Activating Transcription Factor 3 Gene Expression Research Articles

The effect of feeding with different protein levels on internal organ weight and gene expression of MEF2A and ATF3 in crossbred local chicken using RT-PCR

BackgroundMyogenic enhancer transcription factor 2A (MEF2A) is a transcription factor known for its role in controlling skeletal muscle regeneration and metabolic processes, while activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays a role in modulating metabolic processes, immunity, and oncogenesis. Environmental factors, such as dietary protein, can influence gene expression levels. Insufficient protein intake can negatively affect the metabolic performance of internal organs, leading to the abnormal weight of internal organs. A total of 192 non-sexing crossbred local chickens day-old-chick (DOC) with a completely randomized design (CRD) method of 3 treatments and 8 replicates. Real-time polymerase chain reaction (RT-PCR) is used to measure the gene expression levels. This study aimed to determine the effect of feeding with various protein levels on internal organ weight and gene expression of MEF2A and ATF3 in crossbred local chickens. ResultThe analysis of treatment revealed that the results were not significantly different (P > 0.05) on gizzard weight and spleen weight. However, it shows a significantly different result (P < 0.05) on heart weight and a highly significantly different result (P < 0.01) on pancreas weight. These findings suggest that protein levels in the diet had a significant impact on heart and pancreas weights. In terms of gene expression, the increased utilization of protein did not result in an elevation of MEF2A gene expression in both muscle tissue and liver tissue. Specifically, in muscle tissue, MEF2A gene expression was highly expressed at 18% protein feed for the starter phase and 16% for the finisher phase. Conversely, in liver tissue, MEF2A gene expression was highly expressed at 22% protein feed for the starter phase and 20% for the finisher phase. Moreover, ATF3 gene expression in muscle tissue exhibited a negative correlation with increasing feed protein levels. ConclusionThe results indicate that varying protein levels did not lead to abnormal weights in the liver, kidney, heart, and spleen organs. Additionally, the differential gene expression patterns of MEF2A and ATF3 in muscle tissue and liver tissue suggest that these genes respond differently to varying protein-feeding treatments. These findings provide insights into the complex regulatory mechanisms of MEF2A and ATF3 genes in relation to protein levels and organ-specific responses in crossbred local chickens.

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

Although knowledge regarding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has profoundly grown in recent decades, the internal restrictive mechanisms remain largely unknown. We have recently reported that the transcription repressor interferon regulatory factor-2 binding protein 2 (IRF2BP2) is enriched in cardiomyocytes and inhibits pathological cardiac hypertrophy in mice. Notably, IRF2BP2 is abundantly expressed in hepatocytes and dramatically down-regulated in steatotic livers, whereas the role of IRF2BP2 in NAFLD is unknown. Herein, using gain-of-function and loss-of-function approaches in mice, we demonstrated that while hepatocyte-specific Irf2bp2 knockout exacerbated high-fat diet-induced hepatic steatosis, insulin resistance and inflammation, hepatic Irf2bp2 overexpression protected mice from these metabolic disorders. Moreover, the inhibitory role of IRF2BP2 on hepatosteatosis is conserved in a human hepatic cell line in vitro. Combinational analysis of digital gene expression and chromatin immunoprecipitation sequencing identified activating transcription factor 3 (ATF3) to be negatively regulated by IRF2BP2 in NAFLD. Chromatin immunoprecipitation and luciferase assay substantiated the fact that IRF2BP2 is a bona fide transcription repressor of ATF3 gene expression via binding to its promoter region. Functional studies revealed that ATF3 knockdown significantly relieved IRF2BP2 knockout-exaggerated hepatosteatosis in vitro. IRF2BP2 is an integrative restrainer in controlling hepatic steatosis, insulin resistance, and inflammation in NAFLD through transcriptionally repressing ATF3 gene expression.

Carnosol suppresses interleukin-6 production in mouse lungs injured by ischemia-reperfusion operation and in RAW264.7 macrophages treated with lipopolysaccharide.

Carnosol is a naturally occurring herbal compound, known for its antioxidative properties. We previously found that carnosol protected mouse lungs from ischemia-reperfusion injury in ex vivo cultures. To elucidate the molecular mechanisms underpinning carnosol-mediated lung protection, we analyzed modes of interleukin-6 (IL-6) gene expression, which is associated with lung ischemia-reperfusion injury. Microarray analysis of mouse lungs suggested that IL-6 mRNA levels were elevated in the mouse lungs subjected to clamp-reperfusion, which was associated with elevated levels of other inflammatory modulators, such as activating transcription factor 3 (ATF3). Carnosol pretreatment lowered the IL-6 protein levels in mouse lung homogenates prepared after the clamp-reperfusion. On the other hand, the ATF3 gene expression was negatively correlated with that of IL-6 in RAW264.7 cells. IL-6 mRNA levels and gene promoter activities were suppressed by carnosol in RAW264.7 cells, but rescued by ATF3 knockdown. When RAW264.7 cells were subjected to hypoxia-reoxygenation, carnosol treatment lowered oxygen consumption after reoxygenation, which was coupled with a correlation with a transient production of mitochondrial reactive oxygen species and following ATF3 gene expression. These results suggest that carnosol treatment could be a new strategy for protecting lungs from ischemia-reperfusion injury by modulating the ATF3-IL-6 axis.

Larval hemolymph of rhinoceros beetle, Allomyrina dichotoma, enhances insulin secretion through ATF3 gene expression in INS-1 pancreatic β-cells.

Abstract In this study, we show that INS-1 pancreatic β-cells treated for 2 h with hemolymph of larvae of rhinoceros beetle, Allomyrina dichotoma, secreted about twice as much insulin compared to control cells without such treatment. Activating transcription factor 3 (ATF3) was the highest upregulated gene in DNA chip analysis. The A. dichotoma hemolymph dose-dependently induced increased expression levels of genes encoding ATF3 and insulin. Conversely, treatment with ATF3 siRNA inhibited expression levels of both genes and curbed insulin secretion. These results suggest that the A. dichotoma hemolymph has potential for treating and preventing diabetes or diabetes-related complications.

ATF3-Dependent Regulation of EGR1 in vitro and in vivo

Background/Aims: Activating transcription factor 3 (ATF3) and early growth response protein 1 (EGR1) are reported to interact, but their use as prognostic factors in cancer is discussed controversially. Methods: We measured ATF3 and EGR1 gene expression changes in human mini-organ cultures (MOCs) of healthy nasal epithelia, UM-SCC-22B, and FADU_DD cells after acid reflux exposure. Next, ATF3 and EGR1 gene expression was analysed in tumour tissues and related to the median expression of autologous reference tissue samples. Results: ATF3 and EGR1 mRNA expression was significantly reduced after consecutive exposure of MOCs at pH <7.0 to artificial gastric juice (refluxate). In contrast, ATF3 mRNA was upregulated significantly within the first hour of incubation. EGR1 mRNA exhibited no significant changes. The analysed cell lines exhibited a cell line-specific alteration. In FADU_DD cells, the upregulation of EGR1 was significant after refluxate exposure, but in HN-SCC 22B, no significant changes were detected. The analysis of the HNSCC samples confirmed the heterogeneous data of the literature. Conclusion: The data maintain the hypothesis that ATF3 and EGR1 are involved in the beginning of inflammatory processes. Whether these two transcription factors act as tumour suppressors or promoters is context dependent and warrants analysis in further studies.

Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells

Non–small cell lung carcinoma (NSCLC) is the most common cause of cancer deaths, with platin-based combination chemotherapy the most efficacious therapies. Gains in overall survival are modest, highlighting the need for novel therapeutic approaches including the development of next-generation platin combination regimens. The goal of this study was to identify novel regulators of platin-induced cytotoxicity as potential therapeutic targets to further enhance platin cytotoxicity. Employing RNA-seq transcriptome analysis comparing two parental NSCLC cell lines Calu6 and H23 to their cisplatin-resistant sublines, Calu6cisR1 and H23cisR1, activating transcription factor 3 (ATF3) was robustly induced in cisplatin-treated parental sensitive cell lines but not their resistant sublines, and in three of six tumors evaluated, but not in their corresponding normal adjacent lung tissue (0/6). Cisplatin-induced JNK activation was a key regulator of this ATF3 induction. Interestingly, in both resistant sublines, this JNK induction was abrogated, and the expression of an activated JNK construct in these cells enhanced both cisplatin-induced cytotoxicity and ATF3 induction. An FDA-approved drug compound screen was employed to identify enhancers of cisplatin cytotoxicity that were dependent on ATF3 gene expression. Vorinostat, a histone deacetylase inhibitor, was identified in this screen and demonstrated synergistic cytotoxicity with cisplatin in both the parental Calu6 and H23 cell lines and importantly in their resistant sublines as well that was dependent on ATF3 expression. Thus, we have identified ATF3 as an important regulator of cisplatin cytotoxicity and that ATF3 inducers in combination with platins are a potential novel therapeutic approach for NSCLC.

The catalytic topoisomerase II inhibitor dexrazoxane induces DNA breaks, ATF3 and the DNA damage response in cancer cells.

The catalytic topoisomerase II inhibitor dexrazoxane has been associated not only with improved cancer patient survival but also with secondary malignancies and reduced tumour response. We investigated the DNA damage response and the role of the activating transcription factor 3 (ATF3) accumulation in tumour cells exposed to dexrazoxane. Dexrazoxane exposure induced topoisomerase IIα (TOP2A)-dependent cell death, γ-H2AX accumulation and increased tail moment in neutral comet assays. Dexrazoxane induced DNA damage responses, shown by enhanced levels of γ-H2AX/53BP1 foci, ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), Chk1 and Chk2 phosphorylation, and by p53 accumulation. Dexrazoxane-induced γ-H2AX accumulation was dependent on ATM. ATF3 protein was induced by dexrazoxane in a concentration- and time-dependent manner, which was abolished in TOP2A-depleted cells and in cells pre-incubated with ATM inhibitor. Knockdown of ATF3 gene expression by siRNA triggered apoptosis in control cells and diminished the p53 protein level in both control and dexrazoxane -treated cells. This was accompanied by increased γ-H2AX accumulation. ATF3 knockdown also delayed the repair of dexrazoxane -induced DNA double-strand breaks. As with other TOP2A poisons, dexrazoxane induced DNA double-strand breaks followed by activation of the DNA damage response. The DNA damage-triggered ATF3 controlled p53 accumulation and generation of double-strand breaks and is proposed to serve as a switch between DNA damage and cell death following dexrazoxane treatment. These findings suggest a mechanistic explanation for the diverse clinical observations associated with dexrazoxane.

Temporal changes in neurotrophic factors and neurite outgrowth in the major pelvic ganglion following cavernous nerve injury.

Despite nerve-sparing radical prostatectomy, nerve damage and erectile dysfunction (ED) prevail, and preventing neurodegeneration is of great importance. Neurotrophic factors and neurite outgrowth were characterized in major pelvic ganglia (MPG) following bilateral cavernous nerve injury (BCNI). Young male Sprague-Dawley rats underwent sham or BCNI surgery, and the intracavernosal pressure to mean arterial pressure ratio was measured 2, 7, 14, 21, 30, and 60 days following injury (n = 8/group). MPG gene expression (qPCR) and Western blot were performed for glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurturin, neurotrophin (NT)-3, NT4, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor, and activating transcription factor 3 (ATF3). Additional rats were injured, and MPGs were removed 24 hr, 48 hr, 3 days, and 7 days following BCNI (n = 3/group). MPGs were cultured in Matrigel, and neurite outgrowth was measured. Erections were impaired early and improved by 60 days in BCNI rats. GDNF, NGF, BDNF, and ATF3 gene expression was significantly increased and NT3 was decreased in MPGs following BCNI (48 hr to 21 days, P < 0.05). GDNF and NGF protein levels were elevated in 48-hr BCNI rats. MPG neurite outgrowth from 24-hr and 48-hr BCNI was higher than sham (658 ± 19 μm, 607 ± 24 μm, 393 ± 23 μm, respectively, P < 0.05). Further studies examining the roles of neurotrophic factors in modulating signaling pathways may provide therapeutic avenues for neurogenically mediated ED.

MP47-12 TIME COURSE OF APOPTOTIC NEURONAL CELL DEATH IN THE MAJOR PELVIC GANGLION FOLLOWING CAVERNOUS NERVE INJURY

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-12 TIME COURSE OF APOPTOTIC NEURONAL CELL DEATH IN THE MAJOR PELVIC GANGLION FOLLOWING CAVERNOUS NERVE INJURY Johanna Hannan, Maarten Albersen, Joseph Watson, Xiaopu Liu, Petter Hedlund, Arthur Burnett, and Trinity Bivalacqua Johanna HannanJohanna Hannan More articles by this author , Maarten AlbersenMaarten Albersen More articles by this author , Joseph WatsonJoseph Watson More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Petter HedlundPetter Hedlund More articles by this author , Arthur BurnettArthur Burnett More articles by this author , and Trinity BivalacquaTrinity Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1465AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Surgical treatments for prostate, bladder and colorectal cancers often lead to neuronal damage and erectile dysfunction (ED). Despite nerve-sparing techniques, ED prevails and preventing neurodegeneration is of great importance. This study aimed to characterize the markers of neuronal injury and apoptosis in the major pelvic ganglion (MPG) following bilateral cavernous nerve injury (BCNI). Methods Male Sprague-Dawley rats underwent sham or BCNI and MPGs were collected 2, 7, 14, 21, 30 and 60 days following BCNI (n=5/group). MPG gene expression analysis by qPCR was performed for markers of neuronal injury: activating transcription factor 3 (ATF3), glial fibrillary acidic protein (GFAP); apoptotic markers: caspase 1, caspase 3, caspase 9, Bcl2–associated X protein (Bax), BRCA1-associated RING domain protein 1 (BARD1); and neuronal markers: β-tubulin3 (BT3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). Additional injured MPGs were fixed, embedded in paraffin and assessed for apoptosis by TUNEL (n=4/group). Results GFAP and ATF3 gene expression were significantly increased in MPGs from 2-21 days following BCNI (p<0.05). Apoptotic markers caspase1, caspase3, Bax and BARD1 at 2-14 days after BCNI were significantly elevated (p<0.05). There was no change in caspase 9 gene expression. Neuronal markers BT3, TH, and VACht were significantly lower in 2 and 7 day injured MPGs (p<0.05). Furthermore, 2 and 7 days following BCNI nNOS gene expression was 50% lower than sham (p<0.05). Apoptotic positive cells were significantly increased in MPGs 7 and 14 days following BCNI (Sham: 0.3±0.12, 7d: 9.5±1.2, 14d: 9.7±3.5, p<0.05). Conclusions Markers of neuronal injury and apoptosis are significantly increased early following BCNI. Further studies inhibiting the apoptotic pathway at these early time points may provide therapeutic avenues for neurogenic-mediated ED. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e522-e523 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Johanna Hannan More articles by this author Maarten Albersen More articles by this author Joseph Watson More articles by this author Xiaopu Liu More articles by this author Petter Hedlund More articles by this author Arthur Burnett More articles by this author Trinity Bivalacqua More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

3,3′-Diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells

3,3′-Diindolylmethane (DIM) is a major in vivo condensation product of indole-3-carbinol, which is present in cruciferous vegetables. Although these compounds have been widely implicated in antitumorigenic and proapoptotic properties in animal as well as in vitro models of cancer, the underlying cellular mechanisms regulated by DIM are only partially understood. Activating transcription factor 3 (ATF3) is a member of the ATF/c-AMP response element-binding (CREB) subfamily of the basic-region leucine zipper family and has been known to induce apoptosis in human colorectal cancer (CRC) cells. The present study was performed to elucidate the molecular mechanism of ATF3 induction by DIM in human CRC cells. The DIM treatment induced apoptosis and induced ATF3 gene expression at protein and messenger RNA levels. DIM increased ATF3 promoter activity, and the region of −84 to +34 within ATF3 promoter was responsible for promoter activation by DIM. This region contained an ATF binding site. Deletion and point mutation of the ATF binding site (−23 to −16) abolished ATF3 promoter activation by DIM, and overexpression of ATF4 enhanced ATF3 transactivation. Chromatin immunoprecipitation assay confirmed the binding of ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by small interference RNA results in repression of DIM-induced ATF3 expression. The current study demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway and subsequently induces apoptosis in human CRC cells.

Decreased Phosphorylation of Histone H3 serine 10 by Genistein is Associated with the Transcriptional Upregulation of ATF3 in DLD‐1 Colon Cancer Cells

Activating transcription factor 3 (ATF3) is transcriptionally regulated by a variety of bioactive compounds during carcinogenesis. The present study investigated the mechanisms behind the regulation of ATF3 gene expression by genistein (GEN), a soy isoflavone, in DLD‐1 colon cancer cells. We examined the effect of GEN on histone modifications and the binding of transcription factors within the ATF3 promoter using Chromatin immunoprecipitation (ChIP). GEN upregulated ATF3 mRNA expression and the extra‐cellular signal regulated kinase (ERK) in a time‐dependent manner. Inhibition of ERK phosphorylation by U0126 did not abolish GEN‐induced ATF3 upregulation. However, GEN repressed the phosphorylation of Jun‐N‐terminal kinase (JNK). Inhibiting the phosphorylation of JNK (pJNK) by SP600125 resulted in increased ATF3 mRNA, indicating that JNK negatively regulated its expression. GEN reduced the phosphorylation of histone H3 at serine 10 (pH3S10), and the JNK inhibitor experiment confirmed that this was due to the reduction of pJNK. The GEN‐induced increase of ATF3 promoter activity was associated with decreased pH3S10 and increased ATF2 binding at the promoter. Our results indicate that GEN reduces pH3S10 at the promoter of an actively transcribed gene, providing a promising direction for investigating GEN‐mediated gene regulation and its role in colon carcinogenesis.

Role of activating transcription factor 3 (ATF3) in sublytic C5b‐9‐induced glomerular mesangial cell apoptosis

Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell (GMC) apoptosis mediated by these complexes has not been well defined. The activating transcription factor 3 (ATF3) gene is an immediate early gene for the cell to cope with a variety of stress signals and can promote apoptosis of some cells. In this study, ATF3 expression and cell apoptosis in GMCs induced by sublytic C5b-9 were measured, and then the effects of ATF3 gene over-expression or knockdown on GMC apoptosis induced by sublytic C5b-9 were examined at a fixed time. The results showed that both ATF3 expression and GMC apoptosis were markedly increased and ATF3 over-expression obviously increased sublytic C5b-9-induced GMC apoptosis, whereas ATF3 gene silencing had a significant opposite effect. Collectively, these findings indicate that upregulation of ATF3 gene expression is involved in regulating GMC apoptosis induced by sublytic C5b-9 complexes.

Epithelial cell survival by activating transcription factor 3 (ATF3) in response to chemical ribosome-inactivating stress

Ribotoxic stress responses lead to the expression of genes important for cellular homeostasis by modulating cell survival, proliferation and differentiation. ATF3 was investigated for its modulation of the epithelial cellular integrity in response to mucosal ribotoxic stress. ATF3 expression was up-regulated by chemical agents causing ribotoxic stress such as deoxynivalenol and anisomycin in different types of intestinal epithelial cells. Moreover, reduction of ATF3 gene expression promoted ribotoxic stress-triggered programmed cell death, implicating a protective role of ATF3 in epithelial cell survival. Mechanistically, stabilization of ATF3 messenger RNA and protein played a critical role in maintaining enhanced levels of ATF3 production in response to the ribotoxic chemical agent. For ATF3 mRNA stability, p38 specific inhibitor SB203580 was the most efficient agent for suppression, suggesting the involvement of the p38 MAP kinase in ATF3 mRNA stabilization. In addition, the p38 MAP kinase as well as its downstream mediator glycogen synthase kinase 3β (GSK3β) was involved in ATF3 protein stabilization caused by chemical ribotoxic stress in human epithelial cells. As another separate signaling cascade, double-stranded RNA (dsRNA)-activated protein kinase (PKR) was demonstrated to translationally modulate ATF3 expression and contribute to the epithelial cell survival. PKR interference caused cells to be more susceptible to cell death caused by the chemical ribotoxic stress. The results of this study showed that enhanced ATF3 production was associated with cellular defenses by maintaining the epithelial survival after ribotoxic mucosal insults.

Abstract 243: Decreased Adiponectin Levels in Obese Mice with Polygenetic Susceptibility to Type 2 Diabetes Is Linked to Altered miRNA Levels of the ATF3 Gene In Visceral Adipose Tissue

INTRODUCTION: Adiponectin encoded by the APMI gene is an adipocyte-expressed protein with insulin sensitizing, antiatherogenic and antiinflammatory properties. Activating transcription factor 3 (ATF3) is a member of the ATF/cAMP responsive element binding family that acts as a stress-inducible transcriptional repressor. ATF3 was recently shown to repress adiponectin gene expression. HYPOTHESIS: High fat diet with genetic susceptibility for T2D alters the miRNA profile for the adiponectin gene in visceral adipose (VA). METHODS: NONcNZO10 (NZ10) mice with polygenic susceptibility to T2D or NONLt controls (60 per group) were subjected to normal chow (NC; 6% fat), Western diet (WD; high fat/carb) or Mediterranean diet (MD; high protein/unsaturated fat) for 25 weeks. After a 16h fast, VA was isolated from all groups (5 mice per group) and subjected to gene-microarray, micro-RNA and protein analyses. RESULTS: NONLt (control) mice retained normal glycemic control and blood chemistry over 25 weeks with all diets. WD-fed NZ10 mice had increased plasma triglycerides (3-fold, p&lt;0.001), insulin (1.8-fold. P&lt;0.01) and severely impaired glucose tolerance relative to the MD or control groups (p&lt;0.01). NZ10 mice on all diets accumulated 2.5-fold more VA than NONLt controls. Body weights of WD-fed NZ10 mice were 18% greater and fat depots were 22±3.1% larger than SB mice at 25 weeks (p&lt;0.01). Serum adiponectin levels decreased &gt;50% at 25–30 weeks in NZ10 mice fed WD compared with NONLt (p&lt;0.01). The ATF3 gene was upregulated (5-fold, p&lt;0.0001) in the NZ10 mice fed WD compared with MD and 2-fold (p&lt;0.001) compared with NC. NONLt mice did not show any diet-related difference of ATF3 expression. Expression of ATF3 was increased 20-fold in NZ10 mice relative to NONLt mice independently of diet (p&lt;0.001). MiR profiling of the ATF3 gene in VA revealed that miR-203, 685, 191 and 29a were markedly downregulated in NZ10-WD mice (all 2.5-fold, p&lt;0.001). MiR expression in NONLt controls was unchanged and similar to that of NZ10 mice fed MD. CONCLUSIONS: Combined T2D susceptibility and a permissive diet cause selective repression of miR-203, 685, 191 and 29a and up-regulation of ATF3 gene expression. The consequences include defective adiponectin production and predisposition to T2D.

The regulation of ATF3 gene expression by mitogen-activated protein kinases.

ATF3 (activating transcription factor 3) gene encodes a member of the ATF/CREB (cAMP-response-element-binding protein) family of transcription factors. Its expression is induced by a wide range of signals, including stress signals and signals that promote cell proliferation and motility. Thus the ATF3 gene can be characterized as an 'adaptive response' gene for the cells to cope with extra- and/or intra-cellular changes. In the present study, we demonstrate that the p38 signalling pathway is involved in the induction of ATF3 by stress signals. Ectopic expression of CA (constitutively active) MKK6 [MAPK (mitogen-activated protein kinase) kinase 6], a kinase upstream of p38, indicated that activation of the p38 pathway is sufficient to induce the expression of the ATF3 gene. Inhibition of the pathway indicated that the p38 pathway is necessary for various signals to induce ATF3, including anisomycin, IL-1beta (interleukin 1beta), TNFalpha (tumour necrosis factor alpha) and H2O2. Analysis of the endogenous ATF3 gene indicates that the regulation is at least in part at the transcription level. Specifically, CREB, a transcription factor known to be phosphorylated by p38, plays a role in this induction. Interestingly, the ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase)/SAPK (stress-activated protein kinase) signalling pathways are neither necessary nor sufficient to induce ATF3 in the anisomycin stress paradigm. Furthermore, analysis of caspase 3 activation indicated that knocking down ATF3 reduced the ability of MKK6(CA) to exert its pro-apoptotic effect. Taken together, our results indicate that a major signalling pathway, the p38 pathway, plays a critical role in the induction of ATF3 by stress signals, and that ATF3 is functionally important to mediate the pro-apoptotic effects of p38.

TNFalpha-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.

ATF3 (Activating transcription factor 3), a member of the CREB/ATF family, can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Here, we investigated the differential roles of the MAPK pathways involved in TNFalpha (tumour necrosis factor alpha)-induced ATF3 expression in vascular endothelial cells. In human umbilical vein endothelial cells, the expression of constitutively active MKK7 (MAPK kinase 7) increased the number of ATF3-positive cells, and dominant negative MKK7 suppressed the TNFalpha-induced expression of ATF3, indicating a requirement for the JNK pathway. In contrast, the expression of constitutively active or dominant negative MEK1/2 (MAPK/ERK kinase 1/2) suppressed or enhanced TNFalpha-mediated induction of ATF3, respectively. In support of this, the MEK1/2 specific inhibitor U0126 enhanced the expression of ATF3 induced by TNFalpha. Furthermore, the ERK pathway inhibits the TNFalpha-mediated induction of ATF3 mRNA, but not its stability, suggesting the involvement of ERK activity in the transcriptional regulation of the ATF3 gene. Our results suggest that TNFalpha-induced ATF3 gene expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.