The ADAMTS metalloproteinases

Inhibition of the ADAMTSs

Although the TIMPs are, with a few exceptions, broadly effective inhibitors of the MMPs (reviewed in [39]), it is clear that they display much greater selectivity towards both the ADAMs and ADAMTSs. The aggrecanases ADAMTS-4 and ADAMTS-5 are both potently inhibited by TIMP-3, with K_i values in the sub-nanomolar range, although they are essentially insensitive to TIMP-1, -2 and -4 [40–42]. This is supported by data from an ex vivo bovine nasal cartilage explant model of cartilage destruction, where the N-terminal domain of TIMP-3 potently inhibited GAG release at 0.1 μM, whereas neither N-TIMP-1 nor N-TIMP-2 inhibited GAG release, even at 1 μM. Other ADAMTSs may have a different inhibition profile by TIMPs: ADAMTS-1 is partially inhibited by TIMP-2 and TIMP-3 at 500 nM, but TIMP-1 and TIMP-4 have no inhibitory effect at the same concentration [43]. TIMP-3 is thus likely to be the major natural inhibitor of aggrecanase activity in cartilage [44]. Both ADAMTS-4 and -5 cleave α2-macroglobulin at a novel cleavage site within the bait region, at what is also a novel site of cleavage for ADAMTS-4 and -5 [45].

The aggrecanolytic activity of ADAMTS-1, -4 and -5 is effectively inhibited by catechin gallate esters found in green tea: both epigallocatechin gallate and epicatechin gallate potently inhibited all three ADAMTSs, with approximate IC₅₀ values of 100–150 nM for ADAMTS-4 and ADAMTS-5, and 200–250 nM for ADAMTS-1 [46]. Limited studies have been undertaken on the effect of synthetic inhibitors on the ADAMTSs: ADAMTS-1 is inhibited by EDTA, 1,10-phenanthroline [47], BB-94 [43] and MMP inhibitor 2 (from Calbiochem; [29]); ADAMTS-12 is inhibited by BB-94 [20].

It has been suggested that papilin, an essential component of the ECM of C. elegans, may modulate ADAMTSs during organogenesis [48]. Papilin, which exists in mammals as well as in invertebrates, is an ECM glycoprotein containing a conserved sequence (‘papilin cassette’) of non-enzymatic domains that it shares with the ADAMTSs, namely a signal peptide, a TS repeat, a cysteine-rich domain and several more TS repeats, and has been shown to inhibit ADAMTS-2 in vitro [48]. In this respect, papilin is close in structure to the ADAMTSL (ADAMTS-like) proteins, and may suggest a function for the latter. However, unlike either the ADAMTSs or the ADAMTSLs, papilin contains additional domains further towards the C-terminal end: cysteine-rich repeats, a species-dependent variable number of Kunitz domains [homologues of the BPTI (bovine pancreatic trypsin inhibitor)], Ig–C2 loop domains consisting of a unique cysteine-rich sequence, and a C-terminal peptide, and the functions of these domains are uncertain [48]. Despite the sequence homology with BPTI, the Kunitz domains do not inhibit trypsin, chymotrypsin or plasminogen.

Regulation of ADAMTS gene expression

Expression of ADAMTS mRNAs appears to be distributed across a wide range of normal adult tissues. Expression in fetal tissue is generally more limited. A summary of ADAMTS expression analysis is given in Table 2.

Our own expression profiling studies of the 19 ADAMTS genes in breast and prostate cancer using real-time PCR have demonstrated statistically significant dysregulation of several ADAMTSs under these pathological conditions. Eleven of the ADAMTS genes are dysregulated in invasive breast carcinoma: ADAMTS1, 3, 5, 8, 9, 10 and 18 mRNAs are down-regulated by 40–90%, and those for ADAMTS4, 6, 14 and 20 are up-regulated by 80–2000% with respect to normal mammary tissue; in normal breast, expression appears to be predominantly in myoepithelial cells or stroma, and rarely in luminal epithelial cells [49]. Though there was no significant difference in expression of ADAMTS15 between normal breast and mammary cancer, those patients whose tumours had low ADAMTS15 levels showed poorer event-free survival [49]. In prostate cancer, ADAMTS13 and 20 are down-regulated with respect both to samples of benign prostatic hyperplasia and matched normal prostate tissue (S. Porter, A. C. P. Riddick, K. K. Sethia, R. Y. Ball and D. R. Edwards, unpublished work).

Down-regulation of expression of particular ADAMTSs in cancer compared with normal tissue suggests that these proteases may negatively regulate tumour growth, and this would be consistent with the anti-angiogenic properties of ADAMTS-1 and -8 (see the ‘Anti-angiogenesis: ADAMTS-1 and -8’ subsection). Masui et al. [50] have also observed down-regulation of ADAMTS1 mRNA in pancreatic cancer and hepatocellular carcinoma with respect to non-neoplastic tissue. However, ADAMTS1 expression did not correlate with tumour vascularity in either hepatocellular carcinoma or pancreatic cancer. Also, metastatic pancreatic cancer was associated with significantly higher expression of ADAMTS1, and these patients had a poorer prognosis after curative surgery, which suggests that ADAMTS1 may in fact have a positive role in promoting the progression of pancreatic cancer through lymph node metastasis and local invasion. Thus, as with the MMPs, the ADAMTSs may have multiple, perhaps opposing, actions at particular stages during tumour progression.

In cartilage samples obtained from patients with osteoarthritis, eight of the ADAMTS genes showed dysregulation: ADAMTS1, 5, 9 and 15 (all aggrecanases) are down-regulated, and ADAMTS2, 12, 14 and 16 are up-regulated in osteoarthritis cartilage with respect to phenotypically normal cartilage obtained from fracture patients [51]. Although the consistent down-regulation of aggrecanase mRNAs in osteoarthritis cartilage is somewhat surprising, given the catabolic role of the enzymes, it is important to note that the tissues used in this profiling study represent end-stage disease, and therefore it will be important to analyse expression in earlier disease stages and in animal models of arthritis.

Regulation of the ADAMTS genes is still poorly understood, though there is evidence that several are regulated by growth factors, hormones and inflammatory cytokines. Transforming growth factor-β has been shown to induce expression of ADAMTS2 mRNA in MG-63 osteosarcoma cells [52], ADAMTS12 in KMST human fetal fibroblasts [20] and ADAMTS4 (but not ADAMTS5) in fibroblast-like synoviocytes [53]. Consistent with its role as a mediator of cartilage matrix breakdown, IL (interleukin)-1 stimulation of chondrocytes or bovine nasal or articular cartilage led to an increase in detectable aggrecanase activity, without increasing measured ADAMTS-4 protein [54]; however, it is possible that other aggrecanase ADAMTSs may account for the increased activity in this system. In IL-1-treated pig articular cartilage explants, increased ADAMTS-4 protein levels were apparent [23]. In the immortalized chondrocytic cell line TC28a4, ADAMTS1, ADAMTS4 and ADAMTS5 mRNAs were all regulated by a combination of IL-1α and oncostatin M, though with differing magnitude and kinetics [55]. IL-17, a major pro-inflammatory cytokine produced mainly by synovial membranes in rheumatoid arthritis, also up-regulates ADAMTS4 in bovine articular chondrocytes via phosphorylation of ERK (extracellular-signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase) mitogen-activated protein kinases [56]. ADAMTS1 mRNA is also induced in response to IL-1 in N1E-115 rat cells following hypoglossal nerve injury [57]. In human THP-1 monocytic cells, PMA strongly induced ADAMTS4 expression, and this induction was suppressed by both the peroxisome proliferator-activated receptor γ agonist GW7845 and 9-cis-retinoic acid [58], suggesting that ADAMTS4 may be important in activated macrophages in inflammation and various pathologies.

The regulation of some ADAMTS genes is controlled by hormonal stimuli: the thyroid hormone tri-iodothyronine (T3) up-regulates mRNA expression of ADAMTS5, but not ADAMTS4, with subsequent aggrecan degradation, in growth plate cartilage during endochondral ossification [59]. Parathyroid hormone up-regulates ADAMTS1 mRNA expression in bone and osteoblasts [60]. ADAMTS-1 plays a critical role in follicular rupture, and its expression in granulosa cells of pre-ovulatory follicles is induced both by luteinizing hormone and human chorionic gonadotropin, and regulated by progesterone [61,62].

Anti-angiogenesis: ADAMTS-1 and -8

Two ADAMTS proteins that have been proven to be anti-angiogenic are ADAMTS-1 and ADAMTS-8 [9]. Both can inhibit VEGF-induced angiogenesis in a chick chorioallantoic membrane assay and suppress fibroblast-growth-factor-2-induced vascularization in a corneal pocket assay, and they both mediate a greater anti-angiogenic response than either TSP-1 or endostatin on a molar basis, with ADAMTS-1 showing a greater inhibitory capacity than ADAMTS-8.

The anti-angiogenic activity of ADAMTS-1 and -8 is thought to be mediated through their TS motifs. These repeats are a feature of TSP1 and TSP2, and out of the five TSP family members, these are the only two to have anti-angiogenic activity (for a review, see [63]). Of possible relevance to the anti-angiogenic actions of ADAMTS-1 is that TSP1 can interact with CD36, a membrane glycoprotein receptor on endothelial cells [64,65]. This involves the sequence CSVTCG, present in two of the three TS repeats in TSP1, and there is a similar conserved motif, CSRT/SCG, in the central TS repeat of all ADAMTSs. This suggests that ADAMTS-1 might potentially bind to CD36 on the surface of endothelial cells via its central TS motif, as indeed might other ADAMTS proteins, although this has not been demonstrated. However, as mentioned in the ‘C-terminal processing affects substrate specificity and ECM binding’ section above, recent data have shown that it is the region of ADAMTS-1 which contains the two C-terminal TS repeats that is responsible for its anti-angiogenic action, through which the protein can bind VEGF-165 [37]. It is possible that a GWQRRL/TVECRD motif that is common to the first C-terminal TS repeat of both ADAMTS-1 and -8, but absent from any other ADAMTS, may play an important role.

The aggrecanases: ADAMTS-1, -4, -5, -8, -9 and -15

Aggrecan is the major proteoglycan of cartilage and is responsible for the ability of this tissue to resist compression by hydrating and swelling against the type II collagen scaffold [66]. It contains two N-terminal globular domains, G1 and G2, separated by an IGD (interglobular domain), followed by a GAG-attachment region and a C-terminal globular domain, G3 (see Figure 3) [7]. The G1 domain interacts with hyaluronic acid and link protein to form large aggregates that are trapped within the cartilage collagen matrix. Aggrecan protects cartilage collagen from degradation [67]. The major cleavage sites that cause aggrecan depletion from cartilage are in the IGD domain, since aggrecan which lacks the G1 domain is free to exit the matrix and so no longer contributes to cartilage function [68]. Two major proteolytic cleavage sites have been identified in this domain: one at Asn³⁴¹–Phe³⁴², at which all MMPs present in cartilage primarily act, and one at Glu³⁷³–Ala³⁷⁴, which is an aggrecanase cleavage site. There are also additional aggrecanase cleavage sites, but these occur within the GAG-attachment region between the G2 and G3 globular domains: Glu¹⁴⁸⁰–Gly¹⁴⁸¹, Glu¹⁶⁶⁷–Gly¹⁶⁶⁸, Glu¹⁷⁷¹–Ala¹⁷⁷² and Glu¹⁸⁷¹–Leu¹⁸⁷² [38,69].

The most studied aggrecanases are ADAMTS-4 and ADAMTS-5. These enzymes cleave aggrecan at the Glu³⁷³–Ala³⁷⁴ bond, and at the four other sites within the GAG-attachment region mentioned above. Cleavage of these other sites in vitro appears to be more efficient than cleavage within the IGD at the Glu³⁷³–Ala³⁷⁴ bond [38,69]. Aggrecan fragments generated by cleavage at all of these sites by ADAMTS-4 and ADAMTS-5 have been identified in bovine articular cartilage explants treated with IL-1 and tumour necrosis factor to stimulate aggrecan release [70]. ADAMTS-4 and ADAMTS-5 can also cleave the chondroitin sulphate proteoglycans brevican, predominantly expressed in the central nervous system [71], and versican, present in blood vessels [72]. When processed at the C-terminus, ADAMTS-4 is also active against carboxymethylated transferrin, fibromodulin and decorin (discussed above) [23]. ADAMTS-1 is also an aggrecanase [43,46,73], and cleaves versican at the same bond as does ADAMTS-4, although ADAMTS-4 can cleave both versican and aggrecan more efficiently [72]. Recently, ADAMTS-9 was also identified as an aggrecanase and versicanase, cleaving aggrecan at the Glu¹⁷⁷¹–Ala¹⁷⁷² bond and versican at the Glu⁴⁴¹–Ala⁴⁴² bond [11]. According to a patent application filed by the Yamanouchi company in Japan, ADAMTS-15 is also an aggrecanase [74]. Recombinant ADAMTS-8 was also recently demonstrated to possess weak (approx. 3000-fold lower than ADAMTS-4) aggrecanase activity at the Glu³⁷³–Ala³⁷⁴ bond [75].

Outside of a possible role in cartilage metabolism, the aggrecanases may have other functions in physiology or pathology. Adamts1-null mice have growth retardation, malformation of adipose tissue, decreased fertility with changes in histology of uterus and ovaries, and a renal phenotype similar to the human congenital disease ureteropelvic junction obstruction [76–78]. ADAMTS1 is also a transcriptional target of the progesterone receptor during ovulation [61], and is up-regulated in bone and osteoblasts by parathyroid hormone and related agents [60]. ADAMTS1 was down-regulated in endothelial cells derived from the livers of cirrhotic animals [79], and is also reduced in the subcutaneous adipose tissue of obese mice [80]. The ability of ADAMTS-4 to degrade brevican may implicate it in central nervous system physiology and pathology [81]: for example, increased aggrecanase-mediated brevican cleavage is a feature of invasive gliomas [71].

The procollagen N-proteinases: ADAMTS-2, -3 and -14

ADAMTS-2, -3 and -14 are procollagen N-proteinases, involved in the processing of procollagens to collagen by removal of the N-terminus propeptides [31,82–84]. ADAMTS-2 acts on procollagen I, II and III, whereas ADAMTS-3 and ADAMTS-14 have been linked only with procollagen II and procollagen I processing, respectively. Mutations in the ADAMTS2 gene cause dermatosparaxis in sheep and cattle and EDS (Ehlers–Danlos syndrome) type VIIc in humans [85], an autosomal recessive disorder characterized by severe skin fragility, joint laxity (hyperextensibility) and characteristic facies (short stature, micrognathia, epicanthic folds and depressed nasal bridge) [86]. In six individuals with EDS type VIIc so far described, all were homozygous for point mutations in the ADAMTS2 gene, resulting in a premature stop codon. A calf with dermatosparaxis had a 17 bp deletion, which resulted in a frame-shift mutation [85]. Adamts2-null mice, although grossly normal at birth, developed severe skin fragility after 1–2 months. It was noted that males are sterile, although Adamts2-null females have normal fertility [87]. The progressive skin fragility appeared to be caused not only by the abnormal morphology of type I collagen fibrils, distorted by a large proportion of uncleaved N-propeptides, but also by the failure of these fibrils to increase in diameter, a feature essential for their tensile strength. These results suggest that ADAMTS-2 is essential for maturation of type I collagen fibrils in skin, and that neither ADAMTS-3 nor ADAMTS-14 compensate adequately for ADAMTS-2 deficiency in this tissue.

The GON-ADAMTSs: ADAMTS-9 and -20

ADAMTS-9 was first described by Clark et al. [88] in 2000, and was found to share a high degree of sequence homology with the C. elegans ADAMTS gene gon-1 (discussed in the ‘ADAMTS in non-mammalian species’ section below). The gon-1 gene derives its name from its essential role in gonadal development in C. elegans [89]. It was later confirmed that the initially identified human ADAMTS-9 molecule, with three C-terminal TS repeats and no gon domain, was a short isoform when a longer molecule, containing 14 C-terminal TS repeats and a gon domain (a C-terminal region containing ten cysteine residues, similar to the C-terminal domain of gon-1) was identified [11,12], suggesting that the gene is alternatively spliced. Whether or not ADAMTS-9 has a role in mammalian gonadal development is not yet known.

Human ADAMTS9 has been mapped to chromosome 3 at band p14.2–p14.3, a common aphidicolin fragile site (a point on the chromosome that is liable to show gaps and breaks) [90], which is both a translocation breakpoint in hereditary renal cell carcinomas [91,92], and is frequently deleted in various other human cancers, including breast carcinoma [93]. Clark et al. [88] state that this locus is thought to contain one or more tumour suppressor genes, and that ADAMTS9 may be a possible candidate, especially as they describe abrogation of ADAMTS9 expression in ovarian and renal tumours. In our own studies of ADAMTS gene dysregulation in human breast carcinoma, we saw a down-regulation of ADAMTS9 by 83% with respect to non-neoplastic mammary tissue [49].

ADAMTS-20 shares its modular arrangement with that of the long isoform of ADAMTS-9, having 14 C-terminal TS repeats and a gon domain [11,12]. A shorter isoform of ADAMTS-20 has been identified in both humans [12] and the mouse [94], with ten and eight C-terminal TS repeats respectively. Point mutations in the mouse Adamts20 gene cause the pigment defect in belted (bt) mutant mice, a mostly pigmented phenotype with a dorsal non-pigmented region proximal to the hind limbs that appears as a white belt [94].

vWFCP/ADAMTS-13

vWFCP has been identified as ADAMTS-13 [16,95–97]. The existence of vWFCP was known for some years before its identity as an ADAMTS was elucidated [98,99]. Its substrate is vWF, a large multimeric glycoprotein present in plasma, platelets and vascular endothelial cells. vWF is a carrier protein for clotting factor VIII, supports platelet aggregation, and also mediates platelet adhesion to areas of vascular damage by binding both to glycoproteins on the surface of platelets and to exposed ECM components [97,100]. The shear stress to which vWF is subjected in the circulation leads to its enhanced proteolytic susceptibility [101]. Deficiency of vWFCP/ADAMTS-13 is responsible for TTP (thrombotic thrombocytopenic purpura), a pathological condition characterized by the formation of microvascular vWF- and platelet-rich thrombi, and associated with anaemia, renal failure and neurological dysfunction [96]. On the basis of present evidence, there are two basic mechanisms for ADAMTS-13 deficiency: (a) homozygous or compound heterozygous gene mutations; and (b) anti-(ADAMTS-13) autoantibody formation [102].

ADAMTS-13 shows the least homology with any other member of the ADAMTS family (notably, it has a much smaller pro-domain than any other), and is the only human ADAMTS so far described to contain a CUB domain [16]. ADAMTS13 is a large gene comprising 29 exons and encoding a product of 1427 amino acids, with evidence of additional variants due to alternative splicing [96].

Analysis of C-terminal deletion mutants of ADAMTS-13 has shown that the ability to cleave vWF in vitro requires the presence of the spacer region, though the more C-terminal TS and CUB domains were dispensable [103]. However, it seems likely that the C-terminal TS and CUB domains are relevant for the physiological activities of ADAMTS-13 in vivo, since TTP individuals with mutations in these regions of the gene have severe ADAMTS-13 deficiency [96,104]. Another curious feature of ADAMTS-13 is that the unusually short propeptide region seems not to be necessary for secretion, folding or maintenance of enzyme latency [34]. Mutation of the furin cleavage site or deletion of the entire pro-region led to secretion of pro-ADAMTS-13, which was fully functionally active. It is thus not clear at present what function the pro-region of ADAMTS-13 may perform, though it would seem there are no TTP-inherited mutations yet described that affect this region [96,104–106].

To date, 20 mutations (including frame-shifts, splice site mutations and amino acid substitutions) have been identified in the ADAMTS13 gene that lead to a deficient level of protein, thereby causing TTP in affected individuals [96,104,106]. These were spread across many exons, and no clustering of mutations was seen in any particular exon. A study by Kokame et al. [105] investigated four ADAMTS13 mutations in two Japanese families with a history of Upshaw–Schulman syndrome, the congenital form of TTP, and found that one of the four mutations did not completely abrogate enzyme activity. Furthermore, this mutation was found to be present in nearly 10% of the Japanese population. Two of the mutations prevented secretion of ADAMTS-13.

The development of autoantibodies to ADAMTS-13 occurs sporadically, although concordance in monozygotic twins has been reported, which implicates a genetic predisposing factor [107]. A recent study of sera obtained from 25 patients with anti-(ADAMTS-13) autoantibodies showed that, in the majority of cases, autoantibody reaction is directed against several epitopes along the entire ADAMTS-13 molecule, and that the cysteine-rich/spacer region is the only domain to be consistently involved in antibody reactivity [108]. This underlines the functional importance of this region for ADAMTS-13 activity [96,104]. An additional point of interest is that autoantibody reaction was directed against the pro-domain of ADAMTS-13 in 20% of cases, reinforcing the study by Majerus et al. [34] discussed above.

The orphan ADAMTSs

The unofficial term ‘orphan ADAMTS’ has been used by Dr S. S. Apte (http://www.lerner.ccf.org/bme/apte/adamts) to refer to those ADAMTS proteins with no known function or substrate to date. These are currently ADAMTS-6, -7, -10, -12 and -16–19 inclusive. Our studies have shown that ADAMTS6 and ADAMTS18 are dysregulated in breast carcinoma [49], and ADAMTS12 and ADAMTS16 are dysregulated in osteoarthritis [51].

A new study on ADAMTS-7 shows that the large spacer region between TS repeats 4 and 5 of the previously described long isoform, ADAMTS-7B, is a mucin domain that undergoes extensive post-translational O-glycosylation and within which is attached one or more chondroitin sulphate chains. This suggests that ADAMTS-7B may function as a proteoglycan in some tissues and cells, which has implications for its substrate recognition and cellular localization [14]. The sequence of ADAMTS-12 also predicts a mucin domain, and these two proteins therefore constitute a unique subgroup within the ADAMTS family. No substrate has been identified for ADAMTS-7, although aggrecanase activity has been ruled out.

A new paper has described ADAMTS10 mutations in three families with autosomal recessive WMS (Weill-Marchesani syndrome), predicting premature truncation of the protein within the metalloproteinase domain [109]. The clinical features of WMS, which include short stature, brachydactyly, eye and cardiac anomalies and progressive joint stiffness, suggest a role for ADAMTS-10 both in growth and in skin, lens and cardiac development in humans.