Showing papers on "Integrated stress response published in 2012"

Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells

Abstract: In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo These observations have important implications in linking further pathogen detection with the integrated stress response pathways

The bacterial and cellular determinants controlling the recruitment of mTOR to the Salmonella-containing vacuole.

Abstract: Summary Bacterial invasion results in the rapid induction of an acute state of cytosolic amino acid (AA) starvation, provoked by host membrane damage. Bacteria-induced AA starvation, in turn, down-regulates mTOR signaling while triggering autophagy and the integrated stress response pathway dependent on GCN2, eIF2α and ATF3. In Salmonella -infected cells, we now demonstrate that the host AA starvation response program depended on the Salmonella pathogenicity island (SPI)-1, the activity of which was required to damage the Salmonella -containing vacuole (SCV) in the early stage of infection. At a later stage (3–4 hour post-infection), the progressive recruitment of mTOR to the surface of the SCV appeared to be independent of the activity of SPI-2 and of SCV positioning in the cell. Instead, mTOR localization to the SCV required the activity of host AA transporters SLC1A5, SLC3A2 and SLC7A5, resulting in bacterial escape from autophagy. These results expand our understanding of the mechanisms underlying the AA starvation response in Salmonella -infected cells.

Lovastatin induces multiple stress pathways including LKB1/AMPK activation that regulate its cytotoxic effects in squamous cell carcinoma cells.

Abstract: Background Cellular stress responses trigger signaling cascades that inhibit proliferation and protein translation to help alleviate the stress or if the stress cannot be overcome induce apoptosis. In recent studies, we demonstrated the ability of lovastatin, an inhibitor of mevalonate synthesis, to induce the Integrated Stress Response as well as inhibiting epidermal growth factor receptor (EGFR) activation.

Phosphoproteins in stress-induced disease.

Abstract: The integrated stress response (ISR) is an evolutionarily conserved homeostatic program activated by specific pathological states. These include amino acid deprivation, viral infection, iron deficiency, and the misfolding of proteins within the endoplasmic reticulum (ER), the so-called ER stress. Although apparently disparate, each of these stresses induces phosphorylation of a translation initiation factor, eIF2α, to attenuate new protein translation while simultaneously triggering a transcriptional program. This is achieved by four homologous stress-sensing kinases: GCN2, PKR, HRI, and PERK. In addition to these kinases, mammals possess two specific eIF2α phosphatases, GADD34 and CReP, which play crucial roles in the recovery of protein synthesis following the initial insult. They are not only important in embryonic development but also appear to play important roles in disease, particularly cancer. In this chapter, we discuss each of the eIF2α kinases, in turn, with particular emphasis on their regulation and the new insights provided by recent structural studies. We also discuss the potential for developing novel drug therapies that target the ISR.

Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex

Abstract: C. Akay, K. A. Lindl, N. Shyam, B. Nabet, Y. Goenaga-Vazquez, J. Ruzbarsky, Y. Wang, D. L. Kolson and K. L. Jordan-Sciutto (2012) Neuropathology and Applied Neurobiology38, 175–200 Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex Aims: Combined anti-retroviral therapy (cART) has led to a reduction in the incidence of HIV-associated dementia (HAD), a severe motor/cognitive disorder afflicting HIV(+) patients. However, the prevalence of subtler forms of neurocognitive dysfunction, which together with HAD are termed HIV-associated neurocognitive disorders (HAND), continues to escalate in the post-cART era. The microgliosis, astrogliosis, dendritic damage, and synaptic and neuronal loss observed in autopsy cases suggest an underlying neuroinflammatory process, due to the neurotoxic factors released by HIV-infected/activated macrophages/microglia in the brain, might underlie the pathogenesis of HAND in the post-cART era. These factors are known to induce the integrated stress response (ISR) in several neurodegenerative diseases; we have previously shown that BiP, an indicator of general ISR activation, is upregulated in cortical autopsy tissue from HIV-infected patients. The ISR is composed of three pathways, each with its own initiator protein: PERK, IRE1α and ATF6. Methods: To further elucidate the specific ISR pathways activated in the central nervous system of HAND patients, we examined the protein levels of several ISR proteins, including ATF6, peIF2α and ATF4, in cortical tissue from HIV-infected patients. Results: The ISR does not respond in an all-or-none fashion in HAND, but rather demonstrates a nuanced activation pattern. Specifically, our studies implicate the ATF6 pathway of the ISR as a more likely candidate than the PERK pathway for increases in BiP levels in astrocytes. Conclusion: These findings begin to characterize the nature of the ISR response in HAND and provide potential targets for therapeutic intervention in this disease.

ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia

Abstract: Intratumoral hypoxia is associated with poor prognosis, regardless of the mode of therapy. Cancer cells survive this condition through activating several adaptive signaling pathways, including the integrated stress response (ISR) and autophagy. Activating transcription factor 4 (ATF4) is the major transcriptional mediator of the ISR, which we have shown to be involved in autophagy regulation to protect cells from severe hypoxia. Here we demonstrate that ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. We find that the BH3-only proteins HRK, PUMA, and NOXA are transcriptionally induced in severe hypoxia and that their expression is abrogated by RNA interference against ATF4. In particular, we show that the BH3-only protein harakiri (HRK) is transactivated by ATF4 in severe hypoxia through direct binding of ATF4 to the promoter region. Furthermore, we demonstrate through siRNA knockdown that HRK induces autophagy and promotes cancer cell survival in severe hypoxia.

Translation termination efficiency modulates ATF4 response by regulating ATF4 mRNA translation at 5′ short ORFs

Abstract: The activating transcription factor 4 (ATF4) promotes transcriptional upregulation of specific target genes in response to cellular stress. ATF4 expression is regulated at the translational level by two short open reading frames (uORFs) in its 5′-untranslated region (5′-UTR). Here, we describe a mechanism regulating ATF4 expression in translation termination-deficient human cells. Using microarray analysis of total RNA and polysome-associated mRNAs, we show that depletion of the eucaryotic release factor 3a (eRF3a) induces upregulation of ATF4 and of ATF4 target genes. We show that eRF3a depletion modifies ATF4 translational control at regulatory uORFs increasing ATF4 ORF translation. Finally, we show that the increase of REDD1 expression, one of the upregulated targets of ATF4, is responsible for the mTOR pathway inhibition in eRF3a-depleted cells. Our results shed light on the molecular mechanisms connecting eRF3a depletion to mammalian target of rapamycin (mTOR) pathway inhibition and give an example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of eIF2α. We propose that in mammals, in which the 5′-UTR regulatory elements of ATF4 mRNA are strictly conserved, variations in translation termination efficiency allow the modulation of the ATF4 response.

Hyperoxia-induced activation of the integrated stress response in the newborn rat lung

Abstract: Diverse environmental stresses stimulate eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, leading to a stress-resistant state characterized by global attenuation of protein synt...

Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

Abstract: Background: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. Methodology/Principal Findings: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/2 SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/215% to 188+/227% and 100+/28.8% to 176.3+/217.3% respectively, p,0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. Conclusions/Significance: Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

Physiological ER Stress: The Model of Insulin-Secreting Pancreatic b-Cells

Abstract: Endoplasmic reticulum (ER) stress and the consecutive activation of the Unfolded Protein Response (UPR) contribute to the pathogenesis of several diseases including diabetes, neurodegenerative diseases and inflammation. However, the UPR also plays a crucial adaptive role in the acquisition and maintenance of the phenotype of cells that secrete large amounts of proteins. After a brief overview of this physiological role of the UPR in immunoglobulin-secreting plasmocytes and pancreatic acinar cells, this chapter will mainly focus on insulin-secreting pancreatic b-cells that play a critical role in glucose homeostasis. Upon their stimulation with glucose and other nutrients, these cells display a rise in mitochondrial metabolism, ATP production and Ca2+ pumping in the ER, in parallel to the stimulation of protein (preferentially proinsulin) biosynthesis. These metabolic and functional features give rise to a peculiar pattern of acute regulation of the UPR by nutrients. At low non-stimulatory glucose concentrations, when intracellular ATP, [Ca2+]ER and protein synthesis are low, the IRE1-XBP1 branch of the UPR is at its lowest level of activation while the PERK-eIF2α-ATF4 branch of the UPR is maximally activated, with strong upregulation of Integrated Stress Response (ISR) genes. Upon glucose stimulation, the rise in ATP and [Ca2+]ER leads to PERK-eIF2α dephosphorylation, inhibition of the ISR and derepression of protein synthesis. Consequent activation of the IRE1-XBP1 branch of the UPR upregulates the expression of chaperones, foldases, ER to Golgi transport and ER-associated degradation machinery that help the b-cell coping with the large increase in proinsulin biosynthesis. This opposite glucose regulation of the PERK and IRE1 arms of the UPR is rapid and dynamic, suggesting its importance in the physiological adaptation of the b-cell to changes in nutrient supply.

Abstract 4994: An integrated stress response activator causes cellular senescence and inhibits growth in multiple tumor cell types

Abstract: Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Integrated Stress Response (ISR) is a program elicited by mammalian cells to attenuate global translation in response to a variety of stressors. There are four branches of the ISR, including those mediated by PERK, which responds to endoplasmic reticulum stress, and GCN2, which is activated by amino acid deprivation. Activation of these kinases ultimately leads to translational upregulation of the transcription factor ATF4, which modulates the expression of genes involved in amino acid transport and synthesis, oxidative stress, and differentiation. Complete abrogation of the ISR or its overactivation by pharmacological agents in the presence of hypoxic or nutrient stress has been shown to lead to cell death. Here, we employed a small molecule screen to identify modulators of ATF4 expression via a reporter construct with the 5′-UTR of ATF4 fused to the luciferase gene. From this screen, a compound with ATF4-activating properties was identified. Multiple assays have been used to characterize the effects of this drug, referred to as E235, on both transformed and normal cells. Specifically, we have shown that E235 induces the ISR in a dose-dependent manner in tumor cell lines (HT1080 fibrosarcoma, B16F10 melanoma) but not in normal human diploid cells. The drug also reduces cell viability and proliferation at low μM concentrations, which does not appear to be dependent on apoptosis. By performing live cell microscopy, we observed that after prolonged (>24h) treatment with 1μM E235, the majority of cells displayed senescence-like morphology. This was confirmed by additional experiments, such as staining for senescence-associated β-galactosidase activity and immunoblotting for the upregulation of the cdk inhibitor p21. Future experiments will focus on establishing the roles of ATF4, p21 and p53 in E235-mediated senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4994. doi:1538-7445.AM2012-4994

[Cellular stress and eIF-2alpha kinase].

Abstract: The alpha subunit of eukaryotic initiation factor -2 (eIF-2alpha) is a molecule related to the first step of protein synthesis initiation in eukaryotes. eIF-2alpha is phosphorylated in response to a wide variety of stimuli, including viral infection, starvation, ischemia, and heat shock. Four mammalian eIF-2alpha kinases have been reported: PKR (double-stranded RNA dependent protein kinase), HRI (heme-regulated inhibitor), GCN2 (general control non-derepressible 2), and PERK (PKR-like endoplasmic reticulum kinase). Each kinase contains unique domains that recognize a different cellular stress and transmits the signals to eIF-2alpha. Hence, eIF-2alpha is considered to be a key molecule in integrated stress response. Understanding eIF-2alpha as a component of the integrated stress response may be helpful in revealing stress sitmulus and the responses to stress at the cellular level. This knowledge will contribute to the development of preventive and therapeutic approaches to stress mediated diseases.