Tumor Necrosis Factor-α Converting Enzyme Research Articles

A Role for cGMP in Inducible Nitric-oxide Synthase (iNOS)-induced Tumor Necrosis Factor (TNF) α-converting Enzyme (TACE/ADAM17) Activation, Translocation, and TNF Receptor 1 (TNFR1) Shedding in Hepatocytes

We and others have previously shown that the inducible nitric-oxide synthase (iNOS) and nitric oxide (NO) are hepatoprotective in a number of circumstances, including endotoxemia. In vitro, hepatocytes are protected from tumor necrosis factor (TNF) α-induced apoptosis via cGMP-dependent and cGMP-independent mechanisms. We have shown that the cGMP-dependent protective mechanisms involve the inhibition of death-inducing signaling complex formation. We show here that LPS-induced iNOS expression leads to rapid TNF receptor shedding from the surface of hepatocytes via NO/cGMP/protein kinase G-dependent activation and surface translocation of TNFα-converting enzyme (TACE/ADAM17). The activation of TACE is associated with the up-regulation of iRhom2 as well as the interaction and phosphorylation of TACE and iRhom2, which are also NO/cGMP/protein kinase G-dependent. These findings suggest that one mechanism of iNOS/NO-mediated protection of hepatocytes involves the rapid shedding of TNF receptor 1 to limit TNFα signaling.

Abstract 381: Upregulation of Tumor Necrosis Factor α-converting Enzyme (TACE) Protein Expression in db/db Mice is Reversed by Rosiglitazone

TACE, also known as ADAM17, is involved in the ectodomain shedding of several membrane bound proteins. Angiotensin converting enzyme 2 (ACE2), homologue of angiotensin converting enzyme (ACE), is known to be renoprotective by increasing the degradation of vasoactive peptide Angiotensin II (Ang II) to vasodilator peptide Ang - (1-7). It has been shown before that TACE mediates regulated ectodomain shedding of ACE2. PPARγ agonist, rosiglitazone, is known to impart renoprotection by attenuating albuminuria. However, the exact mechanism of renoprotection is not clear. Our previous results and others showed that renal ACE2 protein expression is increased during early stages of diabetes in db/db mice. We also demonstrated increased urinary ACE2 excretion in db/db mice. The goal of this study is to test the hypothesis that TACE is upregulated in db/db diabetic mice and treatment with rosiglitazone imparts renoprotection by attenuating the shedding of renal ACE2 via downregulating TACE protein expression. Male 6 week db/db mice were fed rosiglitazone (20mg/kg/day) for 10 weeks. Metabolic and urinary parameters were monitored weekly. Kidney lysate and urine was used to perform western blot and ACE2 activity (pmols/h/μg protein) respectively. db/db mice demonstrated glucose intolerance, hyperglycemia (348.2±29.9 mg/dL), and albuminuria (0.3±0.05 mg/day) at a very early age. Chronic rosiglitazone treatment normalized blood glucose levels (142.0±12.2 mg/dL), improved glucose tolerance and decreased albuminuria (0.17±0.03 mg/day) in treated db/db mice. Western blot showed increased renal TACE protein expression of db/db mice compared to controls (p<0.05). Treatment with rosiglitazone significantly decreased renal TACE protein expression in treated db/db mice compared to untreated mice (p<0.05). In addition, db/db mice demonstrated a 7 fold increase in urinary ACE2 activity (15.1±4.5) compared to controls (1.74±0.9). Treatment with rosiglitazone significantly attenuated and normalized ACE2 activity in treated db/db mice (0.1±0.06). In conclusion, TACE is upregulated in db/db mice and normalizing hyperglycemia with rosiglitazone could impart renoprotection by decreasing renal TACE protein expression and shedding of renal ACE2.

P12 The complex effects of a slow-releasing hydrogen sulifde donor (GYY4137) in a model of inflammatory arthritis and in primary human cartilage cells

Synthesis of H 2 S is markedly increased in knee joint synovial fluid (SF) joints from patients with inflammatory joint disease (IJD) compared to paired plasma and SF from age-matched disease controls. SF levels also negatively correlated with disease activity, SF total white cell and neutrophil count suggesting a possible anti-inflammatory role for induced H 2 S synthesis in the joint. These observations also highlight the pharmacological potential for H 2 S donor molecules in (IJD). We have assessed the effect of GYY4137 (a slow-releasing H 2 S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro . We also examined the effects of GYY4137 on human recombinant iNOS, COX-1, COX-2 and tumour necrosis factor- α converting enzyme (TACE). We further examined the effect of GYY4137 in a complete Freund’s adjuvant (CFA) model of arthritis in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite ( NO 2 - ) , PGE 2 , TNF- α and IL-6 from HFLS and HAC, reduced the expression and catalytic activity of iNOS, COX-2, and reduced LPS-induced NF-kB activation in vitro . Using recombinant human enzymes, GYY4137 inhibited the activity of COX-1, COX-2, iNOS and TACE. In the CFA treated mouse, adjuvant induced a two fold increase in synovial H 2 S concentrations. GYY4137 (50 mg/kg, i.p.) injected 1 h prior to CFA increased knee joint swelling whilst an anti-inflammatory effect, as evidenced by reduced synovial fluid myeloperoxidase (MPO) and N -acetyl- β - d -glucosaminidase (NAG) activity and decreased TNF- α , IL-1 β , IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 h after CFA. GYY4137 was also anti-inflammatory when given 18 h after CFA. The effects of H 2 S on the inflammatory process are highly complex. Not only is the anti-inflammatory effect determined by the donor used (’fast’ vs ’slow’ release donors) but also determined in vivo by the timing of the donor administration. Thus, whilst GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro , its effect on arthritis in vivo depended on the timing of administration.

Brief Report: Requirement of TACE/ADAM17 for Hair Follicle Bulge Niche Establishment

Hair follicles (HFs) are equipped with stem cell niches that allow regeneration. Tumor necrosis factor-α converting enzyme (TACE), also known as A disintegrin and metalloproteinase 17, is a proteolytic enzyme that regulates a variety of cell surface molecules including TNF-α, via ectodomain shedding. We found TACE expression on mouse HFs and conditionally depleted it in cells that expressed sex-determining region Y-related high-mobility-group box 9 (SOX9) transcription factor, an HF stem cell transcription factor (Tace(flox/flox) -Sox9-Cre, hereafter, "Tace/Sox9"). Tace/Sox9 mice were born with brittle hair with prolonged anagen phase. They underwent diffuse, progressive, and ultimately whole-body hair loss by 20 weeks old. Tace/Sox9 HFs lacked CD34(+) bulge cells as demonstrated via immunofluorescence microscopy and flow cytometry. Real-time PCR revealed downregulation of transcription factors Sox9, Lhx2, and Gata3 and upregulation of Lef1. In vitro colony-forming capacity was abolished in Tace/Sox9 keratinocytes, and HFs exhibited increased proliferation in situ, collectively demonstrating that Tace/Sox9 mice failed to establish the bulge niche and to maintain "stemness" of HF stem cells. Epidermal growth factor receptor (EGFR) signaling was impaired in Tace/Sox9 keratinocytes, and mice depleted of Egfr in SOX9-expressing tissues exhibited hair phenotype nearly identical to Tace/Sox9 mice, demonstrating EGFR signaling as a pathway downstream of TACE in HF homeostasis. This study provides mechanistic implication for human TACE-deficiency and for hair abnormality caused by EGFR inhibitors.

Modulation of Redox Balance Leaves Murine Diabetogenic TH1 T Cells “LAG-3-ing” Behind

Preventing activation of diabetogenic T cells is critical for delaying type 1 diabetes onset. The inhibitory molecule lymphocyte activation gene 3 (LAG-3) and metalloprotease tumor necrosis factor-α converting enzyme (TACE) work together to regulate TH1 responses. The aim of this study was to determine if regulating redox using a catalytic antioxidant (CA) could modulate TACE-mediated LAG-3 shedding to impede diabetogenic T-cell activation and progression to disease. A combination of in vitro experiments and in vivo analyses using NOD mouse strains was conducted to test the effect of redox modulation on LAG-3 shedding, TACE enzymatic function, and disease onset. Systemic treatment of NOD mice significantly delayed type 1 diabetes onset. Disease prevention correlated with decreased activation, proliferation, and effector function of diabetogenic T cells; reduced insulin-specific T-cell frequency; and enhanced LAG-3+ cells. Redox modulation also affected TACE activation, diminishing LAG-3 cleavage. Furthermore, disease progression was monitored by measuring serum soluble LAG-3, which decreased in CA-treated mice. Therefore, affecting redox balance by CA treatment reduces the activation of diabetogenic T cells and impedes type 1 diabetes onset via decreasing T-cell effector function and LAG-3 cleavage. Moreover, soluble LAG-3 can serve as an early T-cell–specific biomarker for type 1 diabetes onset and immunomodulation.

Discovery of Selective Small Molecular TACE Inhibitors for the Treatment of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory disease that afflicts 1-2% of the world population, characterized by an immune mediated inflammatory synovitis that leads to joint destruction, functional impairment, and reduced quality of life. The treatment goals of RA should be longterm substantial relief of pain, arrested joint inflammation and damage, and improved function. Current treatment can be divided into four classes, namely general analgesics and non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease modifying anti-rheumatic drugs (DMARDs) and biological agents (tumor-necrosis factor modifiers). However, gastrointestinal (GI) side effects of NSAIDs cannot be neglected, direct joint injections of glucocorticoids cannot be injected more than once every 3 months, synthetic DMARDs is far from optimal and only minority of patients achieved longterm remission, the biologics are very expensive to manufacture, need to be injected, and can cause allergic reactions. An alternative and good approach to the treatment of this disease is to lower the levels of tumour necrosis factor-α (TNF-α) in RA, which can be achieved by selectively inhibiting the tumour necrosis factor-α converting enzyme (TACE) that generate these cytokines using cheaper small molecules. This review focuses on the current status of selective small molecule inhibitors of TACE, with respect to lead compound search, inhibitors design approach, structure-activity relationship (SAR) and pharmacological studies in animals and humans. Through these methods, new hope is emerging for the treatment of RA through selective inhibition of TACE.

Selective cleavage of ErbB4 by G‐protein‐coupled Gonadotropin‐Releasing Hormone Receptor in Cultured Hypothalamic Neurons

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons). GnRH neurons have a GnRH receptor belonging to the G-protein-coupled receptors. The stimulation of this receptor activates extracellular signal-regulated kinase (ERK). In the present study, we found that epidermal growth factor receptor (EGFR) and ErbB4 were expressed in immortalized GnRH neurons (GT1-7 cells). AG1478, a relatively specific inhibitor of the ErbB family, and small interfering RNA (siRNA) for ErbB4 inhibited the GnRH-induced activation of ERK in GT1-7 cells, suggesting that EGFR and ErbB4 were necessary for the activation. In addition, GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment. After treatment of the cells with 50 nM GnRH for 5 min, about 80% of ErbB4 was cleaved. Biotinylation of cell surface proteins revealed that more than 70% of the cell surface ErbB4 was cleaved by GnRH treatment. A higher concentration and longer treatment were necessary for GnRH to induce ErbB4 cleavage than ERK activation. TAPI-2, an inhibitor of tumor necrosis factor-α-converting enzyme (TACE), and siRNA for TACE inhibited the cleavage of ErbB4, suggesting that TACE was involved. After ErbB4 cleavage, the activation of ERK by neuregulin 1 was almost completely inhibited. These results suggest that the down-regulation of ErbB4 expression is induced by G-protein-coupled receptor stimulation.

ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion

ADAM17, also known as tumor necrosis factor-α converting enzyme (TACE), is involved in proteolytic ectodomain shedding of cell surface molecules and cytokines. Although aberrant expression of ADAM17 has been shown in various malignancies, the function of ADAM17 in prostate cancer has not been clarified. In the present study, we sought to elucidate whether ADAM17 contributes to prostate cancer cell invasion, as well as the mechanism involved in the process. The expression pattern of ADAM17 was investigated in human prostate cancer cells. The results showed that ADAM17 expression levels are correlated with the invasive ability of androgen-independent prostate cancer cell lines. Further, ADAM17 was overexpressed in cells showing high invasion characteristics, activation of the EGFR-MEK-ERK pathway, up-regulation of MMP-2, MMP-9, and an increased TGF-α release into the supernatant. However, AG1478, PD98059 and antibody against TGF-α deactivating the EGFR-MEK-ERK signaling pathway, abolished up-regulation of MMP-2, MMP-9 and prevented cell invasion. In addition, cells with knockdown of ADAM17 by siRNA exhibited low invasive ability, deactivated EGFR-MEK-ERK signaling pathway, reduced TGF-α released and down-regulation of MMP-2, MMP-9. However, these effects could be reversed by simultaneous addition of TGF-α. These data demonstrated that ADAM17 contributes to androgen-independent prostate cancer cell invasion by shedding of EGFR ligand TGF-α, which subsequently activates the EGFR-MEK-ERK signaling pathway, leading finally to overexpression of MMP-2 and MMP-9. This study suggests that the ADAM17 expression level may be a new predictive biomarker of invasion and metastasis of prostate cancer, and ADAM17 could provide a target for treating metastatic PCa.

Reactive Oxygen Species and p38 Mitogen-activated Protein Kinase Mediate Tumor Necrosis Factor α-Converting Enzyme (TACE/ADAM-17) Activation in Primary Human Monocytes

Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.

Tumor Necrosis Factor α-Converting Enzyme Inhibition Reverses Hepatic Steatosis and Improves Insulin Sensitivity Markers and Surgical Outcome in Mice

BackgroundHepatic steatosis is an established risk factor for complications following major hepatic resection. Pharmacological options to reverse steatosis prior to surgery, however, are lacking. We hypothesized that treatment with the pharmacologic tumor necrosis factor-α converting enzyme (TACE)-inhibitor Marimastat would reverse established steatosis, leading to improved outcome following hepatectomy.Methodology/Principal FindingsC57BL/6 male mice were fed a high fat diet for 9 weeks to establish obesity, hepatic steatosis and insulin resistance, and were administered either Marimastat or vehicle for an additional 2 or 4 weeks. Leptin deficient, hyperinsulinemic ob/ob mice were treated with Marimastat for 4 weeks. Hepatic steatosis was quantified by magnetic resonance spectroscopy and confirmed by histology. After two weeks, Marimastat-treated animals significantly improved surrogate markers for insulin sensitivity and liver histology, and experienced a 66% decrease in steatosis (P = 0.010). These findings were confirmed in ob/ob mice. Transcripts related to fatty acid synthesis were significantly downregulated in Marimastat-treated animals. Following pre-treatment with Marimastat or vehicle for two weeks, high fat fed C57BL/6 mice were subjected to two-thirds hepatectomy. Post-operative liver injury as quantified by serum aspartate aminotransferase levels and alanine aminotransferase levels was significantly decreased by 57% (P = 0.020) and 44% (P = 0.032) respectively, compared to controls.Conclusion/SignificanceTreatment with the TACE-inhibitor Marimastat improved surrogate markers for insulin sensitivity and reversed steatosis in mouse models of diet-induced obesity and leptin deficiency, thereby attenuating post-operative injury following hepatectomy. This may suggest a potential therapeutic role in patients with fatty liver disease; especially those who need to undergo hepatic resection.

Placental Growth Factor Regulates Cardiac Inflammation Through the Tissue Inhibitor of Metalloproteinases-3/Tumor Necrosis Factor-α–Converting Enzyme Axis

Background— Heart failure is one of the leading causes of mortality and is primarily the final stage of several overload cardiomyopathies, preceded by an early adaptive hypertrophic response and characterized by coordinated cardiomyocyte growth, angiogenesis, and inflammation. Therefore, growth factors and cytokines have to be critically regulated during cardiac response to transverse aortic constriction. Interestingly, the dual properties of placental growth factor as an angiogenic factor and cytokine make it a candidate to participate in cardiac remodeling in response to hemodynamic overload. Methods and Results— After transverse aortic constriction, placental growth factor knockout mice displayed a dysregulation of cardiac remodeling, negatively affecting muscle growth. Molecular insights underscored that this effect was ascribable mainly to a failure in the establishment of adequate inflammatory response owing to an impaired activity of tumor necrosis factor-α–converting enzyme. Interestingly, after transverse aortic constriction, placental growth factor knockout mice had strongly increased levels of tissue inhibitor of metalloproteinases-3, the main natural TACE inhibitor, thus indicating an unbalance of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α–converting enzyme axis. Strikingly, when we used an in vivo RNA interference approach to reduce tissue inhibitor of metalloproteinases-3 levels in placental growth factor knockout mice during transverse aortic constriction, we obtained a complete phenotype rescue of early dilated cardiomyopathy. Conclusions— Our results demonstrate that placental growth factor finely tunes a balanced regulation of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-α–converting enzyme axis and the consequent TNF-α activation in response to transverse aortic constriction, thus allowing the establishment of an inflammatory response necessary for adaptive cardiac remodeling.

LPA-producing enzyme PA-PLA1α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.

Tumor necrosis factor-α converting enzyme (TACE) increases RANKL expression in osteoblasts and serves as a potential biomarker of periodontitis

Periodontitis is a very prevalent disease. Therefore, biomarkers for the early and standard diagnoses of periodontitis are urgently needed. TACE is a membrane-bound metalloprotease. Although a recent study suggested that TACE levels in GCF are elevated during periodontal disease, the levels of TACE in GCF at different stages of chronic periodontitis have not been determined. Here, we analyzed the protein levels of TACE in GCF from periodontal disease subjects and confirmed that the protein levels of TACE were higher in the moderate periodontitis groups. TACE is known to be a NF-κB ligand that stimulates RANKL secretion in osteoblasts. To understand the effects of TACE on RANKL and OPG in osteoblasts, we treated MG63 cells with TACE. We observed an increase in RANKL protein expression but a decrease in OPG protein expression. Our data suggest that TACE can induce RANKL expression and promote osteoclastogenesis, thus worsening the outcome of periodontitis.

Tumor necrosis factor-α converting enzyme is a key mediator of abdominal aortic aneurysm development

ObjectiveTumor necrosis factor (TNF)-α is known to be elevated in plasma and the aorta in abdominal aortic aneurysm (AAA) patients. We sought to clarify the role of TNF-α converting enzyme (Tace), which cleaves the transmembrane precursor of TNF-α, in AAA development. MethodsWe obtained aortic sample of AAA during surgical operation to assess the histological features and protein expression of human AAA. AAA was induced in mice with temporal systemic deletion of Tace by the inducible Mx-1 Cre transgene (TaceMx1) and in wild-type littermates (CON) by periaortic application of CaCl2 (AAA/TaceMx1, AAA/CON). ResultsTace expression was increased in human AAA samples as compared with normal aorta. Six weeks postoperatively, aortic diameter in AAA/TaceMx1 was decreased than in AAA/CON in association with attenuated TNF-α expression and extracellular matrix disruption. Increased activities of matrix metalloproteinase (MMP)-9 and MMP-2, numbers of Mac-2-positive macrophages, CD3-positive T lymphocytes and CD31-positive vessels in periaortic tissues, mRNA expression of CD68, monocyte chemotactic protein-1, TNF-α, vascular endothelial growth factor-A, p47 and glutathione peroxidases, and protein expression of phospho-c-Jun N-terminal kinase in AAA were all attenuated by Tace deletion. Protein expression of transforming growth factor (TGF)-β1 was upregulated by Tace deletion in sham-operated mice. TGF-β1 expression was further increased in AAA/TaceMx1. ConclusionsTace was overexpressed in the aortic wall in human and experimental AAA. Temporal systemic deletion of Tace prevented AAA development in association with attenuating inflammation, oxidative stress, neoangiogenesis and extracellular matrix disruption, suggesting a crucial role of Tace in AAA development.

TACE cleaves neogenin to desensitize cortical neurons to the repulsive guidance molecule

Neogenin is a receptor for netrins and proteins of the repulsive guidance molecule (RGM) family. It regulates several key developmental processes within the nervous system. The binding of RGMa to neogenin induces the inhibition of neurite outgrowth and the collapse of the growth cone of neurons. Here, we report that a disintegrin and metalloprotease (ADAM) transmembrane protein regulates the sensitivity of neurons to RGMa, by inducing the shedding of the ectodomain of neogenin. The extracellular domain of neogenin is directly associated with and cleaved off by the tumor necrosis factor-α converting enzyme (TACE), also called ADAM17. TACE is endogenously expressed in embryonic cortical neurons and regulates the cleavage of neogenin, and the inhibition of TACE in turn enhances RGMa-induced inhibition of neurite outgrowth and collapse of the growth cone. Conversely, exogenous expression of TACE abolishes the effect of RGMa. Therefore, TACE may play a role in modulating the RGM-induced repulsive behavior of neurons by regulating the expression of neogenin on the cell surface.

Angiotensin-Converting Enzyme 2 Ectodomain Shedding Cleavage-Site Identification: Determinants and Constraints

ADAM17, also known as tumor necrosis factor α-converting enzyme, is involved in the ectodomain shedding of many integral membrane proteins. We have previously reported that ADAM17 is able to mediate the cleavage secretion of the ectodomain of human angiotensin-converting enzyme 2 (ACE2), a functional receptor for the severe acute respiratory syndrome coronavirus. In this study, we demonstrate that purified recombinant human ADAM17 is able to cleave a 20-amino acid peptide mimetic corresponding to the extracellular juxtamembrane region of human ACE2 between Arg(708) and Ser(709). A series of peptide analogues were also synthesized, showing that glutamate subtitution at Arg(708) and/or Arg(710) attenuated the cleavage process, while alanine substitution at Arg(708) and/or Ser(709) did not inhibit peptide cleavage by recombinant ADAM17. Analysis of CD spectra showed a minimal difference in the secondary structure of the peptide analogues in the buffer system used for the ADAM17 cleavage assay. The observation of the shedding profiles of ACE2 mutants expressing CHO-K1 and CHO-P cells indicates that the Arg(708) → Glu(708) mutation and the Arg(708)Arg(710) → Glu(708)Glu(710) double mutation produced increases in the amount of ACE2 shed when stimulated by phorbol ester PMA. In summary, we have demonstrated that ADAM17 is able to cleave ACE2 peptide sequence analogues between Arg(708) and Ser(709). These findings also indicate that Arg(708) and Arg(710) play a role in site recognition in the regulation of ACE2 ectodomain shedding mediated by ADAM17.

TACE/TGF- /EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components

Airborne particulate matter (PM) may induce or exacerbate neutrophilic airway disease by triggering the release of inflammatory mediators, such as CXC chemokine ligand (CXCL)8, from the airway epithelium. It is still unclear which PM components are driving CXCL8 responses, as most candidates occur at low concentrations in the dusts. We therefore hypothesised that different PM constituents may contribute through common mechanisms to induce CXCL8. Human bronchial epithelial cells (BEAS-2B) were exposed to different PM components (Zn²⁺/Fe²⁺ salts, 1-nitropyrene, lipopolysaccharide and diesel exhaust/mineral particles). Gene expression patterns were detected by real-time PCR array. CXCL8 responses were measured by real-time PCR and ELISA. CXCL8 regulation was assessed with a broad inhibitor panel and neutralising antibodies. Epidermal growth factor receptor (EGFR) phosphorylation was examined by immunoprecipitation and Western blotting. Component-induced gene expression was mainly linked to nuclear factor-κB, Ca²⁺/protein kinase C, phospholipase C, low-density lipoprotein and mitogenic signalling. Many inhibitors attenuated CXCL8 release induced by all PM components, but to varying extents. However, EGFR inhibition strongly reduced CXCL8 release induced by all test compounds and selected compounds increased EGFR phosphorylation. Interference with transforming growth factor (TGF)-α or tumour necrosis factor-α-converting enzyme (TACE), which mediates TGF-α ectodomain shedding, also attenuated CXCL8 release. Different PM constituents induced CXCL8 partly through similar signalling pathways but the relative importance of the different pathways varied. However, TACE/TGF-α/EGFR signalling appears to be a convergent pathway regulating innate immune responses of airway epithelial cells upon exposure to multiple airborne pollutants.

CRP enhances soluble LOX-1 release from macrophages by activating TNF-α converting enzyme

Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the inflammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-α converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-α and further treated with CRP in the absence or presence of specific inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dose-dependent manner and that these effects were regulated by Fc γ receptor II (FcγRII)-mediated p47(phox) phosphorylation, reactive oxygen species (ROS) production, and TACE activation. CRP also enhanced sLOX-1 release from macrophages derived from peripheral blood mononuclear cells (PBMC) of patients with acute coronary syndrome (ACS). Pretreatment with antibody against FcγRII or with CD32 siRNA, p47(phox) siRNA, apocynin, N-acetylcysteine, tumor necrosis factor-α protease inhibitor 1 (TAPI-1) or TACE siRNA attenuated sLOX-1 release induced by CRP. CRP also elevated serum sLOX-1 levels in a rabbit model of atherosclerosis. Thus, CRP might stimulate sLOX-1 release, and the underlying mechanisms possibly involved FcγRII-mediated p47(phox) phosphorylation, ROS production, and TACE activation.

Abstract 448: Mechanism of action differences of transmembrane versus secretory tumor necrosis factor-α on tumor growth; role of tumor-associated myeloid cells

Abstract Tumor Necrosis Factor alpha (TNF-α) is a major inflammatory cytokine, produced by a wide variety of cell types including macrophages, T lymphocytes endothelial cells and tumor cells. TNF-α is expressed as a membrane-tethered protein (tmTNF-α), cleaved by tumor necrosis factor-α-converting enzyme (TACE) and subsequently released into extracellular space as a soluble TNF-α (sTNF-α). Recent studies from our laboratory and others have shown that low level expression of TNF-α by tumor cells substantially increases tumor growth, vascularity and decreases tumor necrosis, in part by increasing tumor content of a proangiogenic myeloid subtype (Li et al., Cancer Res. 2009, 1;69(1):338-48). To better understand the mechanism by which TNF-α modulates the myeloid phenotype, we generated murine Lewis lung carcinoma (LLC) and B16F10 melanoma tumor cells transduced with retrovirus expression vectors directing modest overexpression of a membrane tethered moiety, tmTNF-α. Heterotopic implantation of tmTNF-α expressing lines resulted in significantly decreased growth than control tumors in both tumor models. Our studies showed that this was not due to effects on the tumor cells themselves but likely due to effects on tumor stroma as the effect of tmTNF-α was lost in TNF-α receptor deficient mice. Further evaluation showed an absence of tumor associated myeloid cells within the tumor, which suggested apoptosis of migrated myeloid cells or inhibition of myeloid recruitment into the tumor environment. There is a significant gap in our knowledge on the mechanism of transmembrane isoform of TNF-α and its potential anti-tumorogenic role. In this study our goal is to identify the mechanism in which tmTNF-α promotes tumor regression and to understand how it modulates host bone marrow derived myeloid cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2011-448

Pharmacokinetic-Pharmacodynamic Modeling of Apratastat: A Population-Based Approach

Apratastat is an orally active, potent, and reversible dual inhibitor of tumor necrosis factor-α converting enzyme (TACE) and matrix metalloproteinases (MMPs). This study characterizes the pharmacodynamic (PD) effect of apratastat following oral administration on tumor necrosis factor-alpha (TNF-α) release. Data were obtained from 3 clinical studies carried out in healthy subjects. Apratastat was administered orally in these studies as single doses or multiple doses (twice daily). The inhibition of TNF-α release by apratastat was investigated in studies of in vitro, ex vivo, and in vivo. Inhibitory E(max) models were used to characterize the inhibition of TNF-α release in both in vitro and ex vivo studies. Apratastat inhibited TNF-α release with a population mean IC(50) of 144 ng/mL in vitro and of 81.7 ng/mL ex vivo, respectively. The relationship between TNF-α and apratastat plasma concentration in the endotoxin-challenged study in healthy subjects was well characterized by a mechanism-based PD population model with IC(50) of 126 ng/mL. Apratastat can potently inhibit the release of TNF-α in vitro, ex vivo, and in vivo. Even though the dosage provided adequate exposure to inhibit TNF-α release, apratastat was not efficacious in rheumatoid arthritis (RA). This inconsistency between TNF-α inhibition and the clinical response requires further investigation.