关键词： Saturated fatty acid   HDL   Inflammation   Cell signaling   MAP kinase   ATF3  

摘要： Background and aims: Peripheral insulin resistance is associated with several metabolic abnormalities, including elevated serum fatty acids that contribute to vascular injury and atherogenesis. Our goals were to examine whether saturated fatty acids can modify innate immune responses to subclinical concentrations of lipopolysaccharide (LPS) in endothelial cells, and to explore the underlying pathway and determine whether it is modified by high density lipoprotein (HDL) and other factors commonly altered in insulin resistance. Methods: Physiologic concentrations of palmitic acid were added to human aortic endothelial cells with and without a variety of inhibitors or HDL and measures of cell inflammation and function assessed. Results: Palmitic acid significantly amplified human aortic endothelial cell inflammatory responses to LPS. Similar results were obtained from lipolysis products of triglyceride rich lipoproteins. Metabolism of palmitic acid to ceramide and subsequent activation of PKC-zeta, MAPK and ATF3 appeared critical in amplifying LPS induced inflammation. The amplified response to palmitic acid/LPS was decreased by HDL, dose dependently, and this inhibition was dependent on activation of PI3K/AKT and reduction in ATF3. Conclusions: These results indicate that endothelial cell innate immune responses are modified by metabolic abnormalities commonly present in insulin resistance and provide evidence for a novel mechanism by which HDL may reduce vascular inflammation. (C) 2016 Published by Elsevier Ireland Ltd.

关键词： Drosophila   Innate immunity   Intestinal stem cells  

摘要： Epithelial barrier function is maintained by coordination of cell proliferation and cell loss, whereas barrier dysfunction can lead to disease and organismal death. JNK signalling is a conserved stress signalling pathway activated by bacterial infection and tissue damage, often leading to apoptotic cell death and compensatory cell proliferation. Here we show that the stress inducible transcription factor ATF3 restricts JNK activity in the Drosophila midgut. ATF3 regulates JNK-dependent apoptosis and regeneration through the transcriptional regulation of the JNK antagonist, Raw. Enterocyte-specific ATF3 inactivation increases JNK activity and sensitivity to infection, a phenotype that can be rescued by Raw overexpression or JNK suppression. ATF3 depletion enhances intestinal regeneration triggered by infection, but does not compensate for the loss of enterocytes and ATF3-depleted flies succumb to infection due to intestinal barrier dysfunction. In sum, we provide a mechanism to explain how an ATF3-Raw module controls JNK signalling to maintain normal intestinal barrier function during acute infection.

关键词： RNA干扰   p300   亚溶解型C5b-9   凋亡   乙酰化  

摘要： 目的:构建大鼠p300基因短发夹状小干扰RNA(short hairpin RNA,shRNA)真核表达质粒,观察沉默p300基因对亚溶解型(sublytic)C5b-9诱导大鼠肾小球系膜细胞(glomerular messangial cell,GMC)凋亡和激活转录因子3(activating transcription factor 3,ATF3)乙酰化水平的影响。方法:用DNA重组技术针对p300基因不同位点设计3个shRNA序列,然后分别克隆到真核表达载体pGCsi-U6/Neo/GFP/shRNA中,经电转染入GMC,再给予sublytic C5b-9刺激。Western blot筛选干涉效果最佳的p300 shRNA,流式细胞术检测GMC凋亡率,免疫沉淀(immunoprecipitation,IP)和Western blot联合检查ATF3乙酰化水平。结果:核酸测序表明p300 shRNA重组质粒构建成功。Western blot显示p300 shRNA-2具有最佳沉默效率。p300 shRNA处理GMC后,由sublytic C5b-9诱导的GMC凋亡率显著下降,同时ATF3乙酰化水平也明显降低。结论:成功构建了大鼠p300 shRNA真核表达质粒,并初步证实了p300能够促进sublytic C5b-9诱导的GMC凋亡,其机制可能与p300乙酰化修饰ATF3有关。

关键词： alcohol   monocyte   macrophage   LPS   inflammation  

摘要： Discussion on the role of ATF3 in LPS tolerance during binge drinking, and its interaction with cofactor HDAC1.

关键词： innate immunity   binge drinking   TLR4 tolerance   HDAC1  

摘要： Acute ethanol exposure decreases the production of LPS-induced TNF-, and the migration of monocytes and macrophages via ATF3. Binge drinking represses host innate immunity and leads to a high risk of infection. Acute EtOH-pretreated macrophages exhibit a decreased production of proinflammatory mediators in response to LPS. ATF3 is induced and counter-regulates the LPS/TLR4 inflammatory cascade. Here, we investigated the potential role of ATF3 in LPS tolerance in acute ethanol-pretreated macrophages. We found that there was an inverse correlation between ATF3 and LPS-induced TNF- production in acute ethanol-pretreated murine monocytes and macrophages. The knockdown of ATF3 attenuated the inhibitory effects of acute ethanol treatment on LPS-induced TNF- production. Furthermore, ChIP assays and co-IP demonstrated that ATF3, together with HDAC1, negatively modulated the transcription of TNF-. In binge-drinking mice challenged with LPS, an up-regulation of ATF3 and HDAC1 and a concomitant decrease in TNF- were observed. Given that HDAC1 was concomitantly induced in acute ethanol-exposed monocytes and macrophages, we used the HDACi TSA or silenced HDAC1 to explore the role of HDAC1 in acute ethanol-treated macrophages. Our results revealed that TSA treatment and HDAC1 knockdown prevented acute ethanol-induced ATF3 expression and the inhibition of TNF- transcription. These data indicated a dual role for HDAC1 in acute ethanol-induced LPS tolerance. Furthermore, we showed that the induction of ATF3 led to the impaired migration of BM monocytes and macrophages. Overall, we present a novel role for ATF3 in the inhibition of LPS-induced TNF- and in the impairment of monocyte and macrophage migration.

关键词： ATF3   inflammatory   lipopolysaccharide   rHDL  

摘要： Activating transcription factor 3 (ATF3) is a TLR-induced repressor that plays an important role in the inhibition of specific inflammatory signals. We previously constructed recombinant high density lipoproteins (rHDL) (including rHDL(WT), rHDL(M), rHDL(228) and rHDL(74)) and found that rHDL(74) had a strong anti-inflammatory ability. In the present study, we investigate the roles of recombinant apolipoprotein A-I (ApoA-I) (rHDL(WT)) and its cysteine mutant HDLs (rHDL(M), rHDL(228) and rHDL(74)) on ATF3 function in RAW264.7 cells stimulated by lipopolysaccharide. Our results showed that compared with the LPS group, rHDL(74) can decrease the level of TNF-alpha and IL-6, whereas rHDL(228) increases their expression levels. RT-PCR and Western blotting results showed that compared with the LPS group, rHDL(74), rHDL(WT) and rHDL(M) can markedly increase the expression level of ATF3, whereas the level of ATF3 decreases in the rHDL(228) group. In summary, the different anti-inflammatory mechanisms of the ApoA-I cysteine mutants might be associated with the regulation of ATF3 level.

关键词： ATF3   alpha(2)delta-1 subunit   Chronic pain   Formalin test   Neuropathic pain  

摘要： The purpose of this study was to investigate whether 1%, 2% or 5% formalin injection produce hypersensitivity with characteristics of the neuropathic pain induced by spinal nerve injury. Formalin injection (1%, 2% and 5%) produced concentration-dependent long-lasting (at least 14 days) mechanical allodynia and hyperalgesia in both paws. Likewise, L5/L6 spinal nerve ligation induced allodynia and hyperalgesia in both paws. The intensity of hypersensitivity was greater in the ipsilateral than in the contralateral paw in all models. Systemic gabapentin or morphine completely reduced 1% formalin-induced hypersensitivity. In contrast, both drugs were not able to fully diminish 2-5% formalin- and nerve injury-induced hypersensitivity. Indomethacin produced a significant effect in the chronic 1% formalin test. Conversely, this drug did not modify 2 or 5% formalin- and nerve injury induced hypersensitivity. Spinal nerve injury and 2-5%, but not 1%, formalin injection enhanced ATF3 protein expression and immunofluorescence in dorsal root ganglia (DRG) in a time-dependent manner. Furthermore, 2-5%, but not 1%, formalin injection or spinal nerve injury also enhanced alpha(2)delta-1 subunit protein levels in DRG. Our results suggest that 5% and, at lesser extent, 2% formalin injection produces long-lasting hypersensitivity with a pharmacological and molecular pattern that resembles neuropathic pain induced by spinal nerve ligation.

关键词： AP1 transcription factor (AP-1)   arthritis   cell signaling   chondrocyte   inflammation   matrix metalloproteinase (MMP)  

摘要： Irreversible breakdown of cartilage extracellular matrix (ECM) by the collagenase matrix metalloproteinase 13 (MMP13) represents a key event in osteoarthritis (OA) progression. Although inflammation is most commonly associated with inflammatory joint diseases, it also occurs in OA and is thus relevant to the prevalent tissue destruction. Here, inflammation generates a cFOS AP-1 early response that indirectly affects MMP13 gene expression. To ascertain a more direct effect on prolonged MMP13 production we examined the potential molecular events occurring between the rapid, transient expression of cFOS and the subsequent MMP13 induction. Importantly, we show MMP13 mRNA expression is mirrored by nascent hnRNA transcription. Employing ChIP assays, cFOS recruitment to the MMP13 promoter occurs at an early stage prior to gene transcription and that recruitment of transcriptional initiation markers also correlated with MMP13 expression. Moreover, protein synthesis inhibition following early FOS expression resulted in a significant decrease in MMP13 expression thus indicating a role for different regulatory factors modulating expression of the gene. Subsequent mRNA transcriptome analyses highlighted several genes induced soon after FOS that could contribute to MMP13 expression. Specific small interfering RNA-mediated silencing highlighted that ATF3 was as highly selective for MMP13 as cFOS. Moreover, ATF3 expression was AP-1(cFOS/cJUN)-dependent and expression levels were maintained after the early transient cFOS response. Furthermore, ATF3 bound the proximal MMP13 AP-1 motif in stimulated chondrocytes at time points that no longer supported binding of FOS. Consequently, these findings support roles for both cFOS (indirect) and ATF3 (direct) in effecting MMP13 transcription in human chondrocytes.

关键词： 慢性阻塞性肺疾病   活化转录因子3   活化转录因子4   γ-谷氨酰半胱氨酸合成酶  

摘要： 目的探讨慢性阻塞性肺疾病(简称慢阻肺)患者肺组织中活化转录因子3(ATF3)、活化转录因子4(ATF4)的表达变化,以及ATF3和ATF4对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨其在慢阻肺发病中的作用。方法收集伴或不伴慢阻肺并行肺叶切除的肺癌患者临床肺组织标本40例(慢阻肺组和对照组各20例),取材标本均为远离原发肺癌病灶5 cm以上、肉眼观察无肺癌浸润的外周肺组织。所有患者术前均详细询问病史,进行体格检查、胸部X线片或肺部CT以及肺功能检测。应用原位杂交及免疫组化检测患者肺组织中ATF3、ATF4、γ-GCS重链亚基(γ-GCS-HS)m RNA及蛋白的表达,应用免疫共沉淀法(CO-IP)检测ATF3、ATF4与γ-GCS-HS蛋白之间的相互作用,并对ATF3、ATF4 m RNA及蛋白与γ-GCS-HS m RNA及蛋白进行相关分析。结果慢阻肺患者肺组织中ATF3、ATF4、γ-GCS-HS m RNA及蛋白呈强阳性表达,较对照组显著升高(P<0.01)。在γ-GCS-HS抗体捕获的免疫沉淀中,ATF3、ATF4抗体均可杂交出明显的蛋白条带,且慢阻肺组较对照组明显增强(P<0.01)。相关分析显示肺组织中ATF3、ATF4蛋白表达与γ-GCS-HS m RNA及蛋白表达均呈明显正相关(P<0.01);ATF3、ATF4、γ-GCS-HS蛋白表达与FEV1%pred、FEV1/FVC均呈明显正相关(P<0.01)。结论在慢阻肺患者发病过程中,氧化应激诱导转录因子ATF3和ATF4过表达,ATF3和ATF4可能通过影响γ-GCS m RNA和蛋白的表达而发挥抗氧化保护作用。

关键词： 转录激活因子3   急性肺损伤   趋化因子CXCL1   趋化因子CXCL2   巨噬细胞,肺泡   化脓性链球菌M1蛋白  

摘要： 目的探讨转录激活因子3(ATF3)在化脓性链球菌M1蛋白血清型诱导的急性肺损伤小鼠中的作用。方法 20只雌性C57/B6小鼠随机分为对照组(A组)和急性肺损伤组(B组),每组10只。20只雌性ATF3敲基因小鼠(ATF3^(-/-))随机分为对照组(C组)和急性肺损伤组(D组)。应用化脓性链球菌M1蛋白建立小鼠急性肺损伤模型,观察4组小鼠肺组织病理改变,计算肺组织湿重与干重比值,计数支气管肺泡灌洗液(BALF)中中性粒细胞数量,实时PCR方法检测小鼠肺泡巨噬细胞CXC趋化因子(CXCL1和CXCL2)mR NA的表达,ELISA法测定小鼠肺组织中CXCL1和CXCL2的浓度。结果与A组小鼠比较,B组小鼠肺组织病理改变加重、肺组织湿重与干重比值、BALF中中性粒细胞数量、肺泡巨噬细胞中CXCL1和CXCL2 mR NA表达量、肺组织CXCL1、CXCL2浓度均增加(P<0.05);与B组小鼠比较,D组小鼠肺组织病理改变加重、肺组织湿重与干重比值、BALF中中性粒细胞数量、肺泡巨噬细胞中CXCL1和CXCL2 m RNA表达量、肺组织CXCL1、CXCL2浓度均明显增加(P<0.05)。结论 ATF3对化脓性链球菌M1蛋白诱导的急性肺损伤小鼠有保护作用,可能与ATF3减少CXC趋化因子的产生、减轻中性粒细胞肺内聚集有关。