Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.

Identification of nagilactone E as a protein synthesis inhibitor with anticancer activity.

Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg-1·d-1, ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.

Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing.

Activating transcription factor 4 (ATF4), which is ubiquitously expressed, plays a crucial role in regulating various stress-responsive genes under pathophysiological conditions. Further, growth arrest and DNA damage-inducible gene 34 (GADD34), a downstream target of ATF4, has been reported to negatively regulate ATF4 expression. To understand the relationship between intrinsic ATF4 and GADD34 under resting and ER stress conditions, we used a novel gene editing approach, CRISPR/Cas9, to integrate antibiotic-resistant genes into the target genes, ATF4 and GADD34. First, we manipulated the ATF4 gene in the mouse neuroblastoma cell line, Neuro2a, and compared the ER stress responses between parental and ATF4-edited Neuro2a cells. Next, we established Neuro2a cells with edited GADD34 and ATF4/GADD34 genes and found that ATF4 acts as a proapoptotic factor, but GADD34 depletion did not attenuate the expression of cleaved caspase-3 induced by tunicamycin treatment. These findings provide new insights into the ATF4 signaling cascades. Additionally, the rapid establishment of cells lacking multiple genes using this CRISPR/Cas9 system will be a powerful tool for exploring various cellular issues under pathophysiological conditions.

MicroRNA-214 suppresses gluconeogenesis by targeting activating transcriptional factor 4.

Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.

Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury.

BACKGROUND: Understanding the mechanisms underlying neuronal death in spinal cord injury (SCI) and developing novel therapeutic approaches for SCI-induced damage are critical for functional recovery. Here we investigated the role of heme oxygenase-1 (HO-1) in neuroprotection after SCI. METHODS: Adeno-associated virus expressing HO-1 was prepared and injected into rat spinal cords before SCI model was performed. HO-1 expression, inflammasome activation, and the presence of inflammatory cytokines were determined by quantitative polymerase chain reaction, immunohistological staining, immunoblot, and immunoprecipitation. Neuronal apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling. The hindlimb locomotor function was evaluated for extent of neurologic damage. In an in vitro model, hydrogen peroxide was used to induce similar inflammasome activation in cultured primary spinal cord neurons, followed by evaluation of above parameters with or without transduction of HO-1-expressing adeno-associated virus. RESULTS: Endogenous HO-1 expression was found in spinal cord neurons after SCI in vivo, in association with the expression of Nod-like receptor protein 1 (NLRP1) and the formation of NLRP1 inflammasomes. Administration of HO-1-expressing adeno-associated virus effectively decreased expression of NLRP1, therefore alleviating NLRP1 inflammasome-induced neuronal death and improving functional recovery. In the in vitro model, exogenous HO-1 expression protected neurons from hydrogen peroxide-induced neuronal death by inhibiting NLRP1 expression. In addition, HO-1 inhibited expression of activating transcription factor 4 (ATF4), which is a transcription factor regulating NLRP1 expression. CONCLUSIONS: HO-1 protects spinal cord neurons after SCI through inhibiting NLRP1 inflammasome formation.

Unique pharmacological property of ISRIB in inhibition of Aß-induced neuronal cell death.

A pharmacological approach to ameliorate Alzheimer's disease (AD) has not yet been established. In the present study, we investigated the pharmacological characteristics of the recently identified memory-enhancing compound, ISRIB for the amelioration of AD. ISRIB potently attenuated amyloid ß-induced neuronal cell death at concentrations of 12.5-25 nM, but did not inhibit amyloid ß production in the HEK293T cell line expressing the amyloid precursor protein (APP). These results suggest that ISRIB possesses the unique pharmacological property of attenuating amyloid ß-induced neuronal cell death without affecting amyloid ß production.

Activation of the ER stress and calcium signaling in angiomyolipoma.

Renal angiomyolipomas (AMLs) are uncommon benign tumors that occur sporadically or as a part of tuberous sclerosis complex (TSC). Risk of life threatening hemorrhage is the main clinical concern. Although several evidences suggest that hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is crucial for these tumors, modulation of other metabolic pathways might affect tumor growth and progression. Therefore, we aimed to further characterize angiomyolipoma by TSC1/TSC2 expression, hypoxic status, expression of endoplasmic reticulum (ER) stress markers and calcium transport from the ER through the inositol 1,4,5-trisphosphate (IP3) receptors. Despite our expectations, angiomyolipoma were not hypoxic, as determined by absent expression of the carbonic anhydrase IX, which is a reliable marker of hypoxia. This was in accord with very low expression of TSC1 (that is associated with HIF activation) and a high expression of TSC2. Angiomyolipoma specimens also showed a significant upregulation of an anti-apoptotic marker Bcl2 when compared to healthy kidney tissue supporting the induction of pro-survival signaling. Moreover, angiomyolipoma specimens showed the overexpression of the ER stress markers XBP1, CHOP and ATF4 as well as of the mediators of calcium metabolism, namely the type 1 and 2, but not the type 3 IP3 receptors. These data suggest that the ER stress response, survival and calcium metabolism-related pathways but not hypoxia is an important component of the angiomyolipoma pathogenesis.

Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression.

Translational control of transcription factor ATF4 through paired upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While it is typically induced by phosphorylation of eIF2α, ATF4 translation can be also induced by expression of a translational inhibitor protein, eIF5-mimic protein 1 (5MP1, also known as BZW2) in mammals. Here we show that the 5MP gene is maintained in eukaryotes under strong purifying selection, but is uniquely missing in two major phyla, nematoda and ascomycota. The common function of 5MP from protozoa, plants, fungi and insects is to control translation by inhibiting eIF2. The affinity of human 5MP1 to eIF2ß was measured as being equivalent to the published value of human eIF5 to eIF2ß, in agreement with effective competition of 5MP with eIF5 for the main substrate, eIF2. In the red flour beetle, Tribolium castaneum, RNA interference studies indicate that 5MP facilitates expression of GADD34, a downstream target of ATF4. Furthermore, both 5MP and ATF4 are essential for larval development. Finally, 5MP and the paired uORFs allowing ATF4 control are conserved in the entire metazoa except nematoda. Based on these findings, we discuss the phylogenetic and functional linkage between ATF4 regulation and 5MP expression in this group of eukaryotes.

Impact of ER stress-regulated ATF4/p16 signaling on the premature senescence of renal tubular epithelial cells in diabetic nephropathy.

Premature senescence is an important event during diabetic nephropathy (DN) progression. Here, we investigated the role of endoplasmic reticulum (ER) stress-regulated activation of transcription factor 4 (ATF4)/p16 signaling in the premature senescence of renal tubular epithelial cells (RTECs) during DN development. In the renal tissues of Type 2 DN patients, we detected an increased number of senescent cells; elevated deposition of advanced glycation end products (AGEs); upregulated expression of ER stress marker, glucose-regulated protein 78; as well as overexpression of ATF4 and p16. Similarly, these phenomena were also observed in cultured mouse RTECs following AGE treatment. Interestingly, AGE-induced p16 expression and premature senescence were successfully attenuated by ER stress inhibitor and ATF4 gene silencing. Moreover, AGE-induced premature senescence was mimicked by ER stress inducers and ATF4 overexpression, while suppressed by p16 gene silencing. In addition, ER stress inducers can augment ATF4 expression. Therefore, our results demonstrate that the ER stress-regulated ATF4/p16 pathway is involved in the premature senescence of RTECs during DN progression.

Zinc regulates expression of IL-23 p19 mRNA via activation of eIF2α/ATF4 axis in HAPI cells.

Zinc (Zn(2+)) is considered to be one of the factors aggravating brain damage after cerebral ischemia. Since Zn(2+) activates microglia, immune cells in the brain, this metal is proposed to modulate neuroinflammatory responses in the post-ischemic brain. Interleukin (IL)-23 is a heterodimeric cytokine composed of the p19 subunit unique to IL-23 and the p40 subunit common to IL-12. IL-23 has been shown to play a critical role in the progression of ischemic brain injury. However, whether Zn(2+) participates in the expression of IL-23 in microglia remains unknown. In this study, we examined the effect of Zn(2+) on IL-23 p19 mRNA expression using rat immortalized microglia HAPI cells. Exposure to Zn(2+) dose- and time-dependently induced the expression of IL-23 p19 mRNA in HAPI cells. Inhibitors of MAPK and NF-κB pathways failed to suppress this induction. Interestingly, we found that Zn(2+) stimulated the phosphorylation of eIF2α and promoted the nuclear accumulation of activating transcription factor 4 (ATF4). Treatment with salubrinal, an eIF2α dephosphorylation inhibitor, enhanced Zn(2+)-induced ATF4 accumulation and IL-23 p19 mRNA expression. In addition, reporter assay using the IL-23 p19 promoter region revealed that ATF4 directly transactivated IL-23 p19 promoter and that dominant-negative ATF4 suppressed Zn(2+)-induced activation of IL-23 p19 promoter. Taken together, these findings suggest that Zn(2+) up-regulates expression of the IL-23 p19 gene via the eIF2α/ATF4 axis in HAPI cells.

Proapoptotic effects of heme oxygenase-1 inhibitor on Kasumi-1 cells via the ATF4/CHOP/Ire-1α pathway.

We evaluated the effects of down-regulated heme oxygenase (HO)-1 expression on the proliferation of the acute myelocytic leukemia Kasumi-1 cell line by using the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) in combination with daunorubicin (DNR), and evaluated the mechanism. The proliferation rates of cells treated with 10 mg/mL DNR and 10 mM ZnPPIX individually or in combination for different time periods were detected using the MTT assay. The apoptotic outcomes of the blank control, ZnPPIX, DNR, and ZnPPIX groups in combination with the DNR group were detected by flow cytometry. The expression of HO-1, activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α mRNA and proteins were detected by fluorescent quantitative real-time polymerase chain reaction and western blotting, respectively. Combined administration inhibited the cells most potently and time-dependently, decreased the expression of HO-1, and significantly increased the expression of activating transcription factor 4, CCAAT-enhancer-binding protein homologous protein, and inositol-requiring enzyme-α expression levels. The cell apoptotic rates in the blank control, DNR, ZnPPIX, and combined administration groups were 8.32 ± 0.53, 39.16 ± 1.46, 10.46 ± 0.88, and 56.26 ± 2.24%, respectively. Inhibiting HO-1 expression can enhance the damaging effects of DNR on Kasumi-1 cells, providing experimental evidence for the improvement of therapeutic effects on acute myelocytic leukemia in clinical practice.

ATF4 protects against neuronal death in cellular Parkinson's disease models by maintaining levels of parkin.

Parkinson's disease (PD) is a common neurodegenerative disorder, for which there are no effective disease-modifying therapies. The transcription factor ATF4 (activating transcription factor 4) is induced by multiple PD-relevant stressors, such as endoplasmic reticulum stress and oxidative damage. ATF4 may exert either protective or deleterious effects on cell survival, depending on the paradigm. However, the role of ATF4 in the pathogenesis of PD has not been explored. We find that ATF4 levels are increased in neuromelanin-positive neurons in the substantia nigra of a subset of PD patients relative to controls. ATF4 levels are also upregulated in neuronal PC12 cells treated with the dopaminergic neuronal toxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). To explore the role of ATF4 in cell survival in PD-relevant contexts, we either silenced or overexpressed ATF4 in cellular models of PD. In neuronal PC12 cells, silencing of ATF4 enhanced cell death in response to either 6-OHDA or MPP+. Conversely, overexpression of ATF4 reduced cell death caused by dopaminergic neuronal toxins. ATF4 was also protective against 6-OHDA-induced death of cultured mouse ventral midbrain dopaminergic neurons. We further show that parkin, a gene associated with autosomal recessive PD, plays a critical role in ATF4-mediated protection. After treatment with 6-OHDA or MPP+, parkin protein levels fall, despite an increase in mRNA levels. ATF4 silencing exacerbates the toxin-induced reduction of parkin, whereas ATF4 overexpression partially preserves parkin levels. Finally, parkin silencing blocked the protective capacity of ATF4. These results indicate that ATF4 plays a protective role in PD through the regulation of parkin.

IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

Valproate pretreatment protects pancreatic ß-cells from palmitate-induced ER stress and apoptosis by inhibiting glycogen synthase kinase-3ß.

BACKGROUND: Reduction of pancreatic ß-cells mass, major secondary to increased ß-cells apoptosis, is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes (T2D), and saturated free fatty acid palmitate has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting ß-cells apoptosis. Recent literature suggests that valproate, a diffusely prescribed drug in the treatment of epilepsy and bipolar disorder, can inhibit glycogen synthase kinase-3ß (GSK-3ß) activity and has cytoprotective effects in neuronal cells and HepG2 cells. Thus, we hypothesized that valproate may protect INS-1 ß-cells from palmitate-induced apoptosis via inhibiting GSK-3ß. RESULTS: Valproate pretreatment remarkable prevented palmitate-mediated cytotoxicity and apoptosis (lipotoxicity) as well as ER distension. Furthermore, palmitate triggered ER stress as evidenced by increased mRNA levels of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) in a time-dependent fashion. However, valproate not only reduced the mRNA and protein expression of CHOP but also inhibited GSK-3ß and caspase-3 activity induced by palmitate, whereas, the mRNA expression of ATF4 was not affected. Interestingly, TDZD-8, a specific GSK-3ß inhibitor, also showed the similar effect on lipotoxicity and ER stress as valproate in INS-1 cells. Finally, compared with CHOP knockdown, valproate displayed better cytoprotection against palmitate. CONCLUSIONS: Valproate may protect ß-cells from palmitate-induced apoptosis and ER stress via GSK-3ß inhibition, independent of ATF4/CHOP pathway. Besides, GSK-3ß, rather than CHOP, may be a more promising therapeutic target for T2D.

Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells.

In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages.

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response.

INTRODUCTION: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined. METHODS: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion. RESULTS: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids. CONCLUSIONS: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Platelet-derived growth factor-BB mediates cell migration through induction of activating transcription factor 4 and tenascin-C.

The acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-ß. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration.

Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2.

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

Negative regulation of IRF7 activation by activating transcription factor 4 suggests a cross-regulation between the IFN responses and the cellular integrated stress responses.

Cells react to viral infection by exhibiting IFN-based innate immune responses and integrated stress responses, but little is known about the interrelationships between the two. In this study, we report a linkage between these two host-protective cellular mechanisms. We found that IFN regulatory factor (IRF)7, the master regulator of type I IFN gene expression, interacts with activating transcription factor (ATF)4, a key component of the integrated stress responses whose translation is induced by viral infection and various stresses. We have demonstrated that IRF7 upregulates ATF4 activity and expression, whereas ATF4 in return inhibits IRF7 activation, suggesting a cross-regulation between the IFN response and the cellular integrated stress response that controls host innate immune defense against viral infection.