The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.     

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.     

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation

Necrosis is a form of cell death that is detrimental to the affected tissue because the cell ruptures and releases its content (reactive oxygen species among others) into the extracellular space. Clusterin (CLU), a cytoprotective extracellular chaperone has been shown to be upregulated in the face of necrosis. We here show that in addition to CLU upregulation, necrotic cell lysates induce JNK/SAPK signaling, the IRE1α branch of the unfolded protein response (UPR), the MAPK/ERK1/2, and the mTOR signaling pathways and results in an enhanced proliferation of the vital surrounding cells. We name this novel response mechanism: Necrosis-induced Proliferation (NiP).     

Downregulation of hepatic glucose-6-phosphatase-α in patients with hepatic steatosis

Glucose-6-phosphate transporter (G6PT) and microsomal glucose-6-phosphatase-α (G6Pase-α) perform the terminal step in glycogenolysis and gluconeogenesis. Deficiency of these proteins leads to glycogen storage diseases. Partial inhibition of G6Pase in rats results in increased hepatic triglyceride content and de novo lipogenesis leading to hepatic steatosis. Hepatic steatosis represents hepatic manifestation of the metabolic syndrome. We investigated molecular mechanisms that may explain the relationship between fatty liver and G6Pase-α in humans in detail. A total of 27 patients (11 men, 16 women) underwent liver biopsy. Histological diagnosis identified nonfatty liver in seven patients and nonalcoholic fatty liver in 20 patients. We quantified G6Pase-α and G6PT mRNA expression by real-time PCR. Anthropometric measurements and analysis of plasma lipids and liver enzymes were performed. Patients with fatty liver showed no significant differences in age, HOMA(IR) (homeostasis model assessment of insulin resistance), BMI, liver enzymes or waist-to-hip ratio compared to those with nonfatty liver, but total plasma cholesterol levels and liver fat content were higher in patients with fatty liver (P < 0.05). G6Pase-α and G6PT mRNA expressions were significantly downregulated in fatty compared to histologically normal liver (P < 0.05). G6Pase-α and G6PT mRNA expressions correlated positively (R(2) = 0.406 P < 0.05). Both expressions did not correlate with age, BMI, aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase, triglycerides or glucose levels. Our data suggest that expression of hepatic G6Pase-α and G6PT are closely interlinked. Downregulation of G6Pase-α in fatty liver might be associated with hepatic fat accumulation and pathogenesis of hepatic steatosis.     

The unknown face of IRE1α – Beyond ER stress

IRE1α (Inositol Requiring kinase Enzyme 1 alpha), a transmembrane protein localized to the endoplasmic reticulum (ER) is a master regulator of the unfolded protein response (UPR) pathway. The fate determining steps during ER stress-induced apoptosis are greatly attributed to IRE1α’s endoribonuclease and kinase activities. Apart from its role as a chief executioner in ER stress, recent studies have shown that upon activation in the presence or absence of ER stress, IRE1α executes multiple cellular processes such as differentiation, immune response, progression and repression of the cell cycle. Besides its crucial role in protein misfolding, the versatile contributions of IRE1α in other cellular functions are greatly unknown. In this review, we have discussed the structural conservation of IRE1 among eukaryotes, the mechanisms underlying its activation and the recent understandings of the non-apoptotic functions of IRE1 other than ER stress-induced cell death.     

Apocynin protects endothelial cells from endoplasmic reticulum stress-induced apoptosis via IRE1α engagement

Molecular and Cellular Biochemistry - Endoplasmic reticulum (ER) stress-induced endothelial cell (EC) apoptosis has been implicated in a variety of human diseases. In addition to being regarded as...     

Activation of mammalian IRE1α upon ER stress depends on dissociation of BiP rather than on direct interaction with unfolded proteins

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response. Upon ER stress, IRE1 senses the accumulation of unfolded proteins in the ER, and transfers signal from the ER to the cytosol. Recently, it was reported that the luminal domain of yeast Ire1 senses the unfolded proteins via a two-step mechanism, namely dissociation of BiP and direct interaction with unfolded proteins. However, it has been unclear whether a similar mechanism is applicable to mammalian IRE1α. To address this point, we analyzed luminal-domain mutants of mammalian IRE1α in cells, and evaluated the anti-aggregation activity of the luminal fragment of IRE1α in vitro. We generated a mutant that has low affinity for BiP, and this mutant was significantly activated even under normal conditions. Moreover, the luminal fragments of mammalian IRE1α did not exhibit anti-aggregation activity. These results suggest that in contrast to yeast Ire1, the regulation of mammalian IRE1α strongly depends on the dissociation of BiP.     

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.     

Neuronal ER Stress Impedes Myeloid-Cell-Induced Vascular Regeneration through IRE1α Degradation of Netrin-1

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Highlights? Canonical ER stress pathways are activated in central neurons during hypoxia/ischemia ? The ER stress endoribonuclease IRE1α degrades the neurovascular guidance cue netrin-1 ? Neuronal-derived netrin-1 activates a reparative proangiogenic program in microglial cells ? Neuronal ER stress prevents reparative angiogenesis in the ischemic neural retina     

IRE1α dissociates with BiP and inhibits ER stress-mediated apoptosis in cartilage development

Bone morphogenetic protein 2 is known to activate unfolded protein response signaling molecules, including XBP1S, BiP and IRE1α. Endoplasmic reticulum stress is induced in chondrogenesis and activates IRE1α signal pathway, which is associated with ER stress-mediated apoptosis. However, the influence on IRE1α and BiP in BMP2-induced chondrocyte differentiation has not yet been elucidated; the molecular mechanism remains unexplored.In this study, we demonstrate that IRE1α interacts with BiP in unstressed cells and dissociates from BiP in the course of cartilage development. Induction of ER stress-responsive proteins (XBP1S, IRE1α, BiP) was also observed in differentiating cells. IRE1α inhibition ER stress-mediated apoptosis lies in the process of chondrocyte differentiation. Furthermore, knockdown of IRE1α expression by way of the RNAi approach accelerates ER stress-mediated apoptosis in chondrocyte differentiation induced by BMP2, as revealed by enhanced expressions of cleaved caspase3, CHOP and p-JNK1; and this IRE1α inhibition effect on ER stress-mediated apoptosis is required for BiP in chondrogenesis.Collectively, the ER stress sensors were activated during apoptosis in cartilage development, suggesting that selective activation of ER stress signaling was sufficient for induction of apoptosis. These findings reveal a novel critical role of IRE1α in ER stress-mediated apoptosis and the molecular mechanisms involved. These results suggest that activation of p-JNK1, caspase3 and CHOP was detected in developing chondrocytes and that specific ER stress signaling leads to naturally occurring apoptosis during cartilage development.     

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α) in human colon cancer cells

To explore the relationship between UPR and autophagy in intestinal epithelial cells, we investigated whether autophagy was induced by endoplasmic reticulum (ER) stress in colon cancer cell lines. We demonstrated that autophagy was induced by ER stress in HT29, SW480, and Caco-2 cells. In these cells, inositol-requiring enzyme1α (IRE1α) and C/EBP homologous protein (CHOP) were involved in the ER stress–autophagy pathway, and CHOP was a regulator of IRE1α protein expression. Our findings suggest that CHOP promotes IRE1α and autophagy especially in ER stress conditions. This study will provide important insights into the disclosure of the ER stress–autophagy pathway.     

Cdc37/Hsp90 protein-mediated regulation of IRE1α protein activity in endoplasmic reticulum stress response and insulin synthesis in INS-1 cells

IRE1α is an endoplasmic reticulum (ER) localized signaling molecule critical for unfolded protein response. During ER stress, IRE1α activation is induced by oligomerization and autophosphorylation in its cytosolic domain, a process triggered by dissociation of an ER luminal chaperone, binding immunoglobulin-protein (BiP), from IRE1α. In addition, inhibition of a cytosolic chaperone protein Hsp90 also induces IRE1α oligomerization and activation in the absence of an ER stressor. Here, we report that the Hsp90 cochaperone Cdc37 directly interacts with IRE1α through a highly conserved cytosolic motif of IRE1α. Cdc37 knockdown or disruption of Cdc37 interaction with IRE1α significantly increased basal IRE1α activity. In INS-1 cells, Hsp90 inhibition and disruption of IRE1α-Cdc37 interaction both induced an ER stress response and impaired insulin synthesis and secretion. These data suggest that Cdc37-mediated direct interaction between Hsp90/Cdc37 and an IRE1α cytosolic motif is important to maintain basal IRE1α activity and contributes to normal protein homeostasis and unfolded protein response under physiological stimulation.