ADAM17: a molecular switch to control inflammation and tissue regeneration

Review

ADAM17: a molecular switch to control inflammation and tissue regeneration Ju¨rgen Scheller2, Athena Chalaris1, Christoph Garbers1 and Stefan Rose-John1 1 2

Institute of Biochemistry, Christian-Albrechts-University, Olshausenstrasse 40, D-24098 Kiel, Germany Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Du¨sseldorf, Germany

A disintegrin and metalloproteinase 17 (ADAM17), also known as tumor necrosis factor-a converting enzyme (TACE), is a membrane-bound enzyme that cleaves cell surface proteins, such as cytokines (e.g. TNFa), cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGFa and amphiregulin) and adhesion proteins (e.g. Lselectin and ICAM-1). Here we examine how ectodomain shedding of these molecules can alter their biology and impact on immune and inflammatory responses and cancer development. Gene targeting of Adam17 is embryonic lethal, highlighting the importance of ectodomain shedding during development. Tissue-specific deletion, or hypomorphic knock-in, of Adam17 demonstrates an in vivo role for ADAM17 in controlling inflammation and tissue regeneration. The potential of ADAM17 as therapeutic target is also discussed. The A disintegrin and metalloproteinase (ADAM17) is the prototype of the ADAM family of ectodomain shedding proteases It is estimated that on platelets, up to 10% of all cell surface proteins are proteolytically cleaved and released into the extracellular space; this percentage is likely to be in the same range for all somatic cells via a process called ectodomain shedding [1]. Shedding of integral membrane proteins is observed mostly for type I and type II transmembrane or GPI-anchored proteins and the cleavage site is generally located in close proximity to the outer surface of the cell membrane. Ectodomain shedding of transmembrane proteins provides a mechanism for protein downregulation on the cell surface. In addition, production of soluble, functional protein ectodomains, including cytokines and soluble cytokine receptors, can serve to initiate or inhibit autocrine and paracrine signaling. Members of the ADAM family have emerged as major ectodomain shedding proteinases. ADAMs are type I transmembrane proteins that consist of an N-terminal signal sequence followed by a prodomain, a metalloproteinase (catalytic) domain, a disintegrin domain, an EGF-like (cysteine-rich) domain, a single transmembrane domain and a cytoplasmic portion (Figure 1). The prodomain inhibits ADAM17 proteinase activity and is cleaved (to relieve inhibitory effects) by furin proteinases [2–4]. ADAM disintegrin-domains can, in some cases, interact with integrins to influence cell adhesion and cell–cell interactions [5]. The disintegrin- and/or EGF-like-domains are thought to Corresponding author: Rose-John, S. ([email protected])

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be involved in substrate recognition and/or (hetero)-dimerization. Most cleavage events examined are mediated by ADAM17 and its close relative ADAM10 [5]. In all, 76 proteins have been identified as substrates for ADAM17 (Table 1), among them important immunoregulatory cytokines such as TNFa, some ErbB ligands and their receptors, interleukin-6 receptor (IL-6R) and cell adhesion molecules such as L-selectin (CD62L) or ICAM-1. There is overlap and compensation between ADAM proteases for several substrates [4]. Here we discuss recent literature that suggests that ADAM17 can act as a molecular switch to control immune responses, tissue regeneration and cancer development. We also evaluate ADAM17 as a novel therapeutic target in immune-related diseases [6]. ADAM17 function in the immune system Inflammation is characterized by elevated levels of cytokines such as TNFa and IL-6. As outlined below, ADAM17 impacts the biology of TNFa and IL-6, which led to the speculation that inhibition of this metalloprotease might have beneficial effects in autoimmune diseases [6]. In the mouse, blockade of TNFa or inhibition of TNFa shedding results in survival of otherwise lethal LPS-induced septic shock [7]. Unfortunately, use of neutralizing anti-TNFa monoclonal antibodies (mAbs) in clinical trials was not effective in patients with sepsis [8]. Neutralizing TNFa activity with neutralizing mAbs or soluble TNFR was, however, beneficial for patients with chronic (auto)inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease [9]. Today, several TNFaneutralizing agents are approved worldwide for the treatment of inflammatory disorders and far more than one million patients have been treated [9]. In murine experimental autoimmune encephalomyelitis, a model of the human disease multiple sclerosis, membrane-bound TNFa had anti-inflammatory properties [10], leading to the hypothesis that membrane-bound TNFa is anti-inflammatory and that cleavage of TNFa by ADAM17 activity is a prerequisite for pro-inflammatory TNFa activity [11]. Treatment with anti-TNFa agents worsened multiple sclerosis in patients, demonstrating the anti-inflammatory capacity of TNFa and consequently, TNFa inhibitors carry a label from the US Food and Drug Administration warning about the treatment’s potential for worsening multiple sclerosis and other demyelinating disorders [12]. This example shows clearly that membranebound TNFa has anti-inflammatory properties and that cleavage by ADAM17 renders TNFa pro-inflammatory.

1471-4906/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2011.05.005 Trends in Immunology, August 2011, Vol. 32, No. 8

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Pro-domain

Inactivation, chaperone

Metalloprotease domain

Ectodomain shedding

Disintegrin domain

Interaction with integrins; activation?

EGF-like domain

Substrate recognition and activation?

Transmembrane domain Cytoplasmic domain

Subcellular localization, trafficking and activation via phosphorylation, interaction with signaling molecules TRENDS in Immunology

Figure 1. A schematic illustration of the ADAM17 domain structure and (proposed) function(s). ADAM17 is synthesized as a zymogen with the prodomain acting as an inhibitor of the protease activity by a mechanism most likely to be different from the conventional cysteine switch mechanism that occurs when a conserved cysteine residue interacts with the zinc in the active site and prevents binding and cleavage of the substrate [2]. An intramolecular chaperone function has been implicated for the ADAM17 prodomain [3]. During ADAM17 maturation, the prodomain is removed by furin cleavage. The metalloprotease domain contains the zinc-binding consensus motif and forms the active site of the enzyme. The function of the disintegrin and EGF-like domains of ADAM17 is not well understood, although there are implications that the disintegrin domain modulates cell migration by the interaction with the a5b1 integrin [87]. The cytoplasmic tail is a target for kinases and contains putative MAPK and PKC phosphorylation sites. It is unclear whether the cytoplasmic tail of ADAM17 is required for correct trafficking and activation of the enzyme (this figure is modified from [88]).

ADAM17 is the main sheddase of the adhesion protein L-selectin, although leukocyte-specific inactivation of ADAM17 did not affect serum levels of soluble L-selectin, suggesting that ADAM17 is not the only enzyme responsible for shedding this substrate (Figure 2) [13,14]. Mice engineered to express a non-cleavable version of L-selectin have a twofold increase in cell surface L-selectin expression on leukocytes and, in these mice, neutrophils enter the inflamed peritoneum in greater numbers or for a longer duration. In addition, L-selectin serum levels are strongly reduced [15]. Therefore, ADAM17 activity regulates both homeostatic and activation-induced changes in cell surface L-selectin density and, thus, might direct the migration patterns of activated lymphocytes and neutrophils in vivo [15]. ADAM17-mediated shedding of MHC class I chain-related proteins, ligands of the NK cell receptor NKD2D,

represents a strategy by which a cell can evade immune recognition by NK cells and CTLs and might contribute to escape of immune surveillance by cancer cells [16]. Upon antigen uptake, dendritic cells (DCs) migrate to the lymph node. Antigen uptake and TLR engagement in DCs leads to disassembly of podosomes, which are sites of integrinstimulated actin polymerization close to the cell membrane. Loss of podosomes depends on ADAM17 activity. After antigen sampling, e.g. in the skin, DCs need to migrate to the lymph node in order to stimulate T cells. As podosome loss seems to be a prerequisite of migration, these data point to a decisive role of ADAM17 in the early phase of DC activation and therefore in the early phase of the immune response [17]. Apoptosis leads to activation of ADAM17 and shedding of IL-6R from neutrophils, pre-B-cells and hepatoma cells [18]. The agonistic sIL-6R, in a complex with IL-6, induces

Table 1. ADAM17 substrates. Cytokines, growth factors TNF[29,67]1 TGFa[29] [42]1,2 AREG[29] [42]1,2 EREG [29] EPGN, Epigen [29] NRG1, Heregulin [29] HB-EGF [29,42]1,2 Pref1 [29] Fractalkine/CX3CL1 [29] TRANCE/RANKL [76] CSF-1 [39]

SEMA4D [29] LAG-3 [29] DLL1 [29] KL-1 [29] KL-2 [29] MICA [29] MICB [16] Jagged [71] LTA [73] TMEFF2 [77] FLT-3L [66]1

Receptors p55 TNF-a RI [29,67]1 p75 TNF-a RII [29,67]1 p75NTR [29] IL-6Ra [29] IL-1R2 [29] NTRK1, TrkA [29] GHR [29] CSF1R, M-CSFR [29] SORL1, SORLA [29] SORCS1 [29] SORCS3 [29] SORT1 [29] CD91/APOER [82] PTPRF, PTP-LAR [29] EPCR [29] ACE2 [29] LOX-1 [85]

NPR [29] HER4/ErbB4 [29] Notch1 [29] TNFRSF8, CD30 TNFRSF5, CD40 [29] GPIba [29,68]1 GPV [70] GPVI [72] SDC1 [74] SDC4 [74] KDR, VEGFR2 [79] CD89 [80] Ptprz [83] IGF2-R [84] M6P/IGF2R [84]

Adhesion molecules ICAM-1 [29] VCAM-1 [29] NCAM [29] ALCAM [29] L1-CAM [29] EpCAM [69] DSG2 [29] CD62L [13,29]1 Collagen XVII [29] PVRL4, Nectin-4 [29] CD44 [29] F11R, JAM-A [81]

Other molecules APP [29] GP [29] CA9 [29] PRNP, PrPc [29] KL [29] MUC-1 [29] LYPD3, C4.4A [29] VASN [27] CD163 [75] PMEL17 [78]

1

Bold: substrates verified in vivo using genetically engineered mice. In these studies, shedding of the substrate in vivo was demonstrated only indirectly in TIMP3-deficient mice, which show activation of ADAM17.

2

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ErbB Transactivation

TNFR Transactivation

IL-6 Transsignaling

Notch ligand shedding

Biological adhesion buffer system Cell membrane

ErbB

TNFR

GlyCAM-1 CD34 MadCAM-1 PSGL-1

Jagged

gp130 sgp130

sErbB

sJagged sTNFR

sErbB ligands

sIL-6R sTNF

+

ErbB ligands

Intercellular/ Extracelullar

sCD62L

IL-6

TNF

IL-6R

Notch1

Leukocyte migration

CD62L Cell membrane

γ-secretase cleavage

Intracellular

Notch ICD Ligand-independent notch signaling TRENDS in Immunology

Figure 2. ADAM17-induced signal transduction. ADAM17 cleaves various transmembrane proteins, including ErbB ligands, TNFa, IL-6R and L-selectin. ADAM17 induces ErbB transactivation via shedding of ErbB ligands, ‘TNFR-transactivation’ via soluble TNFa, TNFa inhibition via generation of an antagonistic soluble TNFR and IL-6 transsignaling via the agonistic sIL-6R. In addition, ADAM17 induces Notch1 signaling by ligand-independent Notch1 cleavage leading to Notch-ICD generation (RIPing) and transcription of Notch1 target genes. Proteolytic processing of Notch1-ligands, such as Jagged and Dll1, by ADAM17 is reported, which might serve to limit the amount of ligand available at the cell surface to activate Notch1 in a ligand-dependent manner [89,90]. The constitutive cleavage of L-selectin (CD62L) generates a soluble ectodomain, which has an anti-inflammatory effect by inhibiting adhesion of leukocytes to the endothelium. However, it is still unclear if ADAM17 mediates constitutive L-selectin cleavage [13]. Activated endothelium upregulates the expression of adhesion molecules and allows leukocytes to attach and migrate. Thus, stimulated shedding of Lselectin is pro-inflammatory by facilitating leukocyte diapedesis.

IL-6 trans-signaling on cells that express only the IL-6 signal transducer receptor chain gp130 (Figure 2). In a mouse model of acute inflammation, sIL-6R generated by dying neutrophils, together with IL-6, stimulated endothelial cells to secrete MCP-1, a chemokine that leads to attraction of mononuclear cells. Here, ADAM17 is involved in the resolution of inflammation [18,19]. IL-6 responses mediated by IL-6 trans-signaling can also maintain inflammatory states and promote inflammation-associated cancer [20–22], whereas classic IL-6 responses via the membrane-bound IL-6R tend to be rather anti-inflammatory or regenerative [20]. sIL-6R is produced by naive and memory CD4 T cells upon TCR activation via ADAM17-mediated shedding. By contrast, sIL-6R generation by CD8 T cells is negligible [23]. The balance between T regulatory (Treg) cells and T helper (Th)17 cells is regulated via TGFb and IL-6 signaling [24]. Uncommitted T cells differentiate into Treg cells upon TGFb stimulation, whereas triggering of the same cells with TGFb and IL-6 induces Th17 cell differentiation [24,25]. Stimulation of T cells with TGFb in the presence of IL-6 and sIL-6R led to more suppression of Treg cell differentiation than in the presence of IL-6 alone, indicating that IL-6 trans-signaling contributes to regulate the balance between Treg 382

cells and Th17 cells [26]. The involvement of ADAM17 in this balance is further supported by the fact that TGFb activity is regulated by cleavage of vasorin, a protein that binds and neutralizes TGFb in its soluble but not in its cellbound form, thereby influencing the generation of regulatory T cells by TGFb [27]. These data show that ADAM17 activity influences various aspects of T cell biology. Nevertheless, the precise role of ADAM17 activation on T cell differentiation and activation during inflammatory processes in vivo needs to be further addressed in animal studies. ADAM17 and cancer ErbB signaling is involved in the growth of many tumors [28,29]. ADAM17 is upregulated in most tumor cells [28]. In a process called transactivation, shedding of ErbB ligands, e.g. from epithelial cells (Figure 2), by ADAM17 (Table 1) is necessary for appropriate stimulation of ErbB [30]. ADAM17-mediated shedding of ErbB ligands is required for growth of lung carcinoma cells. Surprisingly, on the same cells ADAM17 cleaves Notch1 and the subsequent g-secretase-mediated release of the Notch1-intracellular domain leads to transcriptional upregulation of human ErbB mRNA and increased ErbB expression on the cell surface of human carcinoma cells [31].

Review In addition to regulation at the transcriptional level, ADAM17 activity is controlled post-translationally in cancer cells by oncogenic ras, src and v-src, which leads to increased shedding of the ErbB ligand TGFa [32,33]. In addition, activated ADAM17 and subsequent ErbB transactivation can mediate resistance to chemotherapy [34]. Moreover, the Her2/neu neutralizing antibody Herceptin1 (trastuzumab), which is used in patients with HER2-positive breast cancer, can increase ADAM17 expression and activity via PKB inhibition. As a consequence, ADAM17mediated shedding of soluble ErbB ligands activates other members of the ErbB family in the presence of Herceptin. Therefore, in this scenario, ADAM17 contributes to the escape of tumor cells from antibody blockade [35]. These data show that the involvement of ADAM17 in tumor growth is complex. It remains to be seen whether the role of ADAM17 during cancer development is mostly restricted to shedding of ErbB ligands. Studying ADAM17 activity in vivo ADAM17-deficient mice have defects in the mammary epithelium, vascular system, lung, eye, hair, heart and skin and as a result die between embryonic day 17.5 and the first few days after birth [36]. This is reminiscent of ErbB ligand-deficient mice, indicating the importance of the ADAM17 ErbB axis during development [36]. The small subpopulation of ADAM17-deficient mice that survive have reduced lymphocyte numbers, impaired T and B cell development, reduced body weight and an overall hypermetabolic phenotype [37,38]. Conditional ADAM17-deficient mice and hypomorphic ADAM17 mice were developed to analyze the role of ADAM17 in inflammation and cancer in vivo. Mice with ADAM17-deficient leukocytes, monocytes and granulocytes were protected from fatal LPS-induced endotoxemia due to a failure to cleave TNFa [39]. During Escherichia coli-mediated peritonitis, ADAM17-deficient leukocytes provided mice with a survival benefit, most likely due to the changed time course of neutrophil recruitment into the peritoneal cavity [40]. Furthermore, ADAM17-mediated shedding of L-selectin reduced the recruitment (or infiltration) of neutrophils to the inflamed site in thioglycollateinduced peritonitis (Figure 2) [15]. The role of ADAM17 in skeletal development was studied in mice with a targeted deletion of ADAM17 in osteochondroprogenitor cells [41]. These mice had a reduced life-span and showed increased osteoblast numbers and osteoclasts activation with early onset of bone loss, shorter long bones and osteoporosis due to dysregulated production of G-CSF and IL-17. Mice with a deletion of ADAM17 in endothelial cells exhibited resistance in the B16F0 melanoma model and reduced pathological neovascularization in the oxygen-induced retinopathy model, probably due to inhibition of HB-EGF shedding. A recent study showed that ADAM17 protects hepatocytes from apoptosis. Deletion of ADAM17 in hepatocytes led to increased sensitivity to Fas-induced liver failure. In addition, adenoviral ADAM17 gene delivery prevented acute liver failure in drug-induced toxicity via ErbB activation and subsequent transcription of anti-apoptotic molecules [42].

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Owing to ubiquitous expression of ADAM17, it might be difficult to choose the transgenic Cre recombinase mouse strain appropriate to study the role of ADAM17 in complex disease models, which depend on activation of many cell types. Thus, a mouse model with ADAM17 expression downregulated in all tissues would be desirable. Using a novel gene targeting strategy, so-called exon induced translational stop (EXITS), hypomorphic Adam17ex/ex mice have recently been generated that have reduced amounts of ADAM17 expression in all tissues. Adam17ex/ex mice are viable, show reduced shedding of ADAM17 substrates and have tissue defects similar to those of ADAM17-deficient mice, albeit to a lesser degree [36,43]. Adam17ex/ex mice showed increased susceptibility to intestinal inflammation in response to the irritant dextran sodium sulfate (DSS) due to reduced shedding of ErbB ligands and failure of mucosal regeneration. A mouse strain with impaired ADAM17 activity generated by random mutagenesis also exhibited increased inflammation upon administration of DSS, confirming the protective role of ADAM17 in experimental colitis [43,44]. Regulation of ADAM17 activity Given the importance of cytokine and cytokine receptor shedding for activation of the immune system, it is important to understand how shedding is regulated at the cellular level (reviewed in [45]). ADAM17 is expressed and upregulated in tumor cells almost ubiquitously; however, the involvement of the tumor microenvironment for regulation of ADAM17 expression is not known. The phorbol ester phorbol 12-myristate 13-acetate (PMA) is a potent activator of ADAM17. PMA induces phosphorylation of PKC and ERK and blocking these kinases leads to decreased ADAM17 activity, which leads to shedding of some substrates, such as IL-6R [46]. An early report showed that the ADAM17 intracellular domain (ICD) was dispensable for PMA-induced shedding of the ADAM17 substrates TNFa, p75 TNFR and IL-1RII [47]. Regarding the substrates themselves, the ICD of Lselectin played a role in PMA-induced ADAM17-mediated shedding [48], whereas it was not involved in PMA-induced shedding of IL-6R [49]. L-selectin is linked with the integral cortical actin cytoskeleton via ezrin-radixin-moesin (ERM) proteins and reduced interactions between the ICD of L-selectin with ERM proteins reduced PMA-induced shedding, probably by reducing microvillar localization of L-selectin [48]. Other examples of substrate ICDs involved in ADAM17-mediated shedding, induced by PMA or other stimuli, are still missing. It is tempting to speculate that additional accessory proteins binding either to the ICD of ADAM17 or of the substrates is responsible for regulation of specificity of ADAM17-mediated shedding in vivo. However, it is not clear if ADAM17 acts in vivo like a lawnmower to release any accessible substrate or, more specifically, like a scissor-type instrument to release only selected substrates. Recently, it was demonstrated that direct modification of disulfide bonding within the ADAM17 extracellular domains induced ADAM17 to release L-selectin or HB-EGF after treatment with H2O2 or stimulation by PMA, respectively [50,51]. Modification of disulfide 383

Review bonding was achieved by inactivation of extracellular protein disulfide isomerases (PDIs). The authors of those studies speculated that PMA-induced reactive oxygen species (ROS) alter the cellular redox state, which in turn inactivates PDIs allowing ADAM17 to adopt an’open‘active conformation by rearrangement of disulfide bonding leading to movement of ADAM17 and its substrate(s) within the membrane. ADAM17 was found sequestered in lipid rafts [52] and destruction of the rafts by cholesterol depletion induced ADAM17-dependent shedding [53]. Furthermore, on platelets, ADAM17-mediated shedding of GPIbalpha and GPV was induced after treatment with H2O2 [54]. Whether treatment with H2O2, PDI-inactivation or cholesterol depletion was dependent on the intracellular domains of ADAM17 or of the substrate(s) was not investigated. It is unclear whether treatment with H2O2 and PDIinactivation is restricted to shedding of L-selectin, HB-EGF, GPIbalpha and GPV or if this is transferable to many ADAM17 substrates. Finally, a physiological condition reflecting stimulation by PMA remains to be identified. Intracellular signaling molecules might regulate ADAM17 subcellular localization. The majority of endogenous ADAM17 is stored in perinuclear regions and upon phosphorylation (Thr735) is translocated to the cell surface [55]. ADAM17 and several other ADAM proteins contain a proline-rich PXXP consensus sequences (ADAM17: ProGln-Thr735-Pro) in their cytoplasmic domain, which enables interaction with SH3 domain-containing proteins. The antibiotic anisomycin inhibits protein synthesis and activates stress-activated protein kinases. Anisomycin-induced p38 MAPK activation led to phosphorylation (Thr735) and activation of ADAM17, which resulted in TGFa shedding and subsequent ErbB activation [46]. In vitro, cells expressing a Thr735Ala ADAM17 mutant had a basal shedding activity lower than that of WT cells and shedding was not induced by p38 MAPK. In contrast, PMAinduced shedding was normal in the Thr735Ala mutant. p38 MAPK activation also led to increased expression of ADAM17 on the cell surface, which was independent of ADAM17 catalytic activity [46], showing that the intracellular domain participates in regulation of ADAM17 activity and, under some circumstances, is required for shedding. Furthermore, H2O2-induced ADAM17-mediated shedding of GPIbalpha and GPV from platelets was dependent on p38 activation [54], suggesting that apart from modification of extracellular disulfide bridging of ADAM17, treatment with H2O2 might induce ADAM17 cell surface expression via p38-mediated ADAM17-phosphorylation. TLR3-TRIF-RIP signaling, and the downstream activation of caspase-mediated apoptosis and ROS generation by the NADPH oxidase homolog dual oxidase 2 (DUOX2), was found to contribute to ADAM17-mediated shedding of TNF-receptor 1 (TNFR) [56]. Importantly, p38 MAPK and ERK activation were not required, indicating a novel activation of ADAM17 [56]. Apoptosis activated ADAM17mediated IL-6R [18] and L-selectin shedding [57]. IL-6R shedding during apoptosis was independent of ERK, PKC and p38 MAPK activation [18]. How caspases activate ADAM17 is not known, but this might be linked to the disturbance of membrane homeostasis during apoptosis 384

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[18]. TLR signaling induced L-selectin shedding via ADAM17 activation [58], which might be related to TLRmediated extracellular release of ATP and purinergic receptor activation, which again induced apoptosis and activation of DUOX [56,59]. TLR and ATP-induced TGFa shedding was inhibited by siRNA against DUOX1 and blockade of ERK, consistent with a role of ERK-mediated phosphorylation in the activation of ADAM17 [59]. These data integrate TLR-receptor activation and ADAM17-mediated shedding but the physiological relevance in vivo is not known. ADAM17 activity is regulated also by the extracellular inhibitory protein tissue inhibitor of metallo-proteinase-3 (TIMP3) [60]. The relevance of this inhibitor mechanism was demonstrated in Jun-deficient mice, in which TIMP3 is downregulated. ADAM17-mediated production of soluble TNFa is increased in these mice, which contributes to development of a psoriasis-like inflammatory skin disease [60]. Recently, ADAM17 was found to interact with the tetraspanin web compound CD9 on the surface of leukocytes and endothelial cells. The tetraspanin web organizes cell surface microdomains. Downregulation or blockade of CD9 increased PMA-induced ADAM17-mediated shedding of TNFa and ICAM-1, and represents a novel extracellular negative regulator of ADAM17 activity [61]. It can be concluded that ADAM17 activation is a tightly regulated multistep process that involves modulation of expression, cell surface translocation, disulfide bridging, cell membrane localization and extracellular inhibitory proteins. ADAM17 becomes fully activated only after all these steps have been accomplished. However, it has to be noted that all these studies were done in vitro and functional regulatory data in vivo are still missing. The molecular mechanisms leading to activation of ADAM17 in inflammation and cancer need to be fully understood to evaluate the feasibility of therapeutic intervention. Moreover, the available animal models have taught us that inhibition of ADAM17 can be expected to provoke negative side-effects; e.g. in the intestine and in the liver [43,44]. Recently, it was found that, unlike human IL-6R, murine IL-6R is not shed by ADAM17 [62]. Moreover, apoptosis-induced shedding of IL-6R in mice is mediated by ADAM10 and not ADAM17, indicating that ADAM protease targets are species-specific [18,62]. Shedding of murine and human IL-6R induced by ionomycin or P2X7 receptor stimulation was dependent on murine and human ADAM10, respectively. This unexpected species specificity of ADAM10 and ADAM17 and the identification of ADAM10 as a novel inducible sheddase of IL-6R in mouse and man might have consequences for the interpretation of phenotypes of ADAM17 and ADAM10-deficient mice [62]. It will be of interest to determine if the identity of ADAM proteases responsible for cleaving other ADAM substrates is also species-specific. Finally, a consensus cleavage sequence has not been identified for ADAM17 substrates [63], although a preference for leucine at position P4, alanine at position P3, leucine/glutamine at position P2, alanine at position P1, valine at positions P1’ and P2’, serine at positions P3’ and P4’ has been noted (http://merops.sanger.ac.uk). It is noteworthy that seven of eight amino acids (underlined) fit this

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Box 1. Sheddome analysis and future questions A detailed analysis of the role of ADAM10 and ADAM17 in shedding of the platelet protein GPVI showed that cleavage could be mediated by both proteases in vitro. Mice lacking both, ADAM10 and ADAM17 were still able to shed GPVI, arguing for the existence of additional shedding proteases [72]. The sheddome of human platelets was investigated and led to the identification of 69 shed membrane proteins, 59 of which were not known to be shed from platelets. By using a partially selective ADAM17 inhibitor (blocking MMP3 and MMP12 as well as ADAM with thrombospondin motifs 4 (ADAMTS4)) [6], shedding of 13 out of 69 substrates was inhibited by at least 50% [1]. Only 5 out of 13 proteins were known substrates of ADAM17 and the remaining 8 might represent novel ADAM17 substrates (ephrin B1, PTPRG, CANT1, tetraspanin-9, semaphorin 7A, fibrocystin-L, D-glucuronyl C5 epimerase and TLT-1) [1]. This raises the possibility of using differential sheddome analysis that is generated by ADAM17 depending on different stimulatory conditions using ADAM17-deficient mice and control mice. Such data, however, will have to be interpreted with caution because compensatory shedding of ADAM17-dependent substrates by

TNFα CD62L IL-6R

Regulation of inflammation  Neutrophil macrophage infiltration  Fever induction  T cell activation/anti-apoptosis

ADAM10 has been described in ADAM17-deficient murine embryonic fibroblasts [86]. This example illustrates the fact that even though many reports have elucidated general mechanisms of ADAM17 activation and regulation, the high degree of functional diversity of the 76 reported ADAM17 substrates raises unsolved central questions:  What are the structural requirements for ADAM17 substrate recognition?  Plasticity of the ADAM17 sheddome. How is shedding of 76 or more substrates regulated at the cellular level?  Are the ADAM17 substrates identified in cell culture experiments shed by ADAM17 in vivo?  What are the functional consequences of shedding of ADAM17 substrates in vivo?  Is ADAM17 substrate function best examined in ADAM17-deficient mice or with mice transgenic for soluble or uncleavable substrates? What is the best, most powerful strategy?  How is shedding of ADAM17 substrates differentially regulated in a spatial and temporal manner in vitro and, more importantly, in vivo?

ErbB Notch ligands

Regulation of cell fate  Regeneration  Anti-apoptosis  Differentiation TRENDS in Immunology

Figure 3. ADAM17 regulates inflammatory and regenerative processes. ADAM17 modulates inflammation by activation of TNFR- and IL-6R-mediated signal transduction. Therefore, ADAM17 activation is considered a pro-inflammatory event. ADAM17 mediates shedding of ErbB ligands and Notch-1 and thus potentially contributes to tissue regeneration. Therefore, ADAM17 might balance inflammatory pathways and regeneration in injured tissues. Inhibition of ADAM17 activity during inflammatory processes could block regenerative processes, resulting in unwanted side-effects.

cleavage site preference in the cleavage site of TNFa between alanine and valine (SPLAQA - VRSSSRT). Antibodies directed against substrate epitopes distal from the cleavage site inhibited ADAM17-mediated shedding [64]. This effect might be caused by simple steric hindrance or it could suggest that sites distal to the cleavage site are involved in substrate recognition. A discussion of sheddome analysis and future questions are presented in Box 1. Concluding remarks: ADAM17 and control of inflammatory and regenerative responses Activation of ADAM17 is activated by signals such as MAP kinases, PKCs and activated oncogenes. Activated ADAM17 is capable of controlling pathways that are biologically distinct. On one hand, cleavage of substrates, including TNFa, IL-6R and L-selectin, can be considered pro-inflammatory because they stimulate both innate and acquired immune responses (Figure 3). On the other hand, activation of the ErbB pathway via trans-activation and via Notch1 cleavage has different consequences (Figure 3). Activation of ErbB responses is crucial for regenerative responses of the body during wound healing and in the growth of tumors. The results of studies with Adam17 conditional knock-out animals using the endotoxin shock model and analyzing hematopoiesis and osteoporosis

[39,65,66] have been somewhat less clear than the results of studies in which ADAM17 was downregulated in all tissues [43,44]. These studies in mice have revealed the dominant role of pro-inflammatory TNFa and of pro-regeneratory ErbB ligands as major ADAM17 substrates. In vivo, ADAM17 activation governs a spectrum of proand anti-inflammatory responses leading to the appropriate response of the body to damage and stress. This orchestrating role, however, makes it likely that ADAM17 is involved in driving inflammation-associated cancer, which is caused by a chronic activation of the immune system and often culminates in the excessive activation of members of the ErbB family [43,44]. In this respect it is remarkable that loss of ADAM17 activity led to a complete loss of ErbB signaling in the mouse [43,44], which might point to an alternative to agents that block ErbB directly, which are prone to develop resistance. Acknowledgments The work described in this Review was funded by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB841, project C1 and SFB877, project A1 and A2) and by the Cluster of Excellence ‘Inflammation at Interfaces’.

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