Activating Transcription Factor 3 siRNA Research Articles

Ligustrazine promotes hypoxia/reoxygenation-treated trophoblast cell proliferation and migration by regulating the microRNA-27a-3p/ATF3 axis

ObjectivePreeclampsia (PE) is a pregnancy-specific syndrome. Ligustrazine (LSZ) is involved in hypoxia/reoxygenation (H/R)-treated trophoblast cell regulation, but its mechanism remains elusive. This study explored the mechanism of LSZ in H/R-treated trophoblast cells to provide a theoretical basis for the new treatment method development for PE. MethodsH/R HTR8/SVneo cell model was established for PE simulation to some extent. Trophoblast cell proliferation, apoptosis rate, migration, and invasion were detected by MTT assay, flow cytometry, scratch test, and Transwell assay. miR-27a-3p expression in trophoblast cells was detected by RT-qPCR. Binding sites between miR-27a-3p and ATF3 were predicted using Starbase and verified by dual-luciferase reporter assay. Activating transcription factor 3 (ATF3), β-catenin, Cyclin D1, and c-Myc protein levels were examined using Western blot. After LSZ treatment, H/R-induced HTR8/SVneo cells were delivered with miR-27a-3p mimic or ATF3 siRNA to verify their roles in HTR8/SVneo cells. ResultsLSZ facilitated the proliferation, migration, and invasion of trophoblast cells and inhibited apoptosis. miR-27a-3p was elevated in H/R-induced HTR8/SVneo cells and miR-27a-3p overexpression annulled the effect of LSZ on trophoblast cells. miR-27a-3p targeted ATF3. ATF3 silencing averted the property of LSZ on trophoblast cells. Wnt/β-catenin pathway-related proteins were repressed in H/R-induced HTR8/SVneo cells, and LSZ activated the Wnt/β-catenin pathway by promoting ATF3 expression. ConclusionLSZ mediated the Wnt pathway by regulating the miR-27a-3p/ATF3 axis, thus promoting the proliferation and migration of trophoblast cells. The protective mechanism of LSZ showed the potential application value in the treatment of PE.

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.

Activating transcription factor 3 (ATF3) protects against lipopolysaccharide-induced acute lung injury via inhibiting the expression of TL1A.

Excessive inflammatory responses are critical in the pathogenesis of acute lung injury (ALI). Activating transcription factor 3 (ATF3) is a stress-induced transcriptional regulator that is a negative regulator of inflammatory responses. Therefore, we investigated the role and signaling pathways of ATF3 in lipopolysaccharide (LPS)-induced ALI in mice. The mouse macrophage RAW264.7 cells were cultured on HTS 24-Transwell filter plates in presence of ATF3 siRNA before exposure to LPS. ATF3 knock-out (KO) and wild type (WT) mice were challenged by intra-peritoneal injection of LPS (15 mg/kg). Gene analysis was used to analyze differential gene expression between ATF3 KO and WT mice. LPS increased the expression of ATF3 in RAW264.7 cells and in lung tissues of mice, The concentration of TNFα and IL-6 was significantly increased in ATF3 siRNA-treated RAW264.7 cells compared to control cells after LPS stimulation. The concentration of TNFα, IL-6 and IL-1β in serum and lung tissue of ATF3 KO mice was significantly increased compared to ATF3 WT mice. In addition, the lung wet/dry weight and BALF protein were significantly increased in ATF3 KO mice after LPS injection at 6, 24, and 48 hr. The survival of ATF3 KO mice significantly decreased. Differential gene analysis showed that TL1A was highly expressed in LPS-induced lung tissues of ATF3 KO mice.Moreover, ATF3 down-regulated the expression of TL1A in RAW264.7 cells and in lung tissues. These findings suggest that ATF3 protects against LPS-induced ALI via inhibiting TL1A expression.

Effect of ATF3-deletion on apoptosis of cultured retinal ganglion cells.

To investigate the effect of activating transcription factor-3 (ATF3)-deletion on apoptosis of cultured retinal ganglion cells (RGCs). Three ATF3 siRNA (ATF3-rat-651, ATF3-rat-319, ATF3-rat-520) were constructed, and were transiently transfected into RGC-5 cells. Quantitative real-time polymerase chain reaction (PCR) was used to examine ATF3 expression and the most effective ATF3 siRNA was selected for further studies. Flow cytometry was applied to investigate the effects of ATF3 deletion on RGC-5 apoptosis under elevated hydrostatic pressure. Quantitative real-time PCR and Western blot were performed to validate differentially expressed genes and proteins in ATF3-knockdown RGC-5 cells. ATF3 specific siRNA effectively down-regulated ATF3 expression and significantly inhibited cell apoptosis in RGC-5 cells. Quantitative real-time PCR and Western blot confirmed that ATF3 knockdown remarkably decreased Jun-B and increased c-Jun at both mRNA and protein levels in RGC-5 cells. ATF/cAMP-response element-binding family of transcription factors may be involved in the development of glaucoma and could be novel treatment targets for glaucoma.

ATF3 is a negative regulator of inflammation in human fetal membranes

IntroductionInfection and inflammation stimulate pro-inflammatory cytokines, prostaglandins and matrix metalloproteinase (MMP)-9, which play a central role in myometrial contractions and rupture of fetal membranes. In human and mouse immune cells, activating transcription factor 3 (ATF3) is a negative regulator of inflammation. No studies have examined the role of ATF3 in human labour. MethodsPrimary amnion cells were used to determine the effect of interleukin (IL)-1β and the bacterial product fibroblast-stimulating lipopeptide (fsl-1) on ATF3 expression, and the effect of ATF3 siRNA on pro-labour mediators. ATF3 expression was assessed in fetal membranes from non-labouring and labouring women at term and preterm, and after preterm pre-labour rupture of membranes (PPROM). ResultsIL-1β and fsl-1 significantly increased ATF3 expression. Silencing ATF3 significantly increased IL-1β- or fsl-1-induced expression of pro-inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1 (MCP-1)); cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin PGF2α release; and MMP-9 expression. ATF3 expression was decreased in fetal membranes with term labour. There was no effect of preterm labour or PPROM on ATF3 expression. DiscussionATF3 is a negative regulator of inflammation in human fetal membranes; in primary amnion cells, ATF3 expression is induced by IL-1β and fsl-1, and ATF3 silencing further exacerbates the inflammatory response when stimulated with these factors. Subsequently, ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis, conditions closely associated with inflammation and infection. Our data suggest that ATF3 may play a role in the terminal processes of human labour and delivery.

In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells.

Phlorofucofuroeckol A (PFF-A), one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3) has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB), located between positions −147 and −85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK), glycogen synthase kinase (GSK) 3β, and IκB kinase (IKK)-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose) polymerase (PARP) by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

Anti-cancer activity of Ginger (Zingiber officinale) leaf through the expression of activating transcription factor 3 in human colorectal cancer cells.

BackgroundGinger leaf (GL) has long been used as a vegetable, tea and herbal medicine. However, its pharmacological properties are still poorly understood. Thus, we performed in vitro studies to evaluate anti-cancer properties of ginger leaf and then elucidate the potential mechanisms involved.MethodsCell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA.ResultsExposure of GL to human colorectal cancer cells (HCT116, SW480 and LoVo cells) reduced the cell viability and induced apoptosis in a dose-dependent manner. In addition, GL reduced cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. ATF3 knockdown attenuated GL-mediated apoptosis. GL increased activating transcription factor 3 (ATF3) expressions in both protein and mRNA level and activated ATF3 promoter activity, indicating transcriptional activation of ATF3 gene by GL. In addition, our data showed that GL-responsible sites might be between -318 and -85 region of the ATF3 promoter. We also observed that ERK1/2 inhibition by PD98059 attenuated GL-mediated ATF3 expression but not p38 inhibition by SB203580, indicating ERK1/2 pathway implicated in GL-induced ATF3 activation.ConclusionsThese findings suggest that the reduction of cell viability and apoptosis by GL may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression through ERK1/2 activation in human colorectal cancer cells.

Urotensin II inhibits doxorubicin-induced human umbilical vein endothelial cell death by modulating ATF expression and via the ERK and Akt pathway.

Background and PurposeRegulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Urotensin II (U-II), a potent vasoactive peptide, participates in vascular and myocardial remodeling after injury. We investigated the protective effect of U-II on doxorubicin (DOX)-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved in this process.Experimental ApproachCultured HUVECs were treated with vehicle, DOX (1 µM), U-II, or U-II plus DOX. Apoptosis was evaluated by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells.Key ResultsU-II reduced the quantity of cleaved caspase-3 and cytosol cytochrome c and increased Bcl-2 expression, which results in protecting HUVECs from DOX-induced apoptosis. U-II induced Activating transcription factor 3 (ATF3) at both mRNA and protein levels in U-II-treated cells. Knockdown of ATF3 with ATF3 siRNA significantly reduced ATF3 protein levels and U-II protective effect under DOX-treated condition. U-II downregulated p53 expression in DOX-induced HUVECs apoptosis, and it rapidly activated extracellular signal-regulated protein kinase (ERK) and Akt. The DOX induced change of p53 was not affected by U-II antagonist (urantide) under ATF-3 knockdown. The inhibitory effect of U-II on DOX-increased apoptosis was attenuated by inhibitors of ERK (U0126) and PI3K/Akt (LY294002).Conclusion and ImplicationsOur observations provide evidence that U-II protects HUVECs from DOX-induced apoptosis. ERK-Akt phosphorylation, ATF3 activation, and p53 downregulation may play a signal-transduction role in this process.

ATF3 Confers Resistance to Pneumococcal Infection Through Positive Regulation of Cytokine Production

Activating transcription factor-3 (ATF3) is known as a suppressor of cytokine production after exposure to lipopolysaccharide or during gram-negative bacterial infection. However, the mechanism by which ATF3 regulates innate immunity against gram-positive bacterial infection, particularly Streptococcus pneumoniae, remains unknown. The wild-type and ATF3 knock-out (KO) mice were infected intranasally (i.n) or intraperitoneally with S. pneumoniae, and bacterial colonization or survival rate was determined. Pneumococcal pneumonia was induced by i.n infection, and ATF3 level was determined by Western blot. ATF3 KO cells or ATF3 siRNA transfection were used to determine expression of ATF3 downstream genes. Enzyme-linked immunosorbent assay was used to examine cytokines levels. ATF3 was highly expressed in various cell lines in vitro and in many organs in vivo. Pneumolysin was a novel inducer of ATF3. Pneumococcal infection induced ATF3, which subsequently stimulated production of cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and interferon [IFN]-γ). ATF3-mediated cytokine induction protected the host from pneumococcal infection. In the pneumonia infection model, the bacterial clearance of wild-type mice was more efficient than those of ATF3 KO mice. Taken together, we can conclude that ATF3 regulates innate immunity positively upon pneumococcus infection by enhancing TNF-α, IL-1β, and IFN-γ expression and modulating bacterial clearance.

Salermide up‐regulates death receptor 5 expression through the ATF4‐ATF3‐CHOP axis and leads to apoptosis in human cancer cells

Sirtuins (a class III histone deacetylase) have emerged as novel targets for cancer therapy. Salermide, a reverse amide compound that inhibits Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2), has been shown to induce apoptosis in human cancer cells. The mechanism underlying cellular apoptotic signalling by salermide remains unclear. In this study, we show that salermide up-regulates the expression of death receptor 5 (DR5) in human non-small cell lung cancer (NSCLC) cells. Blocking DR5 expression by gene silencing technology results in a decrease in activated forms of several pro-apoptotic proteins (caspase-8, caspase-9, caspase-3, PARP). Increasing DR5 protein expression correlates with salermide-induced apoptosis in human NSCLC cells. We discovered that IRE-1α, Bip, activating transcription factor 3 (ATF4), activating transcription factor 3 (ATF3) and C/EBP homologous protein (CHOP) are induced by salermide, which suggests that DR5-dependent apoptosis is induced by endoplasmic reticulum stress. Moreover, knockdown of Sirt1 and Sirt2 expression resulted in up-regulation of ATF4, CHOP and DR5. Transfected NSCLC cells with ATF4, ATF3 or CHOP siRNA results in a decline in pro-apoptotic proteins (such as caspase-8, caspase-9, caspase-3 and PARP) despite salermide treatment. We demonstrate that salermide induces expression of ATF4, and ATF4 up-regulates ATF3 and subsequently modulates CHOP. This suggests that DR5 is modulated by the ATF4-ATF3-CHOP axis in NSCLC after Sirt1/2 inhibition or salermide treatment. This study highlights the importance of DR5 up-regulation in apoptosis induced by Sirt1/2 inhibition and elucidates the underlying mechanism in human NSCLC cells.

Role of Activating Transcription Factor 3 on TAp73 Stability and Apoptosis in Paclitaxel-Treated Cervical Cancer Cells

Taxol (paclitaxel) is a potent anticancer drug that has been found to be effective against several tumor types, including cervical cancer. However, the exact mechanism underlying the antitumor effects of paclitaxel is poorly understood. Here, paclitaxel induced the apoptosis of cervical cancer HeLa cells and correlated with the enhanced activation of caspase-3 and TAp73, which was strongly inhibited by TAp73beta small interfering RNA (siRNA). In wild-type activating transcription factor 3 (ATF3)-overexpressed cells, paclitaxel enhanced apoptosis through increased alpha and beta isoform expression of TAp73; however, these events were attenuated in cells containing inactive COOH-terminal-deleted ATF3 [ATF3(DeltaC)] or ATF3 siRNA. In contrast, paclitaxel-induced ATF3 expression did not change in TAp73beta-overexpressed or TAp73beta siRNA-cotransfected cells. Furthermore, paclitaxel-induced ATF3 translocated into the nucleus where TAp73beta is expressed, but not in ATF3(DeltaC) or TAp73beta siRNA-transfected cells. As confirmed by the GST pull-down assay, ATF3 bound to the DNA-binding domain of p73, resulting in the activation of p21 or Bax transcription, a downstream target of p73. Overexpression of ATF3 prolonged the half-life of TAp73beta by inhibiting its ubiquitination and thereby enhancing its transactivation and proapoptotic activities. Additionally, ATF3 induced by paclitaxel potentiated the stability of TAp73beta, not its transcriptional level. Chromatin immunoprecipitation analyses show that TAp73beta and ATF3 are recruited directly to the p21 and Bax promoter. Collectively, these results reveal that overexpression of ATF3 potentiates paclitaxel-induced apoptosis of HeLa cells, at least in part, by enhancing TAp73beta's stability and its transcriptional activity. The investigation shows that ATF3 may function as a tumor-inhibiting factor through direct regulatory effects on TAp73beta, suggesting a functional link between ATF3 and TAp73beta.

Activating Transcription Factor 3 (ATF3) Is Overexpressed in Classical Hodgkin Lymphoma and Is Required for Survival of Hodgkin and Reed-Sternberg Cells in Response to Cellular Stress.

Hodgkin and Reed-Sternberg (HRS) cells display aberrant activity of several transcription factors, including nuclear factor kappa B (NF-kB) and members of the activator protein-1 (AP-1) family, that promote cytokine expression, cellular survival and proliferation. Using oligonucleotide microarrays, we set out to identify additional factors which are responsible for the malignant phenotype of HRS cells. Microarray analysis and subsequent Northern and Western blotting revealed that activating transcription factor 3 (ATF3), a member of the CREB/ATF family of transcription factors that is involved in the cellular response to stress signals, shows a strong constitutive expression in Hodgkin-derived cell lines. In contrast, ATF3 expression could not be detected in several Non-Hodgkin cell lines of distinct differentiation stages, ranging from pre-B cell lines to plasma B cell lines. To investigate the expression pattern of ATF3 in primary tumor cells, we performed immunohistochemistry on tissue microarrays. Immunohistochemistry confirmed a strong expression of ATF3 in primary HRS cells with a nuclear staining pattern. ATF3 expression was observed in almost all cases of classical Hodgkin lymphoma examined (69/71), whereas ATF3 expression was not detected in lymphocyte-predominant Hodgkin lymphoma. In addition, a significant number of anaplastic large cell lymphomas revealed a nuclear ATF3 staining pattern. In contrast, among all other B and T cell Non-Hodgkin lymphomas, ATF3 expression was observed only in rare cases (7/244), indicating that ATF3 expression is almost exclusively restricted to classical Hodgkin lymphoma and to a subset of anaplastic large cell lymphomas. To investigate the role of ATF3 for proliferation and survival of HRS cells, we generated vector-based siRNA constructs to downregulate ATF3 expression. Our vector system carries a puromycin-resistance gene thus allowing selection of siRNA expressing cells. Transfection of Hodgkin-derived cell lines with ATF3 siRNA constructs and subsequent selection of siRNA-expressing cells demonstrated a dramatic loss of viable cells. Our observations indicate that ATF3 is required for the survival of HRS cells exposed to the environmental stress imposed by antibiotic selection.