PGE2 inhibits MMP expression by suppressing MKK4-JNK MAP kinase-c-JUN pathway via EP4 in human articular chondrocytes.

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.

Hyaluronan inhibits IL-1beta-stimulated collagenase production via down-regulation of phosphorylated p38 in SW-1353 human chondrosarcoma cells.

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.

Induction of apoptosis by anti-CD44 antibody in human chondrosarcoma cell line SW1353.

Because chondrosarcoma is resistant to chemotherapy and ionizing radiation, the primary treatment of chondrosarcoma is surgical resection. Effective chemotherapeutic agents for chondrosarcoma are necessary. Although there is evidence that CD44 is involved in apoptosis susceptibility in several cell types, the effectiveness of anti-CD44 treatment on chondrosarcoma has never been studied. This study was aimed to clarify whether anti-CD44 monoclonal antibody induces apoptosis in human chondrosarcoma cell line SW1353. Confocal microscopy revealed that the SW1353 cells expressed CD44 that bound the anti-CD44 antibody IM7. Treatment of the cells with IM7 resulted in a significant decrease in cell viability, compared with that with control IgG. In contrast, IM7 failed to reduce cell viability in human chondrocytes. In SW1353 cells, IM7 induced chromatin condensation, nuclear fragmentation, and apoptotic body formation while control IgG had marginal effect. These data indicate that anti-CD44 treatment could induce apoptosis in chondrosarcoma cells.

Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage.

OBJECTIVE: To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. METHODS: Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. RESULTS: The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels. CONCLUSION: Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.

Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage.

OBJECTIVE: To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin-1beta (IL-1beta)-stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage. METHODS: IL-1beta 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-1beta-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-1beta-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-1beta-stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL-1beta in normal and OA cartilage. CONCLUSION: This study demonstrates that HA effectively inhibits IL-1beta-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-1beta may involve direct interaction between HA and CD44 on chondrocytes.

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture.

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.

Hyaluronan inhibits matrix metalloproteinase-1 production by rheumatoid synovial fibroblasts stimulated by proinflammatory cytokines.

OBJECTIVE: To study the inhibitory effects of hyaluronan (HA) on the production of matrix metalloproteinase-1 (MMP-1) by rheumatoid synovial fibroblasts (RSF) stimulated by proinflammatory cytokines, tumor necrosis factor-a (TNF-a), and interleukin-1beta (IL-1beta). METHODS: HA of various sizes at various concentrations was added to monolayer cultures of RSF in the presence of TNF-a or IL-1beta, with or without pretreatment with a monoclonal antibody against CD44, OS/37. Concentrations of MMP-1 in cell lysates and conditioned media and of CD44 on RSF were assayed by immunoblotting. MMP-1 expression was analyzed by reverse transcriptase-polymerase chain reaction. Binding of HA to RSF was evaluated by confocal microscopy using fluorescein-conjugated HA and OS/37. RESULTS: Treatment with HA (0.3 approximately 3.0 mg/ml) resulted in a significant decrease in the production of MMP-1 induced by TNF-a and IL-1beta, in a dose-dependent manner. HA of 250 approximately 2300 kDa at 3 mg/ml was found to suppress the induction of MMP-1 by TNF-a. HA decreased the cytokine-induced MMP-1 synthesis in RSF at mRNA and protein levels. The monoclonal antibody, which showed abundant expression of CD44 on RSF by immunofluorescein cytochemistry, partially blocked the binding of fluorescein-conjugated HA to RSF. Pretreatment with OS/37 reversed the inhibition of MMP-1 production in TNF-a or IL-1beta-stimulated RSF caused by HA. CONCLUSION: HA suppresses the production of MMP-1 by TNF-a or IL-1beta-stimulated RSF. Based on data from anti-CD44 treatment, HA binding to CD44 is directly involved in the suppression of MMP-1 production. Those results provide the rationale for a therapeutic role of HA in treatment of rheumatoid joints.