Ginette Tardif

The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis.

Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology.

In Vivo Transfer of Interleukin-1 Receptor Antagonist Gene in Osteoarthritic Rabbit Knee Joints: Prevention of Osteoarthritis Progression

The goal of this study was to determine the efficacy of local IL-1Ra gene therapy by intra-articular plasmid injections on structural changes in the meniscectomy rabbit model of osteoarthritis. A partial meniscectomy of the right knee was performed on the rabbits through a medial parapatellar incision. The rabbits were then divided into four experimental groups. Group 1 received no treatment. Group 2 received three consecutive intra-articular injections at 24-hour intervals of 0.9% saline containing a lipid, gammaAP-DLRIE/DOPE, and a DNA plasmid, VR1012. Group 3 received three consecutive injections of saline containing 1000 microg of canine IL-1Ra plasmid and lipid. The injections were given starting 4 weeks post-surgery. Rabbits from Group 1 were killed 4 weeks post-surgery, and all other rabbits 8 weeks post-surgery. The severity of macroscopic and microscopic changes on cartilage on the medial and femoral condyles and tibial plateaus and synovium were graded separately. Specimens were also processed for immunohistochemical staining using a rabbit polyclonal antibody against canine IL-1Ra. The level of canine IL-1Ra in synovial fluid was determined using enzyme-linked immunosorbent assay. The presence of the DNA plasmid in the synovium was tested by polymerase chain reaction. A significant reduction in the width of osteophytes and size of macroscopic lesions (P < 0.04) was observed, and was dependent on the amount of IL-1Ra plasmid injected. A significant reduction was also noted in the severity of histologic cartilage lesions (P < 0.01) in the group that received the highest dosage (1000 microg) of IL-1Ra plasmid. IL-1Ra was detected in synovial fluid by enzyme-linked immunosorbent assay and by immunohistochemical staining in the synovium and cartilage of rabbits that received injections containing the IL-1Ra plasmid. Polymerase chain reaction analysis of synovial DNA revealed the presence of the cloned cDNA dog IL-1Ra up to 4 weeks after the first intra-articular injection. This study demonstrates that direct in vivo transfer of the IL-1Ra gene into osteoarthritis knee cells using intra-articular injections of a plasmid vector and lipids can significantly reduce the progression of experimental osteoarthritis. This avenue may therefore represent a promising future treatment for osteoarthritis.

Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

BackgroundMMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.MethodsGene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.ResultsIGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.ConclusionThis study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.

The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation*

The degradation of osteoarthritic (OA) cartilage is likely related to the synthesis and the release of catabolic factors by chondrocytes. Nitric oxide (NO) has recently been suggested as playing a role in cartilage degradation. Since NO production is largely dependent on stimulation by IL-1, its effects on factors regulating the IL-1 biological activity, such as IL-1ra, are of the utmost importance. This study examined and compared the level of NO production by normal and OA cartilage and chondrocytes, as well as studied the effect of IL-1-induced NO production on the synthesis and steady-state mRNA of interleukin-1 receptor antagonist (IL-1ra). The NO baseline production by normal cartilage explants was undetectable but inducible by rhIL-1 beta. OA cartilage spontaneously produced NO. About a two-fold increase in NO production was found in OA rhIL-1 beta-stimulated (0.5-100 units/ml) cartilage as compared with the similarly stimulated normal cartilage. on chondrocytes rhIL-1 beta-stimulation (0.5-100 units/ml) produced a dose-dependent enhancement of both NO production and IL-1ra synthesis. Treatment with 200 microM N(g)-monomethyl-L-arginine (L-NMA), a well known NO synthase inhibitor, induced over 70% inhibition of the NO production and a marked increased IL-1ra synthesis (average of 84%) and expression (mRNA level). Inhibition of prostaglandin synthesis by indomethacin had no effect on both the NO production or the IL-1ra level. In the present study, we demonstrated the capacity of OA cartilage to produce a larger amount of NO than the normal controls, both in spontaneous and IL-1-stimulated conditions. These data support the notion that, in vivo, OA chondrocytes are stimulated by factors, possibly IL-1, which in turn may induce the expression of NO synthase, thus the synthesis of NO itself. Importantly, our results showed that the elevation of of NO production may be an important factor in the pathophysiology of OA since it can reduce IL-1ra synthesis by chondrocytes. As such, an increased level of IL-1, associated with a decreased IL-1ra level, may be responsible for the stimulation of OA chondrocytes by this cytokine, leading to an enhancement of cartilage matrix degradation.

Interleukin-1β–converting enzyme/caspase-1 in human osteoarthritic tissues: Localization and role in the maturation of interleukin-1β and interleukin-18

OBJECTIVE To study the expression and production of interleukin-1beta-converting enzyme (ICE) in human normal and osteoarthritic (OA) cartilage and synovium, quantitate the level of ICE in OA chondrocytes, and examine the relationship between the topographic distribution of ICE, interleukin-1beta (IL-1beta), and IL-18, as well as apoptosis of chondrocytes. METHODS The expression and synthesis of ICE were investigated in human normal and OA cartilage and synovial membrane using in situ hybridization and immunohistochemical methods. The intracellular level of ICE in OA chondrocytes was also measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the topographic relationship between the presence of ICE and mature IL-1beta and IL-18 was examined by immunohistochemistry, and apoptotic chondrocytes by the TUNEL technique. RESULTS ICE was expressed and synthesized in both human synovial membrane and cartilage, with a significantly greater number of cells staining positive in OA tissue than in normal tissue. ICE production was preferentially located in the superficial and upper intermediate layers of articular cartilage. With a specific ELISA, a level of 230.2+/-22.5 pg/5 x 10(5) cells (mean +/- SEM) of ICE was found in OA chondrocytes. In cartilage, IL-1beta and IL-18 stained positive at a topographic location similar to that of ICE. The production of mature IL-1beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ICE inhibitor, which also markedly diminished the number of IL-18-positive cells. The data show that there was no close relationship between the presence of ICE and the presence of apoptotic chondrocytes in OA cartilage. CONCLUSION This study shows, for the first time, the presence of active ICE in human articular cartilage, with a markedly increased cellular level in OA tissue. The relationship between active IL-1beta and ICE suggests that ICE may promote OA progression by activating this proinflammatory cytokine. The role of IL-18 in pathologic cartilage is discussed.

Collagenase 3 production by human osteoarthritic chondrocytes in response to growth factors and cytokines is a function of the physiologic state of the cells

OBJECTIVE We investigated the response of human osteoarthritic (OA) chondrocytes, in terms of collagenase 3 production, to growth factors and cytokines involved in the anabolism and catabolism of articular cartilage, and explored the major signaling pathways leading to its up-regulation. METHODS Human OA chondrocytes were treated with the following factors: the proinflammatory cytokine interleukin-1beta (IL-1beta), the growth factors basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), transforming growth factor gamma1 (TGFbeta1), and TGFbeta2, the protein kinase (PK) activator antagonists for PKC, PKA, and PKG pathways, and phospholipase A2 and tyrosine kinases, as well as the antiinflammatory cytokines IL-4, IL-10, and IL-13. Collagenase 3 expression and synthesis were determined. Comparison was made with collagenase 1. RESULTS The human OA chondrocyte population could be divided into 2 categories: the L chondrocytes, showing low collagenase 3 basal synthesis levels and high sensitivity to IL-1beta stimulation; and the H chondrocytes, high collagenase 3 basal synthesis levels and low IL-1beta inducibility. In L chondrocytes, all growth factors stimulated collagenase 3 production. In H chondrocytes, PTH, IGF-1, and TGFbeta had little or no impact; bFGF slightly stimulated it and PDGF-BB showed the same pattern as in the L chondrocytes. The effects of all growth factors, except TGFbeta, on collagenase 1 synthesis followed those of collagenase 3, albeit to a higher degree. Interestingly and unlike collagenase 3, the effects of TGFbeta on collagenase 1 could not be related to the state of the cells, but rather, depended on the isoform. Indeed, TGFbeta2 did not induce collagenase 1 synthesis, whereas TGFbeta1 stimulated it. Among the PK activators tested, phorbol myristate acetate was the strongest inducer, suggesting a major involvement of the PKC pathway. IL-13 inhibited collagenase 3 production, IL-4 had little effect, and IL-10 had none. CONCLUSION This study shows that collagenase 3 production in human OA chondrocytes depends on the physiologic state of the cell. TGFbeta might be responsible for the change in cells from the L to the H state. Importantly, our in vitro data implicate TGFbeta2 as a possible in vivo agent capable of specifically triggering collagenase 3 production over that of collagenase 1 in OA cartilage.

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site

Author details 1Osteoarthritis Research Unit, University of Montreal Hospital Centre, NotreDame Hospital, 1560 Sherbrooke Street East, Montreal, Quebec H2L 4M1, Canada. 2Department of Rheumatology, Universitat Autonoma de Barcelona, Hospital del Mar, Passeig Marítim de la Barceloneta, 25, 08005 Barcelona, Spain. 3Genetics Unit, Shriner’s Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec H3G 1A6, Canada. Published: 5 March 2013

Human Adult Chondrocytes Express Hepatocyte Growth Factor (HGF) Isoforms but Not HGF: Potential Implication of Osteoblasts on the Presence of HGF in Cartilage†

HGF is increased in human OA cartilage, possibly from Obs. RT‐PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross‐talk between subchondral bone and cartilage may occur during OA.

Hepatocyte growth factor induction of collagenase 3 production in human osteoarthritic cartilage: Involvement of the stress‐activated protein kinase/c‐Jun N‐terminal kinase pathway and a sensitive p38 mitogen‐activated protein kinase inhibitor cascade

OBJECTIVE Osteoarthritis (OA) involves both a decreased reparative process and an increased degradative phenomenon. Several cytokines and growth factors are known to facilitate the repair of articular cartilage defects. The hepatocyte growth factor (HGF) present in OA cartilage is suggested to be involved in the cartilage repair process as well as in matrix remodeling and chondrocyte migration, leading to partial reconstruction of articular cartilage. Since cell migration is often correlated with metalloprotease activity, the effect of HGF on collagenase 3 production was studied because of its possible implication in OA cartilage remodeling. METHODS We examined HGF-stimulated collagenase 3 production in human OA chondrocytes by Western and Northern blotting. Furthermore, we explored the intracellular signaling pathways through which HGF induced collagenase 3 production. RESULTS This study showed that HGF stimulated collagenase 3 production in human OA chondrocytes at the transcriptional level, and this induction was mediated by activation of the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway, but not the p38 mitogen-activated protein kinase (MAPK). The p44/42 MAPKs were also phosphorylated and the use of their specific inhibitor (PD 98059) did not affect HGF-induced collagenase 3 production in OA chondrocytes. Induced collagenase 3 production via the SAPK/JNK pathway was mediated, at least in part, by the TRE site in the promoter, and in the activator protein 1 complex, c-Jun, JunD, and Fra-1 were activated. Surprisingly, further experiments revealed that the specific p38 MAPK inhibitor SB 202190 also inhibited collagenase 3 production early in the HGF-induced process. The 50% inhibitory concentration was as low as 50 nM, which is unlikely to be related to p38 MAPK inhibition (which is usually in the microM range), suggesting the involvement of another kinase sensitive to SB 202190. CONCLUSION This is the first study to show that HGF has the ability to induce both the expression and synthesis of collagenase 3 in OA chondrocytes. The effect is mediated by kinase cascades involving SAPK/JNK and another, unidentified kinase. This study provides novel information implicating a role for HGF in the pathophysiology of OA through its effect on the production of collagenase 3, which is an enzyme that is possibly involved in OA cartilage remodeling.

Identification of opticin, a member of the small leucine-rich repeat proteoglycan family, in human articular tissues : a novel target for MMP-13 in osteoarthritis

OBJECTIVE One of the proteoglycan families is the small leucine-rich proteoglycans (SLRPs) that are characterized by their association with collagen fibrils and/or some glycosaminoglycans. Opticin is a glycoprotein and class III member of the SLRP family, which was initially identified in the vitreous humour of the eye. In this study, we first investigated whether opticin is expressed and produced in normal and OA human articular tissues/cells. Further, we investigated the ability of the key metalloprotease involved in cartilage pathology, MMP-13, to cleave human cartilage opticin. METHODS Opticin gene expression was investigated in normal and OA human chondrocytes, synovial fibroblasts, and subchondral bone osteoblasts by reverse transcriptase-polymerase chain reaction (RT-PCR). Opticin protein production was determined in normal and OA synovial membrane and cartilage by immunohistochemistry. Opticin was isolated from human cartilage using guanidinium chloride extraction, and human MMP-13-induced opticin degradation analyzed by Western blotting. Finally, the opticin MMP-13 cleavage site was determined. RESULTS Opticin was expressed in human chondrocytes, synovial fibroblasts and subchondral osteoblasts, and the protein identified in synovial membrane and cartilage. At the protein level, OA cartilage showed a slightly higher level of opticin positive stained chondrocytes than normal cartilage; this did not reach statistical significance. However, in contrast with OA, normal cartilage demonstrated a high level of matrix staining in the superficial zone of the tissue, suggesting that in the OA cartilage matrix, opticin is degraded. Data also showed that cartilage opticin could be cleaved by MMP-13 after only 2h of incubation, indicating a preferential substrate compared to other SLRPs for this enzyme. Microsequencing revealed a major cleavage site at the G(104)/L(105)LAAP and a minor at P(109)/A(110)NHPG upon MMP-13 exposure. CONCLUSION We demonstrated, for the first time, that opticin is expressed and produced in human articular tissues. Our data also showed that opticin in OA cartilage is degraded in a process that could be mediated by MMP-13. As opticin may contribute towards the structural stability of cartilage, its cleavage by MMP-13 may predispose cartilage to degeneration, particularly at the surface.