Effect of matrix depleting agents on the expression of chondrocyte metabolism by equine chondrocytes

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Abstract

This study was carried out to investigate the effect of two enzymes (collagenase and chondroitinase) and two cytokines/metabolites (interleukin-1β and retinoic acid) of known catabolic activity on the expression of cartilage metabolism/phenotype in equine articular cartilage. Articular cartilage explants from 11 horses (5–13 years old) were treated for 48 h and assayed for total sulphated glycosaminoglycan (GAG), the incorporation of 35S-sulphate, collagen degradation and mRNA expression of the proteoglycans collagen II, collagen IIA,, collagen III, collagen IX, collagen X, collagen XI and glyceraldehyde-3-phosphate (GAPDH). Purified collagenase and retinoic acid were responsible for increased GAG loss from the tissues. Chondroitinase, responsible for catalysing the elimination of glucuronate residues from chondroitin A, B and C (Chondroitinase ABC) and retinoic acid treatment induced an inhibition of proteoglycan synthesis, whereas collagenase treatment did not. Collagenase activity was correlated with increased appearance of the CB11B epitope and type II collagen denaturation. By RT-PCR there was evidence of expression of altered collagen type IIA in purified collagenase treated tissues.

Introduction

Joint injury and joint disease are the most common causes of lameness and together they represent a major part of the caseload for equine clinicians. Osteoarthritis (OA) (also called degenerative joint disease) represents a group of disorders characterized by deterioration of the articular cartilage accompanied by changes in the bone and soft tissues of the joint including subchondral bone, sclerosis and marginal osteophyte formation. Clinically the disease is characterised by pain and dysfunction of the affected joint (McIlwraith and Vachon, 1988). The typical type1 osteoarthritis affects young racehorses in highly mobile joints such as carpal or metacarpal joints. Naturally present age-related osteoarthritis in horses has also been described by Cantley et al. (1999). Much of the basic work in joint disease was originally done in laboratory animals but more recently considerable original work has been reported in the horse and in vitro work with equine tissues (McIlwraith, 1996).

The functional capability of articular cartilage rests mainly with the three primary components of its extracellular matrix, water, proteoglycan and type II collagen. Glycosaminoglycans, the highly charged polyanionic parts of proteoglycan molecules, create a hydrodynamic force that is resisted by the tension of the three-dimensional network of collagen fibrils. This results in a tissue with unique biochemical properties that provides a shear-resistant, weight-bearing surface essential for joint function. Collagen is generally considered to provide the tensile strength of articular cartilage and proteoglycans provide the main compression-resistant constituent. However, it is the collagen network that controls the instantaneous deformation of the articular cartilage under compression (Blair et al., 2002).

Collagen damage by collagenase treatment represents an exogenous pathway of matrix disruption. Shortly after the discovery of animal collagenases in tadpoles (Gross and Lapiere, 1962), collagenase enzymes were isolated from the cultured fragments of inflamed synovial tissues and also from synovial fluids in patients with inflammatory joint diseases such as rheumatoid arthritis (Evanson et al., 1967; Harris et al., 1969). The role of increased collagenase activity in the cleavage of type II collagen was first suggested by early studies confirming that active enzyme was extractable from osteoarthritic cartilage but not from normal articular cartilage (Blankaert et al., 1989). More recently, the role of collagenases in the direct breakdown of type II collagen has been strengthened considerably and immunohistological studies have confirmed the presence of neoepitopes in osteoarthritic cartilage that correspond to degraded fragments of a purified type II collagen substrate. The neoepitopes correspond to fragments produced by MMP-1 (collagenase-1), MMP-8 (collagenase-2) and MMP-13 (Collagenase-3) (Billinghurst et al., 1997).

The presence of IL-1 in equine osteoarthritic joints was reported in 1990 by Morris et al. In vivo intra-articular administration of IL-1 caused prolonged suppression of cartilage proteoglycan synthesis in rats (Chandrasekhar et al., 1992). By using quantitative PCR analysis, Flannery et al. (1999) reported an approximately two to three fold increase in human and porcine MMP-3 and MMP-13 mRNAs following IL-1 and retinoic acid treatment. Increased activation of matrix metalloproteinases 2 and 9 in equine articular cartilage in the presence of IL-2 has also been described by Clegg and Carter (1999). Yasumoto et al. (2003) also reported the induction of matrix metalloproteinase and aggrecanase enzymes following IL-1 and retinoic acid treatment. Catabolism of aggrecan by explant cultures of human articular cartilage in the presence of retinoic acid has been described by several research workers (Ilic et al., 1995; Ballock et al., 1994; Morales, 1994). Retinoic acid has also been shown to be responsible for a 90% decrease in type II collagen synthesis while a 25% and 30% decrease was observed for IX and XI, respectively (Freyria et al., 1995). Chondroitinase responsible for catalysing the elimination of glucuronate residues from chondroitin A, B and C (Chondroitinase ABC) affects the activity of intracellular enzymes in articular cartilage chondrocytes (Nahir et al., 1995) and has been shown to induce marked proteoglycan depletion without causing any structural damage (Nahir et al., 1995; Kuijer et al., 1988).

Chondrocyte phenotype and survival are regulated by specific cytokines through the expression of SOX9 transcription factor (Kolettas et al., 2001). Sex-determining region Y-type high mobility group Box family of transcription factor (SOX9) is a potent activator of the chondrocyte-specific enhancer of the pro alpha1 (II) collagen and aggrecan genes (Sekiya et al., 2000; Majumdar et al., 2001). Expression of SOX9 and type IIA procollagen during attempted repair of articular cartilage damage in a transgenic mouse model of osteoarthritis has been described by Salminen et al. (2001).

Cartilage explant cultures have been found to be a good model for the analysis of matrix degeneration (Tyler and Sawyer, 1990). The culture of cartilage explants has several advantages over the culture of isolated chondrocytes, including the fact that its extracellular matrix is similar to that found in vivo. Potential phenotypic changes are characteristic of chondrocytes. Many studies have shown that phenotypic changes occur during chondrocyte differentiation in vivo in foetal growth-plate cartilage and of chondrocyte behaviour in vitro. Several factors, such as retinoic acid, bromodeoxyuridine, and IL-1 (Sandell and Aigner, 2001) induce so called dedifferentiation or modulation of the chondrocyte phenotype to a fibroblast-like phenotype. The chondrocytes stop expressing aggrecan and collagen type II, although they are still very active cells and express collagen types I, III, and V, clearly demonstrating the implications of phenotypic alterations of chondrocytes and showing that despite potentially high synthetic activity, dedifferentiated chondrocytes do not express cartilage-specific genes such as aggrecan or type II collagen. Consequently, phenotypic alterations may represent another way that chondrocytes undergo anabolic failure in osteoarthritic cartilage. Classically, chondrocyte phenotype is characterised largely by subtyping of collagen gene expression (Cancedda et al., 1995). Chondroprogenitor cells are characterised by the expression of the alternative splice variant of type II collagen, type IIA procollagen (COL2A) (Sandell et al., 1991). Mature chondrocytes express the typical cartilage collagen type IIB (COL2B), IX and XI as well as aggrecan and link protein.

The present study was carried out to investigate the effects on articular cartilage metabolism and phenotype of the matrix depleting enzymes/cytokines purified collagenase, chondroitinase ABC, retinoic acid and IL-β.

Section snippets

Tissue culture

Articular cartilage explants (2–3 mm in thickness) were collected from the metacarpophalangeal joints of skeletally mature horses (11 animals aged 5–13 years). All joints were free from macroscopic lesions. The cartilage explants were diced, washed three times in serum-free Dulbecco's modified Eagle's Medium (DMEM: Gibco, Paisley, UK) and maintained in DMEM culture medium at 37 °C under 95% air and 5% CO2 for 24 h before the start of any experiment. The explant cultures were treated with the

GAG release

The ability of various agents to activate the process of GAG release and to induce the `recovery' of the cells was tested by culturing the tissue in the presence of a variety of agents for 48 h, followed by a further 6 days to enable the tissue to `recover' from the treatment.

The addition of purified collagenase (Liberase) to the explant cultures resulted in a significant GAG release on day 2 of culture. After the removal of this enzyme, a four-fold increase in GAG loss was observed until day 8

Discussion

There was an increase in the release of glycosaminoglycans (GAG) into the medium following purified collagenase and retinoic acid treatment. An increase in the expression of the aggrecan NITEGE epitope was also apparent in the culture media (by Western blotting) from these experiments (Bird, Personal Communication). We have previously found that the response of equine cartilage to IL-1β is age dependent, diminishing markedly with advancing age (Iqbal et al., 2000). However, no significant

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    This work was supported by the Arthritis Research Campaign, UK.

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