Dataset Information

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.

ABSTRACT: BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich (342)FFGVG-G2 ELISA, (2) competition NITEGE(373)ELISA (3) sandwich G1-NITEGE(373 )ELISA (4) competition (374)ARGSV ELISA, and (5) sandwich (374)ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE(373 )neo-epitopes are delayed compared to the corresponding (374)ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only. CONCLUSION: Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.

SUBMITTER: Wang B

PROVIDER: S-EPMC2803187 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.

Wang Bijue B Chen Pingping P Jensen Anne-Christine Bay AC Karsdal Morten A MA Madsen Suzi H SH Sondergaard Bodil-Cecilie BC Zheng Qinlong Q Qvist Per P

BMC research notes 20091218

<h4>Background</h4>Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fr ...[more]

PMID: 20021645

Similar Datasets

Project description:The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.

Project description:Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

Project description:Although ADAMTS-4 and ADAMTS-5 are the principal aggrecanases in mice and humans, mice lacking both these enzymes (ADAMTS-4/-5Dcat) are healthy, viable, with no overt skeletal phenotype, suggesting the presence of an alternative aggrecanase in these mice. We previously identified a novel aggrecanase activity that was regulated by retinoic acid, but not IL-1a. The upregulated activity cleaved aggrecan at E↓A bonds. This study aimed to identify the alternative aggrecanase. Femoral head cartilage from TS-4/5Dcat mice was stimulated with IL-1a or retinoic acid and total RNA was analysed by microarray. Candidate, upregulated genes identified by Quantile Normalisation included ADAMTS, MMPm cathepsin and calpain families as putative aggrecanases. Three criteria, including quantitative RT-PCR analyses, sensitivity to retinoic acid treatment and sensitivity to TIMP-3 inhibition, were then used as readouts to identify candidate extracellular proteinases whose mRNA expression levels were increased by retinoic acid, but not by IL-1a treatment. Thereafter, gene silencing was used to identify the most likely aggrecanase in chondrocytes from TS-4/5Dcat mice. Aggrecanase activity was monitored by Western blotting and immunohistochemistry with neoepitope antibodies. The microarray analyses identified ADAMTS-9, MMP-11 and calpain-5 as candidate aggrecanases that were upregulated by retinoic acid. While qPCR confirmed this finding, calpain 5 was excluded from the candidate list because it was not inhibited by TIMP-3. MMP-11 was also excluded because it was upregulated by IL-1a. ADAMTS-9 therefore emerged as a candidate for the novel aggrecanase. Gene silencing confirmed ADAMTS-9 as the putative aggrecanase. Immunohistochemistry revealed that ADAMTS-9 in mouse knee joints was expressed in the proliferative zone of both wildtype and TS-4/5Dcat growth plates, and also in the calcified cartilage of the wildtype hypertrophic zone. In conclusion, ADAMTS-9 is an aggrecanase that cleaves at FREEE1467↓1468GLGS in the chondroitin sulphate-rich region of aggrecan.

Dataset Information

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.

Publications

Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets