Bax Inhibitor-1 preserves pancreatic ß-cell proteostasis by limiting proinsulin misfolding and programmed cell death.

The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to ß-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving ß-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, ß-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of ß-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to ß-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in ß-cells limited IRE1α RNase activity from triggering programmed ß-cell death through apoptosis and pyroptosis (caspase-1, IL-1ß) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed ß-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic ß-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on ß-cell function and insulin secretion. In pancreatic ß-cells, BI-1-/- deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, ß-cell programmed cell death, and diabetes.

ER stress signaling at the interphase between MASH and hepatocellular carcinoma.

Hepato-cellular carcinoma (HCC) is the most frequent primary liver cancer with an extremely poor prognosis and often develops on preset of chronic liver diseases. Major risk factors for HCC include metabolic dysfunction-associated steatohepatitis (MASH), a complex multifactorial condition associated with abnormal endoplasmic reticulum (ER) proteostasis. To cope with ER stress, the unfolded protein response (UPR) engages adaptive reactions to restore the secretory capacity of the cell. Recent advances revealed that ER stress signaling plays a critical role in HCC progression. Here we propose that chronic ER stress is a common transversal factor contributing to the transition from liver disease (risk factor) to HCC. Interventional strategies to target the UPR in HCC as cancer therapy are also discussed.

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

A plant receptor domain with functional analogies to animal malectin disables ER stress responses upon infection.

Malectins from the oligosaccharyltransferase (OST) complex in the endoplasmic reticulum (ER) of animal cells are involved in ER quality control and contribute to the Unfolded Protein Response (UPR). Malectins are not found in plant cells, but malectin-like domains (MLDs) are constituents of many membrane-bound receptors. In Arabidopsis thaliana, the MLD-containing receptor IOS1 promotes successful infection by filamentous plant pathogens. We show that the MLD of its exodomain retains IOS1 in the ER of plant cells and attenuates the infection-induced UPR. Expression of the MLD in the ios1-1 knockout background is sufficient to complement infection-related phenotypes of the mutant, such as increased UPR and reduced disease susceptibility. IOS1 interacts with the ER membrane-associated ribophorin HAP6 from the OST complex, and hap6 mutants show decreased pathogen-responsive UPR and increased disease susceptibility. Altogether, this study revealed a previously uncharacterized role of a plant receptor domain in the regulation of ER stress during infection.

Humoral and cellular responses after a third dose of SARS-CoV-2 BNT162b2 vaccine in patients with lymphoid malignancies.

Patients with hematological malignancies have impaired immune response after two doses of BNT162b2 (Pfizer/BioNTech) vaccine against SARS-CoV-2. Here, in this observational study (registration number HDH F20210324145532), we measure SARS-CoV-2 anti-Spike antibodies, neutralizing antibodies and T-cell responses after immune stimulation with a third dose (D3) of the same vaccine in patients with chronic lymphocytic leukemia (n = 13), B cell non-Hodgkin lymphoma (n = 14), and multiple myeloma (n = 16)). No unexpected novel side effects are reported. Among 25 patients with positive anti-S titers before D3, 23 (92%) patients increase their anti-S and neutralizing antibody titer after D3. All 18 (42%) initially seronegative patients remain negative. D3 increases the median IFN-γ secretion in the whole cohort and induces IFN-γ secretion in a fraction of seronegative patients. Our data thus support the use of a third vaccine dose amongst patients with lymphoid malignancies, even though some of them will still have vaccine failure.

SYK-3BP2 Pathway Activity in Parenchymal and Myeloid Cells Is a Key Pathogenic Factor in Metabolic Steatohepatitis.

BACKGROUND & AIMS: Spleen tyrosine kinase (SYK) signaling pathway regulates critical processes in innate immunity, but its role in parenchymal cells remains elusive in chronic liver diseases. We investigate the relative contribution of SYK and its substrate c-Abl Src homology 3 domain-binding protein-2 (3BP2) in both myeloid cells and hepatocytes in the onset of metabolic steatohepatitis. METHODS: Hepatic SYK-3BP2 pathway was evaluated in mouse models of metabolic-associated fatty liver diseases (MAFLD) and in obese patients with biopsy-proven MAFLD (n = 33). Its role in liver complications was evaluated in Sh3bp2 KO and myeloid-specific Syk KO mice challenged with methionine and choline deficient diet and in homozygous Sh3bp2KI/KI mice with and without SYK expression in myeloid cells. RESULTS: Here we report that hepatic expression of 3BP2 and SYK correlated with metabolic steatohepatitis severity in mice. 3BP2 deficiency and SYK deletion in myeloid cells mediated the same protective effects on liver inflammation, injury, and fibrosis priming upon diet-induced steatohepatitis. In primary hepatocytes, the targeting of 3BP2 or SYK strongly decreased the lipopolysaccharide-mediated inflammatory mediator expression and 3BP2-regulated SYK expression. In homozygous Sh3bp2KI/KI mice, the chronic inflammation mediated by the proteasome-resistant 3BP2 mutant promoted severe hepatitis and liver fibrosis with augmented liver SYK expression. In these mice, the deletion of SYK in myeloid cells was sufficient to prevent these liver lesions. The hepatic expression of SYK is also up-regulated with metabolic steatohepatitis and correlates with liver macrophages in biopsy-proven MAFLD patients. CONCLUSIONS: Collectively, these data suggest an important role for the SYK-3BP2 pathway in the pathogenesis of chronic liver inflammatory diseases and highlight its targeting in hepatocytes and myeloid cells as a potential strategy to treat metabolic steatohepatitis.

No association between binge eating disorder and severity of non-alcoholic fatty liver disease in severely obese patients.

BACKGROUND AND AIM: The main aim of this study was to evaluate if the binge eating disorders (BEDs) related to obesity were associated with the severity of non-alcoholic fatty liver disease (NAFLD). METHODS: Severely obese patients who had been referred for bariatric surgery were included in this study at the Nice University Hospital. All patients underwent a liver biopsy at the time of surgery. Between 2008 and 2015, 388 patients had an assessable Bulimia Test (BULIT) self-questionnaire at the time of surgery. A subgroup (n = 183), between 2011 and 2015, also responded to a Beck Depression Inventory, Hospital Anxiety and Depression Scale, and a Fatigue Impact Scale autoquestionnaire. A control group of 29 healthy people matched by age and gender was included. RESULTS: Among the 388 obese patients (median age 40 years, body mass index 41.7 kg/m2, 81% women), 14 patients had a "probable diagnosis" of BED, and 47 patients had a "high risk" of developing a BED according to the BULIT. Obese patients had significantly more severe BED, depression, anxiety, and fatigue compared to controls. Steatosis, non-alcoholic steatohepatitis, or fibrosis was not associated with BED. Similarly, the severity of NAFLD was not associated with depression, anxiety, or fatigue. CONCLUSIONS: Severely obese patients had more severe BED, depression, anxiety, and fatigue than lean subjects independent of the severity of NAFLD.

Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells.

To address unmet clinical need for uveal melanomas, we assessed the effects of BH3-mimetic molecules, the ABT family, known to exert pro-apoptotic activities in cancer cells. Our results uncovered that ABT-263 (Navitoclax), a potent and orally bioavailable BCL-2 family inhibitor, induced antiproliferative effects in metastatic human uveal melanoma cells through cell cycle arrest at the G0/G1 phase, loss of mitochondrial membrane potential, and subsequently apoptotic cell death monitored by caspase activation and poly-ADP ribose polymerase cleavage. ABT-263-mediated reduction in tumor growth was also observed in vivo. We observed in some cells that ABT-263 treatment mounted a pro-survival response through activation of the ER stress signaling pathway. Blocking the PERK signaling pathway increased the pro-apoptotic ABT-263 effect. We thus uncovered a resistance mechanism in uveal melanoma cells mediated by activation of endoplasmic reticulum stress pathway. Therefore, our study identifies ABT-263 as a valid therapeutic option for patients suffering from uveal melanoma.

[Endoplasmic reticulum stress response and pathogenesis of non-alcoholic steatohepatitis]. / La réponse au stress du réticulum endoplasmique dans la physiopathologie des maladies chroniques du foie.

The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. Non-alcoholic fatty liver disease (NAFLD) is currently affecting 20-30% of the general population and 75-100% of obese individuals. NAFLD ranges from simple steatosis to damaging non-alcoholic steatohepatitis (NASH), potentially developing into hepatocellular carcinoma. No efficient pharmacological treatment is yet available. During obesity, the hepatic ER stress response can arise from extracellular stress (lipids, glucose, cytokines) and from intracellular stress including lipid buildup in the hepatocyte (steatosis), a hallmark of NAFLD. The chronic activation of the hepatic ER stress response may be a crucial event in the steatosis-NASH transition, triggering cell death, inflammation and accelerating metabolic disorders. We discuss these aspects and we propose that targeting the ER stress response could be effective in treating NAFLD.

TITLE: La réponse au stress du réticulum endoplasmique dans la physiopathologie des maladies chroniques du foie. ABSTRACT: La prévalence des maladies chroniques du foie ne cesse d'augmenter, du fait de la pandémie de l'obésité. Ces maladies s'étendent de la bégnine stéatose à la stéatopathie non alcoolique (NASH) qui peut évoluer vers le carcinome hépatocellulaire. Il n'existe pas de traitement pour ces maladies. La transition stéatose-NASH apparaît déterminante dans leur progression. Au cours de l'obésité, l'activation chronique de la réponse au stress du réticulum endoplasmique (RE) jouerait un rôle crucial dans cette transition, conduisant à la mort cellulaire, à l'inflammation et à l'aggravation des désordres métaboliques. Dans cette revue, nous discutons ces aspects et proposons que le ciblage de cette réponse au stress du RE puisse être pertinent dans la prise en charge thérapeutique de la NASH.

Hepatic FNDC5 is a potential local protective factor against Non-Alcoholic Fatty Liver.

The proteolytic cleavage of Fibronectin type III domain-containing 5 (FNDC5) generates soluble irisin. Initially described as being mainly produced in muscle during physical exercise, irisin mediates adipose tissue thermogenesis and also regulates carbohydrate and lipid metabolism. The aim of this study was to evaluate the hepatic expression of FNDC5 and its role in hepatocytes in Non-Alcoholic Fatty Liver (NAFL). Here we report that hepatic expression of FNDC5 increased with hepatic steatosis and liver injury without impacting the systemic level of irisin in mouse models of NAFLD (HFD and MCDD) and in obese patients. The increased Fndc5 expression in fatty liver resulted from its upregulation in hepatocytes and non-parenchymal cells in mice. The local production of Fndc5 in hepatocytes was influenced by genotoxic stress and p53-dependent pathways. The down-regulation of FNDC5 in human HepG2 cells and in primary mouse hepatocytes increased the expression of PEPCK, a key enzyme involved in gluconeogenesis associated with a decrease in the expression of master genes involved in the VLDL synthesis (CIDEB and APOB). These alterations in FNDC5-silenced cells resulted to increased steatosis and insulin resistance in response to oleic acid and N-acetyl glucosamine, respectively. The downregulation of Fndc5 also sensitized primary hepatocytes to apoptosis in response to TNFα, which has been associated with decreased hepatoprotective autophagic flux. In conclusion, our human and experimental data strongly suggest that the hepatic expression of FNDC5 increased with hepatic steatosis and its upregulation in hepatocytes could dampen the development of NAFLD by negatively regulating steatogenesis and hepatocyte death.

BAX inhibitor-1: between stress and survival.

Cellular gatekeepers are essential to maintain order within a cell and anticipate signals of stress to promote survival. BCL2 associated X, apoptosis regulator (BAX) inhibitor-1 (BI-1), also named transmembrane BAX inhibitor motif containing-6, is a highly conserved endoplasmic reticulum (ER) transmembrane protein. Originally identified as an inhibitor of BAX-induced apoptosis, its pro-survival properties have been expanded to include functions targeted against ER stress, calcium imbalance, reactive oxygen species accumulation, and metabolic dysregulation. Nevertheless, the structural biology and biochemical mechanism of action of BI-1 are still under debate. BI-1 has been implicated in several diseases, including chronic liver disease, diabetes, ischemia/reperfusion injury, neurodegeneration, and cancer. While most studies have demonstrated a beneficial role for BI-1 in the ubiquitous maintenance of cellular homeostasis, its expression in cancer cells seems most often to contribute to tumorigenesis and metastasis. Here, we summarize what is known about BI-1 and encourage future studies on BI-1's contribution to cellular life and death decisions to advocate its potential as a target for drug development and other therapeutic strategies.

The Differential Expression of Cide Family Members is Associated with Nafld Progression from Steatosis to Steatohepatitis.

Improved understanding of the molecular mechanisms responsible for the progression from a "non-pathogenic" steatotic state to Non-Alcoholic Steatohepatitis is an important clinical requirement. The cell death-inducing DFF45 like effector (CIDE) family members (A, B and FSP27) regulate hepatic lipid homeostasis by controlling lipid droplet growth and/or VLDL production. However, CIDE proteins, particularly FSP27, have a dual role in that they also regulate cell death. We here report that the hepatic expression of CIDEA and FSP27 (α/ß) was similarly upregulated in a dietary mouse model of obesity-mediated hepatic steatosis. In contrast, CIDEA expression decreased, but FSP27-ß expression strongly increased in a dietary mouse model of steatohepatitis. The inverse expression pattern of CIDEA and FSP27ß was amplified with the increasing severity of the liver inflammation and injury. In obese patients, the hepatic CIDEC2 (human homologue of mouse FSP27ß) expression strongly correlated with the NAFLD activity score and liver injury. The hepatic expression of CIDEA tended to increase with obesity, but decreased with NAFLD severity. In hepatic cell lines, the downregulation of FSP27ß resulted in the fractionation of lipid droplets, whereas its overexpression decreased the expression of the anti-apoptotic BCL2 marker. This, in turn, sensitized cells to apoptosis in response to TNF α and saturated fatty acid. Considered together, our animal, human and in vitro studies indicate that differential expression of FSP27ß/CIDEC2 and CIDEA is related to NAFLD progression and liver injury.

Caspase 1/11 Deficiency or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in Mice.

Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.

The mid-infrared spectroscopy: A novel non-invasive diagnostic tool for NASH diagnosis in severe obesity.

There is an urgent medical need to develop non-invasive tests for non-alcoholic steatohepatitis (NASH). This study evaluates the diagnostic performance of an innovative model based on mid-infrared (MIR) spectroscopy for the diagnosis of NASH. METHODS: Severely obese patients who underwent a bariatric procedure at the University Hospital of Nice, France (n = 395) were prospectively recruited. The clinico-biological characteristics were measured prior to surgery. Liver biopsies were collected during the surgical procedure and assessed by a pathologist. A training group (316 patients, NASH: 16.8%) and a validation group (79 patients, NASH: 16.5%) were randomly defined. MIR spectra were acquired by fiber evanescent wave spectroscopy, using chalcogenide glass fiber optic sensors and a spectrometer. This absorption spectroscopic technique delivers a spectrum that identifies the molecular composition of a sample, defining a patient's metabolic fingerprint. RESULTS: The areas under the receiver operating curve (AUROC) for the diagnosis of NASH were 0.82 and 0.77 in the training and validation groups, respectively. The best threshold was 0.15, which was associated with a sensitivity of 0.75 and 0.69, and a specificity of 0.72 and 0.76. Negative predictive values of 0.94 and 0.93 and positive predictive values of 0.35 and 0.36, as well as correctly classified patient rates of 72% and 75% were obtained in the training and validation groups, respectively. A composite model using aspartate aminotransferase level, triglyceride level and waist circumference alongside the MIR spectra led to an increase in AUROC (0.88 and 0.84 for the training and validations groups, respectively). CONCLUSIONS: MIR spectroscopy provides good sensitivity and negative predictive values for NASH screening in patients with severe obesity. LAY SUMMARY: There is an urgent need for tools to non-invasively diagnose and monitor non-alcoholic steatohepatitis (NASH). This study evaluates the performance of a new tool for fast NASH diagnosis based on mid-infrared (MIR) spectroscopy. Using serum samples from severely obese patients who underwent a bariatric procedure, which enabled a concomitant liver biopsy to be performed, the MIR spectroscopy model performed well in screening patients for NASH compared to a traditional, histological diagnosis.

Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease.

The global epidemic of obesity has been accompanied by a rising burden of non-alcoholic fatty liver disease (NAFLD), with manifestations ranging from simple steatosis to non-alcoholic steatohepatitis, potentially developing into hepatocellular carcinoma. Although much attention has focused on NAFLD, its pathogenesis remains largely obscure. The hallmark of NAFLD is the hepatic accumulation of lipids, which subsequently leads to cellular stress and hepatic injury, eventually resulting in chronic liver disease. Abnormal lipid accumulation often coincides with insulin resistance in steatotic livers and is associated with perturbed endoplasmic reticulum (ER) proteostasis in hepatocytes. In response to chronic ER stress, an adaptive signalling pathway known as the unfolded protein response is triggered to restore ER proteostasis. However, the unfolded protein response can cause inflammation, inflammasome activation and, in the case of non-resolvable ER stress, the death of hepatocytes. Experimental data suggest that the unfolded protein response influences hepatic tumour development, aggressiveness and response to treatment, offering novel therapeutic avenues. Herein, we provide an overview of the evidence linking ER stress to NAFLD and discuss possible points of intervention.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.

The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

Bax inhibitor-1 protects from nonalcoholic steatohepatitis by limiting inositol-requiring enzyme 1 alpha signaling in mice.

Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress-dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B-cell lymphoma 2 (BCL2)-associated X protein (Bax) inhibitor-1 (BI-1), a negative regulator of the ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI-1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild-type and BI-1-/- mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high-fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM-treated BI-1-/- mice showed IRE1α-dependent NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI-1 down-regulation parallel to the up-regulation of IRE1α endoribonuclease (RNase) signaling. In HFD-fed BI-1-/- mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase-1/-11. Rises in interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF-083010 or 4µ8c, was evaluated in HFD-induced NAFLD. In BI-1-/- mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte-specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF-083010 or 4µ8c. CONCLUSION: Targeting IRE1α-dependent NLRP3 inflammasome signaling with pharmacological agents or by BI-1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.