University of Groningen the Biology of Adams in Renal Disease

University of Groningen

The biology of ADAMs in renal disease Melenhorst, Wynand Bernhard Willem Henderik

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ADAMALYSINS IN BIOLOGY AND DISEASE

Harry van Goor1 Wynand Melenhorst1 Anthony Turner2 Stephen Holgate3

1. Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, the Netherlands 2. Division of Infection, Inflammation and Repair, Southampton General Hospital, Southampton, United Kingdom 3. Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Journal of Pathology 2009, in press

Chapter 2

Abstract ADAMs (A Disintegrin And Metalloprotease) are membrane-bound enzymes, capable of shedding a multitude of proteins from the surface of the cell. They are therefore considered crucial modulators of physiological and pathophysiological processes. The structure and function of ADAMs is related to those of a family of snake venom metalloproteases which also possess a potential adhesion domain as well as a potential protease domain. Mammalian ADAMs are involved in various biological and disease-related processes like cell-cell fusion, adhesion, and intracellular signalling. Functional involvement has been described in sperm-egg binding and fusion, trophoblast invasion and matrix degradation during pregnancy, angiogenesis, and neovascularization. Clinically, ADAMs are implicated in pathological processes including cancer, inflammation, neurodegeneration and fibrosis through shedding of the apoptosis-inducing FAS ligand, cytokines and growth factors. A second group of proteins within the ADAM family has recently been discovered. These contain several thrombospondin-like repeats in their C-terminal regions, in the absence of the transmembrane domain known to be present in ADAMs. These proteins were called the ADAMTS (ADAM with ThromboSpondin domains) family. The relevance of ADAMTS enzymes has become evident in patients with a deficiency in ADAMTS-13, a von Willebrand factor cleaving protease. These patients develop thrombotic thrombocytopenic purpura, a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). In the present review, we will concentrate on the genetic, developmental, functional and disease-related aspects of ADAMs and ADAMTS. Finally we will discuss the perspectives of the therapeutical potential of ADAMs in disease.

Adamalysins in Biology and Disease

Introduction ADAMs (mnemonic for A Disintegrin And Metalloprotease) are an intriguing group of membrane- bound enzymes that belong to a larger zinc-dependent superfamily called the metzincins. The structure and function of ADAMs is closely related to those of a family of snake venom metalloproteases, which are responsible for the haemorrhage effects and massive tissue necrosis commonly seen after snakebites. These damaging effects are inflicted by massive destruction of the extracellular matrix surrounding capillaries in conjunction with the inhibition of platelet aggregation by disintegrins, which block the function of adhesion molecules and are accompanied by severe inflammation1. These deleterious actions of ADAMs clearly reveal their capacity to modulate (patho)-physiological processes. ADAMs are unique in the sense that they possess a potential adhesion domain as well as a potential protease domain. Mammalian ADAMs are engaged in cell-cell fusion, adhesion, and intracellular signalling. These functions are illustrated by their involvement in various biological processes, like sperm-egg binding and fusion, trophoblast invasion and matrix degradation during pregnancy, angiogenesis, and neovascularization. The clinical significance of ADAMs is evidenced by their implication in pathological processes including cancer, inflammation, neurodegeneration and fibrosis through shedding of the apoptosis-inducing FAS ligand, cytokines and growth factors. More recently, the complexity of the ADAM family of metalloproteases with disintegrin domains has grown considerably after the description of an ADAM-related protein containing several thrombospondin-like repeats in its C- terminal region, in the absence of the transmembrane domain known to be present in ADAMs2. This protein was named ADAMTS-1 (ADAM with ThromboSpondin domains), and is the first member of a new family of 19 metalloproteases with structural and functional properties related to, but distinct from, ADAMs3;4. The clinical relevance of ADAMTS enzymes is most clearly seen in patients with a deficiency in ADAMTS-13, a von Willebrand factor cleaving protease, who develop thrombotic thrombocytopenic purpura, a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). In this review, we discuss the genetic, developmental, functional and disease-related aspects of ADAMs and ADAMTS with recommendations for therapeutic intervention.

Structural and biochemical aspects of ADAMs The first members of the ADAM family of disintegrin-metalloproteases to be discovered (ADAMs 1 and 2) were identified as being critical for the fertilization process5;6. Since that time the family has expanded considerably and now comprises 40 genes in various species and 23 known ADAM genes in humans. A current list of all ADAMs genes and the associated proteins is available on the University of Virginia website: http://people.virginia.edu/~jw7g/. Only around half of the known ADAMs are functionally active as metalloproteases, since significant members of the family lack one or more critical catalytic residues. The protease domain is implicated in many protein- shedding events from the cell-surface, including the release of growth factors, cytokines, receptors and adhesion molecules. The roles of the catalytically inactive ADAMs are mostly

Chapter 2 unknown, but could range from acting as peptide-binding receptors, to mediating cell-cell interactions, influencing cell adhesion or migration and mediating cell-signalling through other functional domains of the protein, particularly the cytoplasmic tail. We will principally focus on the protease-active ADAMs since, for these, there are clearer indications of their (patho)-physiological involvement. Nevertheless, unraveling the substrate repertoire of any individual ADAM or identifying which of the numerous ADAM proteins cleave a particular protein substrate remains a challenging task although newer strategies are emerging to deal with these questions7. These include the use of transgenic models, cellular over-expression or knock-down of individual ADAMs, and the use of ADAM-specific inhibitors. Structurally, ADAMs are single-span, transmembrane proteins that comprise a pro-domain, a zinc metalloprotease domain facing extracellularly, a disintegrin domain, a cysteine-rich region, an EGF-like sequence, a transmembrane region and a cytoplasmic tail (Figure 1). The proteins are related to the matrix metalloproteases (MMPs) and MMP inhibitors often also inhibit the activities of ADAMs. The pro-domain uses a “cysteine-switch” mechanism to hold the catalytic zinc ion and the protease domain in a latent state and to facilitate correct folding of the protein during biosynthesis. However, in the case of ADAM17, the only role for the prodomain appears to be in stabilizing the protein against proteolytic degradation during its biosynthesis and trafficking8. The structures of the catalytic domains of ADAM17 and ADAM33 reveal a central five-stranded b- sheet surrounded by five a-helices with a conserved methionine residue constituting the so-called “methionine-turn”, a distinctive feature of the metzincin proteases9;10. Not all ADAMS are enzymatically active since some lack the characteristic catalytic zinc-binding signature. The related ADAMTS family differs from the ADAMs in lacking the EGF-like sequences and the transmembrane domains and hence function as secreted proteins. They all retain the metalloprotease domain as well as a disintegrin-like domain. The precise functions of the disintegrin-like domain remain unclear as to whether the interactions with integrins seen in vitro are physiologically relevant. Additionally, ADAMTS proteins contain a variable spacer region and one or more C-terminal thrombospondin (TS) repeat motifs. Originally identified as playing a role in procollagen (procollagen N-endopeptidase, ADAMTS2) and aggrecan (ADAMTS4) processing, the ADAMTS proteins are highly relevant to cartilage degeneration in osteoarthritis and inflammatory joint disease3. A comprehensive comparison of the architecture of the ADAM protein domains is to be found in Takeda’s overview11.

Adamalysins in Biology and Disease

Figure 1. The domain structure of a typical ADAM protein. SP, signal peptide; Pro, Pro-domain, including cysteine switch region; Catalytic, catalytic domain including the zinc-binding HEXXH consensus sequence; Dis, disintegrin domain; EGF, epidermal growth factor domain; TM, transmembrane domain; Cyt, cytoplasmic domain. In the ADAMTS family, the EGF, transmembrane and cytosolic domains are absent and are replaced by one or more thrombospondin type-1 (TSP1) motifs. The central TSP1 domain is highly conserved among the ADAMTS proteins but the number of C-terminal TSP1 domains can vary from zero (ADAMTS4) to 14

(ADAMTS 9 and 20). Some ADAMTS contain additional C-terminal extensions (not shown). The disintegrin-like domain in the ADAMTS proteins lacks the typical arginine-glycine-aspartic acid (RGD) integrin-binding motif of

ADAMs proteins and of the snake venom disintegrins.

ADAMs as key modulators of EGF receptor (EGFR) signalling The epidermal growth factor (EGF)/ErbB family of type I receptor tyrosine kinases participates in various cellular processes including proliferation, differentiation, migration, and cellular survival12. EGFR signalling has been implicated in a wide variety of disease conditions that include cancer, inflammation and fibrosis13. The ErbB receptor family is composed of four members (designated HER1, 2 (HER2/Neu), 3, and 4) which can homodimerize or heterodimerize with each other to form several receptor combinations. They can also bind to members of a group of functionally and structurally similar growth factors that mediate cellular responses ranging from cell survival to proliferation and migration, comprising EGF, transforming growth factor-! (TGF-!), heparin- binding EGF-like growth factor (HB-EGF), amphiregulin, epiregulin, betacellulin, the neuregulins (NRG-1,2,3,4) and epigen14. Most of these EGFR ligands are shed by ADAMs, thereby directly implicating them in activation of EGFR signalling pathways. ADAM17 is prominent in this aspect, since mice deficient in ADAM17 display phenotypes similar to mice deficient in EGF and TGF-!. Moreover, ADAM17 was shown to be the principle sheddase of most EGFR ligands15. ADAM17 activity can be stimulated externally by G protein-coupled receptors (GPCRs) to induce shedding of cell-surface ligands (Figure 2). Upon activation of a GPCR, resulting in signalling via mitogen-

Chapter 2 activated protein kinases and protein kinase C, ADAMs are activated to shed EGFR ligands from the cell membrane, thereby enabling ligand binding to the EGFR. Proven agonists for GPCRs include thrombin, angiotensin II, endothelin-1, lysophosphatidic acid, interleukin-8 and the neuropeptide bombesin. The pathway of GPCR-induced, ADAM-mediated, ligand-dependent activation of the EGFR has been termed EGFR transactivation. The recently discovered process of EGFR transactivation by angiotensin II via the AT1 receptor provides the missing link through which Angiotensin II promotes vascular remodeling16. Downstream of the EGFR, a cascade of distinct signal transduction proteins is phosphorylated upon EGFR activation. These include phosphatidylinositol 3-kinase (P13K), mammalian target of rapamycin (mTOR), MEK, and ERK, which, together or independently, determine gene transcription via transcription factors and thus regulate cell growth, proliferation and migration12. This recently identified pathway of ADAM-mediated transactivation of the EGFR has been attributed central roles in fibrotic disease and cancer (see below).

Figure 2. EGFR transactivation. This figure proposes a signalling mechanism in which GPCR-induced ADAM- dependent EGFR transactivation leads to kinase activation and downstream cellular effects. ROS, reactive oxygen species; PI3K, phosphatidylinositol 3-kinase; mTOR mammalian target of rapamycin HB, heparin binding; AR, amphiregulin; TGF-!; transforming growth factor-!; HB-EGF, heparin binding epidermal growth factor.

Adamalysins in Biology and Disease

Functional Role of ADAMs in Biological and Pathophysiological Processes

Embryogenesis ADAMs play cardinal roles in development. At least 18 different ADAMs have been implicated in spermatogenesis and sperm egg fusion17;18, while ADAMTS-1 is involved in ovulation19. ADAMs are also centrally involved in neurogenesis, adipogenesis, neural crest migration, axon extension and branching morphogenesis in the lung, kidney and pancreas (reviewed in 20) (Table 1). Mice lacking ADAM19 die perinatally because of severe cardiac defects including ventricular septal defect, valvular stenosis and abnormalities of the cardiac blood supply21. This is intriguing, because congenital heart disease is among the most common forms of serious birth defects in humans and yet there is still much to learn about the underlying mechanisms of its pathogenesis. The proposed mechanism of ADAM19’s contributions to heart development relate to shedding of critical growth factors or through cell-cell interactions or signalling via its cytoplasmic domain21. The eminent role attributed to ADAM19 in murine heart development warrants further investigation especially in relation to certain types of human congenital heart defects.

Electrolyte homeostasis Among the various ADAM family members, some are ubiquitously expressed in healthy human tissues, while others are restricted to specific organs or cell types. Despite knowledge of their distributional profile, generally little is known about the physiological significance. An example of how insight into the potential physiological functions can be uncovered is illustrated by a recent investigation of a Dutch family with isolated recessive renal hypomagnesemia (IRH), a disorder that leads to hypomagnesemia due to renal Mg2+ wasting22. Genetic analysis revealed a mutation in the pro-EGF gene, which disturbed the basolateral sorting of pro-EGF on the renal tubular epithelial membrane. As a consequence, only decreased amounts of EGF could be released from the cell membrane, thereby seriously hampering EGF-dependent activation of the EGFR. As a consequence, the Mg2+ permeable channel Transient Receptor Potential Melastatin 6 (TRPM6) was insufficiently activated, which resulted in decreased cellular Mg2+ influx as a final outcome. The functional existence of this EGF - EGFR-dependent reabsorption mechanism was supported by a cohort study of 98 colorectal cancer patients who were treated with anti-EGFR monoclonal antibodies, as most patients developed hypomagnesemia resulting from renal Mg2+ wasting23. Since ADAM10 is the responsible sheddase for releasing EGF from the cell membrane and as such enables its binding to and activation of the EGFR15, we speculate that ADAM10 is a key mediator of the process of magnesium reabsorption. The high expression of other ADAM family members on renal tubular epithelial cells, such as ADAM1924, may point to ADAM-mediated processing of other electrolytes besides magnesium as well.

Chapter 2

PROCESS OR DISEASE ADAM AND ADAM TS

Fertility

Spermatogenesis, 1, 2, 3A, 3B, 4, 5 6, 7, 14, 16, 18, 20, 21, 24, 25, 26, sperm-egg fusion 19, 30, 32, 34 Ovulation TS-1 Implantation 8, 12, 15, TS-1, TS-5

Organ development

Heart 9, 17, 19 Kidney 19 Teeth 28 Lung 17, 33 Gut 35 Sensory Organs 35 Bone 12-S, TS-1, TS-4, TS-5 Pancreas 9, 10, 17 Skeletal Muscle 12 Adipose tissue 12, TS-4, TS-5

Single gene diseases

Familial thrombocytopenia (F+P) TS-13 Ehlers Danlos Type VII 2 TS-2 Weill-Marchesani Syndrome TS-10 VSD and valvular Stenosis 19

Complex diseases

Arthritis (RA, JRA, AS) 17 IBD 15, 17 Psoriasis 17 Atherosclerosis 15, 17 Cardiac hypertrophy and failure 10, 12, 15, 17 Alzheimer’s disease 9, 10, 17 Multiple sclerosis TS-14 Asthma 8, 33, TS-12 Cancer: Breast 9, 11, 12, 17, 28 Brain 8, 19, TS-4, TS-5 Pancreatic 17 Glomerulonephritis 15, 17, 19 Allograft nephropathy 19

Table 1. Associations of ADAM Family Members with Development and Disease Abbreviations; RA: Rheumatoid Arthritis; JRA: Juvenile Rheumatoid Arthritis; AS: Ankylosing Spondylitis; IBD: Inflammatory Bowel Disease; VSD: Ventricular septal defect.

Adamalysins in Biology and Disease

Alzheimer’s disease ADAMs exert important functions in chronic CNS disorders, especially in Alzheimer’s disease where neurodegeneration results from the accumulation in the brain of amyloid " peptide (A") derived from amyloid precursor protein (APP). APP is a substrate for three distinct proteases, known as !-, "-, and #-secretase. The consecutive processing of APP by " and # secretases leads to accumulation of A" whereas the action of !-secretase releases a non-amyloidogenic peptide (sAPPa) considered to be neuroprotective. ADAM9, 10 and 17 have all been reported to exhibit !-secretase activity, although most attention has focused on ADAM10 as the “constitutive” a-secretase responsible for shedding of the extracellular domain sAPPa from APP25. ADAM10 was originally described as a membrane proteinase able to degrade myelin basic protein in vitro, and only subsequently identified as an ADAM protein. ADAM10 is homologous with the Drosophila proteinase Kuzbanian, which initiates the Notch developmental signalling pathway and is important in axon guidance. This is the first step in a general signalling process referred to as regulated intramembrane proteolysis or “protein RIPping”, in which the ADAM-mediated shedding is a prerequisite for a second proteolytic step occurring within the membrane, which is carried out by the presenilin/g-secretase complex (Figure 3). Based on this functional profile, overexpression of ADAM10 to enhance APP processing is currently being explored as a new therapeutic approach for Alzheimer’s disease26;27.

Figure 3. Two-stage process of regulated intra-membrane proteolysis initiated by an ADAM. a, cleavage of the extracellular domain of a single-span transmembrane protein by a membrane-bound ADAM proteinase; b, shedding of the ectodomain; c, secondary cleavage, within the membrane, of the residual membrane protein, typically by the gamma-secretase membrane complex; d, release of the intracellular domain which can migrate to the nucleus and regulate transcriptional activity.

Chapter 2

Thrombotic thrombocytopenic purpura A number of inherited diseases can now be traced to mutations in ADAM genes. The von Willebrand factor (VWF)–cleaving protease has been identified as ADAMTS13, in which more than 50 mutations have been described to cause potentially life-threatening familial thrombotic thrombocytopenic purpura (TTP), mainly in young adults28. In vivo, multimeric VWF adheres to endothelial cells or to connective tissue in the wall of blood vessels. Platelets can adhere to VWF through the platelet membrane glycoprotein GPIb. Under physiological conditions i.e.flowing blood, VWF in the platelet-rich thrombus is stretched and cleaved by ADAMTS13, thereby successfully limiting thrombus growth. However, in the absence of ADAMTS13, the VWF- dependent platelet accumulation is a continuing process, leading to to microvascular thrombosis and TTP. The development of auto-antibodies to ADAMTS13 causes an acquired (idiopathic) form of TTP (ITTP), as evidenced by lack of plasma ADAMTS13 activity, and can be effectively treated by plasma exchange29. Unfortunately, some of the acute severe forms of ITTP are refractory to this treatment and in the chronic relapsing form of ITTP it remains a challenge to achieve a sustained remission. More recently, treatment with retuximab, an anti-CD-20 monoclonal antibody that depletes B-cells, has been successfully used as a prophylactic treatment in patients with acute refractory TTP as evidenced by the disappearance of circulating anti-ADAMTS13 antibodies with restoration of ADAMTS13 activity30.

Endotoxic shock and chronic inflammatory diseases TNF-! is a potent proinflammatory cytokine with cardinal impact on the development of endotoxin shock. It is formed as a membrane-anchored precursor (mTNF-!) and subsequently enzymatically shed from the cell surface. ADAM17 was identified through its ability to generate soluble TNF-! from its membrane precursor, and is often referred to as TNF-!-converting enzyme (TACE)31. ADAM17 deficiency in mature neutrophils and macrophages does not result in complete abrogation of TNF-! shedding implicating other ADAMs in this process32. However, ADAM17 inactivation in myeloid cells in mice offers strong protection from endotoxic shock lethality by preventing the release of soluble TNF-! into the circulation33. These findings corroborate that in mouse myeloid cells in vivo, ADAM17 is the major endotoxin-stimulated TNF-! sheddase and reinforces the view that this enzyme is an important therapeutic target in endotoxin shock and other TNF-!-dependent pathologies33. While blockade of TNF-! with antibodies or receptor fusion protein has failed to provide an effective treatment for established endotoxic shock as a result of the complexity of the clinical setting, the success of this approach in chronic inflammatory disease has been truly remarkable (see below).

Asthma Polymorphisms of the ADAM33 gene are associated with asthma and airway hyper- responsiveness34. Although the molecular basis for this association has yet to be identified, the selective expression of ADAM33 mRNA and protein in airway smooth muscle and fibroblasts supports its role in airway remodelling rather than the inflammatory or immunological aspects of

Adamalysins in Biology and Disease the disease35. ADAM33 polymorphism is also associated with impaired lung function early in life and with an accelerated decline in lung function over time in chronic asthma, COPD, as well as in a normal population36-38. While ADAM33 knockout mice fail to express a phenotype either in the absence or presence of antigen sensitization of the lung, human ADAM33 differs from that of the mouse in being expressed in at least six alternatively spliced forms which are highly expressed in airway fibroblasts and smooth muscle cells as well as in primitive mesenchymal cells during human fetal lung development39;40. A 55kD soluble catalytically active form of ADAM33 (sADAM33) is increased in bronchoalveolar lavage fluid from asthmatic subjects in proportion to disease severity and chronicity, raising the possibility that, in this disease, ADAM33 acquires a ‘gain of function’ due to loss of its transmembrane and regulatory cytoplasmic domain allowing inappropriate access to substrates41. sADAM33 and the isolated catalytic domain induce endothelial cell differentiation in vitro, angiogenic sprouting in vivo and enhanced blood vessel formation in human embryonic lungs explants42. Since greatly increased angiogenesis is a key feature of asthma in adults and children, this has immediate relevance to airway remodelling.

Duchenne muscular dystrophy Human muscular dystrophies are a group of over 30 different genetic diseases. Most of the muscular dystrophy genes encode proteins that play a role in the process of cell adhesion and the structural integrity of muscle. X-linked recessive Duchenne muscular dystrophy is the most common form of muscular dystrophy. In these cases there is a mutation(s) in the gene encoding dystrophin and which affects affects 1/3500 boys. ADAM12 was previously shown to markedly attenuate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. This effect is attributed to the ability of ADAM12 to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. Overexpression of ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. These data stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans43.

Cancer ADAMs, being key mediators of cell-cell signalling, cellular migration and angiogenesis, are thought to play crucial roles in tumour biology. By means of proteolysis, ADAMs allow cell membrane-anchored growth factors to become available to cells that are not in direct physical contact. As such, ADAMs can modify the tissue environment in which tumour cells behave. Specifically ADAMs’ role to serve autocrine and paracrine transactivation of the EGFR, an oncogene involved in mitogenesis and proliferation, constitutes an important novel mechanistic concept in cancer biology. It has recently been demonstrated that EGFR transactivation occurs in more than 60 human carcinoma cell lines derived from different tissues44. Additionally, signalling through this pathway has been positively associated with cell proliferation, cell-cycle progression,

Chapter 2 increased migration, tumour growth and metastasis, and negatively associated with treatment success and beneficial patient outcome44-47. The implications of ADAMs in tumour biology have thus far been corroborated by a large number of associative tissue studies and by several in vitro and in vivo interventional experiments. Although no specific mutation in ADAM genes has yet been linked with any type of tumour, an increased expression of ADAM8, 9,10, 12, 15, 17, 19 and 28 was documented in a variety of human cancers (reviewed in48). Most widely expressed and best described is ADAM17, the principle sheddase of EGFR ligands. In colon carcinoma, coexpression of ADAM17 with the activated EGFR was particularly observed in endothelial cells belonging to the tumour stroma, suggesting a role for ADAM17-mediated EGFR transactivation in tumour angiogenesis49. Opposite to the expression of ADAM17, the expression of TIMP-3, the natural inhibitor of ADAM17, was downregulated in various types of cancer50. In the breast, ADAM17 was upregulated in tumour samples when compared with normal mammary tissue51. Moreover, increased ADAM17 expression was detected in high-grade compared with low-grade breast tumours. Patients with high ADAM17 expression had a significantly shorter overall survival compared with those with low ADAM17 expression52;53. Intriguingly, the prognostic impact of ADAM17 was independent of conventional prognostic factors for breast cancer, such as tumour size, grade and lymph node status53. ADAM17 inhibition in vitro using synthetic inhibitors or siRNA reduced EGFR ligand shedding in several breast cancer cell lines52. Moreover, ADAM17 inhibition reverted the malignant phenotype of breast cancer tissue- derived T4-2 cells and restored normal mammary acinar organization. In an in vivo xenograft model, pharmacological ADAM17 inhibition reduced human breast cell tumour growth54. In search for the mechanism by which ADAM17 inhibition exerted its beneficial effects on tumour growth, it was elegantly worked out that cell lines expressing mutant forms of amphiregulin or TGF-! which did not require ADAM17 for their proteolytic release from the cell surface, were susceptible to EGFR inhibition but resistant to ADAM17 inhibition. This indicates that, while the cleavage of all other ADAM17 substrates is inhibited, it is the prevention of the cleavage of EGFR ligands which mediates the anti-tumour effects52. In other experiments, it was shown that ADAM-mediated shedding of EGFR ligands with subsequent EGFR activation and downstream signalling is a central part of the regulatory system that modulates the migratory and invasive behaviour of tumour cells55.

Perspectives for the therapeutic potential of ADAMs in disease

Chronic inflammatory disorders The identification of TNF-! as a major therapeutic target in chronic inflammatory disorders such as rheumatoid arthritis (RA), juvenile arthritis, ankylosing spondylitis, Behcet’s disease, inflammatory bowel disease, psoriasis and sarcoidosis has provided the basis for treating these disorders with TNF-!-blocking monoclonal antibodies and soluble receptors to scavenge TNF-!.

Adamalysins in Biology and Disease

Interest in this approach has further increased with the knowledge that, in addition to inducing disease remission, anti-TNF-! therapy in RA has the potential to prevent radiographic progression 56. While highly effective in some of these chronic inflammatory diseases, the use of biological reagents is expensive. Since soluble TNF-! is produced through the action of ADAM17 (TACE), this enzyme has been selected as a prime target for therapeutic intervention. Initially, broad spectrum MMP inhibitors were developed, but their use was discontinued on account of musculoskeletal pain and increased inflammation. The early ADAM17 inhibitors (such as GI5402) were not sufficiently selective and inhibited other metalloproteases, as well as the cleavage of multiple human membrane expressed molecules, including both the p55 and p75 TNF receptors57. TNF receptors cleaved during inflammation exert a regulatory function by inhibiting further TNF-! signalling and through soluble TNF receptors retaining their ability to bind and neutralise soluble TNF-!. This mechanism has been postulated to explain why the early ADAM17 inhibitors increased rather than decreased the inflammatory response58. Given the promiscuity of ADAM17 in cleaving a broad spectrum of cell membrane-bound factors, it is likely that beneficial effects of ADAM17 inhibition on the target substrate are counterbalanced by undesired alterations with regard to other ADAM17 substrates. However, there remains considerable potential for ADAM17 inhibitors if selectivity could be achieved 59;60. One of the latest described competitive inhibitors of ADAM17 in vivo is WTACE2, a nonpeptide sulfonamide metalloprotease inhibitor which is orally bioavailable61. When it was administered in an experimental model of autosomal recessive polycystic kidney disease, WTACE2 markedly slowed progression with improved renal function. This benefit was initially attributed to modulation of the TGF-!/EGFR axis, but subsequent experiments in TGF-a -/- mice revealed no changes in the disease suggesting that other ADAM17 targets are involved as well62. Indeed, when WTACE2 was combined with an inhibitor of EGFR tyrosine kinase the positive effects on renal damage were markedly enhanced63. In mice treated with angiotensin II, concomitant ADAM17 inhibition using WTACE2 markedly reduced renal fibrotic lesions. The hydroxamate based inhibitor GW280264X is also a potent inhibitor of ADAM17 and to a lesser extent of ADAM10, while the closely related compound GI254023X is a potent inhibitor of ADAM10 but has >100-fold reduced potency toward ADAM1764;65. The metalloprotease activity of ADAM19 is also sensitive to the hydroxamic acid-type metalloprotease inhibitor BB9466. The endogenous tissue inhibitors of metalloproteases (TIMPs) inhibit the activities of some ADAMs as well as that from MMPs, but selectivity among the TIMP members differs to great extent67. For example, TIMP-3 is a potent inhibitor of ADAM10 and ADAM17, whereas TIMP-1 only inhibits ADAM1068. ADAM33 is inhibited moderately by TIMP-3 and TIMP-4, weakly by TIMP-2 but not at all by TIMP-1, a profile distinct from other ADAMs69. Whether these naturally occurring inhibitors can be used in cell systems or in vivo remains to be elucidated70.

Chapter 2

Cancer Clinically relevant, selective inhibition of ADAM family members blocked tumour cell proliferation and invasion in vitro71-73 and in vivo54. The developments in the field of ADAM inhibition as anti- cancer treatment should be appreciated in the light of currently existing EGFR blocking therapies. Targeting the EGFR or receptor family member HER2/Neu by monoclonal antibodies and receptor tyrosine kinase inhibitors has been approved by the Food and Drug Administration for the clinical treatment of colorectal cancer and lung cancer, and its usage has led to a significant increase in patient survival. This beneficial treatment outcome is confronted with the occurrence of drug-related side effects and the development of drug resistance. The latter is probably caused by excessive availability of EGFR ligands, as it was shown that in vitro administration of TGF-! abolished the growth inhibitory effect of Trastuzumab (a monoclonal antibody directed against HER2/Neu), and that excess production of TGF-! as well as amphiregulin conferred resistance to Gefitinib (EGFR tyrosine kinase inhibitor) in lung cancer74. Since ADAMs are responsible for shedding EGFR ligands, ADAM inhibition may form the solution to the problem of drug resistance by reducing the availability of EGFR ligands. Additionally, the theoretical beneficial effect of inhibiting both ADAM17 and EGFR to reduce EGFR signalling has been corroborated by in vitro and in vivo studies that showed a synergistic inhibition of tumour growth upon combined therapy71;75-77. Clinically appealing, ADAM17 inhibition together with Paclitaxel, a traditional chemotherapy drug, attenuated tumour growth in xenograft models of non-small cell lung cancer and breast cancer54;73. The outcome of the currently running phase II clinical trial that elaborates on the value of ADAM10 and 17 inhibition together with Trastuzumab as treatment against breast cancer (www.incyte.com) will provide further insight into the clinical potential of ADAM intervention as anti-cancer therapy.

Concluding remarks The role of ADAMs, as presented in this review, is evident in virtually all the major disease states including inflammation, fibrosis, neurodegeneration and cancer. Its crucial functioning in the shedding of various growth factors, cytokines, and adhesion molecules identifies these cell surface enzymes as critical mediators of various (patho)-physiological processes. The therapeutic potential of ADAMs has only just started to be explored but undoubtedly will be significant. The initial results of targeting this enzyme class are so promising to convince us that development of therapeutics directed to ADAMs will open novel pathways in the treatment of many human disease states - ranging from inflammatory and degenerative to neoplastic disease.

Acknowledgements Supported by Grants from the Dutch Kidney Foundation (HVG), The U.K. M.R.C. and B.B.S.R.C. (AJT). We thank M. Rook for contributions in graphical design.

Adamalysins in Biology and Disease

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