scholarly journals Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine

1998 ◽  
Vol 335 (1) ◽  
pp. 59-66 ◽  
Author(s):  
John D. SANDY ◽  
Dan GAMETT ◽  
Vivian THOMPSON ◽  
Christie VERSCHAREN
Keyword(s):  
Retinoic Acid ◽  
Core Protein ◽  
Interleukin 1 ◽  
Cartilage Explants ◽  
Biosynthetic Activity ◽  
Fragment Analysis ◽  
Analysis System ◽  
Chondrosarcoma Cell Line ◽  
Bovine Cartilage ◽  
Aggrecan Core Protein

A rat chondrosarcoma cell line and bovine cartilage explants have been used to study the control of aggrecan degradation by chondrocytes treated with interleukin-1 (IL-1) or retinoic acid (RA). Aggrecan fragment analysis with anti-neo-epitope antibodies suggests that aggrecanase (an as yet unidentified enzyme) is the only aggrecan-degrading proteinase active in these cultures. With rat cells, aggrecanase converts the aggrecan core protein into two major G1-domain-bearing products (60 kDa with a C-terminal Glu-373, and 220 kDa with a C-terminal Glu-1459). Both products were quantified on a standardized Western analysis system with a G1-specific antibody. Immunoblots were analysed by scanning densitometry and the sensitivity, linearity and reproducibility of the assay were established. With rat cells the aggrecanase response to IL-1 was optimal at about 2 mM glutamine, but was progressively inhibited at higher concentrations, with about 90% inhibition at 10 mM glutamine. Such inhibition by glutamine was not, however, observed with bovine explants. On the other hand, marked inhibition of aggrecanase-dependent cleavage was observed with both rat cells and bovine explants when d(+)-glucosamine was included at concentrations above 2 mM. Inhibition was apparently not due to cytotoxicity or interference with IL-1 signalling, since biosynthetic activity was not inhibited and inhibition of the aggrecanase response was also obtained when RA was used as the catabolic stimulator. Possible mechanisms for the inhibition of the aggrecanase response by glucosamine in chondrocytes treated with IL-1 or RA are discussed.

scholarly journals Monoclonal antibodies that specifically recognize neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro

1995 ◽  
Vol 305 (3) ◽  
pp. 799-804 ◽  
Author(s):  
C E Hughes ◽  
B Caterson ◽  
A J Fosang ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Monoclonal Antibodies ◽  
Retinoic Acid ◽  
Degradation Products ◽  
Interleukin 1 ◽  
Proteolytic Cleavage ◽  
Cartilage Explants ◽  
Reactive Material ◽  
Culture Systems ◽  
Bovine Cartilage

Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, ‘aggrecanase’, and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of ‘aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that ‘aggrecanase’ activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than ‘aggrecanase’ are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited ‘aggrecanase’ activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.

scholarly journals ADAMTS-9 in Mouse Cartilage Has Aggrecanase Activity That Is Distinct from ADAMTS-4 and ADAMTS-5

2019 ◽  
Vol 20 (3) ◽  
pp. 573 ◽  
Author(s):  
Fraser Rogerson ◽  
Karena Last ◽  
Suzanne Golub ◽  
Stephanie Gauci ◽  
Heather Stanton ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Catalytic Domain ◽  
Core Protein ◽  
Skeletal Development ◽  
Normal Growth ◽  
Cartilage Explants ◽  
The Novel ◽  
Proliferative Zone ◽  
Femoral Head Cartilage ◽  
Skeletal Phenotype

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 are the principal aggrecanases in mice and humans; however, mice lacking the catalytic domain of both enzymes (TS-4/5∆cat) have no skeletal phenotype, suggesting there is an alternative aggrecanase for modulating normal growth and development in these mice. We previously identified aggrecanase activity that (a) cleaved at E↓G rather than E↓A bonds in the aggrecan core protein, and (b) was upregulated by retinoic acid but not IL-1α. The present study aimed to identify the alternative aggrecanase. Femoral head cartilage explants from TS-4/5∆cat mice were stimulated with IL-1α or retinoic acid and total RNA was analysed by microarray. In addition to ADAMTS-5 and matrix metalloproteinase (MMP)-13, which are not candidates for the novel aggrecanase, the microarray analyses identified MMP-11, calpain-5 and ADAMTS-9 as candidate aggrecanases upregulated by retinoic acid. When calpain-5 and MMP-11 failed to meet subsequent criteria, ADAMTS-9 emerged as the most likely candidate for the novel aggrecanase. Immunohistochemistry revealed ADAMTS-9 expression throughout the mouse growth plate and strong expression, particularly in the proliferative zone of the TS-4/5-∆cat mice. In conclusion, ADAMTS-9 has a novel specificity for aggrecan, cleaving primarily at E↓G rather than E↓A bonds in mouse cartilage. ADAMTS-9 might have more important roles in normal skeletal development compared with ADAMTS-4 and ADAMTS-5, which have key roles in joint pathology.

scholarly journals Thyroid Hormone Enhances Aggrecanase-2/ADAM-TS5 Expression and Proteoglycan Degradation in Growth Plate Cartilage

Endocrinology ◽  
2003 ◽  
Vol 144 (6) ◽  
pp. 2480-2488 ◽  
Author(s):  
Seicho Makihira ◽  
Weiqun Yan ◽  
Hiroshi Murakami ◽  
Masae Furukawa ◽  
Toshihisa Kawai ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Growth Plate ◽  
Cleavage Site ◽  
Endochondral Ossification ◽  
Core Protein ◽  
Binding Capacity ◽  
Mrna Level ◽  
Cartilage Explants ◽  
Type I ◽  
Proteoglycan Degradation

Abstract Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T4 reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T3 also induced breakdown of proteoglycan. T3 increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T3 treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T3-exposed explants underwent cleavage at Glu373-Ala374, the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T3 was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T3-induced aggrecan degradation and found that T3 enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.

scholarly journals Genome of Deerpox Virus

2005 ◽  
Vol 79 (2) ◽  
pp. 966-977 ◽  
Author(s):  
C. L. Afonso ◽  
G. Delhon ◽  
E. R. Tulman ◽  
Z. Lu ◽  
A. Zsak ◽  
...  
Keyword(s):  
Host Range ◽  
Chemokine Receptors ◽  
Transforming Growth Factor ◽  
Genomic Sequence ◽  
Core Protein ◽  
Interleukin 1 ◽  
Transforming Growth Factor Β1 ◽  
Open Reading Frames ◽  
Host Responses ◽  
Tgf Β1

ABSTRACT Deerpox virus (DPV), an uncharacterized and unclassified member of the Poxviridae, has been isolated from North American free-ranging mule deer (Odocoileus hemionus) exhibiting mucocutaneous disease. Here we report the genomic sequence and comparative analysis of two pathogenic DPV isolates, W-848-83 (W83) and W-1170-84 (W84). The W83 and W84 genomes are 166 and 170 kbp, containing 169 and 170 putative genes, respectively. Nucleotide identity between DPVs is 95% over the central 157 kbp. W83 and W84 share similar gene orders and code for similar replicative, structural, virulence, and host range functions. DPV open reading frames (ORFs) with putative virulence and host range functions include those similar to cytokine receptors (R), including gamma interferon receptor (IFN-γR), interleukin 1 receptor (IL-1R), and type 8 CC-chemokine receptors; cytokine binding proteins (BP), including IL-18BP, IFN-α/βBP, and tumor necrosis factor binding protein (TNFBP); serpins; and homologues of vaccinia virus (VACV) E3L, K3L, and A52R proteins. DPVs also encode distinct forms of major histocompatibility complex class I, C-type lectin-like protein, and transforming growth factor β1 (TGF-β1), a protein not previously described in a mammalian chordopoxvirus. Notably, DPV encodes homologues of cellular endothelin 2 and IL-1R antagonist, novel poxviral genes also likely involved in the manipulation of host responses. W83 and W84 differ from each other by the presence or absence of five ORFs. Specifically, homologues of a CD30 TNFR family protein, swinepox virus SPV019, and VACV E11L core protein are absent in W83, and homologues of TGF-β1 and lumpy skin disease virus LSDV023 are absent in W84. Phylogenetic analysis indicates that DPVs are genetically distinct from viruses of other characterized poxviral genera and that they likely comprise a new genus within the subfamily Chordopoxvirinae.

scholarly journals Modulation of aggrecan and link-protein synthesis in articular cartilage

1992 ◽  
Vol 288 (3) ◽  
pp. 721-726 ◽  
Author(s):  
A J Curtis ◽  
R J Devenish ◽  
C J Handley
Keyword(s):  
Articular Cartilage ◽  
Half Life ◽  
Core Protein ◽  
Calf Serum ◽  
Cartilage Explants ◽  
Dependent Manner ◽  
Link Protein ◽  
The Core ◽  
Explant Cultures ◽  
Igf I

The addition of serum or insulin-like growth factor-I (IGF-I) to the medium of explant cultures of bovine articular cartilage is known to stimulate the synthesis of aggrecan in a dose-dependent manner. The half-life of the pool of proteoglycan core protein was measured in adult articular cartilage cultured for 6 days in the presence and absence of 20 ng of IGF-I/ml and shown to be 24 min under both sets of conditions. The half-life of the mRNA pool coding for aggrecan was also determined and shown to be approx. 4 h in cartilage maintained in culture with or without IGF-I. The pool size of mRNA coding for aggrecan core protein increased 5-6-fold in cartilage explants maintained in culture in medium containing 20% (v/v) fetal-calf serum; however, in tissue maintained with medium containing IGF-I there was no increase in the cellular levels of this mRNA. This suggests that aggrecan synthesis is stimulated by IGF-I at the level of translation of mRNA coding for the core protein of this proteoglycan and that other growth factors are present in serum that stimulate aggrecan synthesis at the level of transcription of the core-protein gene. Inclusion of serum or IGF-I in the medium of cartilage explant cultures induced increases in the amounts of mRNA coding for type II collagen and link protein, whereas only serum enhanced the amount of mRNA for the core protein of decorin.

scholarly journals A potential key role for alpha-haemolysin ofStaphylococcus aureusin mediating chondrocyte death in septic arthritis

2018 ◽  
Vol 7 (7) ◽  
pp. 457-467 ◽  
Author(s):  
I. D. M. Smith ◽  
K. M. Milto ◽  
C. J. Doherty ◽  
S. G. B. Amyes ◽  
A. H. R. W. Simpson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Septic Arthritis ◽  
Cartilage Damage ◽  
Laser Scanning Microscopy ◽  
Cartilage Explants ◽  
Confocal Laser ◽  
Dead Cell ◽  
Chondrocyte Viability ◽  
Chondrocyte Death ◽  
Bovine Cartilage

ObjectivesStaphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability.MethodsBovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy.ResultsHla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect.ConclusionThis study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis. Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457–467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

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