John D. Sandy

The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of approximately 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage of the human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685). These results indicate that the major aggrecan fragments present in both osteoarthritic human synovial fluid and in normal bovine synovial fluid are large, being composed of a short NH2-terminal stretch of the interglobular domain, the G2 domain, the keratan sulfate domain, and variable lengths of the chondroitin sulfate domain(s). We conclude that the release of aggrecan fragments from articular cartilage into the synovial fluid seen at all stages of human osteoarthritis (Lohmander, L. S. 1991. Acta Orthop. Scand. 62:623-632) is promoted by the action of a normal cartilage proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation.

Proteolysis of the hyalectans (aggrecan, versican, brevican) in vivo appears to result from the activity of ADAMTS4 (aggrecanase-1, herein referred to as an hyalectanase). To examine the mode of activation of ADAMTS4, a human chondrosarcoma cell line, JJ012, has been stably transfected with the full-length c-DNA for human ADAMTS4. The cells synthesized a high molecular weight form of the enzyme (p100), which in serum-free culture was processed to three truncated forms, p75, p60, and p50. Treatment of the p100 form with recombinant furin indicated that the p75 form is generated by the removal of the prodomain by a furin-like activity. Analysis with domain-specific antisera showed that the p60 and p50 forms are generated by C-terminal truncation of the p75 form. The appearance of the p60 and p50 forms in culture medium was prevented by inclusion of a furin inhibitor, inhibitors of glycosylphosphatidylinositol synthesis, glucosamine, a hydroxamate-based matrix metalloproteinase (MMP) inhibitor, and TIMP-1, but not by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) or E64. Only medium samples containing the p60/p50 forms exhibited aggrecanase activity, and isolation of the p75, p60, and p50 forms by preparative SDS-PAGE showed that only p60 and p50 were active in aggrecanase and versicanase assays. Pig synovium and human cartilages also contained ADAMTS4 in the p75, p60, and p50 forms. We suggest thatin vivo production of proteolytically active ADAMTS4 requires not only removal of the prodomain by a furin-like activity but also MMP-mediated removal of a portion of the C-terminal spacer domain.

Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants

The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with [35S]sulfate and [3H]proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of [3H]hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules. This study provides a framework for elucidating the biophysical mechanisms involved in the redistribution, catabolism, and loss of macromolecules during cartilage compression.

Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo.

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE(373) and G1-VDIPEN(341)) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu(373)-Ala(374) bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

Aggrecan in bovine tendon

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Aggrecanases and aggrecanase-generated fragments in the human intervertebral disc at early and advanced stages of disc degeneration

Study Design. A comparative study of aggrecanases and aggrecan fragmentation profile in the human intervertebral disc at early and advanced stages of disc degeneration. Objective. To determine differences in the content of the aggrecanases and the profile of aggrecan fragmentation in early and advanced stages of disc degeneration using cadaveric human intervertebral discs. Summary of Background Data. Aggrecanases and aggrecanase-generated aggrecan fragments have been found in human degenerated discs. However, the association between the grade of disc degeneration and the content of the aggrecanases and the profile of aggrecan fragments has not been well studied. Methods. A total of 108 cadaveric donor spines were assessed by MRI T2 imaging and graded based on the Thompson scale. Twelve donor spines (average age, 63 years), each specifically exhibiting 2 different stages (Grade 2 and Grade 4) of disc degeneration at different disc levels, were included in this study. After harvesting the preselected discs, tissue samples were obtained from the center of the nucleus pulposus (NP) and the middle zone of the anulus fibrosus (AF). The amount of the aggrecanases, specifically ADAMTS-4 and ADAMTS-5, and the pattern of aggrecan fragmentation in the isolated tissues were assessed by western blot using specific antibodies. Results. In both NP and the AF tissues, the amount of ADAMTS-4 detected was higher in disc tissues with a higher level of degeneration (Grade 4) than in Grade 2 disc tissues with a lower level of degeneration. However, the amount of ADAMTS-5 detected did not differ between the 2 disc tissue grades. The aggrecan fragmentation analysis of these samples demonstrated the presence of aggrecanase-mediated fragmentation in both groups; however, there was no apparent difference in the aggrecan fragmentation profile between discs at early and advanced stages of disc degeneration. Conclusion. Aggrecanases are involved in aggrecanolysis at both the early and advanced stages of disc degeneration. The aggrecan fragmentation profile analysis demonstrates the involvement of aggrecanases, as well as that of matrix metalloproteinases and/or cathepsins, during disc degeneration.

Recent progress in understanding molecular mechanisms of cartilage degeneration during osteoarthritis.

Osteoarthritis (OA) is a highly prevalent disease affecting more than 20% of American adults. Predispositions include joint injury, heredity, obesity, and aging. Biomechanical alterations are commonly involved. However, the molecular mechanisms of this disease are complex, and there is currently no effective disease‐modifying treatment. The initiation and progression of OA subtypes is a complex process that at the molecular level probably involves many cell types, signaling pathways, and changes in extracellular matrix. Ex vivo studies with tissue derived from OA patients and in vivo studies with mutant mice have suggested that pathways involving receptor ligands such as TGF‐β1, WNT3a, and Indian hedgehog; signaling molecules such as Smads, β‐catenin, and HIF‐2a; and peptidases such as MMP13 and ADAMTS4/5 are probably involved to some degree. This review focuses on molecular mechanisms of OA development related to recent findings.

Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation

Abstract Protease cascades are essential for many biological events, including the LH-induced process of ovulation. ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin-like repeats-1) is expressed and hormonally regulated in the ovary by LH and the progesterone receptor. To determine whether other family members might be expressed and regulated in the rodent ovary, those closely related to ADAMTS1 (ADAMTS4 and ADAMTS5) were analyzed in the mouse ovary by reverse transcription-polymerase chain reaction as well as by Western blot, immunohistochemical, and immunocytochemical analyses using highly specific antibodies. Prior to ovulation, ADAMTS4 and ADAMTS5 were coexpressed in granulosa cells of most follicles, whereas ADAMTS5 was also present in granulosa cells of atretic follicles. Following ovulation, ADAMTS1 and ADAMTS4 (but not ADAMTS5) were expressed in multiple cell types, including those within the highly vascular ovulation cone that marks the site of follicle rupture, endothelial cells of newly forming corpora lutea, and cumulus cells within the ovulated cumulus cell-oocyte complex (COC). Versican, a substrate for ADAMTS1 and ADAMTS4, colocalized with these proteases and hylauronan on the cumulus cell surface. To further characterize induction of these proteases and associated molecules, COCs and granulosa cells were isolated from preovulatory follicles and treated with FSH. In expanded COCs and differentiated granulosa cells, FSH induced expression of ADAMTS4 and versican message and protein, whereas increased levels of ADAMTS1 protein was observed in the media of granulosa cells where it was stabilized by heparin in this in vitro system. These studies provide the first evidence that ADAMTS1, ADAMTS4, and ADAMTS5 are expressed in spatiotemporal patterns that suggest distinct as well as some overlapping functions that relate to the broad expression pattern of versican in granulosa cells of small follicles, expanded COCs, and endothelial cells of the mouse ovary.

Association between protease-specific proteolytic cleavage of brevican and synaptic loss in the dentate gyrus of kainate-treated rats.

Proteolytic fragments generated by ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identified, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEAVESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS-cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate-putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate.Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by specific matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS-induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region.

Degradation of proteoglycan in articular cartilage

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.