关键词： 重组高密度脂蛋白   炎症反应   脂多糖   RAW264.7   细胞   ATF3  

摘要： 背景:ATF3在抑制炎症信号中通过抑制TLR的产生而发挥重要作用,高密度脂蛋白(HDL)或其主要蛋白成份Apo A-I具有显著的抗炎症作用。本研究探讨载脂蛋白A-I(Apo A-I)及其半胱氨酸突变体重组高密度脂蛋白对ATF3表达量的影响。方法:(1)利用pET原核表达系统表达载脂蛋白A-I74,A-I228,A-Im和A-IWT,亲和层析纯化后,加入DPPC,制作成重组高密度脂蛋白。(2)培养RAW264.7细胞,LPS刺激诱导炎症细胞模型,然后给于重组高密度脂蛋白干预,ELISA试剂盒检测炎症因子IL-6和TNF-α的表达水平,观察细胞形态学的改变,RT-PCR和western blotting检测细胞中转录激活因子ATF3 m RNA和蛋白表达水平的变化。结果:细胞形态学观察发现,与正常组比较,LPS组与rHDL228组细胞形态趋向于巨噬细胞化并出现降解,rHDL74、rHDLM和rHDLWT对LPS刺激的细胞有不同的保护作用。ELISA检测发现,rHDL74、rHDLm和rHDLwt中TNF-ɑ与IL-6的表达水平恢复到接近空白对照组水平,rHDL 228组TNF-ɑ与IL-6水平较较空白对照组明显升高(p<0.01)。rHDL74、rHDLM和rHDLWT中TNF-ɑ与IL-6水平较LPS组明显降低(P<0.01)。Western blotting结果显示,与对照组相比,LPS组与rHDL 228组ATF3的表达水平下降,具有显著性差异(p<0.01)。rHDL74、rHDLM和rHDLWT组表达水平明显升高且差异显著(p<0.05)。结论:重组高密度脂蛋白rHDL74、rHDLWT、rHDLM促进ATF3在m RNA和蛋白水平上的表达。而rHDL228下调其表达。

关键词： Thy-1肾炎(Thy-1N)   亚溶解型C5b-9(sublytic C5b-9)   肾小球系膜细胞(GMC)   p300   早期生长应答基因1(Egr-1)   活化转录因子3(ATF3)   生长阻滞和DNA损伤诱导基因45(Gadd45)   细胞凋亡   sublytic C5b-9   Thy-1N   GMC   染色质步移(genome walking)   mRNA稳定性   启动子活性   细胞凋亡   sublytic C5b-9   p300   Egr-1   ATF3   乙酰化修饰   启动子活性   细胞凋亡  

摘要： 第一部分 Thy-1N肾组织和sublytic C5b-9刺激GMC上调的p300、Egr-1、ATF3和Gadd45基因表达时相及其与GMC凋亡的相关性目的:检查Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠肾组织(体内)和体外亚溶解(sublytic)C5b-9刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)中 p300、早期生长应答基因 1(early growth response gene 1,Egr-1)、活化转录因子 3(activating transcription factor 3,ATF3)及生长阻滞和 DNA 损伤诱导基因 45(growth arrest-and DNA-damage-inducible gene 45,Gadd45)的基因表达情况,并探讨p300、Egr-1、ATF3和Gadd45在sublytic C5b-9诱导GMC凋亡中的作用。方法:复制大鼠Thy-1N模型,同时用sublytic C5b-9刺激体外培养的GMC,收集不同时间点和不同分组的肾组织或GMC,行qPCR和western blot(WB)检查p300、Egr-1、ATF3和Gadd45的表达水平,并分别用电镜和流式细胞术检查肾组织中GMC和体外培养GMC的凋亡情况。然后,构建Egr-1、ATF3和Gadd45 的过表达质粒(pIRES2/Egr-1、pIRES2/ATF3、pIRES2/Gadd45α、pIRES2/Gadd45β 和 pIRES2/Gadd45γ)和发卡状小干扰 RNA(short hairpin RNA,shRNA),即 shEgr-1、shATF3、shGadd45α、shGadd45β 和 shGadd45y 以及 p300的shRNA(shp300),将上述质粒分别转染至大鼠GMC中培养48h,过表达组不予处理,而shRNA组再给予sublytic C5b-9刺激3h,收集细胞用流式细胞术检测细胞凋亡百分比。结果:Thy-1N大鼠肾组织和受sublytic C5b-9刺激的大鼠GMC中,p300、Egr-1、ATF3和Gadd45的表达水平显著上调,其中Egr-1蛋白的高峰时间点在体内和体外分别为2h和1h,而p300、ATF3和Gadd45的高峰时间点在体内和体外均为3h。同时观察到发病大鼠的肾小球中GMC呈明显的凋亡改变,且被sublytic C5b-9刺激的GMC凋亡数量也明显增加。此外,过表达Egr-1、ATF3或Gadd45均能促进GMC凋亡,而沉默p300、Egr-1、ATF3或Gadd45则能显著抑制由sublytic C5b-9诱导的凋亡反应。结论:Thy-1N 大鼠肾组织和 sublytic C5b-9 刺激的 GMC 中,p300、Egr-1、ATF3和Gadd45的表达明显上调,且这些分子对sublytic C5b-9诱导的GMC凋亡有促进作用。第二部分 Sublytic C5b-9诱导GMC上调的Egr-1和ATF3之间的上下游关系及其对Gadd45β/γ基因表达的调控目的:检查由sublytic C5b-9诱导上调的Egr-1和ATF3之间的上下游关系,并探讨Egr-1和ATF3对Gadd45基因表达的调控作用与机制。方法:首先用qPCR和WB检查GMC转染pIRES2/Egr-1或shEgr-1后ATF3基因表达水平的变化,再用同样的方法检查GMC转染pIRES2/ATF3或shATF3后Egr-1基因表达水平的改变。接着,在GMC中共转染pIRES2/Egr-1+shATF3(设 shCTR 对照)或 shEgr-1+pIRES2/ATF3(设 pIRES2 对照),后者再予 sublytic C5b-9刺激3h,用流式细胞术检查GMC的凋亡情况。然后,用染色质步移(genome walking)方法获取大鼠ATF3基因近端启动子序列,并构建其启动子荧光素酶报告质粒pGL3-ATF3,将其分别与pIRES2/Egr-1和shEgr-1共转染至GMC，测定ATF3启动子的荧光素酶活性。此外,为研究Egr-1对ATF3转录后调控的机制,我们将shEgr-1转染至GMC后行sublytic C5b-9和蛋白酶体抑制剂MG132处理,用ATF3或泛素抗体进行免疫沉淀(immunoprecipitation,IP),检测ATF3的泛素化水平;另在shEgr-1转染GMC后行sublytic C5b-9刺激1h,再予放线菌素D(actinomycin D)处理的不同时间点,收集RNA用qPCR测定ATF3 mRNA的稳定性。同时,我们用qPCR和WB检查了 GMC在转染Egr-1和ATF3的过表达质粒或相应shRNA后

关键词： Thy-1肾炎（Thy-1N）   E1A结合蛋白p300（p300）   早期生长应答蛋白-1（Egr-1）   激活转录因子3（ATF3）   生长阻滞及DNA损伤诱导基因45（Gadd45）   Thy-1肾炎（Thy-1N）   慢病毒载体（LV）   E1A结合蛋白p300（p300）   早期生长应答蛋白-1（Egr-1）   激活转录因子3（ATF3）   生长阻滞及DNA损伤诱导基因45（Gadd45）   凋亡   增生  

摘要： 第一部分Thy-1肾炎大鼠肾组织中乙酰基转移酶(p300)、转录因子(Egr-1、ATF3)及凋亡基因(Gadd45α/β/γ)的表达情况目的:检查Thy-1肾炎(Thy-1 nephritis，Thy-1N)大鼠肾组织中E1A结合蛋白p300(E1A binding protein p300,p300)、早期生长应答蛋白-1(early growth response protein-1,Egr-1)、激活转录因子 3(activating transcription factor 3,ATF3)和生长阻滞及DNA损伤诱导基因45(growth arrest and DNA-damage-inducible gene 45,Gadd45)α/β/γ的表达情况,并进一步测定 p300与Egr-1或ATF3的相互作用及Egr-1和ATF3的乙酰化水平。方法:给大鼠尾静脉注射抗胸腺细胞血清(anti-thymocyte serum，ATS)复制Thy-1N模型,在肾炎发病的不同时间点(0h、1h、2h、3h、6h、12h)取大鼠肾皮质,通过实时荧光定量聚合酶链式反应(quantitative real-time PCR，qRCR)和蛋白质印迹(western blot，WB)实验检查Thy-1N大鼠肾组织中p300、Egr-1、ATF3和Gadd45α/β/γ的mRNA和蛋白表达水平。然后,通过免疫共沉淀(immunoprecipitation，IP)和WB检查大鼠Thy-1N发病不同时间点肾组织中p300与Egr1或ATF3的相互作用及Egr1和ATF3的乙酰化水平。结果:Thy-1N大鼠肾组织中,p300、Egr-1、ATF3和Gadd45α/β/γ表达水平均以时间依赖性的方式逐渐升高,且在Thy-1N发病1h和3h时(p300为3h,余下因子为1h)mRNA丰度达到峰值,蛋白水平则在2h和3h时(Egr-1为2h,其余蛋白为3h)达到高峰。此外,实验还发现,Thy-1N大鼠肾组织中p300能够与ATF3发生相互作用,且在3h时作用最为显著,但实验并未测到p300与Egr-1间的相互作用。与此同时,ATF3的乙酰化水平也以时间依赖性的方式升高,并在3h达到高峰,但实验亦未测到Egr-1的乙酰化修饰。结论:在Thy-1N大鼠的肾组织中,p300、Egr-1、ATF3和Gadd45α/β/γ的表达均明显升高,且p300能够与ATF3相互作用并使其发生乙酰化。第二部分沉默肾组织中p300、Egr-1、ATF3和Gadd45β/γ基因后对Thy-1肾炎大鼠GMC凋亡和增生病变的影响目的:研究沉默肾组织中的p300、Egr-l、ATF3和Gadd45β/γ基因后对Thy-1N大鼠GMC凋亡和后续增生病变及尿蛋白分泌的影响。方法:分别包装p300、Egr-l、ATF3和Gadd45β/γ shRNA慢病毒载体,形成LV-shp300、LV-shEgr-1、LV-shATF3 和 LV-shGadd45β/γ,通过侵染细胞实验验证其相应慢病毒滴度,并确定其侵染大鼠GMC的能力。然后,通过大鼠肾动脉灌注术将 LV-shp300、LV-shEgr-1、LV-shATF3 和 LV-shGadd45β/γ 悬液分别导入大鼠两侧肾脏,再经尾静脉注射抗胸腺细胞血清(ATS)复制Thy-1N模型。分别在大鼠注射ATS后2h、3h和7d取大鼠肾组织,并留取7d时的24h尿液。通过蛋白质印迹(WB)检查各组大鼠肾组织中相应p300、Egr-l、ATF3和Gadd45β/γ的蛋白表达水平以验证其干扰效果。在确定了相关蛋白能被成功干扰后再用WB检查沉默肾内p300、Egr-1和ATF3后,其肾组织中凋亡相关蛋白Gadd45β/γ的表达情况。另采用免疫共沉淀(IP)和WB实验测定沉默肾内p300后对Thy-1N大鼠肾组织中ATF3乙酰化水平的影响。此外,利用病理学实验技术检查不同处理的Thy-1N大鼠发病3h及7d时,其肾组织的相关病理学改变,并测定各组大鼠7d时的24h尿蛋白总量。结果:包装的 LV-shp300、LV-shEgr-1、LV-shATF3 和 LV-shGadd45β/y 能够侵染大鼠GMC。利用大鼠肾动脉灌注术将LV-shRNAs注入大鼠肾脏,沉默相应p300、Egr-1和ATF3后,Thy-1N大鼠肾组织中凋亡相关蛋白Gadd45β/γ的表达水平显著降低。同样,在干扰了 p300后,Thy-1N大鼠肾组织中的ATF3乙酰化水平也明显下调。此外,沉默相应p300、Egr-1、ATF3和Gadd45β/y后,Thy-1N大鼠发病3h时,其肾组织内肾小球的凋亡细胞数大量减少,并在Thy-1N大鼠发病第7d时,肾组织内GMC增生和细胞外基质(ECM

关键词： 尿道下裂   ATF3   包皮  

摘要： 目的:检测散发性单纯性尿道下裂患者包皮组织中激活转录因子3(Activating transcription factor 3,ATF3)的表达情况,探索ATF3与散发性单纯性尿道下裂之间的关系。方法:1、标本收集:实验组标本为散发性单纯性尿道下裂患者在尿道成形术中的多余的包皮组织(共35例,其中轻度10例,中度15例,重度10例),对照组标本为单纯包皮过长(包括包茎)患者在包皮环切术中去除的多余的包皮组织(共20例)。2、通过反转录聚合酶链反应(RT-PCR)方法检测实验组和对照组ATF3mRNA的表达情况。3、通过免疫组织化学方法检测实验组和对照组ATF3蛋白的表达情况。4、统计学处理:实验过程中采集的数据均进行了严格检查,重复测量对偏出均值3个标准差的数据进行删除。采用SPSS12.0统计学软件进行数据录入,构建数据库。计数资料使用(均值±标准差)的形式表示,计量资料之间的比较采用F/t检验。计数资料用阳性例数或阳性率(%)表示,阳性率之间的比较用卡方检验。检验水准α取值0.05,检验概率为双侧概率。结果:通过RT-PCR方法测得散发性单纯性尿道下裂患者包皮组织中ATF3 mRNA的相对表达量为:0.54±0.06,对照组的包皮组织中ATF3 mRNA的相对表达量为:0.44±0.07,从结果中可以得出在散发性单纯性尿道下裂患者的包皮组织中ATF3的表达情况比正常的包皮组织中明显增加,两组结果比较(t=5.89,P=0.00),差异有统计学意义。比较不同严重程度中尿道下裂患者ATF3mRNA相对表达量,不同严重程度相对表达量差异并不显著(F=0.13,P=0.88)。通过免疫组织化学方法测得ATF3蛋白在散发的单纯性尿道下裂中的阳性表达率为:85.7%(30/35),对照组中阳性率为:15.0%(3/20),两组人群比较,ATF3蛋白阳性表达率差异显著(χ2=7.95,P=0.01),具有统计学意义。比较不同严重程度中尿道下裂患者免疫组化检测ATF3蛋白阳性表达率情况发现,不同严重患者ATF3蛋白阳性表达率呈现递增关系,但是统计学分析差异不明显(χ2=0.65,P=0.72)。结论:在散发性单纯性尿道下裂患者的包皮组织中ATF3的表达比单纯包皮过长(包括包茎)患者的包皮组织中明显增多,提示ATF3和散发性单纯性尿道下裂存在着密切的关系。为将来研究ATF3在尿道下裂中产生的相关信号转导通路提供相关实验基础,及研究尿道下裂的发病机制提供理论依据。

关键词： Activating Transcription Factor 3/biosynthesis   Active Transport, Cell Nucleus   Animals   Aorta/metabolism   Apoptosis   Caspase 3/metabolism   Cells, Cultured   Endothelial Cells/metabolism   Humans   Lipolysis   Lipoproteins/genetics   Lipoproteins/metabolism   Mice   Phosphorylation   Receptors, Transforming Growth Factor beta/metabolism   RNA, Messenger/genetics   RNA, Messenger/metabolism   Signal Transduction   Smad2 Protein/metabolism   Smad4 Protein/metabolism   Stress, Physiological   Transforming Growth Factor beta1/metabolism   Triglycerides/genetics   Triglycerides/metabolism   Tumor Suppressor Protein p53/metabolism  

摘要： Studies have suggested a link between the transforming growth factor beta 1 (TGF-beta 1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-beta at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 mu M), a specific inhibitor of TGF-beta-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-beta 1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-beta and lipolysis product-induced apoptosis is dependent on TGF-beta signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-beta signaling suggesting that TGF-beta signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

关键词： VENTRICULAR remodeling   HEART cells   MACROPHAGES   CARDIAC hypertrophy   PHENYLEPHRINE (Drug)   GENE expression  

关键词： VENTRICULAR remodeling   MACROPHAGES   MANUSCRIPTS   PHENYLEPHRINE (Drug)   CARDIAC hypertrophy   MICE as laboratory animals  

关键词： Macrophage activation   PHYSIOLOGY   Transcription factors -- Physiological aspects   Interleukin receptors -- Therapeutic use   Interleukins -- Therapeutic use   Macrophages  

摘要： Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-beta expression, as well as the magnitude of IFN-beta cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-beta via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Additionally, we observed that ATF3 itself is a type I IFN-inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFN beta, as well as the expression of genes downstream of IFN signaling.

关键词： Pressure overload   Cardiac remodeling   Macrophages   Bone marrow transplantation   Cardiac hypertrophy  

摘要： Rationale: Pressure overload induces adaptive remodeling processes in the heart. However, when pressure overload persists, adaptive changes turn into maladaptive alterations leading to cardiac hypertrophy and heart failure. ATF3 is a stress inducible transcription factor that is transiently expressed following neuroendocrine stimulation. However, its role in chronic pressure overload dependent cardiac hypertrophy is currently unknown. Objective: The objective of the study was to study the role of ATF3 in chronic pressure overload dependent cardiac remodeling processes. Methods and results: Pressure overload was induced by phenylephrine (PE) mini-osmotic pumps in various mice models of whole body, cardiac specific, bone marrow (BM) specific and macrophage specific ATF3 ablations. We show that ATF3-KO mice exhibit a significantly reduced expression of cardiac remodeling markers following chronic pressure overload. Consistently, the lack of ATF3 specifically in either cardiomyocytes or BM derived cells blunts the hypertrophic response to PE infusion. A unique cross-talk between cardiomyocytes and macrophages was identified. Cardiomyocytes induce an ATF3 dependent induction of an inflammatory response leading to macrophage recruitment to the heart. Adoptive transfer of wild type macrophages, but not ATF3-KO derived macrophages, into wild type mice potentiates maladaptive response to PE infusion. Conclusions: Collectively, this study places ATF3 as a key regulator in promoting pressure overload induced cardiac hypertrophy through a cross-talk between cardiomyocytes and macrophages. Inhibiting this cross-talk may serve as a useful approach to blunt maladaptive remodeling processes in the heart. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

关键词： Chaetocin   SUV39H1   CHOP   ATF3   DR5   Apoptosis  

摘要： Epigenetic abnormalities are associated with non-small cell lung cancer (NSCLC) initiation and progression. Epigenetic drugs are being studied and in clinical trials. However, the molecular mechanism underlying the apoptosis by the epigenetic agents remains unclear. SUV39H1 is an important methyl-transferase for lysine 9 on histone H3 and usually related to gene transcriptional suppression, and chaetocin acts as the inhibitor of SUV39H1. We demonstrated here that chaetocin effectively suppressed the growth of multiple lung cancer cells through inducing apoptosis in a death receptor 5 (DR5)-dependent manner. Chaetocin treatment activated endoplasmic reticulum (ER) stress which gave rise to the up-regulation of ATF3 and CHOP. Furthermore, ATF3 and CHOP contributed to the induction of DR5 and subsequent apoptosis. When SUV39H1 was silenced with siRNA, the expression of ATF3, CHOP and DR5 was elevated. Thereafter, knockdown of SUV39H1 induced apoptosis in NSCLC cells. In summary, chaetocin pharmacologically inhibits the activity of SUV39H1 which provokes ER stress and results in up-regulation of ATF3 and CHOP, leading to DR5-dependent apoptosis eventually. These findings provide a novel interpretation on the anti-neoplastic activity of epigenetic drugs as a new therapeutic approach in NSCLC.