基于GTP环化水解酶Ⅰ磷酸化位点的研究探讨roscovitine对脂多糖诱导的一氧化氮的抑制作用机制
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摘要
研究背景
GTP环化水解酶Ⅰ(GCH-1)是四氢生物蝶呤(BH4)生物合成过程中的第一个催化酶,也是重要的限速酶。而BH4又是芳香族氨基酸羟化酶,一氧化氮合酶(NOS)亚基和甘油醚单加氧酶的一种重要的辅助因子。GCH-1突变导致的BH4缺乏已被证明会引起苯丙酮尿症和多巴反应性肌张力障碍(DRD)。因此,BH4在调节NOS活性中起到至关重要的作用。已有文献指出BH4有助于将NOS血红素铁转化成高自旋状态,并且可以增加NOS与精氨酸酶的亲和力。BH4还有利于NOS还原酶的电子传递,以及稳定NOS的二聚体结构。当BH4产生受限时,NOS催化的O2与L-精氨酸的耦合减少,致使NOS催化产生的超氧阴离子自由基(02··-)增多,而一氧化氮(·NO)并未增多。此外,值得注意的是,BH4还容易被氧化为二氢生物蝶呤(BH2),而这种氧化产物对NOS没有任何辅助作用。目前研究已发现,BH4的减少(被消耗或被氧化)与高血压,动脉硬化,糖尿病,心肌肥厚,以及心肌缺血有关。而且,无论在体内还是体外实验中都已证实,GCH-1在心血管生理中对调节BH4的产量和NOS的活性发挥了重要作用。此外,在内皮细胞实验中,GCH-1转基因可以使BH4产量比基础水平增加10倍以上,同时伴随着依赖内皮型NOS(eNOS)催化而产生的·NO的显著增加。在转基因小鼠的血管内皮细胞中过表达人类GCH-1后,小鼠血管内皮的BH4产量升高3倍,同时02··-产量明显降低。因此,与野生型小鼠比较,GCH-1转基因小鼠很好的维持了·NO的生物利用度。但也有文献指出,转基因小鼠中eNOS的过表达可以增加依赖eNOS的O2·-产生,然而有趣的是,当eNOS转基因小鼠与GCH-1转基因小鼠杂交时,O2`-产量又降至正常水平。
大量研究表明,GCH-1可能被磷酸化作用精细地调节着。GCH-1的磷酸化在受到血管紧张素Ⅱ,血小板衍生的生长因子或蛋白激酶C(PKC)激活剂佛波酯(TPA)刺激的细胞内是增加的,而且GCH-1磷酸化的增加趋势与GCH-1的活性和BH4的产生是一致的。同时,过表达的GCH-1是以磷酸化形式存在于肥大细胞中,并且TPA可以刺激肥大细胞中的GCH-1发生磷酸化以及刺激BH4的产生,而这一过程能够被PKC抑制剂所抑制。最近,一项用人类内皮细胞做的实验表明,剪切应力可以激活酪蛋白激酶Ⅱ，从而增加GCH-1的丝氨酸-81位点的磷酸化程度,并提高GCH-1的活性。虽然GCH-1的磷酸化程度可以调节其活性,但是对这些潜在的磷酸化位点是如何调节GCH-1活性的系统性研究尚无报道。
研究目的
GCH-1是BH4生物合成过程中的限速酶,它的突变所导致的BH4的缺乏可引起多种疾病。但目前对GCH-1翻译后的调节机制尚缺乏认识。因此,本研究的目的是找出潜在的GCH-1磷酸化位点,并确定这些特殊磷酸化位点的功能特性。
研究方法
1.质粒的构建：GCH-1的cDNA来自SD大鼠,将cDNA克隆到pcDNA5/FRT/TO/Topo/TA载体中。为了纯化GCH-1蛋白,在其N末端加入FLAG序列。然后,以FLAG-GCH-1为模板,合成FLAG-GCH-1去磷酸化突变体(丝氨酸／苏氨酸[S/T]突变为丙氨酸[A])或模拟磷酸化突变体([S/T]突变为谷氨酸[E]或天门冬氨酸[D])。FLAG-GCH-1突变体被命名为S51A、S51E、S51D等。同时,本课题还构建了GCH-1绿色荧光蛋白(GCH-1-GFP)和T231A-GCH-1绿色荧光蛋白(T231A-GCH-1-GFP)。
2.稳定的细胞株：FLAG-GCH-1和它的突变体与POG44共转染到Flp-InTMT-RExTM-293细胞中建立稳定的细胞系。这些表达野生型(WT)或者突变体的细胞被命名为WT-GCH-1细胞、S51A-GCH-1细胞、S51D-GCH-1细胞等。在所有实验中,细胞用四环素(1μg/mL)刺激24小时来调控细胞内质粒的表达。3.质谱分析：Top-Down质谱用于分析HEK 293细胞中完整的FLAG-GCH-1片段。FLAG-GCH-1是从HEK293细胞中通过免疫沉淀纯化得到。FLAG-GCH-1经过脱盐后,引入ESI/FTMS系统进行质谱分析。Bottom-Up质谱方法需要将FLAG-GCH-1用胰酶消化。消化后产生的多肽混合物通过有C18分离柱的nano-2DLC色谱分析系统而彼此分离,然后用在线LTQ MS仪进行分析。另外,Bottom-Up质谱法还要与胶内消化相配合。FLAG-GCH-1通过凝胶电泳将蛋白质在不同梯度上进行分离并被免疫沉淀。然后将GCH-1带切除、脱色和消化。由其产生的大量的肽段通过固相金属亲和色谱法找出磷酸化肽段,然后使用MALDI-TOF MS对磷酸化肽段进行分析。
4.BH4和BH2是用高效液相色谱法(HPLC)测定。细胞用冷的PBS清洗2遍。将细胞刮下转入试管,离心得到的细胞经过裂解后再次离心,取上清液用于HPLC分析。BH4和BH2测定采用Synergia Polar-RP色谱柱检测,用氩饱和50mM磷酸盐缓冲液(pH 2.6)洗脱。多通道电量检测电压设置为0-600mV。根据0mV和150mV所得到的BH4峰值面积得出BH4的标准曲线,根据280mV和365mV所得到的BH2峰值面积得出BH2的标准曲线。通过BH4和BH2的标准品测定,可以计算出样品细胞内的BH4和BH2浓度。最后,通过细胞内的蛋白定量使BH4和BH2标准化。
5.磷酸化位点预测。利用3种蛋白质磷酸化位点预测软件对GCH-1的磷酸化位点进行预测。PredPhospho软件：http://pred.ngri.re.kr/PredPhospho.htm;NetPhosK2.0软件：http://www.cbs.dtu.dk/services/NetPhos/;Scansites软件：http://scansite.mit.edu/。6.通过免疫沉淀和蛋白印迹(western blot)检测GCH-1不同位点磷酸化水平及其细胞内的定位。
研究结果
1.质谱分析研究表明,在HEK293细胞里过表达的大鼠GCH-1,在其丝氨酸(S)51,S167,和苏氨酸(T)231位点发生了磷酸化。而结合计算机分析结果,共发现了GCH-1共有8个潜在磷酸化位点,即S51、S72、T85、T91、T103、S130、S167和T231。
2.当细胞内转染S72A、T85A、T91A、T103A和S130A去磷酸化突变体时,GCH-1的活性和BH4的产量明显降低；但是当细胞内转染去磷酸化突变体T231A时,GCH-1的活性和BH4的产量却明显增加。
3.BH4和BH2的产量在转染S51E、S72E、T85E、T91E、T103D和T130D突变体的细胞中是显著升高的,但在转染T231D突变体的细胞中是降低的。
4.在转染S167A和S167E突变体的细胞中,BH2的产量也是增加的。
5.此外,本研究还发现细胞中转染T231A突变体后可以降低GCH-1的细胞核定位以及细胞核内GCH-1的活性。
结论
通过本实验,我们确定了8个GCH-1的潜在磷酸化位点,并鉴定了这些位点对GCH-1的活性、BH4和BH2的生物合成,以及GCH-1的定位作用。我们首次发现了GCH-1活性是受多个磷酸化位点调控的,其中S51,S72,T85,T91,T103和S130位点的磷酸化起到正性调节作用,而T231位点的磷酸化起到负性调节作用。此外,该研究还首次提出了S51，T231和S167位点的磷酸化对GCH-1活性调节的重要性。这一发现将会为提高BH4生物利用度提供新的思路。本课题的研究结果将有助于进一步认识在BH4产生过多或过少情况下,细胞水平的调节机制,尤其是该机制在心血管疾病和神经系统疾病(如高血压、冠心病、糖尿病和肌张力障碍性疾病等)中的作用。
研究背景
内毒素是革兰氏阴性细菌细胞壁的组成成份,其化学本质为脂多糖(LPS),为细菌死亡或活跃繁殖时所释放。LPS本身无毒性作用,但能够刺激体内多种细胞合成并释放众多内源性生物活性因子。巨噬细胞积极地参与了宿主防御和炎症过程。当机体暴露于革兰氏阴性菌时,巨噬细胞被细菌细胞壁上的脂多糖激活,从而引发大量·NO和炎性细胞因子的释放。大量产生的·NO的衍生物可以破坏细菌,但也可以引起宿主组织损伤和毒性作用。·NO是在至少3种NOS的催化下产生的,其中诱导型一氧化氮合酶(iNOS)也称为NOS2。iNOS的作用是促进巨噬细胞在LPS刺激下产生大量的·NO。通过与Toll-like receptor 4(TLR4)的相互作用,LPS可以激活细胞内信号通路,包括有丝分裂原活化蛋白激酶(MAPK)和核因子κB(NFκB)通路,同时可以上调巨噬细胞内iNOS的表达。NFκB通路是iNOS介导过程及炎症反应中最重要的信号通路。P50(NFκB1)／p65的(RelA)异源二聚体是NFκB最普遍存在的形式。各种炎症刺激均可诱导NFκBp65(丝氨酸536)发生磷酸化反应,而p65的磷酸化是调节NFκB的激活、细胞核定位和转录活性的关键。
BH4是NOS的一种重要的辅助因子,并且已被证明可以调节iNOS活性及iNOS依赖的·NO的产生。GCH-1是催化BH4生物合成中的第一步,且是该合成过程的限速酶。有资料显示,GCH-1转基因小鼠在LPS的刺激下,肾脏的iNOS的表达与NO含量显著提高。GCH-1的抑制剂可以明显抑制LPS诱导的NO·的产生和干扰素γ诱导的iNOS的表达。
在本实验第一部分关于GCH-1磷酸化调节研究中,通过对GCH-1磷酸化位点的突变,将大鼠GCH-1的85位点苏氨酸(T85)突变为丙氨酸,使其去磷酸化而功能丧失,结果发现GCH-1活性和BH4产生能够被完全抑制。因为GCH-1的T85位点与周期序列依赖性蛋白激酶5(CDK5)有着相同的序列,所以我们提出的假设是CDK5被抑制后可能会抑制GCH-1磷酸化,从而抑制巨噬细胞内·NO的产生。然而,结果显示CDK5抑制剂roscovitine可以直接抑制LPS诱导的GCH-1表达(mRNA和蛋白水平)。
研究目的
根据预实验的结果,我们推测roscovitine抑制LPS诱导的NO·的产生是通过roscovitine沮止了BH4的生物合成及抑制了LPS激活的NFκB途径或MAPK途径。因此,本研究的目的是探讨在LPS刺激的巨噬细胞内,roscovitine对iNOS及GCH-1的表达、·NO的产生、BH4的生物合成以及NFκB途径和MAPK途径的抑制作用。
研究方法
在与炎症有关的疾病中,细胞内毒素的释放所导致的·NO和细胞因子产生过多与组织损伤之间有至关重要的关系。因此,我们用LPS刺激小鼠巨噬细胞株RAW264.7细胞,探究了不同浓度roscovitine对小鼠巨噬细胞中一氧化氮产生的抑制作用的区别。为了确定roscovitine是否有潜在的细胞毒性作用,首先分别用1μM,10μM and 25μM的roscovitine处理RAW 264.7巨噬细胞24个小时,细胞的存活力用MTT方法检测。然后用roscovitine(1μM,10μM and 25μM)预处理巨噬细胞30min,随后用LPS(2μg/m1)刺激12小时后检测巨噬细胞中·NO的产量,并且应用HPLC检测BH4和BH2的产量。为了明确roscovitine对NFκB信号通路的抑制作用,将经过roscovitine干预后的巨噬细胞用LPS刺激,提取其中的RNA和蛋白质,并通过RT-PCR和western blot方法检测NFκB途径和MAPK途径的蛋白酶活性及其表达水平,并与未经roscovitine干预的对照组进行了比较。同时我们也检测了可以调节IκB磷酸化的上游激酶IKK。其次,本实验还分析了受NFκB活性影响的特殊基因(如COX-2,IL-1p,IL-6和TNFα)的表达。再次,由于文献报道roscovitine对CDK1,CDK5和CDK7均有抑制作用,我们用不同种类和不同浓度的CDK抑制剂干预巨噬细胞,从而确定是哪种CDK起作用。最后,考虑到实验用的RAW264.7巨噬细胞系并不能完全代表未经修饰的巨噬细胞,我们又分离了大鼠腹腔巨噬细胞进一步验证了roscovitine对LPS诱导的炎性反应的抑制作用。
研究结果
1.在RAW264.7巨噬细胞中,roscovitine能够抑制LPS诱导的·NO的产生,并明显抑制了由LPS诱导的iNOS的mRNA和蛋白质表达。2.结果显示,roscovitine能够抑制LPS诱导的NFκB活性。同时,roscovitine减弱了LPS诱导的IKKβ、IKB和p65的磷酸化,但增强了ERK、P38和JNK的磷酸化程度。3.RT-PCR实验结果显示LPS明显增加了IL-1β和IL-6的mRNA表达,而当roscovitine剂量为10“M和25μM时,对上调的IL-1β和IL-6有显著的抑制作用,并呈剂量依赖性。但结果显示,roscovitine对LPS诱导的TNFα表达上调没有抑制作用。4.Roscovitine增强了LPS诱导的MAPK活性。Roscovitine不仅对LPS诱导的ERK活性的增加没有抑制作用,相反,roscovitine还使ERK磷酸化程度在LPS刺激后的30min和60min时变得更强。与ERK结果相似,roscovitine增强了LPS刺激后15min和30min时的p38磷酸化程度,同时也增强了LPS刺激后0min和180min时的JNK磷酸化程度。5.经过12个小时LPS的刺激,细胞内BH4的产量高于正常水平的6倍。Roscovitine可以极大程度地抑制LPS诱导的BH4水平的增高,甚至使BH4水平降至低于对照组水平。同时,roscovitine也显著降低了BH4/BH2比值,而这个比值对NOS聚合活性的影响有着与总BH4浓度同样的重要性。此外,LPS也明显增加了GCH-1mRNA和蛋白质的表达,而roscovitine预处理后显著抑制了LPS诱导的GCH-1的表达。
6.我们使用其他CDK抑制剂发现,CDK1,CDK 5和CDK7抑制剂明显抑制了LPS诱导的巨噬细胞中·NO产生,但CDK2的抑制剂对其无明显抑制作用。
7.在分离的大鼠腹腔巨噬细胞中,roscovitine显著地抑制了·NO的生成、iNOS和COX-2的表达、NFκB的活性,以及LPS诱导的GCH-1表达的增加。结论
1.实验结果表明,roscovitine能够通过抑制NFκB的激活和BH4的生物合成,抑制LPS诱导的巨噬细胞内·NO产生。而这种抑制作用可能是由CDK1,CDK5和CDK7所介导的。
2.研究结果还证实了roscovitine可以抑制炎症,而CDKs在其抗炎机制中起到了重要的作用。
3.此外我们发现,roscovitine对LPS诱导的MAPK的活性没有抑制作用,相反是加强作用。这说明MAPK可能没有参与roscovitine对LPS诱导的iNOS和·NO产生的抑制过程。
Background
GTP cyclohydrolase I (GCH-1)is the first and rate limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthase (NOS) isoforms, and glyceryl-ether monooxygenase. Mutations in GCH-1,resulting in BH4 deficiency, have been shown to cause phenylketonuria and Dopa-responsive dystonia (DRD). BH4 critically regulates NOS activity. BH4 shifts the NOS heme iron into a high spin state and increases the affinity of the enzyme for arginine. BH4 also facilitates the electron transfer from NOS reductase and structurally stabilizes NOS dimmers. When BH4 is limited, reduction of O2 by NOS is not coupled to L-arginine oxidation resulting in superoxide anion(O2(?))generation rather than nitric oxide (·NO) production. BH4 is easily oxidized to 6,7,8-trihydrobiopterin or 7,8-dihydrobiopterin (BH2), making it useless for NOS.Increased BH4 depletion or oxidation has been linked to hypertension, atherosclerosis, diabetes, cardiac hypertrophy, and myocardial ischemia. The role of GCH-1 in cardiovascular physiology in regulating BH4 and NOS activity has been investigated in vitro and in vivo. In endothelial cells, GCH-1 gene transfer increases BH4 more than 10-fold over baseline levels, accompanied by a marked increase in eNOS dependent·NO production. Transgenic mice made to express human GCH-1 in vascular endothelial cells have a 3-fold increase in vascular BH4 and a marked decrease in endothelial O2(?) production, which preserves·NO bioavailability compared with wild-type littermates.Overexpression of eNOS in transgenic mice increases eNOS-dependent O2(?) production. Yet, when the eNOS transgenic mice are crossed with GCH-1 transgenic mice, O2(?) generation is normalized.
GCH-1 appears to be finely regulated by phosphorylation. GCH-1 phosphorylation was increased in cells stimulated by ngiotensin II, platelet-derived growth factor, and TPA, and the increase in phosphorylation was correlated with increased enzyme activity and BH4 production. Overexpressed GCH-1 has been shown to exist in a phosphorylated form in mast cells and phorbol ester stimulates GCH-1 phosphorylation and BH4 production which could be inhibited by PKC inhibitors. A more recent study showed that shear stress increases GCH-1 phosphorylation at Serine-81 and enzyme activity in human endothelial cells by a casein kinase II- dependent mechanism.Although phosphorylation of GCH-1 appears to regulate activity, systematic studies examining how these potential phosphorylation sites regulate enzyme activity have not been performed.
Objective
The posttranslational regulation of GTP cyclohydrolase I (GCH-1),the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.
Methods
1.Plasmids Construction. GCH-1 cDNA from Sprague Dawley rats was cloned into pcDNA5/ FRT/TO/Topo/TA (Invitrogen) with a FLAG epitope. FLAG-GCH-1 phospho-defective mutants(Serine/Threonine[S/T] to alanine [A])or phospho-mimic mutants(S/T to glutamic acid [E] or aspartic acid[D])were generated.GCH-1 mutants were named as S51A,S51E or S51D,and so forth. GCH-1-GFP and T231A-GCH-1-GFP were also constructed.
2.Cells.FLAG-GCH-1 and its mutants were cotransfected with POG44(Invitrogen) into Flp-In T-REx-293 cells (Invitrogen) to establish stable cell lines or transiently into bovine aortic endothelial cells (BAECs).The cells expressing wild-type (WT) or its mutants were named as WT-GCH-1,S51A-GCH-1,S51D-GCH-1 cells,and so forth. Cells were treated with/without tetracycline(1μg/mL) for 24 hours in all experiments to regulate the plasmid expression.
3.Mass Spectrometry Analysis. Top down mass spectrometry was used to analyze intact FLAG-GCH-1 from HEK 293 cells. FLAG-GCH-1 was desalted using an offline reverse phase protein microtrap and introduced to the mass spectrometer using an automated chipbased nanoESI source (ESI/FTMS).Bottom-up mass spectrometry was used with in solution trypsin digestion of FLAG-GCH-1.After digestion, the resulting peptide mixture was separated by a nano-2DLC chromatographic system with a C18 column and subsequently analyzed online using an LTQ mass spectrometer. In addition, bottom up mass spectrometry was used with in-gel digestion. FLAG-GCH-1 was immunoprecipitated and isolated by SDS PAGE. The GCH-1 band was excised, destained, and digested. The resulting peptides were enriched for phosphopeptides by immobilized metal ion affinity chromatography and analyzed on a Voyager DE-Pro matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS).
Results
1.Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine(S)51,S167,and threonine(T)231 in HEK293 cells,whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51,S72, T85, T91,T103, S130,S167 and T231).
2. GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant.
3.BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2.
4. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.
Conclusion
We identified 8 candidate phosphorylation sites in GCH-1 and characterized the function of these potential sites with respect to GCH-1 activity, biosynthesis of BH4 and BH2,and enzyme localization. Our report is the first to show that GCH-1 is regulated by multiple phosphorylation sites, both positively and negatively. This is also the first study to identify S51,T231,and S167 as important regulatory phosphorylation sites on GCH-1.These findings should provide new insight into our understanding of the cellular mechanisms regulating BH4 production (or lack thereof) in cardiovascular and neurological diseases such as hypertension, coronary heart disease, diabetes, and DRD.
Backgroud
In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines which are overproduced in response to cellular release of endotoxins. Macrophages actively participate in host defense and inflammation. When exposed to Gram-negative bacteria, macrophages are activated by lipopolysaccharide(LPS)in the bacterial cell wall, which triggers the release of large amounts of nitric oxide (·NO) and inflammatory cytokines. The overproduced·NO may destroy the bacteria but may also cause tissue injury and toxicity in the host by its derivatives.·NO is a free radical produced from L-arginine by at least three·NO synthases (NOS).iNOS is the inducible isoform also known as NOS2, which is responsible for the production of high levels of·NO in macrophages induced by LPS.LPS activates intracellular signaling pathways including the
Mitogen-activated protein kinase (MAPK) and nuclear factorκB (NFκB) pathway to upregulate iNOS expression in macrophages. The NFκB pathway is the most important signaling pathway in TLR4-mediated iNOS induction and inflammatory response. Phosphorylation of NFκB p65(serine536) is physiologically induced in response to a variety of proinflammatory stimuli. Phosphorylation of p65 is critical in regulating the activation, nuclear localization and transcription activity of NFκB.
Tetrahydrobiopterin (BH4) is an essential cofactor for all NOS isoforms and has been shown to be an important regulator of iNOS activity and iNOS dependant·NO production. GTP cyclohydrolase I (GCH-1)is the first step and the rate-limiting enzyme for BH4 biosynthesis. GCH-1 transgenic mice showed significantly enhanced renal iNOS expression and·NO levels with LPS treatment. Inhibition of GCH-1 suppressed LPS-induced·NO production and IFNy-induced iNOS expression.
In our recent study on regulation of GCH-1 by phosphorylation using site-directed mutagenesis, we found that the mutation of threonine at 85 (T85) of rat GCH-1 into alanine (mimic dephosphorylation and loss of function) could completely inhibit GCH-1 activity and BH4 production. Since T85 on GCH-1 has the consensus sequence for cyclin-dependent kinase 5(CDK5), we originally hypothesized that inhibition of CDK5 may suppress GCH-1 phosphorylation and, accordingly,·NO production in macrophages. However, our results indicated that CDK5 inhibitor roscovitine directly inhibited GCH-1 expression (mRNA and protein) induced by LPS.
Objective
Therefore, we hypothesize that roscovitine inhibits LPS-induced·NO production by inhibiting BH4 synthesis and suppressing LPS-activated NFκB pathway and/or MAPK pathway. Accordingly, the aim of this study is to explore the inhibitory effect of roscovitine on the expression of iNOS and GCH-1,production of·NO and BH4, and activation of NFκB pathway and MAPK pathway in macrophage by LPS stimulation.
Methods
Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases(CDKs) on·NO production in mouse macrophages. To determine the potential cell cytotoxicity of roscovitine, RAW 264.7 macrophages were treated with roscovitine at 1μM,10μM and 25μM for 24 hours, and cell viability was measured by MTT assay. Macrophages were pretreated with roscovitine at the above concentrations(1μM,10μM and 25μM) for 30 min followed by stimulation with LPS (2μg/ml) for 12 hours, then test the·NO production in macrophages challenged with LPS. At the same time, BH4 and BH2 were assayed by using HPLC with an electrochemical detector. To confirm the inhibitory effect of roscovitine on the NFκB pathway, we also checked IKK, a kinase upstream of IκB,which regulates the phosphorylation of IκB by real-time PCR and western blot. We also examined the expression of certain genes in response to NFκB activation including COX-2, IL-1p,IL-6 and TNFα. In additional, as roscovitine is reported to inhibit CDK1,CDK5 and CDK7,we used different CDK inhibitors with different concentrations. To overcome the limitation that data from using RAW264.7 macrophage cells may not be able to fully represent the macrophages without modification, rat peritoneal macrophages were isolated and used for examining the effects of roscovitine on LPS-induced inflammatory response.
Results
1.In RAW264.7 cells we found that roscovitine abolished the production of·NO induced by lipopolysaccharide(LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxidase synthase (iNOS)mRNA and protein expression.
2.Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IKKβ, IκB and p65 but enhanced the phosphorylation of ERK, p38 and JNK.
3.In addition, roscovitine dose-dependently inhibited LPS-induced expression of COX-2,IL-1p and IL-6 but not TNFa.
4.Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4/BH2 ratio.
5.Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase 1 (GCH-1),the rate-limiting enzyme for BH4 biosynthesis.
6.Using other CDK inhibitors, we found that CDK1,CDK5,and CDK7, but not CDK2, significantly inhibited LPS-induced·NO production in macrophages.
7.Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited·NO production, iNOS and COX-2 upregulation, activation of NFκB and induction of GCH-1 by LPS.
Conclusion
1.Together, our data indicates that roscovitine abolishes LPS-induced·NO production in macrophages by suppressing NFκB activation and BH4 biosynthesis, which might be mediated by CDK1,CDK5 and CDK7.
2.Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.
GTP环化水解酶Ⅰ(GCH-1)是四氢生物蝶呤(BH4)生物合成过程中的第一个催化酶,也是重要的限速酶。而BH4又是芳香族氨基酸羟化酶,一氧化氮合酶(NOS)亚基和甘油醚单加氧酶的一种重要的辅助因子。GCH-1突变导致的BH4缺乏已被证明会引起苯丙酮尿症和多巴反应性肌张力障碍(DRD)。因此,BH4在调节NOS活性中起到至关重要的作用。已有文献指出BH4有助于将NOS血红素铁转化成高自旋状态,并且可以增加NOS与精氨酸酶的亲和力。BH4还有利于NOS还原酶的电子传递,以及稳定NOS的二聚体结构。当BH4产生受限时,NOS催化的O2与L-精氨酸的耦合减少,致使NOS催化产生的超氧阴离子自由基(02··-)增多,而一氧化氮(·NO)并未增多。此外,值得注意的是,BH4还容易被氧化为二氢生物蝶呤(BH2),而这种氧化产物对NOS没有任何辅助作用。目前研究已发现,BH4的减少(被消耗或被氧化)与高血压,动脉硬化,糖尿病,心肌肥厚,以及心肌缺血有关。而且,无论在体内还是体外实验中都已证实,GCH-1在心血管生理中对调节BH4的产量和NOS的活性发挥了重要作用。此外,在内皮细胞实验中,GCH-1转基因可以使BH4产量比基础水平增加10倍以上,同时伴随着依赖内皮型NOS(eNOS)催化而产生的·NO的显著增加。在转基因小鼠的血管内皮细胞中过表达人类GCH-1后,小鼠血管内皮的BH4产量升高3倍,同时02··-产量明显降低。因此,与野生型小鼠比较,GCH-1转基因小鼠很好的维持了·NO的生物利用度。但也有文献指出,转基因小鼠中eNOS的过表达可以增加依赖eNOS的O2·-产生,然而有趣的是,当eNOS转基因小鼠与GCH-1转基因小鼠杂交时,O2`-产量又降至正常水平。
大量研究表明,GCH-1可能被磷酸化作用精细地调节着。GCH-1的磷酸化在受到血管紧张素Ⅱ,血小板衍生的生长因子或蛋白激酶C(PKC)激活剂佛波酯(TPA)刺激的细胞内是增加的,而且GCH-1磷酸化的增加趋势与GCH-1的活性和BH4的产生是一致的。同时,过表达的GCH-1是以磷酸化形式存在于肥大细胞中,并且TPA可以刺激肥大细胞中的GCH-1发生磷酸化以及刺激BH4的产生,而这一过程能够被PKC抑制剂所抑制。最近,一项用人类内皮细胞做的实验表明,剪切应力可以激活酪蛋白激酶Ⅱ，从而增加GCH-1的丝氨酸-81位点的磷酸化程度,并提高GCH-1的活性。虽然GCH-1的磷酸化程度可以调节其活性,但是对这些潜在的磷酸化位点是如何调节GCH-1活性的系统性研究尚无报道。
研究目的
GCH-1是BH4生物合成过程中的限速酶,它的突变所导致的BH4的缺乏可引起多种疾病。但目前对GCH-1翻译后的调节机制尚缺乏认识。因此,本研究的目的是找出潜在的GCH-1磷酸化位点,并确定这些特殊磷酸化位点的功能特性。
研究方法
1.质粒的构建：GCH-1的cDNA来自SD大鼠,将cDNA克隆到pcDNA5/FRT/TO/Topo/TA载体中。为了纯化GCH-1蛋白,在其N末端加入FLAG序列。然后,以FLAG-GCH-1为模板,合成FLAG-GCH-1去磷酸化突变体(丝氨酸／苏氨酸[S/T]突变为丙氨酸[A])或模拟磷酸化突变体([S/T]突变为谷氨酸[E]或天门冬氨酸[D])。FLAG-GCH-1突变体被命名为S51A、S51E、S51D等。同时,本课题还构建了GCH-1绿色荧光蛋白(GCH-1-GFP)和T231A-GCH-1绿色荧光蛋白(T231A-GCH-1-GFP)。
2.稳定的细胞株：FLAG-GCH-1和它的突变体与POG44共转染到Flp-InTMT-RExTM-293细胞中建立稳定的细胞系。这些表达野生型(WT)或者突变体的细胞被命名为WT-GCH-1细胞、S51A-GCH-1细胞、S51D-GCH-1细胞等。在所有实验中,细胞用四环素(1μg/mL)刺激24小时来调控细胞内质粒的表达。3.质谱分析：Top-Down质谱用于分析HEK 293细胞中完整的FLAG-GCH-1片段。FLAG-GCH-1是从HEK293细胞中通过免疫沉淀纯化得到。FLAG-GCH-1经过脱盐后,引入ESI/FTMS系统进行质谱分析。Bottom-Up质谱方法需要将FLAG-GCH-1用胰酶消化。消化后产生的多肽混合物通过有C18分离柱的nano-2DLC色谱分析系统而彼此分离,然后用在线LTQ MS仪进行分析。另外,Bottom-Up质谱法还要与胶内消化相配合。FLAG-GCH-1通过凝胶电泳将蛋白质在不同梯度上进行分离并被免疫沉淀。然后将GCH-1带切除、脱色和消化。由其产生的大量的肽段通过固相金属亲和色谱法找出磷酸化肽段,然后使用MALDI-TOF MS对磷酸化肽段进行分析。
4.BH4和BH2是用高效液相色谱法(HPLC)测定。细胞用冷的PBS清洗2遍。将细胞刮下转入试管,离心得到的细胞经过裂解后再次离心,取上清液用于HPLC分析。BH4和BH2测定采用Synergia Polar-RP色谱柱检测,用氩饱和50mM磷酸盐缓冲液(pH 2.6)洗脱。多通道电量检测电压设置为0-600mV。根据0mV和150mV所得到的BH4峰值面积得出BH4的标准曲线,根据280mV和365mV所得到的BH2峰值面积得出BH2的标准曲线。通过BH4和BH2的标准品测定,可以计算出样品细胞内的BH4和BH2浓度。最后,通过细胞内的蛋白定量使BH4和BH2标准化。
5.磷酸化位点预测。利用3种蛋白质磷酸化位点预测软件对GCH-1的磷酸化位点进行预测。PredPhospho软件：http://pred.ngri.re.kr/PredPhospho.htm;NetPhosK2.0软件：http://www.cbs.dtu.dk/services/NetPhos/;Scansites软件：http://scansite.mit.edu/。6.通过免疫沉淀和蛋白印迹(western blot)检测GCH-1不同位点磷酸化水平及其细胞内的定位。
研究结果
1.质谱分析研究表明,在HEK293细胞里过表达的大鼠GCH-1,在其丝氨酸(S)51,S167,和苏氨酸(T)231位点发生了磷酸化。而结合计算机分析结果,共发现了GCH-1共有8个潜在磷酸化位点,即S51、S72、T85、T91、T103、S130、S167和T231。
2.当细胞内转染S72A、T85A、T91A、T103A和S130A去磷酸化突变体时,GCH-1的活性和BH4的产量明显降低；但是当细胞内转染去磷酸化突变体T231A时,GCH-1的活性和BH4的产量却明显增加。
3.BH4和BH2的产量在转染S51E、S72E、T85E、T91E、T103D和T130D突变体的细胞中是显著升高的,但在转染T231D突变体的细胞中是降低的。
4.在转染S167A和S167E突变体的细胞中,BH2的产量也是增加的。
5.此外,本研究还发现细胞中转染T231A突变体后可以降低GCH-1的细胞核定位以及细胞核内GCH-1的活性。
结论
通过本实验,我们确定了8个GCH-1的潜在磷酸化位点,并鉴定了这些位点对GCH-1的活性、BH4和BH2的生物合成,以及GCH-1的定位作用。我们首次发现了GCH-1活性是受多个磷酸化位点调控的,其中S51,S72,T85,T91,T103和S130位点的磷酸化起到正性调节作用,而T231位点的磷酸化起到负性调节作用。此外,该研究还首次提出了S51，T231和S167位点的磷酸化对GCH-1活性调节的重要性。这一发现将会为提高BH4生物利用度提供新的思路。本课题的研究结果将有助于进一步认识在BH4产生过多或过少情况下,细胞水平的调节机制,尤其是该机制在心血管疾病和神经系统疾病(如高血压、冠心病、糖尿病和肌张力障碍性疾病等)中的作用。
研究背景
内毒素是革兰氏阴性细菌细胞壁的组成成份,其化学本质为脂多糖(LPS),为细菌死亡或活跃繁殖时所释放。LPS本身无毒性作用,但能够刺激体内多种细胞合成并释放众多内源性生物活性因子。巨噬细胞积极地参与了宿主防御和炎症过程。当机体暴露于革兰氏阴性菌时,巨噬细胞被细菌细胞壁上的脂多糖激活,从而引发大量·NO和炎性细胞因子的释放。大量产生的·NO的衍生物可以破坏细菌,但也可以引起宿主组织损伤和毒性作用。·NO是在至少3种NOS的催化下产生的,其中诱导型一氧化氮合酶(iNOS)也称为NOS2。iNOS的作用是促进巨噬细胞在LPS刺激下产生大量的·NO。通过与Toll-like receptor 4(TLR4)的相互作用,LPS可以激活细胞内信号通路,包括有丝分裂原活化蛋白激酶(MAPK)和核因子κB(NFκB)通路,同时可以上调巨噬细胞内iNOS的表达。NFκB通路是iNOS介导过程及炎症反应中最重要的信号通路。P50(NFκB1)／p65的(RelA)异源二聚体是NFκB最普遍存在的形式。各种炎症刺激均可诱导NFκBp65(丝氨酸536)发生磷酸化反应,而p65的磷酸化是调节NFκB的激活、细胞核定位和转录活性的关键。
BH4是NOS的一种重要的辅助因子,并且已被证明可以调节iNOS活性及iNOS依赖的·NO的产生。GCH-1是催化BH4生物合成中的第一步,且是该合成过程的限速酶。有资料显示,GCH-1转基因小鼠在LPS的刺激下,肾脏的iNOS的表达与NO含量显著提高。GCH-1的抑制剂可以明显抑制LPS诱导的NO·的产生和干扰素γ诱导的iNOS的表达。
在本实验第一部分关于GCH-1磷酸化调节研究中,通过对GCH-1磷酸化位点的突变,将大鼠GCH-1的85位点苏氨酸(T85)突变为丙氨酸,使其去磷酸化而功能丧失,结果发现GCH-1活性和BH4产生能够被完全抑制。因为GCH-1的T85位点与周期序列依赖性蛋白激酶5(CDK5)有着相同的序列,所以我们提出的假设是CDK5被抑制后可能会抑制GCH-1磷酸化,从而抑制巨噬细胞内·NO的产生。然而,结果显示CDK5抑制剂roscovitine可以直接抑制LPS诱导的GCH-1表达(mRNA和蛋白水平)。
研究目的
根据预实验的结果,我们推测roscovitine抑制LPS诱导的NO·的产生是通过roscovitine沮止了BH4的生物合成及抑制了LPS激活的NFκB途径或MAPK途径。因此,本研究的目的是探讨在LPS刺激的巨噬细胞内,roscovitine对iNOS及GCH-1的表达、·NO的产生、BH4的生物合成以及NFκB途径和MAPK途径的抑制作用。
研究方法
在与炎症有关的疾病中,细胞内毒素的释放所导致的·NO和细胞因子产生过多与组织损伤之间有至关重要的关系。因此,我们用LPS刺激小鼠巨噬细胞株RAW264.7细胞,探究了不同浓度roscovitine对小鼠巨噬细胞中一氧化氮产生的抑制作用的区别。为了确定roscovitine是否有潜在的细胞毒性作用,首先分别用1μM,10μM and 25μM的roscovitine处理RAW 264.7巨噬细胞24个小时,细胞的存活力用MTT方法检测。然后用roscovitine(1μM,10μM and 25μM)预处理巨噬细胞30min,随后用LPS(2μg/m1)刺激12小时后检测巨噬细胞中·NO的产量,并且应用HPLC检测BH4和BH2的产量。为了明确roscovitine对NFκB信号通路的抑制作用,将经过roscovitine干预后的巨噬细胞用LPS刺激,提取其中的RNA和蛋白质,并通过RT-PCR和western blot方法检测NFκB途径和MAPK途径的蛋白酶活性及其表达水平,并与未经roscovitine干预的对照组进行了比较。同时我们也检测了可以调节IκB磷酸化的上游激酶IKK。其次,本实验还分析了受NFκB活性影响的特殊基因(如COX-2,IL-1p,IL-6和TNFα)的表达。再次,由于文献报道roscovitine对CDK1,CDK5和CDK7均有抑制作用,我们用不同种类和不同浓度的CDK抑制剂干预巨噬细胞,从而确定是哪种CDK起作用。最后,考虑到实验用的RAW264.7巨噬细胞系并不能完全代表未经修饰的巨噬细胞,我们又分离了大鼠腹腔巨噬细胞进一步验证了roscovitine对LPS诱导的炎性反应的抑制作用。
研究结果
1.在RAW264.7巨噬细胞中,roscovitine能够抑制LPS诱导的·NO的产生,并明显抑制了由LPS诱导的iNOS的mRNA和蛋白质表达。2.结果显示,roscovitine能够抑制LPS诱导的NFκB活性。同时,roscovitine减弱了LPS诱导的IKKβ、IKB和p65的磷酸化,但增强了ERK、P38和JNK的磷酸化程度。3.RT-PCR实验结果显示LPS明显增加了IL-1β和IL-6的mRNA表达,而当roscovitine剂量为10“M和25μM时,对上调的IL-1β和IL-6有显著的抑制作用,并呈剂量依赖性。但结果显示,roscovitine对LPS诱导的TNFα表达上调没有抑制作用。4.Roscovitine增强了LPS诱导的MAPK活性。Roscovitine不仅对LPS诱导的ERK活性的增加没有抑制作用,相反,roscovitine还使ERK磷酸化程度在LPS刺激后的30min和60min时变得更强。与ERK结果相似,roscovitine增强了LPS刺激后15min和30min时的p38磷酸化程度,同时也增强了LPS刺激后0min和180min时的JNK磷酸化程度。5.经过12个小时LPS的刺激,细胞内BH4的产量高于正常水平的6倍。Roscovitine可以极大程度地抑制LPS诱导的BH4水平的增高,甚至使BH4水平降至低于对照组水平。同时,roscovitine也显著降低了BH4/BH2比值,而这个比值对NOS聚合活性的影响有着与总BH4浓度同样的重要性。此外,LPS也明显增加了GCH-1mRNA和蛋白质的表达,而roscovitine预处理后显著抑制了LPS诱导的GCH-1的表达。
6.我们使用其他CDK抑制剂发现,CDK1,CDK 5和CDK7抑制剂明显抑制了LPS诱导的巨噬细胞中·NO产生,但CDK2的抑制剂对其无明显抑制作用。
7.在分离的大鼠腹腔巨噬细胞中,roscovitine显著地抑制了·NO的生成、iNOS和COX-2的表达、NFκB的活性,以及LPS诱导的GCH-1表达的增加。结论
1.实验结果表明,roscovitine能够通过抑制NFκB的激活和BH4的生物合成,抑制LPS诱导的巨噬细胞内·NO产生。而这种抑制作用可能是由CDK1,CDK5和CDK7所介导的。
2.研究结果还证实了roscovitine可以抑制炎症,而CDKs在其抗炎机制中起到了重要的作用。
3.此外我们发现,roscovitine对LPS诱导的MAPK的活性没有抑制作用,相反是加强作用。这说明MAPK可能没有参与roscovitine对LPS诱导的iNOS和·NO产生的抑制过程。
Background
GTP cyclohydrolase I (GCH-1)is the first and rate limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthase (NOS) isoforms, and glyceryl-ether monooxygenase. Mutations in GCH-1,resulting in BH4 deficiency, have been shown to cause phenylketonuria and Dopa-responsive dystonia (DRD). BH4 critically regulates NOS activity. BH4 shifts the NOS heme iron into a high spin state and increases the affinity of the enzyme for arginine. BH4 also facilitates the electron transfer from NOS reductase and structurally stabilizes NOS dimmers. When BH4 is limited, reduction of O2 by NOS is not coupled to L-arginine oxidation resulting in superoxide anion(O2(?))generation rather than nitric oxide (·NO) production. BH4 is easily oxidized to 6,7,8-trihydrobiopterin or 7,8-dihydrobiopterin (BH2), making it useless for NOS.Increased BH4 depletion or oxidation has been linked to hypertension, atherosclerosis, diabetes, cardiac hypertrophy, and myocardial ischemia. The role of GCH-1 in cardiovascular physiology in regulating BH4 and NOS activity has been investigated in vitro and in vivo. In endothelial cells, GCH-1 gene transfer increases BH4 more than 10-fold over baseline levels, accompanied by a marked increase in eNOS dependent·NO production. Transgenic mice made to express human GCH-1 in vascular endothelial cells have a 3-fold increase in vascular BH4 and a marked decrease in endothelial O2(?) production, which preserves·NO bioavailability compared with wild-type littermates.Overexpression of eNOS in transgenic mice increases eNOS-dependent O2(?) production. Yet, when the eNOS transgenic mice are crossed with GCH-1 transgenic mice, O2(?) generation is normalized.
GCH-1 appears to be finely regulated by phosphorylation. GCH-1 phosphorylation was increased in cells stimulated by ngiotensin II, platelet-derived growth factor, and TPA, and the increase in phosphorylation was correlated with increased enzyme activity and BH4 production. Overexpressed GCH-1 has been shown to exist in a phosphorylated form in mast cells and phorbol ester stimulates GCH-1 phosphorylation and BH4 production which could be inhibited by PKC inhibitors. A more recent study showed that shear stress increases GCH-1 phosphorylation at Serine-81 and enzyme activity in human endothelial cells by a casein kinase II- dependent mechanism.Although phosphorylation of GCH-1 appears to regulate activity, systematic studies examining how these potential phosphorylation sites regulate enzyme activity have not been performed.
Objective
The posttranslational regulation of GTP cyclohydrolase I (GCH-1),the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.
Methods
1.Plasmids Construction. GCH-1 cDNA from Sprague Dawley rats was cloned into pcDNA5/ FRT/TO/Topo/TA (Invitrogen) with a FLAG epitope. FLAG-GCH-1 phospho-defective mutants(Serine/Threonine[S/T] to alanine [A])or phospho-mimic mutants(S/T to glutamic acid [E] or aspartic acid[D])were generated.GCH-1 mutants were named as S51A,S51E or S51D,and so forth. GCH-1-GFP and T231A-GCH-1-GFP were also constructed.
2.Cells.FLAG-GCH-1 and its mutants were cotransfected with POG44(Invitrogen) into Flp-In T-REx-293 cells (Invitrogen) to establish stable cell lines or transiently into bovine aortic endothelial cells (BAECs).The cells expressing wild-type (WT) or its mutants were named as WT-GCH-1,S51A-GCH-1,S51D-GCH-1 cells,and so forth. Cells were treated with/without tetracycline(1μg/mL) for 24 hours in all experiments to regulate the plasmid expression.
3.Mass Spectrometry Analysis. Top down mass spectrometry was used to analyze intact FLAG-GCH-1 from HEK 293 cells. FLAG-GCH-1 was desalted using an offline reverse phase protein microtrap and introduced to the mass spectrometer using an automated chipbased nanoESI source (ESI/FTMS).Bottom-up mass spectrometry was used with in solution trypsin digestion of FLAG-GCH-1.After digestion, the resulting peptide mixture was separated by a nano-2DLC chromatographic system with a C18 column and subsequently analyzed online using an LTQ mass spectrometer. In addition, bottom up mass spectrometry was used with in-gel digestion. FLAG-GCH-1 was immunoprecipitated and isolated by SDS PAGE. The GCH-1 band was excised, destained, and digested. The resulting peptides were enriched for phosphopeptides by immobilized metal ion affinity chromatography and analyzed on a Voyager DE-Pro matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS).
Results
1.Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine(S)51,S167,and threonine(T)231 in HEK293 cells,whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51,S72, T85, T91,T103, S130,S167 and T231).
2. GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant.
3.BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2.
4. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.
Conclusion
We identified 8 candidate phosphorylation sites in GCH-1 and characterized the function of these potential sites with respect to GCH-1 activity, biosynthesis of BH4 and BH2,and enzyme localization. Our report is the first to show that GCH-1 is regulated by multiple phosphorylation sites, both positively and negatively. This is also the first study to identify S51,T231,and S167 as important regulatory phosphorylation sites on GCH-1.These findings should provide new insight into our understanding of the cellular mechanisms regulating BH4 production (or lack thereof) in cardiovascular and neurological diseases such as hypertension, coronary heart disease, diabetes, and DRD.
Backgroud
In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines which are overproduced in response to cellular release of endotoxins. Macrophages actively participate in host defense and inflammation. When exposed to Gram-negative bacteria, macrophages are activated by lipopolysaccharide(LPS)in the bacterial cell wall, which triggers the release of large amounts of nitric oxide (·NO) and inflammatory cytokines. The overproduced·NO may destroy the bacteria but may also cause tissue injury and toxicity in the host by its derivatives.·NO is a free radical produced from L-arginine by at least three·NO synthases (NOS).iNOS is the inducible isoform also known as NOS2, which is responsible for the production of high levels of·NO in macrophages induced by LPS.LPS activates intracellular signaling pathways including the
Mitogen-activated protein kinase (MAPK) and nuclear factorκB (NFκB) pathway to upregulate iNOS expression in macrophages. The NFκB pathway is the most important signaling pathway in TLR4-mediated iNOS induction and inflammatory response. Phosphorylation of NFκB p65(serine536) is physiologically induced in response to a variety of proinflammatory stimuli. Phosphorylation of p65 is critical in regulating the activation, nuclear localization and transcription activity of NFκB.
Tetrahydrobiopterin (BH4) is an essential cofactor for all NOS isoforms and has been shown to be an important regulator of iNOS activity and iNOS dependant·NO production. GTP cyclohydrolase I (GCH-1)is the first step and the rate-limiting enzyme for BH4 biosynthesis. GCH-1 transgenic mice showed significantly enhanced renal iNOS expression and·NO levels with LPS treatment. Inhibition of GCH-1 suppressed LPS-induced·NO production and IFNy-induced iNOS expression.
In our recent study on regulation of GCH-1 by phosphorylation using site-directed mutagenesis, we found that the mutation of threonine at 85 (T85) of rat GCH-1 into alanine (mimic dephosphorylation and loss of function) could completely inhibit GCH-1 activity and BH4 production. Since T85 on GCH-1 has the consensus sequence for cyclin-dependent kinase 5(CDK5), we originally hypothesized that inhibition of CDK5 may suppress GCH-1 phosphorylation and, accordingly,·NO production in macrophages. However, our results indicated that CDK5 inhibitor roscovitine directly inhibited GCH-1 expression (mRNA and protein) induced by LPS.
Objective
Therefore, we hypothesize that roscovitine inhibits LPS-induced·NO production by inhibiting BH4 synthesis and suppressing LPS-activated NFκB pathway and/or MAPK pathway. Accordingly, the aim of this study is to explore the inhibitory effect of roscovitine on the expression of iNOS and GCH-1,production of·NO and BH4, and activation of NFκB pathway and MAPK pathway in macrophage by LPS stimulation.
Methods
Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases(CDKs) on·NO production in mouse macrophages. To determine the potential cell cytotoxicity of roscovitine, RAW 264.7 macrophages were treated with roscovitine at 1μM,10μM and 25μM for 24 hours, and cell viability was measured by MTT assay. Macrophages were pretreated with roscovitine at the above concentrations(1μM,10μM and 25μM) for 30 min followed by stimulation with LPS (2μg/ml) for 12 hours, then test the·NO production in macrophages challenged with LPS. At the same time, BH4 and BH2 were assayed by using HPLC with an electrochemical detector. To confirm the inhibitory effect of roscovitine on the NFκB pathway, we also checked IKK, a kinase upstream of IκB,which regulates the phosphorylation of IκB by real-time PCR and western blot. We also examined the expression of certain genes in response to NFκB activation including COX-2, IL-1p,IL-6 and TNFα. In additional, as roscovitine is reported to inhibit CDK1,CDK5 and CDK7,we used different CDK inhibitors with different concentrations. To overcome the limitation that data from using RAW264.7 macrophage cells may not be able to fully represent the macrophages without modification, rat peritoneal macrophages were isolated and used for examining the effects of roscovitine on LPS-induced inflammatory response.
Results
1.In RAW264.7 cells we found that roscovitine abolished the production of·NO induced by lipopolysaccharide(LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxidase synthase (iNOS)mRNA and protein expression.
2.Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IKKβ, IκB and p65 but enhanced the phosphorylation of ERK, p38 and JNK.
3.In addition, roscovitine dose-dependently inhibited LPS-induced expression of COX-2,IL-1p and IL-6 but not TNFa.
4.Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4/BH2 ratio.
5.Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase 1 (GCH-1),the rate-limiting enzyme for BH4 biosynthesis.
6.Using other CDK inhibitors, we found that CDK1,CDK5,and CDK7, but not CDK2, significantly inhibited LPS-induced·NO production in macrophages.
7.Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited·NO production, iNOS and COX-2 upregulation, activation of NFκB and induction of GCH-1 by LPS.
Conclusion
1.Together, our data indicates that roscovitine abolishes LPS-induced·NO production in macrophages by suppressing NFκB activation and BH4 biosynthesis, which might be mediated by CDK1,CDK5 and CDK7.
2.Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.
引文
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