Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer.     

ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk

All ligands of the epidermal growth factor receptor (EGFR) which has important roles in development and disease, are shed from the plasma membrane by metalloproteases. The ectodomain shedding of EGFR ligands has emerged as a critical component in the functional activation of EGFR in the interreceptor cross-talk. Identification of the sheddases for EGFR ligands using mouse embryonic cells lacking candidate sheddases (a disintegrin and metalloprotease; ADAM) has revealed that ADAM10, -12 and -17 are the sheddases of the EGFR ligands in response to various shedding stimulants such as GPCR agonists, growth factors, cytokines, osmotic stress, wounding and phorbol ester. Among the EGFR ligands, heparin-binding EGF-like growth factor (HB-EGF) is a representative ligand to understand the pathophysiological roles of the ectodomain shedding in wound healing, cardiac diseases, etc. Here we focus on the ectodomain shedding of HB-EGF by ADAMs, which is not only a key event of receptor cross-talk but also a novel intercellular signaling by the carboxy-terminal fragment (CTF signal).     

PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.     

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.     

IL-1β and ADAM17 are central regulators of β-defensin expression in Candida esophagitis

Candida albicans resides on epithelial surfaces as part of the physiological microflora. However, under certain conditions, it may cause life-threatening infections, including Candida sepsis. We have recently shown that human β-defensins (hBDs) hBD-2 and hBD-3 are upregulated in Candida esophagitis and that this antifungal host response is distinctly regulated by NF-κB and MAPK/activator protein-1 (AP-1) pathways. Here, we show that C. albicans induces hBD-2 through an autocrine IL-1β loop and that activation of the epidermal growth factor receptor (EGFR) by endogenous transforming growth factor-α (TGF-α) is a crucial event in the induction of hBD-3. To further dissect upstream signaling events, we investigated expression of the central sheddases for EGFR ligands ADAM10 and ADAM17 in the healthy and infected esophagus. Next, we used pharmaceutical inhibitors and small-interfering RNA-mediated knock down of ADAM10 and ADAM17 to reveal that ADAM17-induced shedding of TGF-α is a crucial step in the induction of hBD-3 expression in response to Candida infection. In conclusion, we describe for the first time an autocrine IL-1β loop responsible for the induction of hBD-2 expression and an ADAM17-TGF-α-EGFR-MAPK/AP-1 pathway leading to hBD-3 upregulation in the course of a Candida infection of the esophagus.     

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.     

Shedding light on sheddases: role in growth and development.

The extracellular domains of several integral membrane proteins are released from the cell surface by a group of enzymes known as "sheddases" through a process called "ectodomain shedding". Because many transmembrane growth and differentiation factors, including members of the epidermal growth factor (EGF) family that play a crucial role in development, require ectodomain shedding for proper action in vivo, proteolysis is now viewed as a regulatory mechanism in the developing embryos. Two recent reports by Zhao et al. provide evidence for the role of cell surface proteolysis by an ADAM (a disintegrin and metalloprotease) in the development of murine lung. Inhibition of tumor necrosis factor-alpha converting enzyme (TACE, ADAM17) by the hydroxamic acid-based metalloprotease inhibitor (TAPI), or a targeted mutation in Zn(2+)-binding domain of TACE, disrupts two essential epithelial functions in lung development: branching morphogenesis and cytodifferentiation. Evidence for the role of ADAMs as sheddases in development and growth factor signaling is discussed.     

Interleukin-1 stimulates ADAM17 through a mechanism independent of its cytoplasmic domain or phosphorylation at threonine 735

ADAM17 (a disintegrin and metalloproteinase) is a membrane-anchored metalloproteinase that regulates the release of EGFR-ligands, TNFα and other membrane proteins from cells. ADAM17 can be rapidly activated by a variety of signaling pathways, yet little is known about the underlying mechanism. Several studies have demonstrated that the cytoplasmic domain of ADAM17 is not required for its rapid activation by a variety of stimuli, including phorbol esters, tyrosine kinases and some G-protein coupled receptors. However, phosphorylation of cytoplasmic residue T735 was recently reported as a crucial step for activation of ADAM17 by IL-1β and by the p38 MAP-kinase pathway. One possible mechanism to reconcile these results would be that T735 has an inhibitory role and that it must be phosphorylated as a pre-requisite for the activation of ADAM17, which would then proceed via a mechanism that is independent of its cytoplasmic domain. To test this hypothesis, we performed rescue experiments of Adam17-/- cells with wild type and mutant forms of ADAM17. However, these experiments showed that an inactivating mutation (T735A) or an activating mutation (T735D) of cytoplasmic residue T735 or the removal of the cytoplasmic domain of ADAM17 did not significantly affect the stimulation of ADAM17 by IL-1β or by activation of MAP-kinase with anisomycin. Moreover, we found that the MAP-kinase inhibitor SB203580 blocked activation of cytoplasmic tail-deficient ADAM17 and of the T735A mutant by IL-1β or by anisomycin, providing further support for a model in which the activation mechanism of ADAM17 does not rely on its cytoplasmic domain or phosphorylation of T735.     

ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H₂O₂, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H₂O₂ production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-κB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H₂O₂ scavenging by catalase, these responses were attenuated by intracellular scavengers of H₂O₂ or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H₂O₂-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.     

Mitochondrial Reactive Oxygen Species Mediate GPCR–induced TACE/ADAM17-dependent Transforming Growth Factor-α Shedding

Epidermal growth factor receptor (EGFR) activation by GPCRs regulates many important biological processes. ADAM metalloprotease activity has been implicated as a key step in transactivation, yet the regulatory mechanisms are not fully understood. Here, we investigate the regulation of transforming growth factor-α (TGF-α) shedding by reactive oxygen species (ROS) through the ATP-dependent activation of the P2Y family of GPCRs. We report that ATP stimulates TGF-α proteolysis with concomitant EGFR activation and that this process requires TACE/ADAM17 activity in both murine fibroblasts and CHO cells. ATP-induced TGF-α shedding required calcium and was independent of Src family kinases and PKC and MAPK signaling. Moreover, ATP-induced TGF-α shedding was completely inhibited by scavengers of ROS, whereas calcium-stimulated shedding was partially inhibited by ROS scavenging. Hydrogen peroxide restored TGF-α shedding after calcium chelation. Importantly, we also found that ATP-induced shedding was independent of the cytoplasmic NADPH oxidase complex. Instead, mitochondrial ROS production increased in response to ATP and mitochondrial oxidative complex activity was required to activate TACE-dependent shedding. These results reveal an essential role for mitochondrial ROS in regulating GPCR-induced growth factor shedding.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.     

Stimulation of Platelet-derived Growth Factor Receptor β (PDGFRβ) Activates ADAM17 and Promotes Metalloproteinase-dependent Cross-talk between the PDGFRβ and Epidermal Growth Factor Receptor (EGFR) Signaling Pathways

Binding of the platelet-derived growth factor (PDGF)-B to its receptor PDGFRβ promotes proliferation, migration, and recruitment of pericytes and smooth muscle cells to endothelial cells, serving to stabilize developing blood vessels. The main goals of this study were to determine whether the extracellular domain of the PDGFRβ can be proteolytically released from cell membranes and, if so, to identify the responsible sheddase and determine whether activation of the PDGFRβ stimulates its shedding and potentially that of other membrane proteins. We found that the PDGFRβ is shed from cells by a metalloproteinase and used loss-of-function experiments to identify ADAM10 as the sheddase responsible for constitutive and ionomycin-stimulated processing of the PDGFRβ. Moreover, we showed that ligand-dependent activation of the PDGFRβ does not trigger its own shedding by ADAM10, but instead it stimulates ADAM17 and shedding of substrates of ADAM17, including tumor necrosis factor α and transforming growth factor α. Finally, we demonstrated that treatment of mouse embryonic fibroblasts with PDGF-B triggers a metalloproteinase-dependent cross-talk between the PDGFRβ and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling axis that is also critical for PDGF-B-stimulated cell migration, most likely via ADAM17-dependent release and activation of ligands of the EGFR. This study identifies the principal sheddase for the PDGFRβ and provides new insights into the mechanism of PDGFRβ-dependent signal transduction and cross-talk with the EGFR.     

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.     

Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

A disintegrin and metalloproteinase 17 (ADAM17) regulates key cellular processes including proliferation and migration through the shedding of a diverse array of substrates such as epidermal growth factor receptor (EGFR) ligands. ADAM17 is implicated in the pathogenesis of many diseases including rheumatoid arthritis and cancers such as head and neck squamous cell carcinoma (HNSCC). As a central mediator of cellular events, overexpressed EGFR is a validated molecular target in HNSCC. However, EGFR inhibition constantly leads to tumour resistance. One possible mechanism of resistance is the activation of alternative EGFR family receptors and downstream pathways via the release of their ligands. Here, we report that treating human HNSCC cells in vitro with a human anti-ADAM17 inhibitory antibody, D1(A12), suppresses proliferation and motility in the absence or presence of the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Treatment with D1(A12) decreases both the endogenous and the bradykinin (BK)-stimulated shedding of HER ligands, accompanied by a reduction in the phosphorylation of HER receptors and downstream signalling pathways including STAT3, AKT and ERK. Knockdown of ADAM17, but not ADAM10, also suppresses HNSCC cell proliferation and migration. Furthermore, we show that heregulin (HRG) and heparin-binding epidermal growth factor like growth factor (HB-EGF) predominantly participate in proliferation and migration, respectively. Taken together, these results demonstrate that D1(A12)-mediated inhibition of cell proliferation, motility, phosphorylation of HER receptors and downstream signalling is achieved via reduced shedding of ADAM17 ligands. These findings underscore the importance of ADAM17 and suggest that D1(A12) might be an effective targeted agent for treating EGFR TKI-resistant HNSCC.     

Cell-matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca(2+) influx and PKC activation

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca(2+) influx or the activation of protein kinase C. Here we show that CD44-mediated cell-matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca(2+) influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca(2+) influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell-matrix interaction and cell migration.     

Evaluation of the contribution of different ADAMs to tumor necrosis factor alpha (TNFalpha) shedding and of the function of the TNFalpha ectodomain in ensuring selective stimulated shedding by the TNFalpha convertase (TACE/ADAM17)

Tumor necrosis factor-alpha (TNFalpha), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-alpha converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFalpha in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFalpha cleavage site in vitro, these enzymes are considered to be candidate TNFalpha sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFalpha shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFalpha in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFalpha, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFalpha shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFalpha to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFalpha is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFalpha makes an unexpected contribution to the selective cleavage of TNFalpha by ADAM17: it prevents one or more other enzymes from cleaving TNFalpha following PMA stimulation. Thus, selective stimulated processing of TNFalpha by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.     

Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V

Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.     

Distinct ADAM metalloproteinases regulate G protein-coupled receptor-induced cell proliferation and survival

Cross-talk between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is widely established in a variety of normal and transformed cell types. Here, we demonstrate that the EGFR transactivation signal requires metalloproteinase cleavage of epidermal growth factor-like growth factor precursors in fibroblasts, ACHN kidney, and TccSup bladder carcinoma cells. Furthermore, we present evidence that blockade of the metalloproteinase-disintegrin tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) by a dominant negative ADAM17 mutant prevents angiotensin II-stimulated pro-HB-EGF cleavage, EGFR activation, and cell proliferation in ACHN tumor cells. Moreover, we found that in TccSup cancer cells, the lysophosphatidic acid-induced transactivation signal is mediated by ADAM15, demonstrating that distinct combinations of growth factor precursors and ADAMs (a disintegrin and metalloproteinases) regulate GPCR-EGFR cross-talk pathways in cell lines derived from urogenital cancer. Our data show further that activation of ADAMs results in discrete cellular responses; whereas GPCR agonists promote activation of the Ras/MAPK pathway and cell proliferation via the EGFR in fibroblasts and ACHN cells, EGFR transactivation pathways regulate activation of the survival mediator Akt/protein kinase B and the susceptibility of fibroblasts and TccSup bladder carcinoma cells to proapoptotic signals such as serum deprivation, death receptor stimulation, and the chemotherapeutic drug doxorubicin. Thus, ADAM15 and -17 function as effectors of GPCR-mediated signaling and define critical characteristics of cancer cells.