Differential Expression of Aggrecanase and Matrix Metalloproteinase Activity in Chondrocytes Isolated from Bovine and Porcine Articular Cartilage

Hughes, Claire E.; Little, Christopher B.; Büttner, Frank; Bartnik, Eckart; Caterson, Bruce

doi:10.1074/jbc.273.46.30576

Cited by 64 publications

(61 citation statements)

References 30 publications

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“…A soluble form of IGD-aggrecanase activity was previously reported in a matrix-free agarose culture system (18), and the current study suggests that this activity is attributable to ADAMTS-4. Immunodepletion experiments also suggested that ADAMTS-4 accounts for 75% of the aggrecanase activity in media from IL-1-treated bovine cartilage explants, while ADAMTS-5 accounted for only 15% (22).…”

Section: Discussionmentioning

confidence: 68%

“…Chondrocytes were isolated from the metatarsophalangeal joints of 3-6-month-old pigs, using established procedures (18). Cells were filtered through a 40-m nitex filter and washed, and then cell numbers were established.…”

Section: Sds-page Andmentioning

confidence: 99%

“…Aggrecan fragments in media samples were deglycosylated prior to separation by 4-12% gradient SDS-PAGE and transfer to nitrocellulose membranes (21). Immunoblotting of membranes and incubations with primary and secondary antibodies were performed as previously described (18), using the ProtoBlot Western Blot AP System (Promega) and mAb BC-3 (1:100 dilution) to detect aggrecanase-generated IGD-aggrecan catabolites.…”

Section: Sds-page Andmentioning

confidence: 99%

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Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

Powell

Little

Hughes

2007

Arthritis & Rheumatism

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Objective. The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system.Methods. Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanasegenerated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGDaggrecan catabolism.Results. Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation.Conclusion. In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGDaggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.Degradation of cartilage is one of the major pathologic features of arthropathies such as osteoarthritis and rheumatoid arthritis and involves proteolysis of the major structural elements of cartilage, aggrecan, and type II collagen. Aggrecanolysis has been attributed to ADAMTS-4 and ADAMTS-5 (1). Aggrecan comprises 3 globular domains (G1, G2, and G3) intersected by 2 rod-like segments, the interglobular domain (IGD), and the 2 glycosaminoglycan (GAG) attachment regions, CS1 and CS2, respectively, whose charge density provides the tissue with its water-imbibing properties (2). Aggrecan degradation occurs as an early event in the pathogenesis of osteoarthritis. Cleavage occurs within the IGD at Glu 373 -Ala 374 , resulting in release of the GAG-rich regions to the synovial fluid (3). Both ADAMTS-4 and ADAMTS-5 (and, to a lesser extent, ADAMTS-1, ADAMTS-8, and ADAMTS-9) have demonstrated proteolytic cleavage at this IGD-aggrecan site (4-7).ADAMTS-4 was first isolated from interleukin-1 (IL-1)-stimulated bovine explant culture medium as a

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Section: Discussionmentioning

confidence: 68%

Section: Sds-page Andmentioning

confidence: 99%

Section: Sds-page Andmentioning

confidence: 99%

See 1 more Smart Citation

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

Powell

Little

Hughes

2007

Arthritis & Rheumatism

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“…It was also considered that secondary cleavage of MMP-generated aggrecan metabolites by aggrecanase could mask the detection of any such catabolites in culture media. To test whether such a cleavage could occur, MMP-1-generated metabolites initiating with FFGVG … were incubated with soluble aggrecanase generated in conditioned medium from IL-1-stimulated porcine chondrocytes as previously reported [27]. Western blot analyses of this conditioned medium with MAbs BC-14 and BC-3 showed the lack of endogenous aggrecan catabolites in this preparation ( Figure 2B, lanes 1 and 5).…”

Section: Proteoglycan Catabolism In 4-day Cultures Of Bovine and Porcmentioning

confidence: 71%

“…Immunoblotting of membranes and incubations with primary and secondary antibodies was performed as previously described [27] using monoclonal antibodies (MAbs) or polyclonal antiserum as follows : MAb BC-13 (1 : 250) and MAb BC-3 (1 : 2000 ; [11]) recognizing the aggrecanase generated C-and N-terminal IGD neoepitopes ...ITEGE and ARGSV … respectively ; polyclonal anti-DIPEN (1 : 250 ; [13,19]) and MAb BC-14 (1 : 1000 ; [11,28]) recognizing the MMP-generated C-and N-terminal IGD neoepitopes … DIPEN and FFGVG … respectively ; and MAb 2-B-6 (1 : 10 000) recognizing a chondroitinase-generated chondroitin-4-sulphate disaccharide epitope. All Western blots were scanned and differences in staining intensity of digitized bands were assessed using NIH Image (version 1.55) software.…”

Section: Sds/page and Western Blottingmentioning

confidence: 99%

Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro

Little

Flannery

Hughes

et al. 1999

Biochemical Journal

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115

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The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-α. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD). In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of > 80% of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures. Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected. However, increased expression of these MMPs was not correlated with aggrecan degradation. Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic stimulation when compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.

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MMP-13 is induced during chondrocyte hypertrophy

et al. 2000

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During development, mRNA for matrix metalloproteinase-13 (MMP-13) is found associated with cartilage undergoing hypertrophy, suggesting that this collagenase plays a role in cell enlargement and/or cartilage calcification. Using chondrocytes from prehypertrophic cartilage of chick embryo sternae, we have examined the relationship between MMP-13 expression and the transition to hypertrophy. When hypertrophy was induced by serum-free culture with ascorbate and bone morphogenetic protein-2 (BMP-2), MMP-13 mRNA levels paralleled those for type X collagen. Chondrocytes from the caudal, nonhypertrophying portion of chick sternae expressed neither type X collagen nor MMP-13, confirming that MMP-13 mRNA is a marker for hypertrophy. Zymography with conditioned medium yielded a proteinase band at 59 kDa, which was absent in nonhypertrophic chondrocytes. A polyclonal antibody raised against chick MMP-13 reacted with the 59-kDa protein, confirming that it is MMP-13. Although mRNA for MMP-13 peaked at days 4-5 of culture, only low levels of MMP-13 activity were present, and the activity increased gradually in parallel with later increases in MMP-2. These results suggest that MMP-13 is activated by MMP-2 during chondrocyte maturation, and that the combination of both proteinases is required to prepare cartilage matrix for subsequent calcification, before endochondral ossification.

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Differential Expression of Aggrecanase and Matrix Metalloproteinase Activity in Chondrocytes Isolated from Bovine and Porcine Articular Cartilage

Cited by 64 publications

References 30 publications

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro

MMP-13 is induced during chondrocyte hypertrophy

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