Cop, a caspase recruitment domain-containing protein and inhibitor of caspase-1 activation processing.

The production of bio-active interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves binding of the caspase-1 prodomain to a caspase recruitment domain (CARD)-containing serine/threonine kinase known as RIP2/CARDIAK/RICK. We have identified a novel protein, COP (CARD only protein), which has a high degree of sequence identity to the caspase-1 prodomain. COP binds to both RIP2 and the caspase-1 prodomain and inhibits RIP2-induced caspase-1 oligomerization. COP inhibits caspase- 1-induced IL-1beta secretion as well as lipopolysaccharide-induced IL-1beta secretion in transfected cells. Our data indicate that COP can regulate IL-1beta secretion, implying that COP may play a role in down-regulating inflammatory responses analogous to the CARD protein ICEBERG.     

Regulation of IL-1beta generation by Pseudo-ICE and ICEBERG, two dominant negative caspase recruitment domain proteins

We report here the identification and functional characterization of two new human caspase recruitment domain (CARD) molecules, termed Pseudo-interleukin-1beta converting enzyme (ICE) and ICEBERG. Both proteins share a high degree of homology, reaching 92% and 53% identity, respectively, to the prodomain of caspase-1/ICE. Interestingly, both Pseudo-ICE and ICEBERG are mapped to chromosome 11q22 that bears caspases-1, -4- and -5 genes, all involved in cytokine production rather than in apoptosis. We demonstrate that Pseudo-ICE and ICEBERG interact physically with caspase-1 and block, in a monocytic cell line, the interferon-gamma and lipopolysaccharide-induced secretion of interleukin-1beta which is a well-known consequence of caspase-1 activation. Moreover, Pseudo-ICE, but not ICEBERG, interacts with the CARD-containing kinase RICK/RIP2/CARDIAK and activates NF-kappaB. Our data suggest that Pseudo-ICE and ICEBERG are intracellular regulators of caspase-1 activation and could play a role in the regulation of IL-1beta secretion and NF-kappaB activation during the pro-inflammatory cytokine response.     

INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.     

Nod1, a CARD protein,enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.     

Flightless-I regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity

Caspase-1 and caspase-11 are proinflammatory caspases that regulate cytokine production and leukocyte migration during pathogen infection. In an attempt to identify new intracellular regulators of caspase-11, we found that Flightless-I, a member of the gelsolin superfamily of actin-remodeling proteins, interacts and regulates both caspase-11 and caspase-1. Flightless-I targets caspase-11 to the Triton X-100-insoluble cytoskeleton fraction and the cell leading edge. In addition, Flightless-I inhibits caspase-1 activation and caspase-1-mediated interleukine-1beta (IL-1beta) maturation. The physiological relevance of these findings is supported by the opposing effects of Flightless-I overexpression and knockdown on caspase-1 activity and IL-1beta maturation. Our results suggest that Flightless-I may be a bona fide caspase-1 inhibitor that acts through a mechanism similar to that of cytokine response modifier A, a potent caspase-1 inhibitor from the cowpox virus. Our study provides a new mechanism controlling the localization and activation of proinflammatory caspases.     

IL-1 beta converting enzyme is a target for nitric oxide-releasing aspirin: new insights in the antiinflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs

Caspase-1, the IL-1beta converting enzyme (ICE), is required for intracellular processing/maturation of IL-1beta and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40-80% inhibition of IL-1beta, IL-8, IL-12, IL-18, IFN-gamma, and TNF-alpha release with an EC(50) of 10-20 microM for IL-1beta and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E(2) generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-D-L-penicillamine, inhibited caspase-1 activity with an EC(50) of approximately 20 microM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60-80% reduction of IL-1beta, IL-8, IFN-gamma, and TNF-alpha release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1beta and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.     

Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity

The proteolytic activity of caspases is involved in apoptosis and inflammation. In this regard, caspase-1 is required for pro-interleukin (IL)-1beta and pro-IL-18 maturation. We report here on a novel function of caspase-1 as an activator of nuclear factor of the kappa-enhancer in B-cells (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK). This function is not shared by the murine caspase-1 homologues caspase-11 and -12. In contrast to pro-IL-1beta maturation, caspase-1-induced NF-kappaB activation is not inhibited by the virus-derived caspase-1 inhibitor cytokine response modifier A and is equally induced by the enzymatically inactive caspase-1 C285A mutant. Although the general NF-kappaB-inhibiting protein A20 inhibits caspase-1-derived activation of NF-kappaB, dominant-negative forms of TRAF2 and RIP1 have no effect. We demonstrate that caspase-1 interacts with RIP2 and that dominant-negative forms of RIP2 and IkappaB kinase complex-beta inhibit caspase-1-mediated NF-kappaB activation. Structure-function analysis shows that the caspase recruitment domain of caspase-1 mediates the activation of NF-kappaB and p38 MAPK. These data demonstrate that caspase-1 contributes to inflammation by two distinct pathways: proteolysis of pro-IL-1beta, and RIP2-dependent activation of NF-kappaB and p38 MAPK mediated by the caspase recruitment domain.     

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.     

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.     

Inhibition of nuclear transport of caspase-7 by its prodomain

Apoptosis is a major form of cell death, characterized by a series of morphological changes induced by cleaving cytoplasmic and nuclear proteins via active caspases. The data presented here show, by fluorescence microscopic and immunoblotting analyses, that a prodomain of caspase-7 inhibits its nuclear translocation and apoptosis-inducing activity. This nuclear localization is dependent on the presence of a basic tetrapeptide that is conserved in mammalian and Xenopus caspase-7 and that is located downstream of a cleavage site between a prodomain and a catalytic protease domain. Furthermore, an attachment of the caspase-7 prodomain (31 amino acids) represses the nuclear transport of a fusion protein of a heterologous protein and the caspase-7 nuclear localization signal (19 amino acids), suggesting that the inhibition of nuclear localization by the prodomain is mediated by the interaction of these short peptides.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.     

The estrogen-responsive B box protein: a novel enhancer of interleukin-1beta secretion

The estrogen-responsive B box protein (EBBP) and Pyrin belong to a family of structurally related proteins. While mutations in the pyrin gene cause an autoinflammatory disease, the biological function of EBBP is unknown. In this study, we identified the proinflammatory cytokine interleukin-1beta (IL-1beta) as an EBBP-binding partner. Furthermore, caspase-1 and NACHT, LRR and Pyrin domain containing protein (NALP) 1, two components of the recently identified inflammasome, a platform for the activation of caspase-1, also interact with EBBP. These proteins bind to the RFP domain of EBBP, suggesting that this domain of so far unknown function is an important protein-binding domain. EBBP was secreted in a caspase-1-dependent manner from cultured cells, and its secretion was enhanced by IL-1beta. Vice versa, endogenous and overerexpressed EBBP increased IL-1beta secretion. These results provide evidence for a role of EBBP in innate immunity by enhancing the alternative secretion pathway of IL-1beta.     

Regulation of procaspase-2 by glucocorticoid modulatory element-binding protein 1 through the interaction with caspase recruitment domain

Caspases are the primary executioners of apoptosis. Although procaspases believe to exist as inactive forms in cells, the detailed regulatory system remains unclear. Here we show that glucocorticoid modulatory element-binding protein 1 (GMEB1) is capable of binding to the prodomain of caspase-2. We found that this molecule inhibits the autoproteolytic activation of procaspase-2 by oligomerization on a chemical compound-dependent system. These findings indicated that GMEB1 might be an endogenous inhibitory protein that selectively interacts with prodomain of caspase-2 to disrupt the autoactivation.     

NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages

Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.     

Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release

Aluminum hydroxide (Alum) is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. In this study, we show that Alum activates caspase-1 and induce secretion of mature IL-1beta and IL-18. Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists released only traces of IL-1beta or IL-18, despite the fact that the IL-1beta mRNA was readily induced by both TLR agonists. In contrast, cells costimulated with TLR agonists plus Alum released large amount of IL-1beta and IL-18. Alum-induced IL-1beta and IL-18 production was not due to enhancement of TLR signaling but rather reflected caspase-1 activation and in mouse dendritic cells occurred in a MyD88-independent fashion. Secretion of other proinflammatory cytokines such as IL-8 was not affected by Alum treatments. However, TLR-induced production of IL-10 was increased and that of IFN-gamma-inducible protein decreased by Alum cotreatment. Considering the immunostimulatory activities of these cytokines and the ability of IL-1beta to act as adjuvant, our results suggest a mechanism for the adjuvanticity of Alum.     

Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes

Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.