IRE1 signaling regulates chondrocyte apoptosis and death fate in the osteoarthritis.

IRE1 is an important central regulator of unfolded protein response (UPR) in the endoplasmic reticulum (ER) because of its ability to regulate cell fate as a function of stress sensing. When misfolded proteins accumulated in chondrocytes ER, IRE1 disintegrates with BIP/GRP78 and undergoes dimer/oligomerization and transautophosphorylation. These two processes are mediated through an enzyme activity of IRE1 to activate endoribonuclease and generates XBP1 by unconventional splicing of XBP1 messenger RNA. Thereby promoting the transcription of UPR target genes and apoptosis. The deficiency of inositol-requiring enzyme 1α (IRE1α) in chondrocytes downregulates prosurvival factors XBP1S and Bcl-2, which enhances the apoptosis of chondrocytes through increasing proapoptotic factors caspase-3, p-JNK, and CHOP. Meanwhile, the activation of IRE1α increases chondrocyte viability and reduces cell apoptosis. However, the understanding of IRE1 responses and cell death fate remains controversial. This review provides updated data about the role IRE1 plays in chondrocytes and new insights about the potential efficacy of IRE1 regulation in cartilage repair and osteoarthritis treatment.

PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

Exercise alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice.

The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

Arabidopsis immune-associated nucleotide-binding genes repress heat tolerance at the reproductive stage by inhibiting the unfolded protein response and promoting cell death.

Plants are vulnerable to heat stress, especially during reproductive development. The heat shock response (HSR) in the cytosol and nucleus, as well as the unfolded protein response (UPR) in the endoplasmic reticulum (ER), are two mechanisms that enable plants to survive heat stress. Excessive heat or ER stresses lead to cell death when the UPR cannot repair stress damage, but the means by which cell survival or death is determined remains unclear. In this study, we used a genome-wide association study (GWAS) to identify that a cluster of five Immune-associated nucleotide-binding protein (IAN) genes (IAN2 to IAN6) is responsible for variation in heat tolerance at the reproductive stage in Arabidopsis thaliana. These IAN genes have both unique and overlapping functions in the negative regulation of heat tolerance, and their loss of function singly or in combination confers increased heat tolerance, measured by a lower number of barren siliques and a higher seedling survival rate under heat. The loss of rice IAN1 gene function also leads to enhanced heat tolerance, suggesting a conserved function of plant IANs. Transcriptome analysis revealed enhanced expression of HSR and UPR genes, as well as reduced cell death, under heat and ER stress in the mutant of IAN6, a major effect member in Arabidopsis. Furthermore, the IAN proteins were found to promote cell death induced by heat stress, ER stress, and cell death-inducing molecules. Thus, the Arabidopsis IAN genes repress heat tolerance, probably through the HSR and UPR and by enhancing the cell death pathway. The IAN2 to IAN6 proteins are partially localized to the ER, suggesting a direct role in the UPR and UPR-mediated cell death. In addition, a natural IAN6 variant from more heat-tolerant Arabidopsis accessions confers greater heat tolerance and induces less cell death compared with the natural variant from less heat-tolerant accessions. The heat-tolerant IAN6 variant is associated with a higher maximum temperature of the warmest month at its collection sites compared with the heat-sensitive variant. Taken together, these results reveal an important role of Arabidopsis IAN2 to IAN6 genes in the regulation of the HSR, UPR, and cell death, and suggest that their natural variations have adaptive functions in heat tolerance.

3'quant mRNA-Seq of Porcine Liver Reveals Alterations in UPR, Acute Phase Response, and Cholesterol and Bile Acid Metabolism in Response to Different Dietary Fats.

Animal fats are considered to be unhealthy, in contrast to vegetable fats, which are rich in unsaturated fatty acids. However, the use of some fats, such as coconut oil, is still controversial. In our experiment, we divided experimental animals (domestic pigs) into three groups differing only in the type of fat used in the diet: group R: rapeseed oil (n = 5); group B: beef tallow (n = 5); group C: coconut oil (n = 6). After transcriptomic analysis of liver samples, we identified 188, 93, and 53 DEGs (differentially expressed genes) in R vs. B, R vs. C, and B vs. C comparisons, respectively. Next, we performed a functional analysis of identified DEGs with String and IPA software. We observed the enrichment of genes engaged in the unfolded protein response (UPR) and the acute phase response among genes upregulated in B compared to R. In contrast, cholesterol biosynthesis and cholesterol efflux enrichments were observed among genes downregulated in B when compared to R. Moreover, activation of the UPR and inhibition of the sirtuin signaling pathway were noted in C when compared to R. The most striking difference in liver transcriptomic response between C and B was the activation of the acute phase response and inhibition of bile acid synthesis in the latest group. Our results suggest that excessive consumption of animal fats leads to the activation of a cascade of mutually propelling processes harmful to the liver: inflammation, UPR, and imbalances in the biosynthesis of cholesterol and bile acids via altered organelle membrane composition. Nevertheless, these studies should be extended with analysis at the level of proteins and their function.

Gene Therapy: Contest between Adeno-Associated Virus and Host Cells and the Impact of UFMylation.

Adeno-associated virus (AAV)-based gene therapy is currently limited by (1) decline in therapeutic gene expression over time, (2) immune cell activation and (3) neutralization by pre-existing antibodies. Hence, studying the interaction of AAV vectors with various cellular pathways during the production and transduction process is necessary to overcome such barriers. Post-translational modifications (PTM) of AAV vectors during the production and transduction process is known to limit its transduction efficiency and further evoke the immune response. Further, AAV vectors are known to trigger cellular stress, resulting in an upregulation of distinct arms of the unfolded protein response (UPR) pathway. Recognition of the AAV genome by Toll-like receptor-9 triggers the myeloid differentiation primary response signaling cascade for innate (IL-6, IFN-α, IFN-ß) and adaptive (CD8+ T-cell, B-cell) immune response against the viral capsid and the transgene product. Herein, we highlight a potential intersection of the UPR, PTMs, and intracellular trafficking pathways, which could be fine-tuned to augment the outcome of AAV-based gene delivery.

Transition of the abnormal Savda syndrome to the hepatic carcinoma shifted unfolded protein response to autophagy was partly reversed by Savda Munziq in a rat model.

OBJECTIVES: In Uighur medicine, abnormal Savda Munziq (AMSq) is an adjuvant therapy for cancer patients with abnormal Savda syndrome (ASS) who exhibit the highest degree of pathogenicity and malignancy. The aim of the study was to understand the role(s) of AMSq in cancer patients with ASS. METHODS: A total of 125 rats were divided into groups: control (NC) (n = 15), ASS (n = 25), ASS with hepatic carcinoma (ASSHC) (n = 25) as well as ASSHC treated with low dose (ASSHC-L, n = 20), medium dose (ASSHC-M, n = 20) and high dose (ASSHC-H, n = 20) AMSq. Changes in the unfolded protein response (UPR) and autophagy were analyzed by RT profiler PCR array kits, which covered 84 UPR and 84 autophagy related genes. Protein expression analyses of LC3B, ATG8, GRP78 and CHOP were carried out using western blotting. RESULTS: CHOP and GRP78 expression was enhanced in ASS and ASSHC compared to NC rats and further increased AMSq dose-dependently, indicating an UPR triggering effect of AMSq. The ratios of ATG8/LCB3II-LCB3I protein were reduced in ASSHC rats, an effect which was partly reversed by AMSq. Compared to NC rats in the ASS group, 24 UPR genes were significantly upregulated and 3 downregulated, whereas only 5 autophagy genes were significantly upregulated and 5 downregulated. Compared to NC rats in the ASSHC group, 15 UPR genes were significantly upregulated and 10 downregulated, whereas 16 autophagy genes were significantly upregulated and 8 downregulated. The RT profiler data indicated a shift from UPR in the ASS to the autophagy response in ASSHC rats. ASMq effects on ASSHC rats comprised significant downregulation of 10 autophagy and 2 UPR genes. CONCLUSION: The transformation into hepatic cancer cells included a shift from endoplasmic reticulum stress-related UPR to autophagy gene activation, an effect which could be partly reversed by ASMq.

Expression of genes related to energy metabolism and the unfolded protein response in dairy cow mammary cells is affected differently during dietary supplementation with energy from protein and fat.

Secretory capacity of bovine mammary glands is enabled by a high number of secretory cells and their ability to use a range of metabolites to produce milk components. We isolated RNA from milk fat to measure expression of genes involved in energy-yielding pathways and the unfolded protein response in mammary glands of lactating cows given supplemental energy from protein (PT) and fat (FT) tested in a 2 × 2 factorial arrangement. We hypothesized that PT and FT would affect expression of genes in the branched-chain AA catabolic pathway and tricarboxylic acid (TCA) cycle based on the different energy types (aminogenic versus lipogenic) used to synthesize milk components. We also hypothesized that the response of genes related to endoplasmic reticulum (ER) homeostasis via the unfolded protein response would reflect the increase in milk production stimulated by PT and FT. Fifty-six multiparous Holstein-Friesian dairy cows were fed a basal total mixed ration (34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter basis) for a 28-d control period. Experimental rations were then fed for 28 d, consisting of (1) low protein, low fat (LP/LF); (2) high protein, low fat (HP/LF); (3) low protein, high fat (LP/HF); or (4) high protein and high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on dry matter basis) and 0.68 kg/d hydrogenated palm fatty acids on a dry matter basis. RNA from milk fat samples collected on d 27 of each period underwent real-time quantitative PCR. Energy from protein increased expression of BCAT1 (branched-chain amino acid transferase 1) mRNA, but only at the LF level, and tended to decrease expression of mRNA encoding the main subunit of the branched-chain keto-acid dehydrogenase complex. mRNA expression of malic enzyme, a proposed channeling route for AA though the TCA cycle, was decreased by PT, but only at the LF level. Expression of genes associated with de novo fatty acid synthesis was not affected by PT or FT. Energy from fat had no independent effect on genes related to ER homeostasis. At the LF level, PT activated XBP1 (X-box binding protein 1) mRNA. At the HF level, PT increased mRNA expression of the gene encoding GADD34 (growth arrest and DNA damage-inducible 34). These findings support our hypothesis that mammary cells use aminogenic and lipogenic precursors differently for milk component production when dietary intervention alters AA and fatty acid supply. They also suggest that mammary cells respond to increased AA supply through mechanisms of ER homeostasis, dependent on the presence of FT.

The multiple roles of the unfolded protein response regulator IRE1α in cancer.

Cancer is associated with a number of conditions such as hypoxia, nutrient deprivation, cellular redox, and pH changes that result in accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) and trigger a stress response known as the unfolded protein response (UPR). The UPR is a conserved cellular survival mechanism mediated by the ER transmembrane proteins activating transcription factor 6, protein kinase-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1α (IRE1α) that act to resolve ER stress and promote cell survival. IRE1α is a kinase/endoribonuclease (RNase) with multiple activities including unconventional splicing of the messenger RNA (mRNA) for the transcription factor X-Box Binding Protein 1 (XBP1), degradation of other mRNAs in a process called regulated IRE1α-dependent decay (RIDD) and activation of a pathway leading to c-Jun N-terminal kinase phosphorylation. Each of these outputs plays a role in the adaptive and cell death responses to ER stress. Many studies indicate an important role for XBP1 and RIDD functions in cancer and new studies suggest that these two functions of the IRE1α RNase can have opposing functions in the early and later stages of cancer pathogenesis. Finally, as more is learned about the context-dependent role of IRE1α in cancer development, specific small molecule inhibitors and activators of IRE1α could play an important role in counteracting the protective shield provided by ER stress signaling in cancer cells.

Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.

18ß-Glycyrrhetinic-acid-mediated unfolded protein response induces autophagy and apoptosis in hepatocellular carcinoma.

18ß-Glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicine, Glycyrrhrzae Radix et Rhizoma. Here, we explored the effects of GA on hepatocellular carcinoma (HCC) in vitro and in vivo and the underlying molecular mechanisms. We confirmed that GA suppressed proliferation of various HCC cell lines. Treatment of GA caused G0/G1 arrest, apoptosis and autophagy in HCC cells. GA-induced apoptosis and autophagy were mainly due to the unfolded protein response. We compared the roles of the ATF4/CHOP and IRE1α/XBP1s UPR pathways, which were both induced by GA. The ATF4/CHOP cascade induced autophagy and was indispensable for the induction of apoptosis in GA-treated HCC cells. In contrast, the IRE1α/XBP1s cascade protected HCC cells from apoptosis in vitro and in vivo induced by GA. Despite this, activation of autophagy protected HCC cells from apoptosis induced by GA. We concluded that pharmacological inhibition of autophagy or IRE1α may be of benefit to enhance the antitumor activity of GA.

Lifetime Genistein Intake Increases the Response of Mammary Tumors to Tamoxifen in Rats.

PURPOSE: Whether it is safe for estrogen receptor-positive (ER+) patients with breast cancer to consume soy isoflavone genistein remains controversial. We compared the effects of genistein intake mimicking either Asian (lifetime) or Caucasian (adulthood) intake patterns to that of starting its intake during tamoxifen therapy using a preclinical model. EXPERIMENTAL DESIGN: Female Sprague-Dawley rats were fed an AIN93G diet supplemented with 0 (control diet) or 500 ppm genistein from postnatal day 15 onward (lifetime genistein). Mammary tumors were induced with 7,12-dimethylbenz(a)anthracene (DMBA), after which a group of control diet-fed rats were switched to genistein diet (adult genistein). When the first tumor in a rat reached 1.4 cm in diameter, tamoxifen was added to the diet and a subset of previously only control diet-fed rats also started genistein intake (post-diagnosis genistein). RESULTS: Lifetime genistein intake reduced de novo resistance to tamoxifen, compared with post-diagnosis genistein groups. Risk of recurrence was lower both in the lifetime and in the adult genistein groups than in the post-diagnosis genistein group. We observed downregulation of unfolded protein response (UPR) and autophagy-related genes (GRP78, IRE1α, ATF4, and Beclin-1) and genes linked to immunosuppression (TGFß and Foxp3) and upregulation of cytotoxic T-cell marker CD8a in the tumors of the lifetime genistein group, compared with controls, post-diagnosis, and/or adult genistein groups. CONCLUSIONS: Genistein intake mimicking Asian consumption patterns improved response of mammary tumors to tamoxifen therapy, and this effect was linked to reduced activity of UPR and prosurvival autophagy signaling and increased antitumor immunity. Clin Cancer Res; 23(3); 814-24. ©2017 AACR.

Functional analysis of the mammalian RNA ligase for IRE1 in the unfolded protein response.

The unfolded protein response (UPR) is a conserved signalling pathway activated on the accumulation of unfolded proteins within the endoplasmic reticulum (ER), termed ER stress. Upon ER stress, HAC1/XBP1 undergoes exon/intron-specific excision by inositol requiring enzyme 1 (IRE1) to remove an intron and liberate the 5' and 3' exons. In yeast, the 5' and 3' HAC1 exons are subsequently ligated by tRNA ligase (Rlg1p), whereas XBP1 ligation in mammalian cells is catalysed by a recently identified ligase, RtcB. In the present study, RNA ligase activity of the human RtcB (hRtcB) involved in the unconventional splicing of XBP1/HAC1 mRNA was explored in an rlg1-100 mutant yeast strain. Distinct from Escherichia coli RtcB and Rlg1p, expression of hRtcB alone inefficiently complemented HAC1/XBP1 splicing and the hRtcB cofactor (archease) was required to promote enzymatic activity of hRtcB to catalyse RNA ligation.

AMPK Regulation of Cell Growth, Apoptosis, Autophagy, and Bioenergetics.

In eukaryotic cells, AMP-activated protein kinase (AMPK) generally promotes catabolic pathways that produce ATP and at the same time inhibits anabolic pathways involved in different processes that consume ATP. As an energy sensor, AMPK is involved in the main cellular functions implicated in cell fate, such as cell growth and autophagy.Recently, AMPK has been connected with apoptosis regulation, although the molecular mechanism by which AMPK induces and/or inhibits cell death is not clear.This chapter reviews the essential role of AMPK in signaling pathways that respond to cellular stress and damage, highlighting the complex and reciprocal regulation between AMPK and their targets and effectors. The therapeutic implications of the role of AMPK in different pathologies such as diabetes, cancer, or mitochondrial dysfunctions are still controversial, and it is necessary to further investigate the molecular mechanisms underlying AMPK activation.

Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and the TOR pathway in Saccharomyces cerevisiae.

Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N-terminal protein-interaction and C-terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome-wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N-terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin.

Celastrol induces unfolded protein response-dependent cell death in head and neck cancer.

The survival rate for patients with oral squamous cell carcinoma (OSCC) has not seen marked improvement in recent decades despite enhanced efforts in prevention and the introduction of novel therapies. We have reported that pharmacological exacerbation of the unfolded protein response (UPR) is an effective approach to killing OSCC cells. The UPR is executed via distinct signaling cascades whereby an initial attempt to restore folding homeostasis in the endoplasmic reticulum during stress is complemented by an apoptotic response if the defect cannot be resolved. To identify novel small molecules able to overwhelm the adaptive capacity of the UPR in OSCC cells, we engineered a complementary cell-based assay to screen a broad spectrum of chemical matter. Stably transfected CHO-K1 cells that individually report (luciferase) on the PERK/eIF2α/ATF4/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR pathways, were engineered [1]. The triterpenoids dihydrocelastrol and celastrol were identified as potent inducers of UPR signaling and cell death in a primary screen and confirmed in a panel of OSCC cells and other cancer cell lines. Biochemical and genetic assays using OSCC cells and modified murine embryonic fibroblasts demonstrated that intact PERK-eIF2-ATF4-CHOP signaling is required for pro-apoptotic UPR and OSCC death following celastrol treatment.

Exogenous salicylic acid activates two signaling arms of the unfolded protein response in Arabidopsis.

The unfolded protein response (UPR) is a highly conserved cellular response that prevents abnormal maturation of proteins in the endoplasmic reticulum (ER). The expression of genes encoding ER chaperones is induced during the UPR. In the Arabidopsis UPR, two membrane-bound transcription factors, bZIP60 and bZIP28, activate the expression of those genes. bZIP60 is regulated by unconventional cytoplasmic splicing catalyzed by inositol requiring enzyme 1 (IRE1), which is located in the ER membrane. bZIP28 is regulated by intramembrane proteolysis. Pathogen infection and salicylic acid (SA) have been reported to induce the expression of some UPR genes. Here, we show that UPR genes including BiP3, a marker gene of the Arabidopsis UPR, are induced by exogenous SA treatment and activation of bZIP60 in an IRE1-dependent manner. The induction of gene expression and activation of bZIP60 were independent of NPR1 and HsfB1 under these experimental conditions. We generated antibodies to detect the proteolytic products of bZIP28 after SA treatment. An assay using these antibodies showed that SA activated bZIP28, as well as activating bZIP60 through IRE1. Together, these results show that exogenous SA treatment activates two signaling arms of the Arabidopsis UPR. We propose a possible mechanism of activation of the UPR machinery by SA.

Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila Xbp1.

The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER) stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2--HR2--and the C-terminal translational pausing site) present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. Here, we generated a Drosophila Xbp1 deletion mutant (Excision101) lacking both HR2 and C-terminal region, but not the Ire1 splicing site. We show that Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. Our results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced.

Flavonoids from Litsea coreana decreases TNF-α secretion from peritoneal macrophages in adjuvant-induced arthritis rats via UPR pathway.

Macrophages play a crucial role in rheumatoid arthritis (RA). Their activation is the initial step of RA. This study was designed to detect the effects of total flavonoids from Litsea coreana Levl. (TFLC) on the complete Freund's adjuvant-induced (CFA-induced) arthritis (AA) in rats and to explore whether inflammatory cytokines were induced by the IRE1/mTORC1/TNF-α-dependant mechanism in peritoneal macrophages. In vivo, our data indicated that TFLC (100, 200 mg/kg, i.g. × 10 days) could significantly suppress secondary paw swelling and serum levels of TNF-α and IL-1ß. Histopathological figures showed that TFLC treatment improved the morphologic changes of articular cartilages and synovium. Results of RT-PCR and western blotting demonstrated that TFLC suppressed expression of 78-KD glucose regulated protein (GRP78), X-box binding protein 1 (XBP1), mTOR complex 1 (mTORC1) and TNF-α in peritoneal macrophages of AA rats. Collectively, these results indicate that TFLC is able to ameliorate adjuvant-induced arthritis in a dose-dependent manner by suppressing the IRE1/mTORC1/TNF-α-regulated inflammatory response initiated in peritoneal macrophages.