关键词： DUSP1   缺氧   RAW264.7   炎症反应   ATF3  

摘要： 研究背景:高原病(high altitude disease,HAD)是由高原低压缺氧引起的一种高原特发性疾病,根据发病急缓分为急性高原病和慢性高原病两大类。近年来的研究显示,缺氧诱导的炎症反应参与高原病的发生发展,缺氧引起的炎性血管反应是急性高原病的重要机制,而缺氧诱导的炎性增生则是慢性高原病的重要机制。巨噬细胞是一种重要的固有免疫细胞,广泛存在于机体不同的器官组织中,执行多种不同的功能,包括对抗入侵的病原体和肿瘤细胞、协调伤口愈合、参与获得性免疫应答中的抗原提呈等。许多巨噬细胞参与的病理过程(如炎症、伤口愈合、动脉粥样硬化和肿瘤)也都以缺氧为特征。巨噬细胞对缺氧十分敏感,被激活的巨噬细胞可通过表达和分泌某些炎症细胞因子和趋化因子,活化和招募其它炎症细胞,从而参与缺氧引起的组织和机体损伤或重塑。研究显示,缺氧诱导的巨噬细胞炎症反应与高原病的发生发展密切相关。因此,深入认识缺氧诱导的巨噬细胞炎症反应及其调控机制,对于进一步阐明高原病的发病机制和研发防治药物具有重要意义。目前为止,缺氧诱导的巨噬细胞炎症反应调控的具体机制仍不完全清楚。双特异性磷酸酶1(Dual specificity phosphatase 1,DUSP1),也被称作丝裂素活化蛋白激酶磷酸酶1(Mitogen-activated Protein Kinase Phosphatase-1,MKP-1),是DUSP家族中最早被鉴定并且也是其中最重要的成员。DUSP1主要催化细胞内已激活的丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)家族成员(p38、JNK与ERK)中特异性基序TXY磷酸基团的水解,从而抑制它们的活性,进而阻断其下游相关生物学反应。Dusp1基因最初是作为在培养的小鼠细胞G0/G1转化过程中表达的一组基因之一而被鉴定的,后来的研究发现,多种应激因素如紫外线、病原体蛋白、缺氧等可以诱导DUSP1表达上调。本研究室前期对平原人急进5300m高原前后全血转录组学的研究结果显示,进入高原后DUSP1的转录水平表达较进入高原前明显升高。关于进入高原后DUSP1表达升高在高原病发生发展中的生物学意义尚并不清楚。鉴于DUSP1在负调控TLR配体诱导的巨噬细胞炎症反应中的重要作用及缺氧诱导的巨噬细胞炎症反应在高原病发生发展中的重要作用,DUSP1可能通过调控缺氧诱导的巨噬细胞炎症反应从而参与到高原病发生发展的调节中。目前关于缺氧引起DUSP1表达升高的机制以及DUSP1是否参与调控缺氧诱导的巨噬细胞炎症反应尚不清楚。采用生物信息学分析的结果显示,活化转录因子3(Activating transcription factor 3,ATF3)可能是调节Dusp1表达的关键分子。因此,我们推测缺氧巨噬细胞可能通过转录因子ATF3上调DUSP1的表达,进而调节缺氧RAW264.7巨噬细胞中部分炎症相关细胞因子的表达,从而防止自身过度炎症免疫反应。本研究使用炎症反应研究中广泛使用的小鼠RAW264.7巨噬细胞建立缺氧巨噬细胞模型,初步探讨DUSP1在缺氧巨噬细胞中的表达机制及其在缺氧诱导的巨噬细胞炎症免疫反应调控中的作用,旨在为高原缺氧损伤的发病机制及防治提供新的思路及部分理论和实验依据。方法:以小鼠RAW264.7巨噬细胞作为研究模型。根据不同的实验目的,分为常氧(21%O)组、缺氧(1%O)组;缺氧+干扰序列对照组(si-C)、缺氧+siRNA组。缺氧组细胞置于缺氧工作站(1%O)中培养,常氧(21%O)组则置于普通培养箱中培养。采用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)、蛋白质印迹法(Western blot,WB)检测缺氧(1%O2)处理不同时间RAW264.7巨噬细胞中DUSP1和ATF3的mRNA和蛋白表达水平。采用qRT-PCR和酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)检测缺氧(1%O)处理不同时间RAW264.7巨噬细胞中炎症相关细胞因子IL-1β、IL-10和TNF-α的mRNA和分泌水平。采用RNA干扰技术干扰Dusp1表达,采用qRT-PCR、WB检测干扰效果;采用qRT-PCR和ELISA检测转染si-Dusp1后缺氧(1%O)处理24h RAW264.7巨噬细胞中IL-1β、IL-10和TNF-α的mRNA和分泌水平。采用RNA干扰技术干扰Atf3表达,采用qRT-PCR、WB检测干扰效果;采用qRT-PCR和WB检测转染si-Atf3后缺氧(1%O)处理24h RAW264.7巨噬细胞中DUSP1的mRNA和蛋白表达水平。采用qRT-PCR和ELISA检测转染si-Atf3后缺氧(1%O

关键词： 脂蛋白脂肪酶   藻蓝蛋白   miR-10a-5p   TAK1   JNK   ATF3  

摘要： [背景与目的]动脉粥样硬化（As）是由脂质代谢紊乱等多种因素引起的一种血管退行性病变,严重危害人类健康。在As众多的发病机理中,巨噬细胞源性泡沫细胞的形成构成其核心环节。因此,减少巨噬细胞源性泡沫细胞形成对As的防治具有重要意义。以往研究表明,脂蛋白脂酶（LPL）是水解血浆甘油三酯的关键限速酶,对降低血浆甘油三酯水平至关重要。然而,LPL在巨噬细胞中却通过介导细胞内脂质蓄积促进泡沫细胞形成,从而发挥促As的作用。探索调控LPL表达的作用机制,减少巨噬细胞脂质蓄积,对于防治As具有重要意义。藻蓝蛋白（PC）是一种存在于藻类中的色素蛋白,在螺旋藻中的含量约为10%20%,常温下结构稳定。研究表明,PC在抗氧化、抗肿瘤、免疫应答等方面发挥着多种生物学功能,但其是否通过减少巨噬细胞脂质蓄积从而影响泡沫细胞形成的机制尚未阐明。丝裂原活化蛋白激酶激酶激酶7（TAK1）,能够调节细胞的增殖与凋亡,介导多种促炎症和促存活信号通路的激活,参与As的病理进程。应激活化蛋白激酶（JNK）是位于TAK1下游的因子,TAK1表达升高可增加磷酸化JNK水平,从而引起JNK下游因子的激活或抑制。转录激活因子3（ATF3）,是ATF/环磷腺苷效应元件结合蛋白转录因子家族的成员,位于JNK下游,它是富含甘油三酯的脂蛋白脂解产物的关键反应基因,能够诱导促炎因子聚集,促进As的发生。然而,TAK1/JNK/ATF3信号途径是否参与PC调控巨噬细胞LPL表达及脂质蓄积的机制尚不清楚。miR-10a-5p是新近发现的一个RNA分子,与肿瘤的发生、炎症反应密切相关。研究发现,miR-10a-5p能够靶向结合TAK1,从而增加As斑块的面积,加速As的发生。但miR-10a-5p是否参与PC调控巨噬细胞LPL表达及脂质蓄积的机制尚不明确。因此,本研究的目的是从脂质代谢的角度,探究PC对巨噬细胞LPL表达及脂质蓄积的影响机制。[方法]THP-1细胞置于10%胎牛血清（PBS）的RPMI 1640培养基（含10U/mL青霉素和10U/mL链霉素）中培养,将细胞培养箱温度保持在37℃,同时保证箱内5%的CO2浓度。用160nmol/L的佛波醇（PMA）孵育THP-1细胞48小时,促进其分化,转变为巨噬细胞,50μg/ml的氧化低密度脂蛋白（ox-LDL）无血清培养基处理细胞。首先,PC（5、10、20μg/ml）或PBS孵育巨噬细胞6、12、24小时,然后用RT-PCR和Western Blot检测LPL mRNA及表达,LPL活性比色法定量检测试剂盒检测LPL活性,高效液相色谱（HPLC）用于检测细胞内脂质含量变化情况;染色质免疫共沉淀（ChIP）和荧光素酶报告基因（Luciferase）分别检测PC对转录因子ATF3与LPL启动子区结合以及LPL启动子活性的影响,RT-PCR和Western Blot分别检测PC对ATF3 mRNA及表达,LPL mRNA及表达的影响;接着,使用TAK1过表达质粒,JNK激活剂茴香霉素和ATF3过表达质粒处理后,RT-PCR和Western Blot分别检测PC对TAK1 mRNA及表达、JNK及其磷酸化水平、ATF3 mRNA及表达,LPL mRNA及表达的影响,验证PC是否通过TAK1/JNK/ATF3信号通路影响LPL的表达。最后,利用生物信息学分析手段预测并比对不同物种间miR-10a-5p的序列、保守性及其靶标基因,以此为基础确定miR-10a-5p与TAK1存在结合位点,借助MicroRNA靶标预测网站对两者的结合自由能进行评分。荧光素酶报告基因检测miR-10a-5p与TAK1 3’UTR靶向结合情况,miR-10a-5p抑制剂转染巨噬细胞24小时,同时加入PC进行处理,RT-PCR和Western Blot检测相关基因及蛋白的表达情况。[结果]RT-PCR,Western Blot和HPLC检测发现,PC处理的巨噬细胞中LPL mRNA及表达、总胆固醇、游离胆固醇,胆固醇酯的水平呈浓度和时间依赖性显著降低;通过生物信息学预测分析发现转录因子ATF3与LPL的启动子之间可能存在结合位点;荧光素酶报告基因和ChIP的结果表明,转录激活因子ATF3能够增强LPL启动子的活性,PC明显抑制ATF3与LPL启动子的结合;RT-PCR和Western Blot检测发现,PC减少ATF3 mRNA及表达并下调LPL mRNA及表达,但当过表达ATF3后,能够逆转PC对LPL的抑制作用,表明ATF3是参与调控LPL的关键因子。接下来检测PC对JNK的影响,结果发现PC能够抑制JNK的激活,并且在使用JNK激活剂茴香霉素处理后,恢复了ATF3与LPL mRNA及表达;JNK是MAPK信号通路的另一亚类,其中TAK1是JNK的上游,进一步检测P

关键词： Deoxynivalenol   Cell cycle   ATF3   EGR1   Histone acetylation  

摘要： Deoxynivalenol (DON) is a type B trichothecene mycotoxin that exerts multiple toxic effects on plants, animals and humans. Several reports have shown that DON leads to G2/M cell cycle arrest. However, its molecular mechanism is still unclear. In this study, we showed that DON induced strong G2/M cell cycle arrest in HepG2 cells, and the cell cycle-inhibitory protein p21 was highly upregulated by DON. Further analysis showed that the cell cycle regulating gene EGR1 was highly induced by DON and that EGR1 knockdown abolished the upregulation of p21 and G2/M cell cycle arrest. Furthermore, we showed that the induction of EGR1 was regulated by the stressresponsive transcription factor ATF3. ATF3 Delta Zip2a/2b, which is a DNA binding domain truncated isoform of ATF3, was upregulated by DON. ATF3 knockdown weakened the expression induction of EGR1 and G2/M cell cycle arrest by DON. Moreover, the upregulation of ATF3 Delta Zip2a/2 highly depended on the enhanced presence of histones H3K9ac and H3K27ac. H3K9ac and H3K27ac were enriched at the promoter region of ATF3 following the DON treatment, and the knocking down of the genes responsible for H3K9ac and H3K27ac abolished the upregulation of ATF3 by DON. In summary, we found that DON induced G2/M cell cycle arrest by sequentially inducing the expression of ATF3 Delta Zip2a/2b, EGR1 and p21, and EGR1 played an essential role in this process, which is a novel molecular mechanism of cell cycle arrest by DON and is important for understanding its toxicology.

关键词： ATF3   Breast cancer   Runx2  

摘要： Activating transcription factor 3 (ATF3) is a stress-responsive factor that belongs to the activator protein 1 (AP-1) family of transcription factors. ATF3 expression is stimulated by various factors such as hypoxia, cytokines, and chemotherapeutic and DNA damaging agents. Upon stimulation, ATF3 can form homodimers or heterodimers with other members of the AP-1 family to repress or activate transcription. Under physiological conditions, ATF3 expression is transient and plays a pivotal role in controlling the expression of cell-cycle regulators and tumor suppressor, DNA repair, and apoptosis genes. However, under pathological conditions such as those during breast cancer, a sustained and prolonged expression of ATF3 has been observed. In this review, the structure and function of ATF3, its posttranslational modifications (PTM), and its interacting proteins are discussed with a special emphasis on breast cancer metastasis. (C) 2018 Elsevier B.V. All rights reserved.

关键词： carnosol   IL-6   ATF3   lung ischemia-reperfusion injury  

摘要： Carnosol is a naturally occurring herbal compound, known for its antioxidative properties. We previously found that carnosol protected mouse lungs from ischemia-reperfusion injury in ex vivo cultures. To elucidate the molecular mechanisms underpinning carnosol-mediated lung protection, we analyzed modes of interleukin-6 (IL-6) gene expression, which is associated with lung ischemia-reperfusion injury. Microarray analysis of mouse lungs suggested that IL-6 mRNA levels were elevated in the mouse lungs subjected to clamp-reperfusion, which was associated with elevated levels of other inflammatory modulators, such as activating transcription factor 3 (ATF3). Carnosol pretreatment lowered the IL-6 protein levels in mouse lung homogenates prepared after the clamp-reperfusion. On the other hand, the ATF3 gene expression was negatively correlated with that of IL-6 in RAW264.7 cells. IL-6 mRNA levels and gene promoter activities were suppressed by carnosol in RAW264.7 cells, but rescued by ATF3 knockdown. When RAW264.7 cells were subjected to hypoxia-reoxygenation, carnosol treatment lowered oxygen consumption after reoxygenation, which was coupled with a correlation with a transient production of mitochondrial reactive oxygen species and following ATF3 gene expression. These results suggest that carnosol treatment could be a new strategy for protecting lungs from ischemia-reperfusion injury by modulating the ATF3-IL-6 axis.

关键词： ATF3   microRNA   ossification   osteogenic differentiation   osteoporosis  

摘要： Through their multiple targets, microRNAs (miRNAs) are involved in numerous physiological and pathological processes. In this study, miR-342-3p was found to be deregulated with ossification of ligament or osteoporosis. We demonstrate that silencing miR-342-3p impairs osteoblast activity and matrix mineralization, while over expression of miR-342-3p promotes osteoblast differentiation significantly. Moreover, miR-342-3p directly targets activating transcription factor 3 (ATF3), which inhibits transcription of pro-osteogenic differentiation-associated genes. In addition, there exists a higher frequency of methylation at the CpG island of the Enah/Vasp-Like (EVL) locus in undifferentiated pre-osteoblasts;however, demethylation of the EVL CpG island induces over expression of miR-342-3p during osteogenic differentiation. This study suggests that miR-342-3p may serves as a potential marker for diagnosis and treatment of ossification of ligament and osteoporosis.

摘要： 1327

关键词： 慢性阻塞性肺疾病   ATF3   ATF4   γ-GCS表达  

摘要： 目的探究慢性阻塞性肺疾病(COPD)患者肺组织中活化转录因子3(ATF3)与活化转录因子4(ATF4)对γ-谷氨酰半胱氨酸合酶(γ-GCS)表达的影响,为预防及诊断COPD提供临床资料。方法 63例COPD确诊患者(COPD组)及同期在体检中心进行健康体检的正常居民(正常组) 63例,比较2组患者治疗前后ATF3 mRNA、ATF4 mRNA、γ-GCS mRNA表达含量、ATF3、ATF4、γ-GCS蛋白表达含量及COPD组患者ATF3、ATF4、γ-GCS信使RNA和蛋白及心肺功能指标的相关性。结果 COPD组治疗前ATF3 mRNA、ATF4 mRNA、γ-GCS mRNA、ATF3蛋白、ATF4蛋白、γ-GCS蛋白与正常组比较,差异有统计学意义(P <0. 05),COPD组治疗后ATF4 mRNAγ-GCS蛋白与正常组比较差异有统计学意义(P <0. 05);同时COPD组患者ATF3、ATF4、γ-GCS信使RNA和蛋白与心肺功能相关指标相关性明显,差异有统计学意义(P <0. 05)。结论 COPD患者肺组织中ATF3与ATF4可影响γ-GCS的表达,并且可直接反应患者的心肺功能,临床上可以通过对患者肺组织中ATF3与ATF4对γ-GCS表达的影响,及时的预防及诊断COPD。

关键词： miR-590   Breast cancer   Runx2   ATF3   Proliferation   Apoptosis  

摘要： We previously reported that ATF3 and Runx2 are involved in breast cancer progression and bone metastasis. The expression of these genes can be controlled by post-transcriptional regulators such as microRNAs (miRNAs). In this study, we identified and validated the functional role of miR-590-3p in human breast cancer cells (MDA-MB231). There was an inverse correlation between the expression of miR-590-3p and its putative target genes, ATF3 and Runx2 in these cells. Overexpression of miR-590-3p decreased the expression of ATF3 and Runx2 at the mRNA and protein levels in MDA-MB231 cells. Luciferase reporter assay identified a direct interaction of 3' UTRs of ATF3 and Runx2 with miR-590-3p in these cells. Overexpression of miR-590-3p also decreased proliferation and increased apoptosis of breast cancer cells. Based on our results, we suggest that miR-590-3p might have potential clinical applications towards controlling breast cancer progression and bone metastasis. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.