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.     

Aggrecanases and cartilage matrix degradation

The loss of extracellular matrix macromolecules from the cartilage results in serious impairment of joint function. Metalloproteinases called 'aggrecanases' that cleave the Glu³⁷³–Ala³⁷⁴ bond of the aggrecan core protein play a key role in the early stages of cartilage destruction in rheumatoid arthritis and in osteoarthritis. Three members of the ADAMTS family of proteinases, ADAMTS-1, ADAMTS-4 and ADAMTS-5, have been identified as aggrecanases. Matrix metalloproteinases, which are also found in arthritic joints, cleave aggrecans, but at a distinct site from the aggrecanases (i.e. Asn³⁴¹–Phe³⁴²). The present review discuss the enzymatic properties of the three known aggrecanases, the regulation of their activities, and their role in cartilage matrix breakdown during the development of arthritis in relation to the action of matrix metalloproteinases.     

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.     

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.     

MicroRNAs: Important Epigenetic Regulators in Osteoarthritis

Multiple mechanisms are implicated in the development of primary osteoarthritis (OA), in which genetic and epigenetic factors appear to interact with environmental factors and age to initiate the disease and stimulate its progression. Changes in expression of microRNAs (miRs) contribute to development of osteoarthritis. Numerous miRs are involved in cartilage development, homeostasis and degradation through targeting genes expressed in this tissue. An important regulator of gene expression in human cartilage is miR-140, which directly targets a gene coding aggrecanase ADAMTS-5, that cleaves aggrecan in cartilage. This miR is considered a biological marker for cartilage and its level significantly decreases in OA cartilage. On the other hand, increased expression of miR-146a in early OA inhibits two other cartilage-degrading enzymes: MMP13 and ADAMTS4, and may provide a useful tool in developing treatments for OA. The COL2A1 gene, encoding collagen type II, which is the most abundant structural protein of the cartilage, is silenced by miR-34a and activated by miR-675. Every year, new targets of cartilage miRs are validated experimentally and this opens new possibilities for new therapies that control joint destruction and stimulate cartilage repair. At the same time development of next-generation sequencing technologies allows to identify new miRs involved in cartilage biology.     

Proprotein convertase activation of aggrecanases in cartilage in situ

Proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, is a key pathological event in osteoarthritis (OA). ADAMTS-4 and ADAMTS-5, the primary aggrecanases capable of cartilage aggrecan cleavage, are synthesized as latent enzymes and require prodomain removal for activity. The N-termini of the mature proteases suggest that activation involves a proprotein convertase, but the specific family member responsible for aggrecanase activation in cartilage in situ has not been identified. Here we describe purification of a proprotein convertase activity from human OA cartilage. Through biochemical characterization and the use of siRNA, PACE4 was identified as a proprotein convertase responsible for activation of aggrecanases in osteoarthritic and cytokine-stimulated cartilage. Posttranslational activation of ADAMTS-4 and ADAMTS-5 was observed in the extracellular milieu of cartilage, resulting in aggrecan degradation. These findings suggest that PACE4 represents a novel target for the development of OA therapeutics.     

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.     

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.     

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.     

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future.     

Triptolide suppresses proinflammatory cytokine-induced matrix metalloproteinase and aggrecanase-1 gene expression in chondrocytes

A hallmark of rheumatoid- and osteoarthritis (OA) is proinflammatory cytokine-induced degeneration of cartilage collagen and aggrecan by matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). Effects of the Chinese herb, Tripterygium wilfordii Hook F (TWHF), on cartilage and its anti-arthritic mechanisms are poorly understood. This study investigated the impact of a purified derivative of TWHF, PG490 (triptolide), on cytokine-stimulated expression of the major cartilage damaging proteases, MMP-3, MMP-13, and ADAMTS4. PG490 inhibited cytokine-induced MMP-3, MMP-13 gene expression in primary human OA chondrocytes, bovine chondrocytes, SW1353 cells, and human synovial fibroblasts. Triptolide was effective at low doses and blocked the induction of MMP-13 by IL-1 in human and bovine cartilage explants. TWHF extract and PG490 also suppressed IL-1-, IL-17-, and TNF-alpha-induced expression of ADAMTS-4 in bovine chondrocytes. Thus, PG490 could protect cartilage from MMP- and aggrecanase-driven breakdown. The immunosuppressive, cartilage protective, and anti-inflammatory properties could make PG490 potentially a new therapeutic agent for arthritis.     

Interleukin-4 antagonizes oncostatin M and transforming growth factor beta-induced responses in articular chondrocytes

Oncostatin M (OSM) stimulates cartilage degradation in rheumatoid arthritis (RA) by inducing matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS; a disintegrin and metalloproteinase with thrombospondin motif). Transforming growth factor beta (TGF-beta1) induces cartilage repair in joints but in excessive amounts, promotes inflammation. OSM and TGF-beta1 also induce tissue inhibitor of metalloproteinase-3 (TIMP-3), an important natural inhibitor of MMPs, aggrecanases, and tumor necrosis factor alpha converting enzyme (TACE), the principal proteases involved in arthritic inflammation and cartilage degradation. We studied cartilage protective mechanisms of the antiinflammatory cytokine, interleukin-4 (IL-4). IL-4 strongly (MMP-13 and TIMP-3) or minimally (ADAMTS-4) suppressed OSM-induced gene expression in chondrocytes. IL-4 did not affect OSM-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), protein 38 (p38), c-Jun N-terminal kinase (JNK) and Stat1. Lack of additional suppression with their inhibitors suggested that MMP-13, ADAMTS-4, and TIMP-3 inhibition was independent of these mediators. IL-4 also downregulated TGF-beta1-induced TIMP-3 gene expression, Smad2, and JNK phosphorylation. Additional suppression of TIMP-3 RNA by JNK inhibitor suggests JNK implication. The cartilage protective effects of IL-4 in animal models of arthritis may be due to its inhibition of MMPs and ADAMTS-4 expression. However, suppression of TIMP-3 suggests caution for using IL-4 as a cartilage protective therapy.     

TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5)

The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K(i) values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.     

Expression and regulation of aggrecanase in arthritis: the role of TGF-beta.

Aggrecanases are key matrix-degrading enzymes that act by cleaving aggrecan at the Glu(373)-Ala(374) site. While these fragments have been detected in osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage and synovial fluid, no information is available on the regulation or expression of the two key aggrecanases (aggrecanase-1 and aggrecanase-2) in synovial tissue (ST) or fibroblast-like synoviocytes (FLS). The aggrecanase-1 gene was constitutively expressed by both RA and OA FLS. Real-time PCR demonstrated that TGF-beta significantly increased aggrecanase-1 gene expression in FLS. Aggrecanase-1 induction peaked after 24 h of TGF-beta stimulation. The expression of aggrecanase-1 mRNA was significantly greater in RA ST than in OA or nonarthritis ST. Aggrecanase-2 mRNA and protein were constitutively produced by nonarthritis, OA, and RA FLS but were not increased by IL-1, TNF-alpha, or TGF-beta. Furthermore, OA, RA, and nonarthritis ST contained similar amounts of immunoreactive aggrecanase-2. The major form of the aggrecanase-2 enzyme was 70 kDa in nonarthritis ST, whereas a processed 53-kDa form was abundant in RA ST. Therefore, aggrecanase-1 and -2 are differentially regulated in FLS. Both are constitutively expressed, but aggrecanase-1 is induced by cytokines, especially TGF-beta. In contrast, aggrecanase-2 protein may be regulated by a post-translational mechanism in OA and RA ST. Synovial and FLS production of aggrecanase can contribute to cartilage degradation in RA and OA.     

Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage

Osteoarthritis is a degenerative joint disorder characterized by breakdown of articular cartilage. Degradation of aggrecan, which together with type II collagen provides cartilage with its unique characteristics of compressibility and elasticity, is an early and sustained feature of osteoarthritis. The present work was set up to identify the enzyme(s) responsible for aggrecan breakdown in osteoarthritis. We found that the two cartilage aggrecanases, ADAM-TS4 and ADAM-TS5, are present in osteoarthritic cartilage and that they are responsible for aggrecan degradation without the participation of matrix metalloproteinases. This is based on 1) neoepitopes found on aggrecan fragments in osteoarthritis (OA) cartilage explants in vitro, 2) aggrecan fragments detected in synovial fluid of OA patients, 3) the observation that an aggrecanase inhibitor, BB-16, blocked aggrecan degradation in OA cartilage in vitro, whereas the matrix metalloproteinase inhibitor XS309 did not, and 4) the presence of mRNA and protein for ADAM-TS4 and ADAM-TS5 in OA cartilage. These results suggest that ADAM-TS4 and ADAM-TS5 represent a potential target for the treatment of osteoarthritis.     

Synthesis and biological evaluation of biphenylsulfonamide carboxylate aggrecanase-1 inhibitors

Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of proteoglycan degradation at 10 microg/mL and has an oral bioavailability in rat of 35%.