关键词： Sensory neuron   RNA interference   Axotomy   Cutaneous nerve   Injury   Sry   Penetratin   ROOT GANGLION NEURONS   PROMOTES NEURITE OUTGROWTH   C-JUN   SENSORY NEURONS   MESSENGER-RNA   EXPRESSION   ATF3   GENE   RAT   FAMILY  

摘要： The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury. (C) 2008 Elsevier B.V. All rights reserved.

关键词： CELL-CYCLE CHECKPOINTS   S-PHASE CHECKPOINT   TRANSCRIPTIONAL REPRESSOR ATF3   INDUCED DOWN-REGULATION   G(2) CHECKPOINT   CANCER-CELLS   INDUCED APOPTOSIS   GENOTOXIC STRESS   G2 CHECKPOINT   P53  

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   METASTATIC MALIGNANT-MELANOMA   ENDOTHELIAL GROWTH-FACTOR   CONSTITUTIVE ACTIVATION   TRANSCRIPTION FACTOR   BREAST-CANCER   FACTOR-KB   IN-VITRO   CELLS   INTERLEUKIN-6   CARCINOMA  

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.

关键词： Di-(2-ethylhexyl) phthalate   Hypospadias   Activating transcription factor 3   Apoptosis   Mouse   ACTIVATING TRANSCRIPTION FACTOR-3   INDUCED APOPTOSIS   DIETHYLHEXYL PHTHALATE   SEXUAL-DIFFERENTIATION   DI(N-BUTYL) PHTHALATE   IN-UTERO   CELLS   HYPOSPADIAS   DEHP   EXPOSURE  

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.

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 etal., 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.

关键词： 尿道下裂   DEHP   MEHP   TGF-β1   ATF3  

摘要： 背景:尿道下裂(Hypospadias)是小儿最常见的泌尿生殖道先天畸形。由于阴茎及前尿道发育不全,尿道开口异常伴阴茎下弯畸形,导致患儿不能站立排尿,并影响成年后性功能及生育力。手术矫正畸形是本病的唯一治疗措施,但术后并发症发生率较高,是患儿及家长的心患。为此,有关尿道下裂病因的研究受到学者们关注,以期通过尿道下裂病因学的研究寻找其预防、治疗学突破。 尿道下裂发病率约1/200～1/300,而且有逐年增加趋势,大量研究提示其发病率的增加与环境雌激素污染密切相关。邻苯二甲酸二(2-乙基)己酯(DEHP)是一种广泛应用于塑料及化工产业的增塑剂,在医疗工作和日常生活中被大量应用,已构成我国严重的“白色污染”。DEHP进入环境后不易被降解,能蓄积于人体产生雌激素样生物活性。课题组前期研究已发现DEHP可损害实验动物睾丸及附睾功能导致小鼠隐睾畸形,可同时伴有尿道下裂的产生。虽有文献报道DEHP能诱发实验动物的尿道下裂,但是未就其机制深入研究。因此,本课题针对DEHP对外生殖器的毒性损伤,研究其致畸机制,为进一步防治尿道下裂发生提供科学依据。 目的:利用DEHP的外生殖器的毒性损伤,诱导胎鼠尿道下裂:体内外观察阴茎发育情况,检测细胞凋亡改变、TGF-β1及ATF3等在阴茎组织中的表达;对比尿道下裂患者与正常儿童包皮TGF-β1表达差异,深入探讨尿道下裂发生机制。 方法:1.将C57BL/6及KM孕鼠分为对照组与DEHP处理组,DEHP处理组分为100 mg/kg·d、200 mg/kg·d与500 mg/kg·d三个剂量组(DEHP

关键词： 激活转录因子3(ATF3)   shRNA   肾小球系膜细胞   肾小球系膜细胞   亚溶解剂量的C5b-9复合物(sublytic C5b-9)   激活转录因子3(ATF3)   凋亡  

摘要： 目的:构建并鉴定大鼠野生型激活转录因子3(activating transcription factor 3, ATF3)基因及大鼠ATF3特异性小发夹RNA(small hairpin RNA, shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(glomerular mesangial cells, GMCs)中过表达及沉默ATF3基因的情况。方法:(1)用DNA重组技术构建大鼠ATF3基因真核表达质粒pcDNA3.1/ATF3和构建针对ATF3基因不同位点的五个shRNA序列的真核表达质粒ATF3 shRNA1-5。(2)体外分别将pcDNA3.1/ATF3质粒或ATF3 shRNA1-5质粒转染GMCs,筛选pcDNA3.1/ATF3和ATF3 shRNA转染最佳时间,并将GMCs作相应的分组处理。Western blot检测ATF3蛋白的相对表达量来鉴定pcDNA3.1/ATF3的表达及筛选最佳沉默效率shRNA。结果:(1)核酸序列测定表明,pcDNA3.1/ATF3质粒及ATF3 shRNA质粒构建正确。Western blot证实,pcDNA3.1/ATF3质粒成功表达ATF3蛋白,ATF3 shRNA-1具有最佳沉默效率。结论:(1)成功构建了大鼠野生型ATF3基因真核表达质粒pcDNA3.1/ATF3。(2)成功构建并筛选出最佳沉默效率的大鼠ATF3 shRNA-1。该结果为进一步研究ATF3基因在亚溶解型补体C5b-9(sublytic C5b-9)诱导GMCs凋亡病变中的作用奠定了基础。 目的:检查大鼠GMCs受sublytic C5b-9刺激后,其ATF3基因mRNA丰度和蛋白表达水平的变化,探讨过表达或靶向沉默ATF3基因对sublytic C5b-9诱导GMCs凋亡病变的影响。方法:(1)以sublytic C5b-9刺激GMCs为实验模型,应用RT-PCR、Real-time PCR、Western blot和免疫组化技术检查ATF3 mRNA丰度和蛋白表达水平,用Annexin V(AV)- propidium iodide (PI)双染后流式细胞仪定量分析GMCs凋亡数量。(2)体外将pcDNA3.1/ATF3质粒或ATF3 shRNA质粒瞬时转染GMCs,并将GMCs作相应的分组处理,再用Hoechst 33342染色检查各组GMCs的凋亡形态,并用AV-PI双染后流式细胞仪定量分析GMCs凋亡数量。结果:(1)Sublytic C5b-9刺激GMCs后,ATF3 mRNA及蛋白表达能迅速增高,至3h达到峰值;ATF3蛋白主要分布在GMCs细胞核及核周;Sublytic C5b-9刺激的GMCs,细胞凋亡率较对照组显著增高。(2)Sublytic C5b-9刺激的GMCs和转染pcDNA3.1/ATF3质粒的GMCs均呈现核Hoechst 33342染色致密增强或碎裂等凋亡形态学的改变;同时,GMCs凋亡百分率也明显增高。pcDNA3.1/ATF3质粒转染的GMCs再给予sublytic C5b-9刺激后,上述现象更为明显。与之相反,转染ATF3 shRNA质粒的GMCs再给予sublytic C5b-9刺激后,其GMCs凋亡率则显著降低。结论:Sublytic C5b-9刺激GMCs能诱导GMCs中ATF3表达上调和细胞凋亡,而ATF3表达增加对GMCs的凋亡有促进作用。

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

摘要： The focus of the research performed in this dissertation is on ATF3, a stress inducible transcription factor that plays a central role in the biology of β-cells. The hypothesis of this dissertation is that ATF3 functions by contributing to the expression of target genes that strongly influence β-cell survival and function. Previous studies demonstrated that activating transcription factor 3 (ATF3) is a stress-inducible gene and pro-apoptotic in various cell types, including β-cells. These results support a model that ATF3 plays a role in the pathogenesis of diabetes, partly by its apoptotic effect in β-cells. Based on this information, I set out to investigate the potential functional significance of ATF3 expression in β-cells exposed to two physiologically relevant stress paradigms; islet transplantation and high fat diet induced type 2 diabetes. In chapter 2, I tested whether ATF3 plays a role in the β-cell stress response to islet transplantation. In the course of this study, I compared wild type (WT) and ATF3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation, and hypoxia. I also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. As a result, I found that ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets than KO islets. ATF3 regulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (a) apoptosis (NOXA, bNIP3), and (b) immuno-modulation (TNFα, IL1β, IL6, and CCL2/MCP-1). In vivo, ATF3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in recipients, and reduced caspase 3 activation and macrophage infiltration in the KO grafts. These results identified a role for ATF3 in islet graft rejection by contributing to islet apoptosis and inflammatory