关键词： ATF3   TNF-alpha   Osteoblast   Differentiation   Inflammation  

摘要： Tumor necrosis factor (TNF)-alpha, which is a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions. Activating transcription factor 3 (ATF3), which is 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, the precise interactions between ATF3 and the TNF-alpha signaling pathway in the regulation of osteoblast differentiation remain unclear. In this study, we examined the role of ATF3 in the TNF-alpha-mediated inhibition of osteoblast differentiation and investigated the signaling pathways involved. The treatment of cells with TNF-alpha downregulated osteogenic markers, but significantly upregulated the expression of Atf3. The inhibition of Atf3 by small interfering RNAs rescued osteogenesis, which was inhibited by TNF-alpha. Conversely, the enforced expression of Atf3 enhanced the TNF-alpha-mediated inhibition of osteoblast differentiation, as revealed by the measurement of osteogenic markers and alkaline phosphatase staining. Mechanistically, TNF-alpha-induced Atf3 expression was significantly suppressed by the inhibition of the c-Jun N-terminal kinase (JNK) pathway. Furthermore, the overexpression of Atf3 did not affect the rescue effect that inhibiting TNF-alpha expression using a JNK inhibitor had on alkaline phosphatase activity and mineralization. Taken together, these results indicate that ATF3 mediates the inhibitory action of TNF-alpha on osteoblast differentiation and that the TNF-alpha-activated JNK pathway is responsible for the induction of Atf3 expression. (C) 2018 Elsevier Inc. All rights reserved.

关键词： 成纤维细胞   ATF3   心脏   小鼠   条件性敲除  

摘要： 目的:构建成纤维细胞特异敲除转录因子ATF3基因的小鼠模型。方法:利用胚胎显微注射法构建在ATF3基因携带loxP位点的小鼠(ATF3^(fl/fl)),以成纤维细胞细胞特异性表达的Col1a2-Cre介导ATF3条件性敲除,筛选出成纤维细胞条件性敲除ATF3的小鼠(Col1a2-ERCre:ATF3^(fl/fl));取鼠尾DNA进行PCR鉴定基因型,腹腔注射他莫昔芬诱导该基因敲除;12只敲除小鼠和12只同窝野生型小鼠各分为心脏缺血再灌注(I/R)组和假手术(sham)组,术后2h取心脏组织研磨后流式细胞分选成纤维细胞,进而提取mRNA并反转为cDNA,采用实时荧光定量PCR检测ATF3的表达;另取4只敲除小鼠和4只同窝野生型小鼠复制I/R模型,术后6h取心脏组织进行免疫荧光染色,观察成纤维细胞ATF3表达。结果:鼠尾DNA PCR鉴定基因型结果显示Cre基因型为阳性,ATF3 loxP基因型为纯合阳性;与假手术组相比,野生型小鼠术后心脏成纤维细胞ATF3表达明显升高[(0.02534±0.002654)vs.(0.5982±0.03495),P<0.0001],与术后野生型小鼠相比,术后条件性敲除组心脏成纤维细胞ATF3表达明显降低[(0.5982±0.03495)vs.(0.06456±0.005704),P<0.0001];免疫荧光共定位染色显示野生型小鼠心脏成纤维细胞ATF3蛋白表达,条件性敲除小鼠ATF3蛋白未见表达。结论:成纤维细胞特异敲除ATF3小鼠构建成功,为进一步实验奠定基础。

关键词： ATF3   Particulate matter   Inflammation   Airway epithelial cells   NF-kappa B   AP-1  

摘要： Airborne particulate matter (PM) has been reported to be associated with a wide range of respiratory disorders. However, the mechanisms underlying PM-induced airway inflammation remain largely unknown. Generally, ATF3 negatively regulates pro-inflammatory cytokines production in response to TLR4 signaling. Here we first showed ATF3 has promoting effects in PM-induced airway inflammation in vitro an in vivo. We demonstrated PM significantly upregulated ATF3 expression in HBE cells and in mouse lung tissues. ATF3 siRNA markedly inhibited, while ATF3-recombinant over-expression plasmid significantly increased PM-induced IL-6 expression in cultured HBE cells, and PM-induced IL-6, CXCL2 expression as well as neutrophil infiltration, mucus overproduction in the lung of ATF3(-/-) mice were all notably reduced relative to the wild-type littermates. Furthermore, we showed ATF3 mediated PM-induced inflammatory cytokines expression partly through NF-kappa B and AP-1 pathways. Our data further elucidates the mechanisms underlying PM-induced airway inflammation, and indicates ATF3 may function as different role in response to different stimuli.

摘要： 1194

关键词： Joint inflammatory pain   DRG neurons   ATF3   P2Xreceptors   SGC activation   HSP90 inhibition  

摘要： Purinergic receptors (P2XRs) have been widely associated with pain states mostly due to their involvement in neuron-glia communication. Interestingly, we have previously shown that satellite glial cells (SGC), surrounding dorsal root ganglia (DRG) neurons, become activated and proliferate during monoarthritis (MA) in the rat. Here, we demonstrate that P2X7R expression increases in ipsilateral DRG after 1 week of disease, while P2X3R immunoreactivity decreases. We have also reported a significant induction of the activating transcriptional factor 3 (ATF3) in MA. In this study, we show that ATF3 knocked down in DRG cell cultures does not affect the expression of P2X7R, P2X3R, or glial fibrillary acidic protein (GFAP). We suggest that P2X7R negatively regulates P2X3R, which, however, is unlikely mediated by ATF3. Interestingly, we found that ATF3 knockdown in vitro induced significant decreases in the heat shock protein 90 (HSP90) expression. Thus, we evaluated in vivo the involvement of HSP90 in MA and demonstrated that the HSP90 messenger RNA levels increase in ipsilateral DRG of inflamed animals. We also show that HSP90 is mostly found in a cleaved form in this condition. Moreover, administration of a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), attenuated MA-induced mechanical allodynia in the first hours. The drug also reversed the HSP90 upregulation and cleavage. 17-DMAG seemed to attenuate glial activation and neuronal sensitization (as inferred by downregulation of GFAP and P2X3R in ipsilateral DRG) which might correlate with the observed pain alleviation. Our data indicate a role of HSP90 in MA pathophysiology, but further investigation is necessary to clarify the underlying mechanisms.

关键词： Metabolomics   Toll-like receptors  

摘要： Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring(1) marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite(2). Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines(2), including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase(2,3), yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of I kappa B zeta protein induction. The regulation of I kappa B zeta is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-I kappa B zeta driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-I kappa B zeta regulatory axis could be an important new strategy for the treatment of IL-17-I kappa B zeta-mediated autoimmune diseases.

关键词： JDP2   Cardiac remodeling   AP-1   ATF3  

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

摘要： 目的探究慢性阻塞性肺疾病(COPD)患者肺组织中转录活化因子3(ATF3)与转录活化因子4(ATF4)对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响。方法本研究采用回顾性分析方法,选择2014~2016年收治的160例肺部疾病患者作为研究对象,将经胸部X线或CT检查确诊为COPD者列为观察组(80例),将非COPD者列为对照组(80例)。观察两组光镜下肺病理形态学改变、肺功能指标和吸烟史。经原位杂交分析两组肺组织中的ATF3、ATF4、γ-GCS-HS水平,采用免疫组化法检测肺组织中的ATF3、ATF4、γ-GCS-HS蛋白表达,并采用直线相关法,分析肺组织ATF3、ATF4蛋白与γ-GCS-HS表达的相关性及肺组织ATF3、ATF4、γ-GCS-HS蛋白与肺功能之间的关系。结果经HE法,光镜下可见观察组肺组织标本有病理形态学改变,而对照组无形态学变化;观察组肺功能指标低于对照组(P<0.05),观察组吸烟史多于对照组(P<0.05)。经原位杂交法检验,观察组中的ATF3、ATF4、γ-GCSHS的mRNA表达水平高于对照组,差异有统计学意义(P<0.05)。经免疫组化检测,观察组中的ATF3、ATF4、γ-GCS-HS蛋白的表达水平高于对照组,差异有统计学意义(P<0.05)。经直线相关法分析,ATF3、ATF4蛋白表达与γ-GCS-HSmRNA及其蛋白呈正相关关系(P<0.01);ATF3、ATF4、γ-GCS-HS蛋白与肺功能各指标呈正相关关系(P<0.01)。结论 COPD患者的ATF3、ATF4表达明显上调可影响调控γ-GCS表达,从而使γ-GCS表达上调,以发挥抗氧化作用。

关键词： activating transcription factor 3   aging   ER stress   idiopathic pulmonary fibrosis   mitochondrial dysfunction   PTEN-induced putative kinase 1  

摘要： PINK1 (PTEN-induced putative kinase 1) is a key regulator of mitochondrial homeostasis that is relatively depleted in aging lungs and in lung epithelial cells from patients with idiopathic pulmonary fibrosis (IPF), a disease linked with aging. Impaired PINK1 expression and accumulation of damaged mitochondria in lung epithelial cells from fibrotic lungs were associated with the presence of ER stress. Here, we show that ATF3 (activating transcription factor 3), a member of the integrated stress response (ISR), negatively regulates transcription of the PINK1 gene. An ATF3 binding site within the human PINK1 promoter is located in the first 150 bp upstream of the transcription start site. Induction of ER stress or overexpression of ATF3 inhibited the activity of the PINK1 promoter. Importantly, overexpression of ATF3 causes accumulation of depolarized mitochondria, increased production of mitochondrial ROS, and loss of cell viability. Furthermore, conditional deletion of ATF3 in type II lung epithelial cells protects mice from bleomycin-induced lung fibrosis. Finally, we observed that ATF3 expression increases in the lung with age and, specially, in lung epithelial cells from IPF lungs. These data provide a unique link between ATF3 and PINK1 expression suggesting that persistent stress, driven by ATF3, can dysregulate mitochondrial homeostasis by repression of PINK1 mRNA synthesis.