Objective. To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin-1␤ (IL-1␤)-stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.Methods. IL-1␤ was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800-kd HA to assess its effect on IL-1␤-induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti-CD44 antibody were performed to investigate the mechanism of action of HA.Results. Treatment and pretreatment with 800-kd HA at 1 mg/ml resulted in significant suppression of IL-1␤-stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA-binding blocking experiments using anti-CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti-CD44 antibody, which suppressed IL-1␤-stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL-1␤ in normal and OA cartilage.Conclusion. This study demonstrates that HA effectively inhibits IL-1␤-stimulated production of MMP-1, MMP-3, and MMP-13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL-1␤ may involve direct interaction between HA and CD44 on chondrocytes.Osteoarthritis (OA) is the most prevalent disease of articular joints and is the major cause of disability in the elderly. Pathophysiologic changes occur in OA cartilage due to the excessive expression of cartilagedegrading proteinases, the resultant progressive breakdown of collagen fibers, and the degradation of proteoglycan, mainly aggrecan (1).Matrix metalloproteinases (MMPs) are zinccontaining, calcium-dependent proteinases, which collectively degrade all components of the extracellular matrix. MMPs are considered to be important in the chondrolytic processes that contribute to the degenerative changes in OA cartilage (2-4). Recent studies have identified the messenger RNA (mRNA) for some MMPs, such as MMP-1, in human OA cartilage (4,5), and other investigators have reported specific MMP proteins and collagenasemediated type II collagen degradation products (6,7). There is a consensus that these enzymes play a critical role in intrinsic chondrocyte-mediated degenerative changes of the cartilage matrix in OA. Proinflammatory cytokines such as interleukin-1 (IL-1) strongly stimulate the expression of MMPs by chondrocytes in arthritis (8).Hyaluronan (HA) is a major component of synovial fluid and cartilage matrix, and it play...

HA suppresses IL-1beta-enhanced MMP-1 and MMP-3 synthesis in RSF via ICAM-1 through down-regulation of NF-kappaB and p38. Intra-articular injection of HA of high molecular weight may work through such a mechanism in RA joints.

The purpose of this study was to examine the effects of celecoxib on matrix metalloproteinases (MMP-1 and MMP-3), nitric oxide (NO), and the phosphorylation of nuclear factor-kappaB (NF-kappaB) and three mitogen-activated protein kinases (MAPKs), (p38, JNK and ERK) in human articular chondrocytes from normal, osteoarthritis, and rheumatoid arthritis cartilages. Celecoxib at 100 nM reduced the IL-1beta-induced productions of MMP-1, MMP-3, iNOS, and NO, whereas indomethacin at 100 nM showed no effect. The additional stimulation of prostaglandin E2 (PGE2) failed to restore those productions, while the production of PGE2 were reduced by 1 and 10 microM but not 100 nM of celecoxib. The inhibitors of NF-kappaB, JNK and p38, but not ERK, decreased IL-1beta-enhanced MMP-1, MMP-3 and NO production, respectively, and 100 nM celecoxib down-regulated the phosphorylation of NF-kappaB and JNK but has no effect on either p38 or ERK. Celecoxib has inhibitory effects on MMP-1, MMP-3 and NO productions, suggesting the protective roles directly on articular chondrocytes. Despite the COX-2 selectivity, celecoxib affects those productions via not PGE2 but NF-kappaB and JNK MAPK.

Prostaglandin E2 (PGE2) is one of pro-inflammatory mediators. PGE2 maintains the homeostasis of many organs including articular cartilage, and a previous report showed that continuous inhibition of PGE2 accelerates the progression of osteoarthritis (OA). While PGE2 inhibits matrix metalloprotease (MMP) expression in several types of cells, little is known on direct effects of PGE2 on MMP expression in articular chondrocytes. The objective of this study was to investigate direct effects of PGE2 on IL-1beta-induced MMP-1 and MMP-13 expression and the intracellular signaling in articular chondrocytes. PGE2 showed inhibitory effects on IL-1beta-induced MMP-1 and MMP-13 expression demonstrated by immunoblotting both in OA and normal chondrocytes, which was further confirmed by enzyme-linked immunosorbent assay and immunohistochemistry of explant cultures of articular cartilages. An EP4 agonist, ONO-AE1-329, mimicked the inhibitory effect of PGE2, while an EP4 antagonist, ONO-AE3-208, blocked the effects. PGE2 suppressed the phosphorylation of JNK and ERK MAP kinases, but only knockdown of JNK by specific siRNA mimicked the effect of PGE2. PGE2 further inhibited the phosphorylation of MKK4 without suppression of MKK7 phosphorylation, and of c-JUN to decrease expression levels of MMP-1 and MMP-13. These results demonstrate that PGE2 inhibits IL-1beta-induced MMP-1 and MMP-13 productions via EP4 by suppressing MKK4-JNK MAP kinase-c-JUN pathway.

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The present study clearly demonstrated that HBFN-f stimulated NO production through CD44 in RA cartilage. Increased expression of CD44 in RA cartilage may play a pathological role in joint destruction through enhanced NO production by binding to fibronectin fragments such as HBFN-f.

We investigated the intracellular mechanism for the inhibitory effects of hyaluronan (HA) on interleukin-1beta (IL-1beta)-stimulated collagenase-1 and -3 (matrix metalloproteinases (MMPs)-1 and -13) production in a human chondrosarcoma cell line, SW-1353. MMPs-1 and -13 were induced by IL-1beta at 2 ng/ml in SW-1353 cells for 48 h. HA of 800 kDa, which is used clinically, significantly suppressed IL-1beta-stimulated production of MMPs-1 and -13 by immunoblotting. SW-1353 cells express the standard form of CD44 (CD44H), and immunofluorescent cytochemistry demonstrated the association of HA with CD44 on SW-1353 cells. Phosphorylated p38 (Phos-p38) mitogen-activated protein kinase was stimulated in SW-1353 cells by IL-1beta but not by HA alone. SB203580, a p38 MAPK inhibitor, partially blocked the MMP-1 and -13 production stimulated by IL-1beta. 800-kDa HA suppressed IL-1beta-activated Phos-p38 in a dose-dependent manner. CD44 blocking significantly reversed the inhibitory effects of HA on IL-1beta-activated Phos-p38 production. The present study clearly suggests that HA binds CD44 and inhibits IL-1beta-induced MMP-1 and -13 expression via down-regulation of Phos-p38 in SW-1353 cells.

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH(2)-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1 microM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10 microM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10 microM SB203580 strongly inhibited the collagen cleavage compared with 1 microM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.