关键词： signaling pathway   pulmonary vein-occlusion disease   collagen I   SB203580  

摘要： Pulmonary veno-occlusive disease (PVOD) is a fatal disease of pulmonary vascular lesions leading to right heart failure. Heritable PVOD (hPVOD) is related to biallelic mutation of EIF2AK4 (encoding GCN2), but its molecular mechanism remains unclear. In this study, we aimed to investigate the pathogenesis of PVOD and to find potential drug targets for PVOD. GCN2 dysfunction led to an enhanced transcription of collagen I gene (col1a1 and col1a2) through decreasing ATF3-dependent p38 phosphorylation inhibition in PVOD, which promotes the collagen I synthesis in pulmonary arterial smooth muscle cells (PASMCs) and eventually leads to increased collagen deposition in pulmonary artery. Four GCN2 knockout (KO) cell lines (exon 15 or 33 mutation) were successfully constructed by epiCRISPR system. Two induced pluripotent stem cells (iPSCs) were generated by reprogramming peripheral blood mononuclear cells (PBMCs) of PVOD patient. It was also comfirmed that GCN2 dysfunction could lead to increased expression of collagen I in lateral plate mesoderm lineage-smooth muscle cells (LM-SMCs) differentiated from both GCN2 KO cell lines and iPSCs. SB203580 (a specific inhibitor of p38) improved hemodynamics and pulmonary vascular remodeling in mitomycin C (MMC)-induced PVOD rats by right ventricle echocardiography. On the whole, we proposed that GCN2 deficiency decreased ATF3-dependent p38 phosphorylation inhibition in PVOD development and suggested a potential therapeutic reagent of SB203580 for the treatment of the disease.

关键词： ATF4   ATF3   Brefeldin A   CREB3  

摘要： Brefeldin A (BFA) disrupts the structure of the Golgi apparatus to trigger ER stress signaling pathways. On the other hand, treatment with BFA induces the activation of CREB3, the protein structure of which is similar to that of ATF6. In this study, we established Neuro2a cells in which three different transcription factors, namely, ATF4, ATF3 and CREB3, were deficient using the CRISPR/Cas9 approach, and we investigated the BFA-induced ER and Golgi stress response in these cells. BFA treatment rapidly induced ATF4, ATF3, Herp and GADD153 protein expression in Neuro2a cells. ATF4-deficient Neuro2a cells exhibited significantly decreased mRNA and protein expression of ATF3 and Herp but not GADD153;however, cells deficient in ATF3 exhibited minimal effects on GADD34, GADD153 and Herp expression. The cleavage of CREB3 in Neuro2a cells was triggered by BFA;however, the expression of several ER and Golgi stress-related factors was hardly influenced by the CREB3 deficiency in these Neuro2a cells. This study shows that CREB3 minimally associates with typical ER stress-inducible responses in Neuro2a cells. Therefore, identification and characterization of the downstream transcriptional targets of CREB3 is required to clarify not only Golgi stress response but also its relationship with ER stress signaling pathways.

关键词： Potassium usnate   Usnic acid   RNA-Seq   Endoplasmic Reticulum Stress   Apoptosis   Activating Transcription Factor 3  

摘要： Background: Potassium usnate (KU), a water-soluble form of usnic acid, shows anticancer activity. However, the underlying mechanisms have not been fully elucidated. Purpose: We aimed to identify the pathways involved in anticancer effects of KU in human gastric cancer (GC) and colorectal cancer (CRC) cells using RNA-sequencing (RNA-seq) based transcriptome analysis. Study Design: We analyzed the cytotoxic effects of KU to identify the common molecular events in GC and CRC cells upon KU exposure using unbiased approaches. Methods: Cell viability assays and western blot experiments were used to examine apoptotic changes, cell cycle arrest, and endoplasmic reticulum (ER) stress-induced cellular responses in KU-treated cells. Total RNA from KUtreated human GC and CRC cells was prepared for RNA-seq analysis. Gene ontology term and gene set enrichment analyses were used to identify the key mediators of the cytotoxic effects of KU. The expression of ER stress-induced apoptotic markers was evaluated using quantitative reverse-transcription PCR and western blot analysis. Chromatin immunoprecipitation assays for ATF3 and H3K27ac, and ATF3 knockdown were employed to verify the underlying molecular mechanisms. The inhibitory effect of KU on tumor growth in vivo was validated with metastatic tumor nodule formations in a mouse liver model. Results: KU exerted cytotoxicity in human GC and CRC cells through the activation of the ER stress-induced apoptotic pathway. KU stimulated ATF3 expression, an important mediator of molecular events of apoptosis. ATF3 binds to the promoter region of ATF3, CHOP, GADD34, GADD45A, DR5, and PUMA genes and subsequently promoted apoptotic events. Knockdown of ATF3 significantly reduced the expression of ATF3 target genes and the cytotoxic effects of KU. The intraperitoneal injection of KU induced ATF3 and the apoptosis of implanted colon cancer cells, resulting in reduced metastatic tumor growth in the mouse livers. Conclusion: KU exerts cytotoxic ef

关键词： ATF3   ATF4   serine biosynthesis   serine synthesis pathway   serine metabolism   serine deprivation   PHGDH   PSAT1   PSPH   p300  

摘要： The serine synthesis pathway (SSP) involving metabolic enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) drives intracellular serine biosynthesis and is indispensable for cancer cells to grow in serine-limiting environments. However, how SSP is regulated is not well understood. Here, we report that activating transcription factor 3 (ATF3) is crucial for transcriptional activation of SSP upon serine deprivation. ATF3 is rapidly induced by serine deprivation via a mechanism dependent on ATF4, which in turn binds to ATF4 and increases the stability of this master regulator of SSP. ATF3 also binds to the enhancers/promoters of PHGDH, PSAT1, and PSPH and recruits p300 to promote expression of these SSP genes. As a result, loss of ATF3 expression impairs serine biosynthesis and the growth of cancer cells in the serine-deprived medium or in mice fed with a serine/glycine-free diet. Interestingly, ATF3 expression positively correlates with PHGDH expression in a subset of TCGA cancer samples.

关键词： DNA breaks   ATF3   neuron   regeneration   topoisomerase  

摘要： Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.

关键词： single-cell RNA-seq   exercise   endothelial metabolism   endothelial heterogeneity   amino acid metabolism   muscle angiogenesis  

摘要： Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4(+) mECs are enriched in red oxidative muscle areas while ATF3/4(low) ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4(+) mECs are more angiogenic when compared with white ATF3/4(low) mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4(+)) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.

关键词： Cancer microenvironment   Metastasis  

摘要： The majority of long non-coding RNAs (lncRNAs) have been discovered to be overexpressed in pancreatic cancer (PC) and served as promoters in the tumorigenesis of PC, while the inhibitory functions of lncRNAs in the development of PC have not been fully elucidated yet. LncRNA microarray was adopted to analyze the differential expression of lncRNAs in PC tissues and that in normal peritumoral (NP) tissues. Functional role of lncRNA BM466146.1 on PC was evaluated by gain- and loss-of-function experiments in vivo and in vitro. RNA pull-down, RNA immunoprecipitation, luciferase reporter, and Chromatin-immunoprecipitation assays were performed to assess the mechanism of ZNFTR, respectively. The correlation between the expression of ZNFTR and various clinicopathological characteristics was accessed in PC specimens. This study displayed lncRNA BM466146.1 was downregulated in PC tissues and functioned as a suppressor through regulating the expression of adjacent gene Zinc finger protein 24 (ZNF24), which was assigned as ZNFTR. Mechanistically, ZNFTR interacted with activating transcription factor 3 (ATF3) and sequestered ATF3 away from the ZNF24 promoter, which consequently increased the expression of ZNF24. Further, ZNF24 inhibited the proliferative, metastatic, and pro-angiogenic abilities of PC cells by suppressing transcription of vascular endothelial growth factor A (VEGFA). Therefore, the downregulation of ZNFTR in PC led to the decreased expression of ZNF24, which further resulted in the upregulation of VEGFA to facilitate the development of PC. Meanwhile, ZNFTR was transcriptionally inhibited by the HIF-1 alpha/HDAC1 complex-mediated deacetylation. Clinical results further demonstrated that the low expression of ZNFTR was associated with poor overall survival time. Taken together, our results implicated that ZNFTR was a hypoxia-responsive lncRNA, and functioned as an inhibitor by modulating ATF3/ZNF24/VEGFA pathway in PC.

关键词： Anisomycin   DLD-1   RNA-seq   LAMB3   ATF3  

摘要： Anisomycin is used as a chemical compound that possesses c-Jun N-terminal kinase (JNK)-activating effects. Recently, the potent anti-tumor effects of anisomycin have received much attention. In addition to its JNKactivating effects, anisomycin has been reported to affect gene expression in osteosarcoma, leukemia, hepatocellular carcinoma, ovarian cancer and other cancers. We previously demonstrated that anisomycin induced the degradation of transcription factor GATA-6 in DLD-1 cells (a colorectal cancer cell line) and inhibited their proliferation. However, the details of the gene network involved in the process remain unclear. In this study, we conducted an RNA-seq analysis of differentially expressed genes (DEGs) in anisomycin-treated DLD-1 cells to identify the molecular process of growth-suppressive genes. We found that LAMB3, which regulates cell adhesion and migration, and NFKB2 were down-regulated by anisomycin. In addition, the mRNA expression of several tumor suppressor genes (ATF3, ERRFI1, KLF6, and AKAP12) was transiently enhanced at 3 h after anisomycin treatment. These results suggest that anisomycin blocks a PI3K/Akt-signaling cascade to lead to the suppression of cell growth.

关键词： 脑膜瘤   微小RNA-494   激活转录因子3   预后  

摘要： 目的分析微小RNA-494(miR-494)、激活转录因子3信使RNA(ATF3 mRNA)在脑膜瘤组织中的表达及其意义。方法选取2013年10月—2018年1月于首都医科大学附属北京友谊医院神经外科脑膜瘤切除患者79例为脑膜瘤组,另取因脑外伤切除脑组织患者79例为对照组。采用实时荧光定量PCR(qRT-PCR)法检测2组miR-494、ATF3 mRNA表达水平;分析脑膜瘤组织miR-494、ATF3 mRNA表达水平与脑膜瘤患者临床病理特征及预后的关系;Pearson法分析脑膜瘤组织miR-494表达水平与ATF3 mRNA的相关性;COX回归分析脑膜瘤患者预后的影响因素。结果脑膜瘤组miR-494、ATF3 mRNA表达水平均显著高于对照组(t/P=7.038/0.000、8.271/0.000)。脑膜瘤组织miR-494、ATF3 mRNA低表达亚组有淋巴结转移、WHO分级Ⅲ级、TNM分期Ⅲ~Ⅳ期患者比例低于高表达亚组(miR-494:χ^(2)/P=5.346/0.021、6.627/0.010、12.381/0.000;ATF3 mRNA:χ^(2)/P=12.868/0.000、14.799/0.000、12.166/0.000);脑膜瘤组织miR-494表达水平与ATF3 mRNA水平呈正相关(r/P=0.578/0.000);脑膜瘤患者miR-494、ATF3 mRNA低表达亚组术后36个月累积生存率分别明显高于miR-494、ATF3 mRNA高表达亚组(χ^(2)/P=21.798/0.019、13.015/0.000)。多因素分析结果显示,发生淋巴结转移、TNM分期增加及miR-494、ATF3 mRNA表达水平升高是影响脑膜瘤患者预后的独立危险因素[OR(95%CI)=2.572(1.582~4.182)、2.463(1.554~3.904)、2.257(1.478~3.447)、2.385(1.547~3.678)]。结论脑膜瘤组织中miR-494、ATF3 mRNA表达水平显著升高,两者可能相互作用共同影响脑膜瘤发生发展,检测miR-494、ATF3 mRNA水平有利于判定患者预后情况。

关键词： Ovarian   ATF3   Immune cell infiltration   Bioinformatical analysis  

摘要： 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.