Takao Iwawaki

Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis

ATG16L1T300A, a major risk polymorphism in Crohn’s disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1&agr;. IRE1&agr; accumulates in Paneth cells of Atg16l1&Dgr;IEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1&agr; in intestinal epithelial crypts. In contrast to a protective role of the IRE1&bgr; isoform, hyperactivated IRE1&agr; also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1&Dgr;IEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1&agr;, and optineurin deficiency amplifies IRE1&agr; levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1&Dgr;IEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate–induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1&agr; aggregates during ER stress at this site.

IRE1 signaling exacerbates Alzheimer's disease pathogenesis

Altered proteostasis is a salient feature of Alzheimer’s disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress and abnormal protein aggregation. ER stress triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis or eliminate terminally damaged cells. IRE1 is an ER-located kinase and endoribonuclease that operates as a major stress transducer, mediating both adaptive and proapoptotic programs under ER stress. IRE1 signaling controls the expression of the transcription factor XBP1, in addition to degrade several RNAs. Importantly, a polymorphism in the XBP1 promoter was suggested as a risk factor to develop AD. Here, we demonstrate a positive correlation between the progression of AD histopathology and the activation of IRE1 in human brain tissue. To define the significance of the UPR to AD, we targeted IRE1 expression in a transgenic mouse model of AD. Despite initial expectations that IRE1 signaling may protect against AD, genetic ablation of the RNase domain of IRE1 in the nervous system significantly reduced amyloid deposition, the content of amyloid β oligomers, and astrocyte activation. IRE1 deficiency fully restored the learning and memory capacity of AD mice, associated with improved synaptic function and improved long-term potentiation (LTP). At the molecular level, IRE1 deletion reduced the expression of amyloid precursor protein (APP) in cortical and hippocampal areas of AD mice. In vitro experiments demonstrated that inhibition of IRE1 downstream signaling reduces APP steady-state levels, associated with its retention at the ER followed by proteasome-mediated degradation. Our findings uncovered an unanticipated role of IRE1 in the pathogenesis of AD, offering a novel target for disease intervention.

IRE1α promotes viral infection by conferring resistance to apoptosis

A prosurvival signaling pathway in cells infected with hepatitis C virus overrides interferon responses to impair antiviral resistance. Resisting death by virus The unfolded protein response (UPR) alleviates the cellular stress caused by the accumulation of proteins, such as that occurring during viral infection. During the UPR, processing of Xbp1 mRNA by the nuclease IRE1α generates the transcription factor XBP1, which drives the expression of genes encoding type I interferons (IFNs). Fink et al. found that Xbp1-deficient cells had defective antiviral responses to infection by hepatitis C virus (HCV); however, the cells were not compromised in type I IFN production or responses. Instead, these cells showed increased activation of IRE1α, which cleaved the proapoptotic microRNA miR-125a, leading to decreased apoptosis and increased viral replication. Liver biopsies from HCV-infected patients also showed increased IRE1α activation and decreased miR-125a abundance. Together, these data suggest that IRE1α functions to enhance cell survival in response to viral infection and may provide a potential therapeutic target. The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-folding stresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) promote type I interferon (IFN) responses. We found that Xbp1-deficient mouse embryonic fibroblasts and macrophages had impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather an inability of Xbp1-deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infection in wild-type cells. Xbp1-deficient cells were generally resistant to the intrinsic pathway of apoptosis through an indirect mechanism involving activation of the nuclease IRE1α. We observed an IRE1α-dependent reduction in the abundance of the proapoptotic microRNA miR-125a and a corresponding increase in the amounts of the members of the antiapoptotic Bcl-2 family. The activation of IRE1α by the hepatitis C virus (HCV) protein NS4B in XBP1-proficient cells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1α activation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to those in the livers of healthy controls. Our results reveal a prosurvival role for IRE1α in virally infected cells and suggest a possible target for IFN-independent antiviral therapy.

Crocetin protects ultraviolet A-induced oxidative stress and cell death in skin in vitro and in vivo

Crocetin, the aglycone of crocin, is a carotenoid found in fruits of gardenia (Gardeina jasminoides Ellis) and saffron (Crocus sativus L.). We investigated the protective effects of crocetin against ultraviolet-A (UV-A)-induced skin damage and explored the underlying mechanism. Human skin-derived fibroblasts cells (NB1-RGB) were damaged by exposure to UV-A irradiation (10J/cm(2)). Crocetin protected these cells against cell death and reduced the production of reactive oxygen species induced by UV-A irradiation. Crocetin treatment also suppressed induction of caspase-3 activation by UV-A irradiation. The effects of crocetin against oxidative stress were also examined by imaging of Keap1-dependent oxidative stress detector (OKD) mice. UV-A irradiation upregulated oxidative stress in the OKD mice skin, while crocetin administration (100mg/kg, p.o.) ameliorated this oxidative stress. Crocetin administration also decreased lipid peroxidation in the skin. These findings suggest that crocetin its observed protective effects against UV-A induced skin damage by reducing reactive oxygen species production and cell apoptosis.

Nuclear factor (erythroid derived 2)-like 2 activation increases exercise endurance capacity via redox modulation in skeletal muscles

Sulforaphane (SFN) plays an important role in preventing oxidative stress by activating the nuclear factor (erythroid derived 2)-like 2 (Nrf2) signalling pathway. SFN may improve exercise endurance capacity by counteracting oxidative stress-induced damage during exercise. We assessed running ability based on an exhaustive treadmill test (progressive-continuous all-out) and examined the expression of markers for oxidative stress and muscle damage. Twelve- to 13-week-old Male wild-type mice (Nrf2+/+) and Nrf2-null mice (Nrf2−/−) on C57BL/6J background were intraperitoneally injected with SFN or vehicle prior to the test. The running distance of SFN-injected Nrf2+/+ mice was significantly greater compared with that of uninjected mice. Enhanced running capacity was accompanied by upregulation of Nrf2 signalling and downstream genes. Marker of oxidative stress in SFN-injected Nrf2+/+ mice were lower than those in uninjected mice following the test. SFN produced greater protection against muscle damage during exhaustive exercise conditions in Nrf2+/+ mice than in Nrf2−/− mice. SFN-induced Nrf2 upregulation, and its antioxidative effects, might play critical roles in attenuating muscle fatigue via reduction of oxidative stress caused by exhaustive exercise. This in turn leads to enhanced exercise endurance capacity. These results provide new insights into SFN-induced upregulation of Nrf2 and its role in improving exercise performance.

Deletion of inositol-requiring enzyme-1α in podocytes disrupts glomerular capillary integrity and autophagy

IRE1α is essential for maintaining podocyte and glomerular integrity as mice age and in glomerulonephritis. The mechanism is related, at least in part, to the maintenance of autophagy in podocytes.

ERα promotes murine hematopoietic regeneration through the Ire1α-mediated unfolded protein response

Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.

IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1α. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1α controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1α is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1α expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1α also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1α in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.Urra et al. discover that IRE1α, an ER stress mediator, interacts with filamin A and controls actin dynamics and cell migration in mouse, Drosophila and zebrafish models in a manner independent of its canonical function.

IRE1–XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells

In mammalian pancreatic &bgr; cells, the IRE1&agr;–XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed &bgr; cell–specific Ire1&agr; conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1&agr; was deleted using the Cre–loxP system. Ire1&agr; CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4–20 weeks. Ire1&agr; deletion in pancreatic &bgr; cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1&agr;–XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1&agr; functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic &bgr; cells.

Real-time in vivo imaging reveals localised Nrf2 stress responses associated with direct and metabolism-dependent drug toxicity

The transcription factor Nrf2 coordinates an adaptive response to chemical and oxidative stress characterised by the upregulated expression of cytoprotective target genes. In order to understand the mechanistic relevance of Nrf2 as a marker of drug-induced stress it is important to know if this adaptive response is truly localised in the context of organ-specific drug toxicity. Here, we address this knowledge gap through real-time bioluminescence imaging of transgenic Nrf2-luciferase (Nrf2-luc) reporter mice following administration of the metabolism-dependent hepatotoxin acetaminophen (APAP) or the direct nephrotoxin cisplatin. We detected localised bioluminescence in the liver (APAP) and kidneys (cisplatin) in vivo and ex vivo, whilst qPCR, Taqman low-density array and immunoblot analysis of these tissues further revealed increases in the expression level of several endogenous Nrf2-regulated genes/proteins, including heme oxygenase 1 (Hmox1). Consistent with the toxic effects of APAP in the liver and cisplatin in the kidney, immunohistochemical analysis revealed the elevated expression of luciferase and Hmox1 in centrilobular hepatocytes and in tubular epithelial cells, respectively. In keeping with the role of reactive metabolite formation in APAP-induced chemical stress, both the hepatotoxicity and localised Nrf2-luc response were ameliorated by the cytochrome P450 inhibitor aminobenzotriazole. Together, these findings show that Nrf2 can reflect highly-localised cellular perturbations associated with relevant toxicological mechanisms.