细胞外热休克蛋白70通过与A549人肺癌细胞中的RAGE结合激活ERK1/2、NF-κB和促炎基因转录。
Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells.
作者信息
Somensi Nauana, Brum Pedro Ozorio, de Miranda Ramos Vitor, Gasparotto Juciano, Zanotto-Filho Alfeu, Rostirolla Diana Carolina, da Silva Morrone Maurilio, Moreira José Claudio Fonseca, Pens Gelain Daniel
机构信息
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
出版信息
Cell Physiol Biochem. 2017;42(6):2507-2522. doi: 10.1159/000480213. Epub 2017 Aug 22.
BACKGROUND/AIMS: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE.
METHODS
Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR.
RESULTS
Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited.
CONCLUSIONS
Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.
背景/目的：热休克蛋白70（HSP70）最近被描述具有细胞外作用，在炎症条件下它会被主动释放。细胞外HSP70（eHSP70）作为损伤相关分子模式（DAMPs），与膜受体相互作用并激活炎症信号通路。在此背景下，晚期糖基化终产物受体（RAGE）成为与eHSP70相互作用的一个可能候选者。RAGE是一种模式识别受体，其表达在几种与慢性促炎状态相关的疾病中会增加。RAGE配体结合的主要后果之一是依赖细胞外信号调节激酶1/2（ERK1/2）激活核因子κB（NF-κB），这会导致肿瘤坏死因子α（TNF-α）和其他细胞因子的表达。本研究的目的是使用组成性表达RAGE的A549人肺癌细胞，阐明eHSP70是否能够引发依赖RAGE的信号传导。
方法
利用免疫沉淀和蛋白质邻近分析来证明RAGE与eHSP70之间的联系。为了研究RAGE在细胞对eHSP70反应中的相关性，使用小干扰RNA（siRNA）敲低受体表达。通过蛋白质免疫印迹、基因报告荧光素酶检测和实时定量聚合酶链反应评估信号通路的激活情况。
结果
在我们的细胞模型中，蛋白质eHSP70显示出与受体RAGE发生物理相互作用。用eHSP70处理导致ERK1/2激活以及NF-κB反式激活受到RAGE敲低的损害。此外，在RAGE沉默的细胞中，eHSP70对促炎细胞因子表达的刺激作用受到抑制。最后，当A549细胞的RAGE表达受到抑制时，经eHSP70处理的A549细胞的条件培养基对单核细胞细胞因子表达产生不同影响。
结论
我们的结果证明eHSP70是一种新型的RAGE激动剂，能够影响癌症细胞与免疫细胞之间的相互作用。
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