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Genotoxic agents, that are used in cancer therapy, elicit the reprogramming of the transcriptome of cancer cells. These changes reflect the cellular response to stress and underlie some of the mechanisms leading to drug resistance. Here, we profiled genome-wide changes in pre-mRNA splicing induced by cisplatin in breast cancer cells. Among the set of cisplatin-induced alternative splicing events we focused on COASY, a gene encoding a mitochondrial enzyme involved in coenzyme A biosynthesis. Treatment with cisplatin induces the production of a short isoform of COASY lacking exons 4 and 5, whose depletion impedes mitochondrial function and decreases sensitivity to cisplatin. We identified RBM39 as a major effector of the cisplatin-induced effect on COASY splicing. RBM39 also controls a genome-wide set of alternative splicing events partially overlapping with the cisplatin-mediated ones. Unexpectedly, inactivation of RBM39 in response to cisplatin involves its interaction with the AP-1 family transcription factor c-Jun that prevents RBM39 binding to pre-mRNA. Our findings therefore uncover a novel cisplatin-induced interaction between a splicing regulator and a transcription factor that has a global impact on alternative splicing and contributes to drug resistance.

(1) Background: Obesity and type 2 diabetes have been suspected to impact both intrinsic metabolism and function of circulating immune cells. (2) Methods: To further investigate this immunometabolic modulation, we profiled the phospholipidome of the peripheral blood mononuclear cells (PBMCs) in lean, normoglycemic obese (OBNG) and obese with dysglycemia (OBDysG) individuals. (3) Results: The global PBMCs phospholipidome is significantly downmodulated in OBDysG unlike OBNG patients when compared to lean ones. Multiple linear regression analyses show a strong negative relationship between the global PBMCs phospholipidome and parameters assessing insulin resistance. Even though all classes of phospholipid are affected, the relative abundance of each class is maintained with the exception of Lyso-PC/PC and Lyso-PE/PE ratios that are downmodulated in PBMCs of OBDysG compared to OBNG individuals. Interestingly, the percentage of saturated PC is positively associated with glycated hemoglobin (HbA1c). Moreover, a few lipid species are significantly downmodulated in PBMCs of OBDysG compared to OBNG individuals, making possible to distinguish the two phenotypes. (4) Conclusions: This lipidomic study highlights for the first-time modulations of the PBMCs phospholipidome in obese patients with prediabetes and type 2 diabetes. Such phospholipidome remodeling could disrupt the cell membranes and the lipid mediator's levels, driving an immune cell dysfunction. ispartof: NUTRIENTS vol:13 issue:10 ispartof: location:Switzerland status: published

Photodynamic therapy (PDT) is an approved therapeutic approach and an alternative to conventional chemotherapy for the treatment of several types of cancer with the advantages of reducing the side effects and developing resistance mechanisms. Here, was evaluated the photosensitization capabilities of 5,10,15,20-tetrakis[4-(pyridinium-1-yl-methyl)phenyl]porphyrin (3), its N-confused isomer (4) and of the neutral precursors (1) and (2) and the results were compared with the ones obtained with the cationic 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). Both regular porphyrin derivatives 1 and 3 showed higher efficiency to generate singlet oxygen than TMPyP. The PDT assays towards MCF-7 cells under red light irradiation (λ > 640 nm, 23.7 mW cm−2) demonstrated that the cationic porphyrin 3 is an efficient photosensitizer to kill MCF-7 breast cancer cells. The study of the cell death mechanisms induced by the photodynamic process showed that the studied porphyrin 3 and TMPyP caused cell death by autophagic flux and necrosis.

Growth differentiation factor-15 (GDF-15) and its receptor GFRAL are both involved in the development of obesity and insulin resistance. Plasmatic GDF-15 level increases with obesity and is positively associated with disease progression. Despite macrophages have been recently suggested as a key source of GDF-15 in obesity, little is known about the regulation of GDF-15 in these cells. In the present work, we sought for potential pathophysiological activators of GDF15 expression in human macrophages and identified saturated fatty acids (SFAs) as strong inducers of GDF15 expression and secretion. SFAs increase GDF15 expression through the induction of an ER stress and the activation of the PERK/eIF2/CHOP signaling pathway in both PMA-differentiated THP-1 cells and in primary monocyte-derived macrophages. The transcription factor CHOP directly binds to the GDF15 promoter region and regulates GDF15 expression. Unlike SFAs, unsaturated fatty acids do not promote GDF15 expression and rather inhibit both SFA-induced GDF15 expression and ER stress. These results suggest that free fatty acids may be involved in the control of GDF-15 and provide new molecular insights about how diet and lipid metabolism may regulate the development of obesity and T2D.

Overexpression of the ubiquitous type II melanoma antigen-D2 (MAGED2) in numerous types of cancer suggests that this protein contributes to carcinogenesis, a well-documented characteristic of other MAGE proteins. Modification of MAGED2 intracellular localization during cell cycle phases and following treatment with camptothecin (CPT) and phosphorylation by ATM/ATR following ionizing irradiation led us to investigate the molecular functions of MAGED2 in the cellular response to DNA damage. Cell cycle regulators, cell cycle progression, and bromodeoxyuridine (BrdU) incorporation were compared between MAGED2-sufficient and -depleted U2OS cells following exposure to CPT. At 24 h post-CPT removal, MAGED2-depleted cells had lower levels of p21 and p27, and there was an increase in S phase BrdU-positive cells with a concurrent decrease in cells in G2. These cell cycle modifications were p21-independent, but ATR-, SKP2-, and CDC20-dependent. Importantly, while MAGED2 depletion reduced CHK2 phosphorylation after 8 h of CPT treatment, it enhanced and prolonged CHK1 phosphorylation after a 24 h recovery period, indicating sustained ATR activation. MAGED2 depletion had no impact on cell survival under our experimental conditions. In summary, our data indicate that MAGED2 reduced CPT-related replicative stress, suggesting a role for this protein in genomic stability.

The maintenance of the characteristic lipid compositions and physicochemical properties of biological membranes is essential for their proper function. Mechanisms allowing to sense and restore membrane homeostasis have been identified in prokaryotes for a long time and more recently in eukaryotes. A membrane remodeling can result from aberrant metabolism as seen in obesity. In this review, we describe how such lipid bilayer stress can account for the modulation of membrane proteins involved in the pathogenesis of obesity-linked inflammatory and metabolic disorders. We address the case of the Toll-like receptor 4 that is implicated in the obesity-related low grade inflammation and insulin resistance. The lipid raft-mediated TLR4 activation is promoted by an enrichment of the plasma membrane with saturated lipids or cholesterol increasing the lipid phase order. We discuss of the plasma membrane Na, K-ATPase that illustrates a new concept according to which direct interactions between specific residues and particular lipids determine both stability and activity of the pump in parallel with indirect effects of the lipid bilayer. The closely related sarco(endo)-plasmic Ca-ATPase embedded in the more fluid ER membrane seems to be more sensitive to a lipid bilayer stress as demonstrated by its inactivation in cholesterol-loaded macrophages or its inhibition mediated by an increased PtdCho/PtdEtn ratio in obese mice hepatocytes. Finally, we describe the model recently proposed for the activation of the conserved IRE-1 protein through alterations in the ER membrane lipid packing and thickness. Such IRE-1 activation could occur in response to abnormal lipid synthesis and membrane remodeling as observed in hepatocytes exposed to excess nutrients. Since the IRE-1/XBP1 branch also stimulates the lipid synthesis, this pathway could create a vicious cycle "lipogenesis-ER lipid bilayer stress-lipogenesis" amplifying hepatic ER pathology and the obesity-linked systemic metabolic defects.

NLRP3 inflammasome plays a key role in Western diet-induced systemic inflammation and was recently shown to mediate long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1β secretion while unsaturated ones (UFAs) prevent SFAs-mediated NLRP3 activation. Unlike previous studies using LPS-primed bone marrow derived macrophages, we do not see any ROS or IRE-1α involvement in SFAs-mediated NLRP3 activation in human monocytes-derived macrophages. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their β-oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine (PC) correlated with a loss of membrane fluidity, both events inhibited by UFAs. The silencing of CCTα, the key enzyme in PC synthesis, prevents SFA-mediated NLRP3 activation, demonstrating the essential role of the de novo PC synthesis. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K+ efflux essential and sufficient for NLRP3 activation. This work opens novel therapeutic avenues to interfere with Western diet-associated diseases such as those targeting the glycerolipid pathway. ispartof: BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS vol:1864 issue:7 pages:1017-1030 ispartof: location:Netherlands status: published

The cell wall of mycobacteria is characterised by glycolipids composed of different classes of mycolic acids (MAs; alpha-, keto-, and methoxy-) and sugars (trehalose, glucose, and arabinose). Studies using mutant Mtb strains have shown that the structure of MAs influences the inflammatory potential of these glycolipids. As mutant Mtb strains possess a complex mixture of glycolipids, we analysed the inflammatory potential of single classes of mycolate esters of the Mtb cell wall using 38 different synthetic analogues. Our results show that synthetic trehalose dimycolate (TDM) and trehalose, glucose, and arabinose monomycolates (TMM, GMM, and AraMM) activate bone marrow-derived dendritic cells in terms of the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species, upregulation of costimulatory molecules, and activation of NLRP3 inflammasome by a mechanism dependent on Mincle. These findings demonstrate that Mincle receptor can also recognise pentose esters and seem to contradict the hypothesis that production of GMM is an escape mechanism used by pathogenic mycobacteria to avoid recognition by the innate immune system. Finally, our experiments indicate that TMM and GMM, as well as TDM, can promote Th1 and Th17 responses in mice in an OVA immunisation model, and that further analysis of their potential as novel adjuvants for subunit vaccines is warranted.

The atypical chemokine receptor CXCR7/ACKR3 binds two endogenous chemokines, CXCL12 and CXCL11, and is upregulated in many cancers or following infection by several cancer-inducing viruses, including HHV-8. ACKR3 is a ligand-scavenging receptor and does not activate the canonical G protein pathways but was proposed to trigger β-arrestin-dependent signaling. Here, we identified the human herpesvirus 8-encoded CC chemokine vCCL2/vMIP-II as a third high-affinity ligand for ACKR3. vCCL2 acted as partial ACKR3 agonist, inducing β-arrestin recruitment to the receptor, subsequent reduction of its surface levels and its delivery to endosomes. In addition, ACKR3 reduced vCCL2-triggered MAP kinase and PI3K/Akt signaling through other chemokine receptors. Our data suggest that ACKR3 acts as a scavenger receptor for vCCL2, regulating its availability and activity toward human receptors, thereby likely controlling its function in HHV-8 infection. Our study provides new insights into the complex crosstalk between viral chemokines and host receptors as well as into the biology of ACKR3, this atypical and still enigmatic receptor.

The photoreactive [Ru(TAP)2(phen)]2+(TAP = 1,4,5,8-tetraazaphenanthrene; phen = 1,10-phenanthroline) complex tethered to the cell-penetrating peptide (CPP) TAT was studied in vitro and in cellulo. The tethering of the complex does not affect its behavior under blue-light irradiation in the presence of guanine-containing oligodeoxyribonucleotides (ODNG). Thus, the luminescence is quenched in the presence of ODNG, and gel electrophoresis experiments showed the appearance of products corresponding to the irreversible attachment of the conjugate to ODNGupon illumination. The cellular uptake of the conjugate was examined by flow cytometry, inductively coupled plasma mass spectrometry (ICP-MS), and confocal imaging microscopy. These experiments showed that the [Ru(TAP)2(phen-TAT)] conjugate is readily taken up by HeLa cells and, despite these favorable factors, the cellular survival was 100 %, as measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A possible origin of the inactivity of [Ru(TAP)2(phen-TAT)] under irradiation is proposed on the basis of the fluorescence-activated cell sorting (FACS), ICP-MS, and confocal microscopy results.

Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203-254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248-254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation.

European leadership in the promotion of media education has rapidly been assumed by Great Britain. Within that country, organizations like the Society for Education in Film and Television, the British Film Institute, and the Scottish National Council have been militantly backing the development of media education. The importance assumed by media education is reflected in the policies adopted by other European countries and the Council of Europe. Belgium is a case in point: it has created a teacher-training program designed to induce teachers to make an effective use of television in their classrooms. Canadian involvement in the promotion and development of media education has also been quite conspicuous during the last few years. Media education is in the process of attracting the attention of increasing numbers of leading scholars throughout the world. A cursory examination of media education literature does, however, reveal that relatively few studies focus on the theoretical foundations of media education.

NLRP3 inflammasome plays a key role in Western diet-induced systemic inflammation and was recently shown to mediate long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1β secretion while unsaturated ones (UFAs) prevent SFAs-mediated NLRP3 activation. Unlike previous studies using LPS-primed bone marrow derived macrophages, we do not see any ROS or IRE-1α involvement in SFAs-mediated NLRP3 activation in human monocytes-derived macrophages. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their β-oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine (PC) correlated with a loss of membrane fluidity, both events inhibited by UFAs. The silencing of CCTα, the key enzyme in PC synthesis, prevents SFA-mediated NLRP3 activation, demonstrating the essential role of the de novo PC synthesis. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K

Recent reports have highlighted studies in biomedical research that cannot be reproduced, tending to undermine the credibility, relevance and sustainability of the research process. To address this issue, a number of factors can be monitored to improve the overall probability of reproducibility. These include: (i) shortcomings in experimental design and execution that involve hypothesis conceptualization, statistical analysis, and data reporting; (ii) investigator bias and error; (iii) validation of reagents including cells and antibodies; and (iv) fraud. Historically, research data that have undergone peer review and are subsequently published are then subject to independent replication via the process of self-correction. This often leads to refutation of the original findings and retraction of the paper by which time considerable resources have been wasted in follow-on studies. New NIH guidelines focused on experimental conduct and manuscript submission are being widely adopted in the peer-reviewed literature. These, in their various iterations, are intended to improve the transparency and accuracy of data reporting via the use of checklists that are often accompanied by "best practice" guidelines that aid in validating the methodologies and reagents used in data generation. The present Editorial provides background and context to a newly developed checklist for submissions to Biochemical Pharmacology that is intended to be clear, logical, useful and unambiguous in assisting authors in preparing manuscripts and in facilitating the peer review process. While currently optional, development of this checklist based on user feedback will result in it being mandatory within the next 12 months.

The response of tumors to photodynamic therapy (PDT) largely varies depending upon the intensity of the stress created in the cancer cells and also in the local environment. Singlet oxygen has been demonstrated, in many instances, to be the primary reactive oxygen species generated by PDT and responsible for most of the cellular effects. Cancer cells have developed various sensors which activate signalling pathways in response to PDT, and the nature of the activated pathway varies with the PDT stress intensity. At low doses of PDT, signalling pathways allow cancer cells to both proliferate and switch on pro-survival responses such as autophagy. Above a certain level of PDT stress intensity, cancer cells cannot cope with the heavy damage, and signalling pathways leading to cell death are activated. Two types of regulated cell death have been shown to be induced by PDT: apoptosis and necrosis. Signalling pathways activating NF-κB transcription factors have the peculiar characteristic of being activated both at low and high doses of PDT. These pathways coordinate the cross-talk between the immune system via the release of cytokines and chemokines and an anti-cell death response via the control of apoptosis and necrosis. Therefore, NF-κB induced by PDT appears to play a positive role in educating the immune system to fight tumors but also a negative role in helping cancer cells to survive the stress generated by singlet oxygen. This is why NF-κB cannot easily be considered as a pharmacological target whose inhibition will favor tumor cell eradication by PDT.

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTβR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTβR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

Only a minority of the many genomic clusters of transcription factor binding motifs (TFBM) act as transcriptional enhancers. To identify determinants of enhancer activity, we randomized the spacer sequences separating the ETS and GATA sites of the early neural enhancer of the tunicateCiona intestinalis Otxgene. We show that spacer sequence randomization affects the level of activity of the enhancer, in part through distal effects on the affinity of the transcription factors for their binding sites. A possible mechanism is suggested by the observation that the shape of the DNA helix within the TFBM can be affected by mutation of flanking bases that modulate transcription factor affinity. Strikingly, dormant genomic clusters of ETS and GATA sites are awakened by most instances of spacer randomization, suggesting that the sequence of naturally-occurring spacers ensures the dormancy of a majority of the large reservoir of TFBM clusters present in a metazoan genome.

It is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of obesity-related insulin resistance and type 2 diabetes. Systemic inflammatory markers are risk factors for the development of type 2 diabetes and its macrovascular complications. Adipose tissue, liver, muscle and pancreas are themselves sites of inflammation in presence of obesity. An infiltration of macrophages and other immune cells is observed in these tissues associated with a cell population shift from an anti-inflammatory to a pro-inflammatory profile. These cells are crucial for the production of pro-inflammatory cytokines, which act in an autocrine and paracrine manner to interfere with insulin signaling in peripheral tissues or induce β-cell dysfunction and subsequent insulin deficiency. Particularly, the pro-inflammatory interleukin-1β is implicated in the pathogenesis of type 2 diabetes through the activation of the NLRP3 inflammasome. The objectives of this review are to expose recent data supporting the role of the immune system in the pathogenesis of insulin resistance and type 2 diabetes and to examine various mechanisms underlying this relationship. If type 2 diabetes is an inflammatory disease, anti-inflammatory therapies could have a place in prevention and treatment of type 2 diabetes.

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

Glioblastomas are the deadliest type of brain cancer and are frequently associated with poor prognosis and a high degree of recurrence despite removal by surgical resection and treatment by chemo- and radio-therapy. Photodynamic therapy (PDT) is a treatment well known to induce mainly necrotic and apoptotic cell death in solid tumors. 5-Aminolevulinic acid (5-ALA)-based PDT was recently shown to sensitize human glioblastoma cells (LN-18) to a RIP3 (Receptor Interacting Protein 3)-dependent cell death which is counter-acted by activation of autophagy. These promising results led us to investigate the pathways involved in cell death and survival mechanisms occurring in glioblastoma following PDT. In the present study, we describe a new TSC2 (Tuberous Sclerosis 2)-dependent survival pathway implicating MK2 (MAPKAPK2) kinase and 14-3-3 proteins which conducts to the activation of a pro-survival autophagy. Moreover, we characterized a new RIP3/TSC2 complex where RIP3 is suggested to promote cell death by targeting TSC2-dependent survival pathway. These results highlight (i) a new role of TSC2 to protect glioblastoma against PDT-induced cell death and (ii) TSC2 and 14-3-3 as new RIP3 partners.

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H(2)O(2) in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK-/- cells display disturbed ER morphology and Ca2+ signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK-/- cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress. Cell Death and Differentiation (2012) 19, 1880-1891; doi:10.1038/cdd.2012.74; published online 15 June 2012

Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41 000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are resistant to all current therapies and are associated with a high rate of recurrence. Glioblastoma were previously shown to respond to treatments by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) mainly by activating a necrotic type of cell death. The receptor-interacting protein 3 (RIP3) has recently been outlined as a key mediator of this caspase-independent form of programmed cell death. In the present study, we analyzed the necrotic mechanism induced by 5-ALA-PDT in human glioblastoma cells and explored the role of RIP3 in this context. Our results show that PDT-induced necrosis is dependent on RIP3, which forms aggregates and colocalizes with RIP1 following photosensitization. We demonstrate that PDT-mediated singlet oxygen production is the cause of RIP3-dependent necrotic pathway activation. We also prove that PDT induces the formation of a pro-necrotic complex containing RIP3 and RIP1 but lacking caspase-8 and FADD, two proteins usually part of the necrosome when TNF-α is used as a stimulus. Thus, we hypothesize that PDT might lead to the formation of a different necrosome whose components, besides RIP1 and RIP3, are still unknown. In most cases, glioblastoma are characterized by a constitutive activation of NF-κB. This factor is a key regulator of various processes, such as inflammation, immune response, cell growth or apoptosis. Its inhibition was shown to further sensitize glioblastoma cells to PDT-induced necrosis, however, no difference in RIP3 upshift or aggregation could be observed when NF-κB was inhibited.

Alteration of the genome integrity leads to the activation of a vast network of cellular responses named "DNA damage response". Three kinases from the phosphoinositide 3-kinase-like protein kinase family regulate this network; ATM and DNA-PK both activated by DNA double-strand breaks and ATR activated by replication blocks. "DNA damage response" pathway coordinates cell cycle arrest, DNA repair, and the activation of transcription factors such as p53 and NF-κB. It controls senescence/apoptosis/survival of the damaged cells. Cell death or survival result from a tightly regulated balance between antagonist pro- and anti-apoptotic signals. NF-κB is a key transcription factor involved in immunity, inflammation and cell transformation. When activated by DNA double-strand breaks, NF-κB has most often a pro-survival effect and thereof interferes with chemotherapy treatments that often rely on DNA damage to induce tumor cell death (i.e. topoisomerase inhibitors and ionizing radiation). NF-κB is thus an important pharmaceutical target. Agents leading to replication stress induce a pro-apoptotic NF-κB. The molecular mechanisms initiated by DNA lesions leading to NF-κB nuclear translocation have been extensively studied these last years. In this review, we will focus on ATM, ATR and DNA-PK functions both in the IKKα/IKKβ/NEMO-dependent or -independent signaling pathways and on the regulation they can exercise at the promoter level of NF-κB regulated genes.

PI3K cascade is a central signaling pathway regulating cell proliferation, growth, differentiation, and survival. Tight regulation of the PI3K signaling pathway is necessary to avoid aberrant cell proliferation and cancer development. Together with SHIP-1, the inositol phosphatases PTEN and SHIP-2 are the gatekeepers of this pathway. In this review, we will focus on SHIP-1 functions. Negative regulation of immune cell activation by SHIP-1 is well characterized. Besides its catalytic activity, SHIP-1 also displays non-enzymatic activity playing role in several immune pathways. Indeed, SHIP-1 exhibits several domains that mediate protein-protein interaction. This review emphasizes the negative regulation of immune cell activation by SHIP-1 that is mediated by its protein-protein interaction.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Background and Objective: The receptor-interacting protein 3 (RIP3) has recently been outlined as a key necrosis mediator but is also thought to participate in the regulation of apoptosis. The aim of this study is to compare the cell death profile induced by 5-aminolevulic acid (5-ALA)-mediated photodynamic therapy (PDT) in the RIP3-deficient cell line U2OS and in U2OS cells in which the expression of RIP3 was restored. Materials and Methods: RIP3-expressing U2OS cells (RIP3-U2OS) were obtained after transfection and antibiotic selection. Wild type and RIP3-U2OS cells were treated by 5-ALA-PDT. Overall cell viability was evaluated and different parameters characteristic of apoptosis, autophagy, and necrosis were studied. Results: Surprisingly, the survival of RIP3-U2OS cells was higher compared to that of the wild type cells. In addition, RIP3-U2OS cell death was decreased by a zVADfmk pre-treatment. A higher cleavage of caspase-3, 7, 8, 9, and PARP was also detected in these cells, pointing out to the activation of caspase-dependent apoptosis. In parallel, a thrust of autophagy was clearly identified in the RIP3U2OS cells. Conversely, RIP3-U2OS exhibited a lower level of necrosis than the wild types. Interestingly, necrostatin-1 efficiently decreased necrosis level in RIP3-U2OS but not in wild type cells. Conclusion: Expression of RIP3 in U2OS cells led to a better survival but also to a death profile change in response to PDT. The apoptotic and autophagic pathways were clearly up-regulated compared to the RIP3-deficient wild type cells. However, induction of necrosis was weaker in the RIP3-U2OS cells. In this context, autophagy is likely to play a protective role against PDT-induced cell death and to allow a better survival of RIP3-U2OS cells. This work also highlights the important role played by RIP3 in the apoptotic pathway, although the modalities are still widely unknown. Lasers Surg. Med. 43:557– 564, 2011. 2011 Wiley-Liss, Inc.

Contrary to the T-helper (Th)-2 bias and eosinophil-dominated bronchial inflammation encountered in most asthmatic subjects, other patients may exhibit neutrophil-predominant asthma subphenotypes, along with Th-1 and Th-17 cells. However, the etiology of many neutrophil-dominated asthma subphenotypes remains ill-understood, in part due to a lack of appropriate experimental models. To better understand the distinct immune-pathological features of eosinophilic vs. neutrophilic asthma types, we developed an ovalbumin (OVA)-based mouse model of neutrophil-dominated allergic pulmonary inflammation. Consequently, we probed for particular inflammatory signatures and checkpoints underlying the immune pathology in this new model, as well as in a conventional, eosinophil-dominated asthma model. Briefly, mice were OVA sensitized using either aluminum hydroxide (alum) or complete Freund's adjuvants, followed by OVA aerosol challenge. T-cell, granulocyte, and inflammatory mediator profiles were determined, along with alveolar macrophage genomewide transcriptome profiling. In contrast to the Th-2-dominated phenotype provoked by alum, OVA/ complete Freund's adjuvants adjuvant-based sensitization, followed by allergen challenge, elicited a pulmonary inflammation that was poorly controlled by dexamethasone, and in which Th-1 and Th-17 cells additionally participated. Analysis of the overall pulmonary and alveolar macrophage inflammatory mediator profiles revealed remarkable similarities between both models. Nevertheless, we observed pronounced differences in the IL-12/IFN-γ axis and its control by IL-18 and IL-18 binding protein, but also in macrophage arachidonic acid metabolism and expression of T-cell instructive ligands. These differential signatures, superimposed onto a generic inflammatory signature, denote distinctive inflammatory checkpoints potentially involved in orchestrating neutrophil-dominated asthma.

Viruses are by far the most abundant parasites on earth and they have been found to infect animals, plants and bacteria. However, different types of viruses can only infect a limited range of hosts and many are species-specific. Herpesviruses constitute a large family of DNA viruses that cause diseases in animals, including humans and that are known to undergo lytic or latent infections. Consequently, they developed numerous strategies to counteract host antiviral responses to escape immune surveillance. Innate immune response constitutes the first line of host defence that limits the viral spread and also plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host Pathogen Recognition Receptors (PRRs) which trigger the activation of IRF3, NF-κB and AP-1, three regulators of IFN-β expression. IFN-β is responsible for the induction of Interferon-Stimulated Genes (ISGs) that encode antiviral effectors important to limit the viral spread and to establish an antiviral state as well in the infected cells as in the neighbouring non-infected cells. In this review, we will summarize how host cells recognize viral components and activate downstream signalling pathways leading to the production of IFN-β and ISGs. We will also review the most recent findings in Herpesviruses-encoded proteins involved in host immune evasion.

The signal transduction pathways leading to apoptosis of human keratinocytes responding to ultraviolet B (UVB) irradiation are complex and not completely understood. Previously, we reported that in UVB-irradiated keratinocytes, p38MAPK instigates Bax activation and mitochondrial apoptosis. However, the molecular mechanism underlying the pro-apoptotic function of p38MAPK remained unclear. Here, we show that in UVB-treated human primary keratinocytes the activation of p38MAPK is necessary to upregulate Noxa, a BH3-only pro-apoptotic dominantly induced by UVB and required for apoptosis. Whereas p53-silencing was marginally cytoprotective and poorly affected Noxa expression, p38MAPK inhibition in p53-silenced keratinocytes or in p53-/- cells could still efficiently prevent Noxa induction and intrinsic apoptosis following UVB, indicating that p38MAPK signals mainly through p53-independent mechanisms. Furthermore, p38MAPK was required for the induction and activation of HIF-1 in response to UVB and HIF-1 knockdown reduced Noxa expression and apoptosis. In UVB-irradiated keratinocytes Noxa targeted the anti-apoptotic Mcl-1 for degradation and siRNA-mediated knockdown of Noxa or p38MAPK inhibition restored levels of Mcl-1 and abolished apoptosis. Thus the pro-apoptotic mechanisms orchestrated by p38MAPK in human keratinocytes in response to UVB involve a HIF-1-Noxa axis, which prompts the downregulation of anti-apoptotic Mcl-1, thereby favouring Bax-mediated mitochondrial apoptosis of UVB-damaged keratinocytes. ispartof: Journal of Investigative Dermatology vol:130 issue:9 pages:2269-2276 ispartof: location:United States status: published

Photodynamic therapy (PDT), induced by a photosensitizer (PS) encapsulated in a nanostructure, has emerged as an appropriate treatment to cure a multitude of oncological and non-oncological diseases. Pyropheophorbide-a methyl ester (PPME) is a second-generation PS tested in PDT, and is a potential candidate for future clinical applications. The present study, carried out in a human colon carcinoma cell line (HCT-116), evaluates the improvement resulting from a liposomal formulation of PPME versus free-PPME. Absorption and fluorescence spectroscopies, fluorescence lifetime measurements, subcellular imaging and co-localization analysis have been performed in order to analyze the properties of PPME for each delivery mode. The benefit of drug encapsulation in DMPC-liposomes is clear from our experiments, with a 5-fold higher intracellular drug delivery than that observed with free-PPME at similar concentrations. The reactive oxygen species (ROSs) produced after PPME-mediated photosensitization have been identified and quantified by using electron spin resonance spectroscopy. Our results demonstrate that PPME-PDT-mediated ROSs are composed of singlet oxygen and a hydroxyl radical. The small amounts of PPME inside mitochondria, as revealed by fluorescence co-localization analysis, could maybe explain the very low apoptotic cell death measured in HCT-116 cells.

As a wide variety of pro-inflammatory cytokines are involved in the development of rheumatoid arthritis (RA), there is an urgent need for the discovery of novel therapeutic strategies. Among these, the inhibition of the NF-κB inducing kinase (NIK), a key enzyme of the NF-κB alternative pathway activation, represents a potential interesting approach. In fact, NIK is involved downstream of many tumor necrosis factor receptors (TNFR) like CD40, RANK or LTβR, implicated in the pathogenesis of RA. But, up to now, the number of reported putative NIK inhibitors is extremely limited. In this work, we report a virtual screening (VS) study combining various filters including high-throughput docking using a 3D-homology model and ranking by using different scoring functions. This work led to the identification of two molecular fragments, 4H-isoquinoline-1,3-dione (5) and 2,7-naphthydrine-1,3,6,8-tetrone (6) which inhibit NIK with an IC50 value of 51 and 90 μM, respectively. This study opens new perspectives in the field of the NF-κB alternative pathway inhibition. © 2010 Elsevier Ltd. All rights reserved.

Reactive oxygen species (ROS) concurrently instigate apoptosis and autophagy pathways, but the link between these processes remains unclear. Because cytotoxic ROS formation is exploited in anticancer therapy, such as in photodynamic therapy (PDT), a better understanding of the complex interplay between autophagy and apoptosis is urgently required. Previously, we reported that ROS generated by PDT with an endoplasmic reticulum (ER)-associated sensitizer leads to loss of ER-Ca2+ homeostasis, ER stress and apoptosis. Here we show that PDT prompted Akt-mTOR (mammalian target of rapamycin) pathway down-regulation and stimulated macroautophagy (MA) in cancer and normal cells. Overexpression of the antioxidant enzyme glutathione peroxidase-4 reversed mTOR down-regulation and blocked MA progression and apoptosis. Attenuating MA using Atg5 knockdown or 3-methyladenine, reduced clearance of oxidatively damaged proteins and increased apoptosis, thus revealing a cytoprotective role of MA in PDT. Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling. We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg5−/− cells compensated for MA loss and increased cellular resistance to PDT. CMA-deficient cells were significantly sensitized to photokilling but were protected against the ER stressor thapsigargin. These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.