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

1999 ◽  
Vol 344 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Chris B. LITTLE ◽  
Carl R. FLANNERY ◽  
Clare E. HUGHES ◽  
John S. MORT ◽  
Peter J. ROUGHLEY ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Late Stage ◽  
Core Protein ◽  
Culture Media ◽  
Degradation Products ◽  
Colorimetric Assay ◽  
Late Time ◽  
Human Articular Cartilage ◽  
Primary Cleavage

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.

scholarly journals Link protein as a monitor in situ of endogenous proteolysis in adult human articular cartilage

1991 ◽  
Vol 278 (1) ◽  
pp. 143-147 ◽  
Author(s):  
Q Nguyen ◽  
J Liu ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Articular Cartilage ◽  
Degradation Products ◽  
Proteolytic Processing ◽  
Cathepsin G ◽  
Human Articular Cartilage ◽  
Link Protein ◽  
Adult Human ◽  
Protein Components

The link protein components of proteoglycan aggregates in adult human articular cartilage show heterogeneity due to proteolysis. Cleavages near the N-terminus of the intact link proteins, before residues 17, 19 and 24, generate three proteins of slightly diminished size (LP3). Cleavages within the N-terminal disulphide-bonded loop, before residues 66 and 73 of the intact link proteins, generate proteins that yield smaller degradation products upon reduction (LP fragments). In vitro, modified link protein components of a similar size to LP3 can be generated by a variety of proteinases, but of the physiologically relevant enzymes only stromelysin, cathepsin B and cathepsin G have the ability to yield modified link proteins with N-termini identical with those observed in situ. None of the proteolytic agents tested was able to produce LP fragments with N-termini identical with those observed in situ, and the majority of proteinases were not able to cleave within the disulphide-bonded loops. Cathepsin L and hydroxyl radicals can cleave within the N-terminal disulphide-bonded loop, and have the potential of initially opening the loop to allow further proteolytic processing by other agents to generate the native cleavage sites.

scholarly journals Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Deae Cellulose ◽  
Culture Conditions ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Tissue Slices ◽  
Keratan Sulphate ◽  
Defined Culture

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

scholarly journals Recombinant human gelsolin promotes the migration of human articular cartilage chondrocytes by regulating gene expression in vitro

2020 ◽  
Vol 2 (4) ◽  
pp. 100124
Author(s):  
Jessica Feldt ◽  
Jessica Welss ◽  
Verena Schropp ◽  
Kolja Gelse ◽  
Michael Tsokos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Articular Cartilage ◽  
Human Articular Cartilage

scholarly journals Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

1989 ◽  
Vol 262 (3) ◽  
pp. 823-827 ◽  
Author(s):  
P J Roughley ◽  
R J White
Keyword(s):  
Articular Cartilage ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

scholarly journals Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

1983 ◽  
Vol 209 (2) ◽  
pp. 337-344 ◽  
Author(s):  
J Saklatvala ◽  
S J Sarsfield ◽  
L M C Pilsworth
Keyword(s):  
Articular Cartilage ◽  
Synovial Tissue ◽  
Gel Filtration ◽  
Human Articular Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Mononuclear Leucocytes ◽  
Culture Supernatants

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.

scholarly journals Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro

1985 ◽  
Vol 225 (2) ◽  
pp. 493-507 ◽  
Author(s):  
J A Tyler
Keyword(s):  
Articular Cartilage ◽  
Core Protein ◽  
Fold Increase ◽  
Acid Proteinase ◽  
Binding Region ◽  
Guanidinium Chloride ◽  
The Core ◽  
The Matrix ◽  
Average Size

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The ‘clipped’ monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

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