ADAM15 Participates in Tick-Borne Encephalitis Virus Replication.

Tick-borne encephalitis virus (TBEV), a major tick-borne viral pathogen of humans, is known to cause neurological diseases such as meningitis, encephalitis, and meningoencephalitis. However, the life cycle and pathogenesis of TBEV are not well understood. Here, we show that the knockdown or knockout of ADAM15 (a disintegrin and metalloproteinase 15), a host protein involved in neuroblastoma diseases, leads to TBEV replication and assembly defects. We characterized the disintegrin domain in ADAM15 and found that the ADAM15 subcellular localization was changed following TBEV infection. RNA interference (RNAi) screen analysis confirmed ADAM's nonredundant functions and identified a specific role for ADAM15 in TBEV infection. An RNA-sequencing analysis was also conducted to understand the causal link between TBEV infection and the cellular endomembrane network, namely, the generation of replication organelles promoting viral genome replication and virus production. Our data demonstrated that TBEV infection changes ADAM15 cellular localization, which contributes to membrane reorganization and viral replication.IMPORTANCE Tick populations are increasing, and their geographic ranges are expanding. Increases in tick-borne disease prevalence and transmission are important public health issues. Tick-borne encephalitis virus (TBEV) often results in meningitis, encephalitis, and meningoencephalitis. TBEV causes clinical disease in more than 20,000 humans in Europe and Asia per year. An increased incidence of TBE has been noted in Europe and Asia, as a consequence of climate and socioeconomic changes. The need to investigate the mechanism(s) of interaction between the virus and the host factors is apparent, as it will help us to understand the roles of host factors in the life cycle of TBEV. The significance of our research is in identifying the ADAM15 for TBEV replication, which will greatly enhance our understanding of TBEV life cycle and highlight a target for pharmaceutical consideration.

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

OBJECTIVE: ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. CONCLUSIONS: This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Cut loose and run: The complex role of ADAM proteases during neural crest cell development.

ADAM metalloproteases have been shown to play critical roles during development. In this review, we will describe functional evidence that implicates ADAM proteins during the genesis, migration and differentiation of neural crest cells. We will restrict our analysis to the transmembrane ADAMs as other reviews have addressed the role of extracellular metalloproteases (Christian et al. [2013] Critical Reviews in Biochemistry and Molecular Biology 48:544-560). This review will describe advances that have been obtained mainly through the use of two vertebrate model systems, the frog, and avian embryos. The role of the principal substrates of ADAMs, the cadherins, has been extensively described in other reviews, most recently in (Cousin [1997] Mechanisms of Development 148:79-88; Taneyhill and Schiffmacher [2017] Genesis, 55). The function of ADAMs in the migration of other cell types, including the immune system, wound healing and cancer has been described previously in (Dreymueller et al. [2017] Mediators of Inflammation 2017: 9621724). Our goal is to illustrate both the importance of ADAMs in controlling neural crest behavior and how neural crest cells have helped us understand the molecular interactions, substrates, and functions of ADAM proteins in vivo.

[Effects of integrin metalloproteinases on osteogenic differentiation].

OBJECTIVE: To study the effects of disintegrin and metalloproteinase (ADAM) 9, 15 and 17 on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). METHODS: BMMSCs of ADAM9, ADAM15, ADAM17 conditional knockout mice and wild type mice (WT) were induced and cultured. Alkaline phosphatase (ALP) activity was measured by colorimetry, early osteogenic transcription factors Runx and Osterix were detected by Real-time PCR, and mineral formation was analyzed by alizarin red staining. RESULTS: ALP activity was lower in ADAM9 group (8.08±0.34), ADAM15 group (6.46±3.40), ADAM17 group (9.30±2.30) than that in WT group (9.44±2.50), but there was no significant difference (P>0.05). Stimulated with bone morphogenetic protein 2(BMP2),there was significant difference (P<0.05) between ADAM9 group (14.22±3.25), ADAM15 group (10.14±2.40) and WT group (20.89±3.40), and ADAM 17 group (23.56±2.50) was higher than WT group (20.89±3.40), but no significant difference (P>0.05). Similarly, cultured by osteogenic induction medium (OST), compared with WT group (12.97±1.30), ADAM9 group (9.63±1.00) and ADAM15 group (7.75±1.30) were lower, ADAM17 group (20.09±1.68) was higher, and the difference was statistically significant (P<0.05). Using stimulated culture by BMP2 and OST combined, ADAM9 group (15.75±1.30), ADAM 15 group (12.43±1.30) were less than WT group (26.15 ±1.50), while ADAM17 group (29.55±2.10) was higher than WT group were statistically significant (P<0.05). The expression of Runx2 in ADAM9 group (2.02±0.24), ADAM15 group (3.09±0.19), ADAM17 group (3.89±0.91) had no significant difference compared with WT (2.02±0.21) group (P>0.05). ADAM9 group stimulated by BMP2 (7.00±0.23), ADAM15 group (6.04±0.23) were lower than WT group (12.6±0.23), ADAM17 group (18.52±1.39) was higher than WT group (12.6±0.23), and the difference was statistically significant (P<0.05). In non-stimulating culture, there was no significant difference in Osterix expression between ADAM9 group (9.60±3.87), ADAM17 group (12.40±3.00) and WT group (10.9±1.10, P>0.05), but in ADAM15 group (6.50±1.51) it was slightly lower than that in WT group (P<0.05). After BMP2 stimulation, ADAM9 group (39.20±3.23) and ADAM15 group (20.50±4.80) were less than WT group (60.30±5.93), while ADAM17 group (80.20±3.30) was higher than WT group (P<0.05). Alizarin red staining showed no obvious orange-red mass in the non-induction group. Local calcified nodules could be seen in the BMP2, OST, OST + BMP2 induction culture conditions in all the experimental groups, but there was no significant difference in quantitative analysis (P>0.05). CONCLUSION: ADAM9, 15, 17 took part in the osteogenic differentiation of BMMSCs, and provided new targets for its regulation.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

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

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

ADAMTS13, lucky to have a hydrophobic pocket.

In this issue of Blood, de Groot et al identify a hydrophobic pocket in the Cys-rich domain of ADAMTS13 that appears to interact with the hydrophobic pocket in the central A2 domain of von Willebrand factor (VWF) for its proteolysis.

The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

ADAM23 promotes neuronal differentiation of human neural progenitor cells.

BACKGROUND: ADAM23 is widely expressed in the embryonic central nervous system and plays an important role in tissue formation. RESULTS: In this study, we showed that ADAM23 contributes to cell survival and is involved in neuronal differentiation during the differentiation of human neural progenitor cells (hNPCs). Upregulation of ADAM23 in hNPCs was found to increase the number of neurons and the length of neurite, while its downregulation decreases them and triggers cell apoptosis. RNA microarray analysis revealed mechanistic insights into genes and pathways that may become involved in multiple cellular processes upon up- or downregulation of ADAM23. CONCLUSIONS: Our results suggest that ADAM23 regulates neuronal differentiation by triggering specific signaling pathways during hNPC differentiation.

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.

Role of P2X7 receptor in Clostridium perfringens beta-toxin-mediated cellular injury.

BACKGROUND: Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X(7) receptor, which is an ATP receptor, interacts with beta-toxin. METHODS: We tested the role of P2X(7) receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X(7) receptor was further determined using an in vivo mouse model. RESULTS: Selective P2X(7) receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X(7) receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X(7) receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y(2) or P2Y(6) had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X(7)-transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X(7) receptors in vitro and colocalized with the P2X(7) receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG. CONCLUSIONS: The results of this study indicate that the P2X(7) receptor plays a role in beta-toxin-mediated cellular injury. GENERAL SIGNIFICANCE: P2X(7) receptor is a potential target for the treatment of C. perfringens type C infection.

ADAMTS-7 inhibits re-endothelialization of injured arteries and promotes vascular remodeling through cleavage of thrombospondin-1.

BACKGROUND: ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be significantly associated genomewide with coronary artery disease. However, the mechanisms that link ADAMTS-7 and coronary artery disease risk remain elusive. We have previously demonstrated that ADAMTS-7 promotes vascular smooth muscle cell migration and postinjury neointima formation via degradation of a matrix protein cartilage oligomeric matrix protein. Because delayed endothelium repair renders neointima and atherosclerosis plaque formation after vessel injury, we examined whether ADAMTS-7 also inhibits re-endothelialization. METHODS AND RESULTS: Wire injury of the carotid artery and Evans blue staining were performed in Adamts7(-/-) and wild-type mice. Adamts-7 deficiency greatly promoted re-endothelialization at 3, 5, and 7 days after injury. Consequently, Adamts-7 deficiency substantially ameliorated neointima formation in mice at days 14 and 28 after injury in comparison with the wild type. In vitro studies further indicated that ADAMTS-7 inhibited both endothelial cell proliferation and migration. Surprisingly, cartilage oligomeric matrix protein deficiency did not affect endothelial cell proliferation/migration and re-endothelialization in mice. In a further examination of other potential vascular substrates of ADAMTS-7, a label-free liquid chromatography-tandem mass spectrometry secretome analysis revealed thrombospondin-1 as a potential ADAMTS-7 target. The subsequent studies showed that ADAMTS-7 was directly associated with thrombospondin-1 by its C terminus and degraded thrombospondin-1 in vivo and in vitro. The inhibitory effect of ADAMTS-7 on postinjury endothelium recovery was circumvented in Tsp1(-/-) mice. CONCLUSIONS: Our study revealed a novel mechanism by which ADAMTS-7 affects neointima formation. Thus, ADAMTS-7 is a promising treatment target for postinjury vascular intima hyperplasia.

Knockout of Adamts7, a novel coronary artery disease locus in humans, reduces atherosclerosis in mice.

BACKGROUND: Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. METHODS AND RESULTS: We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7(-/-)/Ldlr(-/-) and Adamts7(-/-)/Apoe(-/-) mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-α stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. CONCLUSIONS: These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Versican: a novel modulator of hepatic fibrosis.

Little is known about the deposition and turnover of proteoglycans in liver fibrosis, despite their abundance in the extracellular matrix. Versican plays diverse roles in modulating cell behavior in other fibroproliferative diseases, but remains poorly described in the liver. Hepatic fibrosis was induced by carbon tetrachloride treatment of C57BL/6 mice over 4 weeks followed by recovery over a 28-day period. Primary mouse hepatic stellate cells (HSCs) were activated in culture and versican was transiently knocked down in human (LX2) and mouse HSCs. Expression of versican, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs (ADAMTS)-1, -4, -5, -8, -9, -15, and -20, and markers of fibrogenesis were studied using immunohistochemistry, real-time quantitative PCR, and western blotting. Immunohistochemistry showed increased expression of versican in cirrhotic human livers and the mouse model of fibrosis. Carbon tetrachloride treatment led to significant increases in versican expression and the proteoglycanases ADAMTS-5, -9, -15, and -20, alongside TNF-α, α-smooth muscle actin (α-SMA), collagen-1, and TGF-ß expression. During recovery, expression of many of these genes returned to control levels. However, expression of ADAMTS-5, -8, -9, and -15 showed delayed increases in expression at 28 days of recovery, which corresponded with decreases in versican V0 and V1 cleavage products (G1-DPEAAE(1401) and G1-DPEAAE(441)). Activation of primary HSCs in vitro significantly increased versican, α-SMA, and collagen-1 expression. Transient knockdown of versican in HSCs led to decreases in markers of fibrogenesis and reduced cell proliferation, without inducing apoptosis. Versican expression increases during HSC activation and liver fibrosis, and proteolytic processing occurs during the resolution of fibrosis. Knockdown studies in vitro suggest a possible role of versican in modulating hepatic fibrogenesis.

Alcohol Decreases Organic Dust-Stimulated Airway Epithelial TNF-Alpha Through a Nitric Oxide and Protein Kinase-Mediated Inhibition of TACE.

BACKGROUND: Farm workers in rural areas consume more alcohol than those who reside in urban areas. Occupational exposures such as agricultural work can pose hazards on the respiratory system. It is established that hog barn dust induces inflammation in the airway, including the release of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8. We have shown that alcohol alters airway epithelial innate defense through changes in both nitric oxide (NO) and cAMP-dependent protein kinase A (PKA). Simultaneous exposure to hog barn dust and alcohol decreases inflammatory mediators, TNF-α, IL-6, and IL-8, in mice. Previously, mice exposed to both alcohol and hog barn dust showed a depleted amount of lymphocytes compared to mice exposed only to hog barn dust. Weakening of the innate immune response could lead to enhanced susceptibility to disease. In addition, mice that were co-exposed to hog barn dust and alcohol also experienced increased mortality. METHODS: Because we recently demonstrated that PKA activation inhibits the TNF-α sheddase, TNF-α-converting enzyme (TACE), we hypothesized that an alcohol-mediated PKA pathway blocks TACE activity and prevents the normative inflammatory response to hog barn dust exposure. To delineate these effects, we used PKA pathway inhibitors (adenylyl cyclase [AC], cAMP, and PKA) to modulate the effects of alcohol on dust-stimulated TNF-α release in the bronchial epithelial cell line, BEAS-2B. Alcohol pretreatment blocked TACE activity and TNF-α release in hog barn dust-treated cells. RESULTS: Alcohol continued to block hog barn dust-mediated TNF-α release in the presence of the particulate AC inhibitor, SQ22,536. The soluble adenylyl cyclase inhibitor, KH7, however, significantly increased the inflammatory response to hog barn dust. phosphodiesterase 4 inhibitors significantly elevated cAMP and enhanced alcohol-mediated inhibition of dust-stimulated TNF-α release. In addition, the NO synthase inhibitor, l-NMMA, also reversed the alcohol-blocking effect on dust-stimulated TNF-α. CONCLUSIONS: These data suggest that alcohol requires a soluble cyclase-generated cAMP-PKA pathway that is dependent upon the action of NO to inhibit TACE and TNF-α release. These findings support our observations that alcohol functions through a dual NO and PKA pathway in bronchial epithelial cells.

iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding.

Protein ectodomain shedding by ADAM17 (a disintegrin and metalloprotease 17), a principal regulator of EGF-receptor signaling and TNFα release, is rapidly and posttranslationally activated by a variety of signaling pathways, and yet little is known about the underlying mechanism. Here, we report that inactive rhomboid protein 2 (iRhom2), recently identified as essential for the maturation of ADAM17 in hematopoietic cells, is crucial for the rapid activation of the shedding of some, but not all substrates of ADAM17. Mature ADAM17 is present in mouse embryonic fibroblasts (mEFs) lacking iRhom2, and yet ADAM17 is unable to support stimulated shedding of several of its substrates, including heparin-binding EGF and Kit ligand 2 in this context. Stimulated shedding of other ADAM17 substrates, such as TGFα, is not affected in iRhom2(-/-) mEFs but can be strongly reduced by treating iRhom2(-/-) mEFs with siRNA against iRhom1. Activation of heparin-binding EGF or Kit ligand 2 shedding by ADAM17 in iRhom2(-/-) mEFs can be rescued by wild-type iRhom2 but not by iRhom2 lacking its N-terminal cytoplasmic domain. The requirement for the cytoplasmic domain of iRhom2 for stimulated shedding by ADAM17 may help explain why the cytoplasmic domain of ADAM17 is not required for stimulated shedding. The functional relevance of iRhom2 in regulating shedding of EGF receptor (EGFR) ligands is established by a lack of lysophasphatidic acid/ADAM17/EGFR-dependent crosstalk with ERK1/2 in iRhom2(-/-) mEFs, and a significant reduction of FGF7/ADAM17/EGFR-stimulated migration of iRhom2(-/-) keratinocytes. Taken together, these findings uncover functions for iRhom2 in the regulation of EGFR signaling and in controlling the activation and substrate selectivity of ADAM17-dependent shedding events.

ADAMTS13 and von Willebrand factor interactions.

PURPOSE OF REVIEW: ADAMTS13 is a zinc-containing metalloprotease that cleaves von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity is accountable for a potentially fatal blood disorder thrombotic thrombocytopenic purpura (TTP). Understanding of ADAMTS13-VWF interaction is essential for developing novel treatments to this disorder. RECENT FINDINGS: Despite the proteolytic activity of ADAMTS13 being restricted to the metalloprotease domain, the ancillary proximal C-terminal domains including the disintegrin domain, first TSP-1 repeat, cysteine-rich region, and spacer domain are all required for cleavage of VWF and its analogs. Recent studies have added to our understandings of the role of the specific regions in the disintegrin domain, the cysteine-rich domain, and the spacer domain responsible for its interaction with VWF. Additionally, regulative functions of the distal portion of ADAMTS13 including the TSP-1 2-8 repeats and the CUB domains have been proposed. Finally, fine mapping of anti-ADAMTS13 antibody epitopes have provided further insight into the essential structural elements in ADAMTS13 for VWF binding and the mechanism of autoantibody-mediated TTP. SUMMARY: Significant progress has been made in our understandings of the structure-function relationship of ADAMTS13 in the past decade. To further investigate ADAMTS13-VWF interactions for medical applications, these interactions must be studied under physiological conditions in vivo.

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases.

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.

Metalloproteinase-dependent and -independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

Higher and lower active circulating VWF levels: different facets of von Willebrand disease.

Most circulating von Willebrand factor (VWF) is normally inactive and incapable of binding platelets, but numerous disorders may modify the proportion of active VWF. We explored active VWF levels in patients with von Willebrand disease (VWD) whose VWF had a higher affinity for platelet glycoprotein (GP)Ib, but different susceptibilities to ADAMTS13 and multimer patterns (9 patients lacking large multimers, 10 with a normal pattern); 12 patients with VWF C2362F and R1819_C1948delinsS mutations, which make VWF resistant to ADAMTS13 were also studied. Type 2B patients with abnormal or normal multimers had significantly more active VWF (3·33 ± 1·6 and 3·74 ± 0·74, respectively; normal 0·99 ± 0·23). The type of VWF mutation influenced VWF activation: V1316M was associated with the highest levels in patients with abnormal multimers, and R1341W in those with normal multimers. Pregnancy induced gradually rising active VWF levels and declining platelet counts in one type 2B VWD patient without large multimers. Active VWF levels dropped significantly in patients homozygous for the C2362F mutation or heterozygous for R1819_C1948delinsS mutations (0·2 ± 0·03 and 0·23 ± 0·1, respectively), and less in cases heterozygous for the VWF C2362F mutation (0·55 ± 0·17). We demonstrate that VWF may be more or less activated, with or without any direct involvement of the A1 domain, and regardless of ADAMTS13.