counterstained with hematoxylin. RESULTS Histologic evaluation of knee joints. At the age of 7 months, articular cartilage of both the UTU17 mice (Figures 1D and E) and their nontransgenic control littermates (Figures 1A and B) appeared normal. Compared with control joints (Figure 1C), the synovial membrane in UTU17 mice was thickened, comprising 3–5 cell layers (Figure 1F). At the age of 12 months, little cartilage degeneration was seen in control joints (Figures 1G, H, and I). In 12-month-old UTU17 mice, the articular surfaces were severely eroded, particularly in the posterior aspect ARTHRITIS RESULTING FROM CATHEPSIN K OVEREXPRESSION Figure 2. Scoring changes in articular cartilage and synovial tissue with age. A, Scoring of degenerative changes of articular cartilage at 7 months (n ⫽ 7 control and 5 UTU17 mice) and at 12 months (n ⫽ 8 control mice and 8 UTU17 mice). Cartilage defects were scored 0–4 according to the depth of penetration. B, Corresponding synovial hyperplasia and fibrosis, scored 0–3. C, Mean ⫾ SD articular cartilage erosion and synovial hyperplasia and fibrosis scores. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01 versus controls, by Mann-Whitney U test. of the femoral and tibial condyles, where proliferative synovial tissue was in contact with cartilage (Figures 1J, K, and L). In addition to cartilage erosion, several other alterations were observed in the knee joints of UTU17 mice with increasing age. Chondrocyte nuclei were pyknotic in defect areas (Figure 1K). Chondrocyte clusters were frequently found at the margins of the articular surface and in menisci (Figures 1K and L). At the age of 12 months, the synovial membrane of control mice was 1–2 cell layers thick (Figure 1I), while in UTU17 mice, moderate to severe hyperplasia was observed (Figure 1L). Proliferation of synovial cells, fibrosis, angiogenesis, and some inflammatory cells were observed in the hyperplastic synovium of UTU17 mice (Figures 1J and L). Scoring changes in articular cartilage and synovial tissue. Scoring of degenerative changes in articular cartilage of knee joints confirmed that only mild OA changes were observed both in UTU17 and control mice at 7 months (Figure 2A), whereas at 12 months severe changes were detected in UTU17 mice (Figure 2A). But scoring of synovial hyperplasia and fibrosis already revealed significant changes in the knee joints of UTU17 mice at 7 months (Figures 2B and C). At the age of 12 months, both synovial and articular cartilage scores were significantly higher in UTU17 mice (Figure 2C). Immunohistochemical detection of cathepsin K in knee joints. In both control mice (Figure 3A) and UTU17 mice (Figures 3B and D–F), a fraction of cells located in the synovium were found to stain positive for 3715 cathepsin K, as shown for 12-month-old mice. In UTU17 mice, intense cathepsin K immunostaining was observed in mononuclear cells in the lining and sublining areas of synovial villi (Figures 3D and F). Because the synovial membrane was thickened in UTU17 mice, the number of cathepsin K–positive cells was considerably greater than in control mice. Strong cathepsin K staining was also observed in mononuclear cells in areas of high proliferation (Figure 3B) and vascularization (Figure 3E). Cells with multiple nuclei, which were in intimate contact with bone and positive for TRAP, were also observed in the synovial tissue of UTU17 mice (Figure 3C). In control knees at 12 months, a fraction of chondrocytes in uncalcified articular cartilage exhibited cathepsin K immunostaining (Figure 3G). In the knee joints of UTU17 mice, pericellular and intracellular staining for cathepsin K was observed in a number of chondrocytes throughout uncalcified cartilage and in the territorial matrix of calcified cartilage (Figures 3H and I). The eroded cartilage surface and chondrocyte clusters in the remaining articular cartilage and menisci of UTU17 mice were also positive for cathepsin K (Figures 3H and I), whereas no immunostaining was observed when primary antibodies were replaced with normal rabbit serum (Figure 3L). TRAP-positive osteoclasts were seen on the surface of endosteal bone in the knee Figure 3. Immunolocalization of cathepsin K and tartrate-resistant acid phosphatase (TRAP) activity in the knee joints of UTU17 mice. A, G, and J, Control mice. B–F, H, I, K, and L, Transgenic UTU17 mice. Immunohistochemistry analysis of TRAP activity (C, J, and K) was performed on sagittal sections of knee joints obtained from UTU17 mice at the age of 12 months. Intense cathepsin K immunostaining was seen in the hyperplastic synovium (B–F) and in articular cartilage at the margins of defect areas (H and I). Cells with multiple nuclei, which were in intimate contact with bone and positive for TRAP, are seen in C, J, and K. As a staining control, normal rabbit serum was used (L). Bar in A ⫽ 25 m (A and B); bar ⫽ 50 m (G–L); bar ⫽ 100 m (C–F). 3716 MORKO ET AL joints of both control mice (Figure 3J) and UTU17 mice (Figure 3K). DISCUSSION The most interesting finding of the present study was that constitutive overexpression of Ctsk under its own promoter in mice makes them susceptible to synovitis, which, upon aging, results in the destruction of articular cartilage and bone. Previously, cathepsin K was considered mainly as the major proteinase in osteoclastic bone resorption (9,10,13). However, recent studies of OA and rheumatic joints have also demonstrated increased expression of cathepsin K in articular cartilage and synovial tissue both in mice and humans (2–5). These observations, and the availability of transgenic UTU17 mice exhibiting constitutive overexpression of Ctsk, prompted us to perform a systematic study on their knee joint phenotype and its association with cathepsin K distribution in these joints. In synovium, cathepsin K overexpression in UTU17 mice resulted in a marked proliferative response, including angiogenesis and moderate infiltration of inflammatory cells. This was associated with widespread distribution of synovial cells expressing cathepsin K. Especially in the hyperplastic synovial membrane of UTU17 mice, the number of cathepsin K–expressing cells was increased. Increased cathepsin K expression has also been observed in the synovial membrane of OA Del1 mice and patients with arthritis, and has been suggested to increase the invasiveness of synovium and to contribute to cartilage and bone erosion (3–5,8). We propose that increased cathepsin K production in the synovial membrane of UTU17 mice may contribute to the development of cartilage and bone erosion in these mice. Strong cathepsin K staining was also detected in deep zones of synovial tissue in UTU17 mice, particularly in mononuclear synoviocytes, around blood vessels, and in multinucleated synovial cells. Similar distribution of cathepsin K has been detected in patients with arthritis, where it has been suggested to facilitate the proliferation and movement of cells in the interstitial matrix, and to contribute to matrix remodeling (4,5,8). Cathepsin K overexpression may also contribute to the development and progression of synovial hyperplasia and fibrosis in UTU17 mice that exhibit features of chronic fibrosing synovitis and rheumatoid pannus, although the underlying mechanism remains unresolved. Furthermore, cathepsin K immunostaining was observed in articular cartilage of control and UTU17 mice. In 12-month-old UTU17 mice, intense cathepsin K staining was detected in areas of cartilage degeneration, particularly in chondrocyte clusters. Increased cathepsin K expression has also been observed in OA cartilage of Del1 mice and human samples, where it has been suggested to contribute to the progression of cartilage degeneration (2,3). It is likely that the high cathepsin K expression in articular cartilage of 12-month-old UTU17 mice has a similar role. Furthermore, since resorption of subchondral bone has been linked to cartilage erosion in arthritic joints (14,15), and cathepsin K expression is significantly increased in subchondral bone of UTU17 mice (9), bone-derived cathepsin K may also play a role in the development of cartilage and bone erosion detected in UTU17 mice. We have previously described increased cathepsin K expression in a transgenic Del1 mouse model for OA during cartilage degeneration (3). However, striking differences exist in the development of arthritis between Del1 mice harboring a deletion mutation in the type II collagen transgene and UTU17 mice. In the former model, the primary defect resides in articular cartilage, and is associated with relatively minor hyperplasia of the synovial tissue during progression of cartilage degeneration (6). In UTU17 mice, synovitis clearly preceded the destruction of articular cartilage and subchondral bone. Because cathepsin K overexpression alone was sufficient to induce the phenotype in UTU17 mice, increased cathepsin K expression in arthritic joints could even serve as the primary event for the disease. However, the relative importance of cathepsin K overexpression in synovium, cartilage, and bone remains to be determined. Further studies are therefore needed to identify the mechanisms whereby increased production of cathepsin K by synovial tissue, articular chondrocytes, and osteoclasts triggers the phenotype in UTU17 mice. Whichever the case, pharmaceutical inhibition of excessive cathepsin K activity might help to prevent or slow the progression of arthritis. ACKNOWLEDGMENTS The authors are grateful to Dr. Dieter Brömme for providing the mouse cathepsin K antibodies. The expert technical help of Ms Maria Ström, Ms Merja Lakkisto, and Ms Tuula Oivanen is also gratefully acknowledged. REFERENCES 1. Kirschke H, Barret AJ, Rawlings ND. Lysosomal cysteine proteinases. 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