General Control Nonderepressible 2 Research Articles

The non-steroidal anti-inflammatory drug indomethacin activates the eIF2α kinase PKR, causing a translational block in human colorectal cancer cells

The NSAID (non-steroidal anti-inflammatory drug) indomethacin, a cyclo-oxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against CRC (colorectal cancer) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. In the present study we show that indomethacin selectively activates the dsRNA (double-stranded RNA)-dependent protein kinase PKR in a cyclo-oxygenase-independent manner, causing rapid phosphorylation of eIF2α (the α-subunit of eukaryotic translation initiation factor 2) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of the eIF2α kinases PERK (PKR-like endoplasmic reticulum-resident kinase), GCN2 (general control non-derepressible-2) and HRI (haem-regulated inhibitor kinase), and induced eIF2α phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in vitro, an effect reversed by the PKR inhibitor 2-aminopurine, suggests a direct effect of the drug in kinase activation. The results of the present study identify PKR as a novel target of indomethacin, suggesting new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.

Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 (general control non-derepressible 2) phosphorylation of eIF2 (eukaryotic initiation factor 2), global protein synthesis reduction and increased ATF4 (activating transcription factor 4). ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 (fibroblast growth factor 21) is induced during fasting in liver and its expression induces a metabolic state that mimics long-term fasting. Thus FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In the present study, we have shown that FGF21 is induced by amino acid deprivation in both mouse liver and cultured HepG2 cells. We have identified the human FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4-binding sequences in the 5' regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional programme initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.

Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK

Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids including DNA, mRNA, and siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and protein kinase RNA-activated (PKR)-like ER kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

細胞性ストレスとeIF-2αキナーゼ

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.

Overexpression of GCN2‐type protein kinase in wheat has profound effects on free amino acid concentration and gene expression

A key point of regulation of protein synthesis and amino acid homoeostasis in eukaryotes is the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by protein kinase general control nonderepressible (GCN)-2. In this study, a GCN2-type PCR product (TaGCN2) was amplified from wheat (Triticum aestivum) RNA, while a wheat eIF2α homologue was identified in wheat genome data and found to contain a conserved target site for phosphorylation by GCN2. TaGCN2 overexpression in transgenic wheat resulted in significant decreases in total free amino acid concentration in the grain, with free asparagine concentration in particular being much lower than in controls. There were significant increases in the expression of eIF2α and protein phosphatase PP2A, as well as a nitrate reductase gene and genes encoding phosphoserine phosphatase and dihydrodipicolinate synthase, while the expression of an asparagine synthetase (AS1) gene and genes encoding cystathionine gamma-synthase and sulphur-deficiency-induced-1 all decreased significantly. Sulphur deficiency-induced activation of these genes occurred in wild-type plants but not in TaGCN2 overexpressing lines. Under sulphur deprivation, the expression of genes encoding aspartate kinase/homoserine dehydrogenase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase was also lower than in controls. The study demonstrates that TaGCN2 plays an important role in the regulation of genes encoding enzymes of amino acid biosynthesis in wheat and is the first to implicate GCN2-type protein kinases so clearly in sulphur signalling in any organism. It shows that manipulation of TaGCN2 gene expression could be used to reduce free asparagine accumulation in wheat grain and the risk of acrylamide formation in wheat products.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.

Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia

Due to the poor prognosis and limited therapeutic options for adult patients with acute lymphoblastic leukemia (ALL), development of novel therapies is much needed to prolong patient survival and increase the efficacy of their treatment. Malignant T cells need high levels of nutrients to maintain their proliferation rate. Borrelidin, a small molecule nitrile-containing macrolide, is an inhibitor of bacterial and eukaryal threonyl-tRNA synthetase. Borrelidin-mediated inhibition of aminoacyl-tRNA synthesis, leads to an induction in the levels of uncharged tRNA, nutritional stress and ultimately inhibition of protein synthesis. The aim of the present study was to investigate whether borrelidin treatment inhibits the proliferation of malignant ALL cell lines, Jurkat and CEM cells, and study the mechanism by which this drug acts. Our results show that borrelidin was able to potently inhibit the proliferation of ALL cell lines with a half maximal inhibitory concentration of 50ng/ml. Borrelidin showed a greater inhibitory effect on ALL cell lines compared to primary fibroblasts. Flow cytometry and western blot analysis indicated that borrelidin was able to increase the level of apoptosis and cause G(1) arrest in ALL cell lines. Activation of the general control nonderepressible-2 (GCN2) kinase stress responsive pathway and induction of CHOP protein was significantly higher in ALL cell lines treated with borrelidin. These findings collectively suggest for the first time that borrelidin targets ALL cell lines by inducing apoptosis and mediating G(1) arrest and that borrelidin treatment in ALL cell lines is correlated with activation of the GCN2 kinase pathway.

Detection of physically interacting proteins with the CC and NB-ARC domains of a putative yellow rust resistance protein, Yr10, in wheat

Gene-for-gene (GFG) resistance is a potent defense mechanism in plants, that is mediated by resistance (R) proteins. In GFG resistance, pathogen effector or avirulence (Avr) proteins are recognised by R-proteins which initiate a series of signal transduction events that lead to hypersensitive cell death. In cereals, many R-proteins are comprised of an N-terminal coiled-coiled (CC) domain, a Nucleotide Binding (NB) domain and a Leucine Rich Repeats (LRR) region associated with effector recognition. NB-LRR immunity proteins are highly conserved across plant and animal taxa. To advance understating of signal transduction events in cereals, we exploited the high level of protein conservation to first identify yeast gene products interacting with the CC and NB domains of the candidate yellow rust R-protein (Yr10). Screening revealed proteins having mostly apoptosis related functions (Signal Recognition Particle 72kDa (SRP72); Chromosome SEgregation 1 (CSE1); ARrest Defective 1 (ARD1), translation initiation control in response to stress conditions (General Control Nonderepressible 2 (GCN2)), and a HSP90 co-chaperone (Cyclosporin-sensitive proline rotamase 7 (CPR7)). We then identified the close homologues of the interactors in barley and show that they were induced at 6h and/or 12 h after infection in Mla3 mediated Powdery Mildew (Blumeria graminis f.sp. hordei, Bgh) disease resistance, suggesting their involvement in pathogen response.

Abstract 2642: Small molecule inhibitor borrelidin induces apoptosis in acute lymphoblastic leukemia cell lines in association with GCN2 kinase pathway activation

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. L-asparaginase (L-ASNase), an important agent in the treatment of ALL, catalyzes the hydrolysis of asparagine (Asn), leading to activation of the general control nonderepressible-2(GCN2) kinase stress-responsive pathway, inhibition of protein synthesis, and apoptotic cell death. Unfortunately, allergic hypersensitivity reactions can occur in patients after repeated administrations of ASNase and are the main reason for discontinuation of ASNase treatment. Borrelidin, a small molecule nitrile-containing macrolide, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). As both ASNase and borrelidin increase the levels of uncharged tRNA, we hypothesize that ASNase may be replaced by borrelidin. Thus, the aim of the present study was to investigate if borrelidin could have a potential role in targeting ALL cell lines and, whether the GCN2 kinase pathway activation is involved as one of the downstream mechanisms by which borrelidin affects lymphoblastic cells. Methods: In order to mediate inhibition of threonyl-tRNA synthesis and to induce nutritional stress in ALL cell lines, Jurkat and CEM cells, cells were treated with borrelidin. Proliferation assay was also performed and the growth rate of these cells was studied. Propidium iodide staining and flow cytometry were performed to study the apoptotic effects of borrelidin on these cells as well as to study the effects of borrelidin on cell cycle progression. The levels of phosphorylated GCN2, total GCN2, phosphorylated eIF2α, total eIF2α, CHOP and cleaved PARP were evaluated by Western blot analysis. Results: Borrelidin was able to potently inhibit the proliferation and growth of ALL cell lines in low concentrations. Borrelidin showed a greater apoptotic effect on T cells compared to primary fibroblasts (half maximal inhibitory concentration (IC50) of 50 ng/ml and 375 ng/ml respectively). Flow cytometry and Western blot analysis indicated that borrelidin was able to increase the level of apoptosis in treated Jurkat cells as compared with non-treated control groups (3.9 ± 1.26 and 0.77 ± 0.06, respectively; p<0.01, n=3). Flow cytometry results showed a significant dose-dependent G1 arrest in Jurkat cells treated with borrelidin (10.88 ± 0.63) compared to that of non-treated control group (4.18 ± 0.24; p<0.001, n=3). Activation of the GCN2 kinase pathway as well as induction of the proapoptotic CHOP protein was significantly higher in ALL cell lines treated with borrelidin relative to the control non-treated cells. Conclusions: These findings collectively suggest for the first time that borrelidin may have promising potentials in treating ALL by activating the GCN2 kinase pathway, inducing apoptosis and mediating G1 arrest in T-cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2011-2642

Leucine Deprivation Increases Hepatic Insulin Sensitivity via GCN2/mTOR/S6K1 and AMPK Pathways

OBJECTIVEWe have previously shown that serum insulin levels decrease threefold and blood glucose levels remain normal in mice fed a leucine-deficient diet, suggesting increased insulin sensitivity. The goal of the current study is to investigate this possibility and elucidate the underlying cellular mechanisms.RESEARCH DESIGN AND METHODSChanges in metabolic parameters and expression of genes and proteins involved in regulation of insulin sensitivity were analyzed in mice, human HepG2 cells, and mouse primary hepatocytes under leucine deprivation.RESULTSWe show that leucine deprivation improves hepatic insulin sensitivity by sequentially activating general control nonderepressible (GCN)2 and decreasing mammalian target of rapamycin/S6K1 signaling. In addition, we show that activation of AMP-activated protein kinase also contributes to leucine deprivation–increased hepatic insulin sensitivity. Finally, we show that leucine deprivation improves insulin sensitivity under insulin-resistant conditions.CONCLUSIONSThis study describes mechanisms underlying increased hepatic insulin sensitivity under leucine deprivation. Furthermore, we demonstrate a novel function for GCN2 in the regulation of insulin sensitivity. These observations provide a rationale for short-term dietary restriction of leucine for the treatment of insulin resistance and associated metabolic diseases.

The eIF2 Kinase GCN2 Is Essential for the Murine Immune System to Adapt to Amino Acid Deprivation by Asparaginase1–3

Amino acid starvation by asparaginase (ASNase) enhances phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible 2 (GCN2) kinase, leading to reduced global mRNA translation rates. This conserves energy and allows cells time to reprogram stress-related gene expression to alleviate cell injury. This study addressed the importance of GCN2 for the immune system to adapt to amino acid starvation by ASNase. GCN2+/+ and GCN2−/− mice were injected once daily with ASNase or saline for up to 7 d. In both thymus and spleen, activation of amino acid stress response genes to ASNase, such as asparagine synthetase and CAAT enhancer binding protein homologous protein, required GCN2. ASNase reduced food intake and body weight in both genotypes, but spleen and thymus wet weights and total cell numbers in thymus, spleen, bone marrow, and mesenteric lymph nodes were less in GCN2−/− mice treated with ASNase (genotype x ASNase, P < 0.05). In the thymus, GCN2−/− mice treated with ASNase demonstrated enhanced apoptosis and fewer cells in all subpopulations examined (CD3+, CD4–8-, CD4+8+, CD4+8-, CD4–8+) compared with GCN2+/+ mice treated with ASNase (genotype x ASNase, P < 0.05). In the spleen, GCN2 deletion magnified ASNase-induced reductions in CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ leukocytes (genotype x ASNase, P < 0.05). These results indicate that loss of GCN2 enhances immunosuppression by ASNase and that this eIF2 kinase is broadly required for amino acid stress management in the immune system.

Stress Signaling in the Methionine-Choline–Deficient Model of Murine Fatty Liver Disease

Stress signaling, both within and outside the endoplasmic reticulum, has been linked to metabolic dysregulation and hepatic steatosis. Methionine-choline-deficient (MCD) diets cause severe fatty liver disease and have the potential to cause many types of cellular stress. The purpose of this study was to characterize hepatic stress in MCD-fed mice and explore the relationship between MCD-mediated stress and liver injury. Stress signaling was examined in mice fed MCD formulas for 4-21 days. Signaling also was evaluated in mice fed MCD formulas supplemented with clofibrate, which inhibits hepatic triglyceride accumulation. The role of the pro-apoptotic stress protein C/EBP homologous protein (CHOP) in MCD-mediated liver injury was assessed by comparing the responses of wild-type and CHOP-deficient mice to an MCD diet. MCD feeding caused steatohepatitis coincident with the activation of cJun N-terminal kinase and caspase-12. In contrast, MCD feeding did not activate inositol-requiring protein-1 and actually suppressed the expression of X-box protein-1s. MCD feeding caused weak stimulation of double-stranded RNA-activated protein kinase-like endoplasmic reticulum-resident kinase, but robust activation of general control nonderepressible-2, followed by the phosphorylation of eukaryotic initiating factor-2α and induction of CHOP. Clofibrate eliminated MCD-mediated hepatic steatosis but did not inhibit diet-induced stress. CHOP deficiency did not alleviate, and in fact worsened, MCD-mediated liver disease. MCD feeding causes an integrated stress response in the liver rather than a classic unfolded protein response. This stress response does not by itself lead to liver injury. CHOP, despite its identity as a mediator of stress-related cell death, does not play a central role in the pathogenesis of MCD-mediated liver disease.

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

Autophagy impairment stimulates PS1 expression and γ-secretase activity

γ-Secretase plays an important role in the development of Alzheimer disease (AD). γ-Secretase activity is enriched in autophagic vacuoles and it augments amyloid-β (Aβ) synthesis. Autophagy-lysosomal dysfunction has been implicated in AD, but whether γ-secretase activity is affected by autophagy remains unclear. Here we report that γ-secretase activity is enhanced in basal autophagy-disturbed cells through the α subunit of eukaryotic translation initiation factor 2 (eIF2α) kinase, general control nonderepressible 2 (GCN2). Presenilin-1 (PS1) expression was increased even in the presence of nutrients in autophagy-related 5 knockdown (Atg5KD) human embryonic kidney (HEK293) cells expressing a short hairpin RNA as well as in chloroquine-treated HEK293 cells. However, PS1 expression induction was prevented in GCN2KD and ATF4KD cells. Furthermore, Atg5KD cells showed an increase in Aβ production and Notch1 cleavage. These were reduced by an autophagy inducer, resveratrol. Thus, we conclude that the autophagy-lysosomal system regulates γ-secretase activity through GCN2.

The interface between metabolic and stress signalling.

It is becoming increasingly clear that stress and metabolic signalling networks interact and that this interaction is important in plant responses to herbivory, pathogen attack, drought, cold, heat and osmotic stresses including salinity. At the interface between these two major signalling systems are the hormone abscisic acid (ABA) and signalling factors including protein kinases and transcription factors. This briefing reviews links between ABA, stress and sugar signalling, focusing on the roles of sucrose non-fermenting-1-related protein kinases (SnRKs), SnRK1-activating protein kinases (SnAKs), calcium-dependent protein kinases (CDPKs) and ABA response element binding proteins (AREBPs, which are transcription factors). Links between stress and nitrogen / amino acid signalling are also described, including the roles of a protein kinase called general control non-derepressible (GCN)-2 in regulating protein synthesis through phosphorylation of the alpha-subunit of translation initiation factor-2 (eIF2alpha) in response not only to decreases in amino acid levels but also to a range of stresses. Evidence of a link between sugar and amino acid signalling is explored, with nitrate reductase being a target for regulation by both SnRK1 and GCN2 through different mechanisms; possible links between SnRK1 and GCN2 via a pathway including the protein kinase target of rapamycin (TOR)-1 are described. The significance of these interactions to the concept of signalling networks as opposed to simple cascades and pathways, and the importance of the subject in the context of the predicted increase in severity and range of stresses that plants will have to withstand as a result of global climate change are discussed.

Amino Acid Deprivation Links BLIMP-1 to the Immunomodulatory Enzyme Indoleamine 2,3-Dioxygenase

Catabolism of tryptophan by IDO1 plays an important role in the control of immune responses. Activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinase general control nonderepressible-2 (GCN2) following tryptophan depletion is a major pathway mediating this effect. However, immunomodulatory target genes of GCN2 activation are poorly defined. The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP-1) is a target of the eIF2alpha kinase1, protein kinase-like ER kinase (PERK) during the unfolded protein response of the endoplasmic reticulum. Thus, BLIMP-1 might also be a mediator of the GCN2 stress response pathway activated by IDO1 and tryptophan depletion. Indeed, in human monocytes BLIMP-1 mRNA and protein are up-regulated in response to both a pharmacological activator of GCN2 and tryptophan-depletion generated by IDO1-transfected cells. This suggests a functional role for BLIMP-1 in the immunomodulatory effects of the IDO1-GCN2 axis. BLIMP-1 has been shown to repress IFN-gamma-regulated promoters. As IDO1 is itself highly responsive to IFN-gamma, we hypothesized that BLIMP-1 functions in a feedback loop to regulate IDO1 expression. We found that BLIMP-1 binds to IFN-responsive sites in the IDO1 promoter and represses IFN-dependent IDO1 activation. We propose that BLIMP-1 acts in a negative feedback loop to successfully balance the outcome of tolerance vs inflammation.

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function.

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)(2) (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-kappaB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 microM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 muM with marked suppression at >100 microM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Indeed, we found a marked (approximately 4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2alpha-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2alpha kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2alpha kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.

EIF2α kinases GCN2 and PERK modulate transcription and translation of distinct sets of mRNAs in mouse liver

In eukaryotes, selective derepression of mRNA translation through altered utilization of upstream open reading frames (uORF) or internal ribosomal entry sites (IRES) regulatory motifs following exposure to stress is regulated at the initiation stage through the increased phosphorylation of eukaryotic initiation factor 2 on its alpha-subunit (eIF2alpha). While there is only one known eIF2alpha kinase in yeast, general control nonderepressible 2 (GCN2), mammals have evolved to express at least four: GCN2, heme-regulated inhibitor kinase (HRI), double-stranded RNA-activated protein kinase (PKR), and PKR-like endoplasmic reticulum-resident kinase (PERK). So far, the main known distinction among these four kinases is their activation in response to different acute stressors. In the present study, we used the in situ perfused mouse liver model and hybridization array analyses to assess the general translational response to stress regulated by two of these kinases, GCN2 and PERK, and to differentiate between the downstream effects of activating GCN2 versus PERK. The resulting data showed that at least 2.5% of mouse liver mRNAs are subject to derepressed translation following stress. In addition, the data demonstrated that eIF2alpha kinases GCN2 and PERK differentially regulate mRNA transcription and translation, which in the latter case suggests that increased eIF2alpha phosphorylation is not sufficient for derepression of translation. These findings open an avenue for more focused future research toward groups of mRNAs that code for the early cellular stress response proteins.