Activating Transcription Factor 4 mRNA Research Articles

MiR-15b-5p promotes HgCl2-induced chicken embryo kidney cells ferroptosis by targeting β-TrCP-mediated ATF4 ubiquitin degradation

Mercuric chloride (HgCl2), a widespread environmental pollutant, induces ferroptosis in chicken embryonic kidney (CEK) cells. Whereas activating transcription factor 4 (ATF4), a critical mediator of oxidative homeostasis, plays a dual role in ferroptosis, but its precise mechanisms in HgCl2-induced ferroptosis remain elusive. This study aims to investigate the function and molecular mechanism of ATF4 in HgCl2-induced ferroptosis. Our results revealed that ATF4 was downregulated during HgCl2-induced ferroptosis in CEK cells. Surprisingly, HgCl2 exposure has no significant impact on ATF4 mRNA level. Further investigation indicated that HgCl2 enhanced the expression of the E3 ligase beta-transducin repeat-containing protein (β-TrCP) and increased ATF4 ubiquitination. Subsequent findings identified that miR-15b-5p as an upstream modulator of β-TrCP, with miR-15b-5p downregulation observed in HgCl2-exposed CEK cells. Importantly, miR-15b-5p mimics suppressed β-TrCP expression and reversed HgCl2-induced cellular ferroptosis. Mechanistically, HgCl2 inhibited miR-15b-5p, and promoted β-TrCP-mediated ubiquitin degradation of ATF4, thereby inhibited the expression of antioxidant-related target genes and promoted ferroptosis. In conclusion, our study highlighted the crucial role of the miR-15b-5p/β-TrCP/ATF4 axis in HgCl2-induced nephrotoxicity, offering a new therapeutic target for understanding the mechanism of HgCl2 nephrotoxicity.

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4.

Accumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer's disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4. We used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses. We show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer's Disease mice model. The same results are seen in a human in vitro model of AD. Together, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt.

G-Quadruplex Structures as a "Switch" Regulate ATF4 Expression in Ferroptotic HepG2 Cells.

G-quadruplex (G4) is a noncanonical structure folded in a widespread manner by guanine-rich tandem repeated sequences. As a key response factor, activating transcription factor 4 (ATF4) has dual functions in managing iron-dependent ferroptosis by regulating amino acid synthesis and antioxidant-related gene expression. In our study, the activity of ATF4 expression was elevated in HepG2 cells induced by erastin. Based on preliminary bioinformatics analyses, the G-tract region, named WT, had high potential to form G4, and it was found that PDS could markedly weaken the increase of ATF4 expression by reducing the sensitivity of HepG2 cells toward erastin. In circular dichroism spectra, WT oligonucleotides showed characteristic molar ellipticity at specific wavelengths of parallel G4 structures, while corresponding single-base mutants possessed a weaker ability to form G4, which were consistent with immunostaining results. In addition, endogenous G4 formed by the WT motif was significantly destroyed in HepG2 cells treated with erastin. After being transfected with WT oligonucleotides, the levels of ATF4 mRNA decreased significantly regardless of being treated with erastin or not. Meanwhile, mutations of G-tracts could advantageously impact the luciferase expression downstream of an ATF4 promoter in reporter assays, manifesting that the decrease of endogenous G4 in the ATF4 promoter was positively associated with the expression enhanced by erastin in HepG2 cells.

Abstract 12592: Nuclear Sirtuin Sirt6, Protects Mice From Endotoxin-Induced Septic Cardiomyopathy

Introduction: Dysregulated systemic inflammatory response from host to infection can result in sepsis and multi-organ failure. Heart is one of the most affected organ. Even with recent medical advances, mortality rate with sepsis is 30-70%. Imbalance of inflammatory and metabolic pathways are crucial in the pathogenesis of sepsis and a multi-target approach is needed. SIRT6 is a key enzyme shown to restore energy homeostasis and to promote cardiac health in various adverse conditions. Hypothesis: We examined whether SIRT6 could alleviate pathogenic effects of sepsis in a murine lipopolysaccharide (LPS)-induced cardiomyopathy model. Methods: By injecting 10mg/kg LPS in wild-type (WT) and SIRT6 whole body overexpressing (T6-Tg) mice, endotoxemia was induced (n=5-6 each). Cardiac functions were assessed by echocardiography. Using heart tissues (n=3 each), TaqMan gene expression arrays were analyzed. Heart protein and serum cytokine levels were also estimated. Rat H9C2 cell line was used for in vitro studies. Results: SIRT6 overexpression in mice prevented LPS-induced hypothermia (p<0.001) and limited mortality to 30% compared to WT. LPS-injected T6-Tg mice showed preserved cardiac functions compared to WT (fractional shortening and ejection fraction, p<0.001). LPS-injected WT mice hearts showed 2- to 6-fold mRNA upregulation in pro-inflammatory transcription factors HIF1α, TLR4 and activating transcription factor 4 (ATF4). These factors were significantly (p<0.05) downregulated in LPS-injected T6-Tg hearts. With four-fold increase in ATF4 mRNA, a known inducer of amino acid transporters, expression of solute carriers (SLC3a2/7a1/7a5/7a8) was significantly upregulated in LPS-treated WT hearts, while reduced with SIRT6 overexpression. Pyruvate dehydrogenase kinase 4, which controls metabolic switch in sepsis, showed 4-fold increase in mRNA expression in LPS-treated H9C2 cells that was downregulated by SIRT6 overexpression. Similarly, upregulated inflammatory cytokines IL6, TNFα and lipocalin-2 were significantly (p<0.05) downregulated with SIRT6 overexpression. Conclusion: Our results indicate that SIRT6 could protect heart from LPS-induced sepsis by restoring metabolic homeostasis via regulation of multiple pathways.

Mechanism of miR-338-3p in sepsis-induced acute lung injury via indirectly modulating ATF4

Sepsis is recognized as an inflammation-related syndrome in response to invading pathogens. Many patients suffer from sepsis including transplant recipients. Lipopolysaccharide (LPS) is known to trigger sepsis-related organ dysfunction. This study expounded on the possible effect of microRNA (miR)-338-3p in sepsis-induced acute lung injury (ALI). Firstly, human bronchial epithelial cell line 16HBE received LPS treatment to establish the cell models of sepsis-induced ALI. The expression patterns of miR-338-3p, long non-coding RNA OPA-interacting protein 5 antisense transcript 1 (lncRNA OIP5-AS1), and activating transcription factor 4 (ATF4) in 16HBE cells were examined. Afterwards, 16HBE cell viability, the apoptosis rate, and the levels of inflammation and lactate dehydrogenase (LDH) were determined to assess the degree of cell injury. We disclosed that LPS treatment triggered 16HBE cell injury, downregulated miR-338-3p, and upregulated OIP5-AS1 and ATF4. miR-338-3p overexpression repressed LPS-induced 16HBE cell injury. miR-338-3p diminished OIP5-AS1 stability via binding to OIP5-AS1 and downregulated OIP5-AS1 expression and OIP5-AS1 can enhance ATF4 mRNA stability and upregulate ATF4 mRNA level. The rescue experiments showed that ATF4 overexpression aggravated LPS-induced 16HBE cell injury. Overall, miR-338-3p overexpression decreased OIP5-AS1 expression and stability and further downregulated ATF4 mRNA level, thereby mitigating LPS-induced 16HBE cell injury.

Specific amino acids regulate Sestrin2 mRNA and protein levels in an ATF4-dependent manner in C2C12 myocytes

BackgroundSestrin2 is a conserved protein in several species, and its expression is upregulated in cells under various environmental stresses. Sestrin2 content is involved in the function of mechanistic target of rapamycin complex 1 (mTORC1) in mouse embryonic fibroblasts and C2C12 cells. MethodsC2C12 cells were treated with amino acid-free DMEM (AF-DMEM) for 5 h. The effects of the addition of specific amino acids to AF-DMEM on Sestrin2 mRNA and protein expression were examined using RT-qPCR and western blotting, respectively. The mechanism by which amino acids regulate Sestrin2 mRNA expression was examined using blocking and siRNA experiments. ResultsAF-DMEM increased the mRNA and protein levels of both Sestrin2 and activating transcription factor 4 (ATF4). The addition of a specific amino acid changed Sestrin2 mRNA and protein levels. The response pattern of Sestrin2 to specific amino acids was similar to that of ATF4. ATF4 siRNA reduced Sestrin2 mRNA levels. AF-DMEM increased eukaryotic initiation factor 2α (eIF2α) phosphorylation as early as 10 min after the treatment; however, ATF4 and Sestrin2 were increased 300 min after the treatment. Nuclear factor erythroid 2-related factor 2 and pancreatic and duodenal homeobox 1 siRNA did not affect ATF4 and Sestrin2 mRNA expression. ConclusionsSpecific Amino acids regulate Sestrin2 levels in an ATF4-dependent manner in C2C12 cells. General significanceThe results of the present study indicate that amino acids regulate levels of Sestrin2, which might cause phenotypic alterations, including mTORC1 activity, in C2C12 cells.

PRMT5 regulates ATF4 transcript splicing and oxidative stress response

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

Translational control by heme-regulated elF2α kinase during erythropoiesis.

HRI is the heme-regulated elF2α kinase that phosphorylates the α-subunit of elF2. Although the role of HRI in inhibiting globin synthesis in erythroid cells is well established, broader roles of HRI in translation have been uncovered recently. This review is to summarize the new discoveries of HRI in stress erythropoiesis and in fetal γ-globin expression. HRI and activating transcription factor 4 (ATF4) mRNAs are highly expressed in early erythroblasts. Inhibition of protein synthesis by HRI-phosphorylated elF2α (elF2αP) is necessary to maintain protein homeostasis in both the cytoplasm and mitochondria. In addition, HRI-elF2αP specifically enhances translation of ATF4 mRNA leading to the repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling. ATF4-target genes are most highly activated during iron deficiency to maintain mitochondrial function, redox homeostasis, and to enable erythroid differentiation. HRI is therefore a master translation regulator of erythropoiesis sensing intracellular heme concentrations and oxidative stress for effective erythropoiesis. Intriguingly, HRI-elF2αP-ATF4 signaling also inhibits fetal hemoglobin production in human erythroid cells. The primary function of HRI is to maintain protein homeostasis accompanied by the induction of ATF4 to mitigate stress. Role of HRI-ATF4 in γ-globin expression raises the potential of HRI as a therapeutic target for hemoglobinopathy.

Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition.

As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy.Methods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model.Results: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N6-methyladenosine (m6A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition.Conclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m6A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment.

Ribosome assembly factor URB1 contributes to colorectal cancer proliferation through transcriptional activation of ATF4.

Ribosome assembly factor URB1 is essential for ribosome biogenesis. However, its latent role in cancer remains unclear. Analysis of The Cancer Genome Atlas database and clinical tissue microarray staining showed that URB1 expression was upregulated in colorectal cancer (CRC) and prominently related to clinicopathological characteristics. Silencing of URB1 hampered human CRC cell proliferation and growth in vitro and in vivo. Microarray screening, ingenuity pathway analysis, and JASPAR assessment indicated that activating transcription factor 4 (ATF4) and X‐box binding protein 1 (XBP1) are potential downstream targets of URB1 and could transcriptionally interact through direct binding. Silencing of URB1 significantly decreased ATF4 and cyclin A2 (CCNA2) expression in vivo and in vitro. Restoration of ATF4 effectively reversed the malignant proliferation phenotype of URB1‐silenced CRC cells. Dual‐luciferase reporter and ChIP assays indicated that XBP1 transcriptionally activated ATF4 by binding with its promoter region. X‐box binding protein 1 colocalized with ATF4 in the nuclei of RKO cells, and ATF4 mRNA expression was positively regulated by XBP1. This study shows that URB1 contributes to oncogenesis and CRC growth through XBP1‐mediated transcriptional activation of ATF4. Therefore, URB1 could be a potential therapeutic target for CRC.

ATF4-modified serum exosomes derived from osteoarthritic mice inhibit osteoarthritis by inducing autophagy.

Activating transcription factor 4 (ATF4) is critical for chondrocyte proliferation and bone formation. Exosomes are considered as promising gene-delivery vehicles for the treatment of osteoarthritis (OA). This study utilized the serum-derived exosomes from OA mice as the gene-delivery vehicles for ATF4 gene therapy and explored their therapeutic effects on OA. Meniscus injury-induced OA model was established by the excision of anterior part of medial meniscus in the right knee of C57BL/6J mice. Exosomes were isolated from serum samples of sham and OA mice, and were referred to as sham-Exo and OA-Exo, respectively. ATF4-overexpressing OA-Exo (ATF4-OA-Exo) was developed by introducing ATF4 mRNA into OA-Exo via electroporation. Four weeks after surgery, OA mice received intra-articular injections of sham-Exo, OA-Exo, and ATF4-OA-Exo, respectively. The results showed that intra-articular injection of ATF4-OA-Exo alleviated articular cartilage degeneration or damage and inflammatory response of OA mice. Autophagy was weakened in knee joint cartilage of OA mice, which was partially restored by intra-articular injection of ATF4-OA-Exo. Further in vitro assays revealed that ATF4-OA-Exo promoted chondrocyte autophagy and inhibited chondrocyte apoptosis in the TNF-α- or tunicamycin-treated chondrocytes. Together, ATF4-modified serum exosomes derived from OA mice protect cartilage and alleviate OA progression by inducing autophagy.

Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

Accumulation of advanced glycation end products in follicles is associated with poor oocyte developmental competence.

Advanced glycation end products (AGEs) affect the follicular microenvironment. The close relationship between AGEs, proinflammatory cytokine production and activation of the unfolded protein response (UPR), which involves activating transcription factor 4 (ATF4), is crucial for regulation of various cellular functions. We examined whether accumulation of AGEs in follicles was associated with proinflammatory cytokine production and activation of the UPR in granulosa cells and decreased oocyte developmental competence. Concentrations of AGEs, soluble receptor for AGE (sRAGE), interleukin (IL)-6 and IL-8 in follicular fluid (FF) were examined by ELISAs in 50 follicles. mRNA expression of ATF4, IL-6 and IL-8 in cumulus cells (CCs) were examined by quantitative RT-PCR in 77 samples. Cultured human granulosa-lutein cells (GLCs) were treated with AGE-bovine serum albumin (BSA) alone or following transfection of ATF4-targeting small interfering RNA. The AGE concentration and the AGE/sRAGE ratio in FF were significantly higher in follicles containing oocytes that developed into poor-morphology embryos (group I) than those with good-morphology embryos (group II). When compared with sibling follicles from the same patients, the AGE/sRAGE and concentrations of IL-6 and IL-8 in FF, as well as ATF4, IL-6 and IL-8 mRNA expression in CCs, were significantly higher in group I follicles than group II. AGE treatment increased mRNA expression of ATF4, IL-6 and IL-8 in cultured GLCs. Knockdown of ATF4 abrogated the stimulatory effects of AGE on mRNA expression and protein secretion of IL-6 and IL-8. Our findings support the idea that accumulation of AGEs in follicles reduces oocyte competence by triggering inflammation via activation of ATF4 in the follicular microenvironment.

SAT-183 Activating Transcription Factor 4 (ATF4) As A Possible Mediator Of Gluconeogenic And Lipogenic Actions Of Fat Mass And Obesity Associated (FTO) In Liver

Hepatic metabolism is critical for the maintenance of whole body glucose and lipid metabolism. Excessive hepatic glucose production and de novo lipogenesis contributes to fasting hyperglycemia in diabetes and hepatic steatosis, respectively. Common genetic variants of the fat mass and obesity associated (FTO) gene are strongly associated with obesity and type 2 diabetes. However, the role of FTO in the regulation of hepatic metabolism is not well understood. We have demonstrated that increased FTO level leads to an increase in gluconeogenic glucose-6-phosphatase (G6Pase) mRNA expression in hepatic cells. Conversely, reduced FTO level or inhibition of FTO activity caused a reduction of G6Pase mRNA expression. The transcription factor activating transcription factor 4 (ATF4) is a positive regulator of the expression of gluconeogenic and lipogenic genes in hepatocytes. These findings led to the hypothesis that increased hepatic FTO expression or activity leads to an increase expression of not only gluconeogenic genes but also lipogenic genes by activating ATF4, while reduced hepatic FTO expression or activity produces opposite effects. To address this hypothesis, we examined the effect of altered FTO expression on ATF4 and lipogenic stearoyl-CoA desaturase (SCD1) mRNA levels in alpha mouse liver 12 (AML12) immortalized hepatic cell line. We also treated AML12 cells with a small molecule FTO inhibitor rhein and measured the expression level of these genes. FTO overexpression resulted in increased ATF4 and SCD1 mRNA levels. FTO knockdown resulted in a reduction of SCD1 mRNA levels. Similarly, rhein treatment (1 μM) for 24 h reduced levels of ATF4 and SCD1 mRNA without causing cytotoxicity. These data support the hypothesis that FTO acts to shift hepatic metabolism toward anabolism, leading to increased gluconeogenesis and de novo lipogenesis and ATF4 is a possible mediator of this action. Our data also suggest that reduced activity of hepatic signaling pathways involving FTO and ATF4 may be effective in reversing hyperglycemic and hepatic steatosis partly by reducing gluconeogenesis and de novo lipogenesis in the liver. Sources of Research Support: Supported by University Research Grants Program (URGP) from University of Manitoba.

Eukaryotic Initiation Factor 5B (eIF5B) Cooperates with eIF1A and eIF5 to Facilitate uORF2-Mediated Repression of ATF4 Translation.

A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B—which can substitute for eIF2 in delivering Met-tRNAi—leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.

The oxido-metabolic driver ATF4 enhances temozolamide chemo-resistance in human gliomas.

Malignant gliomas are devastating neoplasia with limited curative treatment options. Temozolomide (TMZ, Temcat®, Temodal® or Temodar®) is a first-line treatment for malignant gliomas but the development of drug resistance remains a major concern. Activating transcription factor 4 (ATF4) is a critical oxido-metabolic regulator in gliomas, and its role in the pathogenesis of TMZ-resistance remains elusive. We investigated the effect of TMZ on human glioma cells under conditions of enhanced ATF4 expression (ATF4OE) and ATF4 knock down (ATF4KD). We monitored cell survival, ATF4 mRNA expression of ATF4 and xCT (SLC7a11) regulation within human gliomas. TMZ treatment induces a transcriptional response with elevated expression of ATF4, xCT and Nrf2, as a sign of ER stress and toxic cell damage response. ATF4 overexpression (ATF4OE) fosters TMZ resistance in human gliomas and inhibits TMZ-induced autophagy. Conversely, ATF4 suppression by small interfering RNAs (ATF4KD) leads to increased TMZ susceptibility and autophagy in comparison to wild type gliomas. ATF4OE gliomas show reduced cell cycle shift and apoptotic cell death, whereas ATF4KD gliomas reveal higher susceptibility towards cell cycle rearrangements. Hence, the migration capacity of ATF4OE glioma cells is almost not affected by TMZ treatment. In contrast, ATF4KD gliomas show a migratory stop following TMZ application. Mechanistically, xCT elevation is a consequence of ATF4 activation and increased levels of xCT amplifies ATF4-induced TMZ resistance. Our data show that ATF4 operates as a chemo-resistance gene in gliomas, and the tumor promoting function of ATF4 is mainly determined by its transcriptional target xCT. Therefore, therapeutic inactivation of ATF4 can be a promising strategy to overcome chemo-resistance and promote drug efficacy in human gliomas.

Effects of GSK2606414 on cell proliferation and endoplasmic reticulum stress‑associated gene expression in retinal pigment epithelial cells.

GSK2606414 is a novel, highly selective inhibitor of protein kinase R‑like endoplasmic reticulum kinase (PERK). GSK2606414 and its analogues have recently been demonstrated to delay tumor growth and prevent neurodegeneration. The present study investigated the effects of GSK2606414 on proliferation, apoptosis, and the expression of activating transcription factor4 (ATF4), CCAAT/enhancer‑binding protein homologous protein (CHOP) and vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (RPE) cells under endoplasmic reticulum (ER) stress. ARPE‑19 human RPE cells were treated with 0.01‑50µM GSK2606414, and ER stress was induced by thapsigargin (TG) treatment. Cell proliferation was assessed using the Cell Counting kit‑8 cell viability assay. Apoptosis was detected by Annexin‑V/propidium iodide double staining using flow cytometry. Western blot analysis was used to measure eukaryotic initiation factor2α (eIF2α) phosphorylation levels. ATF4, CHOP and VEGF mRNA expression levels were assessed using reverse transcription‑quantitative polymerase chain reaction. GSK2606414 treatment inhibited RPE cell proliferation in a dose‑dependent manner, however it did not induce apoptosis. In addition, GSK2606414 treatment inhibited eIF2α phosphorylation and reduced CHOP and VEGF mRNA expression levels in RPE cells under TG‑induced ER stress. To the best of our knowledge, the present study is the first to demonstrate that GSK2606414 has a potential antiproliferative effect in RPE cells invitro. This effect appeared to be achieved via inhibition of the PERK/ATF4/CHOP signaling pathwayand suppression of VEGF expression levels.

Activation of endoplasmic reticulum stress and its effect on osteogenic differentiation induced by micropit/nanotube topography

Objective: To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropit/nanotube topography (MNT), so as to provide guidance for the topography design of biomaterials. Methods: Four sample groups were fabricated: polishing control group (polished titanium, PT, no treatment), thapsigargin treatment (TG, 0.1 μmol/L TG treated for 9 h), MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching). Scanning electron microscope (SEM) was used to observe the topography of Ti samples. The alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7, 14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation. After 12 h incubation, the shape and size of ER was examined using a transmission electron microscope (TEM), and ERS-related genes including immunoglobulin heavy chain binding protein (BiP), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR). Results: After 7, 14 and 21 d of induction, the ALP production, collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT (P<0.05). The cells grown on TG, MNT5 and MNT20 surfaces displayed gross distortions of the ER. Compared to PT, BiP, PERK, ATF4 mRNA expression in TG was respectively 1.87±0.10, 2.24±0.35, 1.85±0.14; BiP, ATF4 mRNA expression in MNT5 were respectively 1.27±0.09, 1.25±0.04; BiP, PERK, ATF4 mRNA expression in MNT20 were respectively 1.44±0.09, 2.40±0.60, 1.48±0.05 (P<0.05). Conclusions: MNT triggered different degree of ERS, and the activated ERS may promote MNT-induced osteogenic differentiation.

Up-Regulated ATF4 Expression Increases Cell Sensitivity to Apoptosis in Response to Radiation

Background/Aims: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. Methods: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of ⁶⁰Coγ radiation. Cell viability was assayed using a cell counting kit. The cell cycle was analyzed using flow cytometry, and cell apoptosis was assayed using Annexin V-PI double labeling. Small interfering RNA (siRNA) against ATF4 was transfected into ECV304 cells using Lipofectamine 2000. An ATF4 over-expression plasmid (p-ATF4-CGN) was transfected into HEK293 cells that endogenously expressed low levels of ATF4. The levels of intracellular reactive oxygen species (ROS) were measured using CM-H2DCFDA as a probe. Results: ATF4 mRNA and protein expression levels were higher after radiation and increased in a dose- and time-dependent manner in AHH1 lymphoblast cells (P < 0.05). An increase in ATF4 levels was also observed after radiation in primary murine spleen cells, human endothelial ECV304 cells, human liver LO2 cells, breast cancer MCF7 cells, and human hepatocellular carcinoma HEPG2 cells. No change was observed in human embryonic kidney 293 (HEK293) cells. Over-expressing ATF4 in HEK293 cells inhibited cell proliferation, increased cell apoptosis and significantly increased the proportion of cells in G1 phase. Conversely, when ATF4 expression was knocked down using siRNA in ECV304 cells, it protected the cells from radiation-induced apoptosis. These findings suggest that ATF4 may play a role in radiation-induced cell killing by inhibiting cell proliferation and promoting cell apoptosis. Conclusions: In this study, we found that radiation up-regulated the expression of ATF4. We used ATF4 knockdown and over-expression systems to show that ATF4 may play a role in radiation-induced cellular apoptosis.

TBL2 Associates With ATF4 mRNA Via Its WD40 Domain and Regulates Its Translation During ER Stress.

PKR-like ER-resident kinase (PERK) phosphorylates eukaryotic translation initiation factor 2 α (eIF2α) under endoplasmic reticulum (ER) stress; this results in repression of general translation and induction of specific gene expression, such as activating transcription factor 4 (ATF4). We previously showed that, upon ER stress, transducin (β)-like 2 (TBL2) was an ER-localized transmembrane protein and interacted with PERK and that TBL2 was involved in ATF4 expression and cell survival. Here, we show that TBL2 is able to associate with ATF4 mRNA and regulate its translation. The RNA-immunoprecipitation analysis using several TBL2 deletion mutants revealed that the WD40 domain was essential for association with ATF4 mRNA. Importantly, suppression of TBL2 by knockdown or overexpression of the TBL2 mutant with a defective WD40 domain diminished ATF4 induction at the translational level. Thus, our findings indicate that, under ER stress, TBL2 participates in ATF4 translation through its association with the mRNA.