Hyaluronan suppresses mechanical stress-induced expression of catabolic enzymes by human chondrocytes via inhibition of IL-1ß production and subsequent NF-κB activation.

OBJECTIVE: To investigate the inhibitory effect of hyaluronan (HA) on mechanical stress- induced expression of a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS)-4, -5 and matrix metalloproteinase (MMP)-13 by human chondrocytes. MATERIALS AND METHODS: Normal human articular chondrocytes were pre-incubated with or without 1.0 mg/mL HA (2700 kDa) for 12 h at 37 °C in stretch chambers, then they were exposed to uni-axial cyclic tensile strain (CTS, 0.5 Hz, 10% elongation). The expression of ADAMTS-4, -5, and MMP-13 were analyzed by real-time polymerase chain reaction and Immunocytochemistry. The concentration of IL-1ß in the supernatant was measured using enzyme-linked immunosorbent assay (ELISA). The nuclear translocation of runt-related transcription factor 2 (RUNX-2) and nuclear factor-κB (NF-κB) was examined by ELISA and immunocytochemistry, and phosphorylation of NF-κB was examined by western blotting. RESULTS: HA inhibited mRNA expression of ADAMTS-4, -5, and MMP13 after 24 h CTS via inhibition of IL-1ß secretion and NF-κB activation. However, HA failed to inhibit CTS-induced RUNX-2 expression and subsequent expression of ADAMTS-5 and MMP-13 1 h after CTS. CONCLUSIONS: Our results demonstrated that HA significantly suppressed mechanical stress-induced expression of catabolic proteases by inhibition of the NF-κB-IL-1ß pathway, but did not suppress mechanical stress-induced RUNX-2 signaling.

Mechanisms involved in suppression of ADAMTS4 expression in synoviocytes by high molecular weight hyaluronic acid.

Aggrecan degradation is considered to play a key role in the progression of osteoarthritis (OA). Aggrecanases are members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and degrade aggrecan in OA cartilage. The aim of this study was to clarify the mechanisms of expression of ADAMTS4 induced by IL-1ß in human fibroblast-like synoviocyte (HFLS) cells by high molecular weight hyaluronan (HMW-HA), a therapeutic agent used for OA. Monolayer cultures of HFLS cells were incubated with IL-1ß and HMW-HA. In some experiments, cells were pretreated with the CD44 function-blocking monoclonal antibody or inhibitors of signaling pathways prior to addition of IL-1ß and HMW-HA. The expressions of ADAMTS4 mRNA and protein were monitored using real-time RT-PCR, Western blotting, and immunofluorescence microscopy. To further determine the role of HMW-HA in IL-1ß-induced ADAMTS4 expression, activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), Akt, and NF-κB were analyzed by Western blotting. HMW-HA suppressed ADAMTS4 mRNA and protein expressions induced by IL-1ß. Pretreatment with the anti-CD44 monoclonal antibody recovered the inhibitory effect of HMW-HA on expression of ADAMTS4 mRNA induced by IL-1ß. Western blotting analysis revealed that IL-1ß-induced phosphorylation of p38 MAPK and JNK protein were diminished by HMW-HA. Furthermore, inhibition of the p38 MAPK and JNK pathways by chemical inhibitors suppressed ADAMTS4 mRNA expression stimulated by IL-1ß. These results suggest that HMW-HA plays an important role as a regulatory factor in synovial tissue inflammation.

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover.

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

TNF-α and IL-1ß promote a disintegrin-like and metalloprotease with thrombospondin type I motif-5-mediated aggrecan degradation through syndecan-4 in intervertebral disc.

Elevated levels of TNF-α, IL-1ß and a resultant increase in ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type I motifs) expression is seen during disc degeneration. However, if these pro-inflammatory cytokines control ADAMTS activity is not definitively known. The goal of the investigation was to study if TNF-α and IL-1ß regulate syndecan-4 (SDC4) expression, and if SDC4 was responsible for promoting aggrecan degradation through controlling ADAMTS activity in nucleus pulposus cells of the intervertebral disc. Cytokine treatment increased SDC4 expression and promoter activity. Use of inhibitor, SM7368 and co-transfections with IκBα, RelA/p50 showed that NF-κΒ regulated both basal and cytokine-dependent SDC4 transcription. SDC4 promoter harboring RelA binding site mutation was unresponsive to the cytokines. Moreover, cytokines failed to increase SDC4 promoter activity in RelA-null cells. Cytokines increased ADAMTS-4/5 expression and aggrecan degradation and promoted SDC4 interaction with ADAMTS-5. Treatment with heparinase-III and p-nitrophenyl-ß-D-xylopyranoside (PNPX), an inhibitor of heparan sulfate synthesis and transfection with SDC4-shRNA partially blocked cytokine mediated aggrecan degradation. Analysis of human tissues showed increased aggrecan degradation with a concomitant increase in SDC4 and ADAMTS-5 protein expression with severity of disc disease. Likewise, SDC4, TNF-α, IL-1ß, ADAMTS-4, and ADAMTS-5 mRNA expression increased in degenerate tissues. We conclude that in nucleus pulposus, TNF-α and IL-1ß regulate SDC4 expression, which plays a key role in pathogenesis of degenerative disc disease by promoting aggrecan degradation by ADAMTS-5.

Orally active achiral N-hydroxyformamide inhibitors of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) for the treatment of osteoarthritis.

A new achiral class of N-hydroxyformamide inhibitor of both ADAM-TS4 and ADAM-TS5, 2 has been discovered through modification of the complex P1 group present in historical inhibitors 1. This structural change improved the DMPK properties and greatly simplified the synthesis whilst maintaining excellent cross-MMP selectivity profiles. Investigation of structure-activity and structure-property relationships in the P1 group resulted in both ADAM-TS4 selective and mixed ADAM-TS4/5 inhibitors. This led to the identification of a pre-clinical candidate with excellent bioavailability across three species and predicting once daily dosing kinetics.

The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis.

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described.

Structure-activity study on a series of α-glutamic acid scaffold based compounds as new ADAMTS inhibitors.

A series of α-glutamic acid scaffold based 4-(benzamido)-4-(1,3,4-oxadiazol-2-yl) butanoic acids were designed and synthesized as new ADAMTS inhibitors. The compounds dose-dependently inhibited the enzymatic activities of ADAMTS-4 and ADAMTS-5. One of the most active compound 2h potently inhibited ADAMTS-4 and ADAMTS-5 with IC(50) values of 1.2 and 0.8 µM, respectively. These inhibitors may serve as new lead compounds for further development of therapeutics to treat osteoarthritis.

Continued exploration of biphenylsulfonamide scaffold as a platform for aggrecanase-1 inhibition.

Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 µg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.

Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.

We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [-1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [-1A]N-TIMP-3 and [-2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [-1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.

Suppression of aggrecanase: a novel protective mechanism of dehydroepiandrosterone in osteoarthritis?

Aggrecanase-mediated aggrecan degradation is a significant event in the early stages of osteoarthritis (OA). There has been much interest in the possible role of these aggrecanases, mainly aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), as therapeutic targets in OA. The deficiency of current pharmaceutical treatments is that they mainly target the symptoms of OA but do not address the fundamental mechanism behind OA which is the destruction of articular cartilage. Therefore, a treatment which would protect or regenerate cartilage on the cellular level would be desirable. Dehydroepiandrosterone (DHEA), classified as an adrenal androgen, is recently proposed to be "disease-modifying", and has been found to counteract proinflammatory effects of catabolic cytokines, suggesting that it has a protective effect for osteoarthritic cartilage. The suppression by DHEA of some members of the MMP family in OA has been well demonstrated, however, the effect of DHEA on aggrecanases remains unknown. This article reviews recent findings with regard to aggrecanases as critical catabolic enzymes and DHEA as a therapeutic agent in OA, and further discusses the possible relationship between aggrecanase and DHEA in the progression of OA.

Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases.

Degradation of the cartilage proteoglycan aggrecan is a key early event in the development of osteoarthritis. Adamalysin with thrombospondin motifs (ADAMTS) -4 and ADAMTS-5 are considered to be the main enzymes responsible for aggrecan breakdown, making them attractive drugs targets. Here we show that calcium pentosan polysulfate (CaPPS), a chemically sulfated xylanopyranose from beechwood, is a multifaceted exosite inhibitor of the aggrecanases and protects cartilage against aggrecan degradation. CaPPS interacts with the noncatalytic spacer domain of ADAMTS-4 and the cysteine-rich domain of ADAMTS-5, blocking activity against their natural substrate aggrecan with inhibitory concentration 50 values of 10-40 nM but only weakly inhibiting hydrolysis of a nonglycosylated recombinant protein substrate. In addition, CaPPS increased cartilage levels of tissue inhibitor of metalloproteinases-3 (TIMP-3), an endogenous inhibitor of ADAMTS-4 and -5. This was due to the ability of CaPPS to block endocytosis of TIMP-3 mediated by low-density lipoprotein receptor-related protein. CaPPS also increased the affinity of TIMP-3 for ADAMTS-4 and -5 by more than 100-fold, improving the efficacy of TIMP-3 as an aggrecanase inhibitor. Studies with TIMP-3-null mouse cartilage indicated that CaPPS inhibition of aggrecan degradation is TIMP-3 dependent. These unique properties make CaPPS a prototypic disease-modifying agent for osteoarthritis.

Synthesis and biological evaluation of ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides: a class of potent aggrecanase-1 inhibitors.

The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.

[Aggrecanases and osteoarthritis].

Osteoarthritis is mainly caused by the degenerative changes of cartilage and cartilage extracellular matrix, while Aggrecanases degradate Proteoglycans which are the major components of cartilage. This review includes three aspects: (1) We have concluded the major enzymes(ADAMTS-4 and ADAMTS-5) which regulate the metabolism of cartilage extracellular matrix. Meanwhile, we have summarized the structure of aggrecanases(ADAMTS-4 and ADAMTS-5) and introduced the function of each regional structure; (2) We have concluded the way cytokines and glycosaminoglycans regulate the metabolism of aggrecanases, and discussed the regulation and control principle of cytokines and glycosaminoglycan; (3) We have summarized the majority of inhibitors to the aggrecanases, introduced the endogenic inhibitors, and put our emphasis on the extrinsic inhibitors (chelating agents, polypeptides and so on). Through deeper research on the enzymes, it will help us further understand the pathogenesis of osteoarthritis, and open up new avenues to clinical treatment.

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.

Nobiletin, a citrus polymethoxy flavonoid, suppresses gene expression and production of aggrecanases-1 and -2 in collagen-induced arthritic mice.

Aggrecanase-1/a disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS)-4 and aggrecanase-2/ADAMTS-5 have been shown to play crucial roles in cartilage destruction in arthritic diseases, including rheumatoid arthritis and osteoarthritis. In this study, we examined the effects of nobiletin, a citrus polymethoxy flavone, on the expression and production of ADAMTS-4 and -5 in vitro and in vivo. Nobiletin (16-64muM) interfered with the interleukin (IL)-1beta-mediated ADAMTS-4 and -5 mRNA expression in cultured human synovial fibroblasts. Furthermore, intraperitoneal administration of nobiletin (15, 30, and 60mg/kg) also suppressed ADAMTS-4 and -5 mRNA expression in the joint tissues of collagen-induced arthritic (CIA) mice. Immunohistochemical analysis using an antibody against aggrecan neoepitope (NVTEGE(373)) revealed that aggrecanase-mediated degradation of aggrecan in cartilage was effectively inhibited by nobiletin. These results provide novel evidence that nobiletin effectively interferes with gene expression of ADAMTS-4 and -5, and thereby prevents cartilage destruction in CIA mice.

ADAM metallopeptidase with thrombospondin type 1 motif 2 inactivation reduces the extent and stability of carbon tetrachloride-induced hepatic fibrosis in mice.

UNLABELLED: ADAMTS2 belongs to the "ADAM metallopeptidase with thrombospondin type 1 motif" (ADAMTS) family. Its primary function is to process collagen type I, II, III, and V precursors into mature molecules by excising the aminopropeptide. This process allows the correct assembly of collagen molecules into fibrils and fibers, which confers to connective tissues their architectural structure and mechanical resistance. To evaluate the impact of ADAMTS2 on the pathological accumulation of extracellular matrix proteins, mainly type I and III collagens, we evaluated carbon tetrachloride-induced liver fibrosis in ADAMTS2-deficient (TS2(-/-)) and wild-type (WT) mice. A single carbon tetrachloride injection caused a similar acute liver injury in deficient and WT mice. A chronic treatment induced collagen deposition in fibrous septa that were made of thinner and irregular fibers in TS2(-/-) mice. The rate of collagen deposition was slower in TS2(-/-) mice, and at an equivalent degree of fibrosis, the resorption of fibrous septa was slightly faster. Most of the genes involved in the development and reversion of the fibrosis were similarly regulated in TS2(-/-) and WT mice. CONCLUSION: These data indicate that the extent of fibrosis is reduced in TS2(-/-) mice in comparison with their WT littermates. Inhibiting the maturation of fibrillar collagens may be a beneficial therapeutic approach to interfering with the development of fibrotic lesions.

Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro.

AIM: Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation. A major feature is the loss of aggrecan from the cartilage matrix, primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2. The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes. METHODS: Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes. The transfected cells were induced with interleukin-1beta (IL-1beta). Gene mRNA levels were analyzed by RT-PCR. Aggrecan and collagen II content were measured by immunohistochemistry and Western blotting. RESULTS: As the chondrocytes underwent dedifferentiation, aggrecanase-1 increased significantly. The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes. The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly. The alpha-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1. Other genes of the chondrocytic extracellular matrix were not affected. RNAi significantly increased the aggrecan and collagen II content of chondrocytes treated with IL-1beta. CONCLUSION: The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation, without interfering with chondrocytic gene phenotype recovery. RNAi technology can be a useful tool for studying degenerative processes in cartilage.

Aggrecanase and aggrecan degradation in osteoarthritis: a review.

Aggrecanase-mediated aggrecan degradation is a significant event in early-stage osteoarthritis (OA). Aggrecanases belonging to the 'A Disintegrin And Metalloproteinase with ThromboSpondin motifs' (ADAMTS) family of proteinases play a significant role in aggrecan depletion in osteoarthritic cartilage. There has been considerable interest in the possible role of these aggrecanases, especially ADAMTS-4 and ADAMTS-5, as therapeutic targets in OA. This article discusses recent data regarding ADAMTS-4 and ADAMTS-5 in OA, with emphasis on the relationship between aggrecanase and aggrecan degradation as well as the role of aggrecanase in OA.

Drug insight: aggrecanases as therapeutic targets for osteoarthritis.

In healthy cartilage, effective weight-bearing requires a high concentration of intact aggrecan. Degradation and loss of aggrecan are features of osteoarthritis (OA). It is unclear whether ADAMTS-4, ADAMTS-5, or both of these aggrecanases from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzyme family, are responsible for aggrecanolysis in human OA, and at what stage of disease these enzymes are active. Several potential disease-modifying agents for OA include glucosamine and chondroitin sulfate, diacerhein, and pentosan polysulfate; although their mechanisms of action in vivo are unknown, data from in vitro studies and animal models suggest that their efficacy might be partly due to inhibition of proinflammatory pathways that lead to downregulation of ADAMTS enzymes. Some histone deacetylase inhibitors that are successfully used to treat cancer can block ADAMTS-5 expression; however, these inhibitors will only be considered as potential therapies for OA if their toxicity is markedly reduced. ADAMTS inhibitors currently in development are expected to show excellent specificity now that crystal structures for several ADAMTS enzymes are available to guide drug design. ADAMTS-4 and ADAMTS-5 are appropriate targets for OA therapies, but ultimately, inhibitors of these enzymes will form only part of a larger arsenal of therapies.