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Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding σ-1 receptor (σ1R). We previously reported that σ1RE102Q elicits toxicity in cells. The σ1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with σ1R-mCherry (mCh), σ1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. σ1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-α kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with σ1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased σ1RE102Q-mCh insolubility and inhibited apoptosis. Whereas σ1R-mCh formed monomers and dimers, σ1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The σ1RE102Q insolubility was diminished by σ1R-mCh co-expression. These results suggest that the agonist and WT σ1R modify the detergent insolubility, toxicity, and oligomeric state of σ1RE102Q, which may lead to promising new treatments for σ1R-related ALS.

Excess fatty acid accumulation in nonadipose tissues leads to cell dysfunction and cell death that is linked to the pathogenesis of inherited and acquired human diseases. Study of this process, known as lipotoxicity, has provided new insights into the regulation of lipid homeostasis and has revealed new molecular pathways involved in lipid-induced cellular stress. The discovery that disruption of specific small nucleolar RNAs protects against fatty acid–induced cell death and remodels metabolism in vivo opens new opportunities for understanding how nutrient signals influence cellular and systemic metabolic homeostasis through RNA biology.

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

The endoplasmic reticulum (ER) is the entry point to the secretory pathway and major site of protein biogenesis. Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the evolutionarily conserved Sec61 complex, a heterotrimeric channel that comprises the Sec61p/Sec61α, Sss1p/Sec61γ, and Sbh1p/Sec61β subunits. In addition to forming a protein-conducting channel, the Sec61 complex also functions to maintain the ER permeability barrier, preventing the mass free flow of essential ER-enriched molecules and ions. Loss in Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C terminus is juxtaposed to the key gating module of Sec61p/Sec61α, and we hypothesize it is important for gating the ER translocon. The ER stress response was found to be constitutively induced in two temperature-sensitive sss1 mutants (sss1(ts)) that are still proficient to conduct ER translocation. A screen to identify intergenic mutations that allow for sss1(ts) cells to grow at 37 °C suggests the ER permeability barrier to be compromised in these mutants. We propose the extreme C terminus of Sss1p/Sec61γ is an essential component of the gating module of the ER translocase and is required to maintain the ER permeability barrier.

Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress. ER stress did not substantially impact Doa10 or Rkr1 substrates. However, Hrd1-mediated destruction of a protein that aberrantly engages the translocon (Deg1-Sec62) and substrates with luminal degradation signals was markedly impaired by ER stress; by contrast, Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by ER stress. ER stress impaired the degradation of one of two Asi substrates analyzed and caused a translocon-clogging Ste24 substrate to accumulate in a form consistent with persistent translocon occupation. Degradation of Deg1-Sec62 in the absence of stress and stabilization during ER stress were independent of four ER stress–sensing pathways. Our results indicate ER stress differentially impacts degradation of protein quality control substrates, including those mediated by the same ubiquitin ligase. These observations suggest the existence of additional regulatory mechanisms dictating substrate selection during ER stress.

Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and is evolutionarily conserved from yeast to humans. IRE1 possesses both Ser/Thr protein kinase and endoribonuclease (RNase) activities within its cytoplasmic domain and is activated through autophosphorylation and dimerization/oligomerization. It mediates a critical arm of the unfolded protein response to manage ER stress provoked by lumenal overload of unfolded/misfolded proteins. Emerging lines of evidence have revealed that in mammals, IRE1α functions as a multifunctional signal transducer that responds to metabolic cues and nutrient stress conditions, exerting profound and broad effects on metabolic homeostasis. In this review, we cover recent advances in our understanding of how IRE1α integrates a variety of metabolic and stress signals and highlight its tissue-specific or context-dependent metabolic activities. We also discuss how dysregulation of this metabolic stress sensor during handling of excessive nutrients in cells contributes to the progression of obesity and metabolic disorders.

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic β-cells. Loss-of-function mutations in ZnT8 are associated with protection against type-2 diabetes (T2D), but the protective mechanism is unclear. Here, we developed an in-cell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-βH1 cells. Unexpectedly, high glucose and free fatty acids did not alter cellular ZnT8 levels, but proinflammatory cytokines acutely, reversibly, and gradually down-regulated ZnT8. Approximately 50% of the cellular ZnT8 was localized to the endoplasmic reticulum (ER), which was the primary target of the cytokine-mediated ZnT8 down-regulation. Transcriptome profiling of cytokine-exposed β-cells revealed an adaptive unfolded protein response (UPR) including a marked immunoproteasome activation that coordinately degraded ZnT8 and insulin over a 1,000-fold cytokine concentration range. RNAi-mediated ZnT8 knockdown protected cells against cytokine cytotoxicity, whereas inhibiting immunoproteasomes blocked cytokine-induced ZnT8 degradation and triggered a transition of the adaptive UPR to cell apoptosis. Hence, cytokine-induced down-regulation of the ER ZnT8 level promotes adaptive UPR, acting as a protective mechanism that decongests the ER burden of ZnT8 to protect β-cells from proapoptotic UPR during chronic low-grade inflammation.

Knockout of the transcription factor X-box binding protein (XBP1) is known to decrease liver glucose production and lipogenesis. However, whether insulin can regulate gluconeogenesis and lipogenesis through XBP1 and how insulin activates the inositol-requiring enzyme-XBP1 ER stress pathway remains unexplored. Here, we report that in the fed state, insulin-activated kinase AKT directly phosphorylates inositol-requiring enzyme 1 at S724, which in turn mediates the splicing of XBP1u mRNA, thus favoring the generation of the spliced form, XBP1s, in the liver of mice. Subsequently, XBP1s stimulate the expression of lipogenic genes and upregulates liver lipogenesis as previously reported. Intriguingly, we find that fasting leads to an increase in XBP1u along with a drastic decrease in XBP1s in the liver of mice, and XBP1u, not XBP1s, significantly increases PKA-stimulated CRE reporter activity in cultured hepatocytes. Furthermore, we demonstrate that overexpression of XBP1u significantly increases cAMP-stimulated expression of rate-limiting gluconeogenic genes, G6pc and Pck1, and glucose production in primary hepatocytes. Reexpression of XBP1u in the liver of mice with XBP1 depletion significantly increases fasting blood glucose levels and gluconeogenic gene expression. These data support an important role of XBP1u in upregulating gluconeogenesis in the fasted state. Taken together, we reveal that insulin signaling via AKT controls the expression of XBP1 isoforms and that XBP1u and XBP1s function in different nutritional states to regulate liver gluconeogenesis and lipogenesis, respectively.

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Hypoxia occurs in many human solid tumors and activates multiple cellular adaptive-response pathways, including the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Wnt/β-catenin signaling plays a critical role in tumorigenesis, and β-catenin has been shown to enhance hypoxia-inducible factor 1α (HIF1α)-activated gene expression, thereby supporting cell survival during hypoxia. However, the molecular interplay between hypoxic ER stress, Wnt/β-catenin signaling, and HIF1α-mediated gene regulation during hypoxia remains incompletely understood. Here, we report that hypoxic ER stress reduces β-catenin stability, which, in turn, enhances the activity of spliced X-box-binding protein 1 (XBP1s), a transcription factor and signal transducer of the UPR, in HIF1α-mediated hypoxic responses. We observed that in the RKO colon cancer cell line, which possesses a Wnt-stimulated β-catenin signaling cascade, increased ER stress during hypoxia is accompanied by a reduction in low-density lipoprotein receptor-related protein 6 (LRP6), and this reduction in LRP6 decreased β-catenin accumulation and impaired Wnt/β-catenin signaling. Of note, β-catenin interacted with both XBP1s and HIF1α, suppressing XBP1s-mediated augmentation of HIF1α target gene expression. Furthermore, Wnt stimulation or β-catenin overexpression blunted XBP1s-mediated cell survival under hypoxia. Together, these results reveal an unanticipated role for the Wnt/β-catenin pathway in hindering hypoxic UPR-mediated responses that increase cell survival. Our findings suggest that the molecular cross-talks between hypoxic ER stress, LRP6/β-catenin signaling, and the HIF1α pathway may represent an unappreciated mechanism that enables some tumor subtypes to survive and grow in hypoxic conditions.

The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly. Although this condition is generally benign, accumulating evidence now suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); the leading cause of death in developed nations. Despite the well-established role of the liver as a central regulator of circulating low-density lipoprotein (LDL) cholesterol levels, a known driver of CVD, the mechanism(s) by which hepatic steatosis contributes to CVD remains elusive. Interestingly, a recent study has shown that circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels correlate positively with liver steatosis grade. Given that PCSK9 degrades the LDL receptor (LDLR) and prevents the removal of LDL from the blood into the liver, in the present study we examined the effect of hepatic steatosis on LDLR expression and circulating LDL cholesterol levels. We now report that in a manner consistent with findings in patients, diet-induced steatosis increases circulating PCSK9 levels as a result of de novo expression in mice. We also report the finding that steatosis abrogates hepatic LDLR expression and increases circulating LDL levels in a PCSK9-dependent manner. These findings provide important mechanistic insights as to how hepatic steatosis modulates lipid regulatory genes, including PCSK9 and the LDLR, and also highlights a novel mechanism by which liver disease may contribute to CVD.

The 78-kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface–associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here we explored the role of csGRP78 in regulating high glucose (HG)–induced profibrotic AKT Ser/Thr kinase (AKT) signaling and up-regulation of extracellular matrix proteins. Using primary kidney mesangial cells, we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress–dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin β1 and activates focal adhesion kinase and downstream PI3K/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin β1 were also required for extracellular matrix protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy.

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca(2+) into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca(2+) can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.

Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51–like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor–associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.

Alterations in endoplasmic reticulum (ER) calcium (Ca(2+)) levels diminish insulin secretion and reduce β-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca(2+) loss in β cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP(3)R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca(2+) imaging techniques in INS-1 β cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP(3)R function. Our results revealed that the RyR is sensitive mainly to ER stress–induced dysfunction, whereas cytokine stress specifically alters IP(3)R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP(3)R with xestospongin C prevented ER Ca(2+) loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented β-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca(2+) oscillations in tunicamycin-treated INS-1 β cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca(2+) transients that depend on both ER Ca(2+) leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca(2+) dynamics in β cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca(2+) may be an early pathogenic event in diabetes.

The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA-like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA-like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.

High-glucose (HG) levels and hyperglycemia associated with diabetes are known to cause neuronal damage. The detailed molecular mechanisms, however, remain to be elucidated. Here, we investigated the role of transient receptor potential melastatin 7 (TRPM7) channels in HG-mediated endoplasmic reticulum stress (ERS) and injury of NS20Y neuronal cells. The cells were incubated in the absence or presence of HG for 48 h. We found that mRNA and protein levels of TRPM7 and of ERS-associated proteins, such as C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and inducible nitric-oxide synthase (iNOS), increased in HG-treated cells, along with significantly increased TRPM7-associated currents in these cells. Similar results were obtained in cerebral cortical tissue from an insulin-deficiency model of diabetic mice. Moreover, HG treatment of cells activated ERS-associated proapoptotic caspase activity and induced cellular injury. Interestingly, a NOS inhibitor, l-NAME, suppressed the HG-induced increase of TRPM7 expression and cellular injury. siRNA-mediated TRPM7 knockdown or chemical inhibition of TRPM7 activity also suppressed HG-induced ERS and decreased cleaved caspase-12/caspase-3 levels and cell injury. Of note, TRPM7 overexpression increased ERS and cell injury independently of its kinase activity. Taken together, our findings suggest that TRPM7 channel activities play a key role in HG-associated ERS and cytotoxicity through an apoptosis-inducing signaling cascade involving HG, iNOS, TRPM7, ERS proteins, and caspases.

The NAD(+)-dependent deacetylase sirtuin-1 (SIRT1) has emerged as an important regulator of chondrogenesis and cartilage homeostasis, processes that are important for physiological skeletal growth and that are dysregulated in osteoarthritis. However, the functional role and underlying mechanism by which SIRT1 regulates chondrogenesis remain unclear. Using cultured rat metatarsal bones and chondrocytes isolated from rat metatarsal rudiments, here we studied the effects of the SIRT1 inhibitor EX527 or of SIRT1 siRNA on chondrocyte proliferation, hypertrophy, and apoptosis. We show that EX527 or SIRT1 siRNA inhibits chondrocyte proliferation and hypertrophy and induces apoptosis. We also observed that SIRT1 inhibition mainly induces the PERK–eIF-2α–CHOP axis of the endoplasmic reticulum (ER) stress response in growth-plate chondrocytes. Of note, EX527- or SIRT1 siRNA–mediated inhibition of metatarsal growth and growth-plate chondrogenesis were partly neutralized by phenylbutyric acid, a chemical chaperone that attenuates ER stress. Moreover, EX527-mediated impairment of chondrocyte function (i.e. of chondrocyte proliferation, hypertrophy, and apoptosis) was partly reversed in CHOP(−/−) cells. We also present evidence that SIRT1 physically interacts with and deacetylates PERK. Collectively, our findings indicate that SIRT1 deacetylates PERK and attenuates the PERK–eIF-2α–CHOP axis of the unfolded protein response pathway and thereby promotes growth-plate chondrogenesis and longitudinal bone growth.

Heart failure is associated with induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The serine/threonine protein kinase/endoribonuclease IRE1α is a key protein in ER stress signal transduction. IRE1α activity can induce both protective UPR and apoptotic downstream signaling events, but the specific role for IRE1α activity in the heart is unknown. A major aim of this study was to characterize the specific contribution of IRE1α in cardiac physiology and pathogenesis. We used both cultured myocytes and a transgenic mouse line with inducible and cardiomyocyte-specific IRE1α overexpression as experimental models to achieve targeted IRE1α activation. IRE1α expression induced a potent but transient ER stress response in cardiomyocytes and did not cause significant effects in the intact heart under normal physiological conditions. Furthermore, the IRE1α-activated transgenic heart responding to pressure overload exhibited preserved function and reduced fibrotic area, associated with increased adaptive UPR signaling and with blunted inflammatory and pathological gene expression. Therefore, we conclude that IRE1α induces transient ER stress signaling and confers a protective effect against pressure overload-induced pathological remodeling in the heart. To our knowledge, this report provides first direct evidence of a specific and protective role for IRE1α in the heart and reveals an interaction between ER stress signaling and inflammatory regulation in the pathologically stressed heart.

The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress–inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.

Maresin 1 (MAR1), which is derived from docosahexaenoic acid biosynthesized by macrophages, has been reported to improve insulin resistance. Recently, it has been documented that MAR1 could ameliorate inflammation and insulin resistance in obese mice. These findings led us to investigate the effects of MAR1 on hepatic lipid metabolism. We found that MAR1 could stimulate AMP-activated protein kinase (AMPK), thereby augmenting sarcoendoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) expression. This stimulation suppressed lipid accumulation by attenuating the endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. Attenuation was mitigated by knockdown of AMPK or thapsigargin, a SERCA2b inhibitor. We also demonstrated that MAR1 administration resulted in increased hepatic AMPK phosphorylation and Serca2b mRNA expression, whereas hepatic ER stress was reduced in high-fat diet (HFD)-fed mice. Moreover, MAR1 treatment suppressed hepatic lipid synthesis, thereby attenuating hepatic steatosis in HFD-fed mice. In conclusion, our results suggest that MAR1 ameliorates hepatic steatosis via AMPK/SERCA2b-mediated suppression of ER stress. Therefore, MAR1 may be an effective therapeutic strategy for treating non-alcoholic fatty liver disease (NAFLD) via regulation of ER stress-induced hepatic lipogenesis.

The stress-induced unfolded protein response (UPR) in the endoplasmic reticulum (ER) involves various signaling cross-talks and controls cell fate. B-cell receptor (BCR) signaling, which can trigger UPR, induces gammaherpesvirus lytic replication and serves as a physiological mechanism for gammaherpesvirus reactivation in vivo. However, how the UPR regulates BCR-mediated gammaherpesvirus infection is unknown. Here, we demonstrate that the ER stressors tunicamycin and thapsigargin inhibit BCR-mediated murine gammaherpesvirus 68 (MHV68) lytic replication by inducing expression of the UPR mediator Bip and blocking activation of Akt, ERK, and JNK. Both Bip and the downstream transcription factor ATF4 inhibited BCR-mediated MHV68 lytic gene expression, whereas UPR-induced C/EBP homologous protein (CHOP) was required for and promoted BCR-mediated MHV68 lytic replication by suppressing upstream Bip and ATF4 expression. Bip knockout was sufficient to rescue BCR-mediated MHV68 lytic gene expression in CHOP knockout cells, and this rescue was blocked by ectopic ATF4 expression. Furthermore, ATF4 directly inhibited promoter activity of the MHV68 lytic switch transactivator RTA. Altogether, we show that ER stress–induced CHOP inhibits Bip and ATF4 expression and that ATF4, in turn, plays a critical role in CHOP-mediated regulation of BCR-controlled MHV68 lytic replication. We conclude that ER stress–mediated UPR and BCR signaling pathways are interconnected and form a complex network to regulate the gammaherpesvirus infection cycle.

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed non-stop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson’s disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson’s disease pathology.

The up-regulation of chaperones such as the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP or HSPA5) is part of the adaptive cellular response to endoplasmic reticulum (ER) stress. GRP78 is widely used as a marker of the unfolded protein response, associated with sustained ER stress. Here we report the discovery of a proteostatic mechanism involving GRP78 trimethylation in the context of ER stress. Using mass spectrometry–based proteomics, we identified two GRP78 fractions, one homeostatic and one induced by ER stress. ER stress leads to de novo biosynthesis of non-trimethylated GRP78, whereas homeostatic, METTL21A-dependent lysine 585–trimethylated GRP78 is reduced. This proteostatic mechanism, dependent on the posttranslational modification of GRP78, allows cells to differentially regulate specific protein abundance during cellular stress.

The onset of type 2 diabetes is characterized by transition from successful to failed insulin secretory compensation to obesity-related insulin resistance and dysmetabolism. Energy-rich diets in rodents are commonly studied models of compensatory increases in both insulin secretion and β cell mass. However, the mechanisms of these adaptive responses are incompletely understood, and it is also unclear why these responses eventually fail. We measured the temporal trends of glucose homeostasis, insulin secretion, β cell morphometry, and islet gene expression in C57BL/6NTac mice fed a 60% high-fat diet (HFD) or control diet for up to 16 weeks. A 2-fold increased hyperinsulinemia was maintained for the first 4 weeks of HFD feeding and then further increased through 16 weeks. β cell mass increased progressively starting at 4 weeks, principally through nonproliferative growth. Insulin sensitivity was not significantly perturbed until 11 weeks of HFD feeding. Over the first 8 weeks, we observed two distinct waves of increased expression of β cell functional and prodifferentiation genes. This was followed by activation of the unfolded protein response at 8 weeks and overt β cell endoplasmic reticulum stress at 12–16 weeks. In summary, β cell adaptation to an HFD in C57BL/6NTac mice entails early insulin hypersecretion and a robust growth phase along with hyperexpression of related genes that begin well before the onset of observed insulin resistance. However, continued HFD exposure results in cessation of gene hyperexpression, β cell functional failure, and endoplasmic reticulum stress. These data point to a complex but not sustainable integration of β cell-adaptive responses to nutrient overabundance, obesity development, and insulin resistance.

Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9. Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca2+ depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca2+ depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca2+ inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.

Lipids are important nutrients that proliferating cells require to maintain energy homeostasis as well as to build plasma membranes for newly synthesized cells. Previously, we identified nutrient-sensing checkpoints that exist in the latter part of the G1 phase of the cell cycle that are dependent upon essential amino acids, Gln, and finally, a checkpoint mediated by mammalian target of rapamycin (mTOR), which integrates signals from both nutrients and growth factors. In this study, we have identified and temporally mapped a lipid-mediated G1 checkpoint. This checkpoint is located after the Gln checkpoint and before the mTOR-mediated cell cycle checkpoint. Intriguingly, clear cell renal cell carcinoma cells (ccRCC) have a dysregulated lipid-mediated checkpoint due in part to defective phosphatase and tensin homologue (PTEN). When deprived of lipids, instead of arresting in G1, these cells continue to cycle and utilize lipid droplets as a source of lipids. Lipid droplets have been known to maintain endoplasmic reticulum homeostasis and prevent cytotoxic endoplasmic reticulum stress in ccRCC. Dysregulation of the lipid-mediated checkpoint forces these cells to utilize lipid droplets, which could potentially lead to therapeutic opportunities that exploit this property of ccRCC.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-β (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.