关键词： PGF   CL   P-4   ATF3   MAPK   Rat  

摘要： The current study was conducted to evaluate the expression of ATF3, in association with the activation of mitogen-activated protein kinases (MAPK) during prostaglandin F-2 alpha analog (PGF)-induced luteal regression in rats. A sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpora lutea (CL) in rats. Rats were treated with PGF for 0-4 h on day 7 of pseudopregnancy. Results showed that serum progesterone (P-4) concentrations declined in a time dependent manner. Western blot results revealed that ATF3 increased within 2 h post-PGF injection. Phosphorylated ERK1/2 (p-ERK) and JNK (p-JNK) increased within 30 min and then were gradually reduced in response to PGF. In contrast, the levels of phosphorylated p38 MAPK (p-p38) were not significantly altered. The immunostaining density for p-ERK decreased from the periphery to the center of the corpus luteum following treatment with PGF, while ATF3 was expressed uniformly in the nuclei of luteal steroidogenic cells. These results indicated that treatment with PGF in vivo could induce increases in MAPK phosphorylation, especially in p-ERK, which might be correlated with the increases in ATF3 expression and the decline in P-4 concentrations. To our knowledge, this is the first study to provide evidence for temporal relationships between MAPK activation and ATF3 expression during PGF-induced luteal regression in the rat. (C) 2015 Elsevier GmbH. All rights reserved.

关键词： 子宫内膜癌   浆液性癌   突变型P53   预后   子宫内膜癌   浆液性癌   激活转录因子3   突变型P53   预后   子宫内膜癌   浆液性癌   激活转录因子3   SPEC-2细胞   增殖  

摘要： 背景子宫内膜癌是子宫内膜上皮来源的恶性肿瘤,属于女性生殖道三大恶性肿瘤之一,好发于老年妇女。随着人口老龄化、饮食结构和生活方式的改善,其发病率及死亡率在全球范围内呈现上升趋势,且发病年龄趋于年轻化。在欧美国家,子宫内膜癌发病率位于妇科恶性肿瘤首位,在我国位居第二位,但是近年来在北京、上海等发达城市其发病率已超过宫颈癌而位居妇科恶性肿瘤首位。其中浆液性癌是子宫内膜癌中恶性程度较高的一种病理类型,而关于子宫内膜浆液性癌的大宗病例研究少见报道。系统掌握子宫内膜浆液性癌的临床病理学特征、诊断及鉴别诊断,提高对此肿瘤生物学行为的认识,对于妇科、肿瘤科及病理科医师具有重要意义。目的探讨子宫内膜浆液性癌临床病理学特征、诊断、鉴别诊断及预后,进一步提高对该肿瘤的认识。方法回顾性分析烟台毓璜顶医院2005年1月至2013年12月期间确诊为子宫内膜浆液性癌患者的临床病理资料,患者均接受广泛子宫及双侧附件切除术加盆腔淋巴结清扫,术前均未做任何治疗,术后经组织病理学诊断为子宫内膜浆液性癌。对肿瘤组织进行雌激素受体、孕激素受体、突变型P53的免疫组织化学染色,统计分析雌激素受体、孕激素受体、突变型P53表达与临床病理学参数及预后情况的关系。计数资料采用x2检验,P<0.05为差异具有统计学意义。结果1.本组33例子宫内膜浆液性癌占同期子宫内膜癌的比例为5.02%(33/672)。患者发病年龄44-73岁,中位年龄64岁。93.94%患者为绝经后女性(31/33)。肿瘤最大直径1-12cm,平均直径3.87cm。影像学检查以宫腔内占位多见(16/33,48.48%),其次表现为内膜不规则增厚(10/33,30.3%),仅少数患者表现为宫腔积液(7/33,21.21%)。临床症状多表现为绝经后阴道流血或流液(29/33,87.88%),3例患者表现为下腹疼痛或不适(3/33,9.09%),1例表现为外阴脱出肿物伴排尿困难(1/33,3.03%)。14例发生淋巴结转移(14/33,42.42%)；19例患者肿瘤浸润深度<1/2肌层(19/33,57.58%)；15例临床分期Ⅰ-Ⅱ期(15/33,45.45%),18例Ⅲ-Ⅳ期(18/33,54.55%)。其中浸润深度<1/2肌层的19例患者中有7例患者出现淋巴结转移(7/19,36.84%)。本组33例患者均获得随访,随访时间2-64个月,平均26.67个月,其中16例(16/33,48.48%)存活,17例(17/33,51.52%)患者死于本病。2.本组病例共同的组织学特点是肿瘤细胞呈复杂分支状的乳头结构、实性巢状或腺管状排列,乳头结构中央为纤维脉管轴心；肿瘤细胞体积大,边界不清,胞浆嗜酸性或粉染,核大小不一、形态不规则,扭曲分叶,染色质粗颗粒状,核仁显著嗜酸性,核分裂相易见。18例患者(18/33,54.54%)肿瘤组织内可见坏死；7例患者(7/33,24.24%)肿瘤组织内见砂粒体形成。3.子宫内膜浆液性癌中雌激素受体、孕激素受体及突变型P53阳性信号均定位于肿瘤细胞的细胞核。本组病例中27例患者雌激素受体、孕激素受体均为阴性表达(27/33,81.82%),2例(2/33,6.06%)雌激素受体(+)、孕激素受体(+),3例(3/33,9.09%)呈雌激素受体(-)、孕激素受体(+)表达模式,1例(1/33,3.03%)雌激素受体(+)、孕激素受体(-)表达；突变型P53免疫组化染色显示24例(24/33,72.73%)为阳性表达,9例为阴性表达(9/33,27.27%)。雌激素受体、孕激素受体、突变型P53表达差异与患者年龄、肿瘤大小、浸润深度、临床分期及淋巴结转移均无统计学意义(P>0.05)。4.在年龄、肿瘤大小、浸润深度、临床分期及淋巴结转移等因素中,仅淋巴结转移组中患者无病生存率具有显著差异(P<0.05)结论1.子宫内膜浆液性癌是一类少见的子宫内膜癌,以老年女性多见。绝经后阴道流血是常见的临床症状,影像学检查多表现为宫腔内息肉或占位。2.子宫内膜浆液性癌需要与低分化子宫内膜样腺癌、卵巢浆液性癌子宫内膜转移等多种恶性肿瘤鉴别,免疫组织化学染色具有一定帮助。3.子宫内膜浆液性癌为非激素依赖性肿瘤；p53基因突变与子宫内膜浆液性癌发生关系密切。4.子宫内膜浆液性癌生物学行为特殊,早期可发生淋巴结转移；患者预后差,淋巴结转移是预后不良因素。背景子宫内膜浆液性癌恶性程度高,死亡率占全部子宫内膜癌的50%,但子宫内膜浆液性癌确切的病因及发病机制尚不清楚,至今尚无针对性的治疗方法。基于上述情况,深入探讨子宫内膜浆液性癌的发病机制,寻找新的治疗靶点及切实有效的治疗方法,意义重大。子宫内膜浆液性癌的发生与p53基因突变密切相关。p53基因定位于人类第17号染色体短臂,编码393个氨基酸,根据氨基酸组成分为

关键词： 青光眼   视网膜神经节   转录激活因子3   细胞凋亡  

摘要： 目的：　　青光眼(Glaucoma)是一组疾病晚期以特征性视神经凹陷和视野缺损为共同表现的不可逆性、致盲性眼病，严重危害人类的健康和生命质量，影响全球大约70万人。以往，青光眼的早期诊断依赖于眼压的升高，视野的损害和眼底的改变三大指标，但是现已证实，在青光眼患者视野出现特征性改变之前，其RGC轴突的30％-50％已经受损，并且只有大量视网膜神经纤维层(retinal nerve fiberlayer，RNFL)缺损后，临床才能检测到相应的眼底及视野的改变。由此可见，探讨青光眼损伤的早期机制对于疾病的诊断和治疗意义重大，尤其对于正常眼压性青光眼和开角型青光眼。目前普遍认为高眼压(intraocular pressure，IOP)是加速青光眼中视网膜神经节细胞(retinal ganglion cells，RGCs)死亡的最显著危险因素，体外培养的细胞系给予高液体静压(elevated hydrostatic pressure，EHP)通常被用来作为青光眼的细胞模型。已有报道研究证实在高眼压大鼠模型和青光眼患者中RGCs的死亡方式为凋亡。细胞凋亡(apoptosis)是指生物体内细胞在特定的内源和外源信号诱导下，其死亡途径被激活，并在有关基因的调控下发生的程序性死亡过程。细胞凋亡涉及一系列复杂的生化反应，是一个需要一系列酶参与的级联反应，涉及不同基因的表达及调控、信号传导。转录激活因子3(activating transcription factor3，ATF3)作为压力应答反应的早期基因，有研究显示，在大鼠的HIOP和鼠视网膜缺血再灌注模型中，其mRNA表达在损伤早期明显上调，且与损伤程度成线性正相关，而与此同时，尚未检测到凋亡信号标志物的明显改变。另外，丝裂原活化蛋白激酶(mitogen-activated proteinkinases，MAPKs)是细胞内的一类丝氨酸/苏氨酸蛋白激酶，有研究证实，MAPKs信号转导通路存在于大多数细胞内，在将细胞外刺激信号转导至细胞及其核内，并引起细胞生物学反应（如细胞增殖、分化、转化及凋亡等）。研究表明，c-Jun氨基末端激酶(c-Jun N-terminal kinases，JNKs)的激活可诱导细胞发生凋亡，同时JNKs信号通路是轴突损伤后引起RGCs死亡的主要的早期信号通路。综上所述，为了明确压力引起RGCs凋亡的早期分子机制，同时探索ATF3和MAPKs尤其是JNKs通路与青光眼早期损伤的关系，设计并展开了研究。首先本实验通过体外培养RGC-5细胞，给予不同大小的静水压处理，检测其凋亡率和各种因子的表达，明确能够引起RGC-5早期凋亡的压力值和作用时间;然后进一步通过给予ATF3siRNA和不同浓度的JNKs通路抑制剂(SP600125)干扰后，再检测细胞早期凋亡率和相关指标的表达，明确二者在静水压引起的RGCs早期凋亡中的作用，进一步明确静水压引起RGC-5早期凋亡的分子机制。具体实验分为:　　1、时间-压力-早期凋亡曲线的描绘:　　通过给予体外培养的RGC-5不同的静水压处理，分别为0、20、25、30、35、40、45、50、55、60、65、70mmHg，明确引起RGC-5早期凋亡的压力值。　　2、ATF3 siRNA的作用研究:　　选取有效的ATF3 siRNA干扰RGC-5中ATF3 mRNA的表达，探讨siRNA ATF3对RGC-5早期凋亡及ATF3、c-Jun和Jun-B表达的影响。　　3、JNKs通路抑制剂的作用研究:　　在前两部分实验的基础上，通过应用特异性JNKs通路抑制剂SP600125，深入探讨静水压引起RGC-5早期凋亡的机制和ATF3、c-Jun和Jun-B的表达变化。　　方法：　　1、时间-压力-凋亡曲线的描绘:　　体外培养RGC-5，给予不同的静水压处理24小时，通过流式细胞仪检测早期凋亡率，绘制时间-压力-凋亡曲线，明确引起细胞凋亡的静水压值，Real-time PCR检测ATF3、Jun-B和c-Jun mRNA的表达，Western Blot和免疫细胞组化检测ATF3、Jun-B和c-Jun蛋白的表达。　　2、ATF3 siRNA的作用研究:　　构建三种不同siRNA，Real-time PCR检测siRNA和lipo2000的用量和有效干扰ATF3 mRNA表达的siRNA，根据第一部分实验选取的压力值瞬时转染，用流式细胞仪检测早期凋亡率，Real-time PCR、Western Blot检测ATF3、Jun-B和c-JunmRNA和蛋白的表达变化。　　3、JNK通路抑制剂(SP600125)的作用研究:　　不同静水压下分别添加不同浓度的特异性JNK通路抑制剂SP600125，用流式细胞仪检测早期凋亡率，Real-time PCR、We

关键词： Abeliophyllum distichum Nakai   Activating transcription factor 3   Apoptosis   Cancer chemoprevention   Colorectal cancer  

摘要： Background: Recently, Abeliophyllum distichum Nakai (A. distichum) has been reported to exert the inhibitory effect on angiotensin converting enzyme. However, no specific pharmacological effects from A. distichum have been described. We performed in vitro study to evaluate anti-cancer properties of A. distichum and then elucidate the potential mechanisms. Methods: Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA. Results: Exposure of ethyl acetate fraction from the parts of A. distichum including flower, leaf and branch to human colorectal cancer cells, breast cancer cells and hepatocellular carcinoma reduced the cell viability. The branch extracts from A. distichum (EAFAD-B) increased the expression of activating transcription factor 3 (ATF3) and promoter activity, indicating transcriptional activation of ATF3 gene by EAFAD-B. In addition, our data showed that EAFAD-B-responsible sites might be between -147 and -85 region of the ATF3 promoter. EAFAD-B-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. However, the deletion of Ftz sites did not affect ATF3 promoter activity by EAFAD-B. We also observed that inhibition of p38MAPK and GSK3 beta attenuated EAFAD-B-mediated ATF3 promoter activation. Also, EAFAD-B contributes at least in part to increase of ATF3 accumulation. Conclusion: These findings suggest that the anti-cancer activity of EAFAD-B may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression.

关键词： S   pneumoniae   activating transcription factor (ATF3)   pneumonia   pneumococcal infection   TNF-alpha   IL-1 beta   IFN-gamma  

摘要： Background. Activating transcription factor-3 (ATF3) is known as a suppressor of cytokine production after exposure to lipopolysaccharide or during gram-negative bacterial infection. However, the mechanism by which ATF3 regulates innate immunity against gram-positive bacterial infection, particularly Streptococcus pneumoniae, remains unknown. Methods. The wild-type and ATF3 knock-out (KO) mice were infected intranasally (i.n) or intraperitoneally with S. pneumoniae, and bacterial colonization or survival rate was determined. Pneumococcal pneumonia was induced by i.n infection, and ATF3 level was determined by Western blot. ATF3 KO cells or ATF3 siRNA transfection were used to determine expression of ATF3 downstream genes. Enzyme-linked immunosorbent assay was used to examine cytokines levels. Results. ATF3 was highly expressed in various cell lines in vitro and in many organs in vivo. Pneumolysin was a novel inducer of ATF3. Pneumococcal infection induced ATF3, which subsequently stimulated production of cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1 beta, and interferon [IFN]-gamma). ATF3-mediated cytokine induction protected the host from pneumococcal infection. In the pneumonia infection model, the bacterial clearance of wild-type mice was more efficient than those of ATF3 KO mice. Conclusions. Taken together, we can conclude that ATF3 regulates innate immunity positively upon pneumococcus infection by enhancing TNF-alpha, IL-1 beta, and IFN-gamma expression and modulating bacterial clearance.

关键词： Chronic obstructive pulmonary disease   Gene interaction networks   Extracellular matrix-related genes  

摘要： Chronic obstructive pulmonary disease (COPD) is a major public health problem worldwide and is proved to be the number three cause of death in globally. The objective of this study was to explore the molecular mechanism of the progression of COPD. Using the GSE1650 affymetrix microarray data accessible from Gene Expression Omnibus database, we first identified the differentially expressed genes (DEGs) between 18 COPD samples and 12 normal samples, followed by the GO / KEGG pathway analysis and gene interaction networks analysis of the DEGs. Our study identified 134 DEGs which involved in regulation of immune response, vesicle transport system, growth regulator and extracellular matrix (ECM)-related pathways. Gene interaction networks analysis showed that the sub-network involved by activating transcription factor-3 (ATF3) was the most significant sub-network in gene interaction networks. Furthermore, the investigation of extracellular matrix-related genes showed that genes like collagen and insulin-like growth factor binding protein could clearly distinguish the COPD and normal control. The genes regulated by ATF3 transcriptional activator as well as ECM-related genes may play an important role in the process of COPD. Our study provides a comprehensive bioinformatics analysis of genes and pathways which may be involved in the progression of COPD.

关键词： Chronic obstructive pulmonary disease   Gene interaction networks   Extracellular matrix-related genes  

摘要： INTRODUCTION:Chronic obstructive pulmonary disease (COPD) is a major public health problem worldwide and is proved to be the number three cause of death in globally. The objective of this study was to explore the molecular mechanism of the progression of COPD. METHODS:Using the GSE1650 affymetrix microarray data accessible from Gene Expression Omnibus database, we first identified the differentially expressed genes (DEGs) between 18 COPD samples and 12 normal samples, followed by the GO / KEGG pathway analysis and gene interaction networks analysis of the DEGs. Our study identified 134 DEGs which involved in regulation of immune response, vesicle transport system, growth regulator and extracellular matrix (ECM)-related pathways. RESULTS:Gene interaction networks analysis showed that the sub-network involved by activating transcription factor-3 (ATF3) was the most significant sub-network in gene interaction networks. Furthermore, the investigation of extracellular matrix-related genes showed that genes like collagen and insulin-like growth factor binding protein could clearly distinguish the COPD and normal control. CONCLUSIONS:The genes regulated by ATF3 transcriptional activator as well as ECM-related genes may play an important role in the process of COPD. Our study provides a comprehensive bioinformatics analysis of genes and pathways which may be involved in the progression of COPD.

关键词： CGN   MIC-1   ATF3   Epithelial apoptosis  

摘要： Carrageenan (CGN), a widely used food additive, has been shown to injure the epithelial barrier in animal models. This type of damage is a clinical feature of inflammatory bowel disease (IBD) in humans. In the present study, the effects of CGN on pro-apoptotic responses associated with macrophage inhibitory cytokine 1 (MIC-1) regulation in human enterocytes were evaluated. CGN up-regulated the expression of MIC-1 that promoted epithelial cell apoptosis. Although MIC-1 induction was dependent on pro-apoptotic p53 protein, the pro-survival protein activating transcription factor 3 (ATF3) was negatively regulated by p53 expression. However, MIC-1 enhanced the expression of the pro-survival protein ATF3 in enterocytes exposed to CGN. Functionally, MIC-1-mediated epithelial cell apoptosis was counteracted by the pro-survival action of ATF3 in response to CGN exposure. These findings demonstrated that the counterbalance between MIC-1 and ATF3 is critical for deciding the fate of enterocytes under the food chemical stress. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

关键词： Adipocyte differentiation   Activating transcription factor 3 (ATF3)   Peroxisome proliferator activated receptor gamma (PPAR gamma)   Promoter activity   ATF/CREB responsive element (ATF/CRE)  

摘要： Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein a (C/EBP alpha) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3 in negatively regulating peroxisome proliferator activated receptor-gamma (PPAR gamma). ATF3 decreased the expression of PPAR gamma and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of -2.6 Kb promoter of mouse PPAR gamma 2. Overexpression of PPAR gamma significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5' deleted-reporters showed that ATF3 repressed the activity of -2037 bp promoter, whereas it did not affect the activity of -1458 bp promoter, suggesting that ATF3 responsive element is located between the -2037 and -1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5'-TGACGTTT-3') between -1537 and -1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPAR gamma 2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPAR gamma expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPAR gamma expression. Furthermore, overexpression of PPAR gamma prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated inhibition of PPAR gamma expression may contribute to inhibition of adipocyte differentiation during cellular stress including ER stress. (C) 2014 Elsevier Inc. All rights reserved.

关键词： 神经元凋亡   H3甲基化修饰   G9a   ATF3  

摘要： 目的探讨协同c-Jun/ATF2调控ATF3表达的染色质重塑事件及对神经元凋亡的作用。方法小脑颗粒神经元凋亡刺激后检测组蛋白H3乙酰化水平的变化与ATF3表达的变化。G9a抑制剂BIX-01294处理神经元,Western Blot检测ATF3的表达、H3的甲基化变化,核染色检测神经元的凋亡率。结果神经元凋亡时诱导ATF3表达,H3第4位赖氨酸乙酰化修饰(methyl-H3-K4)增加。BIX-01294浓度依赖地抑制H3第4位赖氨酸甲基化修饰,抑制ATF3表达,抑制神经元凋亡(P<0.05)。结论神经元凋亡时,G9a介导的H3第4位赖氨酸甲基化修饰调控ATF3表达及凋亡发生。