关键词： 尿道下裂   氟他胺   雌二醇   生殖结节   器官培养   发病机制   调控机理   动物模型  

摘要： 尿道下裂是泌尿系统做常见的先天性发育异常。目前发病率逐年增高，具体机制尤其是分子生物学机制仍不明确。目前学者们一致认为，尿道下裂是有多因素共同作用引发的。在前期工作中，我们发现了ATF3基因在尿道下裂的发病中起到关键基因的作用。本研究主要探讨尿道下裂的关键基因—ATF3的调控机理。\n 1.尿道下裂动物模型构建:我们用不同剂量的氟他胺和雌激素给孕12.5～17.5d C57小鼠灌胃，结果显示随着雌激素剂量的增加，雄性后代出现尿道下裂的比例增加，肛门与生殖器间距离(ADG)在缩短，同时也产生死胎。氟他胺浓度50mg/kg/d灌胃时，雌激素浓度100ug/kg/d灌胃时，小鼠存活率较高，尿道下裂发生率理想，可作为理想的尿道下裂模型浓度。尿道下裂模型中，死亡胎鼠的性别都是以雄性为主。两者对雄性小鼠尿道发育有重要干扰。\n 2.生殖结节(GT)器官培养体外尿道下裂模型的构建:GT是阴茎的前体，E14.5d是小鼠胚胎前尿道管化发育的开始，该时期的雄性胚胎GT大小＜1mm3，适于体外培养，我们将之切下放于Millicell插入式器官型组织培养板微孔膜上，37℃培养72小时后发现GT在离体培养下出现了明显的生长，其形态结构出现了类似于同期体内阴茎发育的变化，在10nM DHT存在下，GT长度增加了42％，面积增加了45.6％，我们首次建立了体外尿道发育模型，在模型培养液中加入雌激100nM，尿道发育受到抑制，产生尿道下裂。可作为研究尿道下裂或阴茎发育异常等发生机制的新工具。\n 3.免疫组化检测不同时期尿道发育重要信号通路发现:TGF-β1、TGF-β RⅢ、ATF3、AR在E14、E19、尿道下裂标本中，有明显差别。从具体结果看，发生较大较明显变化的是间质。间质的病理性改变是引起尿道发育异常如尿道下裂的主要原因。因此，我们培养成功生殖结节间质细胞进一步实验。\n 4.雌激素刺激间质细胞之后，免疫荧光检测、Western Blot、RT-PCR、Realtime-PCR结果都显示:ATF3蛋白的表达、定量、ATF3mRNA都有了不同程度的表达升高，且荧光素酶分析结果显示:ATF3启动子活性明显升高。这些结果都印证了我们的假说。ATF3作用迅速，在雌激素干预后的45min即开始作用，2h后逐渐消失。定位上看位于细胞质和细胞核，以细胞质为主。从实验结果看，雌激素是通过ATF3进行调控，并引起尿道下裂的。

关键词： ATF3   肿瘤   作用机制  

摘要： ATF3转录激活因子3是转录因子ATF/CREB家族中成员之一,为一适应性反应基因参与细胞内外复杂进程。大量研究表明,其通过精细调控增殖及凋亡间平衡在肿瘤的形成和侵袭转移方面发挥重要作用,然而这种作用依赖靶基因和细胞内外环境的不同表现为双重作用,或抑制肿瘤形成或促进肿瘤发生。现就ATF3在肿瘤方面的研究进展予以综述。

关键词： Nod2   ATF3   chemokines   ribotoxic stress   intestinal epithelial cells   FUSARIUM MYCOTOXINS   NEGATIVE REGULATOR   RESPONSE GENE   IN-VITRO   DEOXYNIVALENOL   KINASE   ATF3   MACROPHAGE   EXPRESSION   STABILITY  

摘要： Chronic exposure to gut bacteria and bacterial products including Nod2 ligands triggers homeostatic regulation in response to various mucosal insults. Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokines via bacterial pattern recognition. On the assumption that ATF3 can be a critical modulator of epithelial inflammation, chronic stimulation of Nod2 was assessed for its effects on ATF3 and proinflammatory signals in response to mucosal ribotoxic insult, which is a critical etiological factor of human intestinal inflammatory disease. Muramyl dipeptide, the minimal moiety of bacterial peptidoglycan, is the Nod2 ligand, and pre-exposure to it enhanced ATF3 expression in ribotoxic stress-exposed human enterocytes. In terms of gene regulation, Nod2 preactivation potentiated ATF3 induction by enhancing stability of the ATF3 transcript, which was particularly linked to the regulation of the 3'-untranslated region of the human ATF3 gene. Moreover, chronic stimulation of Nod2 enhanced both the basal and the ribotoxic stress-stimulated cytoplasmic translocation of the HuR protein, which bound to and stabilized ATF3 messenger RNA (mRNA). Functionally, chronic stimulation of Nod2 also led to superinduction of proinflammatory chemokine genes by the mucoactive ribotoxic stress. However, the chemokine superinduction was not affected by ATF3 gene regulation although Nod2-triggered ATF3 had suppressive effects on the proinflammatory nuclear factor kappa B (NF-kappa B) signal. This paradoxical superinduction of chemokines was also mediated by enhanced mRNA stabilization by HuR protein in spite of ATF3-mediated suppression of NF-kB signal in human intestinal epithelial cells.

关键词： ACTIVATING TRANSCRIPTION FACTOR-3   OXIDATIVE STRESS   MONOCLONAL-ANTIBODY   NEGATIVE REGULATOR   GENETIC-CONTROL   PROTECTS MICE   SEPTIC SHOCK   DOUBLE-BLIND   GLUTATHIONE   INFLAMMATION  

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： JNK knockout   CIP2A   ATF3   c-Myc   Ras transformation   PROTEIN-KINASE ACTIVATION   TRANSCRIPTION FACTOR   HA-RAS   NH2-TERMINAL KINASE   GENE-EXPRESSION   RESPONSE GENE   GROWTH   PHOSPHORYLATION   ATF3   JNK  

摘要： Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is critical for ras transformation in murine embryonic fibroblasts. This cascade is coordinated by ERK and JNK2 MAPKs, whose Ras-mediated activation leads to the enhanced levels of three oncogenic transcription factors, namely, c-Myc, activating transcription factor 2 (ATF2) and ATF3, all of which are essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that counteracts protein phosphatase 2A-mediated dephosphorylation of c-Myc. Here we show that JNK2 regulates Cip2a transcription via ATF2. ATF2 and c-Myc cooperate to activate the transcription of ATF3. Remarkably, not only ectopic JNK2, but also ectopic ATF2, CIP2A, c-Myc and ATF3 are sufficient to rescue the defective ras transformation of JNK2-deficient cells. Thus, these data identify the key signal converging point of JNK2 and ERK pathways and underline the central role of CIP2A in ras transformation. Oncogene (2012) 31, 390-401; doi:10.1038/onc.2011.230; published online 27 June 2011

关键词： HDAC   bZIP   JDP2   ATF3   Histone acetylation   Repressor   N-TERMINAL KINASE   AP-1 REPRESSOR   HISTONE ACETYLATION   MAMMALIAN-CELLS   RESPONSE GENE   JDP2   DIFFERENTIATION   IDENTIFICATION   POLYUBIQUITIN   SUPPRESSES  

摘要： JDP2, is a basic leucine zipper (bZIP) protein displaying a high degree of homology with the stress inducible transcription factor, ATF3. Both proteins bind to cAMP and TPA response elements and repress transcription by multiple mechanisms. Histone deacetylases (HDACs) play a key role in gene inactivation by deacetylating lysine residues on histones. Here we describe the association of JDP2 and ATF3 with HDACs 1, 2-6 and 10. Association of HDAC3 and HDAC6 with JDP2 and ATF3 occurs via direct protein-protein interactions. Only part of the N-terminal bZIP motif of JDP2 and ATF3 basic domain is necessary and sufficient for the interaction with HDACs in a manner that is independent of coiled-coil dimerization. Class I HDACs associate with the bZIP repressors via the DAC conserved domain whereas the Class IIb HDAC6 associates through its C-terminal unique binder of ubiquitin Zn finger domain. Both JDP2 and ATF3 are known to bind and repress the ATF3 promoter. MEF cells treated with histone deacetylase inhibitor, trichostatin A (TSA) display enhanced ATF3 transcription. ATF3 enhanced transcription is significantly reduced in MEF cells lacking both ATF3 and JDP2. Collectively, we propose that the recruitment of multiple HDAC members to JDP2 and ATF3 is part of their transcription repression mechanism. (C) 2012 Elsevier B.V. All rights reserved.

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   FIBROBLASTS   EXPRESSION   FACTOR-3   GENE   CELLS   INVOLVEMENT   REPRESSION   MIGRATION   BIOLOGY  

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： CELL LUNG-CANCER   JS-K   IN-VITRO   ENDOTHELIAL-CELLS   INDUCED APOPTOSIS   DEATH PATHWAY   DNA-DAMAGE   KINASE   GENE   VIVO  

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.