Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.

Through their capacity to sense danger signals and to generate active interleukin-1β (IL-1β), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1β, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1β was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes') in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

Evolutionary analysis of genes of two pathways involved in placental malaria infection

Placental malaria is a special form of malaria that causes up to 200,000 maternal and infant deaths every year. Previous studies show that two receptor molecules, hyaluronic acid and chondroitin sulphate A, are mediating the adhesion of parasite-infected erythrocytes in the placenta of patients, which is believed to be a key step in the pathogenesis of the disease. In this study, we aimed at identifying sites of malaria-induced adaptation by scanning for signatures of natural selection in 24 genes in the complete biosynthesis pathway of these two receptor molecules. We analyzed a total of 24 Mb of publicly available polymorphism data from the International HapMap project for three human populations with European, Asian and African ancestry, with the African population from a region of presently and historically high malaria prevalence. Using the methods based on allele frequency distributions, genetic differentiation between populations, and on long-range haplotype structure, we found only limited evidence for malaria-induced genetic adaptation in this set of genes in the African population; however, we identified one candidate gene with clear evidence of selection in the Asian population. Although historical exposure to malaria in this population cannot be ruled out, we speculate that it might be caused by other pathogens, as there is growing evidence that these molecules are important receptors in a variety of host-pathogen interactions. We propose to use the present methods in a systematic way to help identify candidate regions under positive selection as a consequence of malaria.

From SNPs to pathways: integration of functional effect of sequence variations on models of cell signalling pathways

Background: Single nucleotide polymorphisms (SNPs) are the most frequent type of sequence variation between individuals, and represent a promising tool for finding genetic determinants of complex diseases and understanding the differences in drug response. In this regard, it is of particular interest to study the effect of non-synonymous SNPs in the context of biological networks such as cell signalling pathways. UniProt provides curated information about the functional and phenotypic effects of sequence variation, including SNPs, as well as on mutations of protein sequences. However, no strategy has been developed to integrate this information with biological networks, with the ultimate goal of studying the impact of the functional effect of SNPs in the structure and dynamics of biological networks. Results: First, we identified the different challenges posed by the integration of the phenotypic effect of sequence variants and mutations with biological networks. Second, we developed a strategy for the combination of data extracted from public resources, such as UniProt, NCBI dbSNP, Reactome and BioModels. We generated attribute files containing phenotypic and genotypic annotations to the nodes of biological networks, which can be imported into network visualization tools such as Cytoscape. These resources allow the mapping and visualization of mutations and natural variations of human proteins and their phenotypic effect on biological networks (e.g. signalling pathways, protein-protein interaction networks, dynamic models). Finally, an example on the use of the sequence variation data in the dynamics of a network model is presented. Conclusion: In this paper we present a general strategy for the integration of pathway and sequence variation data for visualization, analysis and modelling purposes, including the study of the functional impact of protein sequence variations on the dynamics of signalling pathways. This is of particular interest when the SNP or mutation is known to be associated to disease. We expect that this approach will help in the study of the functional impact of disease-associated SNPs on the behaviour of cell signalling pathways, which ultimately will lead to a better understanding of the mechanisms underlying complex diseases.

Metabolic intervention on lipid synthesis converging pathways abrogates prostate cancer growth.

One of the most conserved features of all cancers is a profound reprogramming of cellular metabolism, favoring biosynthetic processes and limiting catalytic processes. With the acquired knowledge of some of these important changes, we have designed a combination therapy in order to force cancer cells to use a particular metabolic pathway that ultimately results in the accumulation of toxic products. This innovative approach consists of blocking lipid synthesis, at the same time that we force the cell, through the inhibition of AMP-activated kinase, to accumulate toxic intermediates, such as malonyl-coenzyme A (malonyl-CoA) or nicotinamide adenine dinucleotide phosphate. This results in excess of oxidative stress and cancer cell death. Our new therapeutic strategy, based on the manipulation of metabolic pathways, will certainly set up the basis for new upcoming studies defining a new paradigm of cancer treatment.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Tumor necrosis factor-α activates estrogen signaling pathways in endometrial epithelial cells via estrogen receptor α.

The pro-inflammatory cytokine TNF-α and the female hormone estrogen have been implicated in the pathophysiology of two common gynecological diseases, endometriosis and endometrial adenocarcinoma. Here we describe a novel capacity of TNF-α to activate ER signaling in endometrial epithelial cells. TNF-α induced luciferase expression in the absence and presence of estradiol and also augmented expression of the estrogen-regulated genes c-fos, GREB1, and progesterone receptor. Furthermore, TNF-α mediated ER transcriptional activity is dependent on the Extracellular Regulated Kinase (ERK) 1/2 pathway. Co-treatment with a pure ER antagonist resulted in an inhibition of this TNF-α-induced ERE luciferase activity and gene expression, demonstrating that this cytokine signals through ERs. Additional investigations confirmed that TNF-α acts specifically via ERα. Taken together, these data provide a rationale for the potential use of inhibitors of TNF-α and estrogen production/activity in combination for the treatment of endometrial pathologies.

Modulation of inflammatory pathways by the HIV protease inhibitors

Les inhibiteurs de la protéase du VIH (IP) constituent une des classes de traitements antirétroviraux parmi les plus utilisés au cours de l'infection par le VIH. Leur utilisation est associée à divers effets secondaires, notamment la dyslipidémie, la résistance à l'insuline, la lipodystrophie et certaines complications cardio-vasculaires. Ces molécules ont également des propriétés anti-tumorales, décrites chez des patients non infectés par le VIH. Pourtant, les mécanismes moléculaires à l'origine de ces effets annexes restent méconnus. Dans ce travail, nous démontrons que les IP, comme le Nelfinavir, le Ritonavir, le Lopinavir, le Saquinavir et l'Atazanavir, entrainent la production d'interleukine-lß (IL-lß), une puissante cytokine pro-inflammatoire, connue pour son rôle central dans les maladies inflammatoires. La sécrétion d'IL-lß requiert la formation de l'inflammasome, un complexe protéique intracellulaire servant de plateforme d'activation de la caspase-1 et, par la suite, à la maturation protéolytique de certaines cytokines, dont l'IL-lß. Dans les macrophages murins en culture primaire, ainsi que dans une lignée de monocytes humains, nous démontrons que les IP augmentent la maturation et la sécrétion de l'IL-lß via l'induction d'un inflammasome dépendant de ASC. De plus, nous établissons que les IP induisent spécifiquement l'activation de AIM2, un inflammasome détectant la présence intracytosolique d'ADN viral ou bactérien. Nos résultats démontrent l'existence d'une nouvelle voie d'activation de l'inflammasome AIM2 par un signal endogène dont la nature reste à définir. Ces données suggèrent que AIM2 pourrait jouer un rôle important dans la promotion de l'activité anti-tumorale ainsi que dans les autres effets annexes observés chez les patients traités par IP. -- HIV protease inhibitors (Pis) are among the most often used classes of antiretroviral drugs for HIV infection. Treatment of patients with HIV-PIs is associated with the development of metabolic side effects including dyslipidemia, insulin resistance, lipodystrophy and cardiovascular complications. In addition, these drugs have been reported to have anti¬tumoral properties in non-infected patients, however the molecular mechanisms causing these off-target effects are still unclear. Here we show that the HIV-PIs, such as Nelfinavir, Ritonavir, Lopinavir, Saquinavir and Atazanavir, activate the production of interleukin-lß (IL-lß), a potent pro-inflammatory cytokine that plays a central role in the pathogenesis of inflammatory diseases. The release of IL-lß depends on the activation of the inflammasome, a multiprotein complex that serves as a platform for caspase-1 activation and subsequent proteolytic maturation of cytokines including IL-lß. We found that in mouse primary macrophages as well as in a human monocytic cell line, the HIV-PIs augment the maturation and secretion of IL-lß by triggering an ASC-dependent inflammasome activation. Moreover, we show that the HIV-PIs specifically engage AIM2, a recently characterized inflammasome -forming protein that was described to detect the cytosolic release of bacterial and viral DNA. Our findings demonstrate a new pathway of activation of the AIM2 inflammasome by a yet to be defined endogenous signal and may suggest a possible role for AIM2 in promoting anti¬tumoral activity and off-target effects observed in HIV-PIs treated patients.

Gut symbionts from distinct hosts exhibit genotoxic activity via divergent colibactin biosynthetic pathways.

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is "colibactin", a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway, inflicting DNA damage upon eukaryotic cells and contributing to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees, and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.

Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways.

BACKGROUND: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. OBJECTIVES: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. RESULTS: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. CONCLUSIONS: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner.

Bilateral dorsal and ventral fiber pathways for the processing of affective prosody identified by probabilistic fiber tracking.

Dorsal and ventral pathways for syntacto-semantic speech processing in the left hemisphere are represented in the dual-stream model of auditory processing. Here we report new findings for the right dorsal and ventral temporo-frontal pathway during processing of affectively intonated speech (i.e. affective prosody) in humans, together with several left hemispheric structural connections, partly resembling those for syntacto-semantic speech processing. We investigated white matter fiber connectivity between regions responding to affective prosody in several subregions of the bilateral superior temporal cortex (secondary and higher-level auditory cortex) and of the inferior frontal cortex (anterior and posterior inferior frontal gyrus). The fiber connectivity was investigated by using probabilistic diffusion tensor based tractography. The results underscore several so far underestimated auditory pathway connections, especially for the processing of affective prosody, such as a right ventral auditory pathway. The results also suggest the existence of a dual-stream processing in the right hemisphere, and a general predominance of the dorsal pathways in both hemispheres underlying the neural processing of affective prosody in an extended temporo-frontal network.

Bacterial transcriptional regulators for degradation pathways of aromatic compounds.

Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways.

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases.

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation.