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The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process.To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I- and N-) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis.We found that all I + treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions. (C) 2017 Elsevier Inc. All rights reserved.

Oxidative stress can lead to plant growth retardation, yield loss, and death. The atr7 mutant of Arabidopsis thaliana exhibits pronounced tolerance to oxidative stress. Using positional cloning, confrmed by knockout and RNA interference (RNAi) lines, we identifed the atr7 mutation and revealed that ATR7 is a previously uncharacterized gene with orthologs in other seed plants but with no homology to genes in lower plants, fungi or animals. Expression of ATR7-GFP fusion shows that ATR7 is a nuclear-localized protein. RNA-seq analysis reveals that transcript levels of genes encoding abiotic- and oxidative stress-related transcription factors (DREB19, HSFA2, ZAT10), chromatin remodelers (CHR34), and unknown or uncharacterized proteins (AT5G59390, AT1G30170, AT1G21520) are elevated in atr7. This indicates that atr7 is primed for an upcoming oxidative stress via pathways involving genes of unknown functions. Collectively, the data reveal ATR7 as a novel seed plants-specifc nuclear regulator of oxidative stress response., Published online: 27 June 2019

In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2¿) by one of four members of the eIF2¿ kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress. This work was funded in part by the Health Research Board (Grant Number HRA/2009/59; HRA‐POR‐2014‐643), Belgian Science Policy Office Interuniversity Attraction Poles program (IAP 7/32), and Breast Cancer Campaign grant (2010NovPR13) to A.S. A Science Foundation Ireland (SFI) grant cofunded under the European Regional Development Fund under Grant Number 13/RC/2073. I.K. was funded by an Irish Research Council Scholarship (GOIPG/2014/508) and by the Thomas Crawford Hayes Fund. K.M. is funded by an Irish Research Council Fellowship (GOIPD/2014/53). peer-reviewed 2017-03-14

Necroptosis was initially identified as a backup cell death program when apoptosis is blocked. However, it is now recognized as a cellular defense mechanism against infections and is presumed to be a detrimental factor in several pathologies driven by cell death. Necroptosis is a prototypic form of regulated necrosis that depends on activation of the necrosome, which is a protein complex in which receptor interacting protein kinase (RIPK) 3 is activated. The RIP homotypic interaction motif (RHIM) is the core domain that regulates activation of the necrosome. To date, three RHIM-containing proteins have been reported to activate the kinase activity of RIPK3 within the necrosome: RIPK1, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), and DNA-dependent activator of interferon regulatory factors (DAI). Here, we review and discuss commonalities and differences of the increasing number of activators of the necrosome. Since the discovery that activation of mixed lineage kinase domain-like (MLKL) by RIPK3 kinase activity is crucial in necroptosis, interest has increased in monitoring and therapeutically targeting their activation. The availability of new phospho-specific antibodies, pharmacologic inhibitors, and transgenic models will allow us to further document the role of necroptosis in degenerative, inflammatory and infectious diseases.

The antiapoptotic protein FLIPS is a key suppressor of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)–Akt–atrophin-interacting protein 4 (AIP4) pathway regulates FLIPS ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIPS stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIPS ubiquitination, decreased FLIPS half-life, decreased FLIPS steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)–mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIPS ubiquitination, USP8 seemed to control FLIPS ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIPS E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIPS interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIPS levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIPS stability and TRAIL sensitivity. Cancer Res; 70(12); 5046–53. ©2010 AACR.

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIPS, and confers resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIPS protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIPS polyubiquitination. FLIPS half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIPS E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIPS or with the FLIPS-containing death inducing signal complex. Small interfering RNA–mediated suppression of AIP4 levels in PTEN WT cells decreased FLIPS ubiquitination, prolonged FLIPS half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIPS steady-state levels, and suppressed FLIPS ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIPS ubiquitination, FLIPS stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability. [Cancer Res 2009;69(20):7911–6]

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs' immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-gamma-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2'-5' oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues.

Increased expression of antiapoptotic Bcl-2 proteins confers therapeutic resistance in various cancer types. Targeting Bcl-2 proteins by small molecules or activating alternative pathways to bypass Bcl-2-mediated protection to promote apoptosis are two approaches to overcoming therapeutic resistance. Here, we show that cisplatin triggers a Bak-dependent pathway to induce apoptosis in Bcl-2-overexpressing MCF-7 cells. p53-mediated induction of Noxa expression, generation of lipid peroxidation end products and induction of Noxa-Mcl-1 interaction are necessary for this pathway to function. Although Puma is also induced by cisplatin treatment, it is not required for apoptosis. Similarly, reactive oxygen species production by cisplatin did not have any effect on cisplatin-induced apoptosis in MCF-7 Bcl-2 cells. Furthermore, p53 promotes cisplatin-induced apoptosis by directly binding and counteracting Bcl-x(L) antiapoptotic function. In conclusion, our findings suggest a novel mode of action for cisplatin to overcome Bcl-2-mediated protection against apoptosis, which requires preferential activation of Bak and p53-mediated upregulation of Noxa protein levels and lipid peroxidation.

This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-3, -7 and -9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bim(el) mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bim(el). NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bim(el) protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax.

The efficient functioning of the endoplasmic reticulum ( ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response ( UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress- induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoptosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC exposure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure).

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C-24-ceramide predominated, was observed 6 h after Belt triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BeR triggering. The formation of these very long-chain ceramide species, in which C-24-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which longchain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C-16-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C-24-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.

International audience; The pleiotropic pro-inflammatory cytokine tumour necrosis factor alpha (TNF) is synthesised as a transmembrane protein that is subject to palmitoylation. In this study, the roles of this acylation on TNF-mediated biological effects were investigated. We found that the lipid raft partitioning of TNF is regulated by its palmitoylation. Furthermore, we demonstrated that this palmitoylation process interferes with the cleavage/degradation of TNF intracellular fragments but is not involved in the regulation of its ectodomain shedding. Moreover, we found that the palmitoylation of TNF hinders the binding of soluble TNF to TNFR1 and regulates the integration/retention of TNFR1 into lipid rafts. Finally, we demonstrate that the transmembrane forms of wild-type and palmitoylation-defective TNF interact differently with TNFR1 and regulate NF kappa B activity, Erk1/2 phosphorylation and interleukin-6 synthesis differently, strongly suggesting that palmitoylation of TNF is involved in the regulation of TNFR1 signalling. An evidence for the physiological intervention of this regulation is provided by the fact that in macrophages, the binding of endogenous soluble TNF to TNFR1 is enhanced by inhibition of palmitoylation. Therefore, our data introduce the new concept that palmitoylation of TNF is one of the means by which TNF-producing cells regulate their sensitivity to soluble TNF. (C) 2012 Elsevier B.V. All rights reserved.

Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon.

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings. Background: IAP antagonists sensitize toward apoptosis induced by TNF and other TNFR family ligands. Results: IAP antagonism exerted effects on spontaneous as well as TNF-induced cytokine and chemokine production. Conclusion: IAPs regulate spontaneous as well as TNF-induced cytokine/chemokine production. Significance: IAP antagonists modulate cytokine production as well as apoptosis, which could influence their utility as adjuncts to chemotherapy.

The stimuli for neuronal cell death in neurodegenerative disorders are multi-factorial and may include genetic predisposition, environmental factors, cellular stressors such as oxidative stress and free radical production, bioenergy failure, glutamate-induced excitotoxicity, neuroinflammation, disruption of Ca2+-regulating systems, mitochondrial dysfunction and misfolded protein accumulation. Cellular stress disrupts functioning of the endoplasmic reticulum (ER), a critical organelle for protein quality control, leading to induction of the unfolded protein response (UPR). ER stress may contribute to neurodegeneration in a range of neurodegenerative disorders. This review summarizes the molecular events occurring during ER stress and the unfolded protein response and it specifically evaluates the evidence suggesting the ER stress response plays a role in neurodegenerative disorders.

Necrotrophic fungi, being the largest class of fungal plant pathogens, pose a serious economic problem to crop production. They are the cause of heavy losses in agriculture worldwide. Understanding the process of plant infection by necrotrophic fungi, including subtle interaction networks connecting such evolutionarily distinct organisms has recently been given high research priority. Such studies are now possible mainly because of the utility of the model plant Arabidopsis thaliana. A. thaliana has a sequenced genome and thousands of mutants available, allowing investigation of virtually all aspects of plant pathogenesis. This review focuses on morphological and molecular changes in A. thaliana, which occur during response to infection by necrotrophic fungi. These responses in relation to resistance and susceptibility of the plant will be discussed. Refereed/Peer-reviewed

Diets rich in flavonoids are associated with various positive health effects. Most in vitro research conducted to elucidate the modes of action of flavonoids uses flavonoid aglycones, but not their circulating conjugated metabolites. Conjugation alters the physico-chemical properties of flavonoids and it is widely assumed that this can affect their biological activity. This article gives a state-of-the-art overview of scientific literature reporting on the effect of metabolic conjugation on the biological activity of flavonoids. The biological activity of flavonoid aglycones is compared to that of their conjugates for a broad range of endpoints. Even though there is only limited literature available, it is shown that contrary to common belief, conjugation does not always decrease the biological activity of flavonoids. There are also endpoints which are unaffected by conjugation, and endpoints on which the conjugates have a higher or inverse activity when compared to the aglycone. The effects of conjugation can differ depending on the type and position of conjugation, the flavonoid concentration, the endpoint studied and the assay system used so that no general rules can be deducted. It is concluded that further studies on the effects of conjugation have to be done on a case-by-case basis, and characterization of the stability and metabolic fate of the flavonoids in the assay system under consideration is needed to avoid false positive or false negative outcomes.