关键词： Mycobacterium tuberculosis infection   Alveolar epithelial type II cells   ATF3   TIMP2   NF-kappa B   Autophagy  

摘要： Activating transcription factor 3 (ATF3) is a stress-inducible gene reported with anti-inflammatory response effects against bacterial infections. This study focuses on the function of ATF3 in alveolar epithelial type II cells (A549) following Mycobacterium tuberculosis (MTB) infection. First, RT-qPCR results detected reduced ATF3 expression in broncho-alveolar lavage fluid (BALF) of MTB-infected patients, whereas the ATF3 level was upregulated in A549 cells at early stages after MTB infection but decreased later. The binding relationship between ATF3 and TIMP metallopeptidase inhibitor 2 (TIMP2) promoter was predicted via bioinformatic prediction and validated by ChIP and luciferase assays. ATF3 bound to TIMP2 promoter for transcriptional activation. Overexpression of ATF3 or TIMP2 enhanced autophagy activity, elevated p62 levels and the LC3BII/LC3BI ratio, and decreased IL-6 and TNF-alpha levels in A549 cells. The ATF3/TIMP2 axis suppressed the NF-kappa B pathway to alleviate inflammatory responses in A549 cells. Mice were exposed to MTB aerosol for in vivo experiments. Increased ATF3 expression was correlated with increased autophagy activity, clearance of bacteria as well as inflammation resolution in mouse lung tissues. In conclusion, this study demonstrates that ATF3 promotes cell autophagy and suppresses inflammatory response in MTB-infected A549 cells via TIMP2 activation and NF-kappa B suppression.

关键词： 肺动脉高压   肺动脉平滑肌细胞   自噬   活化转录因子3   热休克蛋白110  

摘要： 目的探讨ATF3调控HSP110表达对低氧刺激下人肺动脉平滑肌细胞增殖及自噬的影响。方法体外培养人肺动脉平滑肌细胞,单独感染ATF3沉默腺病毒或共感染HSP110过表达腺病毒,48 h后加入自噬激活剂雷帕霉素预处理3 h,建立缺氧细胞模型,24 h后,CCK-8法检测细胞增殖能力,流式细胞术分析细胞凋亡,Real-time PCR检测ATF3、HSP110 mRNA的表达,Western blot检测ATF3、HSP110、LC3Ⅱ/Ⅰ、Beclin-1、p62蛋白的表达,免疫荧光观察LC3斑点形成,荧光素酶报告系统鉴定ATF3对HSP110的调控作用。结果缺氧组ATF3和HSP110的表达显著增加(P<0.01),ATF3沉默后,细胞增殖能力下降,凋亡率增加(P<0.01),同时自噬蛋白LC3Ⅱ/Ⅰ、Beclin-1的表达及LC3荧光斑点显著减少(P<0.01),自噬降解底物p62的表达明显增加(P<0.01),而加入雷帕霉素或共感染HSP110过表达腺病毒后,ATF3沉默对细胞增殖和自噬的抑制作用显著减弱(P<0.01)。双荧光素酶报告基因检测结果显示,ATF3可上调HSP110启动子活性。结论ATF3调控HSP110表达增加而促进人肺动脉平滑肌细胞增殖,其机制可能与介导细胞自噬有关。

关键词： 通关藤注射液   乳腺癌   MDA-MB-231细胞   紫杉醇   ATF3   CCL2   TNF-α  

摘要： 目的 探讨ATF3介导的通关藤注射液增强紫杉醇抗乳腺癌的作用机制。方法 体外培养人乳腺癌MDA-MB-231细胞,siRNA干扰ATF3的表达,给予通关藤注射液和紫杉醇单独或联合干预。采用RT-qPCR和Western blot法分别检测ATF3及相关细胞因子CCL2、TNF-α的表达,细胞划痕实验和Transwell实验检测细胞迁移和侵袭能力,Hoechst活细胞染色和流式细胞术检测细胞凋亡情况。结果 在ATF3-siRNA干扰的MDA-MB-231细胞中,紫杉醇可上调ATF3 mRNA和蛋白表达及细胞因子CCL2、TNF-α mRNA表达(P<0.05);而通关藤注射液有抵消紫杉醇引起的ATF3 mRNA和蛋白表达升高的趋势,可抵消细胞因子CCL2、TNF-α mRNA表达的上调(P<0.01)。通关藤注射液可抑制MDA-MB-231细胞的增殖和转移、诱导细胞凋亡,并且联合紫杉醇组的作用更为显著(P<0.01)。结论 通关藤注射液联合紫杉醇可能通过调控ATF3及相关细胞因子的表达,抑制乳腺癌细胞的增殖、转移、侵袭,促进乳腺癌细胞的凋亡,从而发挥增效作用。

关键词： ATF3   NIHL   NRF2/HO-1 pathway   NRF2   Oxidation  

摘要： Background: Excessive oxidative stress of the inner ear as a result of high, intense noise exposure is regarded as a major mechanism underlying the development of noise-induced hearing loss (NIHL). The present study was designed to explore the effect and mechanism of activated transcription factor 3 (ATF3) in reduction/oxidation homeostasis of NIHL.& nbsp;Method: In vitro and in vivo assays were performed to investigate the functional role of ATF3 in the inner ear. Mice hearing was measured using auditory brainstem response. ATF3 short hairpin RNA (shRNA) was transfected into House Ear Institute-Organ of Corti 1 (HEI-OC1) cells to decrease ATF3 expression. Western blotting and quan-titative real-time polymerase chain reaction (RT-qPCR) were performed to quantify ATF3, NRF2, HO-1 and NQO1 expression. Glutathione (GSH) assay was performed to detect intracellular GSH levels. ATF3 immuno-fluorescence analysis was carried out in cochlear cryosectioned samples and HEI-OC1 cells to localize ATF3 expression. Cell counting kit 8 assay and flow cytometry were performed to analyze cell viability.& nbsp;Result: ATF3 was upregulated in noise-exposed cochleae and HEI-OC1 cells treated with H2O2. NRF2 is a key factor regulated by ATF3. NRF2, HO-1, NQO1, and GSH expression was significantly downregulated in shATF3 HEI-OC1 cells. ATF3 silencing promoted reactive oxygen species accumulation and increased apoptosis and necrosis with H2O2 ***: ATF3 functions as an antioxidative factor by activating the NRF2/HO-1 pathway.

关键词： Exercise   Insulin resistance   IRF2bp2, CRLS1, ATF3  

摘要： Purpose: Exercise is an effective way to alleviate insulin resistance (IR). However, the underlying mechanisms remain to be elucidated. Previous studies demonstrated that cardiolipin synthase 1 (CRLS1)/interferon-regulatory factor-2 binding protein 2 (IRF2bp2)-activating transcription factor 3 (ATF3)-adiponectin receptor 2 (AdipoR2)-adaptor protein containing pH domain, PTB domain and leucine zipper motif 1 (APPL1)-protein kinase B (AKT/PKB)-related signaling was closely associated with obesity-induced IR-related diseases, but the correlation between exercise training alleviating obesity-induced IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rats is unknown. Therefore, We want to investigate the effect of exercise training on IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rat. Methods: The male healthy Sprague-Dawley rats were divided into four groups: normal control group (NCG, n = 10), diet-induced obesity-sedentary group (DIO-SG, n = 10), diet-induced obesity-chronic exercise group (DIO-CEG, n = 10) received chronic swim exercise training and diet-induced obesity-acute exercise group (DIO-AEG, n = 10) received acute swim exercise training. We measured the levels of IR-related indicators and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in NCG, DIO-SG, DIO - CEG and DIO-AEG. Results: We found that high-fat diet (HFD)-induced obesity decreased insulin sensitivity in rats accompanied by decreased protein levels of hepatic CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT and increased protein level of hepatic ATF3. The acute exercise and the chronic exercise both increased insulin sensitivity in rats. The chronic exercise decreased hepatic ATF3 protein level and increased CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT protein levels in HFD-fed rats. The acute exercise decreased hepatic ATF3 protein level and increased hepatic IRF2bp2, APPL1 and p-AKT pro

关键词： apoptosis   ATF3   ferroptosis   nanoparticle   nickel oxide  

摘要： 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.

关键词： Sepsis-induced ALI   Ferroptosis   Ubiquitination   FBXW7   AUF1   NRF2   ATF3  

摘要： Background The AU-rich element (ARE)-binding factor 1 (AUF1) acts as a switch for septic shock, although its underlying mechanisms remain largely unknown. In this study, we examined the biological significance and potential molecular mechanism of AUF1 in regulating ferroptosis in sepsis-induced acute lung injury (ALI). Methods Alveolar epithelial cells (AECs) challenged with ferroptosis-inducing compounds and cecum ligation and puncture (CLP)-induced ALI were used as the in vitro and in vivo model, respectively. The stability of AUF1 and its degradation by ubiquitin-proteasome pathway were examined by cycloheximide chase analysis and co-immunoprecipitation assay. The regulation of AUF1 on nuclear factor E2-related factor 2 (NRF2) and activation transcription factor 3 (ATF3) was explored by RNA immunoprecipitation (RIP), RNA pull-down, and mRNA stability assays. Functionally, the effects of altering AUF1, NRF2 or ATF3 on ferroptosis in AECs or ALI mice were evaluated by measuring cell viability, lipid peroxidation, iron accumulation, and total glutathione level. Results AUF1 was down-regulated in AECs challenged with ferroptosis-inducing compounds, both on mRNA and protein levels. The E3 ubiquitin ligase FBXW7 was responsible for protein degradation of AUF1 during ferroptosis. By up-regulating NRF2 and down-regulating ATF3, AUF1 antagonized ferroptosis in AECs in vitro. In the CLP-induced ALI model, the survival rate of AUF1 knockout mice was significantly reduced and the lung injuries were aggravated, which were related to the enhancement of lung ferroptosis. Conclusions FBXW7 mediates the ubiquitination and degradation of AUF1 in ferroptosis. AUF1 antagonizes ferroptosis by regulating NRF2 and ATF3 oppositely. Activating AUF1 pathway may be beneficial to the treatment of sepsis-induced ALI.

关键词： Leucine-rich-alpha-2-glycoprotein 1 (LRG1)   Gastric cancer (GC)   Tumor angiogenesis   Activating transcription factor 3 (ATF3)   SRC   STAT3   VEGFA pathway  

摘要： Background Increasing evidence indicates that leucine-rich-alpha-2-glycoprotein 1 (LRG1) is associated with multiple malignancies, but whether it participates in gastric cancer (GC) angiogenesis remains unclear. Methods The expression levels of LRG1 were assessed in GC samples. Endothelial tube formation analysis, HUVEC migration assay, chorioallantoic membrane assay (CAM), and xenograft tumor model were used to investigate the effect of LRG1 on angiogenesis in gastric cancer. The involvement of activating transcription factor 3 (ATF3) was analyzed by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Western blot and enzyme-linked immunosorbent assay were performed to measure the SRC/STAT3/VEGFA pathway. Results LRG1 was overexpressed in GC tissues and associated with cancer angiogenesis. In addition, LRG1 markedly promoted GC cell proliferation in vitro and in vivo. Moreover, overexpression of LRG1 could stimulate GC angiogenesis in vitro and in vivo. Then, we identified ATF3 promotes the transcription of LRG1 and is a positive regulator of angiogenesis. Additionally, LRG1 could activate VEGFA expression via the SRC/STAT3/ VEGFA pathway in GC cells, thus contributing to the angiogenesis of GC. Conclusions The present study suggests LRG1 plays a crucial role in the regulation of angiogenesis in GC and could be a potential therapeutic target for GC.

关键词： Cell death  

摘要： Intervertebral disc degeneration (IDD), being the predominant root cause of lower back pain, has led to an enormous socioeconomic burden in the world. Ferroptosis is an iron-dependent nonapoptotic and nonpyroptotic programmed cell death associated with an increase in reactive oxygen species (ROS), which has been implicated in the pathogenesis of IDD. Activation transcription factor 3 (ATF3) is widely reported to promote ferroptosis and apoptosis in multiple diseases, but its roles and underlying regulatory mechanism in IDD have not been identified. FAoptosis is defined as a mixed cell death consisting of ferroptosis and apoptosis. The loss- and gain-of-function experiments demonstrated that ATF3 positively regulated tert-butyl hydroperoxide- (TBHP-) induced nucleus pulposus cell (NPC) FAoptosis, ROS production, inflammatory response, and extracellular matrix (ECM) degradation. Furthermore, silencing ATF3 ameliorated the progression of IDD in vivo, whereas its overexpression showed the opposite phenotype. Bioinformatics analysis and molecular experiments corroborated that ATF3 is a direct target of miR-874-3p, suggesting that the upregulation of ATF3 in IDD might be caused at least in part due to the downregulation of miR-874-3p in IDD, thereby relieving the inhibition of ATF3 by miR-874-3p. The findings revealed that ATF3 has the potential to be used as a promising therapeutic target against IDD.

关键词： ATF3   视神经损伤   视网膜   神经节细胞  

摘要： 目的探讨转录激活因子3(ATF3)表达对视神经损伤后大鼠视网膜神经节细胞存活的影响。方法成年雌性SD大鼠65只,随机选取10只大鼠作为对照组,剩余55只建立大鼠视神经损伤模型作为视神经损伤组。采用实时荧光定量PCR检测两组视网膜中ATF3 mRNA的表达;对照组大鼠、视神经损伤组于感染后3,7,14,35 d采用免疫组织化学检测ATF3的表达,免疫荧光组织化学检测胶质原纤维酸性蛋白(GFAP)的表达。取40只视神经损伤大鼠随机分为4组:NC组、ATF3过表达质粒(pIRES2/ATF3)组、阴性对照(shNC)组和ATF3-shRNA组,各组大鼠分别腹腔注射生理盐水、ATF3过表达质粒转染的复合物、shNC的复合物或ATF3-shRNA表达质粒的复合物,然后感染后0,7,14,35 d,采用OCT扫描测量视网膜神经纤维层(RNFL)厚度,CCK-8法检测视网膜神经节细胞存活情况,流式细胞术检测视网膜神经节细胞凋亡情况,免疫印迹法检测Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白的表达。结果视神经损伤组ATF3 mRNA明显高于对照组(P<0.05)。在感染后3,7,14,35 d,视神经损伤组大鼠ATF3和GFAP的表达逐渐增加,并且明显高于对照组(P<0.05)。与NC组比较,在感染后14,35 d,pIRES2/ATF3组RNFL平均厚度显著减少(P<0.05),视网膜神经节细胞存活率降低(P<0.05),pIRES2/ATF3组细胞凋亡率、Bax蛋白水平升高,Bcl-2蛋白水平降低(P<0.05)。与shNC组比较,在感染后14,35 d,ATF3-shRNA组RNFL厚度明显增加,视网膜神经节细胞存活率升高(P<0.05),细胞凋亡率、Bax蛋白水平降低,Bcl-2蛋白水平升高(均P<0.05)。结论ATF3在视神经损伤大鼠视网膜中的表达升高,ATF3能够调控视神经损伤后大鼠视网膜神经节细胞存活和凋亡能力。