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1.
W. Hui A. D. Rowan C. D. Richards T. E. Cawston 《Arthritis \u0026amp; Rheumatology》2003,48(12):3404-3418
Objective
To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.Methods
The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.Results
The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.Conclusion
This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.2.
Karsdal MA Sumer EU Wulf H Madsen SH Christiansen C Fosang AJ Sondergaard BC 《Arthritis and rheumatism》2007,56(5):1549-1558
OBJECTIVE: Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. METHODS: Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 4, 16, or 64 microM). Cartilage degradation was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX-II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography. RESULTS: OSM and TNF induced an 8,000% increase in CTX-II compared with control (P < 0.001). Both forskolin and IBMX dose-dependently inhibited release of CTX-II (P < 0.001). OSM and TNF induced a 6-fold increase in (342)FFGV-G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases. 相似文献
3.
Yuen M. Chung Michael E. Deutscher Karena Last Amanda J. Fosang 《Arthritis \u0026amp; Rheumatology》2010,62(11):3365-3373
Objective
To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes.Methods
Primary chondrocytes were isolated from knee epiphyses of 6–8‐day‐old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin‐6 (IL‐6)/soluble IL‐6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes.Results
Expression of both ADAMTS‐4 and ADAMTS‐5 mRNA was up‐regulated by TNFα and OSM. ADAMTS‐5 mRNA expression was also up‐regulated by IL‐6. Treatment of wild‐type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu373↓374Ala and Glu1670↓1671Gly; in chondrocytes lacking ADAMTS‐5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS‐4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS‐1 or ADAMTS‐9.Conclusion
Despite substantial increases in the expression of ADAMTS‐4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS‐5–deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine‐induced ADAMTS‐4 mRNA expression and the level of aggrecan‐degrading activity in cultured chondrocytes.4.
Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation
J. M. Milner A. D. Rowan S.‐F. Elliott T. E. Cawston 《Arthritis \u0026amp; Rheumatology》2003,48(4):1057-1066
Objective
To investigate the role of furin‐like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation.Methods
Bovine nasal cartilage was stimulated to resorb with the addition of interleukin‐1α (IL‐1α)/oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec‐RVKR‐CH2Cl, or selective matrix metalloproteinase 1 (MMP‐1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using 3H‐acetylated collagen and gelatin zymography, respectively.Results
The addition of Dec‐RVKR‐CH2Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP‐2, suggesting that furin‐like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP‐1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP‐13 resulted in a 50% blockage of collagen release. The addition of Dec‐RVKR‐CH2Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin‐activated enzymes in the pathways leading to proteoglycan degradation.Conclusion
Furin‐like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP‐13, which can be activated by furin‐processed membrane‐type 1 MMP‐1, appears to be a major collagenase involved in collagen degradation induced by IL‐1α/OSM. Furin‐like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.5.
Carole Bougault Marjolaine Gosset Xavier Houard Colette Salvat Lars Godmann Thomas Pap Claire Jacques Francis Berenbaum 《Arthritis \u0026amp; Rheumatology》2012,64(12):3972-3981
Objective
The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin‐1β (IL‐1β) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL‐1β is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the “inflammasome,” a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL‐1β and the inflammasome in cartilage breakdown.Methods
We assessed IL‐1β release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)–, IL‐1α–, and tumor necrosis factor α (TNFα)–induced activity of matrix metalloproteinase 3 (MMP‐3), MMP‐9, and MMP‐13 in NLRP3‐knockout mice and wild‐type mice and the inhibition of caspase 1 with Z‐YVAD‐FMK and the blockade of IL‐1β with IL‐1 receptor antagonist (IL‐1Ra). Cartilage explants from NLRP3‐knockout mice and IL‐1R type I (IL‐1RI)–knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load‐induced glycosaminoglycan (GAG) release and MMP enzymatic activity.Results
Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL‐1β. LPS, IL‐1α, and TNFα dose‐dependently increased MMP‐3, MMP‐9, and MMP‐13 activity in cultured chondrocytes and in NLRP3−/− chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL‐1β. The load‐induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL‐1RI in cartilage explants.Conclusion
OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL‐1β inhibitors observed in previous trials.6.
P. J. T. Koshy C. J. Lundy A. D. Rowan S. Porter D. R. Edwards A. Hogan I. M. Clark T. E. Cawston 《Arthritis \u0026amp; Rheumatology》2002,46(4):961-967
Objective
Previous studies have reported elevated levels of interleukin‐1 (IL‐1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL‐1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM‐TS (ADAM with thrombospondin motifs) family members in human chondrocytes.Methods
T/C28a4 human chondrocytes were stimulated for 2–48 hours with IL‐1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real‐time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM‐TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA.Results
IL‐1 and OSM synergized to markedly induce the expression of the collagenases MMP‐1, MMP‐8, and MMP‐13 as well as MMP‐3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP‐8 mRNA occurred late. Gene expression of MMP‐14, an MMP that degrades collagen and activates proMMP‐13, was elevated by this combination. IL‐1 and OSM also synergized to induce gene expression of the aggrecanase ADAM‐TS4, but not ADAM‐TS5.Conclusion
These data indicate that the potent cartilage‐degrading properties of the combination of IL‐1 and OSM are potentially mediated by a synergistic induction of the aggrecan‐degrading enzyme ADAM‐TS4 and the collagen‐degrading enzymes MMP‐1, MMP‐8, MMP‐13, and MMP‐14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.7.
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9.
H. E. Barksby W. Hui I. Wappler H. H. Peters J. M. Milner C. D. Richards T. E. Cawston A. D. Rowan 《Arthritis \u0026amp; Rheumatology》2006,54(2):540-550
Objective
To identify the genes up‐regulated by interleukin‐1 (IL‐1) in combination with oncostatin M (OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation.Methods
Gene microarray and real‐time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus vectors overexpressing IL‐1 and/or OSM, were analyzed by immunohistochemistry for selected proteins.Results
The combination of IL‐1 and OSM markedly up‐regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines, chemokines, extracellular matrix components, and genes involved in signal transduction. Real‐time PCR confirmed a synergistic induction of several MMPs, activin A, pentraxin 3 (PTX‐3), and IL‐8. The in vivo findings further indicated that stimulation with IL‐1 plus OSM induced protein expression of activin A, PTX‐3, and KC (the murine homolog of IL‐8), as compared with the changes induced by individual cytokine treatment and unstimulated controls.Conclusion
The results confirm that the potent proinflammatory cytokine combination of IL‐1 plus OSM synergistically and coordinately up‐regulates many genes and several MMPs. Moreover, chondrocytes exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are ultimately overwhelmed by degradative processes in the cartilage. This gene‐profiling study provides insight into the complex processes that mediate joint disease in the inflammatory arthritides through the coordinated expression of multiple genes.10.
11.
Thomas Aigner Alexander Zien Angelika Gehrsitz Pia Margarethe Gebhard Louise McKenna 《Arthritis \u0026amp; Rheumatology》2001,44(12):2777-2789
Objective
To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)–array technology.Methods
Nine normal, 6 early degenerated, and 6 late‐stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis.Results
In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late‐stage specimens, reflecting the high matrix‐remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP‐3 appeared to be strongly expressed in normal and early degenerative cartilage and down‐regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP‐2 and MMP‐11, both of which were up‐regulated in late‐stage disease. MMP‐11 was up‐regulated in OA chondrocytes and, interestingly, also in the early‐stage samples. Neither MMP‐1 nor MMP‐8 was detectable, and MMP‐13 and MMP‐2 were significantly detectable only in late‐stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers.Conclusion
This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.12.
13.
Carol G. Chen Daniel Thuillier Emily N. Chin Tamara Alliston 《Arthritis \u0026amp; Rheumatology》2012,64(10):3278-3289
Objective
To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.Methods
A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.Results
Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.Conclusion
Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.14.
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16.
Robert Terkeltaub Bing Yang Martin Lotz Ru Liu‐Bryan 《Arthritis \u0026amp; Rheumatology》2011,63(7):1928-1937
Objective
Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.Methods
Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.Results
Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.Conclusion
Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.17.
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19.
Svetlana Oestergaard Luc Chouinard Nancy Doyle Susan Y. Smith Lszl B. Tank Per Qvist 《Arthritis \u0026amp; Rheumatology》2006,54(9):2886-2890
Objective
To investigate changes in the circulating levels of the C‐telopeptide of type II collagen (CTX‐II) with relation to disease onset and structural damage of cartilage in a rodent model of collagen‐induced arthritis (CIA), and to investigate immunolocalization of the CTX‐II epitope in the articular cartilage of affected joints.Methods
Seven‐week‐old female Lewis rats were immunized with type II collagen and monitored using blood sampling at weekly intervals. At study termination (day 23), the animals were killed, synovial fluid was collected, and the affected joints were scored macroscopically for disease severity and underwent immunohistochemical evaluation.Results
At the time of disease onset (day 15), which was characterized by redness and swelling of the affected joints (mean ± SD macroscopic severity score 9.1 ± 1.6), there was a 355% increase in serum CTX‐II levels. The early change in serum CTX‐II from day 0 to day 15 showed a significant association with the severity of cartilage damage (r = 0.61, P < 0.01). Immunostaining revealed extensive presence of the CTX‐II epitope in the damaged, uncalcified cartilage tissue.Conclusion
The elevation in serum CTX‐II concomitant with the onset of disease and proportional to cartilage damage demonstrates that CTX‐II is a sensitive diagnostic tool for monitoring joint disease in the rodent model of CIA. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum CTX‐II is the damaged articular cartilage.20.
Patrick Garnero Robert Landew Maarten Boers Arco Verhoeven Sjef Van Der Linden Stephan Christgau Dsire Van Der Heijde Annelies Boonen Piet Geusens 《Arthritis \u0026amp; Rheumatology》2002,46(11):2847-2856