IRE1α Pathway Research Articles

DMDD, isolated from Averrhoa carambola L., ameliorates diabetic nephropathy by regulating endoplasmic reticulum stress-autophagy crosstalk

BackgroundStudies have shown that Averrhoa carambola L. possesses therapeutic potential for diabetes and related complications. However, the specific beneficial effects and molecular mechanisms of 2-dodecyl-6-meth-oxycyclohexa-2,5-diene-1,4-dione (DMDD) isolated from Averrhoa carambola L. on diabetic nephropathy (DN) require further investigation.Methods80 C57BL/6 J male mice were subjected to a 1-week adaptive feeding, followed by a high-fat diet and intraperitoneal injection of 100 mg/kg streptozotocin (STZ) to construct an in vivo DN model. Additionally, human renal proximal tubular epithelial cells (HK-2) induced by high glucose (HG) were used as an in vitro DN model. The expression levels of epithelial-mesenchymal transition (EMT), endoplasmic reticulum stress (ERS), and autophagy-related proteins in renal tubular cells were detected by Western Blot, flow cytometry, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) staining. Transcriptome analysis revealed was conducted to elucidate the specific mechanism of by which DMDD mitigates DN by inhibiting ERS and autophagy. HK-2 cells were transfected with IRE1α overexpression lentivirus to reveal the role of IRE1α overexpression in HG-induced HK-2.ResultsThe experimental data showed that DMDD significantly reduced blood glucose levels and improved renal pathological alterations in DN mice. Additionally, DMDD inhibited the calcium (Ca2+) pathway, manifested by decreased autophagosome formation and downregulation of LC3II/I, Beclin-1, and ATG5 expression. Moreover, in HG-induced HK-2 cells, DMDD suppressed the overexpression of GRP78, CHOP, LC3II/I, Beclin1, and ATG5. Notably, IRE1α overexpression significantly increased autophagy incidence; however, DMDD treatment subsequently reduced the expression of LC3II/I, Beclin1, and ATG5.ConclusionDMDD effectively inhibits excessive ERS and autophagy, thereby reducing renal cell apoptosis through the IRE1α pathway and Ca 2+ pathway.

Regulation of lipid metabolism in grass carpprimaryhepatocytes by exosomes derived from fatty hepatocytes though GRP78.

Exosomes regulate lipid metabolism by carrying miRNAs, nucleic acids, and proteins, thereby influencing the function of receptor cells. Glucose-regulated protein 78 (GRP78) is also involved in the regulation of lipid metabolism. However, it remains unclear whether exosomes derived from fatty hepatocytes (OA-Exo) regulate lipid metabolism through the enrichment of GRP78. In this study, we observed the expression of GRP78 was significantly increased in fatty hepatocytes (incubating hepatocytes with oleic acid (OA) for 24h) and OA-Exo (P < 0.05). In addition, OA-Exo (50μg/mL) and GRP78 protein (1μg/mL) significant increasedthe content of triacylglycerol (TG) and total cholesterol (TC), as well as up-regulatedthe expression of GRP78 and inositol-requiring enzyme-1alpha (IRE1α) protein (P < 0.05). We further used YUM70 (an inhibitor of GRP78) to inhibit endogenous GRP78, and compared with the YUM70 group, OA-Exo reversed the effect of YUM70 and increased the content of TG, TC, and the expression of GRP78 protein in hepatocytes (P < 0.05). Furthermore, the inhibition of the IRE1α pathway with 4μ8C resulted in a significant decrease in TG content compared to the control group (P < 0.05). However, when compared with the 4μ8C group, OA-Exo and GRP78 reversed the effect of 4μ8C and significantly increased TG content (P < 0.05). Taken together, these results indicated that OA-Exo activated IRE1α to promote lipid accumulation in hepatocytes through the enrichment of GRP78. This study provided a new perspective for further exploration of exosomal lipid metabolism in fish.

Krüppel-like factor 6 involvement in the endoplasmic reticulum homeostasis of extravillous trophoblasts

IntroductionTrophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis. Materials and methodsProtein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized. ResultsKLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased. DiscussionResults provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.

IRE1α pathway: A potential bone metabolism mediator.

Osteoblasts and osteoclasts collaborate in bone metabolism, facilitating bone development, maintaining normal bone density and strength, and aiding in the repair of pathological damage. Endoplasmic reticulum stress (ERS) can disrupt the intracellular equilibrium between osteoclast and osteoblast, resulting in dysfunctional bone metabolism. The inositol-requiring enzyme-1α (IRE1α) pathway-the most conservative unfolded protein response pathway activated by ERS-is crucial in regulating cell metabolism. This involvement encompasses functions such as inflammation, autophagy, and apoptosis. Many studies have highlighted the potential roles of the IRE1α pathway in osteoblasts, chondrocytes, and osteoclasts and its implication in certain bone-related diseases. These findings suggest that it may serve as a mediator for bone metabolism. However, relevant reviews on the role of the IRE1α pathway in bone metabolism remain unavailable. Therefore, this review aims to explore recent research that elucidated the intricate roles of the IRE1α pathway in bone metabolism, specifically in osteogenesis, chondrogenesis, osteoclastogenesis, and osteo-immunology. The findings may provide novel insights into regulating bone metabolism and treating bone-related diseases.

The Interplay Between Endoplasmic Reticulum Stress and Oxidative Stress in Chondrocyte Catabolism.

Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation. Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM). Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA in vivo. Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.

Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.

ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.

The IRE1α Endonuclease Plays a Dual Role in Regulating the XBP1/miRNA-34a Axis and PD-1 Expression within Natural Killer Cells in Hodgkin Lymphoma

Plain Language SummaryNatural killer (NK) cells play a critical role in our innate immune defense against tumors without prior exposure to cancer cells and, therefore, make an attractive instrument to harness antitumoral immunity as novel immunotherapies. Hodgkin lymphoma (HL) typically lacks major histocompatibility complex class I which is a requirement for an antigen-specific recognition of effector T cells but not for the NK cell antitumoral function. NK cells are dysfunctional in HL patients mainly due to upregulation of immune checkpoint PD-1 expression that inhibits immune cells effector functions. However, the mechanisms underpinning PD-1 expression and NK cell dysfunction remain unknown. In this study, we uncovered the mechanism by which immune checkpoint PD-1 expression is upregulated upon activation of the endonuclease IRE1α to rapidly change the abundance of key regulatory molecules including a small noncoding regulatory RNA (microRNA-34a) and the transcription factor XBP1. The IRE1α endonuclease plays a dual role in regulating the XBP1/miRNA-34a axis and PD-1 expression within NK cells, which is disrupted in patients with HL. IRE1α pathway in NK cells represents a potential target to optimize NK cell immune functions in future HL treatments.

Activation of ACLY by SEC63 deploys metabolic reprogramming to facilitate hepatocellular carcinoma metastasis upon endoplasmic reticulum stress

BackgroundTumor cells display augmented capability to maintain endoplasmic reticulum (ER) homeostasis and hijack ER stress pathway for malignant phenotypes under microenvironmental stimuli. Metabolic reprogramming is a well-known hallmark for tumor cells to provide specific adaptive traits to the microenvironmental alterations. However, it’s unknown how tumor cells orchestrate metabolic reprogramming and tumor progression in response to ER stress. Herein, we aimed to explore the pivotal roles of SEC63-mediated metabolic remodeling in hepatocellular carcinoma (HCC) cell metastasis after ER stress.MethodsThe expression levels of SEC63 in HCC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry and western blot. The regulatory roles of SEC63 in HCC metastasis were investigated both in vitro and in vivo by RNA-sequencing, metabolites detection, immunofluorescence, and transwell migration/invasion analyses. GST pull-down, immunoprecipitation/mass spectrometry and in vivo ubiquitination/phosphorylation assay were conducted to elucidate the underlying molecular mechanisms.ResultsWe identified SEC63 as a new regulator of HCC cell metabolism. Upon ER stress, the phosphorylation of SEC63 at T537 by IRE1α pathway contributed to SEC63 activation. Then, the stability of ACLY was upregulated by SEC63 to increase the supply of acetyl-CoA and lipid biosynthesis, which are beneficial for improving ER capacity. Meanwhile, SEC63 also entered into nucleus for increasing nuclear acetyl-CoA production to upregulate unfolded protein response targets to improve ER homeostasis. Importantly, SEC63 coordinated with ACLY to epigenetically modulate expression of Snail1 in the nucleus. Consequently, SEC63 promoted HCC cell metastasis and these effects were reversed by ACLY inhibition. Clinically, SEC63 expression was significantly upregulated in HCC tissue specimens and was positively correlated with ACLY expression. Importantly, high expression of SEC63 predicted unfavorable prognosis of HCC patients.ConclusionsOur findings revealed that SEC63-mediated metabolic reprogramming plays important roles in keeping ER homeostasis upon stimuli in HCC cells. Meanwhile, SEC63 coordinates with ACLY to upregulate the expression of Snail1, which further promotes HCC metastasis. Metastasis is crucial for helping cancer cells seek new settlements upon microenvironmental stimuli. Taken together, our findings highlight a cancer selective adaption to ER stress as well as reveal the potential roles of the IRE1α-SEC63-ACLY axis in HCC treatment.

IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

Endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR) are involved in various muscle pathological states. The IRE1α arm of UPR can affect immunological properties of myofiber through restraining p38 mitogen-activated protein kinases (MAPK) activation under inflammatory milieu. However, the relevant pathway molecules regulating the initiation of the IRE1α arm in myofiber remain unclear. In this work, expression of transforming growth factor-beta (TGF-β) and TGF-β receptor II (TGF-βr2), and UPR pathway activation were examined in cardiotoxin (CTX)-damaged mouse muscle, which revealed the activation of TGF-β signaling and UPR in CTX-damaged muscle and in regenerating myofibers. Using control or transgenic mice with TGF-βr2 deleted in skeletal muscle (SM TGF-βr2−/−) and the derived primary differentiating myogenic precursor cells (MPCs) treated with/without ERS activator or inhibitor, IRE1α pathway inhibitor, or TGF-β signaling activator, this study further revealed an essential role of intrinsic TGF-β signaling in regulating muscle cell to express inflammation-related molecules including H-2Kb, H2-Eα, TLR3, and special myokines. TGF-β signaling prompted UPR IRE1α arm and restrained p38 MAPK activation in myofiber under inflammatory milieu. This study uncovers a previously unrecognized function of TGF-β signaling acting as an upstream factor controlling myofiber immune capacities in the inflamed state through the UPR–IRE1α–p38 MAPK pathway.

Bushen Yizhi Formula regulates the IRE1α pathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

<b>Background:</b> While the Bushen Yizhi Formula can treat Alzheimer’s disease (AD), the yet to be ascertained specific mechanism of action was explored in this work. <b>Methods:</b> Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide (Aβ) were used to treat rat pheochromocytoma cells (P12) and human neuroblastoma cells (SH-SY5Y). Cell morphological changes were observed to determine the <i>in vitro</i> cell damage. Cell Counting Kit (CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle, respectively. Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress (ERS)-related proteins (GRP78 and CHOP), p-IRE1α, IRE1α, ASK1, p-JNK, JNK, Bax, Bcl-2, XBP-1, and Bim. Fura 2-acetoxymethyl ester (Fura-2/AM) was used to determine the intracellular calcium (Ca²⁺) concentration. Also, an AD model was constructed by injecting Aβ into the CA1 area of the hippocampus in Sprague Dawley rats. AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days. The Morris water maze experiment was conducted to test the learning and memory of rats. Hematoxylin & Eosin (H&E) and Terminal-deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) staining were done to determine histopathological changes in the brain. <b>Results: </b>Bushen Yizhi Formula relieved the Aβ-induced effects including cell injury, decreased viability, increased apoptosis, G0/G1 phase cell cycle arrest, upregulation of GRP78, CHOP, p-IRE1α, p-JNK, Bax, XBP-1 and Bim, as well as down-regulation of Bcl-2. These results were also seen with IRE1α silencing. While Aβ suppressed the learning and memory abilities of rats, the Bushen Yizhi Formula alleviated these effects of Aβ. Brain nerve cell injury induced by Aβ could also be treated with Bushen Yizhi Formula. <b>Conclusion: </b>Bushen Yizhi Formula could influence ERS through the IRE1α signaling pathway to achieve its therapeutic effects on AD.

Thioredoxin-1 regulates IRE1α to ameliorate sepsis-induced NLRP3 inflammasome activation and oxidative stress in Raw 264.7 cell

Objective: Sepsis is life-threatening organ dysfunction caused by the dysregulated host response to infection. Endoplasmic reticulum stress (ERS)-mediated inositol-requiring enzyme 1 α (IRE1α) inflammatory signaling pathway is involved in sepsis. NLRP3 inflammasome plays a key role in the activation of caspase-1 and the maturation of IL-1β and IL-18, and finally enhances the inflammatory response. More and more evidences show that ERS is an endogenous trigger of NLRP3 inflammasome. Thioredoxin-1 (Trx-1) is a small ubiquitous thiol-1 protein with redox/inflammation modulatory properties relevant to sepsis pathogenesis. In this study, we investigated the role of Trx-1 in ERS mediated IRE1α/NLRP3 signaling pathway in Raw 264.7 cells. Methods: Raw 264.7 cells stimulated by LPS were used to construct an inflammation model of sepsis in vitro, and the expression of proteins related to the IRE1α/NLRP3 pathway was detected through using western blot and RT-PCR. The expression of IL-18 and IL-1β in cell supernatant was also measured by ELISA, and caspase 1 activity and ROS expression in cells were detected by kits. Results: Our study shows that IRE1α signaling pathway related to endoplasmic reticulum stress in sepsis can activate inflammation related genes, and stimulate to produce a large number of pro-IL-1β. At the same time, IRE1α can activate NLRP3 inflammasome and promote activation and maturation of pro-IL-1β. Finally leads to excessive inflammatory response and ROS release, and promotes the progress of sepsis. Conclusions: Trx-1 may inhibit NLRP3 activity and pro-IL-Iβ production by inhibit IRE1α pathway of ER stress. So as to inhibit inflammatory response and ROS of cells, and play a protective role in sepsis.

The IRE1α pathway in glomerular diseases: The unfolded protein response and beyond.

Endoplasmic reticulum (ER) function is vital for protein homeostasis ("proteostasis"). Protein misfolding in the ER of podocytes (glomerular visceral epithelial cells) is an important contributor to the pathogenesis of human glomerular diseases. ER protein misfolding causes ER stress and activates a compensatory signaling network called the unfolded protein response (UPR). Disruption of the UPR, in particular deletion of the UPR transducer, inositol-requiring enzyme 1α (IRE1α) in mouse podocytes leads to podocyte injury and albuminuria in aging, and exacerbates injury in glomerulonephritis. The UPR may interact in a coordinated manner with autophagy to relieve protein misfolding and its consequences. Recent studies have identified novel downstream targets of IRE1α, which provide new mechanistic insights into proteostatic pathways. Novel pathways of IRE1α signaling involve reticulophagy, mitochondria, metabolism, vesicular trafficking, microRNAs, and others. Mechanism-based therapies for glomerulopathies are limited, and development of non-invasive ER stress biomarkers, as well as targeting ER stress with pharmacological compounds may represent a therapeutic opportunity for preventing or attenuating progression of chronic kidney disease.

TGFβ1 regulates HRas-mediated activation of IRE1α through the PERK-RPAP2 axis in keratinocytes.

Transforming Growth Factor β1 (TGFβ1) is a critical regulator of tumor progression in response to HRas. Recently, TGFβ1 has been shown to trigger ER stress in many disease models; however, its role in oncogene-induced ER stress is unclear. Oncogenic HRas induces the unfolded protein response (UPR) predominantly via the Inositol-requiring enzyme 1α (IRE1α) pathway to initiate the adaptative responses to ER stress, with importance for both proliferation and senescence. Here, we show a role of the UPR sensor proteins IRE1α and (PKR)-like endoplasmic reticulum kinase (PERK) to mediate the tumor-suppressive roles of TGFβ1 in mouse keratinocytes expressing mutant forms of HRas. TGFβ1 suppressed IRE1α phosphorylation and activation by HRas both in in vitro and in vivo models while simultaneously activating the PERK pathway. However, the increase in ER stress indicated an uncoupling of ER stress and IRE1α activation by TGFβ1. Pharmacological and genetic approaches demonstrated that TGFβ1-dependent dephosphorylation of IRE1α was mediated by PERK through RNA Polymerase II Associated Protein 2 (RPAP2), a PERK-dependent IRE1α phosphatase. In addition, TGFβ1-mediated growth arrest in oncogenic HRas keratinocytes was partially dependent on PERK-induced IRE1α dephosphorylation and inactivation. Together, these results demonstrate a critical cross-talk between UPR proteins that is important for TGFβ1-mediated tumor suppressive responses.

259-LB: Inhibition of the Polyamine-Hypusine Circuit Delimits Islet ß-Cell Stress in the Context of T1D via Suppression of IRE-1a Signaling

Recent data suggest that stress-induced molecular pathways in the β cell may trigger the display of neoantigens that initiate autoimmunity in type 1 diabetes (T1D) . Polyamines (putrescine, spermidine, and spermine) are biogenic amines that participate in stress-induced mRNA translation, in part, through the posttranslational hypusine modification of eIF5A. Rate-limiting enzymes in this circuit include the ODC, which converts Arg to polyamines, and DHPS, which controls eIF5A hypusination. Previously, we showed that inhibition of ODC or DHPS in non-obese diabetic mice using either DFMO or GC7, respectively, resulted in preserved β-cell function, enhanced β-cell mass, and significantly reduced T1D incidence. DFMO treatment subjects with recent-onset T1D was suggestive of reductions in β cell stress. We hypothesized that polyamine depletion with DFMO reduces inflammatory stress in β cells and the consequent accumulation of unhypusinated eIF5A limits stress-related protein production. To test the hypothesis, we treated the human islets with proinflammatory cytokine (PIC) cocktail to mimic T1D in presence or absence of 5 mM DFMO. We observed induction of ER stress as shown by increased expression of unfolded protein response proteins within 24 h of PIC treatment. Treatment with DFMO reduced protein levels of IRE1α and downstream XBP1. Gene expression analysis showed significant downregulation of genes in the IRE1α pathway (CHOP, TNXIP, and XBP1s) . To clarify a mechanism underlying suppression of stress protein production by unhypusinated eIF5A, we treated HEK cells with DFMO or GC7, both of which enhanced the association of unhypusinated eIF5A with GCN2, an eIF2α kinase, suggesting a non-ER stress mediated mechanism of mRNA translational blockade. Collectively, these data suggest that inhibition of the polyamines-hypusine circuit suppresses ER stress via IRE1α signaling and enhances non-ER stress mediated suppression of mRNA translation. Disclosure A. Anderson: None. A. Kulkarni: None. R. G. Mirmira: None.

Gestational cholestasis induced intrauterine growth restriction through triggering IRE1α-mediated apoptosis of placental trophoblast cells.

Epidemiological and animal experimental studies suggest an association between gestational cholestasis and intrauterine growth restriction (IUGR). Here, we explored the mechanism through which gestational cholestasis induced IUGR. To establish gestational cholestasis model, pregnant mice were subcutaneously injected with 17α-Ethynylestradiol (E2) on gestational day 13 (GD13)-GD17. Some pregnant mice were intraperitoneally injected with 4μ8C on GD13-GD17. The results found that the apoptosis of trophoblast cells was elevated in placentas of mice with gestational cholestasis and in deoxycholic acid (DCA)-treated human trophoblast cell lines and primary mouse trophoblast cells. Correspondingly, the levels of placental cleaved caspase-3 and Bax were increased, while placental Bcl2level was decreased in mice with gestational cholestasis and in DCA-treated trophoblast cells. Further analysis found that placental IRE1α pathway was activated in mice with gestational cholestasis and in DCA-treated trophoblast cells. Interestingly, 4μ8C, an IRE1α RNase inhibitor, significantly inhibited caspase-3 activity and apoptosis of trophoblast cells in vivo and in vitro. Importantly, 4μ8C rescued gestational cholestasis-induced placental insufficiency and IUGR. Furthermore, a case-control study demonstrated that placental IRE1α and caspase-3 pathways were activated in cholestasis cases. Our results provide evidence that gestational cholestasis induces placental insufficiency and IUGR may be via triggering IRE1α-mediated apoptosis of placental trophoblast cells.

Caveolin-1-Derived Peptide Reduces ER Stress and Enhances Gelatinolytic Activity in IPF Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor β (TGFβ)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFβ-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFβ-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFβ-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFβ (Ad-TGFβ)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.

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.

Pharmacological Inhibition of IRE-1 Alpha Activity in Herpes Simplex Virus Type 1 and Type 2-Infected Dendritic Cells Enhances T Cell Activation.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections are life-long and highly prevalent in the human population. These viruses persist in the host, eliciting either symptomatic or asymptomatic infections that may occur sporadically or in a recurrent manner through viral reactivations. Clinical manifestations due to symptomatic infection may be mild such as orofacial lesions, but may also translate into more severe diseases, such as ocular infections that may lead to blindness and life-threatening encephalitis. A key feature of herpes simplex viruses (HSVs) is that they have evolved molecular determinants that hamper numerous components of the host’s antiviral innate and adaptive immune system. Importantly, HSVs infect and negatively modulate the function of dendritic cells (DCs), by inhibiting their T cell-activating capacity and eliciting their apoptosis after infection. Previously, we reported that HSV-2 activates the splicing of the mRNA of XBP1, which is related to the activity of the unfolded protein response (UPR) factor Inositol-Requiring Enzyme 1 alpha (IRE-1α). Here, we sought to evaluate if the activation of the IRE-1α pathway in DCs upon HSV infection may be related to impaired DC function after infection with HSV-1 or HSV-2. Interestingly, the pharmacological inhibition of the endonuclease activity of IRE-1α in HSV-1- and HSV-2-infected DCs significantly reduced apoptosis in these cells and enhanced their capacity to migrate to lymph nodes and activate virus-specific CD4+ and CD8+ T cells. These findings suggest that the activation of the IRE-1α-dependent UPR pathway in HSV-infected DCs may play a significant role in the negative effects that these viruses exert over these cells and that the modulation of this signaling pathway may be relevant for enhancing the function of DCs upon infection with HSVs.

Apolipoprotein E4 drives tau propagation through astrocyte-secreted glypican-4.

Apolipoprotein E4 (APOE4) is the most crucial genetic risk factor of late-onset Alzheimer's disease (AD). However, the mechanism through which APOE4 induces AD risk remains unknown. We have utilized post-mortem AD brain tissues of APOE variants, biochemical assays and a series of in vitro and in vivo experiments with transgenic mouse models to study the role of GPC-4 in APOE4 induced tau pathology. Here, we report the astrocyte-secreted protein glypican-4 (GPC-4), as a novel binding partner of APOE4, drives tau pathology. APOE4-carrying post-mortem AD brain tissue display more tau accumulation compared to APOE4-noncarring AD patients. GPC-4 is highly expressed in APOE4 post-mortem AD brains and regulated by microglial factors via NF-κB signaling pathway. The astrocyte-secreted GPC-4 induced both tau accumulation and spreading in vitro and in vivo animal models. Further, GPC-4 is required for APOE4-mediated surface trafficking of low-density lipoprotein receptor-related protein 1 (LRP1) and tau propagation. GPC-4 activates unfolded protein response (UPR) pathway IRE1α, and pharmacological inhibition of IRE1α with KIRA6 blocks GPC-4 induced tau propagation in in vivo animal models. Together, our data demonstrate that one of the APOE4-induced AD risk is directly mediated by GPC-4, and that perturbing GPC-4 induced IRE1α pathway has therapeutic opportunities.

IRE1α-Driven Inflammation Promotes Clearance of Citrobacter rodentium Infection.

ABSTRACTEndoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.