Activating Transcription Factor 3 Deficiency Research Articles

Nickel oxide nanoparticles induce apoptosis and ferroptosis in airway epithelial cells via ATF3.

Exposure to nickel oxide nanoparticles (NiONPs), which have been widely produced and applied in industry, leads to adverse pulmonary and systemic effects. The aim of this study is to investigate the involvement of apoptosis and ferroptosis in NiONPs-induced acute lung injury (ALI). Intratracheal instillation of NiONPs into mice elevated the levels of pro-inflammatory cytokines, neutrophils, and proteins in the bronchoalveolar lavage fluid, and triggered apoptosis and ferroptosis in the lung tissues. Consistently, NiONPs-induced apoptosis and ferroptosis were observed in in vitro experiments using human lung epithelial cells. Activating transcription factor 3 (ATF3), a stress-inducible transcription factor, was upregulated by NiONPs exposure in both murine lung tissues and human lung epithelial cells. Moreover, human lung epithelial cells with ATF3 deficiency exhibited a lower level of apoptosis and ferroptosis when exposed to NiONPs. Collectively, our findings demonstrated that ATF3 was responsive to NiONPs exposure, and promoted NiONPs-induced apoptosis and ferroptosis in lung epithelial cells, indicating that ATF3 is a potential biomarker and therapeutic target for NiONPs-associated ALI.

ATF3 regulates oxidative stress and extracellular matrix degradation via p38/Nrf2 signaling pathway in pelvic organ prolapse

Pelvic organ prolapse (POP) is a common disorder in women, and it is characterized by weakening of pelvic supportive structure with extracellular matrix (ECM) degradation. Activating transcription factor 3 (ATF3) was upregulated in anterior vaginal wall tissues of POP patients. We hypothesized that upregulation of ATF3 might contribute to POP development. This study aims to unveil the role of ATF3 in the pathogenesis of POP using a H2O2-induced in vitro model. Vaginal fibroblasts were isolated from woman with POP-Q stage greater than II and asymptomatic women with normal pelvic floor support. Knockdown of ATF3 enhanced cell viability and decreased cell apoptosis. Flow cytometry and immunnofluorescence showed that ATF3 deficiency inhibited H2O2-induced ROS production and the expression of 8 OHdG and 4-HNE. Western blot and Real-time PCR analysis revealed that ATF3 deficiency attenuated ECM component degradation (increasing collagen I, collagen III and elastin) and MMPs/TIMPs imbalance (decreasing MMP2 and MMP9 and increasing TIMP2). Moreover, knockdown of ATF3 induced the activation of p38/Nrf2/HO-1 signaling pathway. Further treatment with p38 inhibitor SB203580 abolished the protection of ATF3 deficiency against H2O2-induced cell damage, which was reverted by Nrf2 activator TBHQ. Thus, ATF3 likely contributes to POP progression by inducing cell apoptosis, oxidative stress and ECM degradation via regulating p38/Nrf2 pathway, which provides a potential therapeutic target for POP.

ATF3 inhibits arsenic-induced malignant transformation of human bronchial epithelial cells by attenuating inflammation

Arsenic is a naturally occurring metalloid strongly associated with the incidence of lung cancer. Understanding the mechanisms of arsenic-induced carcinogenesis favors the development of effective interventions to reduce the incidence and mortality of lung cancer. In this study, we investigated the role of activating transcription factor 3 (ATF3) in arsenic-induced transformation of human bronchial epithelial cells. ATF3 was upregulated during chronic exposure to 0.25 μM arsenic, and loss of ATF3 promoted arsenic-induced transformation. Moreover, arsenic-transformed ATF3 knockout (ATF3 KO-AsT) cells exhibited more aggressive characteristics, including acceleration in proliferation, resistance to chemotherapy and increase in migratory capacity. RNA-seq revealed that pathways involved in inflammation, cell cycle, EMT and oncogenesis were affected due to ATF3 deficiency during chronic arsenic exposure. Further experiments confirmed the overproduction of IL-6, IL-8 and TNFα as well as enhanced phosphorylation of AKT and STAT3 in ATF3 KO-AsT cells. Our results demonstrate that ATF3 upregulated by chronic low-dose arsenic exposure represses cell transformation and acquisition of malignant characteristics through inhibiting the production of proinflammatory cytokines and activation of downstream proteins AKT and STAT3, providing a new strategy for the prevention of carcinogen-induced lung cancer.

Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis.

Secretory Ig A (sIgA) plays an important role in the maintenance of intestinal homeostasis via cross-talk with gut microbiota. The defects in sIgA production could elicit dysbiosis of commensal microbiota and subsequently facilitate the development of inflammatory bowel disease. Our previous study revealed activating transcription factor 3 (ATF3) as an important regulator of follicular helper T (TFH) cells in gut. ATF3 deficiency in CD4+ T cells impaired the development of gut TFH cells, and therefore diminished sIgA production, which increased the susceptibility to murine colitis. However, the potential role of microbiota in ATF3-mediated gut homeostasis remains incompletely understood. In this study, we report that both Atf3-/- and CD4creAtf3fl/fl mice displayed profound dysbiosis of gut microbiota when compared with their littermate controls. The proinflammatory Prevotella taxa, especially Prevotella copri, were more abundant in ATF3-deficient mice when compared with littermate controls. This phenotype was obviously abrogated by adoptive transfer of either TFH cells or IgA+ B cells. Importantly, depletion of gut microbiota dramatically alleviated the severity of colitis in Atf3-/- mice, whereas transfer of microbiota from Atf3-/- mice to wild-type recipients increased their susceptibility to colitis. Collectively, these observations indicate the importance of IgA-microbiota interaction in ATF3-mediated gut homeostasis.

Transcriptional factor ATF3 protects against colitis by regulating follicular helper T cells in Peyer’s patches

Disruption of mucosal immunity plays a critical role in the pathogenesis of inflammatory bowel disease, yet its mechanism remains not fully elucidated. Here, we found that activating transcription factor 3 (ATF3) protects against colitis by regulating follicular helper T (TFH) cells in the gut. The expression of ATF3 in CD4+ T cells was negatively correlated with the severity of ulcerative colitis in clinical patients. Mice with ATF3 deficiency in CD4+ T cells (CD4creAtf3fl/fl ) were much more susceptible to dextran sulfate sodium-induced colitis. The frequencies of TFH cells, not other T cell subsets, were dramatically decreased in Peyer's patches from CD4creAtf3fl/fl mice compared with Atf3fl/fl littermate controls. The defective TFH cells significantly diminished germinal center formation and IgA production in the gut. Importantly, adoptive transfer of TFH or IgA+ B cells caused significant remission of colitis in CD4creAtf3fl/fl mice, indicating the TFH-IgA axis mediated the effect of ATF3 on gut homeostasis. Mechanistically, B cell lymphoma 6 was identified as a direct transcriptional target of ATF3 in CD4+ T cells. In summary, we demonstrated ATF3 as a regulator of TFH cells in the gut, which may represent a potential immunotherapeutic target in colitis.

ATF3 and JDP2 deficiency in cancer associated fibroblasts promotes tumor growth via SDF-1 transcription

The activating transcription factor 3 (ATF3) and the c-Jun dimerization protein 2 (JDP2) are members of the basic leucine zipper (bZIP) family of transcription factors. These proteins share a high degree of homology and both can activate or repress transcription. Deficiency of either one of them in the non-cancer host cells was shown to reduce metastases. As ATF3 and JDP2 compensate each other’s function, we studied the double deficiency of ATF3 and JDP2 in the stromal tumor microenvironment. Here, we show that mice with ATF3 and JDP2 double deficiency (designated thereafter dKO) developed larger tumors with high vascular perfusion and increased cell proliferation rate compared to wild type (WT) mice. We further identify that the underlying mechanism involves tumor associated fibroblasts which secrete high levels of stromal cell-derived factor 1 (SDF-1) in dKO fibroblasts. SDF-1 depletion in dKO fibroblasts dampened tumor growth and blood vessel perfusion. Furthermore, ATF3 and JDP2 were found to regulate SDF-1 transcription and secretion in fibroblasts, a phenomenon that is potentiated in the presence of cancer cells. Collectively, our results suggest that ATF3 and JDP2 regulate the expression of essential tumor promoting factors expressed by fibroblasts within the tumor microenvironment, and thus restrain tumor growth.

ATF3 Sustains IL-22-Induced STAT3 Phosphorylation to Maintain Mucosal Immunity Through Inhibiting Phosphatases.

In gut epithelium, IL-22 transmits signals through STAT3 phosphorylation (pSTAT3) which provides intestinal immunity. Many components in the IL-22-pSTAT3 pathway have been identified as risk factors for inflammatory bowel disease (IBD) and some of them are considered as promising therapeutic targets. However, new perspectives are still needed to understand IL-22-pSTAT3 signaling for effective clinical interventions in IBD patients. Here, we revealed activating transcription factor 3 (ATF3), recently identified to be upregulated in patients with active IBD, as a crucial player in the epithelial IL-22-pSTAT3 signaling cascade. We found ATF3 is central to intestinal homeostasis and provides protection during colitis. Loss of ATF3 led to decreased crypt numbers, more shortened colon length, impaired ileal fucosylation at the steady state, and lethal disease activity during DSS-induced colitis which can be effectively ameliorated by rectal transplantation of wild-type colonic organoids. Epithelial stem cells and Paneth cells form a niche to orchestrate epithelial regeneration and host-microbe interactions, and IL-22-pSTAT3 signaling is a key guardian for this niche. We found ATF3 is critical for niche maintenance as ATF3 deficiency caused compromised stem cell growth and regeneration, as well as Paneth cell degeneration and loss of anti-microbial peptide (AMP)-producing granules, indicative of malfunction of Paneth/stem cell network. Mechanistically, we found IL-22 upregulates ATF3, which is required to relay IL-22 signaling leading to STAT3 phosphorylation and subsequent AMP induction. Intriguingly, ATF3 itself does not act on STAT3 directly, instead ATF3 regulates pSTAT3 by negatively targeting protein tyrosine phosphatases (PTPs) including SHP2 and PTP-Meg2. Furthermore, we identified ATF3 is also involved in IL-6-mediated STAT3 activation in T cells and loss of ATF3 leads to reduced capacity of Th17 cells to produce their signature cytokine IL-22 and IL-17A. Collectively, our results suggest that via IL-22-pSTAT3 signaling in the epithelium and IL-6-pSTAT3 signaling in Th17 cells, ATF3 mediates a cross-regulation in the barrier to maintain mucosal homeostasis and immunity.

Loss of ATF3 exacerbates liver damage through the activation of mTOR/p70S6K/ HIF-1\u03b1 signaling pathway in liver inflammatory injury

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays important roles in regulating immune and metabolic homeostasis. Activation of the mechanistic target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) transcription factors are crucial for the regulation of immune cell function. Here, we investigated the mechanism by which the ATF3/mTOR/HIF-1 axis regulates immune responses in a liver ischemia/reperfusion injury (IRI) model. Deletion of ATF3 exacerbated liver damage, as evidenced by increased levels of serum ALT, intrahepatic macrophage/neutrophil trafficking, hepatocellular apoptosis, and the upregulation of pro-inflammatory mediators. ATF3 deficiency promoted mTOR and p70S6K phosphorylation, activated high mobility group box 1 (HMGB1) and TLR4, inhibited prolyl-hydroxylase 1 (PHD1), and increased HIF-1α activity, leading to Foxp3 downregulation and RORγt and IL-17A upregulation in IRI livers. Blocking mTOR or p70S6K in ATF3 knockout (KO) mice or bone marrow-derived macrophages (BMMs) downregulated HMGB1, TLR4, and HIF-1α and upregulated PHD1, increasing Foxp3 and decreasing IL-17A levels in vitro. Silencing of HIF-1α in ATF3 KO mice ameliorated IRI-induced liver damage in parallel with the downregulation of IL-17A in ATF3-deficient mice. These findings demonstrated that ATF3 deficiency activated mTOR/p70S6K/HIF-1α signaling, which was crucial for the modulation of TLR4-driven inflammatory responses and T cell development. The present study provides potential therapeutic targets for the treatment of liver IRI followed by liver transplantation.

Role of activating transcription factor 3 in fructose-induced metabolic syndrome in mice.

Activating transcription factor 3 (ATF3) has been implicated in cardiovascular disease and inflammation. This study examined the effects of ATF3 knockout (KO) on blood pressure, glucose intolerance, dyslipidemia, inflammation, and visceral adiposity in mice fed who did and did not consume a high-fructose diet. Male mice were divided into four groups (N = 15 for each group): the Con (control) group (wild-type mice fed a standard chow diet), Fru group (wild-type mice fed a high-fructose [60% fructose] diet), ATF3KO-Con group (ATF3 KO mice fed a standard chow diet), and ATF3KO-Fru group (ATF3 KO mice fed a high-fructose [60% fructose] diet). Experiments were conducted for 8 weeks. Our data demonstrated that ATF3 KO mice have lower systolic blood pressure (SBP) levels than do wild-type mice, and that high-fructose diets increase SBP levels in both wild-type and ATF3 KO mice. ATF3 KO in mice increased the serum levels of glucose, insulin, triglycerides, tumor necrosis factor-alpha, and intercellular adhesion molecule-1, impaired endothelium-dependent aortic relaxation, increased aorta wall thickness and lipid peroxide, and expanded visceral adiposity. These symptoms resembled those exhibited by the wild-type mice fed a high-fructose diet, which caused hyperglycemia, insulin resistance, dyslipidemia, endothelium-dependent aortic dysfunction, inflammation, aorta remodeling, and visceral adiposity. A high-fructose diet among ATF3 KO mice deteriorated metabolic parameters and inflammatory cytokines. The present results therefore suggest that ATF3 deficiency is involved in the pathogenesis of metabolic syndrome and ATF3 might have a therapeutic role in fructose-induced impairment of endothelium-dependent aortic relaxation, a rising of inflammatory cytokines, and metabolic syndrome.

Activating transcription factor 3 protects mice against pseudomonas aeruginosa-induced acute lung injury by interacting with lipopolysaccharide binding protein

Excessive inflammatory response is critical event in the pathogenesis of acute lung injury (ALI). Previous study has shown that activating transcription factor 3 (ATF3) plays a role in downregulate inflammatory responses including ventilation-induced ALI. We hypothesized that ATF3 have a protective effect in ALI induced by pseudomonas aeruginosa. PA was intra-tracheally administrated to ATF3 knock-out (KO) mice to establish ALI model. Inflammatory factors, BALF protein, lung wet to dry ratio, lung injury score and mortality were determined. The activation of NF-κB was detected by western blot and Co-immunoprecipitation (Co-ip) was used to determinate the binding of ATF3 to LBP. Peritoneal macrophages were isolated from ATF3 KO mice and stimulated by PA. PA increased the expression of ATF3 in the lung tissues in ATF3 wild type (WT) mice. ATF3 deficiency significantly increased the concentration of TNFα, IL-6 and IL-1β in the supernatant of peritoneal macrophages, lung tissue and BALF after PA stimulation and also enhanced the activity of NF-κB. ATF3 deficiency also enhanced the BALF protein concentration and increased the lung wet to dry ratio. The lung injury score and mortality were higher in ATF3 KO mice treated with PA. Moreover, ATF3 was demonstrated to bind to LBP These finding suggest ATF3 protect mice against ALI induced by PA partly due to the binding to LBP.

Activating transcription factor 3 represses cigarette smoke-induced IL6 and IL8 expression via suppressing NF-κB activation

Airway and lung inflammation is a fundamental hallmark of chronic obstructive pulmonary disease (COPD). Activating transcription factor 3 (ATF3) has been reported to negatively regulate many pro-inflammatory cytokines and chemokines. However, little is known about the impact of ATF3 on the inflammatory response of COPD. Since cigarette smoke (CS) is considered to be the most important risk factor in the etiology of COPD, we attempted to investigate the effects and molecular mechanisms of ATF3 in CS-induced inflammation. We observed an increase in the expression of ATF3 in the lung tissues of CS-exposed mice and CS extract (CSE)-treated human bronchial epithelial (HBE) cells. In vitro results indicated that ATF3 inhibition significantly increased the expression of proinflammatory cytokines interleukin 6 (IL6) and interleukin 8 (IL8) in CSE-stimulated HBE cells. Furthermore, in vivo data verified that CS induced inflammatory cell recruitment around the bronchus. In addition, neutrophil infiltration in bronchoalveolar lavage fluid (BALF) of CS-exposed Atf3−/− mice was markedly higher than in stimulated WT mice. Finally, ATF3 deficiency increased the in vitro and in vivo expression and phosphorylation of nuclear factor-κB (NF-κB), a positive mediator of inflammation. Thus, this study shows that ATF3 plays an important role in the negative regulation of CS-induced pro-inflammatory gene expression through downregulating NF-κB phosphorylation.

Glucocorticoid Induces Hepatic Steatosis by Inhibiting Activating Transcription Factor 3 (ATF3)/S100A9 Protein Signaling in Granulocytic Myeloid-derived Suppressor Cells

Glucocorticoids (GCs) used as inflammation suppressors have harmful side effects, including induction of hepatic steatosis. The underlying mechanisms of GC-promoted dysregulation of lipid metabolism, however, are not fully understood. GCs could facilitate the accumulation of myeloid-derived suppressor cells (MDSC) in the liver of animals, and the potential role of MDSCs in GC-induced hepatic steatosis was therefore investigated in this study. We demonstrated that granulocytic (G)-MDSC accumulation mediated the effects of GCs on the fatty liver, in which activating transcription factor 3 (ATF3)/S100A9 signaling plays an important role. ATF3-deficient mice developed hepatic steatosis and displayed expansion of G-MDSCs in the liver and multiple immune organs, which shared high similarity with the phenotype observed in GC-treated wild-type littermates. Adoptive transfer of GC-induced or ATF3-deficient G-MDSCs promoted lipid accumulation in the liver, whereas depletion of G-MDSCs alleviated these effects. Mechanistic studies showed that in MDSCs, ATF3 was transrepressed by the GC receptor GR through direct binding to the negative GR-response element. S100A9 is the major transcriptional target of ATF3 in G-MDSCs. Silencing S100A9 clearly alleviated G-MDSCs expansion and hepatic steatosis caused by ATF3 deficiency or GC treatment. Our study uncovers an important role of G-MDSCs in GC-induced hepatic steatosis, in which ATF3 may have potential therapeutic implications.

Induction of activating transcription factor 3 limits survival following infarct-induced heart failure in mice.

Numerous fibrotic and inflammatory changes occur in the failing heart. Recent evidence indicates that certain transcription factors, such as activating transcription factor 3 (ATF3), are activated during heart failure. Because ATF3 may be upregulated in the failing heart and affect inflammation, we focused on the potential role of ATF3 on postinfarct heart failure. We subjected anesthetized, wild-type mice to nonreperfused myocardial infarction and observed a significant induction in ATF3 expression and nuclear translocation. To test whether the induction of ATF3 affected the severity of heart failure, we subjected wild-type and ATF3-null mice to nonreperfused infarct-induced heart failure. There were no differences in cardiac function between the two genotypes, except at the 2-wk time point; however, ATF3-null mice survived the heart failure protocol at a significantly higher rate than the wild-type mice. Similar to the slight favorable improvements in chamber dimensions at 2 wk, we also observed greater cardiomyocyte hypertrophy and more fibrosis in the noninfarcted regions of the ATF3-null hearts compared with the wild-type. Nevertheless, there were no significant group differences at 4 wk. Furthermore, we found no significant differences in markers of inflammation between the wild-type and ATF3-null hearts. Our data suggest that ATF3 suppresses fibrosis early but not late during infarct-induced heart failure. Although ATF3 deficiency was associated with more fibrosis, this did not occur at the expense of survival, which was higher in the ATF3-null mice. Overall, ATF3 may serve a largely maladaptive role during heart failure.

ATF3-Mediated NRF2/HO-1 Signaling Regulates TLR4 Innate Immune Responses in Mouse Liver Ischemia/Reperfusion Injury

Activating transcription factor 3 (ATF3) is a stress-induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)-stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid-derived 2-related factor 2/heme oxygenase-1 (NRF2/HO-1) signaling pathway leading to enhancing Toll-like receptor 4/nuclear factor kappa beta (TLR4/NF-κB) activation, pro-inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti-apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO-1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI-rescued ATF3 anti-inflammatory functions in ATF3-deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMMs) depressed levels of NRF2/HO-1 and PI3K/AKT, resulting in enhanced TLR4/NF-κB activation. Pretreatment of LPS-stimulated BMMs with M2 increased NRF2/HO-1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF-κB-mediated proinflammatory mediators. Thus, our results first demonstrate ATF3-mediated NRF2/HO-1 signaling in the regulation of TLR4-driven inflammatory responses in IR-stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR-induced liver injury.

Loss of ATF3 promotes Akt activation and prostate cancer development in a Pten knockout mouse model.

Activating transcription factor 3 (ATF3) responds to diverse cellular stresses, and regulates oncogenic activities (e.g., proliferation, survival and migration) through direct transcriptional regulation or protein-protein interactions. Although aberrant ATF3 expression is frequently found in human cancers, the role of ATF3 in tumorigenesis is poorly understood. Here we demonstrate that ATF3 suppresses the development of prostate cancer induced by knockout of the tumor suppressor Pten in mouse prostates. Where as the oncogenic stress elicited by Pten loss induced ATF3 expression in prostate epithelium, we found that ATF3 deficiency increased cell proliferation and promoted cell survival, leading to early onset of mouse prostatic intraepithelial neoplasia and the progression of prostate lesions to invasive adenocarcinoma. Importantly, loss of ATF3 promoted activation of the oncogenic AKT signaling evidenced by high levels of phosphorylated AKT and S6 proteins in ATF3-null prostate lesions. In line with these in vivo results, knockdown of ATF3 expression in human prostate cancer cells by sgRNA-mediated targeting activated AKT and increased matrix metalloproteinase-9 expression. Our results thus link ATF3 to the AKT signaling, and suggest that ATF3 is a tumor suppressor for the major subset of prostate cancers harboring dysfunctional Pten.

Activating Transcription Factor 3 Deficiency Promotes Cardiac Hypertrophy, Dysfunction, and Fibrosis Induced by Pressure Overload

Activating transcription factor 3 (ATF3), which is encoded by an adaptive-response gene induced by various stimuli, plays an important role in the cardiovascular system. However, the effect of ATF3 on cardiac hypertrophy induced by a pathological stimulus has not been determined. Here, we investigated the effects of ATF3 deficiency on cardiac hypertrophy using in vitro and in vivo models. Aortic banding (AB) was performed to induce cardiac hypertrophy in mice. Cardiac hypertrophy was estimated by echocardiographic and hemodynamic measurements and by pathological and molecular analysis. ATF3 deficiency promoted cardiac hypertrophy, dysfunction and fibrosis after 4 weeks of AB compared to the wild type (WT) mice. Furthermore, enhanced activation of the MEK-ERK1/2 and JNK pathways was found in ATF3-knockout (KO) mice compared to WT mice. In vitro studies performed in cultured neonatal mouse cardiomyocytes confirmed that ATF3 deficiency promotes cardiomyocyte hypertrophy induced by angiotensin II, which was associated with the amplification of MEK-ERK1/2 and JNK signaling. Our results suggested that ATF3 plays a crucial role in the development of cardiac hypertrophy via negative regulation of the MEK-ERK1/2 and JNK pathways.