关键词： 转录激活因子3   糖皮质激素   邻苯二甲酸二丁酯   尿道下裂   生殖结节  

摘要： 目的:利用糖皮质激素与邻苯二甲酸二丁酯(DBP)联合作用致大鼠尿道下裂,验证转录激活因子3(ATF3)在正常大鼠生殖结节(GT)与不同严重程度尿道下裂大鼠生殖结节中的差异表达,以强调组合因素在尿道下裂病因中的重要性,并探讨ATF3在尿道下裂发生中的作用机制。 方法:SD孕鼠80只,随机分为4组,每组20只,在妊娠14～18天(GD14～18)分别给予:A组:大豆油2ml/d灌胃;B组:地塞米松(DXM)0.1mg/(kg·d)皮下注射;C组:DBP700mg/(kg·d)灌胃;D组:地塞米松0.1mg/(kg·d)皮下注射加DBP700mg/(kg·d)灌胃。于GD19将各组半数孕鼠处死剖腹统计仔鼠数、雄性仔鼠出生体重(BW)及肛门生殖器间距离(AGD),取雄性仔鼠生殖结节,运用免疫印迹法和免疫组织化学方法分析ATF3的表达情况,剩余孕鼠自然分娩后,于出生后第70天(PND70)观察统计尿道下裂和隐睾的发生情况。 结果:地塞米松单独作用于孕鼠仅表现为后代仔鼠出生体重的下降,但与DBP组合作用却能显著增强DBP的致畸作用,导致后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,统计显示隐睾在C组和D组的发生率分别为:58%和88%,尿道下裂的在C组和D组的发生率分别为:37%和60%,而尿道下裂严重程度则根据出生后70天(PND70)大鼠外生殖器观察统计。蛋白质组学结果显示,ATF3在A组的蛋白相对表达量为0.351±0.012(n=10),B组为0.336±0.015(n=10),C组为0.603±0.014(n=10),D组为0.851±0.016(n=10),C组或D组与A组,以及C组与D组之间的差异均有统计学意义(P<0.05);免疫组化结果显示ATF3主要定位于生殖结节的尿道上皮和侧翼间质,C组和D组免疫组化染色强度明显强于A组,而D组染色强于C组。 结论:地塞米松联合DBP进一步增强了DBP的致畸作用,表现为后代雄性仔鼠出生体重及肛门生殖器间距离/出生体重(AGD/BW)明显减少,且尿道下裂发生率、严重程度以及隐睾发生率较DBP单因素作用下更为显著,提示我们尿道下裂的发生是环境中多因素协同作用下的结果;ATF3在对照组与实验组组生殖结节中的表达有明显变化,且随着尿道下裂程度的加重而表达增高,ATF3的高表达影响了生殖结节的发育及尿生殖褶的融合,这可能是尿道下裂发生的机制之一。

关键词： ATF3   c-Jun   STAT3   Sp1   DINE   transcription   axotomy   nerve injury  

摘要： Peripheral nervous system (PNS) neurons survive and regenerate after nerve injury, whereas central nervous system (CNS) neurons lack the capacity to do so. The inability of the CNS to regenerate presumably results from a lack of intrinsic growth activity and a permissive environment. To achieve CNS regeneration, we can learn from successful nerve regeneration in the PNS. Neurons in the PNS elicit dynamic changes in gene expression in response to permissive environmental cues following nerve injury. To switch gene expression on and off in injured neurons, transcription factors and their networks should be carefully orchestrated according to the regeneration program. This is the so-called "intrinsic power of axonal growth." There is an increasing repertoire of candidate transcription factors induced by nerve injury. Some of them potentiate the survival and axonal regeneration of damaged neurons in vivo;however, our knowledge of transcriptional events in injured neurons is still limited. How do these transcription factors communicate with each other? How does the transcriptional machinery regulate the wide variety of regeneration-associated genes (RAGs) in the properly coordinated manner? In this review, we describe our current understanding of the injury inducible transcriptional factors that enhance the intrinsic growth capacity, and propose a potential role for specificity protein 1 (SO), which provides a platform to recruit injury-inducible transcription factors, in simultaneous gene regulation. Finally, we discuss an additional mechanism that is involved in epigenetic modifications in damaged neurons. A comprehensive understanding of the nuclear events in injured neurons will provide clues to clinical interventions for successful nerve regeneration.

关键词： 支气管哮喘   发病机制   氧化应激   Krüppel样转录因子2   红系衍生核因子相关因子-2   γ-谷氨酰半胱氨酸合成酶   激活转录因子  

摘要： 背景:慢性气道炎症是支气管哮喘发病的本质,大量炎细胞刺激产生过多ROS,引起氧化应激,进一步加重炎症反应与气道损伤。谷胱甘肽(glutathione,GSH)是肺内最重要的抗氧化物质,存在于气道分泌物和BALF中。γ-谷氨酰半胱氨酸合成酶催化亚基(γ-glutamylcysteine synthetase catalytic subunit,γ-GCSc)是GSH生物合成的限速酶,具有非常重要的抗氧化作用。红系衍生的核因子2相关因子2(NF-E2-related factor2,Nrf2)是一种抗氧化转录因子,能上调抗氧化反应元件(ARE)介导的靶基因(如γ-GCSc等)抗氧化酶的表达。激活转录因子3( Activating transcription factor3,ATF3)、ATF4和Krüppel样锌指转录因子(Krüppel-like Factor2,KLF2),都是应激反应转录因子。氧化应激时,ATF3、ATF4能与Nrf2结合形成异二聚体;而KLF2能增加Nrf2的核定位及其活性,三者都能作用于Nrf2调控其介导的抗氧化靶基因(如γ-GCSc)的表达。　　目的:探讨ATF3、ATF4、KLF2调控Nrf2/γ-GCSc表达及其在支气管哮喘氧化应激机制中的作用。　　方法:实验分两部分。1.动物实验部分:30只健康雄性豚鼠随机分为对照组(A组)、哮喘组(B组)和地塞米松治疗组(C组),卵清蛋白致敏激发法复制支气管哮喘急性发作期模型;HE染色观察三组豚鼠肺组织(主要为细支气管)病理形态学变化并测支气管肺泡灌洗液(BALF)细胞总数和分类计数;检测三组肺组织匀浆中及 BALF中ROS、丙二醛(MDA)浓度;RT-PCR法、原位杂交法和western blot法、免疫组织化学法分别检测肺组织ATF3、ATF4、KLF2、Nrf2、γ-GCSc mRNA和蛋白质的表达;免疫细胞化学法检测BALF细胞中ATF3、ATF4、KLF2、Nrf2、γ-GCSc蛋白质的表达。2.临床实验部分:分健康对照组、(急性发作期)哮喘患者(治疗前)组、治疗后三组,均行肺功能检查;测三组痰液中ROS、MDA浓度;免疫细胞化学法检测各组痰涂片细胞中ATF3、ATF4、KLF2、Nrf2、γ-GCSc蛋白质的表达。　　结果:　　1.动物实验部分:(1)B组豚鼠BALF中细胞总数、嗜酸性粒细胞百分比(EOS％)显著高于A组(P<0.01),B组肺组织细支气管周围可见大量炎症细胞浸润,及明显气道重塑改变,而C组BALF炎细胞变化及肺组织病理改变较B组明显减轻;(2)豚鼠肺组织、BALF中ROS、MDA浓度,B组明显高于A组,C组较B组降低(P均<0.05);(3)豚鼠肺组织中 ATF4、KLF2、Nrf2、γ-GCScmRNA和蛋白质表达,B组低于 A组和C组(P均<0.05);ATF3 mRNA和蛋白质表达,B组高于A组和C组(P均<0.05);(4)豚鼠BALF细胞中ATF4、KLF2、Nrf2、γ-GCSc蛋白质的表达,B组低于A组和C组(P均<0.05),而ATF3蛋白质表达,B组高于A组和C组(P均<0.05)。　　2.临床实验部分:(5)哮喘患者痰液中ROS、MDA浓度高于健康对照组和治疗后组(P均<0.01);(6)哮喘患者痰中细胞内ATF4、KLF2、Nrf2、γ-GCSc蛋白表达治疗后高于治疗前(P均<0.01),而ATF3蛋白质表达治疗后低于治疗前(P<0.01)。　　3.直线相关性分析:(7)豚鼠肺组织ATF4、KLF2 mRNA及蛋白表达与Nrf2、γ-GCSc mRNA及蛋白表达呈正相关(P均<0.01);ATF3mRNA及蛋白表达与Nrf2、γ-GCSc mRNA及蛋白表达呈负相关(P均<0.01)。(8)豚鼠BALF细胞和哮喘患者痰细胞内 ATF4、KLF2蛋白表达与 Nrf2、γ-GCSc蛋白表达呈正相关(P均<0.01);ATF3蛋白表达与 Nrf2、γ-GCSc蛋白表达呈负相关(P均<0.01)。(9)豚鼠肺组织、BALF细胞和哮喘患者痰液细胞内ATF4、KLF2、Nrf2、γ-GCSc表达水平与ROS、MDA浓度及BALF中炎细胞总数、EOS%均呈负相关,ATF3表达与ROS、MDA浓度及BALF中炎细胞总数、EOS%均呈正相关。　　结论:1.支气管哮喘急性发作期豚鼠模型肺组织及患者痰液细胞中存在氧化应激,刺激Nrf2/γ-GCSc表达抵抗氧化损伤,对组织/细胞起保护作用。2.抗炎治疗后ROS、MDA等氧化物减少,ATF4、KLF2表达增加而ATF3表达降低,可能通过增加Nrf2/γ-GCSc表达,提高抗氧化能力,在支气管哮喘发病与防治中起重要作用。

关键词： 肺疾病,阻塞性   活化转录因子3   活化转录因子4   肺Krüppel样转录因子   红系衍生核因子相关因子-2   γ-谷氨酰半胱氨酸合成酶  

摘要： 【目的】研究转录因子ATF3、ATF4、KLF2、Nrf2及γ-GCS在慢性阻塞性肺疾病(COPD)大鼠及患者肺组织中的表达变化关系;探讨ATF3、ATF4、KLF2与Nrf2蛋白之间的相互作用,以及对γ-GCS-HS基因表达的影响;探索它们在COPD发病中的可能作用,为COPD的防治提供新的理论依据。 【方法】实验分两部分。(1)动物试验部分:将22只雄性SD大鼠按随机数字表法分为对照组和COPD模型组,每组11只。采用每日熏香烟和两次气管内滴入脂多糖(LPS)复制COPD大鼠模型。检测各组大鼠肺功能并观察大鼠肺组织病理形态学改变;原位杂交和逆转录-聚合酶链反应(RT-PCR)检测肺组织ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS mRNA的表达。免疫组化和免疫印迹(Western blot)分别检测ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS蛋白表达水平。并同时利用免疫共沉淀法(CO-IP)探讨ATF3、ATF4、KLF2与Nrf2蛋白之间的相互作用。(2)临床试验部分:肺癌患者手术切除肺组织标本40例,所有患者术前均询问病史,进行体检、X线胸片或肺部CT和肺功能检测,其中COPD组和对照组各20例。原位杂交分析各组患者肺组织中ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS mRNA水平,免疫组化检测肺组织中ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS蛋白的表达。 【结果】 1.动物试验结果 (1)大鼠肺功能指标(FEV0.3、FEV0.3/FVC%、PEF)在COPD组较对照组明显降低(P均<0.01)。COPD组大鼠肺部病理改变符合COPD的形态学改变。 (2) ROS含量在COPD组较对照组显著增高(P<0.01),γ-GCS-HS活性在COPD组较对照组显著升高(P <0.01)。 (3)原位杂交结果显示:ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS mRNA在两组大鼠肺组织中的表达部位基本一致,主要见于支气管上皮细胞、肺泡上皮细胞、平滑肌细胞以及血管内皮细胞,且ATF3、ATF4、KLF2、γ-GCS-HS mRNA在COPD组显著高于对照组(P均<0.05)但两组Nrf2 mRNA比较无明显差异(P >0.05)。RT-PCR结果与原位杂交结果一致。 (4)免疫组化结果显示:ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS主要表达于支气管上皮细胞、肺泡上皮细胞,小血管平滑肌中亦有少许表达。ATF3、ATF4、KLF2、Nrf2在COPD组以胞核表达为主,γ-GCS-HS在胞浆胞核均有免疫着色,其差异具显著性(P均<0.05)。Western blotting结果与免疫组化结果一致(P均<0.05)。 (5)免疫共沉淀显示:在Nrf2抗体捕获的免疫沉淀中,ATF3、ATF4、KLF2抗体均可杂交出明显的蛋白条带,且COPD组明显增强(P<0.01)。 (6) SPSS16.0软件行直线相关分析:大鼠肺组织中ATF3、ATF4、KLF2、Nrf2蛋白表达与γ-GCS-HS mRNA、蛋白及酶活性均呈正相关(均P <0.05);ATF3、ATF4、KLF2蛋白表达与Nrf2蛋白表达均呈正相关(均P <0.05);ATF3、ATF4、KLF2、Nrf2蛋白表达与ROS呈正相关(均P <0.05)。 2.临床试验结果 (1) COPD患者肺组织中ATF3、ATF4、KLF2、γ-GCS-HS mRNA呈强阳性表达,较对照组显著升高(均P <0.01),而Nrf2 mRNA在两组肺组织中均呈阳性表达,两组比较差异无统计学意义(P > 0.05)。 (2) ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS蛋白在COPD患者肺组织中呈强阳性表达,较对照组明显增高(均P < 0.01)。 (3) SPSS16.0软件行直线相关分析:ATF3、ATF4、KLF2蛋白表达与Nrf2蛋白、γ-GCS-HS mRNA及蛋白表达均呈明显正相关(均P <0.01),而与Nrf2 mRNA表达无明显相关性(P >0.05)。另肺组织中ATF3、ATF4、KLF2、Nrf2、γ-GCS-HS蛋白表达与FEV1(%)、FEV1/FVC(%)均呈正相关(均P <0.01)。 【结论】 1.在COPD氧化应激状况下,体内重要抗氧化酶γ-GCS可代偿性上调,而Nrf2是γ-GCS表达上调的关键性转录调控子,可增加GSH合成,降低ROS,对抗氧化损伤。 2.在慢性阻塞性肺疾病发病过程中,氧化应激诱导转录因子ATF3、ATF4过表达可能与Nrf2的转录调控有

关键词： Colon Cancer Cell   Peritoneal Carcinomatosis   Hsp90 Inhibition   HT29 Colon Cancer Cell   HCT116 Colon Cancer Cell  

摘要： Background: Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Methods: Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. Results: The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. Conclusion: In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.

关键词： Transcription factors   Immune receptor signaling   Small interfering RNA   Toll-like receptors   Gene expression   Microarrays   Reverse transcriptase-polymerase chain reaction   Regulator genes  

摘要： Background: Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokine expression in macrophages, and ATF3 deficient mice are more susceptible to endotoxic shock. This study addresses the role of ATF3 in the Kdo(2)-Lipid A-induced Toll-like receptor 4 (TLR4) signaling pathway in mouse embryonic fibroblasts (MEF). Kdo(2)-Lipid A upregulates ATF3 expression in wild type MEF cells and induces both nuclear factor kappa B (NF-kappa B) and c-Jun N-terminal kinase (JNK) activation via the TLR4 signaling pathway, while neither of these pathways is activated in ATF3-/- MEF cells. Interestingly, in contrast to Kdo(2)-Lipid A, the activation of both NF-kappa B and JNK by TNF-alpha was normal in ATF3-/- MEF cells. Methodology/Principal Findings: We found that several genes were dramatically upregulated in ATF3+/+ MEF cells in response to Kdo(2)-Lipid A treatment, while little difference was observed in the ATF3-/- MEF cells. However, we also found that the signal intensities of I kappa B zeta in ATF3-/- MEF cells were substantially higher than those in wild type MEF cells upon microarray analyses, and upregulated I kappa B zeta expression was detected in the cytosol fraction. Conclusions/Significance: Our findings indicate that ATF3 deficiency affects Kdo(2)-Lipid A-induced TLR4 signaling pathways in MEF cells, that it may upregulate I kappa B zeta expression and that the high levels of I kappa B zeta expression in ATF3-/- cells disrupts Kdo(2)-Lipid A-mediated signaling pathways.

关键词： Ischemia reperfusion   Preconditioning   Infarct size  

关键词： Type 1 diabetes   ATF3   STAT1   Streptozotocin (STZ)   Apoptosis  

摘要： It is well established that the IFN-gamma/STAT1 pathway plays an important role in the pancreatic beta-cell apoptosis that is observed in STZ-induced type 1 diabetes;however, the upstream regulatory proteins involved have not been understood. Here, we investigated whether activating transcription factor 3 (AIR) affects STAT1-mediated beta-cell dysfunction and apoptosis in streptozotocin-treated mice. To this, STZ (80 mg/kg, i.p.) was administered to wildtype and STAT1(-/-) or IFN-gamma(-/-) mice for 5 days and the mice were euthanized after 14 days. STZ-induced beta-cell dysfunction and apoptosis were associated with increased STAT1/IRF-1 and ATF3 expression and were correlated with elevated IFN-gamma levels. Genetic depletion using IFN-gamma(-/-) or STAT1(-/-) mice strongly inhibited the reduction of islet cell mass or insulin synthesis/secretion and the increase of beta-cell apoptosis observed in STZ-treated wild-type mice. ATF3 overexpression, especially the C-terminal domain, strongly enhanced beta-cell dysfunction and apoptosis by enhancing STAT1 activation and its accumulation, which were abolished with an ATF3-specific siRNA or C-terminal-deleted ATF3. The STZ induction of ATF3 was completely depleted in IFN-gamma(-/-) mice, but not in STAT1(-/-) mice. Furthermore, STAT1 did not affect ATF3 expression, but STAT1 depletion or its inactivation inhibited STZ-induced ATF3 nuclear translocation and beta-cell apoptosis. Interestingly, ATF3 also increased STAT1 transcription by directly binding to a putative binding region (-116 to -96 bp) in the STAT1 promoter. Our results suggest that ATF3 functions as a potent upstream regulator of STAT1 and ATF3 may play a role in STZ-induced beta-cell dysfunction by enhancing the steady state abundance of STAT1. (C) 2010 Elsevier Inc. All rights reserved.