热休克蛋白70抑制剂PFT-μ对诱导型一氧化氮合成酶诱导表达及蛋白稳定性的影响
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摘要
背景:一氧化氮（Nitric oxide, NO）作为重要的信号分子和效应分子参与多种生理过程。机体通过一氧化氮合成酶（Nitric oxidesynthase, NOS）来合成NO，而诱导型NOS（inducible NOS, iNOS）因其具有的诱导表达及高效产生NO的特性使其成为机体免疫系统用来对抗入侵的微生物及清除自身肿瘤细胞的重要工具。热休克蛋白（Heatshock protein, HSP）是细胞内表达丰度最高的蛋白，在受到热休克等应激刺激时还会大量诱导表达。本课题组前期的研究证实HSP90对iNOS表达、活性及蛋白稳定性有重要影响，而作为与之紧密相关的HSP70是否有相似的作用目前尚不清楚。PFT-μ (Pifithrin-mu)是由George等于2009年首次在Mol Cell上报道的对HSP70具有高度选择性的一种小分子抑制剂，这为我们研究HSP70提供了一个有力的研究工具。到目前为止，利用PFT-μ治疗白血病等癌症方面的研究已有报道，但尚无在脓毒症等炎症方面的研究，另外也无HSP70与iNOS蛋白诱导及蛋白翻译后稳定性维持方面的报道。
目的:拟探讨PFT-μ作为特异性热休克蛋白70抑制剂对IFN-γ诱导的小鼠RAW264.7巨噬细胞NO产生及iNOS表达的影响及分子调控机制；观察PFT-μ对脓毒症小鼠主要组织内iNOS表达的影响，观察PFT-μ是否参与iNOS蛋白翻译后的稳定性。
方法:采用LPS/IFN-γ诱导RAW264.7巨噬细胞株构建炎症反应的细胞模型，Western-Blot检测iNOS等蛋白表达；定量聚合酶链反应(q-PCR)分析iNOS mRNA表达改变，Griess试剂测定培养基中NO的含量；HSP70siRNA转染RAW264.7巨噬细胞检测iNOS等蛋白表达；Western-Blot检测转录因子STAT1磷酸化水平及IRF-1诱导表达及胞核转移情况、染色质免疫共沉淀（Chromosome immunoprecipitate assay,ChIP）检测p-STAT1和IRF-1与iNOS基因启动子区的结合；Western-Blot检测胞浆可溶性及沉淀中iNOS蛋白；构建内毒素血症小鼠模型检测主要组织内iNOS表达水平的变化。
结果:在IFN-γ诱导的RAW264.7小鼠巨噬细胞中，PFT-μ在8-12小时可抑制NO生成（P<0.05）；PFT-μ在8-12小时可下调iNOS蛋白和mRNA表达（P<0.05）；HSP70siRNA转染后可下调iNOS蛋白水平；PFT-μ不影响细胞内STAT1、磷酸化STAT1的水平，PFT-μ不影响IRF-1蛋白的表达及细胞内胞浆与胞核间转移；PFT-μ在2小时可降低p-STAT1和IRF-1对iNOS启动子区的结合（P<0.05）；PFT-μ不影响iNOS mRNA的稳定性（P>0.05）；PFT-μ不影响iNOS的蛋白稳定性；PFT-μ可抑制内毒素血症小鼠主要组织内iNOS的蛋白及mRNA的表达（P<0.05）。
结论：PFT-μ可降低LPS/IFN-γ诱导的RAW264.7中NO的生成、iNOS蛋白和mRNA表达；PFT-μ通过抑制转录因子p-STAT1和IRF-1与iNOS基因启动子结合而减少iNOS生成；PFT-μ对iNOS mRNA及蛋白质稳定性无影响；PFT-μ可在小鼠体内抑制iNOS表达。
Backgroud: Nitric oxide (NO) is a fundamental signaling moleculeand effector involving in a variety of biological processes. In biologicalsystem, NO is produced by nitric oxide synthases (NOSs). Because of itsinducible expression and high-output features, iNOS is taken as aneffective tool to fight against microbe infection and eliminate tumor cells.Although many inflammatory factors can induce iNOS, the molecularmechanism is still largely unknown. Heat shock proteins (HSPs) are one ofthe most abundant protein families in the cytosolic, and they can be furtherinduced under heat shock stimuli. Our previous studies show that HSP90has great impact on iNOS induction, protein activity, and its stability. As arelevant protein, we evaluated whether HSP70has a similar effect on iNOS.George et al reported a small molecule, which strictly inhibits HSP70,called pifithrin-μ (PFT-μ), also called phenylacetylenylsulfonamide at thejournal Molecular Cell in2009. It gives us the possibility to study HSP70in vitro and in vivo. Up to now, many researchers have reported thisinhibitor can effectively at killing various types of tumor cells, but few reports are focused on inflammatory disorders or the role in iNOSinduction and protein quality control.
Objective：To investigate the effects and mechanisms of the specificHSP70inhibtor, PFT-μ on LPS/IFN-γ-induced iNOS induction and nitricoxide production in murine RAW264.7cells; to study the effects of PFT-μon iNOS induction in endotoxemic mice model; to evaluate the effects ofPFT-μ on iNOS protein stability.
Methods： To establish inflammatory cell line model by usingLPS/IFN-γ-stimulating murine RAW264.7cells. The level of iNOS proteinwas determined by Western-Blot, iNOS mRNA level was evaluated by realtime PCR, and nitric oxide production in media was determined by Griessreaction. HSP70siRNA was transfected to evulate the specific inhibition ofPFT-μ on RAW264.7cells. Western-Blot was used to determine thephosphoralation of STAT1, the induction and nuclear translocation of IRF-1.Chromation immunoprecipitaiton assay was used to evaluate the bindingefficiency of p-STAT1and IRF-1to its DNA elements. Western-Blot wasused to evaluate the soluable and insoluable iNOS after PFT-μ pretreatment.The endotoxemic mice model was conducted to evaluate the HSP70inhibition on iNOS induction in vivo.
Results: Nitric oxide production, iNOS mRNA, and protein levelswere blunt after8hours in IFN-γ-stimulating RAW264.7cells bypretreatment with PFT-μ (P<0.05).Transfection with HSP70siRNA could inhibit iNOS protein expression. PFT-μ did not disturb thephosphoralation of STAT1, the induction and nuclear translocation ofIRF-1. In the presence of PFT-μ, IFN-γ-elicited STAT1and IRF-1bingdings to iNOS promoter were largerly abrogated (P<0.05). PFT-μ didnot change the stability of iNOS mRNA nor iNOS protein. HSP70function is essential for iNOS induction in endotoxemic mice model(P<0.05).
Conclusion: PFT-μ prevents iNOS gene transcription, and proteinexpression, and nitric oxide production in LPS/IFN-γ-stimulating murineRAW264.7cell line. PFT-μ could inhibit STAT1and IRF-1bindings toiNOS promoter, and abrogate iNOS gene transcription. PFT-μ did notshorten the half-life of iNOS mRNA. PFT-μ pretreatment did not enhancethe insoluable portion of iNOS protein.
目的:拟探讨PFT-μ作为特异性热休克蛋白70抑制剂对IFN-γ诱导的小鼠RAW264.7巨噬细胞NO产生及iNOS表达的影响及分子调控机制；观察PFT-μ对脓毒症小鼠主要组织内iNOS表达的影响，观察PFT-μ是否参与iNOS蛋白翻译后的稳定性。
方法:采用LPS/IFN-γ诱导RAW264.7巨噬细胞株构建炎症反应的细胞模型，Western-Blot检测iNOS等蛋白表达；定量聚合酶链反应(q-PCR)分析iNOS mRNA表达改变，Griess试剂测定培养基中NO的含量；HSP70siRNA转染RAW264.7巨噬细胞检测iNOS等蛋白表达；Western-Blot检测转录因子STAT1磷酸化水平及IRF-1诱导表达及胞核转移情况、染色质免疫共沉淀（Chromosome immunoprecipitate assay,ChIP）检测p-STAT1和IRF-1与iNOS基因启动子区的结合；Western-Blot检测胞浆可溶性及沉淀中iNOS蛋白；构建内毒素血症小鼠模型检测主要组织内iNOS表达水平的变化。
结果:在IFN-γ诱导的RAW264.7小鼠巨噬细胞中，PFT-μ在8-12小时可抑制NO生成（P<0.05）；PFT-μ在8-12小时可下调iNOS蛋白和mRNA表达（P<0.05）；HSP70siRNA转染后可下调iNOS蛋白水平；PFT-μ不影响细胞内STAT1、磷酸化STAT1的水平，PFT-μ不影响IRF-1蛋白的表达及细胞内胞浆与胞核间转移；PFT-μ在2小时可降低p-STAT1和IRF-1对iNOS启动子区的结合（P<0.05）；PFT-μ不影响iNOS mRNA的稳定性（P>0.05）；PFT-μ不影响iNOS的蛋白稳定性；PFT-μ可抑制内毒素血症小鼠主要组织内iNOS的蛋白及mRNA的表达（P<0.05）。
结论：PFT-μ可降低LPS/IFN-γ诱导的RAW264.7中NO的生成、iNOS蛋白和mRNA表达；PFT-μ通过抑制转录因子p-STAT1和IRF-1与iNOS基因启动子结合而减少iNOS生成；PFT-μ对iNOS mRNA及蛋白质稳定性无影响；PFT-μ可在小鼠体内抑制iNOS表达。
Backgroud: Nitric oxide (NO) is a fundamental signaling moleculeand effector involving in a variety of biological processes. In biologicalsystem, NO is produced by nitric oxide synthases (NOSs). Because of itsinducible expression and high-output features, iNOS is taken as aneffective tool to fight against microbe infection and eliminate tumor cells.Although many inflammatory factors can induce iNOS, the molecularmechanism is still largely unknown. Heat shock proteins (HSPs) are one ofthe most abundant protein families in the cytosolic, and they can be furtherinduced under heat shock stimuli. Our previous studies show that HSP90has great impact on iNOS induction, protein activity, and its stability. As arelevant protein, we evaluated whether HSP70has a similar effect on iNOS.George et al reported a small molecule, which strictly inhibits HSP70,called pifithrin-μ (PFT-μ), also called phenylacetylenylsulfonamide at thejournal Molecular Cell in2009. It gives us the possibility to study HSP70in vitro and in vivo. Up to now, many researchers have reported thisinhibitor can effectively at killing various types of tumor cells, but few reports are focused on inflammatory disorders or the role in iNOSinduction and protein quality control.
Objective：To investigate the effects and mechanisms of the specificHSP70inhibtor, PFT-μ on LPS/IFN-γ-induced iNOS induction and nitricoxide production in murine RAW264.7cells; to study the effects of PFT-μon iNOS induction in endotoxemic mice model; to evaluate the effects ofPFT-μ on iNOS protein stability.
Methods： To establish inflammatory cell line model by usingLPS/IFN-γ-stimulating murine RAW264.7cells. The level of iNOS proteinwas determined by Western-Blot, iNOS mRNA level was evaluated by realtime PCR, and nitric oxide production in media was determined by Griessreaction. HSP70siRNA was transfected to evulate the specific inhibition ofPFT-μ on RAW264.7cells. Western-Blot was used to determine thephosphoralation of STAT1, the induction and nuclear translocation of IRF-1.Chromation immunoprecipitaiton assay was used to evaluate the bindingefficiency of p-STAT1and IRF-1to its DNA elements. Western-Blot wasused to evaluate the soluable and insoluable iNOS after PFT-μ pretreatment.The endotoxemic mice model was conducted to evaluate the HSP70inhibition on iNOS induction in vivo.
Results: Nitric oxide production, iNOS mRNA, and protein levelswere blunt after8hours in IFN-γ-stimulating RAW264.7cells bypretreatment with PFT-μ (P<0.05).Transfection with HSP70siRNA could inhibit iNOS protein expression. PFT-μ did not disturb thephosphoralation of STAT1, the induction and nuclear translocation ofIRF-1. In the presence of PFT-μ, IFN-γ-elicited STAT1and IRF-1bingdings to iNOS promoter were largerly abrogated (P<0.05). PFT-μ didnot change the stability of iNOS mRNA nor iNOS protein. HSP70function is essential for iNOS induction in endotoxemic mice model(P<0.05).
Conclusion: PFT-μ prevents iNOS gene transcription, and proteinexpression, and nitric oxide production in LPS/IFN-γ-stimulating murineRAW264.7cell line. PFT-μ could inhibit STAT1and IRF-1bindings toiNOS promoter, and abrogate iNOS gene transcription. PFT-μ did notshorten the half-life of iNOS mRNA. PFT-μ pretreatment did not enhancethe insoluable portion of iNOS protein.
引文
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