Cleaves the membrane-bound precursor of TNF-alpha to its mature soluble form. Responsible for the proteolytical release of soluble JAM3 from endothelial cells surface. Responsible for the proteolytic release of several other cell-surface proteins, including p75 TNF-receptor, interleukin 1 receptor type II, p55 TNF-receptor, transforming growth factor-alpha, L-selectin, growth hormone receptor, MUC1 and the amyloid precursor protein. Also involved in the activation of Notch pathway (By similarity).
Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells (ECs) that plays a role in tight junction formation, leukocyte adhesion, and transendothelial migration. In the current study, we investigated whether JAM-C is found in soluble form and whether soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared with RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular ECs (HMVECs) and immortalized human dermal microvascular ECs, and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinases 10 and 17. In functional assays, sJAM-C was both chemotactic and chemokinetic for HMVECs and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti-JAM-C Abs inhibited RA synovial fluid-induced HMVEC chemotaxis and sJAM-C-induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C-mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form and suggest that modulation of sJAM-C may provide a novel route for controlling pathological angiogenesis.
MUC1 clearance from the uterine epithelial cell surface is a prerequisite for the creation of an environment conducive to embryo implantation. In some species, reduced mRNA levels along with metabolic turnover account for loss of MUC1 during the receptive phase throughout the uterine epithelium. In other species, MUC1 is rapidly lost solely at the site of blastocyst attachment, suggesting the action of a protease. Correlative studies also indicate the presence of soluble forms of MUC1 in cell culture supernatants in vitro and in bodily fluids in vivo. To characterize the proteolytic activity mediating MUC1 release, shedding of MUC1 was analyzed in a human uterine epithelial cell line (HES) that abundantly expresses and readily sheds MUC1. MUC1 release was stimulated by phorbol 12-myristate 13-acetate and was markedly inhibited by the synthetic peptide hydroxamate metalloprotease inhibitor, tumor necrosis factor-alpha protease inhibitor (TAPI), as well as by an endogenous inhibitor of matrix metalloproteases, tissue inhibitor of metalloproteases (TIMP)-3. These characteristics along with studies conducted with cell lines genetically deficient in various ADAMs (for a disintegrin and metalloprotease) identified tumor necrosis factor-alpha converting enzyme (TACE)/ADAM 17 as a MUC1 sheddase. Furthermore, both TACE and MUC1 were expressed in human uterine epithelia during the receptive phase, and co-immunoprecipitation experiments revealed a physical interaction between TACE and MUC1 in HES cells. These studies establish a proteolytic mechanism for MUC1 clearance from a human uterine epithelial cell line and identify TACE as a MUC1 sheddase.
Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.
Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.
Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.
J. Biol. Chem. 274, 3531-3540 (1999)[PubMed:9920899]
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-alpha convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-alpha-convertase.
Catalysis of the hydrolysis of peptide bonds by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.
The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.
The putative alpha-secretase cleaves the amyloid precursor protein (APP) of Alzheimer's disease in the middle of the amyloid beta peptide (Abeta) domain. It is generally thought that the alpha-secretase pathway mitigates Abeta formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate alpha-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both "constitutive" and "regulated" alpha-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous alpha-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze alpha-secretory cleavage and therefore act as alpha-secretases in A172 cells. This is the first report that to suggest the endogenous alpha-secretase is composed of several ADAM enzymes.
Tumor necrosis factor alpha-convertase (TACE) is a metalloprotease-disintegrin involved in the ectodomain shedding of several proteins and is critical for proper murine development. TACE-mediated ectodomain shedding is regulated, and the cytoplasmic domain of TACE contains several potential signaling motifs, suggesting that this domain may play a role in regulating the metalloprotease activity. Here we report that the protein-tyrosine phosphatase PTPH1, which contains both a band 4.1 domain and a single PDZ domain, can interact with the cytoplasmic domain of TACE. The interaction was initially observed in a yeast two-hybrid screen and was confirmed using an in vitro binding assay and co-immunoprecipitations from eukaryotic cell extracts. The interaction is mediated via binding of the PDZ domain of PTPH1 to the COOH terminus of TACE. The latter represents a novel group I PDZ binding sequence characterized by a terminal cysteine residue. In co-expression experiments, significantly lower levels of TACE were observed in the presence of catalytically active forms of PTPH1 compared with catalytically inactive forms of PTPH1. Furthermore, phorbol ester-stimulated shedding of the TACE substrate tumor necrosis factor-alpha was decreased in cells expressing catalytically active PTPH1 compared with inactive PTPH1. Taken together, these results suggest that PTPH1 may be a negative regulator of TACE levels and function, and thus provide the first evidence for the regulation of TACE through a cytoplasmic protein.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionBHF-UCL
Tumor necrosis factor-alpha (TNF-alpha) is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-alpha-converting enzyme (TACE/ADAM17) is the major sheddase for ectodomain shedding of TNF-alpha. At present, however, it is poorly understood how its catalytic activity is regulated. Here, we show that nardilysin (N-arginine dibasic convertase; NRDc) enhanced TNF-alpha shedding. In a cell-based shedding assay, expression of NRDc synergistically enhanced TACE-induced TNF-alpha shedding. A peptide cleavage assay in vitro showed that recombinant NRDc enhances the cleavage of TNF-alpha induced by TACE. Notably, co-incubation of NRDc completely reversed the inhibitory effect of a physiological concentration of salt on TACE's activity in vitro. Overexpression of NRDc in TACE-deficient fibroblasts resulted in an increase in the amount of TNF-alpha released. Co-expression of NRDc with ADAM10 promoted the release compared with the sole expression of ADAM10. These results suggested that NRDc enhances TNF-alpha shedding through activation of not only TACE but ADAM10. Our results indicate the involvement of NRDc in ectodomain shedding of TNF-alpha, which may be a novel target for anti-inflammatory therapies.
Evidence
2:
Inferred from Physical InteractionBHF-UCL
In HER2-overexpressing mammary epithelial cells, transforming growth factor beta (TGF-beta) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-beta or expression of an activated TGF-beta type I receptor (Alk5 with the mutation T204D [Alk5(T204D)]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-alpha, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-beta-induced activation of Akt and cell invasiveness. Treatment with TGF-beta or expression of Alk5(T204D) in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5(T204D) expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-beta may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.
Evidence
3:
Inferred from Physical InteractionBHF-UCL
TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-alpha converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 A resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.
Evidence
4:
Inferred from Physical InteractionBHF-UCL
A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.
Evidence
5:
Inferred from Physical InteractionIntAct
Biochem. J. 343 Pt 3, 673-680 (1999)[PubMed:10527948]
Metalloprotease-disintegrins are a family of transmembrane glycoproteins that have a role in fertilization, sperm migration, myoblast fusion, neural development and ectodomain shedding. In the present study we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic domain of two metalloprotease-disintegrins, tumour necrosis factor alpha convertase (TACE; ADAM17) and MDC9 (ADAM9; meltrin gamma). We have identified mitotic arrest deficient 2 (MAD2) as a binding partner of the TACE cytoplasmic domain, and a novel MAD2-related protein, MAD2beta, as a binding partner of the MDC9 cytoplasmic domain. MAD2beta has 23% sequence identity with MAD2, which is a component of the spindle assembly (or mitotic) checkpoint mechanism. Northern blot analysis of human tissues indicates that MAD2beta mRNA is expressed ubiquitously. The interaction of the TACE and MDC9 cytoplasmic domains with their binding partners has been confirmed biochemically. The independent identification of MAD2 and MAD2beta as potential interacting partners of distinct metalloprotease-disintegrins raises the possibility of a link between metalloprotease-disintegrins and the cell cycle, or of functions for MAD2 and MAD2beta that are not related to cell cycle control.
Interacting selectively and non-covalently with a SH3 domain (Src homology 3) of a protein, small protein modules containing approximately 50 amino acid residues found in a great variety of intracellular or membrane-associated proteins.
The process in which a precursor cell type acquires the specialized features of a B cell. A B cell is a lymphocyte of B lineage with the phenotype CD19-positive and capable of B cell mediated immunity.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.
The attachment of a cell, either to another cell or to an underlying substrate such as the extracellular matrix, via an integrin, a heterodimeric adhesion receptor formed by the non-covalent association of particular alpha and beta subunits.
Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
A series of molecular signals initiated by binding of a ligand to the tyrosine kinase receptor EGFR (ERBB1) on the surface of a cell. The pathway ends with regulation of a downstream cellular process, e.g. transcription.
Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.
The process in which an epidermal growth factor-activated receptor is activated via signaling events from a G-protein coupled receptor. This is an example of cross-talk between the EGF and GPCR signaling pathways.
Intestinal cells express alpha(2A)-adrenoreceptors that stimulate sodium and peptide absorption and promote cell proliferation. Involved mechanisms are poorly understood and are not fully related to inhibition of cAMP production. Previous study using a clone of CaCo2 cells expressing the human alpha(2A)-adrenoreceptor (CaCo2-3B) showed that alpha(2)-adrenoreceptor agonists cause extracellular signal-regulated kinase (ERK) phosphorylation. Present work examines the signaling pathway triggering ERK activation and investigates the consequence of alpha(2A)-adrenoreceptor stimulation on cell migration. Treatment of CaCo2-3B with the alpha(2)-adrenoreceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline (UK14304) induces not only ERK, but also Akt phosphorylation. Both effects are strongly attenuated by inhibition or desensitization of epidermal growth factor (EGF) receptor, matrix metalloproteinase (MMP) blockade, heparin-binding-EGF neutralization or phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Conditioned medium from UK14304-treated CaCo2-3B stimulates ERK in parental CaCo2 by a mechanism sensitive to EGF receptor and PI3-kinase inhibitors. Exposure of CaCo2-3B to UK14304 accelerates the wound healing. This effect is abolished by heparin-binding-EGF neutralization but not by mitomycin C, indicating that it results probably from increased cell spreading and/or migration. In conclusion, alpha(2A)-adrenoreceptor activates ERK and Akt in intestinal cells by a common pathway which depends on PI3-kinase activation and results from EGF receptor transactivation, via an autocrine/paracrine pathway implying MMP activation and heparin-binding-EGF shedding. Therefore, alpha(2A)-adrenoreceptor could have a positive role in intestinal regeneration in vivo.
The process in which germinal centers form. A germinal center is a specialized microenvironment formed when activated B cells enter lymphoid follicles. Germinal centers are the foci for B cell proliferation and somatic hypermutation.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.
In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.
A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-alpha convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCdeltaDETCy), expressed in baculovirus could cleave preferentially (approximately 12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-alpha to TNF-alpha (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-alpha mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alphaR fused to IgG Fc fragment (TNF-alphaR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-alpha in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE.
Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.
Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the "a disintegrin and metalloproteinase" (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors.
The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
A series of molecular signals initiated by the binding of an extracellular ligand to the receptor Notch on the surface of a target cell, and ending with regulation of a downstream cellular process, e.g. transcription.
The proteolytic cleavage of transmembrane proteins and release of their ectodomain that occurs after induction by phorbol-12-myristate-13-acetate (PMA), a protein kinase C agonist.
Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.
The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.
Glycoprotein V (GPV) is a subunit of the GPIb-IX-V receptor for von Willebrand factor and thrombin and has been shown to modulate platelet responses to the two strongest physiological agonists, thrombin and collagen. Thrombin directly cleaves GPV from the platelet surface, yielding a 69-kDa fragment GPV f1 of unknown function. We show here that a approximately 82-kDa fragment of GPV is shed from the platelet surface upon cellular activation with phorbol 12-myristate 13-acetate or the collagen-related peptide. This shedding was inhibited by the broad range metalloproteinase inhibitor GM6001, the two potent ADAM17 inhibitors GW280264X and TAPI-2, and was absent in mice lacking functional ADAM17 (ADAM17 lacking Zn-binding domain; ADAM17(DeltaZn/DeltaZn)). Furthermore, we show that recombinant ADAM17 ectodomain efficiently releases GPV from the platelet surface. GPV is known to be associated with the intracellular regulatory protein calmodulin, which has previously been shown to be involved in ADAM17-mediated shedding of l-selectin from the surface of leukocytes. As in these reports, inhibition of calmodulin led to rapid GPV shedding from the platelet surface, a process that was again blocked by GM6001 or ADAM17 inhibitors and that was absent in ADAM17(DeltaZn/DeltaZn) mice. Inhibition of outside-in signaling through GPIIb/IIIa did not significantly affect GPV shedding, excluding an essential role of this pathway for the regulation of ADAM17 activity. These results demonstrate that GPV is cleaved upon agonist-induced platelet activation and show that ADAM17 is the major enzyme mediating this process.
The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the "a disintegrin and metalloproteinase" (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors.
In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.
In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.
In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
UVA (315-400 nm), which constitutes approximately 95% of the UV irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here, we show that a low, nonlethal dose of UVA induces dose-dependent cell cycle progression in human HaCaT keratinocytes. We found that UVA induced cyclin D1 accumulation, whereas siRNA knockdown of cyclin D1 blocked the UVA-induced cell cycle progression, indicating that this process is mediated by cyclin D1. UVA irradiation also induced AKT activation; when cells were incubated with phosphatidylinositol-3-OH kinase/AKT inhibitor or infected with dominant-negative AKT, cyclin D1 up-regulation, cell cycle progression, and proliferation were inhibited, suggesting that AKT activation is required for UVA-induced cell cycle progression. In contrast, extracellular signal-regulated kinase (ERK) was not activated by UVA exposure; incubation with ERK/mitogen-activated protein kinase inhibitor had no effect on UVA-induced cyclin D1 up-regulation and cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVA exposure. EGFR kinase inhibitor AG attenuated the UVA-induced AKT/cyclin D1 pathway and cell cycle progression, indicating that EGFR is upstream of AKT/cyclin D1 pathway activation. Furthermore, metalloprotease inhibitor GM6001 blocked UVA-induced cell cycle progression, and siRNA knockdown of a disintegrin and metalloprotease (ADAM)17 had a similar inhibitory effect, demonstrating that ADAM17 mediates the EGFR/AKT/cyclin D1 pathway and cell cycle progression to the S phase induced by UVA radiation. Identification of these signaling pathways in UVA-induced cell proliferation will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.
Intestinal cells express alpha(2A)-adrenoreceptors that stimulate sodium and peptide absorption and promote cell proliferation. Involved mechanisms are poorly understood and are not fully related to inhibition of cAMP production. Previous study using a clone of CaCo2 cells expressing the human alpha(2A)-adrenoreceptor (CaCo2-3B) showed that alpha(2)-adrenoreceptor agonists cause extracellular signal-regulated kinase (ERK) phosphorylation. Present work examines the signaling pathway triggering ERK activation and investigates the consequence of alpha(2A)-adrenoreceptor stimulation on cell migration. Treatment of CaCo2-3B with the alpha(2)-adrenoreceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline (UK14304) induces not only ERK, but also Akt phosphorylation. Both effects are strongly attenuated by inhibition or desensitization of epidermal growth factor (EGF) receptor, matrix metalloproteinase (MMP) blockade, heparin-binding-EGF neutralization or phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Conditioned medium from UK14304-treated CaCo2-3B stimulates ERK in parental CaCo2 by a mechanism sensitive to EGF receptor and PI3-kinase inhibitors. Exposure of CaCo2-3B to UK14304 accelerates the wound healing. This effect is abolished by heparin-binding-EGF neutralization but not by mitomycin C, indicating that it results probably from increased cell spreading and/or migration. In conclusion, alpha(2A)-adrenoreceptor activates ERK and Akt in intestinal cells by a common pathway which depends on PI3-kinase activation and results from EGF receptor transactivation, via an autocrine/paracrine pathway implying MMP activation and heparin-binding-EGF shedding. Therefore, alpha(2A)-adrenoreceptor could have a positive role in intestinal regeneration in vivo.
UVA (315-400 nm), which constitutes approximately 95% of the UV irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here, we show that a low, nonlethal dose of UVA induces dose-dependent cell cycle progression in human HaCaT keratinocytes. We found that UVA induced cyclin D1 accumulation, whereas siRNA knockdown of cyclin D1 blocked the UVA-induced cell cycle progression, indicating that this process is mediated by cyclin D1. UVA irradiation also induced AKT activation; when cells were incubated with phosphatidylinositol-3-OH kinase/AKT inhibitor or infected with dominant-negative AKT, cyclin D1 up-regulation, cell cycle progression, and proliferation were inhibited, suggesting that AKT activation is required for UVA-induced cell cycle progression. In contrast, extracellular signal-regulated kinase (ERK) was not activated by UVA exposure; incubation with ERK/mitogen-activated protein kinase inhibitor had no effect on UVA-induced cyclin D1 up-regulation and cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVA exposure. EGFR kinase inhibitor AG attenuated the UVA-induced AKT/cyclin D1 pathway and cell cycle progression, indicating that EGFR is upstream of AKT/cyclin D1 pathway activation. Furthermore, metalloprotease inhibitor GM6001 blocked UVA-induced cell cycle progression, and siRNA knockdown of a disintegrin and metalloprotease (ADAM)17 had a similar inhibitory effect, demonstrating that ADAM17 mediates the EGFR/AKT/cyclin D1 pathway and cell cycle progression to the S phase induced by UVA radiation. Identification of these signaling pathways in UVA-induced cell proliferation will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.
J. Immunol. 175, 1930-1936 (2005)[PubMed:16034137]
Airway epithelial cells have a major role in initiating inflammation in response to bacterial pathogens. Through the immediate induction of CXCL8 and cytokine expression, polymorphonuclear cells are mobilized and activated to eradicate the infecting organisms. However, the influx of polymorphonuclear cells and the effects of their toxic exoproducts impede respiratory function. We postulated that respiratory epithelial cells must also participate in the regulation of their own proinflammatory signaling. Both Staphylococcus aureus and Pseudomonas aeruginosa were found to potently activate IL-6 expression immediately upon contact with epithelial cells, and by 1 h induced TNF-alpha converting enzyme (TACE) transcription. By 4 h of bacterial exposure, TACE colocalized with IL-6Ralpha on the apical surface of airway cells, and by 24 h, soluble IL-6Ralpha accumulated in the cell culture supernatant. Epithelial IL-6 and soluble IL-6Ralpha were shown to participate in trans-signaling, interacting with membrane-associated gp130 to activate CCL-2 expression and inhibit additional CXCL8 production. Thus, bacteria are physiological activators of TACE expression, which provides a mechanism to regulate inflammatory signaling that is initiated by airway epithelial cells.
In HER2-overexpressing mammary epithelial cells, transforming growth factor beta (TGF-beta) activated phosphatidylinositol-3 kinase (PI3K)/Akt and enhanced survival and migration. Treatment with TGF-beta or expression of an activated TGF-beta type I receptor (Alk5 with the mutation T204D [Alk5(T204D)]) induced phosphorylation of TACE/ADAM17 and its translocation to the cell surface, resulting in increased secretion of TGF-alpha, amphiregulin, and heregulin. In turn, these ligands enhanced the association of p85 with ErbB3 and activated PI3K/Akt. RNA interference of TACE or ErbB3 prevented TGF-beta-induced activation of Akt and cell invasiveness. Treatment with TGF-beta or expression of Alk5(T204D) in HER2-overexpressing cells reduced their sensitivity to the HER2 antibody trastuzumab. Inhibition of Alk5, PI3K, TACE, or ErbB3 restored sensitivity to trastuzumab. A gene signature induced by Alk5(T204D) expression correlated with poor clinical outcomes in patients with invasive breast cancer. These results suggest that by acting on ErbB ligand shedding, an excess of TGF-beta may result in (i) conditioning of the tumor microenvironment with growth factors that can engage adjacent stromal and endothelial cells; (ii) potentiation of signaling downstream ErbB receptors, thus contributing to tumor progression and resistance to anti-HER2 therapies; and (iii) poor clinical outcomes in women with breast cancer.
Any process that increases the rate, frequency or extent of T cell chemotaxis. T cell chemotaxis is the directed movement of a T cell in response to an external stimulus.
In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a drug stimulus. A drug is a substance used in the diagnosis, treatment or prevention of a disease.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a high density lipoprotein particle stimulus.
The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level.
Ectodomain shedding of epidermal growth factor receptor ligands such as transforming growth factor- alpha (TGF-alpha), heparin-binding epidermal growth factor-like growth factor (HBEGF), and amphiregulin (AREG) is considered to be important during implantation. Tumor necrosis factor-alpha converting enzyme (TACE) has been suggested as the major sheddase for these molecules. The objectives of this study are (1) to characterize the expression of TACE in the human placenta throughout gestation; (2) to determine the association between the expression of TACE with TGF-alpha, HBEGF, and AREG; (3) to ascertain whether TACE mediates TGF-alpha, HBEGF, and AREG shedding; and (4) to examine the effect of hypoxia on the expression of TACE. By analyzing a total of 55 villous samples representing different gestational ages, the authors found that TACE was continuously expressed in the placentas throughout gestation and that the levels of TACE were positively correlated with the levels of TGF-alpha, HBEGF, and AREG. Preadministration of a TACE inhibitor in villous explant cultures or transfection of cytotrophoblastic cells with TACE-specific small interference RNA decreased the shedding of HBEGF and AREG. Moreover, hypoxia (2% O(2)) caused an increase in the levels of TACE mRNA and protein in villous explants and primary cytotrophoblastic cells in vitro. These results indicate that oxygen regulates the expression of TACE and that TACE may be important for placental development during human pregnancy.
Any process that results in a change in state or activity of an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a lipopolysaccharide stimulus; lipopolysaccharide is a major component of the cell wall of gram-negative bacteria.
IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.
The process whose specific outcome is the progression of the spleen over time, from its formation to the mature structure. The spleen is a large vascular lymphatic organ composed of white and red pulp, involved both in hemopoietic and immune system functions.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
The process in which a precursor cell type acquires the specialized features of a T cell via a differentiation pathway dependent upon transit through the thymus.
Evidence
1:
Inferred from Sequence or Structural SimilarityBHF-UCL
Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.
Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.
This protein acts as an enzyme. It is known to catalyze the following reaction
EC 3.4.24.86: Narrow endopeptidase specificity. Cleaves Pro-Leu-Ala-Gln-Ala-|-Val-Arg-Ser-Ser-Ser in the membrane-bound, 26-kDa form of tumor necrosis factor alpha (TNF-alpha). Similarly cleaves other membrane-anchored, cell-surface proteins to 'shed' the extracellular domains.
Protein involved in the Notch signaling, a signaling pathway involved in cell-cell communications that regulates a broad spectrum of cell- fate determinations. Notch proteins are transmembrane receptors, which are cleaved by the gamma-secretase complex upon activation and released from the cell membrane and turn into transcriptional activators after their association with SU(H) proteins.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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