Review

Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics

Johanne Dubail and Suneel S. Apte⁎ Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

Correspondence to Suneel S. Apte: Department of Biomedical Engineering (ND20), Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, USA. [email protected] http://dx.doi.org/10.1016/j.matbio.2015.03.001 Edited by R. Iozzo

Abstract The mammalian ADAMTS superfamily comprises 19 secreted metalloproteinases and 7 ADAMTS-like proteins, each the product of a distinct gene. Thus far, all appear to be relevant to extracellular matrix function or to cell–matrix interactions. Most ADAMTS functions first emerged from analysis of spontaneous human and animal mutations and genetically engineered animals. The clinical manifestations of Mendelian disorders resulting from mutations in ADAMTS2, ADAMTS10, ADAMTS13, ADAMTS17, ADAMTSL2 and ADAMTSL4 identified essential roles for each gene, but also suggested potential cooperative functions of ADAMTS proteins. These observations were extended by analysis of spontaneous animal mutations, such as in bovine ADAMTS2, canine ADAMTS10, ADAMTS17 and ADAMTSL2 and mouse ADAMTS20. These human and animal disorders are recessive and their manifestations appear to result from a loss-of-function mechanism. Genome-wide analyses have determined an association of some ADAMTS loci such as ADAMTS9 and ADAMTS7, with specific traits and acquired disorders. Analysis of genetically engineered rodent mutations, now achieved for over half the superfamily, has provided novel biological insights and animal models for the respective human genetic disorders and suggested potential candidate genes for related human phenotypes. Engineered mouse mutants have been interbred to generate combinatorial mutants, uncovering cooperative functions of ADAMTS proteins in morphogenesis. Specific genetic models have provided crucial insights on mechanisms of osteoarthritis (OA), a common adult-onset degenerative condition. Engineered mutants will facilitate interpretation of exome variants identified in isolated birth defects and rare genetic conditions, as well as in genome-wide screens for trait and disease associations. Mammalian forward and reverse genetics, together with genome-wide analysis, together constitute a powerful force for revealing the functions of ADAMTS proteins in physiological pathways and health disorders. Their continuing use, together with genome-editing technology and the ability to generate stem cells from mutants, presents numerous opportunities for advancing basic knowledge, human disease pathways and therapy. © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction The first A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (ADAMTS) proteases were discovered in the late 1990s [1–3], and the first ADAMTS-like proteins (ADAMTSL) were cloned shortly after [4,5]. ADAMTSLs resemble the non-catalytic domains of ADAMTS proteases and lack a protease domain [6]. They are not proteases, nor do they arise by alternative splicing of ADAMTS protease genes. There are 19 ADAMTS genes and 7 ADAMTS-like proteins

(ADAMTSL1-6 and papilin) in humans, mice and other mammals [6]. The gene name ADAMTS11 was given in error to a gene already designated as ADAMTS5 [2,7]; thus the term ADAMTS11 is no longer used. Phylogenetic analysis showed considerable expansion of the mammalian ADAMTS family, compared to a much smaller repertoire in Caenorhabditis elegans, Drosophila melanogaster and Ciona intestinalis [8] (see review by Kim and Nishiwaki in this special issue). Mammalian ADAMTS gene expansion is postulated to have arisen primarily by gene duplication, representing both sub-functionalization

0022-2836/© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Matrix Biol (2015) 44-46, 24–37

25

Insights on ADAMTS proteases and ADAMTS-like proteins

and neo-functionalization of the duplicated genes [8]. Sub-functionalization is suggested by the shared or cooperative function of a subset of ADAMTS proteases in cleavage of proteoglycans such as aggrecan and versican [9], and involvement of several ADAMTS proteins in fibrillin microfibril biology [10]. ADAMTS neo-functionalization is exemplified by ADAMTS13, whose major function seems to be hemostatic [11] and possibly related to evolution of a closed circulatory system. Compared to other metalloprotease families, such as matrix metalloproteinases (MMPs), which have been studied for over half a century [12], the ADAMTS field has thus existed only for a decade and a half. It is important to point out that ADAMTS proteases are genetically distinct from the membrane-anchored A disintegrin and metalloprotease (ADAMs). Whereas membrane localization endows ADAMs with a major role in protein ectodomain shedding from the cell surface [13], ADAMTS proteases are secreted and primarily associated with proteolytic events in extracellular matrix (ECM). There are distinct differences in their respective domain structures, the hallmark of ADAMTS proteases being a C-terminal ancillary domain having a characteristic modular structure that contains one or more thrombospondin type 1 repeats (TSRs). Although there are totally 26 ADAMTS proteins to be considered in defining individual and collective functions of the family, and for determining their shared operational principles in biological networks, genetics has already proved to be the primary source of functional insights. This was inevitable because ADAMTS proteins are not as amenable to biochemical and cellular analysis as MMPs and ADAMs. For example, MMPs are readily expressed, refolded and purified in quantity from eukaryotic expression systems and readily cleave peptide substrates [14]. Analysis of ADAMs was enabled by the finding that they lacked stringent substrate sequence specificity, and mediated ectodomain shedding via a “lawn-mower” effect enabled by their membrane anchorage [15]. Thus, ADAM activities are typically determined by use of transfected constructs in which the target substrate's ectodomain carries an enzymatic reporter such as alkaline phosphatase, while retaining the membrane-proximal segment of the ectodomain [16]. In contrast, ADAMTS protease biochemistry provides many challenges, e.g., these proteases are large, have numerous disulfide bonds, the majority carry heavy and specialized glycosylation, are poorly secreted by cells, sticky and susceptible to autocatalysis, which disrupts the ancillary domain. Unlike MMPs, ADAMTS protease domains expressed in isolation do not typically have specific activity, since the ancillary domains mediate substrate recognition [17–21]. Furthermore, unlike MMPs, most ADAMTS proteases do not cleave short peptide substrates, and some may require substrate

post-translational modifications as a prerequisite for cleavage that are absent in synthetic substitutes. For example, ADAMTS5 proteolysis uses the chondroitin sulfate chains of versican as a crucial determinant for proteolysis [17], and ADAMTS13 processing of von Willebrand factor (vWF) requires shear stress-mediated unfolding of the region containing the scissile bond [22]. Given these challenges, it has been fortunate that genetics has stepped into the breach. In contrast to other metalloproteinases, the ADAMTS field has benefited considerably from forward genetics, i.e., resolution of the genetic basis of spontaneous mutants, which exist for several ADAMTS genes. Reverse genetics, i.e., where genetically engineered or chemically induced mutants are generated and the resulting phenotype analyzed to define gene function, has advanced understanding of the same genes as well as others of previously unknown function. That ADAMTS forward genetics has revealed the basis of extremely rare inherited disorders does not diminish its value, since the mutations revealed previously unsuspected biological pathways. As a case in point, the discovery that rare mutations in the low-density lipoprotein receptor caused hypercholesterolemia paved the way for cholesterol-lowering therapy [23]. ADAMTS genetics has contributed new knowledge of extracellular matrix assembly and degradation in morphogenesis, reproductive biology and many organ systems and processes as reviewed here. Other reviews in this special issue have covered the role of ADAMTS proteases in cancer (see review by Cal and Lopez-Otin) and cancer-related processes such as angiogenesis (see review by Rodriguez-Manzaneque and Iruela-Arispe), as well as fertility (see review by D. Russell), nervous system disorders (see review by P. Gotschall) and procollagen processing (see review by Bekhouche and Colige).

ADAMTS genomics In contrast to a well-defined genomic MMP cluster on human chromosome 11q22 [24], ADAMTS genes are dispersed throughout the human and mouse genomes. However, three pairs of ADAMTS genes are tightly linked in regions of human-mouse synteny [25], and of these, two present potential challenges for mouse genetic engineering. These two pairs include evolutionarily related ADAMTS proteases, namely, ADAMTS1/ADAMTS5 and ADAMTS8/ADAMTS15 on human chromosomes 21 and 11 respectively; the corresponding mouse loci are linked on mouse chromosomes 16 and 9 respectively. Because these genes have a close evolutionary relationship, there is a possibility that they have cooperative functions. However, tight genetic linkage precludes elucidation of cooperative functions by crossing knockout mice (such as of Adamts1 and Adamts5), because the odds

26

Insights on ADAMTS proteases and ADAMTS-like proteins

Table 1. Mendelian conditions resulting from mutations in human ADAMTS protease genes Mendelian condition

MIM number

Gene name, chromosomal locus

Inheritance

225410

ADAMTS2, 5q35.3 [57]

Autosomal recessive

277600

ADAMTS10/19p13.2 [62]

Autosomal recessive

274150

ADAMTS13, 9q34.2 [81]

Autosomal recessive

613195 615458

ADAMTS17, 15q26.3 [68] ADAMTS18, 16q23.1 [82]

Autosomal recessive Autosomal recessive

Ehlers–Danlos syndrome (EDS), dermatosparaxis type or type VIIC Weill–Marchesani syndrome 1/Weill–Marchesani syndrome, autosomal recessive/Mesodermal dysmorphodystrophy, congenital Thrombotic thrombocytopenic purpura, congenital/Upshaw–Schulman syndrome Weill–Marchesani-like syndrome Microcornea, myopic chorioretinal atrophy and telecanthus (MMCAT)

of recombination between closely linked loci are extremely poor. The third genetic linkage involves ADAMTS13, whose locus is linked to that of ADAMTSL2, on human chromosome 9q34 and mouse chromosome 2. These two genes are 120 and 100 kb apart in the human and mouse genomes respectively, with CACFD1 intervening in the human genome, plus two other intervening genes, Slc2a6 and Tmem8c in mouse. In contrast to the first two pairs, the linkage of ADAMTSL2 and ADAMTS13 is thought to be coincidental, or to represent a very distant gene duplication event. ADAMTSL2 is implicated in an inherited connective tissue disorder named geleophysic dysplasia [10,26], which has no clinical overlap with thrombotic thrombocytopenic purpura resulting from ADAMTS13 deficiency. Because of the intervening genes in the syntenic regions, ADAMTS13 and ADAMTSL2 genes probably do not share regulatory regions.

Key biochemical and structural aspects of ADAMTS proteases relevant to understanding genetic defects ADAMTS proteases comprise an N-terminal protease domain containing the catalytic activity and a C-terminal ancillary domain. Biochemical and 3-dimensional structural data indicate that ADAMTS proteases have a similar catalytic mechanism as MMPs and ADAMs, employing a Zn-atom coordinated by three conserved His residues [27,28]. All ADAMTS proteases, but not ADAMTSLs, have an N-terminal propeptide, which like that of MMPs and ADAMs, is thought to be inhibitory to catalytic activity and requires excision. However, ADAMTS9 and ADAMTS13 zymogens appear to be active despite retention of the propeptide, which also happens to be unusually short in ADAMTS13 [29,30]. Propeptide excision in ADAMTS proteases is mediated by proprotein convertases such as furin [30–37]. These serine proteases efficiently cleave after a consensus sequence Arg-Xaa-(Arg/Lys)-Arg, with the cleavage thought to occur in the trans-Golgi, or at the cell surface [35,38]. Upon secretion,

ADAMTS proteins appear to have a propensity for binding to the cell surface/pericellular matrix, and ADAMTS13, which is synthesized in the liver and by vascular endothelium, is present in the circulation, where it processes vWF. During their biosynthesis, ADAMTS proteins undergo extensive post-translational modification, including disulfide bonding, N- and O-glycosylation, and in the case of ADAMTS7, addition of chondroitin–sulfate chains [35]. Most ADAMTS proteins are modified by C-mannosylation of Trp residues, and O-fucosylation of Ser/Thr residues in the TSRs [39–41]. The latter modification, resulting in addition of either a fucose monosaccharide or a fucose–glucose disaccharide, requires sequential action of two specialized glycosyltransferases, POFUT2 and B3GLCT [42,43]. The fate of ADAMTS proteins after release from cells is poorly understood. TIMP-3 and α2-macroglobulin are known endogenous inhibitors of ADAMTS2 [44] the aggrecanases ADAMTS4 and ADAMTS5 [45,46], and others, but these inhibitors have not been tested against the entire family. Some ADAMTS proteins undergo fragmentation, with potentially new functions of the derivative fragments [47], and recent work has identified a cellular clearance mechanism for ADAMTS proteases based on the LRP-1 receptor [48–50]. ADAMTS proteases are implicated in fibrillar procollagen maturation (ADAMTS2, ADAMTS3), versican turnover during embryogenesis (ADAMTS1, ADAMTS5, ADAMTS9, ADAMTS15, ADAMTS20) and ovulation (ADAMTS1), cartilage aggrecan destruction in arthritis (ADAMTS4, ADAMTS5), von Willebrand factor proteolysis and hemostasis (ADAMTS13), and in VEGF-C activation during lymphangiogenesis (ADAMTS3) [51].

Insights from spontaneous animal mutants and human Mendelian disorders Dermatosparaxis, a classic connective tissue disorder, manifests with severe skin fragility. Indeed, affected skin was described as having the consistency of wet blotting paper. It was first identified in

Insights on ADAMTS proteases and ADAMTS-like proteins

inbred cattle, and subsequently in sheep and cats [52–54]. Dermatosparactic skin is characterized by the accumulation of abnormal collagen fibrils having a “hieroglyphic” ultrastructural appearance in the dermis that is the hallmark of this disorder [53]. The underlying mechanism was identified a few decades ago as deficiency of the specific enzymatic activity required for removal of the N-propeptide of procollagen I, the major collagen type in the dermis of skin [55]. Persisting pN-collagen I, the resulting incompletely processed intermediary of collagen maturation, prevents tight packing of collagen molecules by steric hindrance. Hence structurally competent fibrils are not formed, and as a result, the dermis is extremely weak. Later, a human connective tissue disorder, Ehlers– Danlos syndrome type VIIc (or dermatosparaxis type) having similar skin fragility and collagen fibril anomalies was identified [56] (Table 1) and both the bovine and human conditions were attributed to ADAMTS2 mutations in the respective species [57]. One of the persisting conundrums of this disorder is why bone, cartilage, tendons and arteries are not as severely affected as skin, despite containing fibrillar collagens as a quantitatively major structural component, and fibrillar collagen types I, II, III and V being ADAMTS2 substrates [58]. It is possible that this could be due to compensating activities of closely related enzymes with similar activity, such as ADAMTS3 and ADAMTS14 [59,60]. ADAMTS3 is highly expressed in cartilage, where collagen II is a major component, as well as in embryonic bone and tendon, suggesting that it could be a major procollagen processing enzyme in musculoskeletal tissues [60,61]. ADAMTS3 was recently shown to participate in proteolytic activation of the pro-angiogenic and pro-lymphangiogenic factor VEGF-C via binding to a putative co-factor, collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) [51]. The cleavage site in VEGF-C has some similarities to that in procollagens, and the presence of a C-terminal domain with collagenous repeats in CCBE1 may provide a basis for its interaction with ADAMTS3. Unpublished data indicates that embryos lacking Adamts3 do not survive past late gestation (Dubail, J. and Colige, A., unpublished data). Presently, the roles of ADAMTS3 and ADAMTS14 in procollagen processing remain to be fully elucidated, and the role of ADAMTS3 in lymphangiogenesis remains to be validated in vivo. Procollagen N-propeptidases are reviewed extensively by Bekhouche and Colige in this issue. ADAMTS10 mutations lead to Weill–Marchesani syndrome (WMS1), with short stature, brachydactyly, cardiac valve stenosis and ectopia lentis (dislocation of the lens) being the major clinical features (Table 1) [62]. Since WMS is also caused by dominantly inherited fibrillin-1 mutations (WMS2) [63], a functional relationship between ADAMTS10 and tissue microfibrils was suggested by these

27 genetic findings. Tissue microfibrils are supramolecular structures in extracellular matrix that are assembled from large glycoproteins named fibrillins, among which, fibrillin-1 predominates in adult tissues. The potential relationship of ADAMTS10 with microfibrils was confirmed by the finding that ADAMTS10 bound to fibrillin-1 and fibrillin-2 in vitro, and localized to fibrillin microfibrils [64]. ADAMTS10 lacks a perfect consensus sequence for furin processing and its zymogen is inefficiently cleaved. Consistent with this, and despite its specific binding to fibrillin-1, ADAMTS10 cleaves fibrillin-1 poorly [64]. Because ectopia lentis results from a structural weakness of the fibrillin-rich structure called the zonule that centers the lens in the optic path, it is possible that ADAMTS10 is primarily required for formation or stability of the zonule. Indeed, ADAMTS10 was shown to enhance microfibril networks in cultured fibroblasts [64], in which regard it was similar to ADAMTSL4 and ADAMTSL6 [65,66], suggesting it might function as an ADAMTS-like protein rather than as a protease. A WMS-like phenotype in humans and ectopia lentis in dogs result from ADAMTS17 mutations [67,68] (Table 1). Affected individuals have ectopia lentis and short stature, but not brachydactyly or congenital cardiac anomalies [68]. Unlike ADAMTS10, ADAMTS17 has a classic furin consensus site, and its zymogen is probably processed to an active protease. However, little is presently known about ADAMTS17 protein, including whether or not it binds to fibrillins, localizes to microfibrils or affects microfibril biogenesis. In another example of a zonule-related ADAMTS function, recessive isolated ectopia lentis and ectopia lentis et pupillae are caused by ADAMTSL4 mutations [69–71]. A mutation commonly associated with these disorders, (c767_786Del), is thought to represent an ancient founder effect [71]. ADAMTSL4 is widely distributed in various eye components as well as in non-ocular tissues; recent in situ hybridization data (Hubmacher. D., and Apte, S.S., unpublished) show that Adamtsl4 mRNA is strongly expressed in the lens equatorial epithelium whence the zonule attaches [65,72]. An ADAMTSL4 mutation was also found to cause craniosynostosis in association with ectopia lentis, potentially related to its extraocular expression [73]. Like ADAMTSL4 mRNA, ADAMTS10 and ADAMTS17 mRNAs are also widely expressed [34,74,75], suggesting that they function in many organs, where they may overlap or work cooperatively in the context of tissue microfibrils. The specific phenotypes observed in each gene defect may therefore reflect those sites that are the least over-engineered in terms of overlapping expression of a compensating microfibril-related ADAMTS protein. ADAMTSL2 mutations cause geleophysic dysplasia, which presents with short stature, stiff skin, joint

28

Insights on ADAMTS proteases and ADAMTS-like proteins

Table 2. Spontaneous and engineered mutations in rodent ADAMTS genes Phenotype −/−

– Growth retardation [86,132] – Urologic abnormalities possibly underlying ~ 50% post-natal lethality [86,132] – Adrenal abnormalities [86] – Reduced female fertility [86,87,89,133] – Diminished levels of synaptic proteins in the female frontal cortex [134] −/− ; – Urologic abnormalities associated with ~95% post-natal lethality [135] Adamts1 −/− Adamts4 – Skin fragility [136] Adamts2−/− – Male sterility [136] – No apparent phenotype [137] Adamts4−/− – Reduced versican cleavage during spinal cord injury [138] – Reduced cartilage loss in osteoarthritis (OA) models [139–141] Adamts5−/− – Low penetrance of soft tissue syndactyly [101] – Reduced sculpting of pulmonic valves; myxomatous mitral valve [95] – Reduced versican cleavage during myogenesis [142] – Impaired contraction and dermal collagen deposition in an excisional skin wound healing model [143] – Aggrecan accumulation and impaired tendon function [144] A d a m t s 4 − / − ; – Diminished severity of lesions in an induced OA model similar to Adamts5−/− [145] −/− Adamts5 – No reported developmental anomaly. Altered response to vascular injuries [129,130] Adamts7 −/− – Lethal at 7.5 days of gestation [100] Adamts9−/− – Cardiac and aortic anomalies [98] Adamts9+/− – Ocular anterior segment dysgenesis (Dubail, S., and Apte, S.S., unpublished) and spontaneous corneal neovascularization [99] A d a m t s 9 + / − ; – Extensive white spotting [85] Adamts20bt/bt – Soft tissue syndactyly (complete penetrance) [101] – Cleft secondary palate (complete penetrance) [100] +/− ; – Delayed palate closure [100] Adamts9 Adamts20bt/+ A d a m t s 9 + / − ; – High penetrance of soft tissue syndactyly [101] Adamts5+/− A d a m t s 9 f l / f l ; – Soft tissue syndactyly (complete penetrance) [97] Prx-Cre A d a m t s 9 f l / f l ; – Soft tissue syndactyly (complete penetrance; greater severity than Adamts9fl/fl;Prx-Cre) [97] P r x - C r e ; Adamts5−/− Adamts12−/− – Increased tumor growth and angiogenesis [146] – Increased inflammation and delayed recovery in an induced colitis model, endotoxic sepsis and pancreatitis [147] – Heightened bronchial inflammation and hyperresponsiveness in an allergic airways disease model [148] −/− – Thrombocytopenia in CASA/Rb mouse strain with elevated plasma vWF [149] Adamts13 – TTP-like syndrome after shigatoxin challenge [149] – Larger infarct after transient occlusion of left coronary artery [150] Adamts16−/− (rat) – Lower systolic blood pressure [103] – Cryptorchidism and infertility in males [102] – Cataract and reduced body fat (http://www.mousephenotype.org/data/genes/MGI:2442600#section-associations) Adams18−/− – White spotting of the ventral and dorsal lumbar region [85] Adamts20bt/bt – Low penetrance of soft tissue syndactyly [101] – Delayed palate closure [100] bt/bt ; – Soft tissue syndactyly (complete penetrance) [101] Adamts20 Adamts5−/− Adamts1

contractures, liver, tracheo-pulmonary and cardiac anomalies, leading frequently to juvenile death [26]. Interestingly, an ADAMTSL2 point mutation, Arg221Cys, which was found in geleophysic dysplasia and leads to anomalous disulfide bonded dimers, causes Musladin–Lueke syndrome (MLS) in beagle dogs via a founder effect [76]. Joint contractures, small stature and stiff skin are prominent clinical features in affected beagles, but in contrast to geleophysic dysplasia, pulmonary or cardiac abnormalities are absent, and the affected dogs appear to have a normal lifespan. These phenotypes are likely

to be microfibril-related, because ADAMTSL2 bound both to fibrillin-1 and to one of its interacting partners, latent TGFβ-binding protein 1 [26,77]. Intriguingly, Adamtsl2 null mice die shortly after birth with severe lung anomalies associated with a striking increase in fibrillin-2 containing microfibrils in the bronchial wall (Hubmacher and Apte, manuscript submitted). These interspecies differences in the outcome of ADAMTSL2 mutations are not an isolated example, since Adamts10 inactivation in mice did not precisely phenocopy WMS1; although Adamts10 null mice are

Insights on ADAMTS proteases and ADAMTS-like proteins

slightly smaller in size, they lack specific skeletal and cardiac anomalies and did not develop ectopia lentis (Wang. L.W., Kutz, W.E, Apte S.S., manuscript in preparation). The interspecies differences may reflect the lack of fibrillin-3 in mice, the different expression patterns of the ADAMTS proteins in the three species, and the recent observation that the mouse zonule contains both FBN1 and FBN2, whereas the human zonule contains FBN1 [78,79]. ADAMTS13 is the most extensively studied of all ADAMTS proteases but is not directly related to extracellular matrix. It cleaves ultra-large vWF multimers, which are prothrombogenic because they favor platelet adhesion, into an optimal size required for normal hemostasis [22,80]. Recessive ADAMTS13 mutations [81], or inhibitory ADAMTS13 autoantibodies lead to abnormal platelet aggregation and widespread occlusion of microvessels by platelet thrombi, a condition termed thrombotic thrombocytopenic purpura. Recent reviews provided an extensive overview of this disorder [22,80]. ADAMTS18 mutations were described in a syndrome comprising microcornea, myopic chorioretinal atrophy and telecanthus (Table 1) [82,83]. Little is presently known about this protease. An Adamts20 mutant (belted, Adamts20 bt ) (Table 2) occurred spontaneously in a number of inbred mouse colonies, and is a white spotting phenotype affecting the mid-torso [84,85]. Adamts20bt affects proper colonization of hair follicles in that region by neural crest derived melanoblasts. Hair follicles, but not the intervening skin, are the exclusive domain of melanoblasts in mouse skin, and Adamts20 mRNA is expressed by dermal cells around melanoblasts, but not by melanoblasts [84]. ADAMTS20 is not required for neural crest cell migration per se, but acts noncell-autonomously in ensuring the proliferation and survival of melanoblasts once they reach the hair follicles [85].

Insights obtained from targeted gene inactivation in mice and rats Genetic engineering of mice, and uncommonly of rats, has provided null alleles, intragenic reporters for understanding gene expression, a means of conditional mutagenesis, and null cell lines derived from mutant animals. ADAMTS1 deficiency in mice leads to a high incidence of perinatal lethality, together with a high frequency of genitourinary anomalies such as hydronephrosis [86,87]. Surviving female Adamts1 null mice are infertile because ADAMTS1 is required for versican proteolysis during maturation and rupture of the ovarian follicle [88–90]. ADAMTS1 participates in versican proteolysis during myocardial compaction, a process in which cardiac myocytes form a functional myocardium during embryogenesis [91].

29 ADAMTS4 and ADAMTS5 are closely related enzymes termed aggrecanase-1 and -2 respectively, because of their implication in proteolytic destruction of aggrecan in osteoarthritic joint cartilage [92]. Proteolysis of aggrecan is a major pathogenic mechanism of arthritis, since it exposes other cartilage components, e.g., small leucine-rich proteoglycans and collagens to destruction by MMPs and other proteases. Based on the resistance of Adamts4 −/−;Adamts5 −/− mice to surgical or inflammation induced joint cartilage loss, the aggrecanases became major drug targets in arthritis. An attractive aspect of their biology was the apparent lack of critical physiological roles, suggesting that these proteases were excellent drug targets. Accordingly, a number of small-molecule active site inhibitors and function-blocking antibodies were generated and investigated preclinically [92,93]. However, based on its expression pattern obtained from an intragenic lacZ reporter in mice, ADAMTS5 is of potential significance to several organ systems [94]. For example, it is strongly expressed in cardiac outflow tract endocardial cushions, where it is required for versican proteolysis during the formation of valve leaflets, and is implicated in TGFβ signaling [95,96]. Indeed, Adamts5-deficient mice had reduced sculpting of pulmonic valves during embryogenesis, and myxomatous mitral valves in adult hearts [95]. Adamts4 −/− mice are apparently normal, and neither Adamts4 null nor Adamts5 null mice, nor mice lacking both these genes have any anomalies of cartilage turnover, endochondral ossification, or skeletal function. Thus, a physiological aggrecanase, if one exists at all, remains unidentified for now. ADAMTS9 distinguishes itself in several ways. It, along with ADAMTS20, is the largest and most conserved of all metalloproteases, with a clearly recognizable nematode ortholog. It contains a C-terminal domain found only in C. elegans Gon-1 and ADAMTS20. It is also by far the physiologically most crucial metalloprotease to be uncovered, with a startling number of essential functions spanning early, mid and late embryogenesis, and extending into juvenile development and the adult. ADAMTS9 is an absolute requirement for early mouse development, since embryos lacking this protease die during gastrulation [97]. ADAMTS9 haploinsufficiency leads to cardiac and aortic defects, and to a highly penetrant ocular anterior segment dysgenesis [98,99]. In combination with the Adamts20 bt homozygous mutant, Adamts9 haploinsufficiency leads to death at birth from cleft palate [100], and these mice have much greater white spotting than Adamts20 bt/ bt [85]. As further evidence of cooperativity of ADAMTS proteases, mice with combined Adamts5 and Adamts20 deficiency have a higher incidence of soft tissue syndactyly, which results from failure of interdigital web regression in the embryo, than

30 single mutants [101]. Interdigital webs are present not only in aquatic birds and bats, but also during embryogenesis in humans, mice, and other mammals, where they participate in development of digits. They regress by rapid sculpting after digit formation is complete, i.e., by massive apoptosis coupled with ECM proteolysis. ADAMTS proteolysis of versican in the interdigit matrix is required for apoptosis of interdigit mesenchymal cells, suggesting that these ADAMTS proteases couple matrix proteolysis to cell death during web regression [101]. A similar role for Adamts9 in web regression was elucidated first in combination with Adamts5 or Adamts20 and more recently, by its limb-specific conditional inactivation [97,101]. These studies demonstrate the utility of combining deficiency in two or more ADAMTS proteins sharing similar activity to elucidate cooperative and/or compensatory functions. Recently, rats with a targeted mutation of Adamts16 identified its potential role in regulation of blood pressure and male fertility [102,103] and other work has suggested a connection between Adamts16 and renal development [104].

ADAMTS genetic variation and human disease Epigenetic modification of ADAMTS proteins is associated with several cancers and is the subject of another review by Cal and Lopez-Otin in this special issue. Recent genome-wide association studies and transcriptome analysis have connected ADAMTS loci with several common traits and disorders. ADAMTS9 has been associated with multiple anthropometric traits, such as adiposity, age at menarche, adaptation to high altitude, and disorders such as obesity, type 2 diabetes, age-related macular degeneration (AMD), and arrest of cervical dilatation during labor [105–112]. The association with AMD was especially striking because another locus implicated in this condition encodes B3GLCT, the glucosyltransferase that modifies TSRs, which are most numerous in ADAMTS9 [42,113]. Genome-wide transcriptome analysis also identified reduction of ADAMTS9 mRNA in bicuspid aortic valve disease [114]. GWAS of pediatric stroke identified association with ADAMTS2, ADAMTS7, ADAMTS12 and ADAMTS13 [115]. ADAMTSL3 was identified as one of several loci linked to human height [116] and as a candidate locus in schizophrenia [117]. In contrast to causative gene mutations in Mendelian disorders, GWAS associations remain purely suggestive until functionally validated because the majority of single nucleotide polymorphisms (SNPs) map only to the vicinity of a gene locus, i.e., with few exceptions, they are not within the exons of the gene, and usually do not introduce amino acid changes in the proteins. Furthermore, location of the SNP in the intergenic or intronic

Insights on ADAMTS proteases and ADAMTS-like proteins

regions of an ADAMTS gene locus does not imply that it is necessarily in a regulatory region of that gene, since the SNP may actually affect regulation of another locus further away, e.g., if it lies within an enhancer. Thus, SNPs do not directly implicate the ADAMTS protease in that disease unless additional conditions are met, for which there are currently few examples. One such SNP for which this burden of proof has been partially met leads to a Ser 214Pro substitution in the ADAMTS7 propeptide and was associated with coronary artery disease [118]. Biochemical analysis following expression of the Ser and Pro variants suggested that the Pro variant quantitatively impaired ADAMTS7 propeptide excision by furin [119], which is thought to be a prerequisite for proteolytic activity. Thus, the Pro variant potentially has lower activity and individuals with the Pro/Pro ADAMTS7 protein are predicted to have reduced protease activity compared to those with Ser/Ser variants [119]. However, a definitive mechanistic link via an effector substrate, as well as the cell/tissue context in which ADAMTS7 acts to modify coronary artery disease remains elusive for now.

Genetics and rigorous evaluation of ADAMTS substrate significance The genetics and biology of thrombotic thrombocytopenic purpura suggests an exclusive protease-substrate relationship between ADAMTS13 and vWF, no other ADAMTS13 substrates are currently known. For other family members known to have multiple substrates, questions about the substrate's biological significance arise, since the substrates could very well be cleaved in vitro, but not in vivo, e.g., because the enzyme and putative substrate may never normally encounter one another. For example, numerous substrates have been identified for the prototypic ADAMTS protease, ADAMTS1, including chondroitin sulfate proteoglycans such as aggrecan and versican, collagen I, nidogen-1 and nidogen-2, the matricellular proteins thrombospondin-1 and -2, and the cell-anchored EGFR ligands HB-EGF and amphiregulin [120–126]. Of these, however, a significant biological impact, primarily using mouse genetic models has hitherto been established mostly for proteolysis of versican [89–91], which is also cleaved by ADAMTS4, ADAMTS5, ADAMTS9, ADAMTS15 and ADAMTS20 [33,36,85,94,95,97,100,126–128].

Mammalian genetics resources and their applications Many of the engineered and spontaneous mouse genetic models described here are available to users from repositories such as the Jackson Laboratories

31

Insights on ADAMTS proteases and ADAMTS-like proteins

Fig. 1. Schematic showing how Mendelian disorders and engineered rodent ADAMTS mutants, together with genome editing and induced pluripotent stem cells (IPSC), can be integrated into a discovery pipeline for substrates, biological pathway analysis, validation of genome-wide association studies (GWAS) or transcriptome surveys, and drug discovery.

(www.jax.org), or Mutant Mouse Regional Resource Centers (https://www.mmrrc.org). The NIH knockout mouse project (KOMP; www.komp.org), a member of the International Knockout Mouse Consortium (IKMC) (https://www.mousephenotype.org) is completing mutagenesis of all ADAMTS genes, with several mouse mutants already available. The targeting vectors used by IKMC are versatile, since they achieve a “knock-out first” mouse allele, which can be bred to homozygosity to achieve a null phenotype and includes an intragenic reporter such as lacZ for easy readout of gene expression patterns. These alleles can then be rendered suitable for conditional mutagenesis if desired. Another mouse genetic resource that is well established, but has not been widely tapped, and carries ADAMTS mutants, is the International Gene Trap Consortium (www.genetrap.org), which provides trapped mouse ES cell clones for a nominal fee. Gene trap mutants result from random insertions, and thus could potentially generate useful, diverse mutations to further understanding of mammalian biology. For example, our group has recently characterized an Adamts9 gene trap allele, in which this normally secreted molecule is converted into a constitutively membrane-anchored protease (Nandadasa et al., submitted).

Knowledge based resources in genetics keep pace with experimental developments. Information on human Mendelian disorders and individual mouse genes is updated regularly online and is readily accessible at the respective websites, http://www.omim.org and http://www.informatics. jax.org. Initiatives to comprehensively establish phenotypes resulting from mouse mutagenesis are also well under way (http://commonfund.nih. gov/KOMP2/index). Each of the above resources can be searched using the ADAMTS gene symbol.

Future directions and opportunities ADAMTS proteases are presently not as widely known and appreciated as MMPs and ADAMs in the ECM and protease communities. Their immense biological significance revealed by mammalian genetics shows that they are deserving of considerable attention. This review has attempted to show that when mammalian genetics is integrated into experimental biology and biochemistry (Fig. 1), a comprehensive, rigorous evaluation of ADAMTS function will emerge.

32 As the schematic in Fig. 1 illustrates, there are multidirectional information flows between human genetics, genetically engineered models, and in vitro experimental systems that will enrich our understanding of ADAMTS proteases and their role in extracellular matrix. The availability of experimental models for common acquired diseases, such as diabetes, fibrosis, and atherosclerosis, in mice, means that individual ADAMTS genes implicated by GWAS can be tested using knockout mice. For example, recent work used mice with inactivated Adamts7 alleles to explore its role in vascular disease [129,130]. Monoclonal and polyclonal antisera can be produced in viable ADAMTS knockout mice and rats, since they are more likely to mount an immune response to injected antigens derived from the protein product of the inactivated gene. The future holds the promise of using new gene editing technologies such as CRISPR/Cas9 to generate mutants at will in any experimental organism. One of the benefits of this technology is the ability to edit genes in cultured cells, eliminating or minimizing the use of mice to generate null cells. A potential application of this approach relevant to the discovery process is to mutate proteases in cell lines for high throughput proteomics comparing wild type and protease deficient cells (substrate discovery). Another is the testing of ADAMTS substrate determinants by mutating the cleavage sites or altering post-translational modifications for a complete understanding of proteolytic mechanisms. For example, mice with a knock-in mutation of the aggrecanase cleavage site that abrogated ADAMTS processing of aggrecan confirmed that proteolysis at this site was essential for cartilage loss in arthritis [131]. This remains the most rigorous test for identifying definitive, biologically meaningful substrates, and will now become much more accessible, rapid and inexpensive. The widespread utilization of induced pluripotent stem cell technology (IPSC) means that cells obtained from individuals or animals with rare Mendelian disorders or engineered rodents can be expanded and manipulated with ease in culture to understand the underlying biological pathways. Taken together with in vivo studies that analyze tissues and cells in their natural context in genetically engineered mice, these new technologies are likely to advance the ADAMTS field rapidly.

Acknowledgments Work in the authors' laboratory was supported by the National Institutes of Health (NIH) through awards EY021151, EY024943, Program of Excellence in Glycoscience award HL107147, by Sabrina's

Insights on ADAMTS proteases and ADAMTS-like proteins

Foundation, and by the Knights Templar Eye Foundation.

Received 18 February 2015; Received in revised form 3 March 2015; Accepted 4 March 2015 Available online 11 March 2015 Keywords: ADAMTS; ADAMTS-like; Metalloproteinase; Extracellular matrix; Mouse; Knockout; Forward genetics; Reverse genetics; Procollagen; Aggrecanase

References [1] Colige A, Li SW, Sieron AL, Nusgens BV, Prockop DJ, Lapiere CM. cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components. Proc Natl Acad Sci U S A 1997;94:2374–9. [2] Hurskainen TL, Hirohata S, Seldin MF, Apte SS. ADAMTS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family. J Biol Chem 1999;274:25555–63. [3] Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. J Biol Chem 1997;272:556–62. [4] Hirohata S, Wang LW, Miyagi M, Yan L, Seldin MF, Keene DR, et al. Punctin, a novel ADAMTS-like molecule (ADAMTSL-1) in extracellular matrix. J Biol Chem 2002;22:22. [5] Kramerova IA, Kawaguchi N, Fessler LI, Nelson RE, Chen Y, Kramerov AA, et al. Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases. Development 2000;127:5475–85. [6] Apte SS. A disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J Biol Chem 2009;284:31493–7. [7] Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, et al. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem 1999;274: 23443–50. [8] Huxley-Jones J, Apte SS, Robertson DL, Boot-Handford RP. The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into the vertebrate ADAMTS family. Int J Biochem Cell Biol 2005;37:1838–45.

Insights on ADAMTS proteases and ADAMTS-like proteins

[9] Nandadasa S, Foulcer S, Apte SS. The multiple, complex roles of versican and its proteolytic turnover by ADAMTS proteases during embryogenesis. Matrix Biol 2014;35:34–41. [10] Hubmacher D, Apte SS. Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function. Cell Mol Life Sci 2011;68:3137–48. [11] Zheng X, Majerus EM, Sadler JE. ADAMTS13 and TTP. Curr Opin Hematol 2002;9:389–94. [12] Gross J, Lapiere CM. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A 1962;48:1014–22. [13] Blobel CP. ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005;6:32–43. [14] Balbin M, Fueyo A, Knauper V, Lopez JM, Alvarez J, Sanchez LM, et al. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J Biol Chem 2001;276:10253–62. [15] Schlondorff J, Blobel CP. Metalloprotease-disintegrins: modular proteins capable of promoting cell–cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci 1999;112:3603–17. [16] Sahin U, Weskamp G, Zheng Y, Chesneau V, Horiuchi K, Blobel CP. A sensitive method to monitor ectodomain shedding of ligands of the epidermal growth factor receptor. Methods Mol Biol 2006;327:99–113. [17] Foulcer SJ, Nelson CM, Quintero MV, Kuberan B, Larkin J, Dours-Zimmermann MT, et al. Determinants of versican-V1 proteoglycan processing by the metalloproteinase ADAMTS5. J Biol Chem 2014;289:27859–73. [18] Gao W, Anderson PJ, Majerus EM, Tuley EA, Sadler JE. Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease. Proc Natl Acad Sci U S A 2006;103: 19099–104. [19] Gao W, Zhu J, Westfield LA, Tuley EA, Anderson PJ, Sadler JE. Rearranging exosites in noncatalytic domains can redirect the substrate specificity of ADAMTS proteases. J Biol Chem 2012;287:26944–52. [20] Kashiwagi M, Enghild JJ, Gendron C, Hughes C, Caterson B, Itoh Y, et al. Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J Biol Chem 2004; 279:10109–19. [21] Kuno K, Matsushima K. ADAMTS-1 protein anchors at the extracellular matrix through the thrombospondin type I motifs and its spacing region. J Biol Chem 1998;273:13912–7. [22] Zheng XL. Structure-function and regulation of ADAMTS13 protease. J Thromb Haemost 2013;11(Suppl. 1): 11–23. [23] Goldstein JL, Dana SE, Brunschede GY, Brown MS. Genetic heterogeneity in familial hypercholesterolemia: evidence for two different mutations affecting functions of low-density lipoprotein receptor. Proc Natl Acad Sci U S A 1975;72:1092–6. [24] Pendas AM, Matilla T, Estivill X, Lopez-Otin C. The human collagenase-3 (CLG3) gene is located on chromosome 11q22.3 clustered to other members of the matrix metalloproteinase gene family. Genomics 1995;26:615–8. [25] Koo BH, Le Goff C, Jungers KA, Vasanji A, O'Flaherty J, Weyman CM, et al. ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis. Matrix Biol 2007;26:431–41.

33 [26] Le Goff C, Morice-Picard F, Dagoneau N, Wang LW, Perrot C, Crow YJ, et al. ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation. Nat Genet 2008;40: 1119–23. [27] Mosyak L, Georgiadis K, Shane T, Svenson K, Hebert T, McDonagh T, et al. Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5. Protein Sci 2008;17:16–21. [28] Shieh HS, Mathis KJ, Williams JM, Hills RL, Wiese JF, Benson TE, et al. High resolution crystal structure of the catalytic domain of ADAMTS-5 (aggrecanase-2). J Biol Chem 2008;283:1501–7. [29] Koo BH, Longpre JM, Somerville RP, Alexander JP, Leduc R, Apte SS. Regulation of ADAMST9 secretion and enzymatic activity by its propeptide. J Biol Chem 2007; 282:16146–54. [30] Majerus EM, Zheng X, Tuley EA, Sadler JE. Cleavage of the ADAMTS13 propeptide is not required for protease activity. J Biol Chem 2003;278:46643–8. [31] Koo BH, Longpre JM, Somerville RP, Alexander JP, Leduc R, Apte SS. Cell-surface processing of pro-ADAMTS9 by furin. J Biol Chem 2006;281:12485–94. [32] Longpre JM, Leduc R. Identification of prodomain determinants involved in ADAMTS-1 biosynthesis. J Biol Chem 2004;279:33237–45. [33] Longpre JM, McCulloch DR, Koo BH, Alexander JP, Apte SS, Leduc R. Characterization of proADAMTS5 processing by proprotein convertases. Int J Biochem Cell Biol 2009;41: 1116–26. [34] Somerville RP, Jungers KA, Apte SS. ADAMTS10: discovery and characterization of a novel, widely expressed metalloprotease and its proteolytic activation. J Biol Chem 2004;279: 51208–17. [35] Somerville RP, Longpre JM, Apel ED, Lewis RM, Wang LW, Sanes JR, et al. ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain. J Biol Chem 2004;279: 35159–75. [36] Somerville RP, Longpre JM, Jungers KA, Engle JM, Ross M, Evanko S, et al. Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1. J Biol Chem 2003;278: 9503–13. [37] Tortorella MD, Arner EC, Hills R, Gormley J, Fok K, Pegg L, et al. ADAMTS-4 (aggrecanase-1): N-terminal activation mechanisms. Arch Biochem Biophys 2005;444:34–44. [38] Koo BH, Apte SS. Cell-surface processing of the metalloprotease pro-ADAMTS9 is influenced by the chaperone GRP94/gp96. J Biol Chem 2010;285:197–205. [39] Ricketts LM, Dlugosz M, Luther KB, Haltiwanger RS, Majerus EM. O-fucosylation is required for ADAMTS13 secretion. J Biol Chem 2007;282:17014–23. [40] Wang LW, Dlugosz M, Somerville RP, Raed M, Haltiwanger RS, Apte SS. O-fucosylation of thrombospondin type 1 repeats in ADAMTS-like-1/punctin-1 regulates secretion: implications for the ADAMTS superfamily. J Biol Chem 2007;282:17024–31. [41] Wang LW, Leonhard-Melief C, Haltiwanger RS, Apte SS. Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan. J Biol Chem 2009;284:30004–15. [42] Kozma K, Keusch JJ, Hegemann B, Luther KB, Klein D, Hess D, et al. Identification and characterization of

34

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

Insights on ADAMTS proteases and ADAMTS-like proteins

abeta1,3-glucosyltransferase that synthesizes the Glcbeta1,3-Fuc disaccharide on thrombospondin type 1 repeats. J Biol Chem 2006;281:36742–51. Luo Y, Koles K, Vorndam W, Haltiwanger RS, Panin VM. Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats. J Biol Chem 2006;281: 9393–9. Wang WM, Ge G, Lim NH, Nagase H, Greenspan DS. TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2. Biochem J 2006;398:515–9. Kashiwagi M, Tortorella M, Nagase H, Brew K. TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5). J Biol Chem 2001;276: 12501–4. Tortorella MD, Arner EC, Hills R, Easton A, Korte-Sarfaty J, Fok K, et al. Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes. J Biol Chem 2004;279: 17554–61. Li Z, Nardi MA, Li YS, Zhang W, Pan R, Dang S, et al. Cterminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke. Blood 2009;113:6051–60. Troeberg L, Mulloy B, Ghosh P, Lee MH, Murphy G, Nagase H. Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex. Biochem J 2012;443: 307–15. Yamamoto K, Owen K, Parker AE, Scilabra SD, Dudhia J, Strickland DK, et al. Low density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytic clearance of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4): functional differences of non-catalytic domains of ADAMTS-4 and ADAMTS-5 in LRP1 binding. J Biol Chem 2014;289:6462–74. Yamamoto K, Troeberg L, Scilabra SD, Pelosi M, Murphy CL, Strickland DK, et al. LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage. FASEB J 2013;27:511–21. Jeltsch M, Jha SK, Tvorogov D, Anisimov A, Leppanen VM, Holopainen T, et al. CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation. Circulation 2014;129:1962–71. Counts DF, Byers PH, Holbrook KA, Hegreberg GA. Dermatosparaxis in a Himalayan cat: I. Biochemical studies of dermal collagen. J Invest Dermatol 1980;74:96–9. Hanset R, Lapiere CM. Inheritance of dermatosparaxis in the calf. A genetic defect of connective tissues. J Hered 1974;65:356–8. Ramshaw JA, Peters DE, Jones LN, Badman RT, Brodsky BB. Ovine dermatosparaxis. Aust Vet J 1983;60:149–51. Lapiere CM, Lenaers A, Kohn LD. Procollagen peptidase: an enzyme excising the coordination peptides of procollagen. Proc Natl Acad Sci U S A 1971;68:3054–8. Nusgens BV, Verellen-Dumoulin C, Hermanns-Le T, De Paepe A, Nuytinck L, Pierard GE, et al. Evidence for a relationship between Ehlers–Danlos type VII C in humans and bovine dermatosparaxis. Nat Genet 1992;1:214–7. Colige A, Sieron AL, Li SW, Schwarze U, Petty E, Wertelecki W, et al. Human Ehlers–Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

in the procollagen I N-proteinase gene. Am J Hum Genet 1999;65:308–17. Colige A, Ruggiero F, Vandenberghe I, Dubail J, Kesteloot F, Van Beeumen J, et al. Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens type I, II, III and V. J Biol Chem 2005;280:34397–408. Colige A, Vandenberghe I, Thiry M, Lambert CA, Van Beeumen J, Li SW, et al. Cloning and characterization of ADAMTS-14, a novel ADAMTS displaying high homology with ADAMTS-2 and ADAMTS-3. J Biol Chem 2002;277: 5756–66. Fernandes RJ, Hirohata S, Engle JM, Colige A, Cohn DH, Eyre DR, et al. Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis. J Biol Chem 2001;276:31502–9. Le Goff C, Somerville RP, Kesteloot F, Powell K, Birk DE, Colige AC, et al. Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis. Development 2006;133:1587–96. Dagoneau N, Benoist-Lasselin C, Huber C, Faivre L, Megarbane A, Alswaid A, et al. ADAMTS10 mutations in autosomal recessive Weill–Marchesani syndrome. Am J Hum Genet 2004;75:801–6. Faivre L, Gorlin RJ, Wirtz MK, Godfrey M, Dagoneau N, Samples JR, et al. In frame fibrillin-1 gene deletion in autosomal dominant Weill–Marchesani syndrome. J Med Genet 2003;40:34–6. Kutz WE, Wang LW, Bader HL, Majors AK, Iwata K, Traboulsi EI, et al. ADAMTS10 protein interacts with fibrillin1 and promotes its deposition in extracellular matrix of cultured fibroblasts. J Biol Chem 2011;286:17156–67. Gabriel LA, Wang LW, Bader H, Ho JC, Majors AK, Hollyfield JG, et al. ADAMTSL4, a secreted glycoprotein widely distributed in the eye, binds fibrillin-1 microfibrils and accelerates microfibril biogenesis. Invest Ophthalmol Vis Sci 2012;53:461–9. Tsutsui K, Manabe RI, Yamada T, Nakano I, Oguri Y, Keene DR, et al. A disintegrin and metalloproteinase with thrombospondin motifs-like-6 (ADAMTSL-6) is a novel extracellular matrix protein that binds to fibrillin-1 and promotes fibrillin-1 fibril formation. J Biol Chem 2010;285: 4870–82. Farias FH, Johnson GS, Taylor JF, Giuliano E, Katz ML, Sanders DN, et al. An ADAMTS17 splice donor site mutation in dogs with primary lens luxation. Invest Ophthalmol Vis Sci 2010;51:4716–21. Morales J, Al-Sharif L, Khalil DS, Shinwari JM, Bavi P, Al-Mahrouqi RA, et al. Homozygous mutations in ADAMTS10 and ADAMTS17 cause lenticular myopia, ectopia lentis, glaucoma, spherophakia, and short stature. Am J Hum Genet 2009;85:558–68. Aragon-Martin JA, Ahnood D, Charteris DG, Saggar A, Nischal KK, Comeglio P, et al. Role of ADAMTSL4 mutations in FBN1 mutation-negative ectopia lentis patients. Hum Mutat 2010;31:E1622–31. Chandra A, Aragon-Martin JA, Hughes K, Gati S, Reddy MA, Deshpande C, et al. A genotype-phenotype comparison of ADAMTSL4 and FBN1 in isolated ectopia lentis. Invest Ophthalmol Vis Sci 2012;53:4889–96. Christensen AE, Fiskerstrand T, Knappskog PM, Boman H, Rodahl E. A novel ADAMTSL4 mutation in autosomal

35

Insights on ADAMTS proteases and ADAMTS-like proteins

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

recessive ectopia lentis et pupillae. Invest Ophthalmol Vis Sci 2010;51:6369–73. Chandra A, Jones M, Cottrill P, Eastlake K, Limb GA, Charteris DG. Gene expression and protein distribution of ADAMTSL-4 in human iris, choroid and retina. Br J Ophthalmol 2013;97:1208–12. Chandra A, Aragon-Martin JA, Sharif S, Parulekar M, Child A, Arno G. Craniosynostosis with ectopia lentis and a homozygous 20-base deletion in ADAMTSL4. Ophthalmic Genet 2013;34:78–82. Buchner DA, Meisler MH. TSRC1, a widely expressed gene containing seven thrombospondin type I repeats. Gene 2003;307:23–30. Cal S, Obaya AJ, Llamazares M, Garabaya C, Quesada V, Lopez-Otin C. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin1 domains. Gene 2002;283:49–62. Bader HL, Ruhe AL, Wang LW, Wong AK, Walsh KF, Packer RA, et al. An ADAMTSL2 founder mutation causes Musladin–Lueke syndrome, a heritable disorder of beagle dogs, featuring stiff skin and joint contractures. PLoS One 2010;5:e12817. Le Goff C, Mahaut C, Wang LW, Allali S, Abhyankar A, Jensen S, et al. Mutations in the TGFbeta binding-proteinlike domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias. Am J Hum Genet 2011;89:7–14. Shi Y, Tu Y, De Maria A, Mecham RP, Bassnett S. Development, composition, and structural arrangements of the ciliary zonule of the mouse. Invest Ophthalmol Vis Sci 2013;54:2504–15. Beene LC, Wang LW, Hubmacher D, Keene DR, Reinhardt DP, Annis DS, et al. Nonselective assembly of fibrillin 1 and fibrillin 2 in the rodent ocular zonule and in cultured cells: implications for marfan syndrome. Invest Ophthalmol Vis Sci 2013;54:8337–44. Zheng XL. ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura. Annu Rev Med 2015;66:211–25. Levy GG, Nichols WC, Lian EC, Foroud T, McClintick JN, McGee BM, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 2001;413:488–94. Aldahmesh MA, Alshammari MJ, Khan AO, Mohamed JY, Alhabib FA, Alkuraya FS. The syndrome of microcornea, myopic chorioretinal atrophy, and telecanthus (MMCAT) is caused by mutations in ADAMTS18. Hum Mutat 2013;34:1195–9. Peluso I, Conte I, Testa F, Dharmalingam G, Pizzo M, Collin RW, et al. The ADAMTS18 gene is responsible for autosomal recessive early onset severe retinal dystrophy. Orphanet J Rare Dis 2013;8:16. Rao C, Foernzler D, Loftus SK, Liu S, McPherson JD, Jungers KA, et al. A defect in a novel ADAMTS family member is the cause of the belted white-spotting mutation. Development 2003;130:4665–72. Silver DL, Hou L, Somerville R, Young ME, Apte SS, Pavan WJ. The secreted metalloprotease ADAMTS20 is required for melanoblast survival. PLoS Genet 2008;4:1–15. Shindo T, Kurihara H, Kuno K, Yokoyama H, Wada T, Kurihara Y, et al. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J Clin Invest 2000;105:1345–52. Mittaz L, Russell DL, Wilson T, Brasted M, Tkalcevic J, Salamonsen LA, et al. Adamts-1 is essential for the

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

development and function of the urogenital system. Biol Reprod 2004;70:1096–105. Brown HM, Dunning KR, Robker RL, Boerboom D, Pritchard M, Lane M, et al. ADAMTS1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice. Biol Reprod 2010;83:549–57. Brown HM, Dunning KR, Robker RL, Pritchard M, Russell DL. Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis. Dev Biol 2006;300:699–709. Russell DL, Doyle KM, Ochsner SA, Sandy JD, Richards JS. Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. J Biol Chem 2003;278:42330–9. Stankunas K, Hang CT, Tsun ZY, Chen H, Lee NV, Wu JI, et al. Endocardial Brg1 represses ADAMTS1 to maintain the microenvironment for myocardial morphogenesis. Dev Cell 2008;14:298–311. Fosang AJ, Little CB. Drug insight: aggrecanases as therapeutic targets for osteoarthritis. Nat Clin Pract Rheumatol 2008;4:420–7. Gilbert AM, Bursavich MG, Lombardi S, Georgiadis KE, Reifenberg E, Flannery CR, et al. 5-((1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one inhibitors of ADAMTS-5. Bioorg Med Chem Lett 2007;17:1189–92. McCulloch DR, Goff CL, Bhatt S, Dixon LJ, Sandy JD, Apte SS. Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues. Gene Expr Patterns 2009;9:314–23. Dupuis LE, McCulloch DR, McGarity JD, Bahan A, Wessels A, Weber D, et al. Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease. Dev Biol 2011;357:152–64. Dupuis LE, Osinska H, Weinstein MB, Hinton RB, Kern CB. Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves. J Mol Cell Cardiol 2013;60:50–9. Dubail J, Aramaki-Hattori N, Bader HL, Nelson CM, Katebi N, Matuska B, et al. A new Adamts9 conditional mouse allele identifies its non-redundant role in interdigital web regression. Genesis 2014;52:702–12. Kern CB, Wessels A, McGarity J, Dixon LJ, Alston E, Argraves WS, et al. Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies. Matrix Biol 2010;29:304–16. Koo BH, Coe DM, Dixon LJ, Somerville RP, Nelson CM, Wang LW, et al. ADAMTS9 is a cell-autonomously acting, anti-angiogenic metalloprotease expressed by microvascular endothelial cells. Am J Pathol 2010;176: 1494–504. Enomoto H, Nelson C, Somerville RPT, Mielke K, Dixon L, Powell K, et al. Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation. Development 2010;137:4029–38. McCulloch DR, Nelson CM, Dixon LJ, Silver DL, Wylie JD, Lindner V, et al. ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression. Dev Cell 2009;17:687–98. Abdul-Majeed S, Mell B, Nauli SM, Joe B. Cryptorchidism and infertility in rats with targeted disruption of the Adamts16 locus. PLoS One 2014;9:e100967.

36 [103] Gopalakrishnan K, Kumarasamy S, Abdul-Majeed S, Kalinoski AL, Morgan EE, Gohara AF, et al. Targeted disruption of Adamts16 gene in a rat genetic model of hypertension. Proc Natl Acad Sci U S A 2012;109:20555–9. [104] Jacobi CL, Rudigier LJ, Scholz H, Kirschner KM. Transcriptional regulation by the Wilms tumor protein, Wt1, suggests a role of the metalloproteinase Adamts16 in murine genitourinary development. J Biol Chem 2013;288:18811–24. [105] Boesgaard TW, Gjesing AP, Grarup N, Rutanen J, Jansson PA, Hribal ML, et al. Variant near ADAMTS9 known to associate with type 2 diabetes is related to insulin resistance in offspring of type 2 diabetes patients— EUGENE2 study. PLoS One 2009;4:e7236. [106] de Jong EK, Breukink MB, Schellevis RL, Bakker B, Mohr JK, Fauser S, et al. Chronic central serous chorioretinopathy is associated with genetic variants implicated in agerelated macular degeneration. Ophthalmology 2014;122: 562–70. [107] Dong K, Yao N, Pu Y, He X, Zhao Q, Luan Y, et al. Genomic scan reveals loci under altitude adaptation in Tibetan and Dahe pigs. PLoS One 2014;9:e110520. [108] Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, Steinthorsdottir V, et al. Meta-analysis identifies 13 new loci associated with waist–hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution. Nat Genet 2010;42:949–60. [109] Helisalmi S, Immonen I, Losonczy G, Resch MD, Benedek S, Balogh I, et al. ADAMTS9 locus associates with increased risk of wet AMD. Acta Ophthalmol 2014;92:e410. [110] Zeggini E, Scott LJ, Saxena R, Voight BF, Marchini JL, Hu T, et al. Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet 2008;40:638–45. [111] Chaemsaithong P, Madan I, Romero R, Than NG, Tarca AL, Draghici S, et al. Characterization of the myometrial transcriptome in women with an arrest of dilatation during labor. J Perinat Med 2013;41:665–81. [112] Pyun JA, Kim S, Cho NH, Koh I, Lee JY, Shin C, et al. Genome-wide association studies and epistaxis analyses of candidate genes related to age at menarche and age at natural menopause in a Korean population. Menopause 2014;21:522–9. [113] Fritsche LG, Chen W, Schu M, Yaspan BL, Yu Y, Thorleifsson G, et al. Seven new loci associated with agerelated macular degeneration. Nat Genet 2013;45:433–9. [114] Padang R, Bagnall RD, Tsoutsman T, Bannon PG, Semsarian C. Comparative transcriptome profiling in human bicuspid aortic valve disease using RNA-sequencing. Physiol Genomics 2015;47:75–87. [115] Arning A, Hiersche M, Witten A, Kurlemann G, Kurnik K, Manner D, et al. A genome-wide association study identifies a gene network of ADAMTS genes in the predisposition to pediatric stroke. Blood 2012;120:5231–6. [116] Weedon MN, Lango H, Lindgren CM, Wallace C, Evans DM, Mangino M, et al. Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet 2008; 40:575–83. [117] Dow DJ, Huxley-Jones J, Hall JM, Francks C, Maycox PR, Kew JN, et al. ADAMTSL3 as a candidate gene for schizophrenia: gene sequencing and ultra-high density association analysis by imputation. Schizophr Res 2011; 127:28–34. [118] Reilly MP, Li M, He J, Ferguson JF, Stylianou IM, Mehta NN, et al. Identification of ADAMTS7 as a novel locus for

Insights on ADAMTS proteases and ADAMTS-like proteins

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies. Lancet 2011;377:383–92. Pu X, Xiao Q, Kiechl S, Chan K, Ng FL, Gor S, et al. ADAMTS7 cleavage and vascular smooth muscle cell migration is affected by a coronary-artery-disease-associated variant. Am J Hum Genet 2013;92:366–74. Rodriguez-Manzaneque JC, Carpizo D, Plaza-Calonge Mdel C, Torres-Collado AX, Thai SN, Simons M, et al. Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion. Int J Biochem Cell Biol 2009;41:800–10. Rodriguez-Manzaneque JC, Westling J, Thai SN, Luque A, Knauper V, Murphy G, et al. ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun 2002; 293:501–8. Torres-Collado AX, Kisiel W, Iruela-Arispe ML, RodriguezManzaneque JC. ADAMTS1 interacts with, cleaves, and modifies the extracellular location of the matrix inhibitor tissue factor pathway inhibitor-2. J Biol Chem 2006;281: 17827–37. Liu YJ, Xu Y, Yu Q. Full-length ADAMTS-1 and the ADAMTS-1 fragments display pro- and antimetastatic activity, respectively. Oncogene 2006;25:2452–67. Canals F, Colome N, Ferrer C, Plaza-Calonge Mdel C, Rodriguez-Manzaneque JC. Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis. Proteomics 2006;6(Suppl. 1):S28–35. Lee NV, Sato M, Annis DS, Loo JA, Wu L, Mosher DF, et al. ADAMTS1 mediates the release of antiangiogenic polypeptides from TSP1 and 2. EMBO J 2006;25:5270–83. Lind T, Birch MA, McKie N. Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities. Mol Cell Biochem 2006;281: 95–102. Sandy JD, Westling J, Kenagy RD, Iruela-Arispe ML, Verscharen C, Rodriguez-Mazaneque JC, et al. Versican V1 proteolysis in human aorta in vivo occurs at the Glu441–Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem 2001;276:13372–8. Stupka N, Kintakas C, White JD, Fraser FW, Hanciu M, Aramaki-Hattori N, et al. Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion. J Biol Chem 2013;288:1907–17. Kessler T, Zhang L, Liu Z, Yin X, Huang Y, Wang Y, et al. ADAMTS-7 inhibits re-endothelialization of injured arteries and promotes vascular remodeling via cleavage of thrombospondin-1. Circulation 2015 Feb 20 [Epub ahead of print, pii: CIRCULATIONAHA.114.014072]. Wang L, Zheng J, Bai X, Liu B, Liu CJ, Xu Q, et al. ADAMTS-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res 2009;104:688–98. Little CB, Meeker CT, Golub SB, Lawlor KE, Farmer PJ, Smith SM, et al. Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair. J Clin Invest 2007;117:1627–36. Yokoyama H, Wada T, Kobayashi K, Kuno K, Kurihara H, Shindo T, et al. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 null mutant mice develop renal lesions mimicking obstructive nephropathy. Nephrol Dial Transplant 2002;17(Suppl. 9):39–41.

Insights on ADAMTS proteases and ADAMTS-like proteins

[133] Shozu M, Minami N, Yokoyama H, Inoue M, Kurihara H, Matsushima K, et al. ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary. J Mol Endocrinol 2005;35:343–55. [134] Howell MD, Torres-Collado AX, Iruela-Arispe ML, Gottschall PE. Selective decline of synaptic protein levels in the frontal cortex of female mice deficient in the extracellular metalloproteinase ADAMTS1. PLoS One 2012;7:e47226. [135] Boerboom D, Lafond JF, Zheng X, Lapointe E, Mittaz L, Boyer A, et al. Partially redundant functions of Adamts1 and Adamts4 in the perinatal development of the renal medulla. Dev Dyn 2011;240:1806–14. [136] Li SW, Arita M, Fertala A, Bao Y, Kopen GC, Langsjo TK, et al. Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility. Biochem J 2001;355:271–8. [137] Glasson SS, Askew R, Sheppard B, Carito BA, Blanchet T, Ma HL, et al. Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice. Arthritis Rheum 2004;50:2547–58. [138] Demircan K, Topcu V, Takigawa T, Akyol S, Yonezawa T, Ozturk G, et al. ADAMTS4 and ADAMTS5 knockout mice are protected from versican but not aggrecan or brevican proteolysis during spinal cord injury. Biomed Res Int 2014; 2014:693746. [139] Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature 2005; 434:644–8. [140] Botter SM, Glasson SS, Hopkins B, Clockaerts S, Weinans H, van Leeuwen JP, et al. ADAMTS5 −/− mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes. Osteoarthritis Cartilage 2009;17:636–45. [141] Li J, Anemaet W, Diaz MA, Buchanan S, Tortorella M, Malfait AM, et al. Knockout of ADAMTS5 does not eliminate cartilage aggrecanase activity but abrogates joint fibrosis and promotes cartilage aggrecan deposition in murine osteoarthritis models. J Orthop Res 2011;29:516–22.

37 [142] Dancevic CM, Fraser FW, Smith AD, Stupka N, Ward AC, McCulloch DR. Biosynthesis and expression of a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats-15: a novel versican-cleaving proteoglycanase. J Biol Chem 2013;288:37267–76. [143] Velasco J, Li J, Dipietro L, Stepp MA, Sandy JD, Plaas A. ADAMTS5 ablation blocks murine dermal repair through CD44-mediated aggrecan accumulation and modulation of TGFbeta1 signaling. J Biol Chem 2011;286:26016–27. [144] Wang VM, Bell RM, Thakore R, Eyre DR, Galante JO, Li J, et al. Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5. J Orthop Res 2012;30:620–6. [145] Majumdar MK, Askew R, Schelling S, Stedman N, Blanchet T, Hopkins B, et al. Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis. Arthritis Rheum 2007;56:3670–4. [146] El Hour M, Moncada-Pazos A, Blacher S, Masset A, Cal S, Berndt S, et al. Higher sensitivity of Adamts12-deficient mice to tumor growth and angiogenesis. Oncogene 2010; 29:3025–32. [147] Moncada-Pazos A, Obaya AJ, Llamazares M, Heljasvaara R, Suarez MF, Colado E, et al. ADAMTS-12 metalloprotease is necessary for normal inflammatory response. J Biol Chem 2012;287:39554–63. [148] Paulissen G, El Hour M, Rocks N, Gueders MM, Bureau F, Foidart JM, et al. Control of allergen-induced inflammation and hyperresponsiveness by the metalloproteinase ADAMTS-12. J Immunol 2012;189:4135–43. [149] Motto DG, Chauhan AK, Zhu G, Homeister J, Lamb CB, Desch KC, et al. Shigatoxin triggers thrombotic thrombocytopenic purpura in genetically susceptible ADAMTS13deficient mice. J Clin Invest 2005;115:2752–61. [150] De Meyer SF, Savchenko AS, Haas MS, Schatzberg D, Carroll MC, Schiviz A, et al. Protective anti-inflammatory effect of ADAMTS13 on myocardial ischemia/reperfusion injury in mice. Blood 2012;120:5217–23.