Analyzing the impact of ATF3 in tumorigenesis and immune cell infiltration of ovarian tumor: a bioinformatics study.

ATF3 is an essential transcription activator in regulating cancer-related genetic expression. To identify the role of ATF3 in ovarian tumor, we investigated the correlation between ATF3 expression and the clinicopathological properties using multiple database. The cBioPortal and GEPIA database displayed the clinical information of ovarian patients harboring or without harboring ATF3 mutation. Furthermore, we assessed the relationship between survival and ATF3 expression level using Kaplan-Meier plotter, which reveals that the ovarian patients with higher expression of ATF3 suffered the worse overall survival and progression-free survival. The differentially expressed genes were analyzed using gene ontology, protein-protein interaction network, and gene set enrichment analysis to identify the hub gene and critical pathways, significantly affecting the tumorigenesis of ovarian tumor. Finally, we assessed the correlation between ATF3 and immune cell infiltration using Tumor Immunoassay Resource (TIMER) database. The results demonstrated that higher expression has a positive correlation with macrophage infiltration, expression for M1- and M2-type macrophages. Our study suggests that ATF3 can regulate the cell cycle and heme-related oxidative phosphorylation process, and it may be a critical factor to regulate the macrophage cell to be infiltrated into ovarian cancer. ATF3 can be used as a biomarker for diagnosis and therapy of ovarian tumor.

Interference with SRF expression in skeletal muscles reduces peripheral nerve regeneration in mice.

Traumatic injury of peripheral nerves typically also damages nerve surrounding tissue including muscles. Hence, molecular and cellular interactions of neighboring damaged tissues might be decisive for successful axonal regeneration of injured nerves. So far, the contribution of muscles and muscle-derived molecules to peripheral nerve regeneration has only poorly been studied. Herein, we conditionally ablated SRF (serum response factor), an important myofiber transcription factor, in skeletal muscles of mice. Subsequently, the impact of this myofiber-restricted SRF deletion on peripheral nerve regeneration, i.e. facial nerve injury was analyzed. Quantification of facial nerve regeneration by retrograde tracer transport, inspection of neuromuscular junctions (NMJs) and recovery of whisker movement revealed reduced axonal regeneration upon muscle specific Srf deletion. In contrast, responses in brainstem facial motor neuron cell bodies such as regeneration-associated gene (RAG) induction of Atf3, synaptic stripping and neuroinflammation were not overly affected by SRF deficiency. Mechanistically, SRF in myofibers appears to stimulate nerve regeneration through regulation of muscular satellite cell (SC) proliferation. In summary, our data suggest a role of muscle cells and SRF expression within muscles for regeneration of injured peripheral nerves.

Steroidogenic Factor 1 (NR5A1) Activates ATF3 Transcriptional Activity.

Steroidogenic Factor 1 (SF-1/NR5A1), an orphan nuclear receptor, is important for sexual differentiation and the development of multiple endocrine organs, as well as cell proliferation in cancer cells. Activating transcription factor 3 (ATF3) is a transcriptional repressor, and its expression is rapidly induced by DNA damage and oncogenic stimuli. Since both NR5A1 and ATF3 can regulate and cooperate with several transcription factors, we hypothesized that NR5A1 may interact with ATF3 and plays a functional role in cancer development. First, we found that NR5A1 physically interacts with ATF3. We further demonstrated that ATF3 expression is up-regulated by NR5A1. Moreover, the promoter activity of the ATF3 is activated by NR5A1 in a dose-dependent manner in several cell lines. By mapping the ATF3 promoter as well as the site-directed mutagenesis analysis, we provide evidence that NR5A1 response elements (-695 bp and -665 bp) are required for ATF3 expression by NR5A1. It is well known that the transcriptional activities of NR5A1 are modulated by post-translational modifications, such as small ubiquitin-related modifier (SUMO) modification and phosphorylation. Notably, we found that both SUMOylation and phosphorylation of NR5A1 play roles, at least in part, for NR5A1-mediated ATF3 expression. Overall, our results provide the first evidence of a novel relationship between NR5A1 and ATF3.

Benzalkonium chloride-induced direct and indirect toxicity on corneal epithelial and trigeminal neuronal cells: proinflammatory and apoptotic responses in vitro.

Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10-3% BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology.

Circ_0001742 promotes tongue squamous cell carcinoma progression via miR-431-5p/ATF3 axis.

OBJECTIVE: Circular RNAs (circRNAs) have been demonstrated to involve in the development of various cancers. This study aimed to investigate the functions of circ_0001742 on regulating tongue squamous cell carcinoma (TSCC) development and the underlying mechanisms. PATIENTS AND METHODS: The expression of circ_0001742, miR-431-5p and activating transcription factor 3 (ATF3) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of epithelial-mesenchymal transition (EMT)-related proteins and ATF3 were measured by Western blot analysis. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry assay were used to evaluate cell proliferation and apoptosis. Besides, Cell migration and invasion were assessed by transwell assay. The relationships between circ_0001742 and miR-431-5p, miR-431-5p and ATF3 were predicted by online software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), and pull-down assay. RESULTS: The expression of circ_0001742 was upregulated in TSCC tissues and cells. Knockdown of circ_0001742 inhibited proliferation, migration, invasion and EMT and induced apoptosis in TSCC cells. Then, miR-431-5p was identified as a target of circ_0001742, and knockdown of miR-431-5p reversed the effects of circ_0001742 knockdown on proliferation, apoptosis, migration, invasion and EMT of TSCC cells. Moreover, miR-431-5p could bind to ATF3, and overexpression of ATF3 rescued the effects mediated by miR-431-5p in TSCC cells. In addition, circ_0001742 regulated ATF3 expression through miR-431-5p. CONCLUSIONS: Our results demonstrated that circ_0001742 plays a tumor-promoting effect in TSCC cells by serving as a competing endogenous RNA (ceRNA) to regulate miR-431-5p/ATF3 axis, which might provide a potential therapeutic target for TSCC.

Metformin reduces c-Fos and ATF3 expression in the dorsal root ganglia and protects against oxaliplatin-induced peripheral sensory neuropathy in mice.

Oxaliplatin is a third-generation platinum drug commonly used as the first line treatment of metastatic colorectal cancer. Oxaliplatin-based anticancer regimens course with dose-limiting neurotoxicity. The pharmacological strategies used to manage such side effect are not totally effective. Metformin is an anti-diabetic drug that is described to negatively modulate painful diabetic neuropathy. Then, this study aimed to assess the effect of metformin in the oxaliplatin-induced peripheral sensory neuropathy in mice. For that purpose, Swiss male mice were injected with oxaliplatin (1, 2 or 4 mg/kg, i.v., twice a week with a total of nine injections) alone or in combination with daily administration of metformin (250 mg/kg, p.o.). Thermal and mechanical nociceptive tests were performed once a week for five weeks. Then, the animals were euthanized on day 35 post-first injection of oxaliplatin and the dorsal root ganglia were harvested for the assessment of c-Fos and ATF3 expressions. Oxaliplatin caused a nociceptive response accompanied by the increased expression of c-Fos and ATF3 in the dorsal root ganglia and spinal cord. In addition, the oxaliplatin-associated nociception was significantly attenuated by metformin (P < 0.05), which also reduced the expression of c-Fos and ATF3 (P < 0.05). Therefore, metformin protected from the peripheral sensory neuropathy induced by oxaliplatin, which was confirmed by the reduction of c-Fos and ATF3 expression, two known neuronal activation and damage markers, respectively.

Overexpression of activating transcription factor 3 exerts suppressive effects in HepG2 cells.

The present study observed and compared the biological behaviour of HepG2 cells prior and subsequent to the overexpression of activating transcription factor 3 (ATF3). Experiments investigating the cytological function by which ATF3 affects liver cancer cells were also performed. MTT, Transwell and flow cytometry assays were used to observe and detect the biological behaviour of HepG2 cells with and without lentivirus (LV)­ATF3­enhanced green fluorescent protein (EGFP) infection. The effects of ATF3 overexpression on cell proliferation, migration, apoptosis and cell cycle progression were evaluated. The LV­ATF3­EGFP overexpression vector was successfully constructed, and the HepG2 cells were successfully infected with the vector. Following ATF3 overexpression, cell proliferation was decreased, the rate of cell apoptosis was accelerated and cell cycle progression was slowed (P<0.05). There were no marked changes in cell migration (P>0.05), although there was a trend towards a gradual decrease. In conclusion, ATF3 exerted suppressive effects in HepG2 cells, potentially by inhibiting cancer cell growth, accelerating cell apoptosis, and blocking cell cycle progression. Intervention targeting ATF3 expression may represent a novel approach for the prevention and treatment of human liver cancer.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) plays an inhibitory role against inflammation induced by chemical damage to zebrafish hair cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO4, (3) 10 µM CuSO4+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO4 solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated in vivo under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO4-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO4 exposure up-regulated the expression of IL-8, IL-1ß, IL-6 and ATF3, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. Adcyap1r1a and vipr1b appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.

Sufentanil Alleviates Intrathecal Lidocaine Induced Prolonged Sensory and Motor Impairments but not the Spinal Histological Injury in Rats.

Spinal anesthesia has evolved into a safe and widely accepted method of anesthesia. Synergy between opioids and local anesthetics further increases the quality of analgesia and decreases the dose requirement of both local anesthetics and opioids. However, over the last decades compelling evidence suggested that lidocaine could be more neurotoxic than other commonly used local anesthetics. Whether opioids can modify the local anesthetics-induced neurotoxicity is largely unexplored. Here, we investigated the effect of sufentanil on the neurotoxicity induced by intrathecal lidocaine in a rat model. Our data showed that 5 µg/ml sufentanil didn't deteriorate nor reduce the histopathological injuries induced by intrathecal application of 10% lidocaine in a rat model. However, it did alleviate sensory and motor function impairments induced by 10% lidocaine. Repeated intrathecal injection of 5 µg/ml sufentanil also decreased the paw withdraw threshold compared to the baseline. An increase in expression of activating transcription factor 3, a stress response gene, as a marker for injured neurons, was also detected in lidocaine-induced neurotoxicity, while 5 µg/ml sufentanil inhibited lidocaine-induced the upregulation of activating transcription factor 3. These results suggest that sufentanil alleviates lidocaine induced sensory and motor impairments, and did not worsen histopathological injury induced by intrathecal lidocaine.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

ATF3 represses PINK1 gene transcription in lung epithelial cells to control mitochondrial homeostasis.

PINK1 (PTEN-induced putative kinase 1) is a key regulator of mitochondrial homeostasis that is relatively depleted in aging lungs and in lung epithelial cells from patients with idiopathic pulmonary fibrosis (IPF), a disease linked with aging. Impaired PINK1 expression and accumulation of damaged mitochondria in lung epithelial cells from fibrotic lungs were associated with the presence of ER stress. Here, we show that ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. An ATF3 binding site within the human PINK1 promoter is located in the first 150 bp upstream of the transcription start site. Induction of ER stress or overexpression of ATF3 inhibited the activity of the PINK1 promoter. Importantly, overexpression of ATF3 causes accumulation of depolarized mitochondria, increased production of mitochondrial ROS, and loss of cell viability. Furthermore, conditional deletion of ATF3 in type II lung epithelial cells protects mice from bleomycin-induced lung fibrosis. Finally, we observed that ATF3 expression increases in the lung with age and, specially, in lung epithelial cells from IPF lungs. These data provide a unique link between ATF3 and PINK1 expression suggesting that persistent stress, driven by ATF3, can dysregulate mitochondrial homeostasis by repression of PINK1 mRNA synthesis.

Indoxyl sulfate upregulates the cannabinoid type 1 receptor gene via an ATF3/c-Jun complex-mediated signaling pathway in the model of uremic cardiomyopathy.

BACKGROUND: The risk of cardiovascular disease is notably increased in patients with chronic kidney disease (CKD) and cannabinoid receptor type 1 (CB1R) plays an important role in the development of uremic cardiomyopathy. However, the molecular mechanism underlying the uremic toxin-induced upregulation of CB1R remains elusive. METHODS: The expression of the ATF3/c-Jun complex and CB1R in both in vivo and in vitro models of CKD were measured. We also determined the impact of the ATF3/c-Jun complex on CB1R expression by transfecting H9c2 cells with dominant negative mutants of ATF3 or c-Jun. Inhibitors of organic anion transport, specific MAPK pathways and oxidative DNA damage were also used to assess the pathways mediating the effects of indoxyl sulfate (IS). RESULTS: CB1R upregulation was associated with increased ATF3 expression and c-Jun phosphorylation in CKD both in vivo and in vitro. Expression of dominant-negative ATF3 or c-Jun mutants in IS-treated cells significantly reduced CB1R mRNA levels. Moreover, Co-IP revealed that the ATF3/c-Jun complex is formed and ChIP confirmed its binding to the CB1R promoter, suggesting that this complex directly stimulates CB1R transcription in CKD. Blocking the cellular entry of IS using an organic anion transport inhibitor, as well as inhibiting the ERK1/2 and/or JNK pathways, abrogated the effects of IS on CB1R, ATF3, and c-Jun expression. The IS-induced reactive oxygen species (ROS) was observed in the mitochondria. CONCLUSIONS: We demonstrate that uremic toxins induce ATF3/c-Jun complex-mediated CB1R expression both in vivo and in vitro, possibly by modulating the ERK1/2 and JNK signaling pathways and ROS.

Sustained cell body reactivity and loss of NeuN in a subset of axotomized bulbospinal neurons after a chronic high cervical spinal cord injury.

Following central nervous system lesion, the ability of injured axons to regrowth may depend on the level and duration of the injured cell body response (CBR). Therefore, to investigate whether axotomized brainstem neurons maintain a durable growth-competent state after spinal cord injury, we studied the effect of a chronic C2 hemisection in rats on the expression of various CBR markers involved in axon regeneration, such as c-Jun, ATF-3, HSP27, NO synthase (NOS), and also of the neural mature phenotype marker NeuN, in the bulbospinal respiratory neurons as compared to the gigantocellularis nucleus. Both at 7 and 30 days post-lesion (DPL), c-Jun and HSP27 were present in, respectively, ~60 and ~20% of the axotomized respiratory neurons, whereas the apoptotic factor caspase 3 was not detected in these cells. NOS appeared belatedly, and it was detected in ~20% of the axotomized respiratory neurons at 30DPL. At 30DPL, these different CBR markers were strongly colocalized in a sub-population of axotomized respiratory neurons and also in a sub-population of injured neurons within the gigantocellularis nucleus. Such CBR was also accompanied by a sustained alteration of the neural mature phenotype, as indicated by a loss of NeuN immunoreactivity selectively in HSP27+ bulbospinal neurons at 7DPL and 30DPL. Altogether, this study shows that a subset of axotomized medullary respiratory neurons remains in a growth-competent state after a chronic injury, suggesting that they may play a preferential role in long-lasting respiratory neuroplasticity processes.

The Effects of IGF-1 on TNF-α-Treated DRG Neurons by Modulating ATF3 and GAP-43 Expression via PI3K/Akt/S6K Signaling Pathway.

Upregulation of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) is involved in the development and progression of numerous neurological disorders. Recent reports have challenged the concept that TNF-α exhibits only deleterious effects of pro-inflammatory destruction, and have raised the awareness that it may play a beneficial role in neuronal growth and function in particular conditions, which prompts us to further investigate the role of this cytokine. Insulin-like growth factor-1 (IGF-1) is a cytokine possessing powerful neuroprotective effects in promoting neuronal survival, neuronal differentiation, neurite elongation, and neurite regeneration. The association of IGF-1 with TNF-α and the biological effects, produced by interaction of IGF-1 and TNF-α, on neuronal outgrowth status of primary sensory neurons are still to be clarified. In the present study, using an in vitro model of primary cultured rat dorsal root ganglion (DRG) neurons, we demonstrated that TNF-α challenge at different concentrations elicited diverse biological effects. Higher concentration of TNF-α (10 ng/mL) dampened neurite outgrowth, induced activating transcription factor 3 (ATF3) expression, reduced growth-associated protein 43 (GAP-43) expression, and promoted GAP-43 and ATF3 coexpression, which could be reversed by IGF-1 treatment; while lower concentration of TNF-α (1 ng/mL) promoted neurite sprouting, decreased ATF3 expression, increased GAP-43 expression, and inhibited GAP-43 and ATF3 coexpression, which could be potentiated by IGF-1 supplement. Moreover, IGF-1 administration restored the activation of Akt and p70 S6 kinase (S6K) suppressed by higher concentration of TNF-α (10 ng/mL) challenge. In contrast, lower concentration of TNF-α (1 ng/mL) had no significant effect on Akt or S6K activation, and IGF-1 administration activated these two kinases. The effects of IGF-1 were abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These data imply that IGF-1 counteracts the toxic effect of higher concentration of TNF-α, while potentiates the growth-promoting effect of lower concentration of TNF-α, with the node for TNF-α and IGF-1 interaction being the PI3K/Akt/S6K signaling pathway. This study is helpful for interpretation of the association of IGF-1 with TNF-α and the neurobiological effects elicited by interaction of IGF-1 and TNF-α in neurological disorders.

Activating transcription factor 3 is crucial for antitumor activity and to strengthen the antiviral properties of Onconase.

Onconase is a ribonuclease that presents both antitumor and antiviral properties linked to its ribonucleolytic activity and represents a new class of RNA-damaging drugs. It has reached clinical trials for the treatment of several cancers and human papilloma virus warts. Onconase targets different RNAs in the cell cytosol but Onconase-treated cells present features that are different from a simple arrest of protein synthesis. We have used microarray-derived transcriptional profiling to identify Onconase-regulated genes in two ovarian cancer cell lines (NCI/ADR-RES and OVCAR-8). RT-qPCR analyses have confirmed the microarray findings. We have identified a network of up-regulated genes implicated in different signaling pathways that may explain the cytotoxic effects exerted by Onconase. Among these genes, activating transcription factor 3 (ATF3) plays a central role in the key events triggered by Onconase in treated cancer cells that finally lead to apoptosis. This mechanism, mediated by ATF3, is cell-type independent. Up-regulation of ATF3 may also explain the antiviral properties of this ribonuclease because this factor is involved in halting viral genome replication, keeping virus latency or preventing viral oncogenesis. Finally, Onconase-regulated genes are different from those affected by nuclear-directed ribonucleases.

Lupus high-density lipoprotein induces proinflammatory responses in macrophages by binding lectin-like oxidised low-density lipoprotein receptor 1 and failing to promote activating transcription factor 3 activity.

OBJECTIVES: Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation. METHODS: Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA. RESULTS: Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation. CONCLUSIONS: Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk.

The cardiac maladaptive ATF3-dependent cross-talk between cardiomyocytes and macrophages is mediated by the IFNγ-CXCL10-CXCR3 axis.

RATIONAL: Pressure overload induces adaptive and maladaptive cardiac remodeling processes in the heart. Part of the maladaptive process is the cross-talk between cardiomyocytes and macrophages which is dependent on the function of the Activating Transcription Factor 3, ATF3. Yet, the molecular mechanism involved in cardiomyocytes-macrophages communication leading to macrophages recruitment to the heart and cardiac maladaptive remodeling is currently unknown. METHODS AND RESULTS: Isolated peritoneal macrophages from either wild type or ATF3-KO mice were cultured in serum free medium to collect conditioned medium (CM). CM was used to probe an antibody cytokine/chemokine array. The interferon γ induced protein 10kDa, CXCL10, was found to be enriched in wild type macrophages CM. Wild type cardiomyocytes treated with CXCL10 in vitro, resulted in significant increase in cell volume as compared to ATF3-KO cardiomyocytes. In vivo, pressure overload was induced by phenylephrine (PE) infusion using micro-osmotic pumps. Consistently, CXCL11 (CXCL10 competitive agonist) and CXCL10 receptor antagonist (AMG487) attenuated PE-dependent maladaptive cardiac remodeling. Significantly, we show that the expression of the CXCL10 receptor, CXCR3, is suppressed in cardiomyocytes and macrophages derived from ATF3-KO mice. CXCR3 is positively regulated by ATF3 through an ATF3 transcription response element found in its proximal promoter. Finally, mice lacking CXCR3 display a significant reduction of cardiac remodeling processes following PE infusion. CONCLUSIONS: Chronic PE infusion results in a unique cardiomyocytes-macrophages cross-talk that is mediated by IFNγ. Subsequently, macrophages that are recruited to the heart secrete CXCL10 resulting in maladaptive cardiac remodeling mediated by the CXCR3 receptor. ATF3-KO mice escape from PE-dependent maladaptive cardiac remodeling by suppressing the IFNγ-CXCL10-CXCR3 axis at multiple levels.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

Activating transcription factor 3 SUMOylation is involved in angiotensin II-induced endothelial cell inflammation and dysfunction.

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs.