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Item ATF4 regulates arsenic trioxide-mediated NADPH oxidase, ER-mitochondrial crosstalk and apoptosis(Elsevier, 2016-11-01) Srivastava, Ritesh K; Li, Changzhao; Ahmad, Aftab; Abrams, Onika; Gorbatyuk, Marina S.; Harrod, Kevin S.; Wek, Ronald C.; Afaq, Farrukh; Athar, Mohammad; Biochemistry and Molecular Biology, School of MedicineArsenic is a mitochondrial toxin, and its derivatives, such as arsenic trioxide (ATO), can trigger endoplasmic reticulum (ER) and the associated unfolded protein response (UPR). Here, we show that arsenic induction of the UPR triggers ATF4, which is involved in regulating this ER-mitochondrial crosstalk that is important for the molecular pathogenesis of arsenic toxicity. Employing ATF4+/+ and ATF4−/− MEFs, we show that ATO induces UPR and impairs mitochondrial integrity in ATF4+/+ MEF cells which is largely ablated upon loss of ATF4. Following ATO treatment, ATF4 activates NADPH oxidase by promoting assembly of the enzyme components Rac-1/P47phox/P67phox, which generates ROS/superoxides. Furthermore, ATF4 is required for triggering Ca++/calpain/caspase-12-mediated apoptosis following ATO treatment. The IP3R inhibitor attenuates Ca++/calpain-dependent apoptosis, as well as reduces m-ROS and MMP disruption, suggesting that ER-mitochondria crosstalk involves IP3R-regulated Ca++ signaling. Blockade of m-Ca++ entry by inhibiting m-VDAC reduces ATO-mediated UPR in ATF4+/+ cells. Additionally, ATO treatment leads to p53-regulated mitochondrial apoptosis, where p53 phosphorylation plays a key role. Together, these findings indicate that ATO-mediated apoptosis is regulated by both ER and mitochondria events that are facilitated by ATF4 and the UPR. Thus, we describe novel mechanisms by which ATO orchestrates cytotoxic responses involving interplay of ER and mitochondria.,Item Biology of Activating Transcription Factor 4 (ATF4) and Its Role in Skeletal Muscle Atrophy(Elsevier, 2022) Ebert, Scott M.; Rasmussen, Blake B.; Judge, Andrew R.; Judge, Sarah M.; Larsson, Lars; Wek, Ronald C.; Anthony, Tracy G.; Marcotte, George R.; Miller, Matthew J.; Yorek, Mark A.; Vella, Adrian; Volpi, Elena; Stern, Jennifer I.; Strub, Matthew D.; Ryan, Zachary; Talley, John J.; Adams, Christopher M.; Biochemistry and Molecular Biology, School of MedicineActivating transcription factor 4 (ATF4) is a multifunctional transcription regulatory protein in the basic leucine zipper superfamily. ATF4 can be expressed in most if not all mammalian cell types, and it can participate in a variety of cellular responses to specific environmental stresses, intracellular derangements, or growth factors. Because ATF4 is involved in a wide range of biological processes, its roles in human health and disease are not yet fully understood. Much of our current knowledge about ATF4 comes from investigations in cultured cell models, where ATF4 was originally characterized and where further investigations continue to provide new insights. ATF4 is also an increasingly prominent topic of in vivo investigations in fully differentiated mammalian cell types, where our current understanding of ATF4 is less complete. Here, we review some important high-level concepts and questions concerning the basic biology of ATF4. We then discuss current knowledge and emerging questions about the in vivo role of ATF4 in one fully differentiated cell type, mammalian skeletal muscle fibers.Item Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice(Oxford University Press, 2017-06) Pettit, Ashley P.; Jonsson, William O.; Bargoud, Albert R.; Mirek, Emily T.; Peelor, Frederick F., III; Wang, Yongping; Gettys, Thomas W.; Kimball, Scot R.; Miller, Benjamin F.; Hamilton, Karyn L.; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineBackground: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.Item Role of ATF4 in directing gene expression in the basal state and during the unfolded protein response in liver(2016-08) Fusakio, Michael Edward; Wek, Ronald C.Disturbances in membrane composition and protein folding in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR). Three UPR sensory proteins, PERK (PEK/EIF2AK3), IRE1, and ATF6 are each activated by ER stress. PERK phosphorylation of the alpha subunit of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with the preferential translation of ATF4 (CREB2). Results from cultured cells demonstrate that ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterized two ATF4 knockout mouse models and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but rather ATF6 is a primary inducer. RNA-sequence analysis indicated that ATF4 was responsible for a small portion of the PERK-dependent genes in the UPR. This smaller than expected subset of gene expression lends itself to the relevance of UPR crosstalk, with ATF6, XBP1, and CHOP being capable of upregulating UPR genes in the absence of ATF4. RNA-sequence analysis also revealed a requirement for expression of ATF4 for expression of a comparable number of genes basally, including those involved in oxidative stress response and cholesterol metabolism. Consistent with this pattern of gene expression, loss of ATF4 in our mouse model resulted in enhanced oxidative damage and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera. Taken together, this study highlights both an expansion of the role of ATF4 in transcriptional regulation of genes involved in metabolism in the basal state and a more specialized role during ER stress. These findings are important for understanding the variances of the UPR signaling between cell culture and in vivo and for a greater understanding of all the roles ATF4 plays within the cell.Item Time-resolved analysis of amino acid stress identifies eIF2 phosphorylation as necessary to inhibit mTORC1 activity in liver(American Society for Biochemistry and Molecular Biology, 2018-04-06) Nikonorova, Inna A.; Mirek, Emily T.; Signore, Christina C.; Goudie, Michael P.; Wek, Ronald C.; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of MedicineAmino acid availability is sensed by GCN2 (general control nonderepressible 2) and mechanistic target of rapamycin complex 1 (mTORC1), but how these two sensors coordinate their respective signal transduction events remains mysterious. In this study we utilized mouse genetic models to investigate the role of GCN2 in hepatic mTORC1 regulation upon amino acid stress induced by a single injection of asparaginase. We found that deletion of Gcn2 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5'-terminal oligopyrimidine mRNA translation instead of activating transcription factor 4 synthesis. Asparaginase also promoted hepatic mRNA levels of several genes which function as mTORC1 inhibitors, and these genes were blunted or blocked in the absence of Gcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Preconditioning mice with a chemical endoplasmic reticulum stress agent before amino acid stress rescued normal mTORC1 repression in the liver of Gcn2-/- mice but not in livers with both Gcn2 and the endoplasmic reticulum stress kinase, Perk, deleted. Furthermore, treating wildtype and Gcn2-/- mice with ISRIB, an inhibitor of PERK signaling, also failed to alter hepatic mTORC1 responses to asparaginase, although administration of ISRIB alone had an inhibitory GCN2-independent effect on mTORC1 activity. Taken together, the data show that activating transcription factor 4 is not required, but eukaryotic initiation factor 2α phosphorylation is necessary to prevent mTORC1 activation during amino acid stress.Item Transcriptional regulation of ATF4 is critical for controlling the Integrated Stress Response during eIF2 phosphorylation(2012-05) Dey, Souvik; Wek, Ronald C.; Edenberg, Howard J.; Gallagher, Patricia; Turchi, John J.In response to different environmental stresses, phosphorylation of eIF2 (eIF2P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. In this study, we also show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter resulting in its transcriptional repression. The LIP isoform of C/EBPβ, but not the LAP version is regulated following UV exposure and directly represses ATF4 transcription. Loss of the LIP isoform results in increased ATF4 mRNA levels in response to UV irradiation, and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2P and translational control, combined with transcription factors regulated by alternative signaling pathways, can direct programs of gene expression that are specifically tailored to each environmental stress.