Stressors and Stress Responses in Cystic Fibrosis

Cystic fibrosis (CF) is a life-shortening, genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The primary cause of CF is reduced CFTR-mediated chloride and bicarbonate transport, due to mutations in CFTR. However, inflammation and persistent infections influence clinical outcome. Cellular stress response pathways, such as the unfolded protein response (UPR) and the integrated stress response (ISR), referred to here as cellular stress response pathways (SRPs), contribute to the pathology of human disorders. Multiple studies have indicated activation of SRPs in CF tissues. We review our present understanding of how SRPs are activated in CF and their contribution to pathology. We conclude that reduced CFTR function in CF organs establishes a tissue environment in which internal or external insults activate SRPs. SRPs contribute to CF pathogenesis by reducing CFTR expression, enhancing inflammation with consequent tissue remodeling. Understanding the contribution of SRPs to CF pathogenesis is crucial even in the era of CFTR “modulators” that are designed to potentiate, correct or amplify CFTR function, since there is an urgent need for supportive treatments. Importantly, CF patients with established pathology could benefit from the targeted use of drugs that modulate SRPs to reduce the symptoms.

Stress-sensing and regulatory mechanism of the endoplasmic-stress sensors Ire1 and PERK

Ire1 and its family protein PERK are endoplasmic reticulum (ER)-stress sensors that initiate cellular responses against ER accumulation of unfolded proteins. As reviewed in this article, many publications describe molecular mechanisms by which yeast Ire1 senses ER conditions and gets regulated. We also cover recent studies which reveal that mammalian Ire1 (IRE1α) and PERK are controlled in a similar but not exactly the same manner. ER-located molecular chaperone BiP captures these ER-stress sensors and suppresses their activity. Intriguingly, Ire1 is associated with BiP not as a chaperone substrate, but as a unique ligand. Unfolded proteins accumulated in the ER promote dissociation of the Ire1-BiP complex. Moreover, Ire1 is directly bound with unfolded proteins, leading to its cluster formation and potent activation. PERK also captures unfolded proteins and then forms self-oligomers. Meanwhile, membrane-lipid aberrancy is likely to activate these ER-stress sensors independently of ER accumulation of unfolded proteins. In addition, there exist a number of reports that touch on other factors that control activity of these ER-stress sensors. Such a multiplicity of regulatory mechanisms for these ER-stress sensors is likely to contribute to fine tuning of their activity.

Link between endoplasmic reticulum stress and autophagy in neurodegenerative diseases

Increasing evidence suggests that endoplasmic reticulum (ER) stress and autophagy play an important role in regulating brain function. ER stress activates three major branches of the unfolded protein response (UPR) pathways, namely inositol-requiring enzyme-1 (IRE1), double stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) and activating transcription factor 6 (ATF6)-mediated pathways. Recent studies have suggested that these UPR signals may be linked to autophagy. In this review article, we summarize recent evidence and discuss a possible link between ER stress and autophagy with regard to neurodegenerative diseases. Furthermore, possible pharmacological strategies targeting UPR and autophagy are discussed.

Prion disease and endoplasmic reticulum stress pathway correlations and treatment pursuits

Background: Transmissible spongiform encephalopathies are a collection of rare neurodegenerative disorders characterized by loss of neuronal cells, astrocytosis, and plaque formation. The causative agent of these diseases is thought to be abnormally folded prions and is characterized by a conformational change from normal, cellular prion protein (PrPc) to the abnormal form (PrPTSE). Although, there is evidence that normal prion protein can contribute to these disorders. The unfolded protein response, a conserved series of pathways involved in resolving stress associated with unfolded protein accumulation in the Endoplasmic Reticulum (ER), has been shown to play a role in regulating the development of prion diseases. Methods: This review chose papers based on their relevance to current studies involved in prion protein synthesis and transformation, identifies various links between prion diseases and ER stress, and reports on current and potential treatments as they relate to ER stress and prion diseases. Conclusion: For the advancement of prion disease treatment, it is important to understand the mechanisms involved in prion formation, and ER stress is implicated in prion disease progression. Therefore, targeting the ER or pathways involved in response to stress in the ER may help us treat prion diseases.

ER stress in neurodegenerative disease: from disease mechanisms to therapeutic interventions

The conception that protein aggregates composed by misfolded proteins underlies the occurrence of several neurodegenerative diseases suggests that this phenomenon may have a common origin, ultimately driven by disruption of proteostasis control. The unfolded protein response (UPR) embodies a major element of the proteostasis network, which is engaged by endoplasmic reticulum (ER) stress. Chronic ER stress may operate as a possible mechanism of neurodegeneration, contributing to synaptic alterations, neuroinflammation and neuronal loss. In this review we discuss most recent findings relating ER stress and the development of distinct neurodegenerative diseases, and the possible strategies for disease intervention.

Regulation of IRE1α by the small molecule inhibitor 4μ8c in hepatoma cells

The unfolded protein response (UPR) is activated in response to impairments of the folding environment in the endoplasmic reticulum (ER). The most conserved arm of the UPR, inositol-requiring ER-to-nucleus signaling protein (IRE1α), has been linked to the regulation of a diverse array of cellular processes including ER-associated degradation, inflammatory signaling, cell proliferation and membrane biogenesis. Recent studies have utilized the selective, small molecule inhibitor, 4μ8c, to examine the role of IRE1α endoribonuclease (RNase) activity in various cell types including multiple myeloma, mouse embryonic fibroblasts and pancreatic beta cells [1-5]. In the present study we utilized this inhibitor to examine the role of IRE1α RNase activity in hepatoma cells (H4IIE), specifically concentrating on cell proliferation and the identification of potential off target effects under both unstressed and stressed conditions. Experiments were performed in H4IIE hepatoma cells in the absence (control conditions (LG)) or presence (LG + Thapsigargin (Thap)) of ER stress. The presence of 4μ8c decreased IRE1α RNase activity, based on reduced splicing of X-box binding protein-1 (XBP1s) and regulated IRE1α-dependent decay of mRNA in both treatments and at concentrations ranging from 10-90 μM. Cell proliferation was significantly reduced at higher concentrations (> 60 μM 4μ8c) in unstressed cells and displayed a dose-response relationship with 4μ8c in both treatments. The presence of 4μ8c did not promote cytoxicity in either of the treatment conditions but higher concentrations of the inhibitor (60 μM) were associated with apparent off-target or compensatory responses that were not observed at 10 μM. Overall, the small-molecule inhibitor, 4μ8c is an effective inhibitor of IRE1α RNase activity in H4IIE cells. Potential off-target effects associated with this inhibitor require the use of multiple inhibitor concentrations in all experiments.

The role of Endoplasmic Reticulum (ER) stress in pulmonary fibrosis

The activation of Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) was first observed in patients with familial interstitial pneumonia (FIP) carrying mutations in the C-terminal BRICHOS domain of surfactant protein C (SFTPC). Here, aggresome formation and severe ER stress was demonstrated in type-II alveolar epithelial cells (AECII), which specifically express this very hydrophobic surfactant protein. In subsequent studies, FIP-patients with mutations in the gene encoding surfactant protein A2 (SFTPA2) were discovered, whose overexpression in epithelial cells in vitro also resulted in significant induction of ER stress. Moreover, prominent ER stress in AECII was also observed in FIP-patients not carrying the SFTPC/SFTPA2 mutations, as well as in patients with the more common sporadic forms of IP. Additionally, cases of adult-onset FIP with mutations in Telomerase genes and other telomereassociated components were reported. These mutations were associated with telomere shortening, which is a potential cause for triggering a persistent DNA damage response and replicative senescence in affected cells. Moreover, shortened telomeres were observed directly in the AECII of FIP-patients, and even sporadic IP cases, in the absence of any gene mutations. Here, we try to figure out the possible origins of ER stress in sporadic IP cases and non-SFTPC/SFTPA2-associated FIP.

IRE-1α regulates expression of ubiquitin specific peptidases during hypoxic response in U87 glioma cells

IRE-1α (inositol requiring enzyme-1α), the most evolutionarily conserved of the endoplasmic reticulum stress signaling pathways, is highly implicated in sustaining the proliferation of glioma cells and subsequent tumor growth, which is decreased by the inhibition of IRE-1α. To explore the IRE-1α mediated regulation of ubiquitin system in glioma cells, the expression of a subset of ubiquitin specific peptidases (USP) and of ubiquitin activating enzyme E1-like protein/autophagy related 7 (GSA7/ATG7) genes was studied, during hypoxic stress in wild type and U87 glioma cells with inhibited IRE-1α. Hypoxic treatment of wild type glioma cells leads to the up-regulation of USP25 and the concomitant downregulation of USP1, USP10, USP14, and GSA7 genes. USP4 and USP22 genes expression did not significantly change with hypoxic treatment. Inhibition of IRE-1α activity led to up-regulation of USP1, USP4, USP10, USP22, and USP25, while USP14 and GSA7 genes were down-regulated. Therefore, IRE-1α activity modifies substrate-targeting specificity to proteasome during hypoxic stress, which in turn can affect cell survival. Inhibition of IRE-1α correlates directly with deregulation of ubiquitin specific peptidases and GSA7 in a fashion that ultimately slows tumor growth.

α1-antitrypsin Deficiency: A Misfolded Secretory Protein Variant with Unique Effects on the Endoplasmic Reticulum

In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (ER) of liver cells and so ATD has come to be considered a prototypical ER storage disease . It is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (COPD) by lossof- function mechanisms and hepatic cirrhosis and carcinogenesis by gain-of-function mechanisms. The lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of α1-anitrypsin (AT) in the circulating blood and body fluids. Cigarette smoking is a powerful disease-promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non-smoking homozygotes. The liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. Liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the ER and recent studies of iPS-derived hepatocyte-like cells from individuals with ATD indicate that the changes in the ER directly vary with the hepatic phenotype i.e there is much lesser alteration in the ER in cells derived from homozygotes that do not have clinically significant liver disease. From a signaling perspective, studies in mammalian cell line and animal models expressing the classical α1-antitrypsin Z variant (ATZ) have found that ER signaling is perturbed in a relatively unique way with powerful activation of autophagy and the NFκB pathway but relatively limited, if any, UPR signaling. It is still not known how much these unique structural and functional changes and the variation among affected homozygotes relate to the tendency of this variant to polymerize and aggregate and/ or to the repertoire of proteostasis mechanisms that are activated.

Inhibition of IRE1 signaling affects expression of a subset genes encoding for TNF-related factors and receptors and modifies their hypoxic regulation in U87 glioma cells

Inhibition of IRE1 (inositol requiring enzyme-1), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth and proliferation of glioma cells. To elucidate the role of IRE1- mediated glioma growth, we studied the expression of a subset genes encoding for TNF (tumor necrosis factor)- related factors and receptors and their hypoxic regulation in U87 glioma cells overexpressing dominant-negative IRE1 (dnIRE1). We demonstrated that the expression of TNFAIP1, TNFRSF10D, TNFRSF21, TNFRSF11B, TNFSF7, and LITAF genes is increased in glioma cells with modified IRE1; however, TNFRSF10B, TRADD, and TNFAIP3 is down-regulated in these cells as compared to their control counterparts. We did not find TNFRSF1A gene expression to change significantly under this experimental condition. In control glioma cells, hypoxia leads to the up-regulated expression of TNFAIP1, TNFAIP3, TRADD, and TNFRSF10D genes and the concomitant down-regulation of TNFRSF21, TNFRSF11B, and LITAF genes; while, TNFRSF10B and TNFRSF1A genes are resistant to hypoxic treatment. However, inhibition of IRE1 modifies the hypoxic regulation of LITAF, TNFRSF21, TNFRSF11B, and TRADD genes and introduces hypoxia-induced sensitivity to TNFRSF10B, TNFRSF1A, and TNFSF7 gene expressions. Furthermore, knockdown by siRNA of TNFRSF21 mRNA modifies the hypoxic effect on the IRE1-dependent rate of proliferation and cell death in U87 glioma cells. The present study demonstrates that fine-tuned manipulation of the expression of TNF-related factors and receptors directly relating to cell death and proliferation, is mediated by an effector of endoplasmic reticulum stress, IRE1, as well as by hypoxia in a gene-specific manner. Thus, inhibition of the kinase and endoribonuclease activities of IRE1 correlates with deregulation of TNF-related factors and receptors in a manner that is gene specific and thus slows tumor growth.