关键词： Cardiac remodeling   Pressure overload   Phenylephrine   ATF3   Macrophages   Cardiomyocytes  

摘要： Rational: Pressure overload induces adaptive and maladaptive cardiac remodeling processes in the heart. Part of the maladaptive process is the cross-talk between cardiomyocytes and macrophages which is dependent on the function of the Activating Transcription Factor 3, ATF3. Yet, the molecular mechanism involved in cardiomyocytes-macrophages communication leading tomacrophages recruitment to the heart and cardiac maladaptive remodeling is currently unknown. Methods and results: Isolated peritoneal macrophages from either wild type or ATF3-KO mice were cultured in serum free medium to collect conditioned medium (CM). CM was used to probe an antibody cytokine/chemokine array. The interferon. induced protein 10 kDa, CXCL10, was found to be enriched in wild type macrophages CM. Wild type cardiomyocytes treated with CXCL10 in vitro, resulted in significant increase in cell volume as compared to ATF3-KO cardiomyocytes. In vivo, pressure overload was induced by phenylephrine (PE) infusion using micro-osmotic pumps. Consistently, CXCL11 (CXCL10 competitive agonist) and CXCL10 receptor antagonist (AMG487) attenuated PE-dependent maladaptive cardiac remodeling. Significantly, we show that the expression of the CXCL10 receptor, CXCR3, is suppressed in cardiomyocytes andmacrophages derived from ATF3-KO mice. CXCR3 is positively regulated by ATF3 through an ATF3 transcription response element found in its proximal promoter. Finally, mice lacking CXCR3 display a significant reduction of cardiac remodeling processes following PE infusion. Conclusions: Chronic PE infusion results in a unique cardiomyocytes-macrophages cross-talk that is mediated by IFN gamma. Subsequently, macrophages that are recruited to the heart secrete CXCL10 resulting in maladaptive cardiac remodeling mediated by the CXCR3 receptor. ATF3-KO mice escape from PE-dependent maladaptive cardiac remodeling by suppressing the IFN gamma-CXCL10-CXCR3 axis at multiple levels. (C) 2016 Elsevier Ireland Ltd. All rights r

关键词： Type 2 diabetes   Cardiac remodeling   Cytokines   High fat diet   ATF3  

摘要： Aims Obesity and type 2 diabetes (T2D) trigger a harmful stress-induced cardiac remodeling process known as cardiomyopathy. These diseases represent a serious and widespread health problem in the Western world;however the underlying molecular basis is not clear. ATF3 is an ` immediate early' gene whose expression is highly and transiently induced in response to multiple stressors such as metabolic, oxidative, endoplasmic reticulum and inflammation, stressors that are involved in T2D cardiomyopathy. The role of ATF3 in diabetic cardiomyopathy is currently unknown. Our research has aimed to study the effect of ATF3 expression on cardiomyocytes, heart function and glucose homeostasis in an obesity-induced T2D mouse model. Methods and results We used wild type mice (WT) as well as mutant mice with a cardiac-specific ATF3 deficiency (ATF3-cKO). Mice were fed a high-fat diet (HFD) for 15 weeks. HFD induced high ATF3 expression in cardiomyocytes. Mice were examined for cardiac remodeling processes and the diabetic state was assessed. HFD-fed ATF3-cKO mice exhibited severe cardiac fibrosis, higher levels of heart hypertrophic markers, increased inflammation and worse cardiac function, as compared to WT mice. Interestingly, HFD-fed ATF3-cKO mice display increased hyperglycemia and reduced glucose tolerance, despite higher blood insulin levels, as compared to HFD-fed WT mice. Elevated levels of the cardiac inflammatory cytokines IL-6 and TNFa leading to impaired insulin signalling may partially explain the peripheral glucose intolerance. Conclusions Cardiac ATF3 has a protective role in dampening the HFD-induced cardiac remodeling processes. ATF3 exerts both local and systemic effects related to T2D-induced cardiomyopathy. This study provides a strong relationship between heart remodeling processes and blood glucose homeostasis.

关键词： Osteoclasts   RANKL   Activating transcription factor 3   Calcium signaling  

摘要： Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, whether ATF3 is involved in osteoclast differentiation and activity has not been well-studied. In the present study, we examined the role of ATF3 in osteoclast differentiation and function. ATF3 expression was down regulated during RANKL-induced osteoclast differentiation. Overexpression of ATF3 in bone marrow-derived monocyte/macrophage lineage cells (BMMs) promoted osteoclast differentiation and activity and strongly induced the expression of osteoclast genes encoding nuclear factor of activated T-cells cl (NFATc1) and tartrate-resistant acid phosphatase (TRAP) compared to that in the control group. In contrast, small interfering RNA mediated knockdown of ATF3 prevented the formation of multinucleated osteoclasts and markedly abrogated the expression of osteoclast marker genes. Mechanistically, ATF3 synergistically enhanced c-Fos- or NFAT-mediated transcriptional activity of the NFATc1 or TRAP promoter, respectively. Furthermore, ATF3 physically interacted with c-Fos and NFATc1 and enhanced the binding affinity of c-Fos and NFATc1 to the promoters. Interestingly, ATF3 is involved in calcium signaling during osteoclastogenesis. Taken together, these results suggest that ATF3 is a new co-factor of c-Fos and NFATc1 to activate osteoclast differentiation and activity. (C) 2016 Elsevier Inc. All rights reserved.

摘要： Endothelial dysfunction caused by the combined action of disturbed flow, inflammatory mediators and oxidants derived from cigarette smoke is known to promote coronary atherosclerosis and increase the likelihood of myocardial infarctions and strokes. Conversely, laminar flow protects against endothelial dysfunction, at least in the initial phases of atherogenesis. We studied the effects of TNF alpha and cigarette smoke extract on human coronary artery endothelial cells under oscillatory, normal laminar and elevated laminar shear stress for a period of 72 hours. We found, firstly, that laminar flow fails to overcome the inflammatory effects of TNF alpha under these conditions but that cigarette smoke induces an anti-oxidant response that appears to reduce endothelial inflammation. Elevated laminar flow, TNF alpha and cigarette smoke extract synergise to induce expression of the transcriptional regulator activating transcription factor 3 (ATF3), which we show by adenovirus driven overexpression, decreases inflammatory gene expression independently of activation of nuclear factor-kappa B. Our results illustrate the importance of studying endothelial dysfunction in vitro over prolonged periods. They also identify ATF3 as an important protective factor against endothelial dysfunction. Modulation of ATF3 expression may represent a novel approach to modulate proinflammatory gene expression and open new therapeutic avenues to treat proinflammatory diseases.

摘要： Glioblastoma is associated with poor survival and a high recurrence rate in patients due to inevitable uncontrolled infiltrative tumor growth. The elucidation of the molecular mechanisms may offer opportunities to prevent relapses. In this study we investigated the role of the activating transcription factor 3 (ATF3) in migration of GBM cells in vitro. RNA microarray revealed that gene expression of ATF3 is induced by a variety of chemotherapeutics and experimental agents such as the nitric oxide donor JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate). We found NF kappa B and STAT3 to be downstream targets inhibited by overexpression of ATF3. We demonstrate that ATF3 is directly involved in the regulation of matrix metalloproteinase expression and activation. Overexpression of ATF3 therefore leads to a significantly reduced migration capacity and induction of tissue inhibitors of matrix metalloproteinases. Our study for the first time identifies ATF3 as a potential novel therapeutic target in glioblastoma.

关键词： Apoptosis   Activating Transcription Factor 3   Bcl-2   Colorectal Cancer   Vitex rotundifolia L  

摘要： Fruit from Vitex rotundifolia L. (VF) has been reported to initiate apoptosis in human colorectal cancer cells through the accumulation of reactive oxygen species. Since various regulatory factors are involved in the apoptotic pathway, further study of the potential mechanisms of VF associated with the induction of apoptosis may be important despite the fact that the molecular target of VF for apoptosis has already been elucidated. In this study, we showed a new potential mechanism for the relationship between VF-mediated ATF3 expression and apoptosis to better understand the apoptotic mechanism of VF in human colorectal cancer cells. VF reduced the cell viability and induced apoptosis in human colorectal cancer cells. VF treatment increased both the protein and mRNA level of ATF3 and upregulated ATF3 promoter activity. The cis-element responsible for ATF3 transcriptional activation by VF was CREB which is located between -147 to -85 of ATF3 promoter. Inhibitions of ERK1/2, p38, JNK and GSK3 beta blocked VF-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of PARP by VF, while ATF3 overexpression increased VF-mediated cleaved PARP. ATF3 knockdown also attenuated VF-mediated cell viability and cell death. In addition, VF downregulated Bcl-2 expression at both protein and mRNA level. ATF3 knockdown by ATF3 siRNA blocked VF-mediated downregulation of Bcl-2. In conclusion, VF may activate ATF3 expression through transcriptional regulation and subsequently suppress Bcl-2 expression as an anti-apoptotic protein, which may result in the induction of apoptosis in human colorectal cancer cells.

关键词： 猪血凝性脑脊髓炎病毒   ATF2   表达与调控  

摘要： 血凝性脑脊髓炎病毒主要感染1～3周龄的哺乳仔猪,常被称为猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV),仔猪感染后可出现明显的神经症状,病死率可达20%～100%。小鼠可感染PHEV,常作为感染PHEV的病理模型。本课题组前期研究表明小鼠感染PHEV后,激活转录因子3(Activating transcription factor 3,ATF3)的表达也增加。ATF3可作为ATF2的下游靶基因,ATF2为JNK信号通路中的一员。除JNK信号通路外,ATF3还可由P53、TGF-β/smad和c-Myc等信号通路诱导激活。但是前期研究中仅发现JNK信号通路相关因子被激活。据此推测PHEV感染小鼠后,大脑中ATF3的高表达可能是由JNK信号通路所诱导的。若此推论成立,作为调控ATF3的上游基因ATF2也应呈现高表达。为验证上述假设,本实验对感染PHEV后不同时间段的小鼠大脑中的ATF2和ATF3的表达水平进行检测。本实验首先构建了 PHEV急性感染小鼠的模型,采用颅内接种方式,以小鼠在接毒后第5天全部死亡的接种剂量最为最佳接种剂量。选取4周龄SPF雌性昆明小鼠30只,随机分两组,接毒组和对照组,每组15只。接毒组以最佳接种剂量颅内接种PHEV,对照组以相同剂量颅内接种PBS。于接毒后的第1天,2天,3天,4天,5天每组随机选取3只小鼠进行剖杀。采取大脑,一部分固定于福尔马林溶液,另一部分冻存于液氮中。固定于福尔马林溶液中的大脑用于病理组织学病变的观察;冻存于液氮中的大脑,采用Real-time PCR和Western Blot法检测ATF2和ATF3 mRNA和蛋白在大脑内的表达情况。采用2-△△Ct相对表达量检测ATF2、ATF3和PHEVmRNA的表达水平,采用半定量法检测其蛋白的表达水平,并采用SPSS 17.0软件进行数据分析和处理。结果显示,小鼠感染PHEV后主要表现为非化脓性脑炎的病理组织学变化,且随着感染时间的延长,病理损伤逐渐加重。同时,随着感染时间的延长,ATF2、ATF3与PHEV的mRNA和蛋白表达水平均呈逐渐上升趋势。经相关性分析,ATF2和ATF3的表达呈正相关(p<0.01),ATF2表达与PHEV之间呈正相关(p<0.01),ATF3和PHEV之间表达呈正相关(p<0.01)。接毒第5天达到峰值,经One-Way ANOVA分析均具有显著性差异(p<0.01)。说明小鼠感染PHEV后大脑中的病理损伤逐步加重,且随着ATF2的表达量的增高,ATF3表达和PHEV病毒载量也增高。小鼠感染PHEV后ATF3的高表达可能是JNK信号通路激活后由ATF2诱导调控的。本研究为进一步探明PHEV感染的神经细胞过程中ATF3高表达的机制指引了新方向,为深入解析PHEV的分子致病机制提供重要的理论依据。

关键词： Activating transcription factor 3   Early growth response protein 1   HNSCC   Human mini-organ culture model   Laryngopharyngeal reflux  

摘要： Background/Aims: Activating transcription factor 3 (ATF3) and early growth response protein 1 (EGR1) are reported to interact, but their use as prognostic factors in cancer is discussed controversially. Methods: We measured ATF3 and EGR1 gene expression changes in human mini-organ cultures (MOCs) of healthy nasal epithelia, UM-SCC-22B, and FADU DD cells after acid reflux exposure. Next, ATF3 and EGR1 gene expression was analysed in tumour tissues and related to the median expression of autologous reference tissue samples. Results: ATF3 and EGR1 mRNA expression was significantly reduced after consecutive exposure of MOCs at pH <7.0 to artificial gastric juice (refluxate). In contrast, ATF3 mRNA was upregulated significantly within the first hour of incubation. EGR1 mRNA exhibited no significant changes. The analysed cell lines exhibited a cell line-specific alteration. In FADU DD cells, the upregulation of EGR1 was significant after refluxate exposure, but in HN-SCC 22B, no significant changes were detected. The analysis of the HNSCC samples confirmed the heterogeneous data of the literature. Conclusion: The data maintain the hypothesis that ATF3 and EGR1 are involved in the beginning of inflammatory processes. Whether these two transcription factors act as tumour suppressors or promoters is context dependent and warrants analysis in further studies. (C) 2017 S. Karger AG, Basel

关键词： 尿道下裂   WT1蛋白   ATF3基因   细胞病理学  

摘要： 目的：　　研究WT1(Wilms tumor1)基因，ATF3（Activating Transcription Factor3）基因在儿童尿道下裂包皮中的表达及它们与尿道下裂严重程度的联系。　　方法：　　尿道下裂组22例（I°，II°，III°分别为11例，7例，4例）的包皮组织来自在温州医科大学附属第二医院行“尿道成形术”的患儿，以及对照组22例非尿道下裂（包括10例包茎，5例包皮过长，7例隐匿阴茎）分别在我院儿童外科行“包皮环切术”，“阴茎延长术”的患儿。　　结果：　　WT1蛋白在尿道下裂患儿中的表达显著高于对照组（包茎，包皮过长，隐匿阴茎）的表达，I°、II°、III°的平均光密度值分别为（1.23E-03±5.11E-05）、（1.97E-03±4.46E-05）、（3.32E-03±2.63E-05），且两组差异具有统计学意义（P＜0.05），而且阳性表达位于尿道下裂腹侧包皮基底层的细胞核中。ATF3蛋白在重度尿道下裂患儿的表达高于在对照组（包茎，包皮过长，隐匿阴茎）的表达，I°、II°、III°的平均光密度值分别为（6.51E-06±3.21E-05）、（1.30E-03±4.51E-05）、（2.72E-03±2.23E-05），两组之间差别具有统计学意义（P＜0.05），尿道下裂腹侧包皮基底层的细胞核中能看到棕褐色颗粒。而对照组未见明显阳性表达结果。　　结论：　　WT1蛋白在尿道下裂患儿腹侧包皮组织中的表达明显上调，提示WT1基因的过表达可能与人类尿道下裂严重程度存在相关性。ATF3蛋白在II°、III°尿道下裂患儿中的表达量显著高于在对照组中的表达量，I°尿道下裂与对照组之间无明显差异（P＞0.05），病例组之间ATF3蛋白的表达量随着病情的加重，表达量明显上升。

关键词： 猪血凝性脑脊髓炎病毒   ATF3   JNK抑制剂   细胞周期与凋亡  

摘要： 猪血凝性脑脊髓炎是由猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV)引起的,主要感染3周龄的仔猪,临床以呕吐衰竭和明显的神经症状为特征。前期研究结果表明,小鼠感染PHEV后大脑中ATF3呈高表达,为了探明ATF3在神经细胞感染PHEV后ATF3是否呈现高表达,以及其高表达的机制,本实验对神经细胞感染PHEV前后ATF3的表达进行了检测,同时探索JNK信号通路在猪血凝性脑脊髓炎病毒感染神经细胞过程中对ATF3表达的影响。本试验以N2a细胞作为研究PHEV感染神经细胞的模型,根据试验要求,设置4组:对照组、接毒组、JNK抑制剂组和JNK抑制剂接毒组,每组3个重复。于六孔板中培养N2a细胞到单层,以吸附法接种PHEV,然后分别在2h、12h、24h时提取细胞总RNA和总蛋白,通过荧光定量PCR和蛋白质印迹法(Western Blot)分别测定PHEV感染前后及JNK抑制剂作用前后N2a细胞中ATF3 mRNA和蛋白的表达量。同时设置6组:对照组、接毒组、DMSO组、DMSO接毒组、JNK抑制剂组和JNK抑制剂接毒组,每组3个重复,通过Annexin V-FITC/PI双染法和PI染色法检测12h和24h的细胞凋亡和细胞周期,以测定JNK抑制剂作用前后对PHEV感染的神经细胞的凋亡和细胞周期的影响。结果表明,ATF3基因在正常N2a细胞中的处于低表达,随着PHEV mRNA和蛋白表达量的降低或升高ATF3 mRNA和蛋白的表达量也随着降低或升高,两者之间的变化趋势一致且呈正相关;JNK抑制剂作用N2a细胞后,ATF3的mRNA和蛋白表达量明显降低。通过Annexin V-FITC/PI双染法标记凋亡细胞,再经流式细胞仪进行检测,结果发现与对照组相比,JNK抑制剂作用后接毒组细胞凋亡率明显上升,差异极显著(P<0.01),此时ATF3表达量降低,说明ATF3在细胞内起到抑制凋亡的作用;同时,与对照组相比,JNK抑制剂作用后接毒组细胞周期发生阻滞。N2a细胞感染PHEV后,ATF3的表达量增高。JNK抑制剂作用于N2a细胞后,ATF3表达量下降,细胞凋亡率上升,细胞周期阻滞,DNA合成受阻,细胞活力下降,抗病毒能力下降。表明神经细胞感染PHEV后JNK信号通路可调控ATF3的表达,进而影响神经细胞凋亡和细胞周期。