Disintegrin Metalloproteinase Research Articles

Retinoids as a Perspective in Treatment of Alzheimer’s Disease

Background: In the past, we demonstrated that the disintegrin metalloproteinase ADAM10 has α-secretase activity in vitro and in cultured cells. We also found out that moderate overexpression of this proteinase inhibits Aβ peptide production and prevents the formation of amyloid plaques in an Alzheimer’s disease (AD) mouse model. Moreover, it corrects early hippocampal defects like LTP impairment and increases cortical synaptogenesis. Objective: Upregulation of ADAM10 might be an alternative approach concerning AD therapy. Our current research therefore focuses on substances and/or pathways which regulate ADAM10 gene expression. Methods: Analyses of ADAM10 promoter activity were performed in human and murine neuroblastoma cell lines and important findings were validated in an AD mouse model. Results: The outcome of our investigations indicates that a heterodimeric retinoid receptor mediates the activation of the ADAM10 promoter by all-trans-retinoic acid. This then results in enhanced ADAM10 expression, pronounced APPs-α secretion and diminished proteolytic processing products of the amyloidogenic pathway (APPs-β, Aβ). Conclusion: Nontoxic synthetic retinoids – like acitretin – offer a valuable therapeutic approach in treatment of AD by moderately enhancing ADAM10 gene expression.

Conservation and divergence of ADAM family proteins in the Xenopus genome

BackgroundMembers of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species.ResultsBased on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region.ConclusionsOur genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease activities are absent, while other novel ADAM proteins in this species are predicted by this study. The conservation and unique divergence of ADAM genes in Xenopus probably reflect the particular selective pressures these amphibian species faced during evolution.

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke

AbstractAnti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385–amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti–GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti–ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.

Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases

AbstractJunctional adhesion molecule A (JAM-A) is a transmembrane adhesive glycoprotein that participates in the organization of endothelial tight junctions and contributes to leukocyte transendothelial migration. We demonstrate here that cultured endothelial cells not only express a cellular 43-kDa variant of JAM-A but also release considerable amounts of a 33-kDa soluble JAM-A variant. This release is enhanced by treatment with proinflammatory cytokines and is associated with the down-regulation of surface JAM-A. Inhibition experiments, loss/gain-of-function experiments, and cleavage experiments with recombinant proteases indicated that cleavage of JAM-A is mediated predominantly by the disintegrin and metalloproteinase (ADAM) 17 and, to a lesser extent, by ADAM10. Cytokine treatment of mice increased JAM-A serum level and in excised murine aortas increased ADAM10/17 activity correlated with enhanced JAM-A release. Functionally, soluble JAM-A blocked migration of cultured endothelial cells, reduced transendothelial migration of isolated neutrophils in vitro, and decreased neutrophil infiltration in a murine air pouch model by LFA-1– and JAM-A–dependent mechanisms. Therefore, shedding of JAM-A by inflamed vascular endothelium via ADAM17 and ADAM10 may not only generate a biomarker for vascular inflammation but could also be instrumental in controlling JAM-A functions in the molecular zipper guiding transendothelial diapedesis of leukocytes.

Lack of evidence to support the association of polymorphisms within the alpha- and beta-secretase genes (ADAM10/BACE1) with Alzheimer's disease

Cleavage of the amyloid precursor protein (APP) occurs through either an amyloidogenic or a non-amyloidogenic pathway. The first results in the generation of beta-amyloid (Aβ) and is initiated through cleavage by the beta-site amyloid beta A4 precursor protein-cleaving enzyme 1 (BACE1). The second precludes the formation of Aβ through cleavage by alpha-secretase, an enzyme's activity demonstrated in a disintegrin metalloproteinase, ADAM10. To assess the contribution of variants in the BACE1 and ADAM10 genes we used a detailed fine mapping approach. Genotyping of 11 single nucleotide polymorphisms covering the complete BACE1 gene, and 27 covering the entire ADAM10 gene, revealed no single-marker or haplotypic association with AD. We conclude that, in this present study, neither ADAM10 nor BACE1 present with any evidence to suggest that they are major candidate genes involved in conferring risk for AD.

ADAM13 is Required for Xenopus Head Development by Regulating Class B Ephrin and Wnt Signaling

Protein ectodomain shedding by disintegrin metalloproteinases (ADAMs) has emerged as a key regulatory event in cell signaling. We previously showed that an ADAM metalloproteinase, ADAM13, is expressed in cranial neural crest (CNC) cells in Xenopus laevis, and that overexpression of a protease dead mutant of ADAM13 prevents CNC migration. To further understand the roles of this gene in vertebrate development, we carried out loss‐of‐function studies in X. tropicalis, a closely related frog species with a diploid genome. Knockdown of ADAM13 by antisense morpholinos (MOs) resulted in abnormal head development, including reduced CNC induction and perturbed eye field patterning. The metalloproteinase activity of ADAM13 was required for these functions in vivo. Using cultured cells, we identified membrane‐bound ephrins (Efns) B1 and B2 as substrates for ADAM13. Knockdown of ADAM13 protected endogenous EfnB1/B2 in developing embryos, indicating that these two ephrins are in vivo substrates for ADAM13. The defects caused by ADAM13 MO could be rescued by blocking forward EfnB signaling, restoring canonical Wnt signaling, or by ectopic expression of the transcription factor Slug. Our data suggest a novel EfnB‐Wnt‐Slug signaling cascade through which ADAM13 controls CNC induction and eye field patterning.

The role of the disintegrin metalloproteinase ADAM15 in prostate cancer progression

The metalloproteinase ADAM15 is a multi-domain disintegrin protease that is upregulated in a variety of human cancers. ADAM15 mRNA and protein levels are increased in prostate cancer and its expression is significantly increased during metastatic progression. It is likely that ADAM15 supports disease progression differentially through the action of its various functional domains. ADAM15 may downregulate adhesion of tumor cells to the extracellular matrix, reduce cell-cell adhesion, and promote metastasis through the activity of its disintegrin and metalloproteinase domains. Additionally, ADAM15 can influence cell signaling by shedding membrane-bound growth factors and other proteins that interact with receptor tyrosine kinases, leading to receptor activation. There is also evidence supporting a role for ADAM15 in angiogenesis and angioinvasion of tumor cells, which are critical for unrestrained tumor growth and metastatic spread. Given its diverse functions, ADAM15 may represent a pivotal regulatory component of tumor progression, an important target for therapeutic intervention, or emerge as a biomarker of disease progression.

The Orphan Receptor GPR31 Is the Platelet and HUVEC Receptor for 12(S)-HETE

Activation of platelet 12-lipoxygenase (12-LO) generates large quantities of 12(S)- HETE, an oxidized metabolite of arachidonic acid. For decades, the function of this lipid was unknown. Recently, we demonstrated that platelets undergo oxidative fragmentation when exposed to nanomolar concentrations of 12(S)-HETE (JCI 43:973, 2004). 12-LO activation occurs upon binding of human or rabbit anti-platelet GPIIIa to a specific platelet epitope GPIIIa49-66 (Cell 106:551, 2001) which is regulated by a specific IgG conformation (JBC 283: 3224, 2008). Fragmentation requires activation of the NADPH oxidase pathway downstream of the release of 12(S)-HETE since NADPH oxidase deficient (gp91 phox −/−) platelets fail to respond to exogenous 12(S)-HETE, whereas 12-LO−/− platelets are fully reactive. Since several lipid mediator receptors have been identified, including the leukotriene BLT1 and BLT2 receptors, we reasoned that the stereospecific action of the 12(S)-HETE was also likely mediated by a G-protein coupled receptor (GPCR). Recent data published in an abstract has suggested that some tumor cells might respond to 12(S)- HETE through the orphan GPCR, GPR31 (Guo, et al., IMPaCT, 2007). We searched for evidence of this receptor in platelets and in HUVEC. GPR31 mRNA was found in highly purified platelet and HUVEC preparations by RT-PCR. Further, incubation of gel filtered platelets with [3H]12(S)-HETE demonstrated saturation binding that was 30- fold above background. Binding of the radioligand was inhibited by a 100-fold molar excess of non-radioactive 12(S)-HETE, but was not effected by addition of 12(R)-HETE, a stereoisomer of the 12-LO product (n=3). In two experiments the Kd for 12(S)-HETE binding to platelets was ~20 nM with a Bmax of ~16 fmol/106 platelets. Oxidative platelet fragmentation induced by anti-GPIIIa49-66 Ab is a 12(S)-HETE-dependent process. We now show that this can be inhibited by 13(S)-HODE with an IC50 of ~400 nM (n=3). 13- HODE has been suggested to be a competitive inhibitor of 12(S)-HETE (Pidgeon, et al., Cancer Metastasis Rev 26:503, 2007). CHO cells which do not express an endogenous 12-(S)-HETE receptor were transfected with a GPR31-bearing plasmid. [3H]12(S)-HETE binding to GPR31-CHO cells was dose-dependent and saturable while the radioligand did not bind to mock transfected cells. Radioligand binding to the GPR31-CHO cells was inhibited by both 13(S)-HODE as well as cold 12(S)-HETE with an IC50 of <50 nM. No inhibition was noted with 100-fold molar excess of the inactive stereoisomer, 12(R)- HETE. Employing a peptide phage display library with GPIIIa49-66 as bait, we have recently found 70% homology with a peptide within the C-terminal end of ADAMTS-18 (a Disintegrin Metalloproteinase with Thrombospondin Motifs which is present in endothelial cells). An active 45kD C-terminal fragment is formed from ADAMTS-18 after thrombin cleavage. This fragment binds to platelets and induces oxidative fragmentation via the activation of 12(S)-HETE synthesis (Blood 110:127A, 2007). Fragmentation was inhibited by an anti-ADAMTS-18 Ab against the C-terminal end of the protein, as well as a C-terminal 18-mer peptide that mimics the platelet binding region of ADAMTS-18 (n=6). We next tested the ability of 12(S)-HETE to stimulate the release of ADAMTS-18 from HUVEC. Employing a highly sensitive immunoassay for ADAMTS-18, we observed a constitutive secretion of ADAMTS-18 that was enhanced ~15-fold with 12(S)-HETE (300 nM), but not by 12(R)-HETE (n=6). ADAMTS-18 secretion was also inhibited from 10- to 15-fold by 13-HODE (300 nM) as well as by an anti-GPR31 Ab (n=3) while 12(R)- HETE had no effect. Using siRNA to knock down GPR31 in HUVEC (n=3) resulted in an 85% inhibition of 12(S)-HETE-induced secretion, indicating that GPR31 is required to transduce the 12(S)-HETE effect in HUVEC. Thus, the orphan GPCR, GPR31, appears to be the long-sought 12(S)-HETE receptor on platelets and endothelial cells. In platelets, it stimulates activation of NADPH oxidase coupled to oxidative platelet fragmentation. In endothelial cells, it stimulates the release of ADAMTS-18 which binds to platelets and activates the production of platelet 12(S)-HETE leading to further oxidative platelet fragmentation. These observations also support a role for endothelial cell ADAMTS-18 regulation of platelet thrombus formation.

The ADAMs: signalling scissors in the tumour microenvironment

Over the last few years disintegrin metalloproteinases of the Adam (a disintegrin and metalloproteinase) family have been associated with the process of proteolytic 'shedding' of membrane-associated proteins and hence the rapid modulation of key cell signalling pathways in the tumour microenvironment. Furthermore, numerous members of the Adam family have been associated with tumorigenesis and tumour progression. The question now arises of whether pharmacological manipulation of their functions would be a useful adjunct to therapies targeting intercellular communications. To learn from the lessons of matrix metalloproteinase inhibitors as anticancer agents, there are many facets of the biological and clinical relevance of the ADAMs that need to be understood before embarking with confidence on such an approach.

Expression of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs) in experimental systems of cartilage matrix degradation

The chondrocytes of articular cartilage synthesise a number of proteinases which are capable of degrading the component molecules of this specialized extracellular matrix. The use of class-specific proteinase inhibitors indicates that major activities responsible for catabolism of proteoglycan (aggrecan) and collagen are attributable to zinc-dependent metalloproteinases. Amino acid sequence analysis of aggrecan catabolites isolated from human cartilage and synovial fluids has identified two major sites of proteolytic cleavage which occur in vivo at Asn341-Phe342 and Glu373-Ala374. Whilst cleavage at the latter site is attributed to an unknown ‘aggrecanase’, the Asn341-Phe342 bond is readily cleaved by many of the known matrix metalloproteinases. While considerable attention has focused in recent years on the expression and activity of MMPs in articular cartilage, it has now been shown (McKie et al. 1997; Patel et al. 1998) that chondrocytes can also express other metalloproteinases (e.g. the ADAM family of disintegrin-metalloproteinases, most of which have a consensus hydrophobic transmembrane sequence at their C-terminus, and many of which possess a consensus zinc-binding catalytic site which is also present in the MMPs). In this study, we have compared and quantified the mRNA expression profiles of two matrix metalloproteinases (MMP-3 and MMP-13) and five disintegrin metalloproteinases (ADAM-10, ADAM-9, ADAM-15, TNF-alpha converting enzyme and decysin) in chondrocytes (human, porcine and bovine) from fresh cartilage and in cartilage explant cultures and isolated cells cultured in monolayer or in agarose gels.

The Ectodomain Shedding of E-cadherin by ADAM15 Supports ErbB Receptor Activation

The zinc-dependent disintegrin metalloproteinases (a disintegrin and metalloproteinases (ADAMs) have been implicated in several disease processes, including human cancer. Previously, we demonstrated that the expression of a catalytically active member of the ADAM family, ADAM15, is associated with the progression of prostate and breast cancer. The accumulation of the soluble ectodomain of E-cadherin in human serum has also been associated with the progression of prostate and breast cancer and is thought to be mediated by metalloproteinase shedding. Utilizing two complementary models, overexpression and stable short hairpin RNA-mediated knockdown of ADAM15 in breast cancer cells, we demonstrated that ADAM15 cleaves E-cadherin in response to growth factor deprivation. We also demonstrated that the extracellular shedding of E-cadherin was abrogated by a metalloproteinase inhibitor and through the introduction of a catalytically inactive mutation in ADAM15. We have made the novel observation that this soluble E-cadherin fragment was found in complex with the HER2 and HER3 receptors in breast cancer cells. These interactions appeared to stabilize HER2 heterodimerization with HER3 and induced receptor activation and signaling through the Erk pathway, supporting both cell migration and proliferation. In this study, we provide evidence that ADAM15 catalyzes the cleavage of E-cadherin to generate a soluble fragment that in turn binds to and stimulates ErbB receptor signaling.

The ADAM10 Prodomain Is a Specific Inhibitor of ADAM10 Proteolytic Activity and Inhibits Cellular Shedding Events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.

PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid

Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.

Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

Alpha-Secretase As a Therapeutic Target

In the non-amyloidogenic pathway the alpha-secretase cleaves the amyloid precursor protein (APP) within the sequence of Abeta-peptides and precludes their formation. In addition, alpha-secretase cleavage releases an N-terminal extracellular domain with neurotrophic and neuroprotective properties. The disintegrin metalloproteinase ADAM10 has been shown to act as alpha-secretase in vivo, to prevent amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. An increase in alpha-secretase activity therefore is an attractive strategy for treatment of AD and may be achieved by modulating selective signalling pathways. Functional characterization of the human ADAM10 promoter showed that it contains several binding elements for transcription factors which are regulated by extracellular ligands. Retinoic acid (RA) was identified as an inducer of human ADAM10 promoter activity. In human neuroblastoma cell lines RA treatment upregulated the expression of both the alpha-secretase ADAM10 and its substrates APP and the related APP-like-protein 2 (APLP2), and led to an enhanced secretion of their extracellular domains. Furthermore, G protein-coupled receptors (GPCRs) localized in brain areas affected by AD were investigated. Activation of the PAC1 receptor by the neuropeptide PACAP was identified as an approach for upregulation of the alpha-secretase pathway.

Effects of metoprolol treatment on a disintegrin metalloproteinase expression and extracellular matrix remodeling after myocardial infarction in rats

Effects of metoprolol treatment on a disintegrin metalloproteinase expression and extracellular matrix remodeling after myocardial infarction in rats

Role of metalloproteinases in platelet function

Platelets contain and release matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and disintegrin metalloproteinases (ADAMs) including MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP (MMP-14), ADAM-10, ADAM-17, ADAMTS-13, TIMP-1, TIMP-2 and TIMP-4. These proteins exert several effects regulating platelet functions such as agonist-stimulated platelet adhesion and aggregation, tumour cell-induced platelet aggregation and platelet-leukocyte aggregation. In this review, mechanisms of MMPs, TIMPs and ADAMs on platelets are discussed.

Polyductin undergoes notch-like processing and regulated release from primary cilia

Mutations at a single locus, PKHD1, are responsible for causing human autosomal recessive polycystic kidney disease (ARPKD). Recent studies suggest that the cystic disease might result from defects in planar cell polarity, but how the 4074 amino acid ciliary protein encoded by the longest open reading frame of this transcriptionally complex gene may regulate this process is unknown. Using novel in vitro expression systems, we show that the PKHD1 gene product polyductin/fibrocystin undergoes a complicated pattern of Notch-like proteolytic processing. Cleavage at a probable proprotein convertase site produces a large extracellular domain that is tethered to the C-terminal stalk by disulfide bridges. This fragment is then shed from the primary cilium by activation of a member of the ADAM metalloproteinase disintegrins family, resulting in concomitant release of an intra-cellular C-terminal fragment via a gamma-secretase-dependent process. The ectodomain of endogenous PD1 is similarly shed from the primary cilium upon activation of sheddases. This is the first known example of this process involving a protein of the primary cilium and suggests a novel mechanism whereby proteins that localize to this structure may function as bi-directional signaling molecules. Regulated release from the primary cilium into the lumen may be a mechanism to distribute signal to down-stream targets using flow.

ADAM10 Inhibition of Human CD30 Shedding Increases Specificity of Targeted ImmunotherapyIn vitro

CD30 is a transmembrane protein selectively overexpressed on many human lymphoma cells and therefore an interesting target for antibody-based immunotherapy. However, binding of therapeutic antibodies stimulates a juxtamembrane cleavage of CD30 leading to a loss of target antigen and an enhanced release of the soluble ectodomain of CD30 (sCD30). Here, we show that sCD30 binds to CD30 ligand (CD153)-expressing non-target cells. Because antibodies bind to sCD30, this results in unwanted antibody binding to these cells via sCD30 bridging. To overcome shedding-dependent damage of normal cells in CD30-specific immunotherapy, we analyzed the mechanism involved in the release. Shedding of CD30 can be enhanced by protein kinase C (PKC) activation, implicating the disintegrin metalloproteinase ADAM17 but not free cytoplasmic calcium. However, antibody-induced CD30 shedding is calcium dependent and PKC independent. This shedding involved the related metalloproteinase ADAM10 as shown by the use of the preferential ADAM10 inhibitor GI254023X and by an ADAM10-deficient cell line generated from embryonically lethal ADAM10(-/-) mouse. In coculture experiments, the antibody-induced transfer of sCD30 from the human Hodgkin's lymphoma cell line L540 to the CD30-negative but CD153-expressing human mast cell line HMC-1 was inhibited by GI254023X. These findings suggest that selective metalloproteinase inhibitors blocking antibody-induced shedding of target antigens could be of therapeutic value to increase the specificity and reduce side effects of immunotherapy with monoclonal antibodies.

TIMP-3 Ameliorates Hepatic Ischemia/Reperfusion Injury Through Inhibition of Tumor Necrosis Factor-Alpha-Converting Enzyme Activity in Rats

Tumor necrosis factor (TNF)-alpha and its receptors play a critical role in the inflammatory cascade after hepatic ischemia/reperfusion injury. TNF-alpha converting enzyme (TACE) or disintegrin and metalloproteinase (ADAM)-17 is a metalloproteinase disintegrin that specifically cleaves precursor TNF-alpha to its mature form and is involved in the ectodomain shedding of TNF receptors. The regulation of TACE is poorly understood and its role in liver injury and/or regeneration is unknown. Male Wistar rats were subjected to 10 or 30 min of partial warm hepatic ischemia followed by 3 to 24 hr of reperfusion. Serum and/or hepatic TACE, TNF-alpha, TNF receptor 1 (TNFR1), and interleukin (IL)-6 levels were assessed by enzyme-linked immunosorbent assay, real-time reverse-transcriptase polymerase chain reaction, and/or Western blot. Low levels of TACE were detected in normal liver tissue. Both 10 and 30 min warm ischemia resulted in a rise in TACE expression which peaked six hr after reperfusion. TNF-alpha, TNFR1, and IL-6 levels were up-regulated in a pattern similar to TACE messenger RNA (mRNA) levels. Moreover, selective inhibition of TACE activity by specific inhibitor tissue inhibitor of metalloproteinase (TIMP)-3 at dosages of 100 or 1000 ng/kg body weight showed significant decrease of circulating TNF-alpha and serum alanine transferase (ALT) levels and histological improvement of hepatic ischemia/reperfusion injuries. TACE expression and its activity, as measured by increases in TNF-alpha, TNFR1, and IL-6 levels, are increased following hepatic ischemia/reperfusion injury, implying that TACE plays an important role in hepatic ischemia/reperfusion injury. Amelioration of hepatic ischemia/reperfusion injury after inhibition of TACE activity by TIMP-3 suggests that TACE inhibition may play an important role in preventing liver ischemia/reperfusion injury warranting further experimental and clinical study.