<i>N</i>-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage

Loulakis, Pat; Shrikhande, Alka; Davis, Gary L.; Maniglia, Charles A.

doi:10.1042/bj2840589

Cited by 132 publications

(109 citation statements)

References 39 publications

(36 reference statements)

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“…Thus, in all cultures in which an increase in the release of aggrecan was detected using the DMMB assay (Table 1), there was an increase in BC-3-reactive metabolites. These results confirm previous findings demonstrating that catabolic stimulation of cartilage explants results in an increase in aggrecanase-generated aggrecan metabolites in the culture media [12,[20][21][22][23][24][25]. No BC-3-positive bands were observed above 250 kDa, indicating that the highmolecular-mass bands observed with MAb 2-B-6 had not been cleaved within the IGD.…”

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

confidence: 91%

“…In attempts to elucidate the actual sequence of catabolic events, researchers have studied in itro models of normal and accelerated aggrecan catabolism using either cartilage explants or chondrocyte monolayer cultures exposed to catabolic agents such as retinoic acid (RA), interleukin-1 (IL-1) or tumour necrosis factor-α (TNF) [12,[20][21][22][23][24][25]. However, no studies have simultaneously evaluated the generation of the N-and C-terminal neoepitopes which result from the action of both aggrecanase and MMPs in itro.…”

Section: Introductionmentioning

confidence: 99%

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

Little

Flannery

Hughes

et al. 1999

Biochemical Journal

113

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

confidence: 91%

Section: Introductionmentioning

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

113

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“…under conditions of normal and IL-1 stimulated turnover cartilage explants release aggrecan fragments with N-terminal sequences corresponding to cleavage between E373 and A374 [20][21][22]. The putative enzyme responsible for this cleavage has been named aggrecanase but its identity remains unknown.…”

Section: *Corresponding Author Fax: (61) (3) 9345 6668mentioning

confidence: 99%

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

Fosang

Knäuper²,

Murphy³

et al. 1996

FEBS Letters

344

217

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“…However, the two that have been most extensively characterized are the metalloproteinase and aggrecanase cleavage sites located within the interglobular domain, involving the Asn$%"-Phe$%# and Glu$($-Ala$(% peptide bonds, respectively (Figure 1) [6][7][8]. These cleavage events are likely to be of significant functional consequence because they uncouple the G1 domain from the glycosaminoglycan-rich domains, which are then no longer anchored in the cartilage and hence might be lost.…”

Section: Introductionmentioning

confidence: 99%

Aggrecan degradation in human intervertebral disc and articular cartilage

Sztrolovics

Alini

Roughley

et al. 1997

Biochemical Journal

241

172

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Aggrecan degradation in human intervertebral disc and articular cartilage has been studied by using anti-neoepitope antibodies specific for the N-terminal degradation products generated by cleavage within the interglobular domain at the metalloproteinase and aggrecanase sites. Immunoblot analysis of extracts of annulus fibrosus, nucleus pulposus and articular cartilage demonstrated age-related patterns in the abundance of both degradation products. In all three tissues the metalloproteinase-generated fragment was present at very low levels in young individuals but increased in abundance with age. In the disc tissues, the abundance of this degradation product levelled off in the juvenile; for cartilage this occurred in early adulthood. Despite these temporal differences, the levels attained in adults were comparable for the three tissues. In contrast, the aggrecanase-generated degradation product exhibited tissue-specific differences in the variation of its abundance with age. Whereas this degradation product increased with age in annulus fibrosus and articular cartilage and had levelled off by adulthood, in nucleus pulposus it was present in greatest abundance in young individuals and decreased to very low levels with age. Examination of discs exhibiting various degrees of degeneration did not reveal any differences in the levels of the metalloproteinase and aggrecanase-generated cleavage products that could not be accounted for by differences in age. In adults the product of aggrecanase action was much more abundant in articular cartilage than in either of the disc tissues, despite the age-related increase also observed for annulus fibrosus. Analysis of tissue extracts with an antibody recognizing the G1 domain of aggrecan identified two major degradation products whose abundance and size were correlated with the fragments detected by the anti-neoepitope antibodies. Taken together, these results indicate that cleavage at the metalloproteinase and aggrecanase sites are quantitatively important events in aggrecan catabolism in both articular cartilage and intervertebral disc in vivo. Moreover the two enzyme systems act independently and exhibit differences in the degree to which they contribute to aggrecan degradation in these tissues.

show abstract

N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage

Cited by 132 publications

References 39 publications

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

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

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

Aggrecan degradation in human intervertebral disc and articular cartilage

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