关键词： SEPTICEMIA   IMMUNOSUPPRESSION   ACTIVE oxygen   GLUTATHIONE   TRANSCRIPTION factors   ENDOTOXINS  

摘要： Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)ll6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

关键词： ATF3   Mapin   MMP2   RNA干扰   胶质瘤   胶质母细胞瘤   U373MG  

摘要： 胶质母细胞瘤是神经系统最常见的高致命性恶性肿瘤,尽管目前治疗手段已经有了很多进展,其预后仍然很差。它占所有神经胶质肿瘤的60%,患者的总体生存率在过去20年中却没有发生显著变化。即使进行了积极的治疗,基本上所有的胶质母细胞瘤都会复发,最终导致死亡。肿瘤的侵袭是一种不同组织学类型的细胞恶性转化时的复杂行为,涉及一系列分子间、细胞间、细胞与胞外基质间相互作用的多机制过程,这些过程使得肿瘤细胞迁移并侵袭到周围健康脑组织中。以往的研究显示,蛋白酶、胞外基质成分、粘连细胞分子的相互作用及激活相关的多种信号通路在神经胶质瘤细胞侵袭和转移中起重要作用,每一成分都可以被视为潜在神经胶质瘤治疗靶点。近来的研究提示某些在应激状态下被激活的信号分子,在特定情况下发挥促进肿瘤细胞迁移的作用,很可能促进了胶质母细胞的侵袭。ATF3(activating transcription factor 3,转录激活因子3)是细胞应激反应中最早表达的转录因子之一,是介导细胞外信号如细胞应激、生长因子和激素等产生生物效应作用的关键调节者,是一种早期反应基因。许多研究都证实ATF3还是一种适应性反应基因,是细胞对那些扰乱机体内稳态信号的适应性反应网络的中心,它使细胞增殖加速,诱导细胞从静止期进入细胞周期,在多种恶性肿瘤组织中高表达,与肿瘤侵袭、转移及预后关系密切。例如,在乳腺癌组织[44]、恶性前列腺癌组织[45]以及何杰金氏病患者中[46]发现均ATF3高度表达。因此,其成为研究恶性肿瘤侵袭过程中的重要热点。ATF3对恶性胶质瘤侵袭作用的研究在国内外尚属空白,为探讨其在恶性胶质瘤发展侵袭过程中的作用以及其是否可能成为恶性胶质瘤治疗的靶点,本研究采用了RNA干扰和分子生物学技术对胶质瘤母细胞瘤进行了体内和体外的相关研究。实验目的:1.观察ATF3、Maspin和MMP2在人脑各级别胶质瘤中的表达情况,评价三者在m RNA和蛋白水平的表达趋势和可能的关联性,推测ATF3的表达在胶质瘤中的可能意义。2.构建ATF3-si RNA真核表达载体,观察ATF3-si RNA对人脑胶质母细胞瘤U373细胞株活性、细胞周期、凋亡和侵袭情况及Maspin和MMP2表达水平的影响,评价ATF3-si RNA在体外对U373细胞的抑制作用。3.观察ATF3-si RNA对裸鼠体内人脑胶质瘤移植瘤的抑制作用及对Maspin和MMP2表达水平的影响,评价ATF3-si RNA在体内对U373细胞的抑制作用。主要内容:第一部分:ATF3、Maspin和MMP2在人脑胶质瘤中表达的实验研究方法:1.运用免疫组织化学检测人正常脑组织和各级胶质瘤组织中ATF3、Maspin和MMP2蛋白的表达情况,观察三者在细胞中的定位和分布。2.实时运用荧光定量PCR检测人正常脑组织和各级胶质瘤组织中ATF3、Maspin和MMP2的m RNA相对表达量,评价三者在m RNA水平的表达趋势和关联性。3.以western blot检测人正常脑组织和各级胶质瘤组织中ATF3、Maspin和MMP2蛋白表达相对量,评价三者在蛋白水平的表达趋势和关联性。结果:***3和MMP2蛋白表达随胶质瘤病理分级的增高而增强,Maspin蛋白表达随胶质瘤病理分级的增高而减弱。***3和MMP2 m RNA相对表达量随胶质瘤病理分级的增高而增多,II-IV胶质瘤和正常脑组织相比,有统计学意义(P<0.05);Maspin m RNA相对表达量随胶质瘤病理分级的增高而减少,I-IV胶质瘤和正常脑组织相比,有统计学意义(P<0.05)。***3和MMP2蛋白相对表达量随胶质瘤病理分级的增高而增多,II-IV胶质瘤和正常脑组织相比,有统计学意义(P<0.05);Maspin蛋白相对表达量随胶质瘤病理分级的增高而减少,I-IV胶质瘤和正常脑组织相比,有统计学意义(P<0.05)。第二部分:ATF3-si RNA体外抑制胶质母细胞瘤的实验研究第一章U373MG细胞中ATF3 si RNA的筛选与鉴定方法:1.设计合成三对完全互补的63bp序列,两端带有Hind III和Bam H I粘性末端酶切位点,便于与p Silencer2.1 U6载体克隆连接。将ATF3-si RNA寡核苷酸片段退火,与p Silencer2.1 U6载体连接,筛选阳性菌落,构建p Silencer/si R-ATF3表达载体。2.转染人脑胶质母细胞瘤U373GM细胞株,荧光显微镜观察GFP的表达,确定最佳转染效率。3.转染48h后,提取RNA,实时荧光定量PCR检测p Silencer/si R-ATF3表达载体对ATF3表达水平的影响。4.转染72h后,收集细胞,western blot检测p Silencer/si R-ATF3表达载体对ATF3表达

关键词： 非小细胞肺癌   ATF3   突变   甲基化   风险  

摘要： 背景与目的：肺癌是世界上死亡率极高的恶性肿瘤之一，而非小细胞肺癌占据肺癌的85%。高达86%的非小细胞肺癌死亡率是由于缺乏早期诊断方法，因此，深入理解非小细胞肺癌（NSCLC）的发生发展过程和分子变异机制非常重要。活化转录因子3（Activating transcription factor3,ATF3）是ATF/CREB家族的一员，已被证实在几种癌症发病过程起着很大作用。然而，ATF3在非小细胞肺癌中表达是否异常以及其甲基化状态和基因突变情况尚未明确。本研究以正常支气管上皮细胞（HBEpiC）为对照，检测分析4株非小细胞肺癌细胞株中ATF3基因在蛋白水平表达情况;并检测ATF3基因启动子区甲基化状态及其启动子区和编码区基因突变情况。 方法：β-Actin作为内参, western blot检测ATF3基因在非小细胞肺癌细胞株（A549,NCI-H292,95C,95D）以及HBEpiC中的表达情况，Quantity1.0软件对ATF3蛋白表达作相对定量分析。亚硫酸氢钠处理后测序法（BSP）检测5株细胞以及20例NSCLC癌组织和癌旁组织中ATF3基因启动子区甲基化状态。DNA直接测序法分析ATF3启动子区1500bp的突变情况，同时使用单链构象多态性分析（single strand conformation polymorphism, SSCP）检测ATF3编码区突变情况。 结果：与相比HBEpiC,ATF3在NSCLC细胞株中蛋白表达量显著增高（P＜0.01）。Methyl Primer软件分析显示ATF3基因启动子区-1175到-1039以及-496到-288处共有27个CpG位点;且均位于转录因子结合位点之上。检测结果显示5株细胞株中27个CpG位点均未见甲基化，测序显示启动子区1500bp无遗传突变，且20例非小细胞肺癌组织中检测结果均支持细胞株检测结果。60例非小细胞肺癌组织ATF3编码区也未见遗传突变。 结论：研究显示ATF3蛋白表达在NSCLC细胞株（A549,NCI-H292,95C,95D）中比正常支气管上皮细胞显著增高，显示该基因表达上调可能导致非小细胞肺癌发生。遗传以及表观遗传分析显示ATF3基因启动子区甲基化异常或者基因突变不是ATF3蛋白表达增高的主要原因。另外，ATF3基因编码区基因突变和非小细胞肺癌发生发展没有关系。

关键词： Activating Transcription Factor 3/physiology   Blotting, Western   Blotting, Western   Cell Compartmentation   Cell Line, Tumor   Cell Proliferation   Disease Progression   Gene Expression Regulation, Neoplastic   Humans   Neoplasms/genetics   Neoplasms/metabolism   Neoplasms/pathology   Real-Time Polymerase Chain Reaction   Stromal Cells/metabolism   Transcription, Genetic/physiology  

摘要： Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.

关键词： TRANSCRIPTION factors   ANTINEOPLASTIC agents   PRODRUGS   CANCER cells   CELL culture   JNK mitogen-activated protein kinases   GENETIC regulation   DRUG activation  

摘要： Improved therapies are needed for nonsmall cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O-2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, 1) is effective against nonsmall cell lung cancer (NSCLC) cells in culture and in vivo. Here we report mechanistic studies with compound 1 and its homopiperazine analogue and structural modification of these into more stable prodrugs. Compound 1 and its., homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have antitumor activity. The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by these prodrugs.

关键词： Type 2 diabetes   ER stress   ATF3   PDX-1   beta-Cell dysfunction  

摘要： Chronic endoplasmic reticulum (ER) stress leads to p-cell failure via reduction of pancreatic and duodenal homeobox-1 (PDX-1) activity, which contributes to the pathogenesis of type 2 diabetes. However, the exact mechanisms by which ER stress reduces PDX-1 activity in pancreatic beta-cells are unclear. Previously, we showed that ATF3 downregulates PDX-1 gene expression in MIN6N8 pancreatic beta-cells. Here, we investigated another role of ATF3 on the regulation of PDX-1 activity. ATF3 significantly inhibited PDX-1-stimulated transactivation of reporter plasmid containing promoters for PDX-1 binding element and the PDX-1 target gene glucokinase, which is dependent on C-terminal domain of ATF3. ATF3 interacted with PDX-1, and effectively inhibited p300-mediated transcriptional coactivation of the PBE-containing promoter, whereas C-terminal domain-deleted ATF3 did not inhibit the transcoactivation of p300. ATF3 decreased the interaction of p300 with PDX-1 in MIN6N8 cells coexpressing PDX-1 and ATF3. In addition, chromatin immunoprecipitation analysis demonstrated that both tunicamycin treatment and ATF3 overexpression inhibited the recruitment of p300 to PDX-1 on the insulin promoter in MIN6N8 cells. Taken together, these results suggest that ATF3 inhibits PDX-1-mediated transactivation through the inhibition of p300-stimulated coactivation, which may lead to beta-cell dysfunction by ER stress. (C) 2011 Elsevier Inc. All rights reserved.

关键词： Activating Transcription Factor 3/genetics   Apoptosis/genetics   Apoptosis Regulatory Proteins/genetics   Cell Line, Tumor   DNA Damage/genetics   Gene Regulatory Networks   Humans   Male   Neoplasms/genetics   Neoplasms/metabolism   Oxidative Stress/genetics   Transcription Factors/genetics   Tumor Suppressor Protein p53/metabolism  

摘要： Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development.

关键词： 肺疾病,慢性阻塞性   活化转录因子3   活化转录因子4   红系衍生核因子相关因子2  

摘要： 目的:研究活化转录因子3(ATF3)、活化转录因子4(ATF4)和红系衍生核因子相关因子2(Nrf2)在慢性阻塞性肺疾病(COPD)患者体内的表达变化,探讨ATF3、ATF4与Nrf2蛋白之间的相互作用。方法:肺组织标本取自40例肺肿瘤行肺叶切除者,于远离病灶处取材,诊断符合COPD者入选COPD组(20例),不符合者入选对照组(20例)。取两组患者的肺组织,原位杂交和免疫组化检测ATF3、ATF4和Nrf2 mRNA及蛋白表达水平。复制COPD大鼠模型,应用免疫共沉淀法(Co-IP)研究ATF3、ATF4与Nrf2蛋白之间的相互关系。结果:(1)COPD组患者第1秒用力呼气容积/用力肺活量(FEV1/FVC)和第1秒用力呼气容积占预计值百分比(FEV1%预计值)明显低于对照组(均P<0.01)。(2)COPD大鼠肺功能(FEV0.3、FEV0.3/FVC和PEF)较对照组显著恶化(均P<0.01)。(3)免疫组化显示COPD组ATF3、ATF4和Nrf2蛋白水平较对照组升高(均P<0.01)。(4)原位杂交结果显示ATF3和ATF4 mRNA表达水平在COPD组显著高于对照组(均P<0.01),而Nrf2 mRNA在两组表达无明显差异(P>0.05)。(5)Co-IP结果显示在Nrf2抗体捕获的免疫沉淀中,ATF3和ATF4抗体均可杂交出明显的蛋白条带(P<0.05)。结论:氧化应激状况下ATF3和ATF4表达明显上调,通过与Nrf2之间的相互作用,可能转录调控Nrf2靶基因的表达,在COPD的氧化/抗氧化失衡中发挥生物学作用。

关键词： ATF3   Atlantic cod   Innate immune response   Polyriboinosinic polyribocytidylic acid (pIC)   Teleost fish  

摘要： Activating transcription factor 3 (ATF3) participates in cellular processes to adapt to various extra- and intra-cellular changes including the modulation of immunity to prevent uncontrolled immune responses to pathogens. In teleost fishes, the involvement of ATF3 in immune response has not been documented. In this study, the putative Atlantic cod (Gadus morhua) ATF3 transcript was identified by performing rapid amplification of cDNA ends (RACE) based on unknown expressed sequence tags (ESTs) that are potentially inducible by polyriboinosinic polyribocytidylic acid (pIC, a synthetic double-stranded RNA viral mimic) in Atlantic cod. ATF3-like ESTs were the most abundant unknown transcript (i.e. lacking significant BLAST hits) generated from a previously constructed cDNA library enriched for pIC inducible transcripts in Atlantic cod spleen. The full-length cDNA of cod ATF3 consists of 2329 nucleotides with an open reading frame (ORF) of 735 bp encoding 244 amino acids. The deduced amino acid sequence of Atlantic cod ATF3 shares over 45% identity with its putative orthologs from other vertebrates. In addition, the presence of a conserved basic region leucine zipper (bZIP) domain in the deduced Atlantic cod ATF3-like protein further supports its identity as an ATF3 homolog. In the spleen of Atlantic cod challenged with intraperitoneal (IP) injections of pIC, the time-course transcript expression of ATF3 was studied using quantitative reverse transcription-polymerase chain reaction (QPCR). At 6 h following the pIC injection, the relative expression level of ATF3 mRNA was significantly up-regulated in comparison to a pre-injected control (61.9-fold) and its time-matched saline-injected control (97.3-fold). At 24 h following the pIC injection, the mRNA expression level of cod ATF3 had subsided and was no longer significantly different from its pre-injected control, but significantly higher (1.88-fold) than its time-matched saline-injected control. Collectively, these resul

关键词： Pancreatic and duodenal homeobox factor-1 (PDX-1)   Activating transcription factor 3 (ATF3)   Promoter   ATF/CREB responsive element (ATF/CRE)   Hyperglycemia   Hyperlipidemia  

摘要： The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic beta-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic beta-cells, MIN6N8. Furthermore, they also repressed the activity of -4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of 0.9 Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the -903 and -702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from -759 to -738. Moreover, mutation of the putative ATF/CRE site between -752 and -745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic beta-cells function. (C) 2011 Elsevier Inc. All rights reserved.