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author:("iwasaki, T")
1.  Saturated fatty acids engage an IRE1α-dependent pathway to activate the NLRP3 inflammasome in myeloid cells 
Cell reports  2016;14(11):2611-2623.
Diets rich in saturated fatty acids (SFAs) produce a form of tissue inflammation driven by “metabolically activated” macrophages. We show that SFAs, when in excess, induce a unique transcriptional signature in both mouse and human macrophages that is enriched for a subset of ER stress markers, particularly IRE1α and many adaptive downstream target genes. SFAs also activate the NLRP3 inflammasome in macrophages, resulting in IL-1β secretion. We found that IRE1α mediates SFA-induced IL-1β secretion by macrophages, and that its activation by SFAs does not rely on unfolded protein sensing. We show instead that the ability of SFAs to stimulate either IRE1α activation or IL-1β secretion can be specifically reduced by preventing their flux into phosphatidylcholine (PC) or by increasing unsaturated PC levels. IRE1α is thus an unrecognized intracellular PC sensor critical to the process by which SFAs stimulate macrophages to secrete IL-1β, a driver of diet-induced tissue inflammation.
eTOC Blurb
Excessive saturated fat consumption promotes tissue inflammation driven by “metabolically activated” macrophages. Here, Robblee et al. use transcriptomic profiling to identify the ER stress sensor IRE1α as a key component of metabolic activation that senses phospholipid saturation to mediate inflammatory activation in macrophages exposed to saturated fat.
PMCID: PMC5242525  PMID: 26971994
2.  2-Phenyl-APB-144-Induced Retinal Pigment Epithelium Degeneration and Its Underlying Mechanisms 
Purpose: To investigate the efficacy of 2-phenyl-APB-144 (APB)-induced retinopathy in a rat model and its underlying mechanisms, with a particular focus on retinal pigment epithelium (RPE) degeneration.
Methods: Electroretinograms (ERGs) were evaluated in APB-administered rats. In ARPE-19 cells, cathepsin, and autophagy marker LC3 were analyzed by western blotting or immunohistochemistry. Organelle pH alterations were detected by Acridine Orange Staining. Endoplasmic reticulum stress-dependent or -independent cell death signaling was analyzed by reporter gene assays of activating transcription factor 4 (ATF4), immunoglobulin heavy-chain binding protein (BiP), inositol-requiring enzyme 1α (IRE1α), quantitative reverse transcription-polymerase chain reaction of CHOP mRNA, and the effects of pharmacological eukaryotic initiation factor 2α (eIF2α) dephosphorylation inhibitor, Salubrinal. The pharmacological effects of Salubrinal were examined by fluorophotometry, electrophysiology, and histopathology.
Results: APB-induced ERG amplitude reduction and fluorescein permeability enhancement into the vitreous body of rats were determined. In ARPE-19 cells, APB-induced organelle pH alterations, imbalances of procathepsin and cathepsin expression, the time-dependent accumulation of LC3-II, and the translational activation of ATF4 were determined. Salubrinal protected against APB-induced cell death and inhibited ATF4 downstream factor CHOP mRNA induction. In APB-induced rat retinopathy, systemic Salubrinal alleviated the enhanced fluorescein permeability into the vitreous body from the RPE, the reductions in ERG amplitudes, and RPE degeneration.
Conclusions: Organelle pH alterations and autophagy impairments are involved in APB-induced RPE cell death. Inhibition of eIF2α dephosphorylation protected the RPE in vivo and in vitro. These findings suggested that APB-induced retinopathy is a valuable animal model for exploring the mechanism of RPE-driven retinopathy.
PMCID: PMC4642833  PMID: 26313169
3.  Physical and Psychological Effects of Head Treatment in the Supine Position Using Specialized Ayurveda-Based Techniques 
Objective: To clarify the physical and psychological effects of head massage performed in the supine position using Ayurveda-based techniques (head treatment).
Design: Twenty-four healthy female students were included in the study. Using a crossover study design, the same participants were enrolled in both the head treatment intervention group and control group. There was an interval of 1 week or more between measurements.
Outcome measures: The physiologic indices measured included blood pressure and heart rate fluctuations (high frequency and low frequency/high frequency). The psychological markers measured included liveliness, depression, and boredom using the visual analogue scale method. State anxiety was measured using the State-Trait Anxiety Inventory method.
Results: The parasympathetic nerve activity increased immediately after head treatment. Upon completion of head treatment, the parasympathetic nerve predominance tended to gradually ease. Head treatment boosted freshness and relieved anxiety.
Conclusions: The results suggest that head treatment has a relaxing and refreshing effect and may be used to provide comfort.
PMCID: PMC4939366  PMID: 27163344
4.  IRE1α is an endogenous substrate of endoplasmic reticulum-associated degradation 
Nature cell biology  2015;17(12):1546-1555.
Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both intramembrane hydrophilic residues of IRE1α and lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signaling in vivo by managing its protein turnover.
PMCID: PMC4670240  PMID: 26551274
ER; Sel1L; Hrd1; ERAD; UPR; intestinal epithelium; inflammation; colitis
5.  In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse 
Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes.
We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway.
We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age.
Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.
We demonstrate that fluorescence ophthalmoscopy enables in vivo visualization of rodent retinas undergoing ER stress using the ERAI transgenic reporter line.
PMCID: PMC4627472  PMID: 26513501
ER stress; GFP; funduscopy
6.  Density and temperature characterization of long-scale length, near-critical density controlled plasma produced from ultra-low density plastic foam 
Scientific Reports  2016;6:21495.
The ability to produce long-scale length (i.e. millimeter scale-length), homogeneous plasmas is of interest in studying a wide range of fundamental plasma processes. We present here a validated experimental platform to create and diagnose uniform plasmas with a density close or above the critical density. The target consists of a polyimide tube filled with an ultra low-density plastic foam where it was heated by x-rays, produced by a long pulse laser irradiating a copper foil placed at one end of the tube. The density and temperature of the ionized foam was retrieved by using x-ray radiography and proton radiography was used to verify the uniformity of the plasma. Plasma temperatures of 5–10 eV and densities around 1021 cm−3 are measured. This well-characterized platform of uniform density and temperature plasma is of interest for experiments using large-scale laser platforms conducting High Energy Density Physics investigations.
PMCID: PMC4770428  PMID: 26923471
7.  Transgenic mouse model for imaging of interleukin-1β-related inflammation in vivo 
Scientific Reports  2015;5:17205.
Inflammation is a biological response associated with symptoms of various diseases, and its study is important in gaining an understanding of the pathological conditions of such diseases and in making strategic plans for promoting healing. It is therefore essential to develop technologies for the detection of inflammatory conditions. Interleukin-1β (IL-1β) is a proinflammatory cytokine produced and secreted mainly by monocytes and macrophages in response to inflammatory stimulation. The activation of IL-1β is regulated through transcriptional induction by the promoter and post-translational processing by the inflammasome. Here we have developed a reporter gene to monitor the activation status of IL-1β by using a dual regulation system and, by using the reporter gene, we have established a mouse model that permits low-invasive visualization of the inflammatory status. Previous reporter systems dependent on the transcription or processing of IL-1β show problems in terms of background noise or signal specificity. Our reporter system overcomes these weaknesses by combining advantages from regulation by a promoter and processing of IL-1β. Our mouse model detected specific physiological inflammation in the liver and pancreas caused by hepatitis or pancreatitis models, respectively. Our reporter gene and mouse model are therefore expected to become useful bioresources for future medical science.
PMCID: PMC4657042  PMID: 26598133
8.  IRE1α/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis 
The Journal of Clinical Investigation  2015;125(8):3269-3279.
The unfolded protein response (UPR) is a cellular adaptive mechanism that is activated in response to the accumulation of unfolded proteins in the endoplasmic reticulum. The inositol-requiring protein-1α/X-box–binding protein–mediated (IRE1α/XBP1-mediated) branch of the UPR is highly conserved and has also been shown to regulate various cell-fate decisions. Herein, we have demonstrated a crucial role for the IREα/XBP1-mediated arm of the UPR in osteoclast differentiation. Using murine models, we found that the conditional abrogation of IRE1α in bone marrow cells increases bone mass as the result of defective osteoclastic bone resorption. In osteoclast precursors, IRE1α was transiently activated during osteoclastogenesis, and suppression of the IRE1α/XBP1 pathway in these cells substantially inhibited the formation of multinucleated osteoclasts in vitro. We determined that XBP1 directly binds the promoter and induces transcription of the gene encoding the master regulator of osteoclastogenesis nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, activation of IRE1α was partially dependent on Ca2+ oscillation mediated by inositol 1,4,5-trisphosphate receptors 2 and 3 (ITPR2 and ITPR3) in the endoplasmic reticulum, as pharmacological inhibition or deletion of these receptors markedly decreased Xbp1 mRNA processing. The present study thus reveals an intracellular pathway that integrates the UPR and osteoclast differentiation through activation of the IRE1α/XBP1 pathway.
PMCID: PMC4563737  PMID: 26193638
9.  Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress 
Scientific Reports  2015;5:14812.
Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.
PMCID: PMC4597200  PMID: 26446798
10.  The co-chaperone and reductase ERdj5 facilitates rod opsin biogenesis and quality control 
Human Molecular Genetics  2014;23(24):6594-6606.
Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110–C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.
PMCID: PMC4240209  PMID: 25055872
11.  Paneth cells as a site of origin for intestinal inflammation 
Nature  2013;503(7475):272-276.
Autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn’s disease (CD)1. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity causes Paneth cell dysfunction2,3. As Atg16l1HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells (IECs), whose human orthologue harbors rare inflammatory bowel disease (IBD) risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis4. Unresolved ER stress is a common feature of IBD epithelium4,5, and several genetic risk factors of CD affect Paneth cells2,4,6-9. Here we show that impairment in either UPR (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) in IECs results in each other’s compensatory engagement, and severe spontaneous CD-like transmural ileitis if both mechanisms are compromised. Xbp1ΔIEC mice exhibit autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased IEC death, inositol requiring enzyme 1α (IRE1α)-regulated NFκB activation and tumor necrosis factor signaling which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity and augmentation of autophagy in IECs ameliorates ER stress-induced intestinal inflammation and eases NFκB overactivation and IEC death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal CD as a specific disorder of Paneth cells.
PMCID: PMC3862182  PMID: 24089213
12.  ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells 
The Journal of Experimental Medicine  2013;210(10):2041-2056.
X-box–binding protein 1 suppresses tumor formation in the gut by regulating Ire1α and Stat3-mediated regenerative responses in the epithelium as a consequence of ER stress.
Unresolved endoplasmic reticulum (ER) stress in the epithelium can provoke intestinal inflammation. Hypomorphic variants of ER stress response mediators, such as X-box–binding protein 1 (XBP1), confer genetic risk for inflammatory bowel disease. We report here that hypomorphic Xbp1 function instructs a multilayered regenerative response in the intestinal epithelium. This is characterized by intestinal stem cell (ISC) expansion as shown by an inositol-requiring enzyme 1α (Ire1α)–mediated increase in Lgr5+ and Olfm4+ ISCs and a Stat3-dependent increase in the proliferative output of transit-amplifying cells. These consequences of hypomorphic Xbp1 function are associated with an increased propensity to develop colitis-associated and spontaneous adenomatous polyposis coli (APC)–related tumors of the intestinal epithelium, which in the latter case is shown to be dependent on Ire1α. This study reveals an unexpected role for Xbp1 in suppressing tumor formation through restraint of a pathway that involves an Ire1α- and Stat3-mediated regenerative response of the epithelium as a consequence of ER stress. As such, Xbp1 in the intestinal epithelium not only regulates local inflammation but at the same time also determines the propensity of the epithelium to develop tumors.
PMCID: PMC3782039  PMID: 24043762
13.  Silencing of lipid metabolism genes through IRE1α-mediated mRNA decay lowers plasma lipids in mice 
Cell metabolism  2012;16(4):487-499.
XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1 deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.
PMCID: PMC3475419  PMID: 23040070
14.  Oral Factors Affecting Titanium Elution and Corrosion: An In Vitro Study Using Simulated Body Fluid 
PLoS ONE  2013;8(6):e66052.
Ti, which is biocompatible and resistant to corrosion, is widely used for dental implants, particularly in patients allergic to other materials. However, numerous studies have reported on Ti allergy and the in vitro corrosion of Ti. This study investigated the conditions that promote the elution of Ti ions from Ti implants.
Specimens of commercially pure Ti, pure nickel, a magnetic alloy, and a gold alloy were tested. Each specimen was immersed in a simulated body fluid (SBF) whose pH value was controlled (2.0, 3.0, 5.0, 7.4, and 9.0) using either hydrochloric or lactic acid. The parameters investigated were the following: duration of immersion, pH of the SBF, contact with a dissimilar metal, and mechanical stimulus. The amounts of Ti ions eluted were measured using a polarized Zeeman atomic absorption spectrophotometer.
Eluted Ti ions were detected after 24 h (pH of 2.0 and 3.0) and after 48 h (pH of 9.0). However, even after 4 weeks, eluted Ti ions were not detected in SBF solutions with pH values of 5.0 and 7.4. Ti elution was affected by immersion time, pH, acid type, mechanical stimulus, and contact with a dissimilar metal. Elution of Ti ions in a Candida albicans culture medium was observed after 72 h.
Elution of Ti ions in the SBF was influenced by its pH and by crevice corrosion. The results of this study elucidate the conditions that lead to the elution of Ti ions in humans, which results in implant corrosion and Ti allergy.
PMCID: PMC3676376  PMID: 23762461
15.  Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor, IRE1β 
PLoS ONE  2012;7(12):e51290.
IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1α and IRE1β, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1β senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1β clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1β complex is robustly increased upon ER stress. Moreover, unlike IRE1α, the luminal domain of IRE1β showed anti-aggregation activity in vitro, and IRE1β was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1β, while IRE1α was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1α, the luminal event mediated by IRE1β involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.
PMCID: PMC3517461  PMID: 23236464
16.  IRE1α activation protects mice against acetaminophen-induced hepatotoxicity 
Mice lacking the transcription factor XBP1 exhibit constitutive activation of the stress sensor IRE1α and are protected from acetaminophen overdose–induced acute liver failure.
The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism.
PMCID: PMC3280871  PMID: 22291093
17.  Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing 
Blood Cancer Journal  2012;2(7):e79-.
The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.
PMCID: PMC3408640  PMID: 22852048
multiple myeloma; ER stress; IRE1α; XBP1; toyocamycin; adenosine analog
18.  A transgenic mouse model for monitoring oxidative stress 
Scientific Reports  2012;2:229.
Oxidative stress conditions enhance the production of reactive oxygen species resulting from a variety of stimuli, and are associated with various human diseases, including neurodegenerative disorders, inflammation, and various cancers. Though such associations have been closely studied using animal models, there has been no in vivo system for monitoring oxidative stress. We have developed an oxidative stress indicator that is dually regulated by induction at the transcriptional level, and by protein stabilisation at the post-translational level in Keap1-Nrf2 pathway. In vitro, our indicator elicited an intense and specific signal to oxidative stress among various agents, in a Keap1-Nrf2-dependent manner. Moreover, the transgenic animal expressing the indicator exhibited significant signals upon oxidative stress. These results indicate the usefulness of our system as an indicator of oxidative stress both in vitro and in vivo.
PMCID: PMC3261443  PMID: 22355743
19.  IRE1α Disruption Causes Histological Abnormality of Exocrine Tissues, Increase of Blood Glucose Level, and Decrease of Serum Immunoglobulin Level 
PLoS ONE  2010;5(9):e13052.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress. As a cellular adaptive response to ER stress, unfolded protein response (UPR) activates molecules for the quality control of ER proteins. One enzyme that plays an important role in UPR is Inositol Requiring Enzyme-1 (IRE1), which is highly conserved from yeast to humans. In particular, mammalian IRE1α activates X-box-binding protein 1 (XBP1) by unconventional splicing of XBP1 mRNA during ER stress. From analysis of knockout mice, both IRE1α and XBP1 have been shown to be essential for development and that XBP1 is necessary for the secretory machinery of exocrine glands, plasma cell differentiation, and hepatic lipogenesis. However, the essentiality of IRE1α in specific organs and tissues remains incompletely understood. Here, we analyzed the phenotype of IRE1α conditional knockout mice and found that IRE1α-deficient mice exhibit mild hypoinsulinemia, hyperglycemia, and a low-weight trend. Moreover, IRE1α disruption causes histological abnormality of the pancreatic acinar and salivary serous tissues and decrease of serum level of immunoglobulin produced in the plasma cells, but not dysfunction of liver. Comparison of this report with previous reports regarding XBP1 conditional knockout mice might provide some clues for the discovery of the novel functions of IRE1α and XBP1. (196 words)
PMCID: PMC2946364  PMID: 20885949
20.  Identification of a consensus element recognized and cleaved by IRE1α 
Nucleic Acids Research  2010;38(18):6265-6273.
IRE1α is an endoplasmic reticulum (ER)-located transmembrane RNase that plays a central role in the ER stress response. Upon ER stress, IRE1α is activated and cleaves specific exon–intron sites in the mRNA encoding the transcription factor X-box-binding protein 1 (XBP1). In addition, previous studies allow us to predict that IRE1α targets several RNAs other than the XBP1. In fact, we have identified CD59 mRNA as a cleavage target of IRE1α. However, it is not yet clear how IRE1α recognizes and cleaves target RNAs. To address this question, we devised a unique method that combines an in vitro cleavage assay with an exon microarray analysis, and performed genome-wide screening for IRE1α cleavage targets. We identified 13 novel mRNAs as candidate IRE1α cleavage targets. Moreover, an analysis of the novel cleavage sites revealed a consensus sequence (CUGCAG) which, when accompanied by a stem-loop structure, is essential for IRE1α-mediated cleavage. The sequence and structure were also conserved in the known IRE1α cleavage targets, CD59 and XBP1. These findings provide the important clue to understanding the molecular mechanisms by which IRE1α recognizes and cleaves target RNAs.
PMCID: PMC2952871  PMID: 20507909

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