TNF-alpha Converting Enzyme Research Articles

Porphyromonas gingivalis stimulates TACE production by T cells

Tumour necrosis factor-alpha converting enzyme (TACE), also known as ADAM17, is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T-cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. P. gingivalis 6-day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell-associated TACE levels were measured by enzyme-linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real-time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat-inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. P. gingivalis challenge resulted in a concentration-dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell-bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.

Multiple shRNA‐mediated knockdown of TACE reduces the malignancy of HeLa cells

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) is a key enzyme involved in the proteolytic shedding of the ectodomain of several membrane-bound growth factors, cytokines and receptors. Here, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against TACE. We found that in HeLa cells our multiple shRNAs vector produced a higher level of TACE knockdown than any single shRNA vector containing only one TACE shRNA. Silencing TACE expression in HeLa cells decreased their malignancy by decreasing the proliferation, adhesion and migration, as well as inducing apoptosis in these cells. Furthermore, our data suggest that the effects of TACE on the malignancy of HeLa cells may be mediated via activation of the EGFR (epidermal growth factor receptor) signaling pathway. Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy for tumors.

Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

BackgroundNuclear localization of proteolytically formed intracellular fragment of ErbB4 receptor tyrosine kinase has been shown to promote cell survival, and nuclear localization of ErbB4 receptor has been described in human breast cancer. Tumor necrosis factor alpha converting enzyme (TACE) initiates the proteolytic cascade leading to ErbB4 intracellular domain formation. Interactions between matrix metalloproteases and heparan sulfate have been described, but the effect of cell surface heparan sulfate on TACE activity has not been previously described.ResultsAs indicated by immunodetection of increased ErbB4 intracellular domain formation and direct enzyme activity analysis, TACE activity was substantially amplified by enzymatic removal of cell surface heparan sulfate but not chondroitin sulfate.ConclusionIn this communication, we suggest a novel role for cell surface heparan sulfate. Removal of cell surface heparan sulfate led to increased formation of ErbB4 intracellular domain. As ErbB4 intracellular domain has previously been shown to promote cell survival this finding may indicate a novel mechanism how HS degradation active in tumor tissue may favor cell survival.

Involvement of caspases and transmembrane metalloproteases in sulphur mustard-induced microvesication in adult human skin in organ culture: Directions for therapy

While skin is a major target for sulphur mustard (HD), a therapy to limit HD-induced vesication is currently not available. Since it is supposed that apoptotic cell death and proteolytic digestion of extracellular matrix proteins by metalloproteases are initiating factors for blister formation, we have explored whether inhibition of these processes could prevent HD-induced epidermal–dermal separation using adult human skin in organ culture. Involvement of the caspase and the metalloprotease families was confirmed by the observation that their respective broad spectrum inhibitors, Z-VAD-fmk and GM6001, each suppressed HD-induced microvesication. The lowest effective concentrations were 10 and 100 μM, respectively. Using specific inhibitors for caspase-8 (≥10 μM) and caspase-9 (≥10 μM) we learned that HD-induced apoptosis is initiated by the death receptor pathway as well as by the mitochondrial pathway. Remarkably, blocking caspase-8 activity resulted in morphologically better conserved cells than blocking caspase-9 activity. We zoomed in on the role of metalloproteases in HD-induced microvesication by testing the effects of two inhibitors: dec-RVKR-cmk and TAPI-2. Dec-RVKR-cmk is an inhibitor of furin, which activates transmembrane enzymes of the ‘a disintegrin and metalloproteinase’ (ADAM)-family as well as the membrane-type metalloproteases (MTx-MMP). TAPI-2 specifically inhibits TNFalpha-converting enzyme (TACE/ADAM17), which is involved in pericellular proteolysis. Both inhibitors prevented microvesication at concentrations of ≥500 and ≥20 μM, respectively. This confirms that ADAMs and MT-MMPs play a role in HD-induced epidermal–dermal separation, with a particular role for TACE/ADAM17. Since TACE is involved not only in degradation of cell-matrix adhesion structures, but also in ectodomain shedding of ligands for epidermal growth factor receptor (EGFR) and in release of TNFα, these results imply TACE-mediated pathways as a new concept in HD toxicity. In conclusion, transmembrane metalloproteases probably form a main target for treatment of blisters in HD casualties. The observation that microvesication in the ex vivo human skin model still could be prevented when the metalloprotease inhibitor GM6001 was applied up to 8 h after exposure to HD opens perspectives for non-urgent cure of HD casualties.

Serotonin 5-HT 2B Receptor in Cardiac Fibroblast Contributes to Cardiac Hypertrophy

See related article, pages 113–123 Cardiac hypertrophy is often accompanied by cardiac remodeling characterized by loss of cardiac myocytes, interstitial fibroblasts, and collagen deposition, leading to decreased ventricular compliance and an increased risk for heart failure. The mortality for patients with heart failure is still high, although some improvements have been demonstrated in patients with systolic heart failure.1–3 To improve the therapeutic strategy for patients with heart failure, especially diastolic heart failure, further research and investigations to better understand the molecular and biochemical mechanism are needed. Serotonin (5-hydroxytryptamine [5-HT]) affects many physiological functions through the interaction with specific G-coupled membrane receptors, 5-HT receptors. There are 4 classes of 5-HT receptors (5-HT1/5, 5-HT2, 5-HT3, and 5-HT4/6/7).4 Serotonin, via the 5-HT2B receptor, regulates cardiac development and function.5 Transgenic mice with a 5-HT2B receptor gene ablation show embryonic and neonatal death caused by lack of trabeculae in the heart.6 5-HT2B receptors are essential for isoproterenol-induced cardiac hypertrophy, which involves the regulation of interleukin-6, interleukin-1β, and tumor necrosis factor-α cytokine production by cardiac fibroblasts.7 The 5-HT2B receptor has been shown functionally coupled to reactive oxygen species synthesis through NAD(P)H oxidase stimulation in neuronal cells8 and in angiotensin II and isoproterenol-induced cardiac hypertrophy.9 Recently, 5-HT2B receptor blockade has been shown to prevent the cardiac hypertrophy induced by angiotensin II or isoproterenol infusion.9 …

Les deux visages d’ADAM17 dans l’inflammation

ADAM17 was initially characterized as the TNF Alpha Converting Enzyme (TACE) and, until now, has been the most studied member of the ADAM family. It is a type I transmembrane metalloproteinase involved in the shedding of the extracellular domain of several transmembrane proteins (at least 40) such as cytokines, growth factors, receptors or adhesion molecules. As a consequence, depending on the transmembrane molecule cleaved, one may expect possible opposite effects of ADAM17 activity on inflammation (e.g. TNF and its receptors). The role of ADAM17 in regulating inflammatory cellular processes is clearly demonstrated in cells deficient in active ADAM17 or expressing substrates mutated for the ADAM17 cleavage site. As ADAM17-deficient mice died at birth, mice overexpressing the mutated uncleavable form of some substrates and recently conditional knock-out of ADAM17 are used to approach in vivo the role of this metalloprotease in regulating inflammation. Arguments are provided that ADAM17 plays a role in atherosclerosis, in adipose tissue metabolism, insulin resistance and diabetes. The multitude of substrates cleaved by ADAM17 makes this enzyme an attractive candidate to study its role in inflammation-driven pathologies.

Development of Predictive 3D‐QSAR CoMFA and CoMSIA Models for β‐aminohydroxamic Acid‐Derived Tumor Necrosis Factor‐α Converting Enzyme Inhibitors

A three-dimensional quantitative structure-activity relationship study was performed on a series of beta-aminohydroxamic acid-derived tumor necrosis factor-alpha converting enzyme inhibitors employing comparative molecular field analysis and comparative molecular similarity indices analysis techniques to investigate the structural requirements for the inhibitors, and derive a predictive model that could be used for the design of novel tumor necrosis factor-alpha converting enzyme inhibitors. log P was used as an additional descriptor in the comparative molecular field analysis analysis to study the effects of lipophilic parameters on activity. Inclusion of log P did not improve the models significantly. The statistically significant model was established with 45 molecules, which were validated by a test set of 11 compounds. Ligand molecular superimposition on the template structure was performed by the atom-/shape-based root mean square fit and database alignment methods. Docked conformer based alignment (V) yielded the best predictive comparative molecular field analysis model = 0.673, = 0.860, F-value = 86.073, predictive r (2) = 0.642, with two components, standard error of prediction = 0.394 and standard error of estimates = 0.243 while the comparative molecular similarity indices analysis model yielded = 0.635, = 0.858, F-value = 84.451, predictive r (2) = 0.441 with three components, standard error of prediction = 0.393 and standard error of estimates = 0.245. The contour maps obtained from three-dimensional quantitative structure-activity relationship studies were appraised for activity trends for the molecules analyzed. The comparative molecular field analysis models exhibited good external predictivity as compared with that of comparative molecular similarity indices analysis models. The model generated through comparative molecular field analysis was validated with the IK-682. The data generated from this study may guide our efforts in designing and predicting the tumor necrosis factor-alpha converting enzyme inhibitory activity of novel molecules.

P38 MAPK Inhibition Prevents TACE-Mediated Receptor Shedding and Improves the Hemostatic Function of Stored Platelets.

Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding from platelet stored for 18 hours at 37ºC or 22ºC. Using pharmacological inhibitors, we show that TACE -dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during the storage of mouse platelets also markedly improved their posttransfusion recovery and hemostatic function in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved their adhesion to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed after platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

The Histone Deacetylase-8 (HDAC8) Selective Inhibitor PCI-34051 Decreases Interleukin-1 Beta Secretion in Vitro and Reduces Inflammation in Vivo

Inhibitors of histone deacetylases (HDACs) which are currently in clinical testing for treating various cancers typically inhibit multiple isoforms of the 11-member HDAC family. We have developed an isoform-selective HDAC inhibitor, PCI-34051, that inhibits HDAC8 with a Ki of 10 nM and greater than 200-fold selectivity over other HDAC isoforms (Balasubramanian et al. (2008) Leukemia,22:1026–34). We have shown that PCI-34051 selectively induced apoptosis in cell lines derived from T-cell lymphomas and leukemias, but not in other tumor or normal cell types. Here we show that it potently inhibits the secretion of the pro-inflammatory cytokine interleukin-1 beta (IL-1b) in lipopolysaccharide (LPS)-stimulated peripheral mononuclear blood cells (PBMC) and isolated monocytes. PCI-34051 inhibited IL-1b secretion (by 80% compared to control) from LPS-stimulated human PBMC with an IC50 of 0.6 uM, which is much lower than the growth inhibitory concentrations of 2.4–4 uM required in T-cell lymphomas. We found that PCI-34051 also inhibited the secretion of interleukin-18 (IL-18) to a similar extent as IL-1b, but secretion of other pro-inflammatory cytokines, including MIP-1b, MCP-1, TNFa, and IL-6, was inhibited to a smaller extent. Interestingly, IL-18, like IL-1b, is synthesized without a signal peptide, and also utilizes the same non-classical endosomal secretory pathway as IL-1b including cleavage of the pro-form by caspase-1. Thus, we theorized that the modulatory effect of PCI-34051 is likely to involve modulation of the post-translational secretory process. In accordance, we found that the IL-1b mRNA levels were reduced by only 20% compared to control, but the intracellular protein levels of the pro-form was increased by >50% in primary monocytes after treatment with PCI-34051, indicating that the mechanism was due to inhibition of the processing from the pro- to the mature form of the cytokine. We showed that this was not due to a direct inhibition of caspase-1 or TACE (TNF-alpha converting enzyme), but is likely due to an as-yet unidentified substrate of HDAC8. In vivo, PCI-34051 inhibited ear swelling induced by oxazolone in a model of contact hypersensitivity in mice, and we showed that this was accompanied by a reduction in IL-1b at both the protein and mRNA levels. Based on this result, we examined the effect of PCI-34051 on IL-1b secretion in human keratinocytes, as well as in PBMC from psoriasis patients, and found that it could reduce IL-1b secretion in both. We found that PCI-34051 decreased IL-1b by 60% in LPS-stimulated PBMC from rheumatoid arthritis (RA) patients, but the pan-HDAC inhibitors which were only weakly inhibitory to HDAC8 did not have this effect, indicating a specific role for HDAC8 in the secretory process. Finally, we found that in unstimulated PBMC from RA patients that had basal production of IL-1b that this could be decreased by 90% by treatment with PCI-34051. Taken together, these findings indicate that PCI-34051 is an active drug that could be useful for the treatment of T-cell lymphoma as well as for autoinflammatory diseases such as RA and psoriasis.

Tissue Inhibitor of Metalloproteinase 3 Deficiency Causes Hepatic Steatosis and Adipose Tissue Inflammation in Mice

Obesity-driven, low-grade inflammation affects systemic metabolic function and can lead to insulin resistance, hepatic steatosis, and atherosclerosis. Decreased expression of tissue inhibitor of metalloproteinase 3 (Timp3) is a catalyst for insulin resistance and inflammation. Timp3 is a natural inhibitor of matrix metalloproteinases, tumor necrosis factor-alpha-converting enzyme (TACE), and vascular endothelial growth factor receptor 2, and therefore could affect signaling processes involved in inflammation and angiogenesis. We assessed the effects of Timp3 on inflammation, tissue remodeling, and intermediary metabolism in mice, under conditions of environmental stress (high-fat diet), genetic predisposition to insulin resistance (insulin receptor [Insr] haploinsufficiency), and varying levels of inflammation (Timp3 or Tace deficiencies). Metabolic tests, immunohistochemistry, real-time polymerase chain reaction, and immunoblotting were used to compare data from wild-type, Insr(+/-), Timp3(-/-), Insr(+/-)Timp3(-/-), and Insr(+/-)Tace(+/-) mice placed on high-fat diets for 10 weeks. Insr(+/-)Timp3(-/-) mice showed a higher degree of adipose and hepatic inflammation compared with wild-type, Insr(+/-), Timp3(-/-), and Insr(+/-)Tace(+/-) mice. In particular, the Insr(+/-)Timp3(-/-) mice developed macrovesicular steatosis and features of severe nonalcoholic fatty liver disease, including lobular and periportal inflammation, hepatocellular ballooning, and perisinusoidal fibrosis. These were associated with increased expression of inflammatory and steatosis markers, including suppressor of cytokine signaling 3 and stearoyl CoA desaturase 1, in both liver and adipose tissue. Interestingly, Insr(+/-)Tace(+/-) mice had a nearly opposite phenotype. Timp3, possibly through its regulation of TACE, appears to have a role in the pathogenesis of fatty liver disease associated with obesity.

Metalloproteinase- and γ-Secretase-mediated Cleavage of Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/gamma-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system.

Upregulation of tumor necrosis factor receptor 1 and TNF-α converting enzyme during corneal wound healing

To elucidate tumor necrosis factor (TNF) receptor-1 (TNFR1) and TNF-alpha converting enzyme (TACE) involvement during corneal wound healing. The corneas of BALB/c mice cornea were scarred by alkali burns using filter paper dipped in 1N NaOH solution. TACE and TNFR1 expression in the alkali-burned corneas were evaluated by immunohistochemistry (total, 16 eyes). Using cultured fibroblasts (human) and macrophages (mice), we evaluated the release of soluble TNF-alpha (sTNF-alpha) and soluble TNFR1 (sTNFR1) in the supernatant by enzyme-linked immunosorbent assay after stimulating TACE activity with phorbol myristate acetate (PMA). In alkali-burned corneas, both TACE and TNFR1 expression were observed in the stromal cells after the acute phase of wound healing response. In macrophage-cultured supernatant, both sTNF-alpha and sTNFR1 release were promoted by PMA stimulation. On the other hand, only sTNFR1 released by PMA stimulation was observed in fibroblast-cultured supernatant. TACE and TNFR1 were expressed mainly after the acute phase of corneal wound healing. The TACE-dependent extracellular release of sTNFR1 was recognized in cultured fibroblasts and macrophages.

Neuronal RA175/SynCAM1 isoforms are processed by tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17-like proteases

RA175/SynCAM1, a member of immunoglobulin superfamily 4 (Igsf4; recently named Cadm1), is a cell adhesion molecule involved in the formation of a functional synapse. Little is known about the modulation of RA175/SynCAM1-mediated synaptic formation and plasticity. Neurons express two major isoforms containing exons 7-8a-8b-9 and exons 7-8b-9. We found that these isoforms were processed within an 11-amino acid sequence, encoded by exon 8b, near the transmembrane domain. TNF-alpha protease inhibitor-1 (TAPI-1) blocked the processing of RA175/SynCAM1 (exons 7-8a-8b-9). Furthermore, TAPI-1 increased the number of synaptophysin and RA175/SynCAM1 colocalization on the dendrites of neurons. Non-cleaved RA175/SynCAM1 was located at the synapse and membrane-bound, cleaved fragments were detected at the non-synaptic region of dendrites. These results suggest that tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17-like proteases play a role in synaptic formation to generate specific neuronal connections by processing the excess amount of RA175/SynCAM1 located in the non-synaptic region.

Polymorphisms of the tumor necrosis factor-alpha (TNF) and the TNF-alpha converting enzyme (TACE/ADAM17) genes in relation to cardiovascular mortality: the AtheroGene study

Tumor necrosis factor (TNF) is a major cytokine involved in inflammatory reaction and a mortality predictor in patients with coronary artery disease (CAD). Plasma levels of soluble TNF (sTNF) depend on the rate of its synthesis but also on its shedding from cell surface, a mechanism mainly regulated by the TNF alpha converting enzyme (TACE or ADAM17). We investigated the relationship between ADAM17 and TNF polymorphisms, circulating levels of shed ADAM17 substrates (sTNF, sTNFR1 and sTNFR2), and cardiovascular risk in a prospective cohort of CAD patients. Five tag single-nucleotide polymorphisms (SNPs) of the ADAM17 gene as well as four previously described TNF SNPs were genotyped in the Atherogene Study composed of 1,400 CAD patients among which 136 died from a cardiovascular (CV) cause. sTNF, sTNFR1, and sTNFR2 concentrations were all significantly elevated in patients with future CV death, independently of other clinical/biological variables. While none of the studied TNF SNPs was associated with sTNF, sTNFR1, nor sTNFR2 levels, the ADAM17 -154A allele was found associated with a 14% increase of sTNF levels as compared to the -154C allele (p = 0.0066). Moreover, individuals carrying the 747Leu allele displayed a borderline increased risk of future cardiovascular death [odds ratio, 2.06 (1.05-4.04), p = 0.03]. These results suggest a role of ADAM17 in the regulation of sTNF plasma levels and identifies ADAM17 gene as a candidate for CAD. Tumor necrosis factor (TNF) is a major cytokine involved in inflammatory reaction and a mortality predictor in patients with coronary artery disease (CAD). We have studied the association of ADAM17 and TNF polymorphisms with circulating levels of shed ADAM17 substrates (sTNF, sTNFR1 and sTNFR2) and with cardiovascular risk in a large population of individuals with CAD (Atherogene Study, n = 1,400). Two newly identified polymorphisms, obtained by a systematic sequencing of the ADAM17 gene, C-154A and Ser747leu, slightly influence respectively sTNF plasma levels and the risk of cardiovascular death.

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

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

Kuz and TACE can activate Notch independent of ligand.

A central mechanism in activation of the Notch signaling pathway is cleavage of the Notch receptor by ADAM metalloproteases. ADAMs also cleave Delta, the ligand for Notch, thereby downregulating Notch signals. Two ADAMs, Kuzbanian (Kuz) and TNF-alpha converting enzyme (TACE), are known to process both Delta and Notch, yet the role of these cleavages in signal propagation has remained controversial. Using an in vitro model, we show that Kuz regulates Notch signaling primarily by activating the receptor and has little overall effect on signaling via disabling Delta. We confirm that Kuz-dependent activation of Notch requires stimulation of Notch by Delta. However, over-expression of Kuz gives ligand-independent Notch activation. In contrast, TACE, which is elevated in expression in the developing Drosophila nervous system, can efficiently activate Notch in a ligand-independent manner. Altogether, these data demonstrate the potential for Kuz and TACE to participate in context- and mechanism-specific modes of Notch activation.

Expression of Tumor Necrosis Factor α Converting Enzyme in Endocrine Cancers

Tumor necrosis factor alpha converting enzyme (TACE) mediates shedding of human epidermal growth factor receptor-4 (HER4). Recent data suggest that released HER4 intracellular domain (4ICD) induces apoptosis in breast cancer. TACE expression, as measured by immunohistochemical analysis, was observed in 183 of 383 breast carcinomas, 39 of 217 ovarian carcinomas, and 16 of 24 and 17 of 24 hormonesensitive and hormone-insensitive prostate carcinomas, respectively. HER4 expression was detected in breast carcinomas by using 2 antibodies recognizing an extracellular or intracellular epitope. TACE expression was predominantly seen in tumors with high levels of 4ICD and membranous HER4. Apoptotic activity was measured by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and cleaved caspase-3 staining in breast carcinomas. There was no significant association between cleaved caspase-3 or TUNEL positivity and 4ICD, whereas TUNEL positivity was seen predominantly in tumors with high levels of internalized HER4. The data presented herein show TACE expression in endocrine cancers and further support a role for TACE in breast cancer apoptosis.

Polymorphisms in the Tumor Necrosis Factor/Lipopolysaccharides Pathway in Crohn Disease in the Jewish Ashkenazi Population

Tumor necrosis factor (TNF)-alpha plays a role in the inflammatory process in Crohn disease, a disease with an apparent polygenic basis. We investigated whether polymorphisms in multiple genes involved in the lipopolysaccharide-TNF inflammatory pathway are independently associated with Crohn disease in the Jewish Ashkenazi population. Polymorphisms in CD14, Toll-like receptor 4 (TLR4), and TNF-alpha were studied. In addition, we investigated polymorphisms in the TNF-alpha converting enzyme (TACE) gene, which to date has not been studied for an association with Crohn disease. To examine whether TLR4 Asp299Gly, CD14-260C/T, TNF-1031T/C, TNF-863C/A, TNF-857C/T, TACE-172C/T, and TACE-154C/A polymorphisms are associated with Crohn disease in the Ashkenazi Jewish population, we analyzed families with at least 1 child with Crohn disease for association with these mutations using a family-based association test (transmission disequilibrium test) for analysis. The allelic frequency in the patient population of TLR4 G allele was 8.0%, CD14 T allele was 51.3%, TNF-1031C was 18.8%, TNF-863A was 14.2%, TNF-857T was 25.2%, TACE172T was 20.7%, and TACE154A was 24.5%. The transmission disequilibrium test transmitted:untransmitted (T:U) result for TLR4G was T:U = 32:20, for CD14T was T:U = 103:88, for TNF-1031C was T:U = 48:56, for TNF-863A was T:U = 39:42, for TNF-857T was T:U = 63:62, for TACE-172C/T was T:U = 48:59, and for TACE-154C/A was T:U = 52:55. No statistically significant associations were observed. The transmission disequilibrium test did not demonstrate preferential transmission of these variants in Jewish Ashkenazi patients with Crohn disease. These results suggest that these polymorphisms in the TNF/lipopolysaccharide pathway play little or no role in susceptibility to Crohn disease in the Jewish Ashkenazi population.

Interleukin‐10 regulates TNF‐α−converting enzyme (TACE/ADAM‐17) involving a TIMP‐3 dependent and independent mechanism

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.

Genetics of humoral and cytokine activation in heart failure and its importance for risk stratification of patients

The study objective is to prove an association among plasma concentration of big endothelin and endothelin-1, other clinical parameters and two frequent polymorphisms – G8002A and − 3A/− 4A – in the endothelin-1 (EDN-1) coding gene (6p21–23), and among plasma concentration of TNF alpha and gene polymorphisms TNF alpha − 308 A/G, − 238 A/G, TNF beta Ncol and 3′TACE (tumour necrosis factor alpha converting enzyme) in patients with chronic heart failure (CHF). The second objective is to find an association between polymorphisms G8002A and − 3A/4A EDN-1 with diabetes mellitus (DM), peripheral artery disease (PAD) and myocardial infarction (MI) in patients with chronic heart failure (CHF). The study population included 266 patients with symptomatic CHF and proven dysfunction of the left ventricle (LV). Genotyping and plasma concentrations of humoral substances were examined in 224 patients with ejection fraction (EF) below 40%. No associations between plasma concentrations of endothelin-1 and big endothelin and polymorphisms G8002A ( p = 0.87, p = 0.81) and − 3A/− 4A ( p = 0.871, p = 0.749) in the gene coding endothelin-1 were found. No associations were observed between plasma concentration of TNF alpha and genotypes in four polymorphisms in TNF alpha, beta and TACE genes. A significant correlation was seen between plasma concentration of big endothelin and pulmonary congestion. Patients with ischemic heart disease (IHD) and previous MI showed a difference in the distribution of genotype G8002A for endothelin-1: allele G 0.718 and A 0.282 vs those without MI: allele G 0.882 and A 0.118, ( p < 0.05). Patients with IHD and DM had allele G in 0.67 and A 0.33, while those without DM had allele G in 0.790 and A in 0.209 ( p < 0.03). Patients with IHD and concomitant PAD had allele G in 0.718 and A in 0.282 vs those without PAD allele G in 0.882 and A in 0.118 ( p < 0.0004). Patients with dilative cardiomyopathy (DCMP) showed no differences in genotype G8002A and presence of DM or PAD. It might be speculated that in the case of endothelin-1 and TNF alpha in CHF the genetic determination is not important, and plasma concentrations are influenced more by the disease severity. Ischemics with previous MI, concomitant DM or PAD showed more frequently allele A and less often allele G than those without these diseases. A genotype with allele A is associated with higher risk of concomitant diseases.