关键词： Excitotoxicity   Neuroprotection   Plasminogen   Tissue-type plasminogen activator  

摘要： Abstract: The release of the serine proteinase tissue-type plasminogen activator (tPA) from cerebral cortical neurons has a neuroprotective effect in the ischemic brain. Because excitotoxicity is a basic mechanism of ischemia-induced cell death, here we investigated the effect of tPA on excitotoxin-induced neuronal death. We report that genetic overexpression of neuronal tPA or treatment with recombinant tPA renders neurons resistant to the harmful effects of an excitotoxic injury in vitro and in vivo. We found that at concentrations found in the ischemic brain, tPA interacts with synaptic but not extrasynaptic NMDARs. This effect is independent of tPA''s proteolytic properties and leads to a rapid and transient phosphorylation of the extracellular signal regulated kinases1/2 (ERK1/2), with ERK1/2-mediated activation of the cAMP response element binding protein (CREB) and induction of the neuroprotective CREB-regulated activating transcription factor 3 (Atf3). In line with these observations, Atf3 down-regulation abrogates the protective effect of tPA against excitotoxin-induced neuronal death. Our data indicate that tPA preferentially activates synaptic NMDARs via a plasminogen-independent mechanism turning on a cell signaling pathway that protects neurons from the deleterious effects of excitotoxicity. [Copyright &y& Elsevier]

关键词： hemin   HK-2 renal proximal tubular cell   microarray   ATF3   siRNA  

摘要： Objective: To identify gene expression changes and the role of activating transcription factor 3 (ATF3) in hemin toxicity in renal tubular epithelial cells, then elucidate molecular mechanisms of hemin toxicity on renal tubular epithelial cells. Methods: An oligo array comprising 35,035 genes was used to compare differential gene expression in hemin-treated and non-treated HK-2 cells (human renal proximal tubular epithelial cells), and the role of ATF3 in hemin toxicity was assessed using siRNA technique. Results: A total of 128 mRNAs were at least twofold up-regulated and 101 mRNAs were at least twofold down-regulated after hemin treatment. Expression levels of ATF3, heat shock protein 70, c-fos, and c-jun were remarkably increased. Hemin also suppressed nuclear factor-kappa B inhibitor alpha, beta-2 adrenergic receptor, and interleukin-6 mRNA amounts more than twofold. We further demonstrated the protective role of ATF3 in hemin cytotoxicity. Conclusions: The data suggest that hemin caused multiple changes of gene expression in HK-2 cells, and ATF3 protects against hemin cytotoxicity.

关键词： 前列腺癌   双联苄化合物   蛋白酶体   自噬性死亡   ATF3  

摘要： 前列腺癌(prostate cancer, PCa)是欧美国家老年男性最常见的恶性肿瘤,其死亡率仅次于肺癌。虽然我国前列腺癌的发病率明显低于西方国家,但随着人口老龄化、饮食结构的改变等,我国前列腺癌发病率,特别是晚期前列腺癌的发病率呈显著上升趋势。据预计10年后,前列腺癌在我国的发病率可能会超过60-70/10万,已成为威胁我国老年男性健康的恶性疾病之一。 前列腺癌是激素依赖性肿瘤,因此内分泌治疗是治疗前列腺癌的首选标准方案。然而绝大多数前列腺癌患者经内分泌治疗1-2年后易发展为激素难治性前列腺癌(Hormone Refractory Prostate Cancer,HRPC),对雄激素撤除不敏感,抵抗凋亡并伴随侵袭转移,预后差,死亡率高,目前临床尚无有效的治疗方法。由于HRPC的演变是复杂的多因素、多信号途径参与的过程,其机制尚不完全清楚,因此寻找有效的治疗靶点及机制,产生多种途径的协同效应,是治疗前列腺癌的有效策略之一。 第一部分地钱素M抑制蛋白酶体的活性,诱导细胞内质网应激和凋亡 泛素-蛋白酶体系统(Ubiquitin-proteasome system, UPS)是真核生物细胞内重要的蛋白质质量控制体系之一。UPS通过将小分子蛋白泛素与靶蛋白的赖氨酸残基共价结合,从而将其泛素化。泛素标记的蛋白被蛋白酶体特异性地识别并迅速降解。UPS通常降解受损变性蛋白、短寿命的调控蛋白等,对于细胞周期运转、凋亡与分化、信号转导等生理过程起着重要的调控作用。恶性肿瘤细胞内蛋白代谢紊乱,蛋白酶体活性升高,UPS调节失衡,使得肿瘤细胞更加依赖UPS,以维持细胞内稳态。因此,肿瘤细胞对蛋白酶体抑制剂更加敏感,蛋白酶体也成为肿瘤治疗的新靶点,开发针对UPS的抑制剂在肿瘤治疗中具有广阔的前景,而天然化合物以其独特的结构、多样的生物学活性更是备受关注。 双联苄(Bisbibenzyls)是苔藓植物中分离得到的一类大环多酚类化合物,因联苄之间的连接方式不同、取代基不同而具有结构新颖、多样的特点,具有抗真菌、抗炎、抗肿瘤等多种生物活性。本课题组前期研究发现双联苄类化合物地钱素M(Marchantin M,Mar)显著抑制前列腺癌细胞的增殖、促进其凋亡。由于地钱素M的结构与茶多酚的功能区域相似,而茶多酚已被证明是蛋白酶体的不可逆抑制剂。我们推测地钱素M可能具有抑制蛋白酶体活性的功能。为此,我们研究了地钱素M对重组纯化的20S蛋白酶体、前列腺癌细胞内蛋白酶体活性的影响,并对其生物学效应进行了分析,取得了以下研究结果： 一、地钱素M显著抑制蛋白酶体活性,是新的可逆性蛋白酶体抑制剂 1.地钱素M显著抑制蛋白酶体胰凝乳样蛋白酶(ChT-L)和肽基谷氨酰多肽解酶(PGPH)活性。Mar显著抑制重组纯化的20S蛋白酶体中ChT-L和PGPH活性,呈剂量依赖性。其半数抑制率分别为6.99和5.33μM。但不影响胰蛋白酶样活性。细胞实验表明,地钱素M对激素非依赖性前列腺癌PC3,DU145细胞和激素依赖性前列腺癌LNCaP细胞内26S蛋白酶体显示同样的抑制作用,且呈时间和剂量依赖性。表达差异谱基因芯片数据显示,地钱素M并不影响UPS途径中各组分的基因表达。 2.地钱素M是新的可逆性蛋白酶体抑制剂。通过Autodock vina软件分析发现,地钱素M能与蛋白酶体的β1(PGPH)和β5(ChT-L)亚基结合。二者主要依靠氢键、疏水键等非共价键相互作用,其自由能分别为：-7.4kcal/mol和-6.5kcal/mol。林-贝氏双倒数作图法表明,地钱素M对蛋白酶体的抑制呈现较典型的非竞争性抑制的方式。上述结果表明,地钱素M是一种新的蛋白酶体可逆的抑制剂。 3.地钱素M抑制蛋白酶体活性,导致前列腺癌细胞内多泛素化蛋白累积,且呈时间、剂量依赖性。 二、地钱素M抑制蛋白酶体活性,导致内质网相关蛋白降解途径(ERAD)受阻,诱导内质网应激和细胞凋亡 1.地钱素M介导的蛋白酶体抑制,抑制ERAD,诱导内质网应激。地钱素M处理前列腺癌细胞后,导致经ERAD降解的蛋白如突变蛋白CFTRAF508、SPCA4在内质网堆积；电镜下观察到内质网肿胀,激光共聚焦结果显示高浓度的地钱素M可引起内质网空泡化；Western blotting结果表明,内质网伴侣蛋白GRP78表达显著上调、PERK及其下游eIF2α磷酸化水平升高；定量PCR分析显示应激分子***4及其靶基因ATF3表达显著增加；IRE1激活并剪接XBP1的mRNA,激活XBP1。上述结果表明,地钱素M抑制蛋白酶体活性导致内质网内未折叠或错误折叠的蛋白累积,诱导内质网应激。 2.地钱素M诱导前列腺癌细胞凋亡。地钱素M呈剂量依赖性抑制激素依赖

关键词： ANALYSIS of variance   ANIMAL experimentation   GENE expression   GENES   INTERLEUKINS   MICE   PROTEINS   RESEARCH -- Finance   RNA   SEPTICEMIA   T-test (Statistics)   TUMOR necrosis factor   LIPOPOLYSACCHARIDES   DESCRIPTIVE statistics   KAPLAN-Meier estimator  

摘要： Lipopolysaccharide (LPS) triggers innate immunity mainly via TLR4 signaling. ATF3 is a negative regulator of TLR4 signaling. HMGB1 plays a critical role in the final step of sepsis. However, the mechanisms of ATF3 and the role of HMGB1 in regulating innate immunity-induced sepsis are incompletely understood. In this study, we found that serum HMGB1 levels were 10-fold higher in patients with sepsis than normal controls. We further demonstrated that ATF3 gene knockout in mice subjected to LPS-induced endotoxemia correlates with an increase in the mortality rate and the elevated expression of IL-6, TNF-alpha, NO, MCP-1, and HMGB1 in the lung tissues or serum. The biochemical effects of ATF3 were observed in in vitro macrophages and blocked by ATF3 siRNA treatment. We have also shown that adeno-associated virus-mediated ATF3 gene transfer protected ATF3 knockout mice from LPS-induced mortality. In addition, ATF3 knockdown increased LPS-induced release of HMGB1. In conclusion, upregulation of ATF3 contributes to the reduced release of inflammatory molecules, especially HMGB1, which induced lung injury and increased the survival rate of mice after LPS challenge. Therefore, suppressing LPS-induced inflammation with ATF3 induction or ATF3 mimetics may be an important strategy for sepsis therapy.

关键词： COX-2   肝星状细胞   Atf3   Serpinb2   Pgk1   Thra  

摘要： 目的 研究靶向COX-2shRNA对大鼠HSC-T6COX-2的抑制作用，观察Atf3、Serpinb2、Pgk1、Thra基因表达的影响，进一步探讨COX-2shRNA抑制HSC增殖的作用机制。 方法 实验将HSC-T6分为三组，空白对照组（以PBS液替代质粒），空载体组（未插入目的基因的DNA片段，HK质粒组）及转染组（pYr-1.1-hU6-EGFP-COX-2shRNA1组），利用LipofectamineTM2000将质粒转染入大鼠肝星状细胞，在荧光显微镜下观察其转染效率，然后提取转染后24h，48h，72h各组细胞中RNA，应用RT-PCR技术检测各组各时间段COX-2、Atf3、Serpinb2、Pgk1、Thra基因表达情况。 结果 1、荧光显微镜下观察结果：COX-2shRNA1转染大鼠肝星状细胞后24h、48h、72h，荧光显微镜下观察，转染成功的细胞质中可见绿色的荧光，转染效率均可达70%以上，空白组与空载体组细胞质中未见绿色荧光。 2、转染后COX-2表达的影响：应用RT-PCR方法检测转染后24h、48h、72h转染组COX-2mRNA的表达都低于空白对照组和空载体组（P<0.05），其中以72h抑制最为明显，空白对照组和空载体组无沉默效应。 3、转染后Atf3、Serpinb2、Thra表达的影响：应用RT-PCR方法检测转染后24h、48h、72h转染组Atf3、Serpinb2mRNA的表达都低于空白对照组和空载体组（P<0.05），其中以72h抑制最为明显，空白对照组和空载体组无沉默效应。 4、转染后Pgk1表达的影响：应用RT-PCR方法检测转染后24h转染组Pgk1mRNA的表达与空白对照组和空载体组之间无差异（P﹥0.05），48h、72h低于空白对照组和空载体组（P<0.05），以72h抑制最为明显，空白对照组和空载体组无沉默效应。 结论 1.靶向COX-2shRNA转染入大鼠HSC-T6可沉默HSC中COX-2的表达。 2.靶向COX-2shRNA可以下调HSC增殖相关基因Serpinb2的表达。 3.靶向COX-2shRNA可以下调HSC脂肪代谢相关基因Pgk1、Thra的表达。 4.靶向COX-2shRNA抑制HSC增殖可能通过调节Serpinb2、Pgk1、Thra等基因的表达起作用。

关键词： 结直肠肿瘤/病理学   ATF3   CyclinD1   Maspin   免疫组织化学  

摘要： 目的:检测转录激活因子3(ATF3)及其靶基因Cyclin D1与Maspin在结直肠癌组织芯片中的表达,探讨ATF3表达在结直肠癌发生、发展中的意义。方法:构建含结直肠癌及癌旁相对正常肠黏膜的组织芯片,应用免疫组织化学检测ATF3、Cyclin D1及Maspin的表达情况。结果:ATF3、Cyclin D1与Maspin在结直肠癌中阳性表达率分别为70.3%、51.4%和59.5%,明显高于癌旁正常组织的21.6%、2.7%和13.5%,差异有统计学意义,χ2值分别为35.252、44.411和33.689,P值均为0.000;ATF3与结直肠癌患者的性别、年龄、肿瘤部位、肿瘤大小及肿瘤分化程度无关,而与浸润程度和淋巴结有无转移有关,浆膜层表达(83.3%)高于肌层(38.9%),有淋巴结转移(92.3%)高于无淋巴结转移(45.7%),χ2值分别为13.290和17.110,P值均为0.000;Cyclin D1与结直肠癌患者的性别、年龄、肿瘤部位及肿瘤大小无关,而与病理学分级、浸润程度和淋巴结有无转移有关,低分化组织表达(71.4%)高于高中分化组织(39.1%),浆膜层表达(61.1%)高于肌层(28.8%),有淋巴结转移(69.2%)高于无淋巴结转移(31.4%),χ2值分别为7.268、6.019和10.550,P值分别为0.007、0.014和0.001;Maspin与结直肠癌患者的性别、年龄、肿瘤部位、肿瘤大小、肿瘤分化程度、浸润深度及有无淋巴结转移均无关,P值均>0.05。ATF3、Cyclin D1与Maspin在结直肠癌中均高表达,ATF3与Cyc-lin D1在结直肠癌中表达呈正相关(r=0.362,P=0.000),与Maspin表达无明显相关(r=0.075,P=0.513)。结论:ATF3表达与结直肠癌的发生密切相关,ATF3可能通过调控Cyclin D1表达而促进结直肠癌的发生发展及侵袭转移。

关键词： HDAC   bZIP   JDP2   ATF3   Histone acetylation   Repressor  

摘要： JDP2, is a basic leucine zipper (bZIP) protein displaying a high degree of homology with the stress inducible transcription factor, ATF3. Both proteins bind to cAMP and TPA response elements and repress transcription by multiple mechanisms. Histone deacetylases (HDACs) play a key role in gene inactivation by deacetylating lysine residues on histones. Here we describe the association of JDP2 and ATF3 with HDACs 1, 2-6 and 10. Association of HDAC3 and HDAC6 with JDP2 and ATF3 occurs via direct protein-protein interactions. Only part of the N-terminal bZIP motif of JDP2 and ATF3 basic domain is necessary and sufficient for the interaction with HDACs in a manner that is independent of coiled-coil dimerization. Class I HDACs associate with the bZIP repressors via the DAC conserved domain whereas the Class IIb HDAC6 associates through its C-terminal unique binder of ubiquitin Zn finger domain. Both JDP2 and ATF3 are known to bind and repress the ATF3 promoter. MEF cells treated with histone deacetylase inhibitor, trichostatin A (TSA) display enhanced ATF3 transcription. ATF3 enhanced transcription is significantly reduced in MEF cells lacking both ATF3 and JDP2. Collectively, we propose that the recruitment of multiple HDAC members to JDP2 and ATF3 is part of their transcription repression mechanism. (C) 2012 Elsevier B.V. All rights reserved.

关键词： Gene Regulation   Gene Transcription   Histidine   Histone Deacetylase Inhibitors   Nutrition   ATF3   Asparagine Synthetase   Dietary Protein   Histone Demethylase   Nutrient Sensing  

摘要： Following amino acid deprivation, the amino acid response (AAR) induces transcription from specific genes through a collection of signaling mechanisms, including the GCN2-eIF2-ATF4 pathway. The present report documents that the histone demethylase JMJD3 is an activating transcription factor 4 (ATF4)-dependent target gene. The JMJD3 gene contains two AAR-induced promoter activities and chromatin immunoprecipitation (ChIP) analysis showed that the AAR leads to enhanced ATF4 recruitment to the C/EBP-ATF response element (CARE) upstream of Promoter-1. AAR-induced histone modifications across the JMJD3 gene locus occur upon ATF4 binding. Jmjd3 transcription is not induced in Atf4-knock-out cells, but the AAR-dependent activation was rescued by inhibition of histone deacetylation with trichostatin A (TSA). The TSA rescue of AAR activation in the absence of Atf4 also occurred for the Atf3 and C/EBP homology protein (Chop) genes, but not for the asparagine synthetase gene. ChIP analysis of the Jmjd3, Atf3, and Chop genes in Atf4 knock-out cells documented that activation of the AAR in the presence of TSA led to specific changes in acetylation of histone H4. The results suggest that a primary function of ATF4 is to recruit histone acetyltransferase activity to a sub-set of AAR target genes. Thus, absolute binding of ATF4 to these particular genes is not required and no ATF4 interaction with the general transcription machinery is necessary. The data are consistent with the hypothesis that ATF4 functions as a pioneer factor to alter chromatin structure and thus, enhance transcription in a gene-specific manner.

关键词： BIOCHEMICAL markers   ADENYLATE kinase   LUNGS -- Cancer   CANCER invasiveness   METASTASIS   TRANSCRIPTION factors  

摘要： Biomarkers predicting metastatic capacity might assist the development of better therapeutic strategies for aggressive cancers such as lung cancer. In this study, we show that adenylate kinase-4 (AK4) is a progression-associated gene in human lung cancer that promotes metastasis. Analysis of published microarray data showed that AK4 was upregulated in lung adenocarcinoma compared with normal cells. High AK4 expression was associated with advanced stage, disease recurrence and poor prognosis. Loss of AK4 expression suppressed the invasive potential of lung cancer cell lines, whereas AK4 overexpression promoted invasion in vitro and in vivo. Mechanistically, the transcription factor ATF3 was identified as a pivotal regulatory target of AK4. Simultaneous reduction in AK4 and ATF3 expression abolished the inhibitory effects of ATF3 on invasion. ATF3 overexpression in AK4-overexpressing cells limits invasion activity. Furthermore, patients with high AK4 and low ATF3 expression showed unfavorable outcomes compared with patients with low AK4 and high ATF3 expression. Taken together, our findings indicated that AK4 promotes malignant progression and recurrence by promoting metastasis in an ATF3-dependent manner. Cancer Res;72(19);5119-29. (C)2012 AACR.

关键词： TRPV1   Pain   Capsaicin   Vanilloid   Nociception   Resiniferatoxin   ATF3   Dorsal root ganglion   MRS1477   Adelta fiber  

摘要： Background: The prevalence of long-term opiate use in treating chronic non-cancer pain is increasing, and prescription opioid abuse and dependence are a major public health concern. To explore alternatives to opioid-based analgesia, the present study investigates a novel allosteric pharmacological approach operating through the cation channel TRPV1. This channel is highly expressed in subpopulations of primary afferent unmyelinated C-and lightly-myelinated Ad-fibers that detect low and high rates of noxious heating, respectively, and it is also activated by vanilloid agonists and low pH. Sufficient doses of exogenous vanilloid agonists, such as capsaicin or resiniferatoxin, can inactivate/deactivate primary afferent endings due to calcium overload, and we hypothesized that positive allosteric modulation of agonist-activated TRPV1 could produce a selective, temporary inactivation of nociceptive nerve terminals in vivo. We previously identified MRS1477, a 1,4-dihydropyridine that potentiates vanilloid and pH activation of TRPV1 in vitro, but displays no detectable intrinsic agonist activity of its own. To study the in vivo effects of MRS1477, we injected the hind paws of rats with a non-deactivating dose of capsaicin, MRS1477, or the combination. An infrared diode laser was used to stimulate TRPV1-expressing nerve terminals and the latency and intensity of paw withdrawal responses were recorded. qRT-PCR and immunohistochemistry were performed on dorsal root ganglia to examine changes in gene expression and the cellular specificity of such changes following treatment. Results: Withdrawal responses of the capsaicin-only or MRS1477-only treated paws were not significantly different from the untreated, contralateral paws. However, rats treated with the combination of capsaicin and MRS1477 exhibited increased withdrawal latency and decreased response intensity consistent with agonist potentiation and inactivation or lesion of TRPV1-containing nerve terminals. The loss of n