Activation of the endoplasmic reticulum stress response in skeletal muscle of G93A*SOD1 amyotrophic lateral sclerosis mice.
ABSTRACT: Mutations in Cu/Zn superoxide dismutase (SOD1) are one of the genetic causes of Amyotrophic Lateral Sclerosis (ALS). Although the primary symptom of ALS is muscle weakness, the link between SOD1 mutations, cellular dysfunction and muscle atrophy and weakness is not well understood. The purpose of this study was to characterize cellular markers of ER stress in skeletal muscle across the lifespan of G93A*SOD1 (ALS-Tg) mice. Muscles were obtained from ALS-Tg and age-matched wild type (WT) mice at 70d (pre-symptomatic), 90d and 120-140d (symptomatic) and analyzed for ER stress markers. In white gastrocnemius (WG) muscle, ER stress sensors PERK and IRE1? were upregulated ~2-fold at 70d and remained (PERK) or increased further (IRE1?) at 120-140d. Phospho-eIF2?, a downstream target of PERK and an inhibitor of protein translation, was increased by 70d and increased further to 12.9-fold at 120-140d. IRE1? upregulation leads to increased splicing of X-box binding protein 1 (XBP-1) to the XBP-1s isoform. XBP-1s transcript was increased at 90d and 120-140d indicating activation of IRE1? signaling. The ER chaperone/heat shock protein Grp78/BiP was upregulated 2-fold at 70d and 90d and increased to 6.1-fold by 120-140d. The ER-stress-specific apoptotic signaling protein CHOP was upregulated 2-fold at 70d and 90d and increased to 13.3-fold at 120-140d indicating progressive activation of an apoptotic signal in muscle. There was a greater increase in Grp78/BiP and CHOP in WG vs. the more oxidative red gastrocnemius (RG) ALS-Tg at 120-140d indicating greater ER stress and apoptosis in fast glycolytic muscle. These data show that the ER stress response is activated in skeletal muscle of ALS-Tg mice by an early pre-symptomatic age and increases with disease progression. These data suggest a mechanism by which myocellular ER stress leads to reduced protein translation and contributes to muscle atrophy and weakness in ALS.
Project description:IL-1? and TNF-? are important proinflammatory cytokines that respond to mutated self-antigens of tissue damage and exogenous pathogens. The endoplasmic reticulum (ER) stress and unfolded protein responses are related to the induction of proinflammatory cytokines. However, the detailed molecular pathways by which ER stress mediates cytokine gene expression have not been investigated. In this study, we found that ER stress-induced inositol-requiring enzyme (IRE)1? activation differentially regulates proinflammatory cytokine gene expression via activation of glycogen synthase kinase (GSK)-3? and X-box binding protein (XBP)-1. Surprisingly, IL-1? gene expression was modulated by IRE1?-mediated GSK-3? activation, but not by XBP-1. However, IRE1?-mediated XBP-1 splicing regulated TNF-? gene expression. SB216763, a GSK-3 inhibitor, selectively inhibited IL-1? gene expression, whereas the IRE1? RNase inhibitor STF083010 suppressed only TNF-? production. Additionally, inhibition of GSK-3? greatly increased IRE1?-dependent XBP-1 splicing. Our results identify an unsuspected differential role of downstream mediators GSK-3? and XBP-1 in ER stress-induced IRE1? activation that regulates cytokine production through signaling cross-talk. These results have important implications in the regulation of inflammatory pathways during ER stress, and they suggest novel therapeutic targets for diseases in which meta-inflammation plays a key role.
Project description:Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.
Project description:IRE1 is a conserved dual endoribonuclease/protein kinase that is indispensable for directing the endoplasmic reticulum (ER) stress response in yeast, flies, and worms. In mammalian systems, however, the precise biological activities carried out by IRE1alpha are unclear. Here, molecular and chemical genetic approaches were used to control IRE1 activity in a number of prostate cancer cell lines and the resulting impact on gene transcription, cell survival, and proliferation was examined. Modulating IRE1alpha activity had no transcriptional effect on the induction of genes classically associated with the ER stress response (Grp78 and CHOP) or cell survival when confronted with ER stress agents. Rather, IRE1alpha activity was positively correlated to proliferation. Since Xbp-1 mRNA is the sole known substrate for IRE1 endoribonuclease activity, the role of this transcription factor in mediating proliferation was examined. Repressing total Xbp-1 levels by siRNA techniques effectively slowed proliferation. In an effort to identify IRE1/XBP-1 targets responsible for the cell cycle response, genome-wide differential mRNA expression analysis was performed. Consistent with its ability to sense ER stress, IRE1alpha induction led to an enrichment of ER-Golgi, plasma membrane, and secretory gene products. An increase in cyclin A1 expression was the only differentially expressed cell cycle regulatory gene found. Greater cyclin A protein levels were consistently observed in cells with active IRE1alpha and were dependent on XBP-1. We conclude that IRE1alpha activity controls a subset of the ER stress response and mediates proliferation through tight control of Xbp-1 splicing.
Project description:Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1? (IRE1?), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1? interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1? with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1?, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1? RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.
Project description:Evidence implicating dysregulation of the IRE1/XBP-1s arm of the unfolded protein response (UPR) in cancer pathogenesis (e.g., multiple myeloma) has prompted the development of IRE1 RNase inhibitors. Here, effects of cyclin-dependent kinase (CDK) inhibitor SCH727965 (dinaciclib) on the IRE1 arm of the UPR were examined in human leukemia and myeloma cells. Exposure of cells to extremely low (e.g., nmol/L) concentrations of SCH727965, a potent inhibitor of CDKs 1/2/5/9, diminished XBP-1s and Grp78 induction by the endoplasmic reticulum (ER) stress-inducers thapsigargin and tunicamycin, while sharply inducing cell death. SCH727965, in contrast to IRE1 RNase inhibitors, inhibited the UPR in association with attenuation of XBP-1s nuclear localization and accumulation rather than transcription, translation, or XBP-1 splicing. Notably, in human leukemia cells, CDK1 and 5 short hairpin RNA (shRNA) knockdown diminished Grp78 and XBP-1s upregulation while increasing thapsigargin lethality, arguing for a functional role for CDK1/5 in activation of the cytoprotective IRE1/XBP-1s arm of the UPR. In contrast, CDK9 or 2 inhibitors or shRNA knockdown failed to downregulate XBP-1s or Grp78. Furthermore, IRE1, XBP-1, or Grp78 knockdown significantly increased thapsigargin lethality, as observed with CDK1/5 inhibition/knockdown. Finally, SCH727965 diminished myeloma cell growth in vivo in association with XBP-1s downregulation. Together, these findings demonstrate that SCH727965 acts at extremely low concentrations to attenuate XBP-1s nuclear accumulation and Grp78 upregulation in response to ER stress inducers. They also highlight a link between specific components of the cell-cycle regulatory apparatus (e.g., CDK1/5) and the cytoprotective IRE1/XBP-1s/Grp78 arm of the UPR that may be exploited therapeutically in UPR-driven malignancies.
Project description:The aged kidney is susceptible to acute injury due presumably to its decreased ability to handle additional challenges, such as endoplasmic reticulum (ER) stress. This was tested by giving tunicamycin, an ER stress inducer, to either old or young mice. Injection of high dose caused renal failure in old mice, not in young mice. Moreover, injection of low dose resulted in severe renal damage in old mice, confirming the increased susceptibility of aged kidney to ER stress. There existed an abnormality in ER stress response kinetics in aged kidney, characterized by a loss of XBP-1 splicing and decreased PERK-eIF2? phosphorylation at late time point. The presence of excessive oxidative stress in aged kidney may play a role since high levels of oxidation increased ER stress-induced cell death and decreased IRE1 levels and XBP-1 splicing. Importantly, treatment with antioxidants protected old mice from kidney injury and normalized IRE1 and XBP-1 responses. Furthermore, older mice (6 months old) transgenic with antioxidative stress AGER1 were protected from ER stress-induced kidney injury. In conclusion, the decreased ability to handle ER stress, partly due to the presence of excessive oxidative stress, may contribute to increased susceptibility of the aging kidney to acute injury.
Project description:The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
Project description:The ER stress-mediated apoptosis has been implicated in several neurodegenerative diseases; however, its role in HIV/neuroAIDS remains largely unexplored. The present study was undertaken to assess the involvement and detailed mechanism of IRE1? pathway in HIV-1?gp120-mediated ER stress and its possible involvement in cell death. Various signaling molecules for IRE1? pathway were assessed using SVGA cells, primary astrocytes and gp120 transgenic mice, which demonstrated gp120-mediated increase in phosphorylated JNK, XBP-1 and AP-1 leading to upregulation of CHOP. Furthermore, HIV-1 gp120-mediated activation of IRE1? also increased XBP-1 splicing. The functional consequence of gp120-mediated ER stress was determined via assessment of gp120-mediated cell death using PI staining and MTT assay. The gp120-mediated cell death also involved caspase-9/caspase-3-mediated apoptosis. These findings were confirmed with the help of specific siRNA for IRE1?, JNK, AP-1, BiP and CHOP showing significant reduction in gp120-mediated CHOP expression. Additionally, silencing all the intermediates also reduced the gp120-mediated cell death and caspase-9/caspase-3 activation at differential levels. This study provides ER-stress as a novel therapeutic target in the management of gp120-mediated cell death and possibly in the treatment of neuroAIDS.
Project description:Subclinical hypothyroidism (SCH) and diabetes mellitus are closely related and often occur together in individuals. However, the underlying mechanism of this association is still uncertain. In this study we re-analyzed the data of a mature database (NHANES, 1999 ~ 2002) and found that both fasting plasma glucose levels and the proportion of hyperglycemic subjects among SCH patients were higher than that found in euthyroid controls. SCH was also associated with a 2.29-fold increased risk for diabetes. Subsequently, we established an SCH mouse model and subjected it to an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT). SCH mice exhibited impaired glucose and insulin tolerance. Increased HOMA-IR and decreased ISI indexes, indicating insulin resistance (IR), were also observed in the SCH state. Hepatic ERp29 and Bip, as well as IRE1? and XBP-1s, were induced significantly in SCH mice, suggesting the induction of endoplasmic reticulum (ER) stress, particularly involving the IRE1?/XBP-1s pathway. Interestingly, when we relieved ER stress using 4-phenyl butyric acid, abnormal glucose metabolism, and IR status in SCH mice were improved. Our findings suggest that ER stress, predominantly involving the IRE1?/XBP-1s pathway, may play a pivotal role in abnormal glucose metabolism and IR in SCH that may help develop potential strategies for the prevention and treatment of diabetes.
Project description:ER stress results in activation of the unfolded protein response and has been implicated in the development of fibrotic diseases. In this study, we show that inhibition of the ER stress-induced IRE1? signaling pathway, using the inhibitor 4?8C, blocks TGF?-induced activation of myofibroblasts in vitro, reduces liver and skin fibrosis in vivo, and reverts the fibrotic phenotype of activated myofibroblasts isolated from patients with systemic sclerosis. By using IRE1?(-/-) fibroblasts and expression of IRE1?-mutant proteins lacking endoribonuclease activity, we confirmed that IRE1? plays an important role during myofibroblast activation. IRE1? was shown to cleave miR-150 and thereby to release the suppressive effect that miR-150 exerted on ?SMA expression through c-Myb. Inhibition of IRE1? was also demonstrated to block ER expansion through an XBP-1-dependent pathway. Taken together, our results suggest that ER stress could be an important and conserved mechanism in the pathogenesis of fibrosis and that components of the ER stress pathway may be therapeutically relevant for treating patients with fibrotic diseases.