Activating Transcription Factor 3 mRNA Research Articles

Deubiquitinating enzyme OTUD4 stabilizes RBM47 to induce ATF3 transcription: a novel mechanism underlying the restrained malignant properties of ccRCC cells.

Dysregulation of deubiquitination contributes to various diseases, including cancer, and aberrant expression of deubiquitinating enzymes is involved in carcinoma progression. As a member of the ovarian tumor (OTU) deubiquitinases, OTUD4 is considered a tumor suppressor in many kinds of malignancies. The biological characteristics and mechanisms of OTUD4 in clear cell renal cell carcinoma (ccRCC) remain unclear. The downregulation of OTUD4 in ccRCC was confirmed based on the TCGA database and a validation cohort of 30-paired ccRCC and para-carcinoma samples. Moreover, OTUD4 expression was detected by immunohistochemistry in 50 cases of ccRCC tissues, and patients with lower levels of OTUD4 showed larger tumor size (p = 0.015). TCGA data revealed that patients with high expression of OTUD4 had a longer overall survival rate. In vitro and in vivo studies revealed that downregulation of OTUD4 was essential for tumor cell growth and metastasis in ccRCC, and OTUD4 overexpression inhibited these malignant phenotypes. We further found that OTUD4 sensitized ccRCC cells to Erastin-induced ferroptosis, and ferrostain-1 inhibited OTUD4-induced ferroptotic cell death. Mechanistic studies indicated that OTUD4 functioned as an anti-proliferative and anti-metastasic factor through the regulation of RNA-binding protein 47 (RBM47)-mediated activating transcription factor 3 (ATF3). OTUD4 directly interacted with RBM47 and promoted its stability via deubiquitination events. RBM47 was critical in ccRCC progression by regulating ATF3 mRNA stability, thereby promoting ATF3-mediated ferroptosis. RBM47 interference abolished the suppressive role of OTUD4 overexpression in ccRCC. Our findings provide mechanistic insight into OTUD4 of ccRCC progression and indicate a novel critical pathway OTUD4/RBM47/ATF3 may serve as a potential therapeutic pathway for ccRCC.

N6 -methyladenosine-modified circSTX6 promotes hepatocellular carcinoma progression by regulating the HNRNPD/ATF3 axis and encoding a 144 amino acid polypeptide.

Circular RNAs (circRNAs) play a significant role in the initiation and progression of various cancers, including hepatocellular carcinoma (HCC). Circular syntaxin 6 (circSTX6, also known as hsa_circ_0007905) has been identified as a microRNA (miRNA) sponge in pancreatic adenocarcinoma. However, its full range of functions in terms of protein scaffold and translation remain largely unexplored in the context of HCC. The expression of circSTX6 and its encoded protein was examined in HCC tumour tissues. N6 -methyladenosine (m6 A) on circSTX6 was verified and quantified by methylated RNA immunoprecipitation (Me-RIP), RIP and dual luciferase reporter assays. The biological functions of circSTX6 and its encoded protein in HCC were clarified by in vitro and in vivo experiments. Mechanistically, the interaction between circSTX6 and heterogeneous nuclear ribonucleoprotein D (HNRNPD) was investigated by RNA pull-down, RIP and fluorescence in situ hybridization (FISH)/IF. The regulatory effects of circSTX6 and HNRNPD on activating transcription factor 3 (ATF3) mRNA were determined by mRNA stability and RIP assays. Furthermore, the presence of circSTX6-encoded protein was verified by mass spectrometry. CircSTX6 and its encoded 144 amino acid polypeptide, circSTX6-144aa, were highly expressed in HCC tumour tissues and served as independent risk factors for overall survival in HCC patients. The expression of circSTX6 was regulated by METTL14 in an m6 A-dependent manner. Functionally, circSTX6 accelerated HCC proliferation and tumourigenicity and reinforced tumour metastasis in vitro and in vivo. Mechanistically, circSTX6 acted as a sponge for HNRNPD protein, facilitating its binding to ATF3 mRNA, consequently promoting ATF3 mRNA decay. Meanwhile, circSTX6-144aa promoted HCC proliferation, migration and invasion independent of circSTX6 itself. Collectively, our study reveals that m6 A-modified circSTX6 drives malignancy in HCC through the HNRNPD/ATF3 axis, while its encoded circSTX6-144aa contributes to HCC progression independent of circSTX6. CirSTX6 and its encoded protein hold promise as potential biomarkers and therapeutic targets in HCC.

Activating Transcription Factor 3 Diminishes Ischemic Cerebral Infarct and Behavioral Deficit by Downregulating Carboxyl-Terminal Modulator Protein.

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor and a familiar neuronal marker for nerve injury. This factor has been shown to protect neurons from hypoxic insult in vitro by suppressing carboxyl-terminal modulator protein (CTMP) transcription, and indirectly activating the anti-apoptotic Akt/PKB cascade. Despite prior studies in vitro, whether this neuroprotective pathway also exists in the brain in vivo after ischemic insult remains to be determined. In the present study, we showed a rapid and marked induction of ATF3 mRNA throughout ischemia-reperfusion in a middle cerebral artery (MCA) occlusion model. Although the level of CTMP mRNA was quickly induced upon ischemia, its level showed only a mild increase after reperfusion. With the gain-of-function approach, both pre- and post-ischemic administration of Ad-ATF3 ameliorated brain infarct and neurological deficits. Whereas, with the loss-of-function approach, ATF3 knockout (KO) mice showed bigger infarct and worse functional outcome after ischemia. In addition, these congenital defects were rescued upon reintroducing ATF3 to the brain of KO mice. ATF3 overexpression led to a lower level of CTMP and a higher level of p-Akt(473) in the ischemic brain. On the contrary, ATF3 KO resulted in upregulation of CTMP and downregulation of p-Akt(473) instead. Furthermore, post-ischemic CTMP siRNA knockdown led to smaller infarct and better behaviors. CTMP siRNA knockdown increased the level of p-Akt(473), but did not alter the ATF3 level in the ischemic brain, upholding the ATF3→CTMP signal cascade. In summary, our proof-of-principle experiments support the existence of neuroprotective ATF3→CTMP signal cascade regulating the ischemic brain. Furthermore, these results suggest the therapeutic potential for both ATF3 overexpression and CTMP knockdown for stroke treatment.

Activating transcription factor 3 (ATF3) as a perspective biomarker of Crohn’s disease

Introduction:Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the intestinal tract. Known types are Crohn’s disease (CD) and ulcerative colitis (UC), but their cause remains unclear and there is no convenient biomarker for IBD. The present study aimed to demonstrate an association between the onset of CD and activating transcription factor 3 (ATF3); as a new biomarker, measurement of blood ATF3 mRNA would be useful for distinguishing between CD and UC.Methods:First, in a mouse model of IBD in which damage to the intestinal mucosa was chemically induced with dextran sulfate sodium (DSS), intestinal ATF3 mRNA was evaluated. Next, in human subjects, CD and UC patients, blood ATF3 mRNA and intestinal ATF3 protein production were evaluated.Results:In the mouse model of IBD, intestinal ATF3 mRNA was elevated compared with the control ( P < 0.0001). In CD patients, blood ATF3 mRNA was elevated as compared with normal controls (NCs) and UC patients ( P < 0.05). In addition, we observed an increase in ATF3 production in the intestinal tract specific to CD.Conclusion:ATF3 is involved in the onset of CD, and blood ATF3 mRNA measurements would be useful for distinguishing it from UC.

Downregulated ATF3 expression inhibited growth of adrenocortical carcinoma cells and investigation of its mechanisms

Objective To investigate the effect of downregulated activating transcription factor 3 (ATF3) expression on proliferation of adrenocortical carcinoma cells and its mechanisms. Methods Immunohistochemistry and Western blotting were used to detect the expression of ATF3 in human adrenocortical tumor tissues and cells. Adrenocortical carcinoma cells, Sw-13, and NCI-H259R cells, were transfected with siATF3 using lipidosome 2000, and expression of ATF3 mRNA was determined using RT-PCR; expression of ATF3, cleaved caspase 3, caspase 3, cleaved PARP, and PARP proteins were detected using Western blotting; cell growth inhibition rate and apoptosis rate were monitored using MTT and AnnexinV-FITC/PI, respectively. Sw-13 and NCI-H259R cells were treated with NVP-BEZ235, Perifosine, BKM120, IWP-2, PP2, KN93, Everolimus respectively followed by detected expression of ATF3 mRNA by realtime PCR. The effect of ATF3 on cell proliferation after inhibition of related signaling pathways were detected by MTT. Results The ATF3 in human adrenocortical gland tumor tissues and cells showed high expression. The levels of ATF3 mRNA and protein in Sw-13 and NCI-F259R cells transfected with siATF3 were significantly reduced. Compared with the negative control group (NC siRNA), siATF3 transfection significantly inhibited the proliferation of Sw-13 and NCI-F259R cells (P<0.05), and increased the apoptosis rate (P<0.05). Western blotting shown that the levels of cleaved caspase 3 and cleaved PARP protein in siATF3 transfected cells increased significantly; and realtime PCR results indicated that the expression of ATF3 mRNA was dramatically inhibited by PP2, KN93, and IWP-2 in NCI-F259R cells compared with control group (DMSO); but ATF3 significantly promoted the proliferation activity of NCI-F259R cells which treated by PP2, KN93, and IWP-2 signaling inhibitors. Conclusion High expression of ATF3 is existed in adrenocortical carcinoma cells. Downregulated ATF3 expression may inhibit cell proliferation and activate apoptosis pathway, resulting in apoptosis in Sw-13 and NCI-F259R cells, this mechanism of action is related to activating Wnt/β-catenin, CaMKI, and SRC pathway. (Chin J Endocrinol Metab, 2018, 34: 738-745) Key words: Adrenocortical carcinoma; Activating transcription factor 3; Signal pathway; Apoptosis; Mechanism

Activating Transcription Factor 3 Is Reduced in Preeclamptic Placentas and Negatively Regulates sFlt-1 (Soluble fms-Like Tyrosine Kinase 1), Soluble Endoglin, and Proinflammatory Cytokines in Placenta.

Preeclampsia is a major pregnancy complication associated with poor placental perfusion and placental hypoxia. Systemic and placental inflammation and elevated placental secretion of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin) are hallmarks of preeclampsia, causing endothelial dysfunction and multiorgan injury. A molecule that links placental hypoxia, inflammation, and antiangiogenic factor release has not been described. ATF3 (activating transcription factor 3) is highly expressed in placenta. We assessed whether placental ATF3 is dysregulated in preterm preeclampsia, is altered by hypoxia, and regulates proinflammatory cytokine and antiangiogenic factor production. ATF3 mRNA and protein expression was significantly reduced in preterm preeclamptic placentas compared with gestation-matched controls. Hypoxia reduced ATF3 expression in primary cytotrophoblast and placental explants. Silencing ATF3 in primary cytotrophoblast increased proinflammatory cytokine (IL-6 [interleukin 6], TNF-α [tumor necrosis factor α]) and NF-κB (nuclear factor κB) expression. In silico analysis identified an ATF3-binding site in the promoter of Flt-1 (the transcript from which sFlt-1 is produced). Silencing ATF3 increased sFlt-1 and sEng secretion from primary cytotrophoblast possibly by increasing Rab11a and Arf1, cargo proteins that facilitate exosomal release of sFlt-1. ATF3 knockout mice did not have a preeclampsia phenotype, suggesting that these pathways may be specific to humans (preeclampsia is a uniquely human condition). To conclude, we have shown that ATF3 is decreased in preeclamptic placentas and that this decrease is likely to occur after prolonged hypoxia. We show that ATF3 is a regulator of placental proinflammatory cytokines and antiangiogenic factors sFlt-1 and sEng. Therefore, reduced ATF3 may be centrally involved in the pathology of preeclampsia.

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.

Activating transcription factor 3 promotes spinal cord regeneration of adult zebrafish

Zebrafish is an excellent model to study the mechanisms underlying successful central nervous system (CNS) regeneration. Previous study shows that activating transcription factor 3 (ATF3) promotes neurite outgrowth and is involved in optic nerve regeneration in zebrafish. Here, we used zebrafish model to investigate the role of ATF3 in regeneration following spinal cord injury (SCI). Quantitative polymerase chain reaction (qPCR) and in situ hybridization revealed that ATF3 mRNA levels increased at 12 h and 6 d following SCI. Double labeled immunofluorescence showed that ATF3 expressed in motoneurons. Treatment of anti-sense ATF3 morpholino (MO) inhibited locomotor recovery and decreased axon regeneration of spinal cord injured zebrafish. Further, inhibition of ATF3 up-regulated the expression of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These data suggest that ATF3 could promote locomotor recovery and axon regrowth in zebrafish SCI model possibly by regulating inflammatory response.

TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-β Receptor Pathway in Human Aortic Endothelial Cells.

Studies have suggested a link between the transforming growth factor beta 1 (TGF-β1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-β at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 μM), a specific inhibitor of TGF-β-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-β1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-β and lipolysis product-induced apoptosis is dependent on TGF-β signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-β signaling suggesting that TGF-β signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

The contribution of activating transcription factor 3 to apoptosis of human colorectal cancer cells by protocatechualdehyde, a naturally occurring phenolic compound

Protocatechualdehyde (PCA) is one of the important compounds found in barley, green cavendish bananas and grapevine leaves. PCA shows anti-cancer activities in breast, leukemia and colorectal cancer cells. Previous study reported that PCA exerts anti-cancer activity through down-regulating cyclin D1 and HDAC2 in human colorectal cancer cells. However, the underlying mechanisms for the expression of activating transcription factor 3 (ATF3) by PCA has not been studied. Thus, we performed in vitro study to investigate if treatment of PCA affects ATF3 expression and ATF3-mediated apoptosis in human colorectal cancer cells. PCA decreased cell viability in a dose-dependent manner in HCT116 and SW480 cells. In addition, PCA reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. Exposure of PCA activated the levels of ATF3 protein and mRNA in HCT116 and SW480 cells. Inhibition of ERK1/2/ by PD98059 and p38 by SB203580 inhibited PCA-induced ATF3 expression and transcriptional activation. ATF3-knockdown inhibited PCA-induced apoptosis and cell viability. In addition, ATF3 overexpression enhanced PCA-mediated cleavage of PARP. These findings suggest that inhibition of cell viability and apoptosis by PCA may be result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

Characterization of the regulatory mechanisms of activating transcription factor 3 by hypertrophic stimuli in rat cardiomyocytes.

AimsActivating transcription factor 3 (ATF3) is a stress-activated immediate early gene suggested to have both detrimental and cardioprotective role in the heart. Here we studied the mechanisms of ATF3 activation by hypertrophic stimuli and ATF3 downstream targets in rat cardiomyocytes.Methods and ResultsWhen neonatal rat cardiomyocytes were exposed to endothelin-1 (ET-1, 100 nM) and mechanical stretching in vitro, maximal increase in ATF3 expression occurred at 1 hour. Inhibition of extracellular signal-regulated kinase (ERK) by PD98059 decreased ET-1– and stretch–induced increase of ATF3 protein but not ATF3 mRNA levels, whereas protein kinase A (PKA) inhibitor H89 attenuated both ATF3 mRNA transcription and protein expression in response to ET-1 and stretch. To characterize further the regulatory mechanisms upstream of ATF3, p38 mitogen-activated protein kinase (MAPK) signaling was investigated using a gain-of-function approach. Adenoviral overexpression of p38α, but not p38β, increased ATF3 mRNA and protein levels as well as DNA binding activity. To investigate the role of ATF3 in hypertrophic process, we overexpressed ATF3 by adenovirus-mediated gene transfer. In vitro, ATF3 gene delivery attenuated the mRNA transcription of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1), and enhanced nuclear factor-κB (NF-κB) and Nkx-2.5 DNA binding activities. Reduced PAI-1 expression was also detected in vivo in adult rat heart by direct intramyocardial adenovirus-mediated ATF3 gene delivery.ConclusionsThese data demonstrate that ATF3 activation by ET-1 and mechanical stretch is partly mediated through ERK and cAMP-PKA pathways, whereas p38 MAPK pathway is involved in ATF3 activation exclusively through p38α isoform. ATF3 activation caused induction of modulators of the inflammatory response NF-κB and Nkx-2.5, as well as attenuation of pro-fibrotic and pro-inflammatory proteins IL-6 and PAI-1, suggesting cardioprotective role for ATF3 in the heart.

Age-Related Brain Expression and Regulation of the Chemokine CCL4/MIP-1β in APP/PS1 Double-Transgenic Mice

The detrimental effect of activation of the chemokine CCL4/MIP-1β on neuronal integrity in patients with HIV-associated dementia has directed attention to the potential role of CCL4 expression and regulation in Alzheimer disease. Here, we show that CCL4 mRNA and protein are overexpressed in the brains of APPswe/PS1ΔE9 (APP/PS1) double-transgenic mice, a model of cerebral amyloid deposition; expression was minimal in brains from nontransgenic littermates or single-mutant controls. Increased levels of CCL4 mRNA and protein directly correlated with the age-related progression of cerebral amyloid-β (Aβ) levels in APP/PS1 mice. We also found significantly increased expression of activating transcription factor 3 (ATF3), which was positively correlated with age-related Aβ deposition and CCL4 in the brains of APP/PS1 mice. Results from chromatin immunoprecipitation-quantitative polymerase chain reaction confirmed that ATF3 binds to the promoter region of the CCL4 gene, consistent with a potential role in regulating CCL4 transcription. Finally, elevated ATF3 mRNA expression in APP/PS1 brains was associated with hypomethylation of the ATF3 gene promoter region. These observations prompt the testable hypothesis for future study that CCL4 overexpression, regulated in part by hypomethylation of the ATF3 gene, may contribute to neuropathologic progression associated with amyloid deposition in Alzheimer disease.

Genistein‐induced histone modifications on ATF3 may contribute to cell cycle arrest and apoptosis in human colon cancer cell line SW620 (800.8)

Genistein, a soy‐derived isoflavone, has been shown to suppress colon cancer progression by regulating different cellular properties. The aim of the study is to investigate the role of Activating Transcription Factor 3 (ATF3), an immediate‐early response gene, in genistein‐modulated cell cycle arrest and apoptosis in a human colon cancer cell line SW620. Results showed that genistein induced G2 phase arrest and increased late apoptosis in SW620. Genistein also increased the expression of ATF3 mRNA and protein, as well as its transcriptional activity in SW620. mRNA expression of downstream genes of ATF3, such as cell cycle inhibitors p53, p21, CyclinD1, and apoptotic enzymes Casp8, and Casp9, were all induced by genistein. Chromatin immunoprecipitation (ChIP) showed decreased phosphorylated histone H3 serine 10 (H3S10p) at both the promoter and coding region of ATF3. In addition, genistein increased trimethylated histone H3 lysine 36 (H3K36me3) level at a coding region and dimethylated histone H3 lysine 4 (H3K4me2) level at the promoter region. Methylation sensitive PCR showed that genistein did not change the level of DNA methylation at various regions of ATF3 gene. In conclusion, genistein‐mediated histone modifications of ATF3 may lead to G2 phase arrest and apoptosis in SW620.

Activating transcription factor 3 (ATF3) in the human adrenal cortex: its possible involvement in aldosterone biosynthesis.

The activating transcription factor 3 (ATF3) is a member of the cAMP-responsive element-binding (CREB) protein family of transcription factors. ATF3 is expressed in H295R human adrenocortical carcinoma cells and considered a rapid-responder gene to angiotensin-II stimulation. However, the functions of ATF3 in human adrenocortical tissues have remained unknown. In this study, we analyzed the localization and possible regulatory mechanisms of ATF3 in human adrenocortical cells and tissues. The expression levels of ATF3 mRNA were analyzed in 66 aldosterone-producing adenomas (APA) and 14 cortisol-producing adenomas (CPA) using real-time RT-PCR. To localize the ATF3 protein, we performed immunohistochemical analysis in 20 non-pathological adrenal glands, 9 adrenal glands with idiopathic hyperaldosteronism (IHA), 20 APA, and 5 CPA using a mouse monoclonal antibody against human ATF3. We showed that ATF3 mRNA levels were higher in APA compared to CPA (P = 0.0053). ATF3 was immunolocalized to the zona glomerulosa of non-pathological adrenal glands and adrenal glands with IHA, and diffusely detected in the tumor cells of APA and CPA. Subsequently, H295R cells were treated for 6 h with each inhibitor of Src kinase (SRC), PKC, JAK2, and calcium-dependent calmodulin kinase-II (CaMKII) in the presence or absence of angiotensin-II. The expression levels of ATF3 mRNA were increased by angiotensin-II (about 3.5-fold induction), but the magnitude of the induction was significantly decreased in the presence of an inhibitor for SRC (PP2) or CaMKII (KN93). These results suggest that ATF3 is a downstream target of SRC and CaMKII signaling, and may be involved in adrenocortical aldosterone synthesis.

ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression†

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.

Lovastatin‐induced apoptosis is mediated by activating transcription factor 3 and enhanced in combination with salubrinal

We have previously demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to induce tumor-specific apoptosis. The apoptotic effects of lovastatin were regulated in part by the integrated stress response (ISR) that regulates cellular responses to a wide variety of stress inducers. A key regulator of the ISR apoptotic response is activating transcription factor 3 (ATF3) and its target gene CHOP/GADD153. In our study, we demonstrate that in multiple lovastatin-resistant clones of the squamous cell carcinoma (SCC) cell line SCC9, lovastatin treatment (1-25 μM, 24 hr) in contrast to the parental line failed to significantly induce ATF3 expression. Furthermore, the SCC-derived cell lines SCC25 and HeLa that are sensitive to lovastatin-induced apoptosis also preferentially induce ATF3 expression compared to resistant breast (MCF-7) and prostate carcinoma (PC3)-derived cell lines. In HeLa cells shRNA targeting ATF3 expression as well as in ATF3-deficient murine embryonic fibroblasts, lovastatin-induced cytotoxicity and apoptosis were attenuated. In ex vivo HNSCC tumors, lovastatin also induced ATF3 mRNA expression in two of four tumors evaluated. Salubrinal, an agent that can sustain the activity of a key regulator of the ISR eIF2α, further increased the expression of ATF3 and demonstrated synergistic cytotoxicity in combination with lovastatin in SCC cells. Taken together, our results demonstrate preferential induction of ATF3 in lovastatin-sensitive tumor-derived cell lines that regulate lovastatin-induced apoptosis. Importantly, combining lovastatin with salubrinal enhanced ATF3 expression and induced synergistic cytotoxicity in SCC cells.

Induction of ATF3 Gene Network by Triglyceride-Rich Lipoprotein Lipolysis Products Increases Vascular Apoptosis and Inflammation

Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Vasopressin increases ATF3 mRNA expression in mouse renal inner medulla

In renal inner medulla (IM) the concentration of urea is high and variable depending on the concentrating mechanism. During water restriction (WR), the level of vasopressin increases, followed by further increase of urea concentration in IM. Expression of activating transcription factor 3 (ATF3) is increased by high urea in vitro. To determine whether ATF3 is regulated by high urea in vivo, we water restricted mice for 48 hours and found that ATF3 mRNA increased by 3.81 ± 0.24 fold vs. control 1.00 ± 0.06 (p≤0.05). 7 day infusion of AVP increased ATF3 mRNA by 1.69 ± 019 fold vs. control 1.00 ± 0.15 (p≤0.05). However, 7 day infusion of dDAVP, an agonist of type 2 vasopressin receptor, did not cause significant change in the expression of ATF3 mRNA. ATF3 is an important component in the pathway of endoplasmic reticulum (ER) stress. We previously found that GCN2, a kinase in ER stress pathway, was involved in high urea associated increase of ATF3 in vitro. However, there was no difference in the basal level of ATF3 mRNA between GCN2 null and wild type mice. As in wild type mice, 48 hours of WR increased ATF3 mRNA in IM of GCN2 null mice by 4.14 ± 0.09 fold vs. control GCN2 null mice 1.00 ± 0.06 (p≤0.05). Knocking down GRP78, an important ER chaperone, in mIMCD3 cells did not affect high urea induced increase of ATF3 protein. We concluded that deficiency of ER stress genes does not affect vasopressin induced increase of ATF3 in renal IM.

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.

Identification and molecular cloning of Atlantic cod ( Gadus morhua) activating transcription factor 3 (ATF3) transcript and its induction in spleen following intraperitoneal polyriboinosinic polyribocytidylic acid injection

Activating transcription factor 3 (ATF3) participates in cellular processes to adapt to various extra- and intra-cellular changes including the modulation of immunity to prevent uncontrolled immune responses to pathogens. In teleost fishes, the involvement of ATF3 in immune response has not been documented. In this study, the putative Atlantic cod ( Gadus morhua) ATF3 transcript was identified by performing rapid amplification of cDNA ends (RACE) based on unknown expressed sequence tags (ESTs) that are potentially inducible by polyriboinosinic polyribocytidylic acid (pIC, a synthetic double-stranded RNA viral mimic) in Atlantic cod. ATF3-like ESTs were the most abundant unknown transcript (i.e. lacking significant BLAST hits) generated from a previously constructed cDNA library enriched for pIC inducible transcripts in Atlantic cod spleen. The full-length cDNA of cod ATF3 consists of 2329 nucleotides with an open reading frame (ORF) of 735 bp encoding 244 amino acids. The deduced amino acid sequence of Atlantic cod ATF3 shares over 45% identity with its putative orthologs from other vertebrates. In addition, the presence of a conserved basic region leucine zipper (bZIP) domain in the deduced Atlantic cod ATF3-like protein further supports its identity as an ATF3 homolog. In the spleen of Atlantic cod challenged with intraperitoneal (IP) injections of pIC, the time-course transcript expression of ATF3 was studied using quantitative reverse transcription-polymerase chain reaction (QPCR). At 6 h following the pIC injection, the relative expression level of ATF3 mRNA was significantly up-regulated in comparison to a pre-injected control (61.9-fold) and its time-matched saline-injected control (97.3-fold). At 24 h following the pIC injection, the mRNA expression level of cod ATF3 had subsided and was no longer significantly different from its pre-injected control, but significantly higher (1.88-fold) than its time-matched saline-injected control. Collectively, these results suggest that ATF3 may be involved in the modulation of innate anti-viral response in Atlantic cod.