关键词： Ribotoxic stress   Activating transcription factor 3 (ATF3)   Epithelial cell survival   p38 MAP kinase   Double-stranded RNA (dSRNA)-activated protein kinase (PKR)  

摘要： Ribotoxic stress responses lead to the expression of genes important for cellular homeostasis by modulating cell survival, proliferation and differentiation. ATF3 was investigated for its modulation of the epithelial cellular integrity in response to mucosal ribotoxic stress. ATF3 expression was up-regulated by chemical agents causing ribotoxic stress such as deoxynivalenol. and anisomycin in different types of intestinal epithelial cells. Moreover, reduction of ATF3 gene expression promoted ribotoxic stress-triggered programmed cell death, implicating a protective role of ATF3 in epithelial cell survival. Mechanistically, stabilization of ATF3 messenger RNA and protein played a critical role in maintaining enhanced levels of ATF3 production in response to the ribotoxic chemical agent. For ATF3 mRNA stability, p38 specific inhibitor SB203580 was the most efficient agent for suppression, suggesting the involvement of the p38 MAP kinase in ATF3 mRNA stabilization. in addition, the p38 MAP kinase as well as its downstream mediator glycogen synthase kinase 30 (GSK3 beta) was involved in ATF3 protein stabilization caused by chemical ribotoxic stress in human epithelial cells. As another separate signaling cascade, double-stranded RNA (dsRNA)-activated protein kinase (PKR) was demonstrated to translationally modulate ATF3 expression and contribute to the epithelial cell survival. PKR interference caused cells to be more susceptible to cell death caused by the chemical ribotoxic stress. The results of this study showed that enhanced ATF3 production was associated with cellular defenses by maintaining the epithelial survival after ribotoxic mucosal insults. (C) 2009 Published by Elsevier Inc.

关键词： Transcription  

摘要： Eukaryotic cells respond to DNA damage and stalled replication forks by activating signaling pathways that promote cell cycle arrest and DNA repair. A systematic screening of the protein kinase small interfering RNA library reveals that Chk1 and ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) are the main kinases responsible for intra-S-phase checkpoint upon topoisomerase I inhibitor camptothecin-induced DNA damage. It is well known that ATR-Chk1-mediated protein degradation of Cdc25A protein phosphatase is a crucial mechanism conferring this checkpoint activation. Here we describe another mechanism underlying Cdc25A down-regulation in response to DNA damage that occurs at the transcriptional level. We show that activation of tumor suppressor p53 by DNA damage results in inhibition of Cdc25A transcription as a result of activation of transcriptional repressor ATF3 that directly binds to the Cdc25A promoter. In cells deficient in both Chk1 and p53, Cdc25A down-regulation upon camptothecin-induced DNA damage is completely abolished, leading to severe defects in cell cycle checkpoints and remarkable cell death in mitosis. Our findings reveal two independent mechanisms acting in concert in regulation of Cdc25A in DNA damage response. Although Chk1 affects Cdc25A via rapid phosphorylation and protein turnover, inhibition of Cdc25A transcription by p53-ATF3 is required for the maintenance of cell cycle arrest.

关键词： Atf3   妊娠   胰岛β细胞   细胞增殖   催乳素  

摘要： 目的:探讨Atf3在大鼠妊娠期胰岛β细胞中的表达及其对体外催乳素作用下INS-1细胞增殖的影响。方法:运用RT-PCR,Real-timePCR和Westernblotting检测大鼠妊娠期Atf3mRNA及蛋白的表达;MTT法测定不同浓度催乳素作用下INS-1细胞的增殖情况;RT-PCR检测催乳素作用不同时间后INS-1细胞Atf3 mRNA的表达;Western blotting检测相应蛋白表达。结果:与正常未孕组相比,孕10.5天组Atf3 mRNA表达明显增强,孕14.5天时降至正常未孕组水平。Atf3蛋白在孕期表达亦呈一过性增强,于孕10.5天达高峰。体外不同浓度催乳素(50～1000ng/ml)作用24h后,INS-1细胞增殖显著增加,且呈浓度依赖性。200ng/ml催乳素作用3h,Atf3 mRNA表达开始增加,6h达高峰,其后又复降低;蛋白表达则从6h开始上调,12h显著增加,一直持续至24h。结论:转录因子Atf3在大鼠妊娠期表达一过性增强,且时相上早于胰岛增殖的高峰(孕14.5天),提示它可能与胰岛再生相关。另外,催乳素可促进INS-1细胞增殖,并伴随Atf3表达的相应增加,提示Atf3在催乳素介导的细胞增殖效应中发挥重要作用,且于早期即启动。

关键词： NF-kappa B p50   melanoma   angiogenesis   IL-6   ATF3  

摘要： Nuclear factor kappa B (NF-kappa B) signaling is deregulated in many tumor types, resulting in aberrant expression and/or activation of NF-kappa B transcriptional complexes. We have previously reported that nuclear expression of the NF-kappa B subunit p50 is strongly correlated with melanoma progression and poor 5-year patient survival. In this study, we used cDNA microarray to analyze the gene expression profiles of melanoma cells overexpressing NF-kappa B p50. We found that NF-kappa B p50 expression strongly induced interleukin-6 (IL-6) upregulation in melanoma cells at both the transcriptional and translational levels and that IL-6 production by melanoma cells enhanced the growth of endothelial cells in vitro. Expression of activating transcription factor 3 (ATF3), a negative regulator of IL-6 gene transcription, inhibited p50-mediated IL-6 upregulation. Knockdown of p50 expression using lentiviral-based shRNA abrogated IL-6 induction in melanoma cells and inhibited its effects on endothelial cell growth. Finally, we used an in vivo matrigel plug assay to show that NF-kappa B p50 overexpression promotes angiogenesis, while silencing NF-kappa B p50 inhibits blood vessel formation. Our results demonstrate for the first time that the NF-kappa B p50 subunit mediates melanoma angiogenesis by specifically upregulating IL-6, highlighting a novel and important role for the NF-kappa B p50/IL-6 signaling axis in melanoma progression. (c) 2008 Wiley-Liss, Inc.

摘要： Overall, our studies are consistent with Atf3 acting directly on the IL6 promoter to inhibit further transcription of IL6 mRNA following stimulation with ET-1, given that Atf3 protein binds directly to the promoter (Fig. 5) and knockdown of the protein enhances IL6 mRNA expression (Fig. 4). Klf2 also appears to operate in a negative feedback system to limit IL6 mRNA expression since its knockdown also enhances IL6 mRNA expression (Fig. 6), although further studies are required to determine whether its effects are directly mediated at the level of the IL6 promoter. Interestingly, Ereg appears to be co-regulated with IL6 with a similar pattern of expression in response to ET-1 and similar enhancement of expression by cycloheximide (Fig. 2), Atf3 knockdown (Fig. 4) or Klf2 knockdown (Fig. 5). However, for both IL6 and Ereg, other factors may further contribute to the downregulation of mRNA expression following the increase induced by ET-1, possibly by increasing its rate of degradation. It is interesting that ET-1 also increased expression of Zfp36 (Table 1) which is associated with regulation of mRNA stability (Carrick et al., 2004). This and other factors may be required to limit the expression of IL6, Ereg and other selected IEGs responding to stimuli such as ET-1, and ensure that they are expressed only transiently. Understanding how these early changes associated with intercellular or intracellular signalling and gene/protein expression relate to the changing environment of a cell will help us understand how a complex phenotypic response such as cardiac hypertrophy may develop in vivo and how such an initially beneficial response may deteriorate into (for the heart) cardiac failure. © 2009 Elsevier Ltd. All rights reserved.

摘要： To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing > 1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation. The Journal of Immunology, 2009, 182: 466-476.

关键词： Di-(2-ethylhexyl) phthalate   Hypospadias   Activating transcription factor 3   Apoptosis   Mouse  

摘要： Di-(2-ethylhexyl) Phthalate Upregulates ATF3 Expression and Suppresses Apoptosis in Mouse Genital Tubercle: Xing Liu, et al. Department of Pediatric Urology, Chongqing Children's Hospital, Chongqing Medical University, China-Objectives: To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) on the expression of activating transcription factor 3 (ATF3) and apoptosis of fetal mouse genital tubercle (GT). Methods: In this developmental toxicity study, pregnant C57BL/6 mice were exposed to corn oil or DEHP (100 or 500 mg/kg/day) from embryonic day 12 (ED12) to ED16. Apoptosis was characterized by Terminal transferase dUTP nick end labeling (TUNEL) assay. Using RT-PCR and western blot, the expressions of ATF3 and apoptosis-related genes (P53, Bcl-2 and Bax) were investigated. Results: Apoptosis of fetal mouse GT cells notably decreased after DEHP treatment. DEHP activated ATF3 both at the mRNA and protein levels in GT. Furthermore, pro-apoptotic P53 was downregulated and the ratio of anti-apoptotic (Bcl-2)/pro-apoptotic (Bax) was not significantly changed. Conclusions: These results suggest that DEHP may induce external genital defects via a mechanism involving apoptosis, which might correlate with the regulation of ATF3 and P53 expressions.

关键词： 原发性肝癌   ATF3   MMP-2   maspin   肿瘤转移   组织芯片   免疫组织化学   EMSA   图像分析  

摘要： 目的:从蛋白水平上,检测肝细胞癌(hepatocellular carcinoma,HCC)组织芯片中ATF3、MMP-2及maspin的表达情况和手术切除新鲜肝癌标本中ATF3的DNA结合活性,探讨组织芯片中ATF3、MMP-2及maspin的表达与HCC侵袭转移的关系及其意义,及新鲜肝癌标本中ATF3的DNA结合活性与HCC侵袭转移的关系及其意义。 方法:对手工制作组织芯片的方法进行改良,构建含有HCC、癌旁肝组织、远癌肝组织、正常肝组织及肝内转移灶的组织芯片,应用免疫组化及彩色图像分析技术从蛋白质水平上检测了126例HCC、126例癌旁肝组织、81例远癌肝组织、20例正常肝组织和33例肝内转移灶中ATF3、MMP-2和maspin的表达情况,同时收集新鲜肝癌标本,利用凝胶电泳迁移率分析法( Electrophoretic Mobility Shift Assay, EMSA)检测其中的26例HCC、10例癌旁肝组织中ATF3的DNA结合活性情况,并综合分析ATF3、MMP-2和maspin的表达,ATF3的DNA结合活性与各临床病理参数的关系及其意义。 结果: 1、利用改良的手工制作组织芯片方法构建7个含有HCC、癌旁肝组织、远癌肝组织、正常肝组织以及肝内转移灶共185个点的芯片蜡块。其中中间列5个点为阳性对照点及标志点。所有蜡块点阵排列整齐,无裂纹,能完整切下。芯片经H-E、免疫组化染色后切片有效率达95%以上,芯片组织符合镜下组织学观察和进一步研究的需要。 2、免疫组织化学染色结果显示ATF3及MMP-2在HCC中表达高于癌旁肝组织、远癌肝组织和正常肝组织(P<0.01),maspin在HCC中的表达明显低于癌旁肝组织、远癌肝组织和正常肝组织(P<0.01)。 3、ATF3蛋白在转移组中表达高于无转移组(P<0.01),在女性HCC患者中的表达量高于男性患者(P<0.01)。此外,ATF3蛋白的表达与肿瘤大小、包膜侵犯、组织学类型以及病理学分级均无关(P>0.05)。MMP-2蛋白在包膜侵犯组中表达高于无包膜侵犯组(P<0.05),肝内转移灶表达量高于原发灶(P<0.05),在直径大于3cm的HCC中的表达量高于直径小于3cm的(P<0.05),但是MMP-2蛋白随HCC病理学分级的升高,相对表达量降低(P<0.05)。此外,MMP-2蛋白的表达与性别、组织学类型均无关(P>0.05)。ATF3随着HCC的转移进展表达量逐渐增高,MMP-2亦随着HCC的包膜侵袭、转移进展表达量逐渐增高,然而maspin却随着HCC病理学分级的升高,相对表达量降低(P<0.05)。此外,maspin蛋白的表达与性别、肿瘤大小、包膜侵犯、组织学类型以及转移情况均无关(P>0.05)。 4、ATF3蛋白表达与MMP-2表达呈正相关(P<0.05),与maspin表达呈负相关(P<0.01);MMP-2蛋白表达与maspin表达呈负相关(P<0.01)。 5、EMSA实验结果显示:ATF3蛋白在HCC中的DNA结合活性明显高于癌旁肝组织(P<0.01);在HCC组织中,转移组ATF3的DNA结合活性显著高于无转移组(P<0.05);此外,ATF3的DNA结合活性与HCC患者年龄、性别、组织学类型、肿瘤大小、包膜侵犯、病理学分级均无明显相关性(P>0.05)。 结论: 1、ATF3蛋白在转移组中表达高于无转移组,在女性HCC患者中的表达量高于男性患者。此外,ATF3蛋白表达与肿瘤大小、包膜侵犯、组织学类型以及病理学分级均无关。 2、MMP-2蛋白在包膜侵犯组中表达高于无包膜侵犯组,肝内转移灶表达量高于原发灶,在直径大于3cm的HCC中的表达量高于直径小于3cm的,但是MMP-2蛋白随HCC病理学分级的升高,相对表达量降低。此外,MMP-2蛋白的表达与性别、组织学类型以及转移情况均无关。ATF3及MMP-2均随着HCC进展表达量升高,然而maspin却随着HCC病理学分级的升高,相对表达量降低。此外,maspin蛋白的表达与性别、肿瘤大小、包膜侵犯、组织学类型以及转移情况均无关。 3、ATF3蛋白和MMP-2蛋白在肝癌组织中高表达,而maspin蛋白在肝癌组织中低表达。ATF3蛋白表达与MMP-2表达呈正相关,与maspin表达呈负相关;MMP-2蛋白表达与maspin表达呈负相关。在肝癌的侵袭转移过程中,ATF3和MMP-2、maspin之间存在相互调控机制。 4、在EMSA检测结果中,ATF3的DNA结合活性在HCC中高于癌旁肝组织,转移组ATF3的DNA结合活性高于无转移组。提示ATF3的DNA结合活性与HCC的侵袭转移