Profile of Matrix-Remodeling Proteinases in Osteoarthritis: Impact of Fibronectin.

The extracellular matrix (ECM) is a complex and specialized three-dimensional macromolecular network, present in nearly all tissues, that also interacts with cell surface receptors on joint resident cells. Changes in the composition and physical properties of the ECM lead to the development of many diseases, including osteoarthritis (OA). OA is a chronic degenerative rheumatic disease characterized by a progressive loss of synovial joint function as a consequence of the degradation of articular cartilage, also associated with alterations in the synovial membrane and subchondral bone. During OA, ECM-degrading enzymes, including urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), cleave ECM components, such as fibronectin (Fn), generating fibronectin fragments (Fn-fs) with catabolic properties. In turn, Fn-fs promote activation of these proteinases, establishing a degradative and inflammatory feedback loop. Thus, the aim of this review is to update the contribution of ECM-degrading proteinases to the physiopathology of OA as well as their modulation by Fn-fs.

Male mice lacking ADAMTS-16 are fertile but exhibit testes of reduced weight.

Adamts16 encodes a disintegrin-like and metalloproteinase with thrombospondin motifs, 16, a member of a family of multi-domain, zinc-binding proteinases. ADAMTS-16 is implicated in a number of pathological conditions, including hypertension, cancer and osteoarthritis. A large number of observations, including a recent report of human ADAMTS16 variants in cases of 46,XY disorders/differences of sex development (DSD), also implicate this gene in human testis determination. We used CRISPR/Cas9 genome editing to generate a loss-of-function allele in the mouse in order to examine whether ADAMTS-16 functions in mouse testis determination or testicular function. Male mice lacking Adamts16 on the C57BL/6N background undergo normal testis determination in the fetal period. However, adult homozygotes have an average testis weight that is around 10% lower than age-matched controls. Cohorts of mutant males tested at 3-months and 6-months of age were fertile. We conclude that ADAMTS-16 is not required for testis determination or male fertility in mice. We discuss these phenotypic data and their significance for our understanding of ADAMTS-16 function.

Morphogenesis of the kidney and lung requires branch-tip directed activity of the Adamts18 metalloprotease.

Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.

Adamts17 is involved in skeletogenesis through modulation of BMP-Smad1/5/8 pathway.

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.

Inhibition of MMPs and ADAM/ADAMTS.

Matrix metalloproteinases (MMPs), A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) are zinc-dependent endopeptidases that play a critical role in the destruction of extracellular matrix proteins and, the shedding of membrane-bound receptor molecules in various forms of arthritis and other diseases. Under normal conditions, MMP, ADAM and ADAMTS gene expression aids in the maintenance of homeostasis. However, in inflamed synovial joints characteristic of rheumatoid arthritis and osteoarthritis. MMP, ADAM and ADAMTS production is greatly increased under the influence of pro-inflammatory cytokines. Analyses based on medicinal chemistry strategies designed to directly inhibit the activity of MMPs have been largely unsuccessful when these MMP inhibitors were employed in animal models of rheumatoid arthritis and osteoarthritis. This is despite the fact that these MMP inhibitors were largely able to suppress pro-inflammatory cytokine-induced MMP production in vitro. A focus on ADAM and ADAMTS inhibitors has also been pursued. Thus, recent progress has identified the "sheddase" activity of ADAMs as a viable target and the development of GW280264X is an experimental ADAM17 inhibitor. Of note, a monoclonal antibody, GLPG1972, developed as an ADAMTS-5 inhibitor, entered a Phase I OA clinical trial. However, the failure of many of these previously developed inhibitors to move beyond the preclinical testing phase has required that novel strategies be developed that are designed to suppress both MMP, ADAM and ADAMTS production and activity.

Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.

Mutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-ß signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-ß binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.

Epigenetic changes in preterm birth placenta suggest a role for ADAMTS genes in spontaneous preterm birth.

Preterm birth (PTB) affects approximately 1 in 10 pregnancies and contributes to approximately 50% of neonatal mortality. However, despite decades of research, little is understood about the etiology of PTB, likely due to the multifactorial nature of the disease. In this study, we examined preterm and term placentas, from unassisted conceptions and those conceived using in vitro fertilization (IVF). IVF increases the risk of PTB and causes epigenetic change in the placenta and fetus; therefore, we utilized these patients as a unique population with a potential common etiology. We investigated genome-wide DNA methylation in placentas from term IVF, preterm IVF, term control (unassisted conception) and preterm control pregnancies and discovered epigenetic dysregulation of multiple genes involved in cell migration, including members of the ADAMTS family, ADAMTS12 and ADAMTS16. These genes function in extracellular matrix regulation and tumor cell invasion, processes replicated by invasive trophoblasts (extravillous trophoblasts (EVTs)) during early placentation. Though expression was similar between term and preterm placentas, we found that both genes demonstrate high expression in first- and second-trimester placenta, specifically in EVTs and syncytiotrophoblasts. When we knocked down ADAMTS12 or ADAMTS16in vitro, there was poor EVT invasion and reduced matrix metalloproteinase activity, reinforcing their critical role in placentation. In conclusion, utilizing a population at high risk for PTB, we have identified a role for ADAMTS gene methylation in regulating early placentation and susceptibility to PTB.

Metalloproteinase Adamts16 Is Required for Proper Closure of the Optic Fissure.

Purpose: Coloboma is a sight-threatening congenital eye disease caused by a failure in optic fissure (OF) closure. The aim of this study was to investigate the role of Adamts16, a metalloproteinase, in OF closure. Methods: RNA in situ hybridization was used to examine the expression of Adamts16 in developing mouse and zebrafish eyes. Morpholino knockdowns were performed to study adamts16 function during zebrafish eye development. Additionally, immunofluorescent staining, RNA in situ hybridization, bromodeoxyuridine (BrdU) labeling, TUNEL assays, and high-throughput sequencing were used to examine altered cellular and molecular events in adamts16-morphant optic cups (OCs). Results: Adamts16 is expressed at the edges of the closing OF in both mice and zebrafish eyes. Zebrafish adamts16 knockdown resulted in coloboma formation. In adamts16-morphant eyes, the basement membrane failed to disassemble at the closing OF edges, OC cells exhibited decreased proliferation and increased apoptosis, and fibroblast growth factor 8 (fgf8) was ectopically upregulated in the OC. Conclusions: adamts16 is required for proper OF closure in zebrafish eyes. adamts16 controls OF closure possibly through the combined functions of degrading the basement membrane at the closing OF edges, promoting cell proliferation and survival, and restricting fgf8 expression. Our study linked a metalloproteinase to OF closure, which may facilitate future etiologic studies on human coloboma cases.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs 18 Deficiency Leads to Visceral Adiposity and Associated Metabolic Syndrome in Mice.

Visceral adiposity is of greater risk than obesity in s.c. adipose tissue for diabetes and cardiovascular disease. Its pathogenesis remains unclear, but it is associated with extracellular matrix (ECM) remodeling. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are a family of secreted zinc-dependent metalloproteinases that play crucial roles in development and various diseases because of their ECM remodeling activity. ADAMTS18 is an orphan ADAMTS whose function and substrate remain unclear. Herein, we showed that Adamts18 mRNA was abundantly expressed in visceral (gonadal) white adipose tissue (vWAT) during the early stage of development after birth. Adamts18 knockout (KO) mice showed increased body fat percentage and larger adipocyte size in vWAT relative to wild-type littermates. These findings may be partly attributed to ECM remodeling, especially increased expression of laminin 1 and adipokine thrombospondin 1 in vWAT. Attenuated extracellular signal-regulated kinase 1 and 2 activity, along with increased expression of adipocyte-specific transcription factors peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein ß, and marker gene Fabp4, was detected in vWAT of Adamts18 KO mice. Furthermore, Adamts18 KO mice showed early metabolic syndrome, including hyperlipidemia, blood glucose metabolic disorder, and hypertension. ADAMTS18 deficiency promotes atherosclerosis in apolipoprotein E-deficient mice. These results indicate a novel function of ADAMTS18 in vWAT development and associated metabolic disorders.

ADAMTS and ADAM metalloproteinases in osteoarthritis - looking beyond the 'usual suspects'.

INTRODUCTION: Matrix metalloproteinases (MMPs) and 'aggrecanase' a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are well established to play key roles in osteoarthritis (OA) through degradation of extracellular matrix (ECM) type II collagen and aggrecan, and are thus potential targets for development of OA therapies. OBJECTIVE: This paper aims to provide a comprehensive review of the expression and potential roles of other, lesser-known ADAMTSs and related adamalysins (or a disintegrin and metalloproteinases (ADAMs)) in cartilage, with a view to identifying potentially protective or homeostatic metalloproteinases in the joint and informing consequent selective inhibitor design. DESIGN: A comprehensive literature search was performed using PubMed terms 'osteoarthritis' and 'ADAMTS' or 'ADAM'. RESULTS: Several ADAMTSs and ADAMs were identified as having reportedly increased expression in OA. These include enzymes likely to play roles in cartilage matrix anabolism (e.g., the procollagen N-proteinases ADAMTS-2, ADAMTS-3 and ADAMTS-14), chondrocyte differentiation and proliferation (e.g., ADAM9, ADAM10, ADAM12), as well as enzymes contributing to cartilage catabolism (e.g., Cartilage oligomeric protein (COMP)-degrading ADAMTS-7 and ADAMTS-12). CONCLUSIONS: In addition to the well-characterised MMPs, ADAMTS-4 and ADAMTS-5, many other ADAMTSs and ADAMs are expressed in cartilage and several show significantly altered expression in OA. Studies aimed at elucidating the pathophysiological roles of these enzymes in cartilage will contribute to our understanding of OA pathogenesis and enable design of targeted inhibitors that effectively target metalloproteinase-mediated cartilage degradation while sparing cartilage repair pathways.

Systems Genetics Approach Identifies Gene Pathways and Adamts2 as Drivers of Isoproterenol-Induced Cardiac Hypertrophy and Cardiomyopathy in Mice.

We previously reported a genetic analysis of heart failure traits in a population of inbred mouse strains treated with isoproterenol to mimic catecholamine-driven cardiac hypertrophy. Here, we apply a co-expression network algorithm, wMICA, to perform a systems-level analysis of left ventricular transcriptomes from these mice. We describe the features of the overall network but focus on a module identified in treated hearts that is strongly related to cardiac hypertrophy and pathological remodeling. Using the causal modeling algorithm NEO, we identified the gene Adamts2 as a putative regulator of this module and validated the predictive value of NEO using small interfering RNA-mediated knockdown in neonatal rat ventricular myocytes. Adamts2 silencing regulated the expression of the genes residing within the module and impaired isoproterenol-induced cellular hypertrophy. Our results provide a view of higher order interactions in heart failure with potential for diagnostic and therapeutic insights.