Breakdown of Phospholipid Asymmetry Triggers ADAM17-Mediated Rescue Events in Cells Undergoing Apoptosis.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.

Influence of Anoctamin-4 and -9 on ADAM10 and ADAM17 Sheddase Function.

Ca2+-activated Cl- channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Scramblases as Regulators of Proteolytic ADAM Function.

Proteolytic ectodomain release is a key mechanism for regulating the function of many cell surface proteins. The sheddases ADAM10 and ADAM17 are the best-characterized members of the family of transmembrane disintegrin-like metalloproteinase. Constitutive proteolytic activities are low but can be abruptly upregulated via inside-out signaling triggered by diverse activating events. Emerging evidence indicates that the plasma membrane itself must be assigned a dominant role in upregulation of sheddase function. Data are discussed that tentatively identify phospholipid scramblases as central players during these events. We propose that scramblase-dependent externalization of the negatively charged phospholipid phosphatidylserine (PS) plays an important role in the final activation step of ADAM10 and ADAM17. In this manuscript, we summarize the current knowledge on the interplay of cell membrane changes, PS exposure, and proteolytic activity of transmembrane proteases as well as the potential consequences in the context of immune response, infection, and cancer. The novel concept that scramblases regulate the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface.

Role of ADAM10 and ADAM17 in Regulating CD137 Function.

Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway.

Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.

Mutagenesis of the ADAM17-phosphatidylserine-binding motif leads to embryonic lethality in mice.

ADAM17, prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Several of these play central roles in oncogenesis and inflammation, yet despite its importance, the mechanism by which ADAM17 is activated is not fully understood. We recently presented evidence that surface exposure of phosphatidylserine (PS) is the penultimate event required for sheddase activation, which occurs upon binding of a membrane-proximal, cationic binding motif to the anionic phospholipid headgroup. Here, we show that mutagenesis of the 3 amino acids constituting the PS-binding motif leads to embryonic lethality in mice. Heterozygotes showed no abnormalities. Primary hepatocytes and fibroblasts were analysed and found to express the mutant protease on the cell surface. However, PMA-stimulated release of ADAM17 substrates was completely abolished. The results directly support the novel concept of transiently externalised PS as essential trigger of extracellular protease function in vivo.

ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly.

Oncogenic human papillomaviruses (HPV) are small DNA viruses that infect keratinocytes. After HPV binding to cell surface receptors, a cascade of molecular interactions mediates the infectious cellular internalization of virus particles. Aside from the virus itself, important molecular players involved in virus entry include the tetraspanin CD151 and the epidermal growth factor receptor (EGFR). To date, it is unknown how these components are coordinated in space and time. Here, we studied plasma membrane dynamics of CD151 and EGFR and the HPV16 capsid during the early phase of infection. We find that the proteinase ADAM17 activates the extracellular signal-regulated kinases (ERK1/2) pathway by the shedding of growth factors which triggers the formation of an endocytic entry platform. Infectious endocytic entry platforms carrying virus particles consist of two-fold larger CD151 domains containing the EGFR. Our finding clearly dissects initial virus binding from ADAM17-dependent assembly of a HPV/CD151/EGFR entry platform.

ADAM10 sheddase activation is controlled by cell membrane asymmetry.

Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.

Anoctamin-6 regulates ADAM sheddase function.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca2+-dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca2+-mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.

Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers.

BACKGROUND: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. METHODS: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. RESULTS: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. CONCLUSIONS: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

Regulation of TMEM16A/ANO1 and TMEM16F/ANO6 ion currents and phospholipid scrambling by Ca2+ and plasma membrane lipid.

KEY POINTS: TMEM16 proteins can operate as Ca2+ -activated Cl- channels or scramble membrane phospholipids, which are both highly relevant mechanisms during disease. Overexpression of TMEM16A and TMEM16F were found to be partially active at 37°C and at resting intracellular Ca2+ concentrations. We show that TMEM16 Cl- currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids, through reactive oxygen species and phospholipase A2. While phospholipids and Cl- ions are likely to use the same pore within TMEM16F, TMEM16A only conducts Cl- ions. Lipid regulation of TMEM16 proteins is highly relevant during inflammation and regulated cell death such as apoptosis and ferroptosis. ABSTRACT: TMEM16/anoctamin (ANO) proteins form Ca2+ -activated ion channels or phospholipid scramblases. We found that both TMEM16A/ANO1 and TMEM16F/ANO6 produced Cl- currents when activated by intracellular Ca2+ , but only TMEM16F was able to expose phosphatidylserine to the outer leaflet of the plasma membrane. Mutations within TMEM16F or TMEM16A/F chimeras similarly changed Cl- currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl- and phospholipids. When overexpressed, TMEM16A and TMEM16F produced spontaneous Cl- currents at 37°C even at resting intracellular Ca2+ levels, which was abolished by inhibition of phospholipase A2 (PLA2 ). Connversely, activation of PLA2 or application of active PLA2 , as well as lipid peroxidation induced by reactive oxygen species (ROS) using staurosporine or tert-butyl hydroperoxide, enhanced ion currents by TMEM16A/F and in addition activated phospholipid scrambling by TMEM16F. Thus, TMEM16 proteins are activated by an increase in intracellular Ca2+ , or independent of intracellular Ca2+ , by modifications occurring in plasma and intracellular membrane phospholipids. These results may help to explain why regions distant to the TMEM16 pore and the Ca2+ binding sites control Cl- currents and phospholipid scrambling. Regulation of TMEM16 proteins through modification of membrane phospholipids occurs during regulated cell death such as apoptosis and ferroptosis. It contributes to inflammatory and nerve injury-induced hypersensitivity and generation of pain and therefore provides a regulatory mechanism that is particularly relevant during disease.

Extracellular sphingomyelinase activity impairs TNF-α-induced endothelial cell death via ADAM17 activation and TNF receptor 1 shedding.

ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.

The plasma membrane: Penultimate regulator of ADAM sheddase function.

BACKGROUND: ADAM10 and ADAM17 are the best characterized members of the ADAM (A Disintegrin and Metalloproteinase) - family of transmembrane proteases. Both are involved diverse physiological and pathophysiological processes. ADAMs are known to be regulated by posttranslational mechanisms. However, emerging evidence indicates that the plasma membrane with its unique dynamic properties may additionally play an important role in controlling sheddase function. SCOPE OF REVIEW: Membrane events that could contribute to regulation of ADAM-function are summarized. MAJOR CONCLUSIONS: Surface expression of peptidolytic activity should be differentiated from ADAM-sheddase function since the latter additionally requires that the protease finds its substrate in the lipid bilayer. We propose that this is achieved through horizontal and vertical reorganization of membrane nanoarchitecture coordinately occurring at the sites of sheddase activation. Reshuffling of nanodomains thereby guides traffic of enzyme and substrate to each other. For ADAM17 phosphatidylserine exposure is required to then induce its shedding function. GENERAL SIGNIFICANCE: The novel concept that physicochemical properties of the lipid bilayer govern the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays.

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.

Phosphatidylserine exposure is required for ADAM17 sheddase function.

ADAM17, a prominent member of the 'Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca(2+) elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function.

Extracellular Juxtamembrane Segment of ADAM17 Interacts with Membranes and Is Essential for Its Shedding Activity.

A wide variety of biological processes including differentiation, regeneration, and cancer progression are regulated by shedding of membrane-anchored proteins. One of the major sheddases is A Disintegrin And Metalloprotease-17 (ADAM17) whose extracellular region consists of a pro-, a catalytic, a disintegrin-, and a membrane-proximal domain (MPD) as well as a short juxtamembrane segment of 17 amino acid residues that has been named "Conserved ADAM-seventeeN Dynamic Interaction Sequence" (CANDIS). This segment is involved in substrate recognition. Key mediators of inflammation including interleukin-6 receptor (IL-6R) and tumor necrosis factor (TNF-α) are substrates of ADAM17. The shedding activity of ADAM17 is regulated by the conformation of the membrane-proximal domain preceding the CANDIS segment. Here, we show that CANDIS, besides being involved in substrate recognition, is able to interact with lipid bilayers in vitro and that this property could be involved in regulating ADAM17 shedding activity.

The Antiatherogenic Effect of Fish Oil in Male Mice Is Associated with a Diminished Release of Endothelial ADAM17 and ADAM10 Substrates.

BACKGROUND: Growing evidence suggests that disintegrin and metalloprotease (ADAM) 17 (ADAM17) and ADAM10 contribute to the pathogenesis of vascular diseases. ADAM17 promotes inflammatory processes by liberating tumor necrosis factor α, interleukin 6 receptor (IL-6R), and tumor necrosis factor receptor 1 (TNFR1). ADAM17 and ADAM10 modulate vascular permeability by cleaving endothelial adhesion molecules such as junctional adhesion molecule A (JAM-A) and vascular endothelial cadherin (VE-cadherin), respectively. OBJECTIVE: This study was designed to investigate whether a link might exist between the protective effects of fish oil (FO) supplementation against atherosclerosis and ADAM function. METHODS: Male LDL receptor knockout (LDLR(-/-)) mice and male wild-type (WT) mice were fed a Western diet (200 g/kg fat, 1.5 g/kg cholesterol) containing either 20% lard (LDLR(-/-)-lard and WT-lard groups) or 10% lard combined with 10% FO (LDLR(-/-)-FO and WT-FO groups) for 12 wk. Atherosclerotic lesion development and fatty acid composition of liver microsomes were evaluated. ADAM10 and ADAM17 expression was determined by quantitative real-time polymerase chain reaction and immunoblot analyses. Concentrations of soluble ADAM substrates in plasma and liver extracts were measured by ELISA. RESULTS: Diets supplemented with FO markedly reduced development of early atherosclerotic lesions in LDLR(-/-) mice (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 29.6 ± 6.1% vs. 22.5 ± 4.2%, P < 0.05). This was not accompanied by changes in expression of ADAM17 or ADAM10 in the aorta or liver. No dietary effects on circulating TNFR1 (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.22 ± 0.23 vs. 1.39 ± 0.28, P > 0.2) or IL-6R (1.06 ± 0.12 vs. 0.98 ± 0.09 fold of WT-lard group, P > 0.1), classical substrates of ADAM17 on macrophages, and neutrophil granulocytes were observed. However, a reduction in atherosclerotic lesions in the LDLR(-/-)-FO group was accompanied by a significant reduction in the circulating endothelial cell adhesion molecules JAM-A (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.42 ± 0.20 vs. 0.95 ± 0.56 fold of WT-lard group, P < 0.05), intercellular adhesion molecule 1 (1.15 ± 0.14 vs. 0.88 ± 0.17 fold of WT-lard group, P < 0.05), and VE-cadherin (0.88 ± 0.12 vs. 0.72 ± 0.15 fold of WT-lard group, P < 0.05), reflecting reduced ADAM activity in endothelial cells. CONCLUSION: FO exerted an antiatherogenic effect on male LDLR(-/-) mice that was accompanied by a reduced release of ADAM17 and ADAM10 substrates from endothelial cells. It is suggested that FO-decreased ADAM activity contributes to improved endothelial barrier function and thus counteracts intimal lipoprotein insudation and macrophage accumulation.

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.

Malarial anemia: digestive vacuole of Plasmodium falciparum mediates complement deposition on bystander cells to provoke hemophagocytosis.

The digestive vacuole (DV) of Plasmodium falciparum, which is released into the bloodstream upon rupture of each parasitized red blood cell (RBC), was recently discovered to activate the alternative complement pathway. In the present work, we show that C3- and C5-convertases assembling on the parasitic organelle are able to provoke deposition of activated C3 and C5b-9 on non-infected bystander erythrocytes. Direct contact of DVs with cells is mandatory for the effect, and bystander complement deposition occurs focally, possibly at the sites of contact. Complement opsonization promotes protracted erythrophagocytosis by human macrophages, an effect that is magnified when ring-stage infected RBCs with reduced CD55 and CD59, or paroxysmal nocturnal hemoglobinuria (PNH)-RBCs lacking these complement inhibitors are employed as targets. Bystander attack can also directly induce lysis of PNH-RBCs. Direct evidence for complement activation and bystander attack mediated by DVs was obtained through immunohistochemical analyses of brain paraffin sections from autopsies of patients who had died of cerebral malaria. C3d and the assembled C5b-9 complex could be detected in all sections, colocalizing with and often extending locally beyond massive accumulations of DVs that were identified under polarized light. This is the first demonstration that a complement-activating particle can mediate opsonization of bystander cells to promote their antibody-independent phagocytosis. The phenomenon may act in concert with other pathomechanisms to promote the development of anemia in patients with severe malaria.