Elsevier

Matrix Biology

Volume 21, Issue 3, April 2002, Pages 251-262
Matrix Biology

Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes

https://doi.org/10.1016/S0945-053X(02)00007-0Get rights and content

Abstract

Interleukin-1 (IL-1), the main cytokine instigator of cartilage degeneration in arthritis, induces matrix metalloproteinase-3 (MMP-3) and MMP-13 RNA and protein in chondrocytes. The molecular mechanisms of this induction were investigated with specific inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and activating protein (AP-1) and nuclear factor kappa B (NF-κB) transcription factors. IL-1 rapidly induced the activation of extracellular-signal regulated kinase (ERK), protein 38 (p38) and c-Jun N-terminal kinase (JNK) MAPKs in the first-passage human femoral head OA chondrocytes. The ERK-MAPK pathway inhibitor, PD98059, attained 46–53% (MMP-3) and 59–66% (MMP-13) inhibition of RNA induction in human OA and 47–52% (MMP-3) and 69–73% (MMP-13) inhibition in bovine chondrocytes. U0126 conferred 37–77% (MMP-3) and 43–73% (MMP-13) suppression in human and 77–100% (MMP-3) and 96–100% (MMP-13) in bovine chondrocytes. P38 and JNK inhibitor, SB203580 caused 35–37% reduction of MMP-3 and MMP-13 RNA in human and 36–46% (MMP-3) and 60–88% (MMP-13) in bovine chondrocytes. Inhibitor of JNK, AP-1 and NF-κB, curcumin, achieved 48–99% suppression of MMP-3 and 45–97% of MMP-13 in human and 8–100% (MMP-3) and 32–100% (MMP-13) in bovine chondrocytes. NF-κB inhibitor, pyrrolidine dithiocarbamate yielded 83–84% reduction of MMP-3 and 38–55% for MMP-13 in human chondrocytes. In bovine chondrocytes, the induction decreased by 54–64% for MMP-3 and 74–93% for MMP-13 RNA. These results suggest the involvement of MAPKs, AP-1 and NF-κB transcription factors in the IL-1 induction of MMPs in chondrocytes. Inhibition of IL-1 signal transduction by these agents could be useful for reducing cartilage resorption by MMPs in arthritis.

Introduction

Interleukin-1 (IL-1) is a major proinflammatory cytokine implicated in arthritic joint damage. Its levels are increased in the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) (reviewed in Arend and Dayer, 1995, Westacott and Sharif, 1996). It is expressed at high levels in OA cartilage (Tetlow et al., 2001). Interestingly, IL-1beta converting enzyme expression is also increased in OA cartilage relative to normal tissue (Saha et al., 1999). OA chondrocytes display increased expression of IL-1 receptor and are more sensitive to this cytokine (Martel-Pelletier et al., 1992, Ismaiel et al., 1992). IL-1 significantly alters the physiology of joints and cartilage metabolism by inducing a variety of genes in chondrocytes (Margerie et al., 1997). It decreased the cartilage-specific, type II collagen but increased matrix metalloproteinases (MMP) mRNA expression in immortalized and primary human chondrocytes (Goldring et al., 1994, Mort et al., 1993). Anti-IL-1 therapies in the collagen-induced mouse models of arthritis prevent cartilage and bone loss, suggesting its key role in the pathogenesis. Tumor necrosis factor alpha (TNF-α) is mostly involved in joint inflammation (Joosten et al., 1999). Inhibition of IL-1 and its actions by IL-1 receptor antagonist via gene therapy and antibodies against IL-1 are beneficial in reducing the symptoms of arthritis including invasion of cartilage by synovium (Oligino et al., 1999, Neidhart et al., 2000, Mehraban and Kasturi, 1998). Some of these effects of IL-1 are mediated by MMPs.

MMPs are a family of proteases comprising of collagenases (e.g. MMP-1 and MMP-13), stromelysins (e.g. MMP-3), gelatinases and membrane-type MMPs (reviewed in Nagase and Woessner, 1999). They are secreted as an inactive form by cytokine-stimulated cells. Activated MMPs can cleave most of the components of cartilage extracellular matrix (ECM) including type II collagen and aggrecan. Stromelysin-1 (MMP-3) cleaves proteoglycans, collagens, gelatins and link protein of aggrecan. Collagenase-3 or MMP-13 has particular affinity for type II collagen but can also cleave aggrecan at specific sites (Fosang et al., 1996). Increased MMP-3 in the serum and synovium of RA patients is an indicator of inflammation and associated joint damage (Yoshihara et al., 2000, Cheung et al., 2000, Yamanaka et al., 2000). The serum levels of MMP-3 are related to disease activity in RA (Ribbens et al., 2000). MMP-3 is in 20-fold excess over MMP-1 in the synovial fluids of patients with RA (Walakovits et al., 1992) and was localized in the superficial zone of cartilage and in the synovium of patients with OA (Okada et al., 1992). The synovia of OA patients have elevated MMP-3 RNA (Zafarullah et al., 1993). IL-1 preferentially induces MMP-3 expression in rabbit and human cartilage (Hutchinson et al., 1992, Nguyen et al., 1992). MMP-3 RNA is elevated in the cartilage of damaged human knee and ankle (Chubinskaya et al., 1999).

MMP-13 from chondrocytes selectively enhances cleavage and denaturation of type II collagen in OA cartilage (Mitchell et al., 1996, Billinghurst et al., 1997) and aggrecan at the specific sites (Fosang et al., 1996). Its expression is also increased in rheumatoid synovium in 58% of the patients and can be correlated with systemic markers of inflammation (Lindy et al., 1997, Westhoff et al., 1999). Chondrocyte-derived IL-1 and MMP-13 could promote invasion of cartilage by RA synovial fibroblasts (Pap et al., 2000). MMP-13 levels are high in remodeling rabbit synovial connective tissue (Hellio Le Graverand et al., 2000). Human OA cartilage has elevated expression of MMP-13 RNA and protein that can be induced by IL-1 and TNF-α through c-fos in chondrocytes (Reboul et al., 1996, Borden et al., 1996, Shlopov et al., 1997). MMP-13 is a major target for developing cartilage-protective drugs. Thus blocking IL-1-induced MMP-3 and MMP-13 enzymes or their gene expression by novel physiologic and pharmacological inhibitors (van Den Berg, 2000) is an important therapeutic approach for arthritis. The mechanisms through which IL-1 increases the expression of MMPs have not been fully elucidated. A better understanding of these signaling and regulatory mechanisms in chondrocytes may lead to novel strategies for suppressing the catabolic actions of IL-1. Here we show that blocking ERK, p38 and JNK-MAP kinase signal transduction pathways as well as AP-1 and NF-κB transcription factors in the first-passage human or bovine femoral head chondrocytes leads to down-regulation of the IL-1-induced MMP-3 and MMP-13 gene expression.

Section snippets

Primary cultures of human and bovine chondrocytes and treatments

Human cartilage was from the femoral heads of the OA patients who underwent hip-replacement surgery at the Notre Dame Hospital. Normal bovine articular cartilage was obtained from the knee and femoral head of adult animals from a local abattoir. Chondrocytes were released by pronase (1 mg/ml) digestion for 60 min and collagenase (Sigma type IA) digestion for 9 h in Dulbecco's modified Eagle's medium (DMEM) at 37 °C. The cells were washed five times with phosphate buffered saline (PBS) and grown

IL-1 time-dependently stimulates the activation of mitogen-activated protein kinases

Previous work has shown that both IL-1 and MMPs are increased in arthritic tissues and synovial fluid (see Section 1). In preliminary experiments we ascertained that the primary or first-passage human femoral head chondrocytes from different OA patients responded to 24 h stimulation with IL-1 (10 ng/ml) by potently inducing MMP-3 and MMP-13 RNA and protein expression.

To investigate the mechanisms of IL-1 signal transduction leading to increased MMP gene expression, primary human femoral head

Discussion

IL-1 is the most important proinflammatory cytokine involved in triggering the catabolism of cartilage in arthritis by MMPs. We have shown here that IL-1 induces MMP-3, MMP-13 and MAPKs in chondrocytes. Inhibition of MMP gene expression by PD98059, U0126, SB203580, curcumin and PDTC suggests that ERK-, p38- and JNK-MAPK pathways as well as AP-1 and NF-κB transcription factors are mediators of MMP induction by IL-1.

Induction of MMP-3 and MMP-13 gene expression in human femoral head chondrocytes

Acknowledgments

This work was supported by The Arthritis Society and in part by the Canadian Arthritis Network (CAN) and Canadian Institutes of Health Research. We thank Drs Faramaze Dehnade, Julio Fernandes and Nicolas Duval for human cartilage and Ms Anna Chelchowska for preparing the figures.

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