Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.     

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.     

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.     

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 that in 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.     

VIP and CRF reduce ADAMTS expression and function in osteoarthritis synovial fibroblasts

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family is known to play an important role in the pathogenesis of osteoarthritis (OA), working on aggrecan degradation or altering the integrity of extracellular matrix (ECM). Thus, the main purpose of our study was to define the role of vasoactive intestinal peptide (VIP) and corticotrophin‐releasing factor (CRF), as immunoregulatory neuropeptides, on ADAMTS production in synovial fibroblasts (SF) from OA patients and healthy donors (HD). OA‐ and HD‐SF were stimulated with pro‐inflammatory mediators and treated with VIP or CRF. Both neuropeptides decreased ADAMTS‐4, ‐5, ‐7 and ‐12 expressions, aggrecanase activity, glycosaminoglycans (GAG), and cartilage oligomeric matrix protein (COMP) degradation after stimulation with fibronectin fragments (Fn‐fs) in OA‐SF. After stimulation with interleukin‐1β, VIP reduced ADAMTS‐4 and ‐5, and both neuropeptides decreased ADAMTS‐7 production and COMP degradation. Moreover, VIP and CRF reduced Runx2 and β‐catenin activation in OA‐SF. Our data suggest that the role of VIP and CRF on ADAMTS expression and cartilage degradation could be related to the OA pathology since scarce effects were produced in HD‐SF. In addition, their effects might be greater when a degradation loop has been established, given that they were higher after stimulation with Fn‐fs. Our results point to novel OA therapies based on the use of neuropeptides, since VIP and CRF are able to stop the first critical step, the loss of cartilage aggrecan and the ECM destabilization during joint degradation.     

ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan

ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including Delta Sp (Delta Arg(693)-Lys(837), lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC(50) = 110 nm), whereas no inhibition was observed with Delta Sp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC(50) = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.     

Versican/PG-M is essential for ventricular septal formation subsequent to cardiac atrioventricular cushion development

Versican (Vcan)/proteoglycan (PG)-M is a large chondroitin sulfate proteoglycan which forms a proteoglycan/hyaluronan (HA) aggregate in the extracellular matrix (ECM). We tried to generate the Vcan knockout mice by a conventional method, which resulted in mutant mice Vcan(Δ3/Δ3) whose Vcan lacks the A subdomain of the G1 domain. The Vcan knockout embryos died during the early development stage due to heart defects, but some Vcan(Δ3/Δ3) embryos survived through to the neonatal period. The hearts in Vcan(Δ3/Δ3) newborn mice showed normal cardiac looping, but had ventricular septal defects. Their atrioventricular canal (AVC) cushion was much smaller than those of wild-type (WT) embryos, and the extracellular space for cardiac jelly was narrow. The Vcan deposition in the Vcan(Δ3/Δ3) AVC cushion had decreased, whereas the HA deposition was maintained and condensed. In the tip of ventricular septa, both Vcan and HA had decreased. The cell proliferation based on the number of Ki67-positive cells had remarkably increased in both the AVC cushion and ventricular septa, compared with that of WT embryos. Vcan(Δ3/Δ3) seemed to have endocardial and mesenchymal mixed characteristics. When the ex vivo explant culture of these regions was performed on the collagen gel, hardly any migration to make sufficient space for the ECM construction was apparent. Our results suggest that the proteoglycan aggregates are necessary in both the AVC cushion and ventricular septa to fuse interventricular septa, and the Vcan A subdomain plays an essential role for the interventricular septal formation by constituting the proteoglycan aggregates.     

A quantitative assay for aggrecanase activity.

Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N-terminal ARGSVIL sequence and a second anti-aggrecan peptide antibody. For higher sensitivity of the assay, P1-P5 residues of the aggrecanase site within the aggrecan substrate were changed by in vitro mutagenesis. Specific activities of recombinant truncated ADAMTS1 and ADAMTS4 estimated with authentic aggrecan interglobular domain amounted to 2.4 +/- 0.4 and 21.7 +/- 9.5 nmoles hydrolyzed substrate/min.mg, respectively. The values were 10.3 +/- 5.1 and 151.5 +/- 93.5 nmoles/min.mg for hydrolysis of the modified substrate. The aggrecanase activity assay can be used for (1) kinetic characterization of aggrecanase activities of human and animal ADAMTS, (2) screening of inhibitors for aggrecan hydrolyzing ADAMTS, and (3) estimation of aggrecanase activities in biological samples.     

Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4)

ADAMTS4 (aggrecanase-1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP-3 inhibited the activity most efficiently with an IC(50) value of 7.9 nM, which was at least 44-fold lower than that of TIMP-1 (350 nM) and TIMP-2 (420 nM) and >250-fold less than that of TIMP-4 (2 microM for 35% inhibition). These results suggest that TIMP-3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo.     

Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes

Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.     

Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

A Disintegrin and Metalloproteinase with ThromboSpondin motif (ADAMTS) 5 functions as an anti-angiogenic and anti-cancer protein independent of its metalloproteinase activity. Both full-length ADAMTS5 and TS5-p45, the autocatalytically cleaved C-terminal 45 kDa truncate of ADAMTS5, inhibits angiogenesis, and induces endothelial cell (EC) apoptosis. However, how ADAMTS5 triggers EC apoptosis remains unclear. This work shows that caspase-8 (Cas-8) and caspase-9 (Cas-9) are involved in TS5-p45-induced EC apoptosis. We identify cell surface nucleolin (NCL) as a novel high-affinity receptor for TS5-p45 in ECs, mediating TS5-p45’s cell surface binding and pro-apoptotic function. We show that the central RNA-binding domain (RBD) of NCL is essential and sufficient for its binding to TS5-p45. Upon interacting with EC surface NCL, TS5-p45 is internalized through clathrin- and caveolin-dependent endocytosis and trafficked to the nucleus via late endosomes (LEs). We demonstrate that the nuclear trafficking of TS5-p45 is important for its pro-apoptotic activity as disruption of LE membrane integrity with an endosomolytic peptide suppressed both nuclear trafficking and pro-apoptotic activity of TS5-p45. Through cell surface biotinylation, we revealed that cell surface NCL shuttles extracellular TS5-p45 to the nucleus to mediate apoptosis. Furthermore, blocking the importin α1/ß1 receptor hindered the nuclear trafficking of TS5-p45, suggesting the involvement of the nuclear importing machinery for this nuclear translocation. RNA-seq identified many apoptosis-related genes that are differentially expressed at least two-fold in TS5-p45-treated ECs, with 10 of them qRT-PCR-validated and at least 5 of these genes potentially contributing to TS5-p45-NCL-induced apoptosis. Altogether, our work identifies NCL as a novel cell surface receptor for ADAMTS5 and demonstrates the critical role of NCL-mediated internalization and nuclear trafficking for ADAMTS5-induced EC apoptosis. These findings reveal novel mechanistic insights of the secreted metalloproteinase ADAMTS5 in angiogenesis inhibition.Subject terms: Tumour angiogenesis, Apoptosis, Protein translocation     

Adamts5 deletion blocks murine dermal repair through CD44-mediated aggrecan accumulation and modulation of transforming growth factor β1 (TGFβ1) signaling

ADAMTS5 has been implicated in the degradation of cartilage aggrecan in human osteoarthritis. Here, we describe a novel role for the enzyme in the regulation of TGFβ1 signaling in dermal fibroblasts both in vivo and in vitro. Adamts5(-/-) mice, generated by deletion of exon 2, exhibit impaired contraction and dermal collagen deposition in an excisional wound healing model. This was accompanied by accumulation in the dermal layer of cell aggregates and fibroblastic cells surrounded by a pericellular matrix enriched in full-length aggrecan. Adamts5(-/-) wounds exhibit low expression (relative to wild type) of collagen type I and type III but show a persistently elevated expression of tgfbRII and alk1. Aggrecan deposition and impaired dermal repair in Adamts5(-/-) mice are both dependent on CD44, and Cd44(-/-)/Adamts5(-/-) mice display robust activation of TGFβ receptor II and collagen type III expression and the dermal regeneration seen in WT mice. TGFβ1 treatment of newborn fibroblasts from wild type mice results in Smad2/3 phosphorylation, whereas cells from Adamts5(-/-) mice phosphorylate Smad1/5/8. The altered TGFβ1 response in the Adamts5(-/-) cells is dependent on the presence of aggrecan and expression of CD44, because Cd44(-/-)/Adamts5(-/-) cells respond like WT cells. We propose that ADAMTS5 deficiency in fibrous tissues results in a poor repair response due to the accumulation of aggrecan in the pericellular matrix of fibroblast progenitor cells, which prevents their transition to mature fibroblasts. Thus, the capacity of ADAMTS5 to modulate critical tissue repair signaling events suggests a unique role for this enzyme, which sets it apart from other members of the ADAMTS family of proteases.     

High resolution crystal structure of the catalytic domain of ADAMTS-5 (aggrecanase-2)

Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4A resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.     

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.     

Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.     

Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.     

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.     

CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice

Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.