【作者】 张晓静；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 一氧化氮(nitric oxide ,NO)作为体内气体信号分子在多种生理和病理条件下对细胞功能具有重要调节作用。NO发挥生物学作用不依赖膜受体而直接作用于效应分子的特定靶点。以往关于NO与蛋白质直接作用的研究主要集中于NO与金属离子的结合,例如NO可以与可溶性鸟苷酸环化酶(soluble guanylyl cyclase ,sCG)中血红素卟啉环的Fe2+结合,改变酶的构,使酶活性升高,生成环磷酸鸟苷(3′-5′guanosine monophosphate ,cGMP),发挥舒张血管和抑制血小板聚集等生物学效应。近年来NO对巯基的氧化修饰作用逐步受到关注。Stamler等发现, NO可作用于半胱氨酸巯基(-SH)生成低分子量亚硝基硫醇(S-nitrosothiol ,SNO)或亚硝基化蛋白质(S-nitrosylated protein ,PSNO),改变蛋白质构象及蛋白质功能,这一作用被定义为蛋白质巯基亚硝基化(protein S-nitrosylation)。亚硝基化是一种依赖NO的巯基氧化修饰,可直接或通过引起谷胱甘肽化,二硫键形成等与NO相关的巯基氧化修饰方式调节蛋白质功能。细胞内影响亚硝基化修饰的因素包括:细胞内氧化还原状态,蛋白质或巯基所处的微环境,催化氧化还原反应的酶的水平和分布以及NO的浓度和活性氧族(reactive oxygen species ,ROS)的特性等。目前,亚硝基化修饰正逐渐被认为是一种与磷酸化相似的新的信号转导调控机制。类风湿性关节炎(rheumatoid arthritis ,RA)是一种以滑膜组织增生和关节炎症反应为病理特征的慢性自身免疫性疾病。成纤维细胞样滑膜细胞(fibroblast-like synoviocytes ,FLS)是滑膜组织的主要组成细胞,在RA患者的病变关节内过度增殖并侵入邻近组织,同时生成大量细胞因子和趋化因子引起局部炎症细胞的聚集和活化,在炎症的起始和持久化以及关节结构的破坏过程中起重要作用。有研究发现,在RA患者关节滑液中,NO含量增高,可诱导FLS凋亡并抑制其增殖,提示在RA发病过程中FLS所处的关节腔内环境中有NO存在,NO在调节FLS功能方面可能发挥重要作用。研究发现,亚硝基化修饰参与NO对核因子κB(nuclear factor-kappa B,NF-κB)和活性蛋白-1(activating protein-1,AP-1)活性的抑制作用,提示亚硝基化修饰可从转录水平调节细胞的功能和状态。Takeba1等证实,FLS中cAMP反应元件结合蛋白(cAMP response element binding protein ,CREB)可被白介素-1β(IL-1β)等细胞因子激活,在RA发病过程中,介导FLS过度增殖。与CREB相似,信号转导和转录激活因子(signal transducer and activator of transcription , STAT-1)也参与RA的发病。Kasperkovitz等发现,磷酸化STAT-1在RA患者滑膜组织中表达升高,主要分布在滑膜衬里层的FLS、局部浸润的T细胞和B细胞等。STAT-1主要被IFN-γ激活,通过诱导趋化因子、补体等炎症基因的表达,促进滑膜炎症反应。CREB和STAT-1在介导滑膜组织增生和关节炎症反应方面可能发挥重要作用。有研究结果表明,NO对CREB和STAT-1的活性具有调节作用。大量关于巯基氧化修饰的研究证实,含半胱氨酸的转录因子对依赖NO的巯基氧化修饰具有敏感性。CREB的激酶诱导区和STAT-1的DNA结合区中含有自由巯基,NO是否可以引起FLS内发生蛋白质亚硝基化;蛋白质亚硝基化是否能导致CREB和STAT-1发生巯基氧化修饰;对二者的磷酸化水平、DNA结合活性以及下游基因表达有何调节作用,均未见文献报道。亚硝基化蛋白质(PSNO)中的S-NO键不稳定,在还原性物质和强光的作用下极易分解,所以直接研究蛋白质巯基亚硝基化在方法学上存在一定困难。最近,Jaffrey等提出的生物素转化技术(biotin switch method)可特异性将生物素标记在蛋白质分子中发生亚硝基化的巯基上,将亚硝基化转化为稳定的生物素化,通过检测生物素化蛋白表达水平来反映亚硝基化蛋白表达水平,成为研究亚硝基化修饰的新方法。本文采用biotin switch method和Western blotting法相结合检测成纤维细胞样滑膜细胞RSC-364蛋白质亚硝基化水平;利用电泳迁移率改变分析(Electropehoresis mobility shift assay , EMSA)技术观察蛋白质亚硝基化对CREB和STAT-1活性的调节作用;同时采用Western blotting方法检测亚硝基化作用对CREB和STAT-1磷酸化水平的影响;并进一步研究亚硝基化对CREB和STAT-1启动的下游基因蛋白表达的调节作用,以探讨蛋白质巯基亚硝基化在RA发病中的调控作用,为揭示NO快速调节效应提供实验依据。1 NO对RSC-364细胞蛋白质亚硝基化水平的影响1.1 NO与RSC-364细胞提取的总蛋白直接作用后对亚硝基化水平的影响本部分实验提取RSC-364总蛋白与NO供体直接孵育,采用biotin switch method结合Western blotting对RSC-364细胞总蛋白亚硝基化水平进行探索性研究。方法:提取RSC-364细胞总蛋白进行细胞外亚硝基化反应。将蛋白分为四组:①对照组②还原型谷胱甘肽(GSH)组③NO供体亚硝基化谷胱甘肽(GSNO)组④GSNO+DTT(还原剂)组。各组反应结束后均用丙酮沉淀法除去多余试剂,进行biotin switch method。在biotin switch method中以上各组均设无生物素标记组为阴性对照。生物素标记后用Western blotting法检测生物素化蛋白表达水平以反映亚硝基化蛋白表达水平。结果:在标记生物素的四个组中,对照组没有见到清晰的蛋白条带,GSNO组可明显见到一组由低分子量到高分子量的蛋白条带,表明GSNO使RSC-364细胞提取的总蛋白亚硝基化水平增高。与GSNO组相比,GSNO+DTT组亚硝基化反应被显著抑制。与对照组相比,GSH对亚硝基化水平无明显影响。无生物素标记组均在分子量为39KDa和58KDa之间有弱的,光密度一致的内源性生物素化蛋白的表达。小结:本部分实验在细胞外水平证实,GSNO释放的NO可引起RSC-364细胞提取的总蛋白发生亚硝基化修饰。1.2 NO对RSC-364细胞内蛋白质亚硝基化水平的影响在第一部分(一)中,我们成功利用biotin switch method结合Western blotting证实,NO与RSC-364细胞提取的蛋白质直接作用可发生亚硝基化修饰。本部分实验应用GSNO孵育RSC-364细胞或采用细胞因子联合刺激RSC-364细胞,检测细胞内蛋白质亚硝基化水平。方法:实验分为九组:①对照组I;②GSH组;③GSNO组;④GSNO+DTT组;⑤对照组II;⑥IFN-γ+IL-1β组;⑦iNOS抑制剂氨基胍(aminoguanidine ,AG)组;⑧IFN-γ+IL-1β+AG组;⑨IFN-γ+IL-1β+DTT组。各组孵育相应时间后提取细胞总蛋白立即进行biotin switch method,并进一步用Western blotting法检测亚硝基化蛋白的表达。其中⑤-⑧组在孵育相应时间后,收集细胞培养液上清进行NO含量测定。结果显示如下:(1) GSNO孵育细胞对亚硝基化水平的影响在标记生物素的四个组中,对照组I没有见到清晰条带,GSNO组可明显见到一组由低分子量到高分子量的蛋白条带,表明GSNO孵育细胞后使细胞内蛋白质亚硝基化水平增高。与GSNO组相比,GSNO+DTT组亚硝基化反应被显著抑制。与对照组I相比,GSH对亚硝基化水平无明显影响。无生物素标记组均在分子量为39KDa和58KDa之间有弱的,光密度一致的内源性生物素化蛋白的表达。(2) IFN-γ和IL-1β联合刺激细胞对细胞内亚硝基化水平的影响在标记生物素的四个组中,对照组II没有见到清晰的条带,IFN-γ、IL-1β联合孵育细胞24h后可明显见到一组由低分子量到高分子量的蛋白条带,表明IFN-γ、IL-1β联合孵育细胞使细胞内蛋白质亚硝基化水平增高。DTT可显著抑制IFN-γ和IL-1β共同孵育引起的亚硝基化反应。同时加入AG也可抑制IFN-γ和IL-1β引起的亚硝基化反应。无生物素标记组均在分子量为39KDa和58KDa之间有弱的,光密度一致的内源性生物素化蛋白的表达。(3) NO含量检测结果与对照组II (4.34±2.82μm/L)相比,细胞因子联合孵育细胞使培养液上清中NO含量显著增高(30.93±4.8μm/L ,p<0.01),而同时应用iNOS抑制剂AG可逆转IFN-γ与IL-1β的作用(4.7±2.63μm/L ,p<0.01)。单独应用AG对培养液上清中NO含量无明显影响(5.4±2.47μm/L ,p>0.05)。以上研究结果表明,外源性应用GSNO可引起RSC-364细胞内发生蛋白质巯基亚硝基化。IFN-γ、IL-1β联合应用可诱导RSC-364细胞产生NO增多,并引起RSC-364细胞内发生蛋白质亚硝基化。Biotin switch method可成功应用在RSC-364细胞中检测蛋白质亚硝基化水平。2蛋白质巯基亚硝基化对RSC-364细胞CREB活性的影响2.1细胞外亚硝基化修饰对RSC-364细胞CREB活性的影响在第一部分中,我们证实用NO供体GSNO直接孵育RSC-364细胞总蛋白可引起蛋白质巯基亚硝基化,本部分实验首先采用IL-1β诱导RSC-364细胞CREB活化,然后提取核蛋白,用GSNO对细胞核蛋白直接进行亚硝基化修饰,最后用EMSA法观察亚硝基化修饰对CREB活性的影响。方法:实验分为六组:①对照组;②IL-1β组;③IL-1β+GSNO组;④IL-1β+GSNO+DTT组;⑤DTT组;⑥IL-1β+DTT组。各组反应结束后立即进行EMSA实验。结果如下:在对照组,RSC-364细胞核内未检测到与特异性寡核苷酸探针结合的CREB。用IL-1β孵育细胞2h后细胞核内CREB活性明显高于对照组(p<0.01),而用GSNO与核蛋白反应后明显抑制了IL-1β诱导的CREB活性(p<0.01),GSNO的作用可被DTT所逆转(p<0.01)。DTT单独孵育核蛋白对CREB活性无明显影响(p>0.01)。用同源性寡核苷酸(含CREB结合位点)和异源性寡核苷酸(含AP-2结合位点)作为竞争物证实了DNA-蛋白结合的特异性。小结:本部分实验证实,NO与蛋白质直接作用可通过引起蛋白质巯基亚硝基化,导致CREB发生巯基氧化修饰,可逆性抑制IL-1β诱导的CREB活性。2.2细胞内亚硝基化修饰对RSC-364细胞CREB活性的影响我们已经证实,NO与蛋白质直接作用可通过引起亚硝基化,导致CREB发生巯基氧化修饰,抑制IL-1β诱导的CREB活性,同时也表明CREB对依赖NO的巯基氧化修饰具有敏感性。本部分实验采用NO供体或联合应用细胞因子孵育RSC-364细胞,研究细胞内亚硝基化对CREB磷酸化水平和DNA结合活性的影响。方法:实验分为十组:①对照组;②IL-1β组;③GSNO组;④GSNO+IL-1β组;⑤NEM组;⑥NEM+GSNO+IL-1β组;⑦GSNO+IL-1β+DTT组;⑧IL-1β+IFN-γ组;⑨AG+IL-1β+IFN-γ组;⑩AG组。各组于相应时间点提取细胞核蛋白进行EMSA实验检测CREB活性;①-⑥组提取总蛋白进行western blotting实验测定CREB和磷酸化CREB(P-CREB)的表达水平。结果显示如下:(1) GSNO孵育细胞对CREB活性的影响在对照组,RSC-364细胞核内未检测到与特异性寡核苷酸探针结合的CREB。用IL-1β孵育细胞2h后细胞核内CREB活性明显高于对照组(p<0.01),而用GSNO预先孵育细胞明显抑制了IL-1β诱导的CREB活性(p<0.01),GSNO的作用可被DTT所逆转(p<0.01)。在应用GSNO之前预先加入NEM也可逆转GSNO对IL-1β诱导的CREB活性的抑制作用(p<0.01)。NEM单独孵育细胞对CREB活性无明显影响(p>0.05)。用同源性寡核苷酸(含CREB结合位点)和异源性寡核苷酸(含AP-2结合位点)作为竞争物证实了DNA-蛋白结合的特异性。(2)联合应用细胞因子IL-1β和IFN-γ对CREB活性的影响IL-1β孵育细胞2h后,细胞核内CREB活性明显高于对照组(p<0.01),而IL-1β和IFN-γ共同孵育细胞12h后可显著抑制IL-1β诱导的CREB活性(p<0.01),预先加入iNOS抑制剂AG可减弱IL-1β和IFN-γ共同孵育对CREB活性的抑制作用(p<0.01),但较IL-1β组仍呈下降趋势(p<0.01)。AG单独孵育细胞对CREB活性无明显影响(p>0.05)。(3) GSNO孵育细胞对CREB和磷酸化CREB蛋白表达的影响与对照组相比,IL-1β组CREB表达水平无明显变化(p>0.05),而P-CREB表达水平明显升高(p<0.01)。与IL-1β相比,GSNO+IL-1β组和NEM+GSNO+IL-1β组CREB和P-CREB表达水平均无明显变化(p均>0.05)。与对照组相比,GSNO或NEM单独孵育细胞对CREB和P-CREB水平也均无明显影响(p均>0.05)。以上结果表明:GSNO可通过引起RSC-364细胞内蛋白质亚硝基化,导致CREB发生巯基氧化修饰,并抑制其活性,但对其磷酸化水平无明显影响。细胞因子联合刺激RSC-364细胞可通过诱导内源性NO产生,引起亚硝基化水平升高,导致CREB发生巯基氧化修饰,抑制CREB活性。3蛋白质巯基亚硝基化对RSC-364细胞STAT-1活性的影响3.1细胞外亚硝基化对修饰RSC-364细胞STAT-1活性的影响本部分实验首先采用IFN-γ诱导STAT-1活化,然后提取核蛋白,用GSNO对细胞核蛋白直接进行亚硝基化修饰,最后进行EMSA实验观察亚硝基化修饰对STAT-1活性的影响。方法:实验分为六组:①对照组;②IFN-γ组;③IFN-γ+GSNO组;④IFN-γ+GSNO+DTT组;⑤DTT组;⑥IFN-γ+DTT组。各组反应结束后立即进行EMSA实验。结果:在对照组,RSC-364细胞核内未检测到与特异性寡核苷酸探针结合的STAT-1。用IFN-γ孵育细胞30min后细胞核内STAT-1活性明显高于对照组(p<0.01),而用GSNO与核蛋白反应后明显抑制了IFN-γ诱导的STAT-1活性(p<0.01),GSNO的作用可被DTT所逆转(p<0.01)。DTT单独孵育核蛋白对STAT-1活性无明显影响(p>0.05)。用同源性寡核苷酸(含STAT-1结合位点)和异源性寡核苷酸(含AP-2结合位点)作为竞争物证实了DNA-蛋白结合的特异性。小结:本部分实验证实,NO与蛋白直接作用可引起亚硝基化,导致STAT-1发生巯基氧化修饰,抑制IFN-γ诱导的STAT-1活性。3.2细胞内亚硝基化修饰对RSC-364细胞STAT-1活性的影响我们已经证实,NO与蛋白直接作用可通过引起亚硝基化,导致STAT-1发生巯基氧化修饰,抑制IFN-γ诱导的STAT-1活性,同时也表明STAT-1对依赖NO的氧化修饰具有敏感性。NO孵育RSC-364可引起细胞内发生亚硝基化,能否导致STAT-1在细胞内发生巯基氧化修饰,并抑制其活性;能否调节STAT-1磷酸化水平均不清楚。此外,内源性产生的NO能否对STAT-1活性进行调节,也未见文献报道。本部分实验对这些问题进行了探讨。方法:实验分为十组:①对照组;②IFN-γ组;③GSNO组;④GSNO+IFN-γ组;⑤NEM组;⑥NEM+GSNO+IFN-γ组;⑦GSNO+IFN-γ+DTT组;⑧IL-1β+IFN-γ组;⑨AG+IL-1β+IFN-γ组;⑩AG组。各组于相应时间点提取细胞核蛋白进行EMSA实验检测STAT-1活性;①-⑥组提取细胞总蛋白进行Western blotting实验测定STAT-1和磷酸化STAT-1(P-STAT-1)的蛋白表达水平。结果显示如下:(1) GSNO孵育细胞对STAT-1活性的影响在对照组,RSC-364细胞核内未检测到与特异性寡核苷酸探针结合的STAT-1。IFN-γ孵育细胞30min后细胞核内STAT-1活性明显高于对照组(p<0.01),而GSNO预先孵育细胞明显抑制了IFN-γ诱导的STAT-1活性(p<0.01),GSNO的作用可被DTT所逆转(p<0.01)。在给与GSNO之前预先加入NEM也可逆转GSNO对IFN-γ诱导的STAT-1活性的抑制作用(p<0.01)。NEM单独孵育细胞对STAT-1活性无明显影响(p>0.05)。用同源性寡核苷酸(含STAT-1结合位点)和异源性寡核苷酸(含AP-2结合位点)作为竞争物证实了DNA-蛋白结合的特异性。(2)细胞因子联合刺激对STAT-1活性的影响IFN-γ孵育细胞30min后细胞核内STAT-1活性明显高于对照组(p<0.01),而IL-1β和IFN-γ共同孵育细胞12h后可明显抑制STAT-1活性(p<0.01),预先加入iNOS抑制剂AG可部分逆转IL-1β和IFN-γ共同孵育对STAT-1活性的抑制作用(p<0.01)。AG单独孵育细胞对STAT-1活性无明显影响(p>0.05)。(3) GSNO孵育细胞对STAT-1和磷酸化STAT-1蛋白表达的影响与对照组相比,IFN-γ组STAT-1表达水平无明显变化(p>0.05),而P-STAT-1表达水平明显升高(p<0.01)。与IFN-γ相比,GSNO+IFN-γ组和NEM+GSNO+IFN-γ组STAT-1和P-STAT-1表达水平均无明显变化(p均>0.05)。与对照组相比,GSNO或NEM单独孵育细胞对STAT-1和P-STAT-1水平也均无明显影响(p均>0.05)。以上结果证实:GSNO可通过引起RSC-364细胞内蛋白质亚硝基化,导致STAT-1发生巯基氧化修饰,并抑制其活性,但对其磷酸化水平无明显影响。细胞因子联合刺激RSC-364细胞可通过诱导内源性NO产生,引起亚硝基化水平升高,导致STAT-1发生巯基氧化修饰,抑制STAT-1活性。4蛋白质巯基亚硝基化对RSC-364细胞CREB和STAT-1启动的下游基因蛋白表达的影响4.1蛋白质巯基亚硝基化对RSC-364细胞IL-6蛋白表达的影响本课题第二部分已证实,NO可通过引起细胞内亚硝基化,导致CREB发生巯基氧化修饰,抑制IL-1β诱导的CREB活性,是否能进一步影响CREB启动的下游基因IL-6的表达,尚不清楚。因此,本部分实验将探讨细胞内蛋白质亚硝基化对RSC-364细胞分泌IL-6水平的影响。方法:用DMEM培养基调整细胞浓度为5×105/ml,接种于24孔细胞培养板(1.0ml/well)。实验分为六组:①对照组;②IL-1β组;③GSNO组;④GSNO+ IL-1β组;⑤NEM组;⑥NEM+GSNO+IL-1β组。结果:RSC-364细胞在静息状态下可分泌少量IL-6 (66.92±8.2pg/ml)。IL-1β孵育RSC-364细胞24h后,细胞培养液上清中IL-6含量较对照组明显增多(431.69±14.15pg/ml,p<0.01)。与IL-1β组相比,GSNO+IL-1β组IL-6含量显著下降(143.96±14.7pg/ml,p<0.01),单独应用NO培养液上清中IL-6含量较对照组无明显变化(68.49±4.7pg/ml,p>0.05)。NEM+GSNO+IL-1β组培养液上清中IL-6含量较GSNO+IL-1β组明显增多(433.27±14.4pg/ml,p<0.01)。单独应用NEM IL-6含量较对照组没有明显变化(70.06±10.21pg/ml,p>0.05)。小结:蛋白质亚硝基化可通过引起CREB发生巯基氧化修饰抑制其活性,减少其下游基因转录产物IL-6的表达。4.2蛋白质巯基亚硝基化对RSC-364细胞IP-10蛋白表达的影响本课题第三部分已证实,NO可通过引起细胞内蛋白质亚硝基化,导致STAT-1发生巯基氧化修饰,抑制IFN-γ诱导的STAT-1活性,是否能进一步影响STAT-1启动的下游基因IP-10的表达,尚不清楚。因此,本部分实验观察了细胞内蛋白质亚硝基化对RSC-364分泌IP-10水平的影响。方法:实验分为六组:①对照组;②IFN-γ组;③GSNO组;④GSNO+IFN-γ组;⑤NEM组;⑥NEM+GSNO+IFN-γ组。结果:与对照组相比,IFN-γ孵育RSC-364细胞3h后IP-10表达水平明显升高(p<0.01),而GSNO可抑制IFN-γ的作用(p<0.01),预先应用NEM可逆转GSNO对IFN-γ诱导IP-10表达的抑制作用(p<0.01)。NEM或GSNO单独孵育RSC-364对IP-10表达水平均无明显影响(p>0.05)。小结:蛋白质亚硝基化可通过引起STAT-1发生巯基氧化修饰抑制其活性,减少其下游基因转录产物IP-10的表达。结论本课题探讨了蛋白质巯基亚硝基化对RSC-364细胞转录因子CREB和STAT-1的磷酸化水平、DNA结合活性以及启动的下游基因转录产物表达的影响。结论如下:1 NO可通过蛋白质亚硝基化导致CREB和STAT-1发生巯基氧化修饰,抑制二者DNA结合活性和下游基因的表达,这可能是NO在RA发病过程中调节FLS稳态,抑制RA发病进程的机制之一。2 NO是一种气体信号分子,其产生和消失是短暂的过程,进入细胞不需要特殊通道和受体。本课题证实,NO可通过蛋白质亚硝基化调节转录因子的活性,提示亚硝基化修饰是机体快速反应调节机制,与神经-内分泌等调节机制相比,可能是最快速的应激反应。3 GSNO广泛分布于包括血液系统在内的多种组织和器官,是NO的储存器和运输载体。本实验证实,GSNO可释放NO进入细胞调节转录因子的活性,提示NO不仅以自分泌和旁分泌还可能以内分泌方式调节细胞稳态。更多还原

【Abstract】 Nitric oxide (NO), as a gaseous messenger, regulates cell function in a variety of physiologic and pathophysiologic states. NO may act on target site of protein directly, in a membrane receptor-independent way, and plays multiple biological roles. Previous studies on direct action of NO on protein were mainly focused on the metal ion of the target protein. For example, NO can bind to heme iron of soluble guanylyl cyclase (sGC), produce the second messenger 3′-5′guanosine monophosphate (cGMP) and plays important roles in relaxation of vasculum and inhibition of platelets aggregation. Recently, NO-dependent cysteine thiols (Cys-SH) oxidative modification was paid attention to. In 1994, Stamler proposed the concept of protein S-nitrosylation: the coupling of a NO moiety to a reactive cysteine thiol and formation of an S-nitrosothiol (SNO) or S-nitrosylated protein (PSNO) can regulate conformation and function of protein. Protein S-nitrosylation, as a reversible cysteine redox modification, may regulate protein function directly or indirectly through formation of other NO-dependent thiols redox modifications including formation of the mixed disulfide or glutathiolation et al. Protein S-nitrosylation is tightly linked to cell type, cellular rodox status, hydrophobic environment around the target thiols, the colocalization of NOS with target protein, the concentration of NO and the characteristic of reactive oxygen species (ROS) et al. Protein S-nitrosylation has been recognized gradually as an ubiquitous regulatory mechanism in cell signal transduction comparable to phosphorylation.Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synoviocytes proliferation and synovium inflammation. Fibroblast-like synoviocytes (FLS) isolated from synovium of RA play a major role in the pathogenesis of RA by uncontrolled proliferation and secreting effector molecules including cytokines, chemokines, and proteolytic enzymes, which promote inflammation and joint destruction. NO is elevated in the synovial fluids of patients with RA, may inhibit FLS proliferation and induce FLS apoptosis. These studies indicate that NO exists in the micro-environment around FLS and may regulate function of FLS in pathogenesis of RA.Marshall et al. showed that protein S-nitrosylation may inhibit nuclear factor-kappa B (NF-κB) activities, Nikitovic et al. found that NO inhibited activating protein-1(AP-1) DNA binding activities by protein S-nitrosylation. These studies suggested that protein S-nitrosylation of transcription factors may serve as one of the mechanisms by which NO regulates cell homeostasis. Takeba et al. confirmed that cAMP response element-binding protein (CREB) is involved in the FLS proliferation in patients with RA. Analogous to CREB,the transcription factor signal transducer and activator of transcription-1(STAT-1) is activated in RA synovium by increased phosphorylation level. STAT-1 mainly distributes in FLS, infiltrated T cell et al. and plays a pivotal role in the expression of inflammatory gene products involved in RA. Both CREB and STAT-1 may play important roles in the pathogenesis of RA. It has been confirmed that NO may modulate CREB and STAT-1 activities. A number of researches demonstrated that most of transcription factors contained cysteine residues are sensitive to NO-dependent thiols redox modification. There are several conserved cysteine residues in kinase induced domain of CREB and DNA binding domain of STAT-1, whether NO may result in protein S-nitrosylation in FLS; whether protein S-nitrosylation can modulate CREB or STAT-1 activities by thiols redox modification; whether protein S-nitrosylation may regulate phosphorylation levels and downstream genes transcription of CREB or STAT-1 are all still unclear.Nitrosothiols are exceptionally labile as a result of reactivity with reducing agents and degradation in light, so it is difficult to detect proteins contained nitrosothiols. Recently, Jaffrey et al. developed the biotin switch method to specifically transform S-nitrosylated thiols in biotinylated thilos. The expression levels of S-nitrosylated proteins were evaluated by detection the expression levels of biotinylated proteins. Biotin switch method becomes a new method for research protein S-nitrosylation.To explore the role of protein S-nitrosylation and to reveal the fast regulatory effect of NO in pathogenesis of RA, we assaied the expression levels of S-nitrosylated proteins using the biotin switch method and Western blotting, investigated the effect of protein S-nitrosylation on DNA binding activities of CREB or STAT-1 using electrophoresis mobility shift assay (EMSA)and detected the effect of protein S-nitrosylation on phosphorylation levels and downstream genes transcription of CREB or STAT-1 in RSC-364(a kind of murine FLS).1 The effect of NO on protein S-nitrosylation in RSC-364 cells.1.1 The effect of NO on protein S-nitrosylation in RSC-364 cells extracts.To investigate the effect of NO on protein S-nitrosylation in RSC-364 cells extracts, RSC-364 cell extracts were prepared and were divided randomly into four groups:①control group;②glutathione (GSH) group;③S-nitroso-glutathione (GSNO) group;④GSNO+dithiothreitol (DTT) group. After S-nitrosylation reaction in vitro, extracts were precipitated with acetone to remove the GSNO or GSH. The resulting S-nitrosylated proteins were subjected to the biotin switch assay and some samples were treated with DMSO vehicle solution in non-biotin added groups as a control. After biotin switch assay, the expression of S-nitrosylated proteins was assayed by Western blotting. Results: in samples that were treated with biotin-HPDP, there was a wide range of biotinylated proteins and theoretically, S-nitrosylated proteins, in GSNO group, but none were present in unstimulated control group. After incubated with GSNO, addition of DTT as a reducing agent results in ablation of all signal. In samples that were not treated with biotin-HPDP, the endogenous biotinylated proteins are detectable. In conclusion, the present study confirmed that treating RSC-364 cell extracts with GSNO lead to protein S-nitrosylation . 1.2 The effect of NO on protein S-nitrosylation in RSC-364 cells.Above experiments showed that treating RSC-364 cell extracts with GSNO lead to protein S-nitrosylation, which indicated that there are a wide range of S-nitrosylation targets in RSC-364 cells. The purpose of the present study was to observe the effect of NO on protein S-nitrosylation in RSC-364 cells. The cultured RSC-364 cells were divided randomly into nine groups treated with different agents:①control group I;②GSH group;③GSNO group;④GSNO+DTTgroup;⑤control group II;⑥interferon-γ(IFN-γ)+ interleukin-1(IL-1β) group;⑦iNOS inhibitor aminoguanidine (AG) group;⑧IFN-γ+IL-1β+AG group;⑨IFN-γ+IL-1β+DTT group. The cell extracts from each group were prepared and were subjected to the biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. The contents of NO in RSC-364 cells culture supernatant in⑤-⑧groups were measured according protocols. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. The results were as follows: (1) GSNO obviously increased the expression of S-nitrosylated proteins in RSC-364 cells, which was reversed by DTT. Administration of GSH alone has no effect on the expression of nitrosylated proteins in cell. In samples that were not treated with biotin-HPDP, the endogenous biotinylated proteins were detectable. (2) In samples that were treated with biotin-HPDP, there was a wide range of biotinylated proteins in IFN-γ+IL-1βgroup, but none were present in control group and AG group. Interestingly, the pattern of IFN-γ+IL-1β+AG group was not different from that noted in control group. (3) Compared with control group, NO content in culture supernatant in IFN-γ+IL-1βgroup increased significantly (4.34±2.82μmol/L vs. 30.93±4.8μmol/L, p<0.01). Co-incubation with AG and cytokines mixture (IFN-γand IL-1β) resulted in a significant decrease of NO content in culture supernatant (4.7±2.63μmol/L, p<0.01). AG alone did not affect NO production (5.4±2.47μmol/L, p>0.05).These data confirmed that: 1 GSNO resulted in protein S-nitrosylation in RSC-364 cells. 2 This combination stimulation of cytokines (IFN-γand IL-1β) lead to protein S-nitrosylation in RSC-364 cells by increasing NO production.2 The effect of protein S-nitrosylation on CREB activities in RSC-364 cells2.1 The effect of protein S-nitrosylation in vitro on CREB activities in RSC-364 cellsAbove study showed that NO lead to protein S-nitrosylation by reaction with directly RSC-364 cell extracts. Whether protein S-nitrosylation is involved in regulation of CREB activity by NO has remained unclear, the aim of the present study was to investigate the effect of protein S-nitrosylation in vitro on CREB activies in RSC-364 cells. The nuclear extracts from RSC-364 cells treated with vehicle (NS) or IL-1βfor 2h were prepared and were incubated with GSNO in the absence or presence of DTT for 15 min at room temperature. After S-nitrosylation reaction in vitro, the nuclear extracts in each groups were subjected to electropehoresis mobility shift assay (EMSA) to explore the CREB binding activity. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). Treating the nuclear extracts from RSC-364 cells stimulated with IL-1βwith GSNO decreased CREB activation. If DTT was included in the nuclear extracts after GSNO exposure, the effect of GSNO on CREB activation was reversed. DTT treatment alone had no effect on the CREB binding activity (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors. These results showed that, NO resulted in protein S-nitrosylation in vitro, which could inhibit CREB activity by thiols oxidative modification.2.2 The effect of protein S-nitrosylation on CREB activity in RSC-364 cellsAbove experiments showed that NO could result in protein S-nitrosylation in RSC-364 cell extracts and inhibit CREB activity by thiols oxidative modification in vitro. It is remained unclear that the effect of protein S-nitrosylation in RSC-364 cells on CREB/DNA binding activity and on CREB phosphorylation. In the present study, the cultured RSC-364 cells were divided randomly into ten groups after treatment with different agents:①control group;②IL-1βgroup;③GSNO group;④GSNO+IL-1βgroup;⑤the thiol-alkylating agent N-ethylmaleimide (NEM) group;⑥NEM+GSNO+ IL-1βgroup;⑦GSNO+IL-1β+DTT group;⑧IL-1β+IFN-γgroup;⑨AG+ IL-1β+IFN-γgroup;⑩AG group. The CREB binding activity was analyzed by EMSA; the CREB and phosphorylated CREB levels in①-⑥groups were detected by western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: (1) The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). Pre-treatment RSC-364 cells with GSNO decreased IL-1β-induced CREB activation (p<0.01). If DTT was added in the nuclear extracts from the RSC-364 cells incubated with GSNO and IL-1β, the effect of GSNO on CREB activation was reversed (p<0.01). If RSC-364 cells were treated with NEM for 30 min followed by stimulation with GSNO and IL-1β, the inhibition effect of GSNO on CREB activation was also reversed (p<0.01). Pre-treatment with NEM alone had no effect on the CREB binding activity (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors.(2) The CREB binding activity was significantly higher in RSC-364 cells stimulated with IL-1βin comparison with unstimulated cells (p<0.01). The combination stimulation of cytokines (IFN-γand IL-1β) for 12h resulted in a obvious decrease in CREB activation induced by IL-1β(p<0.01), but co-incubation with AG and cytokines increased CREB activation (p<0.01). AG alone had no significant effect on CREB activation (p>0.05). The binding specificity was confirmed by using homologous (CREB) and nonhomologous (AP-2) oligonucleotides as competitors.(3) The phosphorylated CREB level in RSC-364 cells was markedly increased 2h after incubation with IL-1βin comparison with the control group (p<0.01). Pre-treatment with GSNO had no obvious effect on the phosphorylated CREB level in RSC-364 cells in comparison with the IL-1βgroup (p>0.05), pre-treatment with NEM had no significant effect on the phosphorylated CREB level in RSC-364 cells in comparison with the GSNO+IL-1βgroup (p>0.05). Treatment with GSNO or NEM alone had no effect on the phosphorylated CREB level in comparison with the control group (both p>0.05). NO significant changes in CREB level were observed in all groups (all p>0.05). These results showed that: (1) NO resulted in protein S-nitrosylation in RSC-364 cell, which might CREB inhibit activity by thiols oxidative modification. (2) Protein S-nitrosylation induced by NO had no significant effect on the phosphorylated CREB level. (3) This combination stimulation of cytokines (IFN-γand IL-1β) might inhibit CREB activity by increasing S-nitrosylation level.3 The effect of protein S-nitrosylation on STAT-1 activities in RSC-364 cells3.1 The effect of protein S-nitrosylation in vitro on STAT-1 activities in RSC-364 cellsWhether protein S-nitrosylation is involved in regulation of STAT-1 activity by NO has remained unproved, the aim of the present study was to investigate the effect of protein S-nitrosylation in vitro on STAT-1 activies in RSC-364 cells. The nuclear extracts from RSC-364 cells treated with vehicle (NS) or IFN-γfor 30min were prepared and were incubated with GSNO in the absence or presence of DTT for 15 min at room temperature. After S-nitrosylation reaction in vitro, the nuclear extracts in each groups were subjected to EMSA to assay the STAT-1 binding activity. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with control group (p<0.01). Treating the nuclear extracts from RSC-364 cells stimulated with IFN-γwith GSNO decreased STAT-1 activation induced by IFN-γ(p<0.01). If DTT was included in the the nuclear extracts after GSNO exposure, the effect of NO on STAT-1 activation was reversed (p<0.01). Treatment with DTT alone had no effect on the STAT-1 binding activity (p>0.05). The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors. These results showed that, NO resulted in protein S-nitrosylation in vitro, which might inhibit STAT-1 activity by thiols oxidative modification.3.2 The effect of protein S-nitrosylation in cells on STAT-1 activity in RSC-364 cellsAbove experiments showed that NO could result in protein S-nitrosylation in cell extracts from RSC-364 cells and inhibit STAT-1 activity by cysteine oxidative modification, which suggested that STAT-1 may be sensitive to thiols oxidative modification. It is unclear that the effect of protein S-nitrosylation in cell on STAT-1/DNA binding activity and STAT-1 phosphorylation. To elucidate the effect of protein S-nitrosylation in cells on STAT-1/DNA binding activity and STAT-1 phosphorylation, the cultured RSC-364 cells were divided randomly into ten groups treated with different agents:①control group;②IFN-γgroup;③GSNO group;④GSNO+IFN-γgroup;⑤NEM group;⑥NEM+GSNO+IFN-γgroup;⑦GSNO+IFN-γ+DTT group;⑧IL-1β+IFN-γgroup;⑨AG+IL-1β+ IFN-γgroup;⑩AG group. The STAT-1 binding activity was analyzed by EMSA; the STAT-1 and phosphorylated STAT-1 levels in①-⑥groups were detected by western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: (1) The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with unstimulated cells (p<0.01). Pre-treatment with GSNO decreased IFN-γ-induced STAT-1 activation (p<0.01). If DTT was added in the nuclear extracts from the RSC-364 cells incubated with GSNO and IFN-γ, the effect of GSNO on STAT-1 activation was reversed (p<0.01). Pre-treatment with NEM also reversed the effect of GSNO on STAT-1 activation (p<0.01). Treatment with NEM alone had no effect on the STAT-1 binding activity (p>0.05).The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors. (2) The STAT-1 binding activity was significantly higher in RSC-364 cells stimulated with IFN-γin comparison with unstimulated cells (p<0.01), the combination stimulation of cytokines (IFN-γand IL-1β) for 12h resulted in a obvious decrease in STAT-1 activation induced by IFN-γ(p<0.01), while co-incubation with AG and cytokines increased STAT-1 activation (p<0.01). AG alone had no significant effect on STAT-1 activation (p>0.05). The binding specificity was confirmed by using homologous (STAT-1) and nonhomologous (AP-2) oligonucleotides as competitors.(3)The phosphorylated STAT-1 level in RSC-364 cells was markedly increased 30min after incubation with IFN-γin comparison with the control group (p<0.01).Pre-treatment with GSNO had no obvious effect on the phosphorylated STAT-1 level in RSC-364 cells in comparison with the IFN-γgroup (p>0.05), pre-treatment with NEM had also no significant effect on the phosphorylated STAT-1 level in RSC-364 cells in comparison with the GSNO+IFN-γgroup (p>0.05). GSNO or NEM alone had no effect on the phosphorylated STAT-1 level in comparison with the control group (both p>0.05). NO significant changes in STAT-1 level were observed in all groups (all p>0.05).These results shown that: 1 NO resulted in protein S-nitrosylation in cells, which might inhibit STAT-1 activity by thiols oxidative modification. 2 protein S-nitrosylation had no effect on phosphorylated STAT-1. 3 The combination stimulation of cytokines (IFN-γand IL-1β) inhibited STAT-1 activity by increasing protein S-nitrosylation level.4 The effects of protein S-nitrosylation on interleukin-6 (IL-6) and IFN-γinducible protein of 10kDa (IP-10) protein expression in RSC-364 cells4.1 The effect of protein S-nitrosylation on the expression of IL-6 in RSC-364 cellsAbove experiments showed that NO might inhibit CREB activity by thiols oxidative modification. Because IL-6 gene transcription was mainly mediated by CREB, the present study was undertaken to determine the effect of CREB thiols oxidative modification on the protein expression of IL-6 induced by IL-1β. The cultured RSC-364 cells were divided randomly into six groups treated with different agents:①control group;②IL-1βgroup;③GSNO group;④GSNO+IL-1βgroup;⑤NEM group;⑥NEM+GSNO+IL-1βgroup. The IL-6 protein expression level was detected by ELISA. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: Stimulating RSC-364 cells with IL-1βfor 24h resulted in an obvious increase in IL-6 production in supernatant in comparison with control group (431.69±14.15pg/ml vs. 66.92±8.2 pg/ml, p<0.01). Compared with IL-1βgroup, the IL-6 content in supernatant in GSNO+IL-1βgroup decreased (143.96±14.7pg/ml, p<0.01). But the IL-6 content in supernatant NEM+ GSNO+IL-1βgroup significantly increased in comparison with GSNO+IL-1βgroup(433.27±14.4pg/ml,p<0.01). NEM alone had no effect on the IL-6 level (70.06±10.21 pg/ml, p>0.05). These results confirmed that CREB thiols oxidative modification inhibited the expression level of IL-6 induced by IL-1β.4.2 The effect of protein S-nitrosylation on the expression of IP-10 in RSC-364 cellsAbove experiments showed that NO resulted in protein S-nitrosylation in vivo, which might inhibit STAT-1 activity by thiols oxidative modification. Because IP-10 gene transcription was mainly mediated by STAT-1, the present study was undertaken to determine the effect of cysteine thiols modification of STAT-1 on the protein expression of IP-10 induced by IFN-γ, The cultured RSC-364 cells were divided randomly into six groups after treated with different agents:①control group;②IFN-γgroup;③GSNO group;④GSNO+IFN-γgroup;⑤NEM group;⑥NEM+GSNO+IFN-γgroup. The expression of IP-10 was detected by Western blotting. Data were presented as x±s and analyzed with ANOVA and LSD using SPSS statistical program. Results: The IP-10 level in RSC-364 cells was markedly increased 3h after incubation with IFN-γin comparison with the control group (p<0.01). Pre-treatment with GSNO resulted in a decrease in the IP-10 level in RSC-364 cells in comparison with the IFN-γgroup (p<0.01). But pre-treatment with NEM reversed the effect of GSNO (p<0.01), NEM alone had no effect on the IP-10 level (p>0.05). These results demonstrated that thiols oxidative modification of STAT-1 inhibited the expression level of IP-10 induced by IFN-γ. CONCLUSIONSIn the present study, we investigated the modulatory effects of protein S-nitrosylation on phosphorylation levels of CREB and STAT-1, the DNA/binding activities of these two transcription factors and the downstream gene expression, the conclusions were as follows:1 NO could lead to CREB and STAT-1 thiols oxidative modification by protein S-nitrosylation, and as a result the DNA/binding activities of CREB and STAT-1 were inhibited, and the downstream gene expression were reduced. It is an important mechanism for NO to regulate FLS homeostasis and inhibit RA progression.2 As a gaseous messenger, the production and disappearance of NO is rapid. NO can freely enter into cells without the existence of receptors or channels. The present study demonstrated that NO could regulate the DNA/binding activities of transcription factors, which suggested that protein S-nitrosylation is a fast regulatory mechanism. Compared with other regulation mechanism such as neuroendocrine pathway, protein S-nitrosylation may be the most fast stress reaction.3 GSNO, as the conservator and transport agent of NO, distributes in many varieties of tissues and organs including hematological system. The present study showed that NO released by GSNO could enter into cells and regulate the DNA/binding activities of transcription factors, which suggested that NO played biological roles not only in autocrine or paracrine way but also in endocrine way.更多还原