Expression and function of tumour necrosis factor-alpha-converting enzyme in the central nervous system

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE/ADAM17) is a membrane protein belonging to the ADAM (a disintegrin and a metalloprotease) family able to cleave various membrane proteins, including the transmembrane form of TNF-alpha at its physiological processing site. Being an ADAM, TACE may mediate not only proteolysis but also adhesive interactions; however, the role of the disintegrin domain of TACE has not been studied. In the central nervous system (CNS), little is known about the physiological role of TACE, but some important pathophysiological functions have been reported recently, with both neurotoxic and neuroprotective repercussions. This article discusses and reviews the main contributions to this field of investigation addressing the expression and function of TACE in the CNS.     

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

Cutting edge: TNF-alpha-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock

TNF-alpha, a potent proinflammatory cytokine, is synthesized as a membrane-anchored precursor and proteolytically released from cells. Soluble TNF is the primary mediator of pathologies such as rheumatoid arthritis, Crohn's disease, and endotoxin shock. The TNF-alpha converting enzyme (TACE), a disintegrin and metalloprotease 17 (ADAM17), has emerged as the best candidate TNF sheddase, but other proteinases can also release TNF. Because TACE-deficient mice die shortly after birth, we generated conditional TACE-deficient mice to address whether TACE is the relevant sheddase for TNF in adult mice. In this study, we report that TACE inactivation in myeloid cells or temporal inactivation at 6 wk offers strong protection from endotoxin shock lethality in mice by preventing increased TNF serum levels. These findings corroborate that TACE is the major endotoxin-stimulated TNF sheddase in mouse myeloid cells in vivo, thereby further validating TACE as a principal target for the treatment of TNF-dependent pathologies.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exons. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33.     

The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17

The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype.     

The mouse homolog of the human amyloid beta protein (AD-AP) gene is located on the distal end of mouse chromosome 16: further extension of the homology between human chromosome 21 and mouse chromosome 16

The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21.     

The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.     

Molecular cloning of mouse beta 2-glycoprotein I and mapping of the gene to chromosome 11.

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.     

The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are tandemly arranged on chromosome 17 of the mouse

We have isolated clones containing the gene for tumor necrosis factor (TNF-alpha) from a mouse genomic library. Four out of five clones containing the TNF-alpha gene also hybridized to a human lymphotoxin (TNF-beta) probe. We constructed a restriction enzyme cleavage map of a 6.4 kb region from one of the genomic clones. From partial sequencing data and hybridizations with exon-specific oligonucleotide probes, we conclude that this region contains the mouse TNF-alpha and TNF-beta genes in a tandem arrangement, that they are separated by only about 1100 bases, and that their intron-exon structure is very similar to that seen in man. We probed genomic blots of DNA from human/mouse hybrids containing single mouse chromosomes for the presence of the mouse TNF genes. The results show that the genes are located on mouse chromosome 17, which also contains the major histocompatibility complex. Therefore, both the mouse and the human TNF genes are tandemly arranged and located on the same chromosome as the MHC.     

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.     

The ADAM1a and ADAM1b genes,instead of the ADAM1 (fertilin alpha) gene,are localized on mouse chromosome 5

Fertilin is reported to be a heterodimeric protein composed of A Disintegrin And Metalloprotease 1 (ADAM1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface. In the process of clarifying the molecular basis of mouse ADAM1, we have identified two intron-less mouse genes encoding different isoforms of ADAM1, termed ADAM1a and ADAM1b. The amino acid sequences of ADAM1a and ADAM1b deduced from the DNA sequences were homologous to each other (99% identity) in the pro- and metalloprotease domains, whereas the C-terminal half region of ADAM1a, including the disintegrin and Cys-rich domains, shared only a low degree of identity (37%) with that of ADAM1b. These two genes were both localized on mouse chromosome 5 as a single copy gene, and were expressed specifically in the testis. These data demonstrate the presence of the ADAM1a (Adam1a) and ADAM1b (Adam1b) genes in mouse, instead of the ADAM1 gene, and may imply different roles of ADAM1a and ADAM1b in spermatogenesis, sperm maturation, and/or fertilization.     

Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model

Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose-response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6 h after etoposide addition in GC-2 cells, whereas the same was observed 18 h after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18 h after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12 h after etoposide was removed. ADAM10 mRNA increased after 3 h and then steadily decreased up to 12 h after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM10 and TACE/ADAM17 have a role in etoposide-induced apoptosis.     

Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsolin and dopamine beta-hydroxylase map to mouse chromosome 2.

The mapping of human chromosome 9 (HSA9) and mouse chromosome 2 (MMU2) has revealed a conserved syntenic region between the distal end of the long arm of chromosome 9 and proximal mouse chromosome 2. Two genes that map to human chromosome 9q34, gelsolin (GSN) and dopamine beta-hydroxylase (DBH), have not previously been located in the mouse. We have used an interspecific backcross to map each of these genes, by Southern blot analysis, to mouse chromosome 2. Gelsolin (Gsn) is tightly linked to the gene for complement component C5 (Hc), and dopamine beta-hydroxylase (Dbh) is just proximal to the Abelson leukemia virus oncogene (Abl) and alpha-spectrin 2 (Spna-2). The loci for gelsolin and dopamine beta-hydroxylase therefore form part of the conserved synteny between HSA9q and MMU2.     

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.     

Involvement of TACE/ADAM17 and ADAM10 in etoposide-induced apoptosis of germ cells in rat spermatogenesis

Germ cell apoptosis is important to regulate sperm production in the mammalian testis, but the molecular mechanisms underlying apoptosis are still poorly understood. We have recently shown that in vitro, etoposide induces upregulation of TACE/ADAM17 and ADAM10, two membrane-bound extracellular metalloproteases. Here we show that in vivo these enzymes are involved in etoposide-, but not in heat shock-, induced apoptosis in rat spermatogenesis. Germ cell apoptosis induced by DNA damage was associated with an increase in protein levels and cell surface localization of TACE/ADAM17 and ADAM10. On the contrary, apoptosis of germ cells induced by heat stress, another cell death stimulus, did not change levels or localization of these proteins. Pharmacological in vivo inhibition of TACE/ADAM17 and ADAM10 prevents etoposide-induced germ cell apoptosis. Finally, Gleevec (STI571) a pharmacological inhibitor of p73, a master gene controlling apoptosis induced by etoposide, prevented the increase of TACE/ADAM17 levels. Our results strongly suggest that TACE/ADAM17 participates in in vivo apoptosis of male germ cells induced by DNA damage.     

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

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