关键词： Activating Transcription Factor 3/genetics   Activating Transcription Factor 3/physiology   Animals   Cells, Cultured   Chemokine CXCL1/metabolism   Immune System Diseases/genetics   Leukocyte Disorders/genetics   Lipopolysaccharides/pharmacology   Lung/cytology   Lung/immunology   Lung/metabolism   Mice   Mice, Inbred C57BL   Mice, Knockout   Mice, Knockout   Neutrophil Infiltration/genetics   Neutrophil Infiltration/genetics   Respiratory Mucosa/drug effects   Respiratory Mucosa/metabolism  

摘要： Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3(-/-) neutrophil recruitment to wild-type lungs. In vitro, ATF3(-/-) neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3(-/-) neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3(-/-) and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

关键词： HDACI   ATF3   DR5   ER stress   Agonistic anti-DR5 antibody  

摘要： Histone deacetylase inhibitors (HDACIs) are promising agents for cancer therapy. However, the mechanism(s) responsible for the efficacy of HDACIs have not yet to be fully elucidated. Death receptor 5 (DR5) is a transmembrane receptor containing death domain that triggers cell death upon binding to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or agonistic anti-DR5 monoclonal antibody, and the combination of TRAIL/agonistic anti-DR5 monoclonal antibody and agents that increase the expression of DR5 is expected as a novel anticancer therapeutic strategy. Here we report that six different HDACIs activated endoplasmic reticulum (ER) stress sensor PERK and eIF2 alpha and induced the ATF4/ATF3/CHOP pathway in p53-deficient human colon cancer cells. This resulted in an increased expression of DR5 on the cell surface and sensitized cells to apoptosis by agonistic anti-DR5 monoclonal antibody. Stress response gene ATF3 was required for efficient DR5 induction by HDACIs, and DR5 reporter assay showed that ATF3 play crucial role for the HDACIs-induced activation of DR5 gene transcription. These provide important mechanistic insight into how HDACIs exhibit pro-apoptotic activity in clinical anti-cancer treatments when they are used in combination with other therapeutic strategies. (C) 2014 Elsevier Inc. All rights reserved.

关键词： HIGH density lipoproteins   ANTI-inflammatory agents   MACROPHAGES   GENETIC transcription -- Regulation   TOLL-like receptors   CHOLESTEROL  

摘要： High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

关键词： AMYOTROPHIC lateral sclerosis   MOTOR neurons   MOTOR ability   NEURODEGENERATION   TRANSCRIPTION factors   AXONS   MICE as laboratory animals  

摘要： ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and atrophy of distal axon terminals in muscle, resulting in loss of motor function. Motor end plates denervated by axonal retraction of dying motor neurons are partially reinnervated by remaining viable motor neurons;however, this axonal sprouting is insufficient to compensate for motor neuron loss. Activating transcription factor 3 (ATF3) promotes neuronal survival and axonal growth. Here, we reveal that forced expression of ATF3 in motor neurons of transgenic SOD1(G93A) ALS mice delays neuromuscular junction denervation by inducing axonal sprouting and enhancing motor neuron viability. Maintenance of neuromuscular junction innervation during the course of the disease in ATF3/SOD1(G93A) mice is associated with a substantial delay in muscle atrophy and improved motor performance. Although disease onset and mortality are delayed, disease duration is not affected. This study shows that adaptive axonal growth-promoting mechanisms can substantially improve motor function in ALS and importantly, that augmenting viability of the motor neuron soma and maintaining functional neuromuscular junction connections are both essential elements in therapy for motor neuron disease in the SOD1(G93A) mice. Accordingly, effective protection of optimal motor neuron function requires restitution of multiple dysregulated cellular pathways.

关键词： Activating transcription factor 3 (ATF3)   Bone morphogenetic protein 2 (BMP2)   Tunicamycin   Osteoblast differentiation  

摘要： Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts. (C) 2013 Elsevier Inc. All rights reserved.

关键词： BCL2   MCL1   BCLXL   NOXA   Reactive oxygen species   ATF3  

摘要： S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.

关键词： p53   ATF3   骨肉瘤   基因治疗  

摘要： 目的：骨肉瘤(osteosarcoma)是较常见的发生在20岁以下的青少年或儿童的一种恶性骨肿瘤,在小儿骨恶性肿瘤中最多见,约为小儿肿瘤的5%。骨肉瘤起源于间叶组织,恶性程度高,侵袭性强,较早出现肺部转移,因而骨肉瘤患者的预后非常差,患者生存率很低,截肢后5年存活率仅为5-15%。现如今分子靶向治疗的出现,给骨肉瘤患者带来了新的福音。应激反应基因ATF3是P53的一个重要靶基因,在多种癌症中具有抑癌基因的作用,ATF3基因能调控细胞死亡,细胞周期。RITA (reactivation of p53and induction of tumor cell apoptosis)是一种P53靶向小分子抗癌化合物,诱导P53及其靶基因表达,在许多细胞系中导致大量细胞凋亡。本课题研究了ATF3基因在抗癌化合物RITA诱导的骨肉瘤细胞(U20S)凋亡中起到的调节作用。 方法：不同浓度及时间长度RITA刺激骨肉瘤细胞系(U20S),通过Westerblot及荧光定量PCR测定ATF3蛋白及mRNA表达水平。将ATF3沉默载体转入骨肉瘤细胞系(U20S),建立稳定细胞系,RITA处理细胞,用MTT、流氏细胞术测定细胞凋亡水平,通过Westerblot及荧光定量PCR测定ATF3下游凋亡调控基因DR5、PUMA的蛋白及mRNA水平。 结果： 1、用0.2uM RITA处理U20S细胞后,取0,2,4,8,12,24小时收取蛋白及mRNA后,用westernblot及q-RTPCR检测ATF3蛋白及mRNA水平。结果显示：第0小时(即空白对照组)ATF3蛋白及mRNA表达水平极低,从第2小时开始ATF3蛋白及mRNA水平显著升高,而且随着时间延长ATF3蛋白及mRNA水平也随之增高,说明RITA对ATF3蛋白及mRNA水平的诱导具有时间依赖性。选取OuM,0.05uM,0.1uM,0.2uM,0.5uM,1uM RITA处理U20S细胞24小时后收取蛋白及mRNA,用westernblot及q-RTPCR检测ATF3蛋白及mRNA水平。结果显示：未用RITA处理的细胞(即空白对照组)ATF3蛋白及mRNA表达水平极低,而随着RITA浓度增加,ATF3蛋白及mRNA水平显著升高,说明RITA对ATF3蛋白及mRNA水平的诱导具有浓度依赖性。 2、 ATF3siRNA转染U20S细胞系,分为：空白对照组,空白对照组+RITA, siATF3组,siATF3+RITA组,0.2uM RITA诱导时间为24小时,westernblot结果显示ATF3siRNA转染U20S细胞后,对比空白对照组及空白对照组+RITA组,siATF3组及siATF3+RITA组均很难检测到ATF3蛋白表达。 3、用不同浓度RITA处理空白对照组及两个ATF3沉默组,MTT结果显示,RITA的不同浓度在固定的时间(72h)对骨肉瘤细胞的抑制作用存在差异,抑制作用随浓度的增大而增加；在同一浓度中,ATF3沉默组细胞较空白对照组对RITA的抵抗力显著增强。 4、流式细胞术测定细胞凋亡siATF3组对比空白对照组,细胞凋亡率差别无统计学意义,siATF3+RITA组对比空白对照组+RITA组,细胞凋亡率显著降低。 5、westernblot结果显示：siATF3组对比空白对照组,DR5、PUMA蛋白水平均显著降低,siATF3+RITA组对比空白对照组+RITA组,DR5、PUMA蛋白水平也显著降低。q-RTPCR结果显示：siATF3组对比空白对照组,DR5、PUMA mRNA水平无明显差异；siATF3+RITA组对比空白对照组·+RITA组,DR5、PUMA mRNA水平显著降低。 结论： 1、RITA诱导ATF3基因在骨肉瘤细胞U2OS中高表达。 2、骨肉瘤细胞系U2OS中ATF3沉默后RITA无法诱导ATF3蛋白表达。 3、ATF3基因沉默后骨肉瘤细胞U2OS对RITA抵抗力增强。 4、ATF3基因沉默后骨肉瘤细胞U2OS对RITA抵抗力增强是通过调控下游凋亡基因实现的。

关键词： 组蛋白去乙酰化酶抑制剂   ATF3   死亡受体5   内质网应激   抗DR5竞争性抗体  

摘要： 组蛋白去乙酰化酶抑制剂（Histone deacetylase inhibitors, HDACIs）是一类极具潜力的抗癌治疗药物，但它们发挥作用的机制，迄今为止仍未得到充分完整的阐述。死亡受体5（DR5）是一种包含死亡结构域的膜受体，这类受体能够与Apo2L/TRAIL或者抗DR5单克隆竞争性抗体结合，促进细胞的凋亡。目前，将TRAIL或者抗DR5单克隆竞争性抗体与其它能够增加DR5表达的药物联合应用治疗癌症，被期望能成为一种新的有效的抗癌治疗手段，已经成为研究的热点。在本研究中，我们首次提出6种结构不同的HDACIs都能刺激p53突变的人结肠癌细胞发生内质网应激，激活非折叠蛋白反应的PERK-eIF2α路径，进而诱导ATF4/ATF3/CHOP通路，最终上调细胞表面DR5的表达同时增加细胞对抗DR5竞争性单克隆抗体诱导凋亡的敏感性；应激反应因子ATF3在HDACIs有效诱导DR5表达和细胞死亡过程中扮演重要的功能性角色；并且通过荧光素酶报告基因分析实验我们证实了ATF3和CHOP在HDACIs激活DR5基因转录的过程中扮演重要的功能性角色。这是HDACIs机制研究的又一进展，将为未来临床上将HDACIs单独或与其它药物联合应用治疗癌症提供新的重要理论依据。

关键词： 骨肉瘤   ATF3基因   基因治疗   细胞凋亡   P53蛋白  

摘要： 目的:骨肉瘤(osteosarcoma)是较常见的发生在20岁以下的青少年或儿童的一种恶性骨肿瘤，在小儿骨恶性肿瘤中最多见，约为小儿肿瘤的5％。骨肉瘤起源于间叶组织，恶性程度高，侵袭性强，较早出现肺部转移，因而骨肉瘤患者的预后非常差，患者生存率很低，截肢后5年存活率仅为5-15％。现如今分子靶向治疗的出现，给骨肉瘤患者带来了新的福音。应激反应基因ATF3是P53的一个重要靶基因，在多种癌症中具有抑癌基因的作用，ATF3基因能调控细胞死亡，细胞周期。RITA(reactivation of p53 and induction of tumor cell apoptosis)是一种P53靶向小分子抗癌化合物，诱导P53及其靶基因表达，在许多细胞系中导致大量细胞凋亡。本课题研究了ATF3基因在抗癌化合物RITA诱导的骨肉瘤细胞(U2OS)凋亡中起到的调节作用。　　方法:不同浓度及时间长度RITA刺激骨肉瘤细胞系(U2OS)，通过Westerblot及荧光定量PCR测定ATF3蛋白及mRNA表达水平。将ATF3沉默载体转入骨肉瘤细胞系(U2OS)，建立稳定细胞系，RITA处理细胞，用MTT、流氏细胞术测定细胞凋亡水平，通过Westerblot及荧光定量PCR测定ATF3下游凋亡调控基因DR5、PUMA的蛋白及mRNA水平。　　结果:　　1、用0.2uM RITA处理U2OS细胞后，取0，2，4，8，12，24小时收取蛋白及mRNA后，用westernblot及q-RTPCR检测ATF3蛋白及mRNA水平。结果显示:第0小时（即空白对照组）ATF3蛋白及mRNA表达水平极低，从第2小时开始ATF3蛋白及mRNA水平显著升高，而且随着时间延长ATF3蛋白及mRNA水平也随之增高，说明RITA对ATF3蛋白及mRNA水平的诱导具有时间依赖性。选取0uM，0.05uM，0.1uM，0.2uM，0.5uM，1uM RITA处理U2OS细胞24小时后收取蛋白及mRNA，用westernblot及q-RTPCR检测ATF3蛋白及mRNA水平。结果显示:未用RITA处理的细胞（即空白对照组）ATF3蛋白及mRNA表达水平极低，而随着RITA浓度增加，ATF3蛋白及mRNA水平显著升高，说明RITA对ATF3蛋白及mRNA水平的诱导具有浓度依赖性。　　2、ATF3 siRNA转染U2OS细胞系，分为:空白对照组，空白对照组+RITA，siATF3组，siATF3+RITA组，0.2uM RITA诱导时间为24小时，westernblot结果显示ATF3 siRNA转染U2OS细胞后，对比空白对照组及空白对照组+RITA组，siATF3组及siATF3+RITA组均很难检测到ATF3蛋白表达。　　3、用不同浓度RITA处理空白对照组及两个ATF3沉默组，MTT结果显示，RITA的不同浓度在固定的时间(72h)对骨肉瘤细胞的抑制作用存在差异，抑制作用随浓度的增大而增加;在同一浓度中，ATF3沉默组细胞较空白对照组对RITA的抵抗力显著增强。　　4、流式细胞术测定细胞凋亡siATF3组对比空白对照组，细胞凋亡率差别无统计学意义，siATF3+RITA组对比空白对照组+RITA组，细胞凋亡率显著降低。　　5、westernblot结果显示:siATF3组对比空白对照组，DR5、PUMA蛋白水平均显著降低，siATF3+RITA组对比空白对照组+RITA组，DR5、PUMA蛋白水平也显著降低。q-RTPCR结果显示:siATF3组对比空白对照组，DR5、PUMA mRNA水平无明显差异;siATF3+RITA组对比空白对照组+RITA组，DR5、PUMA mRNA水平显著降低。　　结论:　　1、RITA诱导ATF3基因在骨肉瘤细胞U2OS中高表达。　　2、骨肉瘤细胞系U2OS中ATF3沉默后RITA无法诱导ATF3蛋白表达。　　3、ATF3基因沉默后骨肉瘤细胞U2OS对RITA抵抗力增强。　　4、ATF3基因沉默后骨肉瘤细胞U2OS对RITA抵抗力增强是通过调控下游凋亡基因实现的。

关键词： HCT116   ATF3   慢病毒过表达   增殖   肿瘤起始能力   迁移侵袭   EMT   Carfilzomib  

摘要： 【目的】全球范围内，结直肠癌致死位居肿瘤死因第四位，尽管如今结直肠癌的诊断以及治疗水平有所提高，其5年生存率也只有53.8-65.2%。ATF3蛋白属于ATF/CREB转录因子家族成员具有碱性亮氨酸拉链结构（bZip）,作为一种转录活化因子在肿瘤中起着致癌基因还是抑癌基因作用存在争论。本研究通过慢病毒介导ATF3在结直肠癌细胞HCT116中过表达，探讨ATF3在HCT116细胞中对其增殖、肿瘤起始能力、转移侵袭以及上皮间质转化（EMT）生物学作用，并初步探讨可能的分子机制,探讨蛋白酶体抑制剂Carfilzomib对HCT116的细胞毒作用以及对ATF3的调节。 【方法】构建慢病毒介导的ATF3表达载体并感染HCT116，puro筛选出稳定ATF3过表达的HCT116细胞株HCT116/ATF3和阴性对照HCT116/Control。应用MTS细胞活性分析、FACS细胞周期检测、FACS AnexinⅤ-7-AAD、克隆形成实验来检测细胞增殖能力；应用细胞球囊形成实验以及FACS检测细胞肿瘤起始能力；应用划痕实验、Transwell转移分析和Transwell侵袭分析来检测细胞转移侵袭能力；应用光学显微镜观察细胞形态变化鉴定细胞EMT；用Western Bloting和real-time qPCR来检测相关基因的mRNA及蛋白表达情况。 【结果】MTS细胞活性检测增殖能力以及克隆形成实验克隆数在HCT116/ATF3细胞明显低于对照细胞HCT116/Contro（lp<0.05）；real-time qPCR和Western Blot检测到HCT116/ATF3细胞survivin、cyclinD1、PCNA表达下调以及PARP表达上调。HCT116/ATF3细胞形成的细胞球囊平均直径和数量以及CD133+CD24+细胞群的百分比明显低于对照细胞（p<0.05）；qRT-PCR和Western Bloting检测到HCT116/ATF3细胞CD133、 CD24、Ep-CAM、 CD166、 ID1、beta-catenin和EZH2表达下调。HCT116/ATF3细胞创伤愈合划痕实验创伤愈合程度、Transwell迁移分析与Transwell侵袭实验的细胞穿过数明显低于对照细胞（p<0.05）；real-time qPCR和Western Blot检测到HCT116/ATF3细胞MMP2和MMP9表达下调以及TIMP2和TIMP1表达上调。光学显微镜观察细胞形态HCT116/ATF3细胞形态由间质细胞向上皮细胞转化（MET）；Western Blot检测到HCT116/ATF3细胞E-cadherin表达上调和snail、slug和twist表达下调。通过MTS细胞活性检测HCT116生长随Carfilzomib剂量增加而受到抑制，细胞周期检测Carfilzomib作用后HCT116主要停滞在G0/G1期；FACS AnexinⅤ-7-AAD检测HCT116凋亡随Carfilzomib剂量增加而增加。通过real-time qPCR和Western Blot检测到Carfilzomib诱导ATF3表达具有时间依赖性。 【结论】应用慢病毒介导基因转染成功构建ATF3稳定表达HCT116细胞模型HCT116/ATF3和对照细胞HCT116/Control；ATF3通过下调survivin、cyclinD1、PCNA以及上调PARP来抑制HCT116细胞增殖； ATF3通过下调CD133、 CD24、Ep-CAM、 CD166、 ID1、beta-catenin和EZH2来抑制HCT116肿瘤起始能力；ATF3通过下调MMP2和MMP9以及上调TIMP1和TIMP2来抑制HCT116细胞迁移侵袭能力； ATF3通过上调E-cadherin和下调snail、slug和twist逆转HCT116EMT；蛋白酶体抑制剂Carfilzomib可上调HCT116中ATF3的表达并抑制HCT116细胞生长。