High ATF5 expression is a favorable prognostic indicator in patients with hepatocellular carcinoma after hepatectomy.

Activating transcription factor 5 (ATF5) is a member of the ATF/CREB family involved in diverse physiological functions, ranging from metabolite homeostasis to cellular differentiations and regulation of the cell cycle. However, the clinical significance of ATF5 expression in patients with hepatocellular carcinoma (HCC) is unknown. We investigated ATF5 mRNA expression in tumors and adjacent non-tumor hepatic tissues from 119 HCC patients using quantitative real-time PCR. Association between the ATF5 mRNA expression level and clinical and pathological parameters, including recurrence-free survival (RFS) and overall survival, were also evaluated using appropriate statistical methods. ATF5 was downregulated significantly (P < 0.001) in the tumor versus matched HCC non-tumor hepatic tissue, with average mRNA expression levels of 0.25 ± 0.92 and 0.41 ± 1.19, respectively. Low ATF5 expression was correlated significantly with liver cirrhosis, intrahepatic metastasis, and TNM stage (P < 0.05). Additionally, both Kaplan-Meier survival curve and multivariate analyses showed a relationship between ATF5 and RFS. In conclusion, a low expression level of ATF5 in HCC indicated aggressive tumor behavior and predicted a worse clinical outcome. ATF5 may be useful as a novel prognostic indicator in hepatocellular carcinoma.

Oxidative stress-related transcription factors in the regulation of secondary metabolism.

There is extensive and unequivocal evidence that secondary metabolism in filamentous fungi and plants is associated with oxidative stress. In support of this idea, transcription factors related to oxidative stress response in yeast, plants, and fungi have been shown to participate in controlling secondary metabolism. Aflatoxin biosynthesis, one model of secondary metabolism, has been demonstrated to be triggered and intensified by reactive oxygen species buildup. An oxidative stress-related bZIP transcription factor AtfB is a key player in coordinate expression of antioxidant genes and genes involved in aflatoxin biosynthesis. Recent findings from our laboratory provide strong support for a regulatory network comprised of at least four transcription factors that bind in a highly coordinated and timely manner to promoters of the target genes and regulate their expression. In this review, we will focus on transcription factors involved in co-regulation of aflatoxin biosynthesis with oxidative stress response in aspergilli, and we will discuss the relationship of known oxidative stress-associated transcription factors and secondary metabolism in other organisms. We will also talk about transcription factors that are involved in oxidative stress response, but have not yet been demonstrated to be affiliated with secondary metabolism. The data support the notion that secondary metabolism provides a secondary line of defense in cellular response to oxidative stress.

Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus.

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5ß, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

Interference with ATF5 function enhances the sensitivity of human pancreatic cancer cells to paclitaxel-induced apoptosis.

BACKGROUND: Past work has established that human glioblastomas and breast cancer cells invariably express the activating transcription factor 5 (ATF5) and that loss of function of ATF5 caused massive apoptotic death of all cancer cell lines tested. ATF5 expression and function in pancreatic cancer cells have not been investigated. MATERIALS AND METHODS: Quantitative real-time/reverse transcription-polymerase chain reaction (QRT/RT-PCR), western blotting (WB), immunohistochemistry (IHC) and promoter reporter assay were used for gene expression analysis. MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS (fluorescence-activated cell sorting) analysis were used to monitor cell viability/apoptosis. RESULTS: ATF5 is highly expressed in pancreatic cancer cells as compared with non-tumor tissues. Both paclitaxel treatment and loss of function of ATF5 elicited apoptosis of SW1990 cells. Interference with ATF5 function in SW1990 cells resulted in down-regulation of BCL-2 and up-regulation of BAX, resulting in enhanced sensitivity to apoptosis induced by paclitaxel treatment. CONCLUSION: ATF5 is highly expressed in pancreatic cancer cells. Targeting ATF5 significantly enhances paclitaxel-induced apoptosis in human pancreatic cancer cells. ATF5 could be an important therapeutic target for pancreatic cancer treatment.

Endoplasmic reticulum stress is involved in granulosa cell apoptosis during follicular atresia in goat ovaries.

Follicular atresia is primarily induced by granulosa cell apoptosis, but description of the apoptotic pathway in granulosa cells is incomplete. In this study, we explored the possibility that endoplasmic reticulum (ER) stress could be involved in granulosa cell apoptosis during goat follicular atresia. Immunohistochemical analysis revealed that DNA damage-inducible transcript 3 (DDIT3) and glucose-regulated protein 78 (Grp78) were observed in scattered apoptotic granulosa cells of atretic follicles. Grp78 and DDIT3 mRNA and protein were upregulated in granulosa cells during follicular atresia, although DDIT3 was not significantly different between early atretic and progressed atretic follicles. Spontaneous apoptosis was also observed in vitro in granulosa cells induced by serum deprivation or by the ER stress agent tunicamycin, both inducing similar increases in DDIT3 mRNA. Activating transcription factor-6 (ATF6) and ATF4 mRNAs were significantly increased during granulosa cell apoptosis in vivo; in contrast to ATF6, ATF4 mRNA was attenuated after 16 hr of culture despite the persistence of ER stress. Taken together, ER stress-dependent DDIT3 pathways may play an important role in the regulation of selective granulosa cell apoptosis in goat ovaries during early follicular atresia. Serum deprivation could also increase apoptosis of cultured granulosa cells through the ER stress pathway as both ATF6 and PERK/eIF2α/ATF4 signaling have been implicated in the granulosa cell apoptosis of atretic follicles.

A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation.

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

Amino acid limitation induces expression of ATF5 mRNA at the post-transcriptional level.

ATF5 is a transcription factor in the cAMP response element (CRE)-binding protein/activating transcription factor (CREB/ATF) family. We studied the effect of amino acid limitation on ATF5 mRNA levels in a mammalian cell line. Northern-blot analysis demonstrated that limitation of a single amino acid, glutamine, methionine, or leucine, resulted in increased ATF5 mRNA levels in HeLaS3 cells. This resulted, at least in part, from increased half-life of the ATF5 mRNA transcript. Cycloheximide inhibited the increase in ATF5 mRNA expression induced by glutamine limitation, indicating that it was dependent on de novo protein synthesis. Moreover, rapamycin had no effect on basal ATF5 mRNA expression or on increased expression induced by glutamine limitation. These results indicate that amino acid limitation regulates ATF5 mRNA expression during post-transcription in a rapamycin-independent manner. The potential role for ATF5 in protecting cells from amino acid-limitation is of considerable interest.

Astrocyte-derived transgene GDNF promotes complete and long-term survival of adult facial motoneurons following avulsion and differentially regulates the expression of transcription factors of AP-1 and ATF/CREB families.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MNs). We have previously demonstrated that overexpression of GDNF in astrocytes of GFAP-GDNF mice promotes long-term survival of neonatal MNs after facial nerve axotomy. In the present study, we investigated whether astrocyte-derived GDNF could also have a neuroprotective effect on adult MNs following facial nerve avulsion. We also examined avulsion- and GDNF-induced changes in the expression pattern of several members of the AP-1 and ATF/CREB families of transcription factors, which are involved in the fate determination of neurons following injury. We demonstrated that GDNF promotes complete rescue of avulsed MNs for at least 4 months post-injury. Transgene GDNF significantly upregulates c-Jun expression in naive MNs, further upregulates injury-induced c-Jun expression in facial MNs, and results in its activation in most surviving MNs. No significant changes were found in c-Fos expression. We found that GDNF has an opposing effect on ATF2 and ATF3 expression. It dramatically downregulates increased levels of ATF3 in response to injury, whereas the expression of ATF2, which is normally reduced after injury, is completely preserved in GFAP-GDNF mice. Our data suggest that maintenance of high levels of ATF2 in injured MNs could be crucial in modulating c-Jun function, and c-Jun/ATF2 signaling could be involved in GDNF-mediated survival of mature MNs.

Expression of transcription factors c-Fos, c-Jun, CREB-1 and ATF-2, and caspase-3 in relation with abnormal tau deposits in Pick's disease.

Hyper-phosphorylated tau deposition in Pick bodies and neuron loss are major hallmarks of Pick's disease (PiD). However, there is no regional correlation between neuron loss and Pick bodies, as illustrated in dentate gyrus, where Pick bodies are present in almost every neuron, whereas cell death, if present, is not a major event. In order to better understand the possible role of selected transcription factors and members of the caspase family in cell death and cell survival, immunohistochemistry to c-Fos, c-Jun, CREB-1, ATF-2; c-Fos(P), c-Jun(P) and CREB-1(P); and procaspase-8, procaspase-3 and active (cleaved) caspase-3 immunohistochemistry was carried out in the frontal cortex and hippocampus. Increased expression of c-Fos, c-Jun, CREB-1 and ATF-2 was observed in PiD cases. Increased c-Fos(P), c-Jun(P) and CREB-1(P) was also found in the nuclei of neurons in diseased brains. Interestingly, c-Fos but not c-Fos(P) co-localized in many Pick bodies, as observed by double labelling-immunofluorescence and confocal microscopy. Pro-caspase-8 and pro-caspase-3 were increased in PiD. Moreover, granular active caspase-3 was observed in the nuclei as was aggregated active caspase-3 in the cytoplasm of neurons in PiD. Finally, double-labelling immunofluorescence and confocal microscopy disclosed co-localization of cytoplasmic active caspase-3 only in neurons with Pick bodies. Together, these findings show an increased expression of selected transcription factors and active (phosphorylated) forms in PiD, c-Fos sequestration in Pick bodies, and increased active caspase-3 expression in relation with Pick bodies. Since all these findings were observed equally in neurons of both vulnerable regions (frontal cortex) and resistant regions (dentate gyrus), it may be suggested that transcription factors are only barely related with cell death. Active caspase-3 is associated with tau deposition in Pick bodies, but it is not a marker of cell death in the dentate gyrus in PiD. The present findings are in line with the previous studies showing tau products cleaved by caspase-3, as recognized by specific tau-cleaved antibodies, in Alzheimer's disease and other tauopathies.