Interleukin-1β Converting Enzyme Research Articles

Characterization of ATP-gated P2X 7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1β

Mammalian interleukin-1β (IL-1β) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1β-converting enzyme (ICE) after the activation of P2X 7 receptor by extracellular ATP. The mechanism of IL-1β release in non-mammalian vertebrates is largely unknown, although most of the IL-1β gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X 7 receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X 7 receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X 7 receptors. We used this information to further investigate the mechanisms of IL-1β release induced by mammalian and fish P2X 7 receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1β remained unprocessed within the cell. However, activation of rat P2X 7 receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1β. In contrast, neither seabream nor zebrafish P2X 7 receptors induced the secretion of mammalian or fish IL-1β when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X 7 and the intracellular region of its rat counterpart did so. These findings indicate that P2X 7 receptor-mediated activation of ICE and release of IL-1β result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1β secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1β converting enzyme (ICE) inhibitors

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1β converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC 50 values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1β production in a whole cell assay (IC 50 < 500 nM).

Fas Signaling in Thyroid Carcinomas Is Diverted from Apoptosis to Proliferation

The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor kappaB (NF-kappaB) inhibitor IkappaB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-kappaB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase, NF-kappaB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

FAS Death Domain Deletions and Cellular FADD-like Interleukin 1β Converting Enzyme Inhibitory Protein (Long) Overexpression: Alternative Mechanisms for Deregulating the Extrinsic Apoptotic Pathway in Diffuse Large B-Cell Lymphoma Subtypes

Large B-cell lymphomas (LBCL) arise from normal antigen-exposed B cells at germinal center (GC) or post-GC stages of differentiation. Negative selection of normal low-affinity or self-reactive GC B-cells depends on CD95 (FAS)-mediated apoptosis. FAS mutations that result in deletion of the cytoplasmic death domain destabilize the trimeric receptor and inhibit FAS-mediated apoptosis. This apoptotic pathway is also inhibited when the nuclear factor kappaB (NFkappaB) target, cellular FADD-like interleukin 1beta converting enzyme inhibitory protein (cFLIP), interacts with the death-inducing signaling complex, assembled around the FAS death domain. Herein, we ask whether FAS death domain mutations and NFkappaB-mediated overexpression of cFLIP represent alternative mechanisms for deregulating the extrinsic apoptotic pathway in LBCL subtypes defined by gene expression profiling [oxidative phosphorylation, B-cell receptor/proliferation, and host response diffuse LBCLs and primary mediastinal LBCLs]. The FAS receptor was sequenced, FAS death domain mutations identified, and cFLIP expression assessed in a series of primary LBCLs with gene expression profiling-defined subtype designations and additional genetic analyses [t(14;18) and t(3;v)]. FAS death domain deletions were significantly more common in oxidative phosphorylation tumors, which also have more frequent t(14;18), implicating structural abnormalities of either the extrinsic or intrinsic pathway in this diffuse LBCL subtype. In marked contrast, host response tumors, which have up-regulation of multiple NFkappaB target genes and increased NFkappaB activity, express significantly higher levels of cFLIP(long). These data suggest that the gene expression profiling-defined LBCL subtypes have different mechanisms for deregulating FAS-mediated cell death and, more generally, that these tumor groups differ with respect to their underlying genetic abnormalities.

Enantioselective Total Synthesis of the Polyketide Natural Product (‐)‐EI‐1941‐2: A Novel Interleukin‐1β Converting Enzyme (ICE) Inhibitor.

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A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis

There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.

Reduction of Caspase-8 and -9 Cleavage Is Associated With Increased c-FLIP and Increased Binding of Apaf-1 and Hsp70 After Neonatal Hypoxic/Ischemic Injury in Mice Overexpressing Hsp70

Caspase-8 and caspase-9 are essential proteases of the extrinsic and intrinsic apoptotic pathways, respectively. We investigated whether neuroprotection associated with overexpression of heat-shock protein 70 (Hsp70), a natural cellular antiapoptotic protein, is mediated by caspase-8 and caspase-9 signaling in the neonatal mouse brain after hypoxia/ischemia (H/I) injury. Postnatal day 7 transgenic mice overexpressing rat Hsp70 (Hsp70 Tg) and their wild-type (Wt) littermates underwent unilateral common carotid artery ligation followed by 30 minutes of exposure to 8% O2. The expression of apoptotic proteins was quantified by Western blot analysis, and the specific interaction between Hsp70 and apoptotic protease activating factor 1 (Apaf-1) was determined by coimmunoprecipitation. Hsp70 overexpression reduced cytosolic translocation of cytochrome c without affecting the levels of Apaf-1 and pro-caspase-9 24 hours after H/I. The expression of these apoptotic proteins in the naïve neonatal brains was also not affected by Hsp70 overexpression. Reduced caspase-9 cleavage occurred in Hsp70 Tg mice compared with Wt littermates 24 hours after H/I and correlated with increased binding of Hsp70 and Apaf-1. Increased cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) expression and decreased caspase-8 cleavage were also observed in Hsp70 Tg compared with Wt mice 24 hours after H/I. Our results suggest that the extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of Hsp70 overexpression in neonatal H/I, specifically by upregulating FLIP and sequestering Apaf-1, leading to reduced cleavage of caspase-8 and caspase-9.

Caspase Family Proteases and Apoptosis

Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1beta-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain, and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

Synthesis and evaluation of novel 8,6-fused bicyclic peptidomimetic compounds as interleukin-1β converting enzyme inhibitors

Two novel 8,6-fused bicyclic peptidomimetic ring systems were synthesized utilizing olefin metathesis as the key reaction for the formation of the eight-membered ring. Both peptidomimetic scaffolds were further elaborated into potent ICE inhibitors, with numerous compounds exhibiting caspase-1 IC 50s less than 10 nM.

Enantioselective total synthesis of the polyketide natural product (−)-EI-1941-2: a novel interleukin-1β converting enzyme (ICE) inhibitor

An enantioselective total synthesis of the novel bioactive epoxyquinone natural product (−)-EI-1941-2, exhibiting promising ICE inhibiting activity, has been accomplished from a readily available chiron derived from the Diels–Alder adduct of cyclopentadiene and p-benzoquinone.

Synthesis and evaluation of tricyclic pyrrolopyrimidinones as dipeptide mimetics: Inhibition of interleukin-1β-converting enzyme

The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-1β-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhibitors were accomplished in 4–5 steps. In vitro biological evaluation of the tricyclic pyrrolopyrimidinones revealed fair to good ICE inhibitors, with the most active compound exhibiting an IC 50 of 14 nM in a caspase-1 enzyme binding assay.

Interleukin-18 is expressed in rat testis and may promote germ cell growth

Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1β converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.

Small molecular anti-cytokine agents

The recent successful introduction of the anti-cytokine biologicals Etanercept, Infliximab, Adalimumab, and Anakinra has stimulated the search for anti-cytokine small-molecules. A number of molecular targets have been identified for the development of such small molecular anti-cytokine agents. The focus of this review will be on those inhibitors of cytokine production, which target either p38 mitogen activated protein (MAP) kinase, TNF-alpha converting enzyme (TACE), or IL-1beta converting enzyme (ICE). P38 MAP kinase occupies a central role in the signaling network responsible for the upregulation of proinflammatory cytokines like interleukin 1beta (IL-1beta) and TNF-alpha, and regulates their biosynthesis at both the transcriptional and translational level. TACE and ICE are two proteases required for the processing of proTNF-alpha and proIL-1beta, respectively into their mature, proinflammatory form. Since the mid-1990s, a plethora of inhibitors of p38 MAP kinase, TACE, and ICE has been characterized in vitro, and individual representatives from all three inhibitor classes have in the meantime been advanced into clinical trials. This review will highlight the correlation between effective inhibition at the molecular target and cellular activity in functional assays of cytokine, particularly TNF-alpha and IL-1beta, production. Structure-activity relationships (SAR) will be discussed regarding activity at the respective enzyme target, but also with regard to properties required for efficient in vitro and in vivo cellular activity (e.g., oral availability, solubility, cell penetration, etc.).

Insulin-like growth factor-I decreased etoposide-induced apoptosis in glioma cells by increasing bcl-2 expression and decreasing CPP32 activity

Aims: In a variety of tumors, the susceptibility of the tumor cells to apoptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Gliomas are resistant to most chemotherapeutic agents, and its mechanism is not known in detail. In an attempt to understand the mechanism of chemo-resistance, we investigated the roles of insulin-like growth factor-I (IGF-I), IGF-I receptors (IGF-IR), and their relationship with the apoptotic response of two glioma cell lines to etoposide, a chemotherapeutic agent for malignant gliomas.Methods: Two human glioma cell lines, U-87MG and KNS-42, were used. Etoposide-induced cell growth inhibition was quantified using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), colorimetric assay. Hoechst 33258 staining, DNA fragmentation assay, and western blot were used for the evaluation of apoptosis. ApoAlertTM caspase assay was used for measuring the activity of caspase-3 (CPP32) and interleukin-1 β -converting enzyme (ICE) protease. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines.Results: Etoposide inhibited the growth of U-87MG and KNS-42 cells in a concentrationdependent manner. Etoposide increased the expression of wild-type p53, activated CPP32 (but not ICE) activity, and induced apoptosis in these cells. IGF-I prevented etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32. IGF-IR antisense enhanced the apoptotic effect of etoposide. Conclusions IGF-I decreased etoposideinduced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The antisense of IGF-IR increased etoposide-induced apoptosis. The antiapoptotic effect of IGF-I and IGF-IR might be related to the chemo-resistance of glioma to chemotherapeutic agents.

Structure Elucidation of EI‐1941‐1 and ‐2, Novel Interleukin‐1β Converting Enzyme Inhibitors Produced by Farrowia sp. E‐1941.

Abstract EI-1941-1 ( 1a ) and EI-1941-2 ( 2a ) accompanied by EI-1941-3 ( 3 ) have been isolated from culture broth of Farrowia sp. E-1941 as the inhibitors of interleukin-1β converting enzyme. The structures of 1a , 2a , and 3 were elucidated by the analysis of NMR and MS data, and finally the absolute stereochemistries of 1a and 2a were confirmed by optical rotation data, or X-ray crystallographic analysis of p -bromobenzoate, 2b , respectively.

Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1beta-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2alpha or kinase-inactive CK2alpha (K68M) or CK2alpha' (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2alpha expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.

Structure elucidation of EI-1941-1 and -2, novel interleukin-1β converting enzyme inhibitors produced by Farrowia sp. E-1941

EI-1941-1 (1a) and EI-1941-2 (2a) accompanied by EI-1941-3 (3) have been isolated from culture broth of Farrowia sp. E-1941 as the inhibitors of interleukin-1β converting enzyme. The structures of 1a, 2a, and 3 were elucidated by the analysis of NMR and MS data, and finally the absolute stereochemistries of 1a and 2a were confirmed by optical rotation data, or X-ray crystallographic analysis of p-bromobenzoate, 2b, respectively.

TNF-related apoptosis-inducing ligand is involved in neutropenia of systemic lupus erythematosus

AbstractNeutropenia is a common laboratory finding in systemic lupus erythematosus (SLE). However, the molecular mechanism of SLE neutropenia has not been fully explained. In this study, we examined whether TNF-related apoptosis-inducing ligand (TRAIL) is involved in the pathogenesis of SLE neutropenia using samples from SLE patients. Serum TRAIL levels in SLE patients with neutropenia were significantly higher than those of SLE patients without neutropenia and healthy volunteers. Serum TRAIL levels showed a significant negative correlation with neutrophil counts in SLE patients. The expression of TRAIL receptor 3 was significantly lower in SLE patients with neutropenia than in patients without neutropenia or in healthy volunteers. Treatment with glucocorticoids negated the decrease of TRAIL receptor 3 expression on neutrophils of SLE patients. TRAIL may accelerate neutrophil apoptosis of neutrophils from SLE patients, and autologous T cells of SLE patients, which express TRAIL on surface, may kill autologous neutrophils. Interferon gamma and glucocorticoid modulated the expression of TRAIL on T cells of SLE patients and also modulated the expression of cellular Fas-associating protein with death domain–like interleukin-1β–converting enzyme (FLICE)–inhibitory protein (cFLIP), an inhibitor of death receptor signaling, in neutrophils. Thus, our results provide a novel insight into the molecular pathogenesis of SLE neutropenia.

Nuclear factor of activated T cells balances angiogenesis activation and inhibition.

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis.We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export.We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.

Synthesis and Structure of Diastereomers of Pentenocin B Produced by Trichoderma hamatum FO‐6903.

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