XBP1 mRNA Splicing Research Articles

Endoplasmic reticulum stress in bipolar disorder? – BiP and CHOP gene expression- and XBP1 splicing analysis in peripheral blood

BackgroundEndoplasmic Reticulum stress activates the Unfolded Protein Response, which is partially impaired in Bipolar Disorder (BD) according to previous in-vitro studies. Thus, BiP and CHOP gene expression and XBP1 splicing were analyzed in peripheral blood of study participants with BD and controls. MethodsRNA was isolated from fasting blood of study participants with BD (n = 81) and controls (n = 54) and reverse transcribed into cDNA. BiP and CHOP gene expression was analyzed with quantitative RT-PCR. Atypical splicing of XBP1 mRNA was measured by semi-quantitative RT-PCR, gel-electrophoresis and densitometry. ANCOVAs with the covariates age, BMI, sex, lithium and anticonvulsants intake were used with SPSS. Bonferroni correction was used to correct for multiple testing (adjusted p = 0.0083). ResultsBiP gene expression was significantly higher in BD than in controls (F(1/128) = 10.076, p = 0.002, Partial η2 = 0.073). Total XBP1 (F(1/126) = 9.550, p = 0.002, Partial η2 = 0.070) and unspliced XBP1 (F(1/128)= 8.803, p= 0.004, Patial η2 = 0.065) were significantly decreased in BD. Spliced XBP1 (F(1/126) = 5.848, p = 0.017, Partial η2 = 0.044) and the ratio spliced XBP1/ unspliced XBP1 did not differ between BD and controls (F(1/126) = 0.599, p = 0.441, Partial η2 = 0.005). Gene expression did not differ between euthymia, depression and mania. DiscussionBiP gene expression was significantly higher in BD compared to controls. Total and unspliced XBP1 were significantly lower in BD than in the control group. Thus, both genes may be considered as putative trait markers. Nevertheless, XBP1 splicing itself did not differ between both groups.

The unfolded protein response, microRNA-214, and expression of the transcription factor XBP1

Abstract The transcription factor X-box binding protein 1 (XBP1) plays critical roles in the immune system. For example, XBP1 is required for plasma cell development and can disrupt proper function of tumor-associated dendritic cells. A number of post-transcriptional mechanisms regulate XBP1, but a complete understanding of the processes that govern its expression and activity in distinct situations is lacking. The active form of XBP1, termed XBP1s, is dependent upon cytoplasmic splicing of Xbp1 mRNA by IRE1, a transmembrane endoribonuclease/kinase positioned in the endoplasmic reticulum (ER) membrane. IRE1 is a key signal transducer in the cellular response to ER stress, also known as the unfolded protein response (UPR). The IRE1-XBP1 pathway coordinates a broad program of gene transcription that promotes functions of the secretory pathway and alters various aspects of lipid homeostasis under conditions that perturb the ER environment and/or increase demand on its protein folding capacity. ER stress also activates the UPR signal transducer PERK, an ER transmembrane kinase that mediates efficient repression of protein synthesis. Previous studies have shown that microRNA(miR)-214 can negatively regulate XBP1 expression and is down-regulated during ER stress. Using PERK-deficient mouse embryo fibroblasts, we have found that ER stress-mediated diminishment of miR-214 is partially dependent on the PERK pathway, suggesting a novel mechanism of regulatory crosstalk between the PERK and IRE1-XBP1 branches of the UPR. The significance of miR-214 in the cellular response to ER stress and to XBP1-mediated events is under investigation.

Analysis of Endoplasmic Reticulum (ER) Stress Induced during Somatic Cell Nuclear Transfer (SCNT) Process in Porcine SCNT Embryos.

ABSTRACTThis study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

Marine bis-γ-pyrone polypropionates of onchidione family and their effects on the XBP1 gene expression

Two additional new members of the onchidione family, 16-epi-onchidione (1) and 4-epi-onchidione (2), co-occurring with six previously reported bis-γ-pyrone polypropionates including onchidione (3), were isolated from the marine pulmonate Onchidium sp. Their structures were determined by extensive spectroscopic analysis and by comparison with 3 and onchidione-related derivatives. The absolute configuration of 1 was established by X-ray diffraction analysis employing graphite monochromated Cu Kα radiation (λ = 0.71073 Å) with small Flack parameter 0.08. In addition, the absolute stereochemistry of previously reported onchidionol (6) was confirmed by the X-ray diffraction analysis. Some of the isolated compounds showed significant activation effects on the splicing of XBP1 mRNA as ER stress modulators to inhibit the growth of tumors.

The double‐edged sword of endoplasmic reticulum stress in uremic sarcopenia through myogenesis perturbation

BackgroundSarcopenia is the age‐related degeneration characterized with the decline of skeletal muscle mass, strength, and function. The imbalance of protein synthesis and degradation which jeopardizes immune, hormone regulation, and muscle‐motor neuron connection is the main cause of sarcopenia. There is limited knowledge regarding molecular mechanism of sarcopenia. As the endoplasmic reticulum is the control centre of the protein syntheses and degradation, we hypothesized that endoplasmic reticulum stress and unfolded protein response (UPR) play an important in the development of sarcopenia. Understanding the sarcopenia molecular mechanisms may benefit the therapeutic diagnosis and treatment in the future.MethodsMouse myoblast C2C12 cells are exposed to designated time and concentration of indoxyl sulfate (IS), a uremic toxin of chronic kidney disease. The proliferation, differentiation, and the expression of atrogin 1 are examined. The protein and mRNA expression of IS treated‐C2C12 cells are inspected to distinguish the role of ER stress and oxidative stress underlying the sarcopenia.ResultsIndoxyl sulfate inhibits myoblast differentiation. We demonstrate that as the number of multi‐nuclei myotube decreased, the differentiation markers including myoD, myoG, and myosin heavy chain are also suppressed. Indoxyl sulfate inhibits myoblast proliferation and induces the myotubular atrophy marker atrogin‐1 protein expression. Indoxyl sulfate stimulates eIF2α phosphorylation and XBP1 mRNA splicing in UPR. Interestingly, the oxidative stress is related to eIF2α phosphorylation but not XBP1 mRNA splicing. The eIF2α phosphorylation triggered by IS reduces myoD, myoG, and myosin heavy chain protein expression, which represents the anti‐myogenic modulation on the early differentiation event. The XBP1 mRNA splicing induced by IS, however, is considered the adaptive response to restore the myogenic differentiation.ConclusionsOur studies indicated that the ER stress and UPR modulation are critical in the chronic kidney disease uremic toxin‐accumulated sarcopenia model. We believe that UPR‐related signals showed great potential in clinical application.

Dual IRE1 RNase functions dictate glioblastoma development

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1‐dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1‐dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

Fatty acid synthase knockout impairs early embryonic development via induction of endoplasmic reticulum stress in pigs.

Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction.

Xbp-1 mRNA splicing is attenuated under prolonged exposure to ER stress.

xbp-1 mRNA splicing is attenuated under prolonged exposure to ER stress.

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish.

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.

Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells

Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases.

XBP1-FoxO1 interaction regulates ER stress-induced autophagy in auditory cells

The purpose of this study was to clarify the relationship among X-box-binding protein 1 unspliced, spliced (XBP1u, s), Forkhead box O1 (FoxO1) and autophagy in the auditory cells under endoplasmic reticulum (ER) stress. In addition, the relationship between ER stress that causes unfolded protein response (UPR) and autophagy was also investigated. The present study reported ER stress induction by tunicamycin treatment that resulted in IRE1α-mediated XBP1 mRNA splicing and autophagy. XBP1 mRNA splicing and FoxO1 were found to be involved in ER stress-induced autophagy. This inference was based on the observation that the expression of LC3-II was suppressed by knockdown of IRE1α, XBP1 or FoxO1. In addition, XBP1u was found to interact with XBP1s in auditory cells under ER stress, functioning as a negative feedback regulator that was based on two important findings. Firstly, there was a significant inverse correlation between XBP1u and XBP1s expressions, and secondly, the expression of XBP1 protein showed different dynamics compared to the XBP1 mRNA level. Furthermore, our results regarding the relationship between XBP1 and FoxO1 by small interfering RNA (siRNA) paradoxically showed negative regulation of FoxO1 expression by XBP1. Our findings revealed that the XBP1-FoxO1 interaction regulated the ER stress-induced autophagy in auditory cells.

The requirement of IRE1 and XBP1 in resolving physiological stress during Drosophila development.

ABSTRACTIRE1 mediates the unfolded protein response (UPR) in part by regulating XBP1 mRNA splicing in response to endoplasmic reticulum (ER) stress. In cultured metazoan cells, IRE1 also exhibits XBP1-independent biochemical activities. IRE1 and XBP1 are developmentally essential genes in Drosophila and mammals, but the source of the physiological ER stress and the relative contributions of XBP1 activation versus other IRE1 functions to development remain unknown. Here, we employed Drosophila to address this question. Explicitly, we find that specific regions of the developing alimentary canal, fat body and the male reproductive organ are the sources of physiological stress that require Ire1 and Xbp1 for resolution. In particular, the developmental lethality associated with an Xbp1 null mutation was rescued by transgenic expression of Xbp1 in the alimentary canal. The domains of IRE1 that are involved in detecting unfolded proteins, cleaving RNAs and activating XBP1 splicing were all essential for development. The earlier onset of developmental defects in Ire1 mutant larvae compared to in Xbp1-null flies supports a developmental role for XBP1-independent IRE1 RNase activity, while challenging the importance of RNase-independent effector mechanisms of Drosophila IRE1 function.

Piceatannol attenuates homocysteine-induced endoplasmic reticulum stress and endothelial cell damage via heme oxygenase-1 expression

A growing body of evidence implicates endoplasmic reticulum (ER)-induced cellular dysfunction and apoptosis as important factors to a variety of diseases. In endothelial cells (ECs), the sulfur-containing amino acid homocysteine (Hcy) causes EC apoptosis and reactive oxygen species (ROS) generation through induction of ER stress. Here, we have investigated whether piceatannol (Pic), a resveratrol analog, could protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress, with specific emphasis on heme oxygenase-1 (HO-1). In human ECs, we determined the effects of Hcy and Pic on annexin V positivity, glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression, X-box binding protein 1 (Xbp-1) mRNA slicing, and ROS-sensitive dihydroethidium (DHE) oxidation. Hcy increased annexin V-positive cells, DHE oxidation, GRP78 and CHOP expression and Xbp-1 mRNA splicing, indicating that Hcy induces apoptosis, oxidative stress and ER stress. Pretreatment of ECs with Pic significantly inhibited Hcy-induced apoptosis, ROS generation and ER stress. Pic also increased HO-1 expression via activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, the inhibitory effects of Pic on Hcy-induced apoptosis, ROS generation and ER stress were abolished by down-regulation of HO-1 expression, while mimicked by treatment of ECs with the HO-1 inducer hemin. Overall, these results suggest that Pic may protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress via Nrf2-dependent HO-1 expression.

Chlorpyrifos induces endoplasmic reticulum stress in JEG-3 cells

Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50μM or 100μM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53.

Mycobacterium bovis Induces Endoplasmic Reticulum Stress Mediated-Apoptosis by Activating IRF3 in a Murine Macrophage Cell Line

Mycobacterium bovis (M. bovis) is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults. However, the host cells in turn deploy a multipronged defense mechanism to control bacterial infection. Endoplasmic reticulum (ER) stress-mediated apoptosis is one such primary defense mechanism. However, the role of interferon regulatory factor 3 (IRF3) between ER stress and apoptosis during M. bovis infection is unknown. Here, we demonstrate that M. bovis effectively induced apoptosis in murine macrophages. Caspase-12, caspase-9, and caspase-3 were activated over a 48 h infection period. The splicing of XBP-1 mRNA and the level of phosphorylation of eIF2α, indicators of ER stress, significantly increased at early time points after M. bovis infection. The expansion of the ER compartment, a morphological hallmark of ER stress, was observed at 6 h. Pre-treatment of Raw 264.7 cells with 4-PBA (an ER stress-inhibitor) reduced the activation of the ER stress indicators, caspase activation and its downstream poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylation of TBK1 and IRF3 and cytoplasmic co-localization of STING and TBK1. M. bovis infection led to the interaction of activated IRF3 and cytoplasmic Bax leading to mitochondrial damage. Role of IRF3 in apoptosis was further confirmed by blocking this molecule with BX-795 that showed significant reduction expression of caspase-8 and caspase-3. Intracellular survival of M. bovis increased in response to 4-PBA and BX-795. These findings indicate that STING-TBK1-IRF3 pathway mediates a crosstalk between ER stress and apoptosis during M. bovis infection, which can effectively control intracellular bacteria.

287 - The Redox Active Anti-Cancer Drug, Dp44mT, Induces Endoplasmic Reticulum Stress Activating Pro-Apoptotic Pathways of the Unfolded Protein Response in Cancer Cells

In the stressful tumor microenvironment, endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are activated in cancer cells promoting cancer cell survival. A novel strategy for anti-cancer drugs is to re-sensitise cancer cells to ER stress-induced apoptosis by altering the kinetics of the UPR. Chelators of the DpT thiosemicarbazone class form cellular redox active metal complexes and demonstrate potent in vitro and in vivo anti-cancer and anti-metastatic activity. The most potent of these agents is currently being examined in clinical trials for the treatment of patients with advanced solid tumors (NCT02688101). However, the mechanism by which these drugs alter the ER stress response and induce apoptosis is unknown. Consequently, studies assessed the mechanisms by which the potent DpT redox active agent, Dp44mT, alters the ER stress response, relative to the redox inactive chelator, DFO, and the classical ER stress-inducing agent, tunicamycin, in SK-N-MC neuroepithelioma and PANC1 pancreatic cancer cells. Our studies demonstrate that Dp44mT significantly: (1) increased activation of ER stress-associated pro-apoptotic signaling (i.e., p-eIF2α, ATF4 and CHOP); (2) increased phosphorylation of IRE1α and XBP1 mRNA splicing; (3) reduced the expression of molecules involved in ER stress-associated cell survival signaling ( e.g. , XBP1s and p58IPK); (4) activated the transcription factor, ATF6, and its downstream targets ( i.e. CHOP and BiP); and (5) activated pro-apoptotic p-CaMKII. In contrast, redox inactive DFO and the ER stress-inducing agent, tunicamycin, had little or no effect on the expression or phosphorylation of these proteins. Considering the role of reactive oxygen species (ROS) generation in the effects observed, it was demonstrated that the ROS scavenger, N -acetyl-l-cysteine, that supplements cellular glutathione could inhibit Dp44mT-mediated activation of the UPR, while the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced Dp44mT activity. In conclusion, ER stress is induced after iron sequestration by chelators, and this effect was enhanced by drugs such as Dp44mT that produce ROS. These results demonstrate that Dp44mT activates the pro-apoptotic pathways of the UPR, re-sensitizing cancer cells to apoptosis while inhibiting cell survival signals.

Peroxisome Proliferator-activated Receptor-γ Activation Augments the β-Cell Unfolded Protein Response and Rescues Early Glycemic Deterioration and β Cell Death in Non-obese Diabetic Mice

Type 1 diabetes is an autoimmune disorder that is characterized by a failure of the unfolded protein response in islet β cells with subsequent endoplasmic reticulum stress and cellular death. Thiazolidinediones are insulin sensitizers that activate the nuclear receptor PPAR-γ and have been shown to partially ameliorate autoimmune type 1 diabetes in humans and non-obese diabetic (NOD) mice. We hypothesized that thiazolidinediones reduce β cell stress and death independently of insulin sensitivity. To test this hypothesis, female NOD mice were administered pioglitazone during the pre-diabetic phase and assessed for insulin sensitivity and β cell function relative to controls. Pioglitazone-treated mice showed identical weight gain, body fat distribution, and insulin sensitivity compared with controls. However, treated mice showed significantly improved glucose tolerance with enhanced serum insulin levels, reduced β cell death, and increased β cell mass. The effect of pioglitazone was independent of actions on T cells, as pancreatic lymph node T cell populations were unaltered and T cell proliferation was unaffected by pioglitazone. Isolated islets of treated mice showed a more robust unfolded protein response, with increases in Bip and ATF4 and reductions in spliced Xbp1 mRNA. The effect of pioglitazone appears to be a direct action on β cells, as islets from mice treated with pioglitazone showed reductions in PPAR-γ (Ser-273) phosphorylation. Our results demonstrate that PPAR-γ activation directly improves β cell function and survival in NOD mice by enhancing the unfolded protein response and suggest that blockade of PPAR-γ (Ser-273) phosphorylation may prevent type 1 diabetes.

OS1.8 IRE1-mediated immunomodulation in glioblastoma

AbstractIntroduction:Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion, tumor vascularization and recruitment of inflammatory or immune cells. Indeed, IRE1 RNase promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival and this activity also contributes to the degradation of mRNA and microRNA, an activity called RIDD. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information.Results:Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered IRE1 RNase activity. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures from human tumors, (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice and (iii) the modulation of immune cells recruitment to the tumor.Conclusions:This study provides the first evidence that the selectivity of IRE1 RNase can be modulated naturally and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells by reprogramming the tumor stroma.

Drug Synergism of Proteasome Inhibitors and Mitotane by Complementary Activation of ER Stress in Adrenocortical Carcinoma Cells.

Mitotane is the only drug approved for treatment of the orphan disease adrenocortical carcinoma (ACC) and was recently shown to be the first clinically used drug acting through endoplasmic reticulum (ER)-stress induced by toxic lipids. Since mitotane has limited clinical activity as monotherapy, we here study the potential of activating ER-stress through alternative pathways. The single reliable NCI-H295 cell culture model for ACC was used to study the impact MG132, bortezomib (BTZ) and carfilzomib (CFZ) on mRNA and protein expression of ER-stress markers, cell viability and steroid hormone secretion. We found all proteasome inhibitors alone to trigger expression of mRNA (spliced X-box protein 1, XBP1) and protein markers indicative of the inositol-requiring enzyme 1 (IRE1) dependent pathway of ER-stress but not phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker of the PRKR-like endoplasmic reticulum kinase (PERK)-dependent pathway. Whereas mitotane alone activated both pathways, combination of BTZ and CFZ with low-dose mitotane blocked mitotane-induced eIF2α phosphorylation but increased XBP1-mRNA splicing indicating that proteasome inhibitors can commit signalling towards a single ER-stress pathway in ACC cells. By applying the median effect model of drug combinations using cell viability as a read out, we determined significant drug synergism between mitotane and both BTZ and CFZ. In conclusion, combination of mitotane with activators of ER-stress through the unfolded protein response is synergistic in an ACC cell culture model. Since proteasome inhibitors are readily available clinically, they are attractive candidates to study for ACC treatment in clinical trials in combination with mitotane.

Effects of muscular dystrophy, exercise and blocking activin receptor IIB ligands on the unfolded protein response and oxidative stress

Protein homeostasis in cells, proteostasis, is maintained through several integrated processes and pathways and its dysregulation may mediate pathology in many diseases including Duchenne muscular dystrophy (DMD). Oxidative stress, heat shock proteins, endoplasmic reticulum (ER) stress and its response, i.e. unfolded protein response (UPR), play key roles in proteostasis but their involvement in the pathology of DMD are largely unknown. Moreover, exercise and activin receptor IIB blocking are two strategies that may be beneficial to DMD muscle, but studies to examine their effects on these proteostasis pathways are lacking. Therefore, these pathways were examined in the muscle of mdx mice, a model of DMD, under basal conditions and in response to seven weeks of voluntary exercise and/or activin receptor IIB ligand blocking using soluble activin receptor-Fc (sAcvR2B-Fc) administration. In conjunction with reduced muscle strength, mdx muscle displayed greater levels of UPR/ER-pathway indicators including greater protein levels of IRE1α, PERK and Atf6b mRNA. Downstream to IRE1α and PERK, spliced Xbp1 mRNA and phosphorylation of eIF2α, were also increased. Most of the cytoplasmic and ER chaperones and mitochondrial UPR markers were unchanged in mdx muscle. Oxidized glutathione was greater in mdx and was associated with increases in lysine acetylated proteome and phosphorylated sirtuin 1. Exercise increased oxidative stress when performed independently or combined with sAcvR2B-Fc administration. Although neither exercise nor sAcvR2B-Fc administration imparted a clear effect on ER stress/UPR pathways or heat shock proteins, sAcvR2B-Fc administration increased protein expression levels of GRP78/BiP, a triggering factor for ER stress/UPR activation and TxNIP, a redox-regulator of ER stress-induced inflammation. In conclusion, the ER stress and UPR are increased in mdx muscle. However, these processes are not distinctly improved by voluntary exercise or blocking activin receptor IIB ligands and thus do not appear to be optimal therapeutic choices for improving proteostasis in DMD.