Tribble3在动脉粥样硬化巨噬细胞凋亡中的作用及其信号转导通路
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摘要
背景
动脉粥样硬化(AS)及其并发症引起的急性心血管病事件已成为人类的头号杀手,其防治一直是世界性公共卫生的重点和难题。急性冠状动脉综合征(acute coronary syndrome, ACS)是冠状动脉粥样硬化性心脏病最重要的死因。ACS主要是由于冠状动脉内粥样斑块的破裂及其引起的血栓形成,导致冠状动脉急性管腔狭窄或闭塞所致。从病理生理角度而言,ACS最主要的深层原因是易损斑块,即易于发生血栓或可能迅速进展成为罪犯病变的斑块。
近年来的研究表明,动脉粥样斑块内的巨噬细胞凋亡是易损斑块形成的重要原因。尽管巨噬细胞减少导致其分泌基质金属蛋白酶下降,斑块细胞外胶原降解减少而具有稳定斑块的作用,但大量研究证实,巨噬细胞凋亡可通过以下的机制促进易损斑块的形成：1.巨噬细胞数量减少,凋亡的平滑肌细胞和巨噬细胞不能被有效吞噬,发生继发性坏死,胞浆内脂质内容物释放,堆积形成斑块的脂质核心,促进粥样斑块脂质核心的形成；2.巨噬细胞凋亡后释放出胆固醇,导致斑块脂质核心针形胆固醇结晶增多,损伤纤维帽；3.巨噬细胞凋亡后释放促血栓形成物质,尤其是组织因子,促进斑块血栓形成。
巨噬细胞凋亡的信号调控是一个复杂的网络体系,在各种动脉粥样硬化危险因子如氧化低密度脂蛋白(ox-LDL)等的作用下,Akt、核因子-KB(NF-κB)、促分裂原活化蛋白激酶(MAPKs)等信号分子均参与巨噬细胞凋亡调控过程,但其具体的调节机制还不清楚。
Tribbles (TRBs)基因是Groβhans等在果蝇体内发现的一类丝氨酸／苏氨酸蛋白激酶样蛋白基因家族,是抑制有丝分裂、诱导细胞凋亡的核基因,也被称为神经细胞死亡诱导蛋白激酶。人类TRBs基因家族包括TRB1、TRB2、TRB3,果蝇TRB3是哺乳动物的同系物,因此目前有关TRBs基因的研究主要集中在TRB3。TRB3可能具有广泛的生物活性,在肝脏、脾、胸腺、前列腺、胰腺和心脏均有表达。最近的研究认为,TRB3基因是一类重要的信号通路调节蛋白,至少可以通过CDC25/String、Akt和MAPKs信号途径通路发挥作用,并能调节NF-κB信号通路。
因此,本研究提出如下假说：TRB3可能参与了ox-LDL诱导的巨噬细胞的凋亡过程,并通过Akt、MAPKs、NF-kB信号转导通路介导巨噬细胞凋亡,从而在动脉粥样硬化易损斑块的发生发展中发挥重要的调节作用。
目的
1.人单核源性巨噬细胞中TRBs的表达情况；
2. ox-LDL对巨噬细胞TRB3的影响；
3.TRB3在ox-LDL诱导的巨噬细胞凋亡中的作用及其信号转导机制。
方法
本研究以原代培养的人单核源性巨噬细胞为研究对象,采用实时定量逆转录聚合酶链反应(real time RT-PCR)、蛋白免疫印迹(western blot)、酶联免疫吸附(ELISA)等实验方法,分别观察：
1.在巨噬细胞自然分化第1、3、5、7、9、11天检测TRB1、TRB2、TRB3 mRNA表达水平；
2.不同浓度(0、5、10、25、50μg／m1)的ox-LDL、LDL处理巨噬细胞不同时间(0、4、8、12、24、48h)后,检测TRB3 mRNA和蛋白含量；
3.TRB3基因克隆及腺病毒表达载体的构建：根据Genebank人TRB3基因序列,设计引物,提取人全血DNA, PCR扩增TRB3基因,回收目的基因并亚克隆至表达载体,经测序鉴定正确后,应用BD Adeno-XTM Expression System 2腺病毒载体构建系统,构建含人TRB3基因的腺病毒质粒。经转染293细胞行病毒包装、扩增及纯化,得到人TRB3腺病毒载体；
4.设计、合成、筛选TRB3 siRNA;
5.TRB3腺病毒及siRNA分别转染巨噬细胞,观察ox-LDL刺激后巨噬细胞凋亡的变化(MTT检测细胞存活率,ELISA法检测凋亡细胞ssDNA含量,western blot检测caspase-3);
6.TRB3腺病毒及siRNA分别转染巨噬细胞,观察Akt、MAPKs及NF-κB信号通路的变化。
结果
1.人单核源性巨噬细胞中TRBs的表达
三种TRBs均能在巨噬细胞中表达,其中以TRB3表达为主,而TRB1、TRB2仅微量表达。在分化的第3天,TRB3 mRNA表达水平即明显上升,第7天表达量达到最高。
2. ox-LDL对巨噬细胞TRB3的影响
分别以0μg／ml、5μg／ml、10μg／ml、25μg／ml、50μg／ml的ox-LDL、LDL孵育巨噬细胞24h后发现,ox-LDL能有效刺激巨噬细胞TRB3 mRNA表达上调,而LDL作用不明显。ox-LDL刺激巨噬细胞TRB3 mRNA表达的上调呈剂量依赖性。在50μg／ml浓度下,与LDL处理组相比,ox-LDL处理组TRB3 mRNA表达显著升高(P<0.01)。
50μg/ml ox-LDL、LDL分别处理巨噬细胞0h、4h、8h、12h、24h、48h后,发现ox-LDL刺激巨噬细胞TRB3 mRNA表达上调还呈时间依赖性。与LDL处理组相比,ox-LDL处理细胞24h后TRB3 mRNA开始明显增加(P<0.05),48h后TRB3 mRNA增加更明显(P＜0.01)。Western Blot进一步证实,50μg/m1L ox-LDL处理巨噬细胞48h后,TRB3蛋白表达量亦显著增加。
3.TRB3基因克隆及腺病毒载体构建
根据人TRB3基因序列,自行设计引物,成功克隆出了人TRB3基因。经测序分析与已知人TRB3序列完全相符。利用BD Adeno-XTM Expression System 2腺病毒构建系统,成功构建了含人TRB3基因的腺病毒载体(pLP-Adeno/TRB3)。
4.TRB3对ox-LDL诱导的巨噬细胞凋亡的影响
与对照组相比,巨噬细胞过表达TRB3后,细胞存活率降低(P<0.01),凋亡增加(P<0.05),caspase-3表达量增加,即细胞凋亡增加；过表达TRB3的巨噬细胞经ox-LDL处理后细胞凋亡也增加,即细胞存活率明显下降(P<0.01),凋亡增加(P<0.01),caspase-3表达量增加；但TRB3过表达和ox-LDL处理之间无交互作用。
TRB3 siRNA转染巨噬细胞后,与对照组相比,TRB3基因沉默的巨噬细胞凋亡减少,TRB3 RNAi组巨噬细胞存活率升高(P0.01),凋亡减少(P<0.05)；经ox-LDL处理后凋亡进一步增加,TRB3 RNAi/ox-LDL组巨噬细胞存活率亦明显降低(P<0.01),凋亡增加(P<0.01),TRB3基因沉默与ox-LDL处理之间有交互作用(P=0.048,P=0.001),为拮抗作用。
5.TRB3对Akt的影响
与对照组相比,巨噬细胞过表达TRB3后,p-Akt表达量降低。过表达TRB3的巨噬细胞经ox-LDL处理后细胞p-Akt表达量比单纯ox-LDL刺激组降低。
TRB3 siRNA转染巨噬细胞后,与对照组相比,TRB3基因沉默的巨噬细胞p-Akt表达量增加；再经ox-LDL孵育后,与单纯ox-LDL处理组相比其p-Akt表达量也增加,但与TRB3基因沉默组相比p-Akt表达量降低。
6.TRB3对MAPKs的影响
巨噬细胞过表达TRB3后,与对照组相比,p38的磷酸化水平明显减低；与ox-LDL处理的对照组相比,过表达TRB3的巨噬细胞经ox-LDL处理后p-p38表达量降低。ox-LDL刺激过表达TRB3的巨噬细胞24h后,与空载体对照组相比,c-Fos的活性明显降低约22%,c-Jun的活性也明显降低。
与对照组相比,siTRB3组的p38的磷酸化水平表达量明显增加；与ox-LDL处理的对照组相比,转染siTRB3的巨噬细胞经ox-LDL处理后p38磷酸化水平明显增加。ELLISA显示与对照组相比,TRB3基因沉默后c-Fos的活性增加1.3倍,c-Jun的活性增加1.3倍。
7.TRB3对NF-κB的影响
ox-LDL呈时间依赖性降低NF-κB的活性。Ox-LDL处理细胞24h时NF-κB的活性开始降低,与LDL对照组相比,p50、p65的活性分别降低40%(P<0.05)和21%(P<0.05)。48h时p50、p65的活性分别降低40%(P<0.01)和21%(P<0.01)。但是巨噬细胞过表达TRIB3后,与对照组相比,p50、p65的活性变化差异无统计学意义。
结论
1.人单核源性巨噬细胞中TRBs的表达以TRB3为主,TRB3主要分布于细胞核内；
2. ox-LDL呈剂量和时间依赖性刺激巨噬细胞合成TRB3,；
3.TRB3部分参与ox-LDL诱导巨噬细胞凋亡过程；
4.TRB3主要是通过抑制Akt、p38 MAPK/AP-1通路参与ox-LDL诱导的巨噬细胞凋亡；
5. NF-κB参与ox-LDL诱导的巨噬细胞凋亡,但不参与TRB3介导的巨噬细胞凋亡过程
背景
内质网位于细胞核附近的胞质区域,是哺乳动物细胞重要的Ca2+贮存器,也是蛋白质合成与翻译后修饰、多肽链正确折叠与装配的重要场所。多种因素如低氧、高血糖、化学毒物等均可使内质网腔内Ca2+耗竭、内质网蛋白质糖基化抑制、二硫键错配、内质网蛋白质向高尔基体转运减少,导致未折叠或错误折叠的蛋白质在内质网腔内积蓄等,均可使内质网功能发生改变,统称内质网应激(endoplasmic reticulum stress, ER stress)。内质网应激与多种疾病相关,最近的研究显示,内质网应激参与了动脉粥样硬化的发生和发展。
C/EBP同源蛋白(CCAAT/enhancer binding proteins homologus protein, CHOP)是C/EBPs家族成员之一,具有一个转录激活区和一个与DNA结合的亮氨酸拉链结构,可以调控细胞应激反应过程中众多基因的表达。在非应激的基础状态下,CHOP的表达水平很低,而在细胞外和内质网应激中,其表达量大大增加。在内质网应激中,PERK、ATF6以及IRE-1都能够诱导CHOP的转录,然而PERK-eIF2a-ATF4是CHOP蛋白表达所必需的。通过CHOP基因过表达和CHOP基因敲除的小鼠,CHOP已被证实在内质网应激中作为细胞周期停滞和凋亡的一个诱导物。
TRB3是晚近在果蝇体内发现一种抑制有丝分裂的蛋白激酶,TRB3可能不仅是糖尿病糖耐量异常的原因,而且作用于MAPKs及NF-KB等信号通路,在细胞生长、发育、分裂、凋亡等生理反应过程具有相当重要的作用。最近的研究认为,TRB3参与内质网应激的信号转导过程。衣霉素、大麻素、毒胡萝卜素等内质网应激诱导剂,能够刺激TRB3基因的表达,并可以同时上调ATF4和CHOP的表达,但TRB3的mRNA变化要晚于ATF4和CHOP。有研究表明,在内质网应激通路中,TRB3属于下游基因,应激反应时ATF4和CHOP所形成的复合物,通过与TRB3启动子区域的一段33bp碱基重复序列结合后促进TRB3基因的表达。Ox-LDL被认为是AS发生发展过程中的一个危险因素,本研究前期实验发现,ox-LDL呈剂量和时间依赖性刺激人单核源性巨噬细胞中TRB3的表达,并且TRB3部分参与了ox-LDL诱导巨噬细胞凋亡的过程。
因此,本研究提出如下假说：ATF4-CHOP可能参与了ox-LDL诱导的巨噬细胞的TRB3表达增加的过程,并通过TRB3参与巨噬细胞凋亡,从而在动脉粥样硬化易损斑块的发生发展中发挥重要的调节作用。
目的
1.探讨ox-LDL对人单核源性巨噬细胞CHOP、ATF4的调控作用；
2.观察ER stress时巨噬细胞TRB3的表达变化；
3.探讨巨噬细胞中TRB3与ATF4-CHOP的相互调节作用。
方法
1.不同浓度(0、2.5、5、10、25、50μg／m1)的ox-LDL、LDL处理巨噬细胞不同时间(0、4、8、12、24、48h)后,检测CHOP、ATF4 mRNA含量；
2.2μg／ml衣霉素刺激人单核源性巨噬细胞不同时间(0h、4h、8h、12h、24h、48h)后,检测TRB3 mRNA的表达；
3.CHOP表达质粒转染巨噬细胞后,观察ox-LDL刺激后TRB3 mRNA表达的变化；
4.构建TRB3重组腺病毒和TRB3siRNA,转染巨噬细胞后,观察TRB3过表达或抑制表达后CHOP mRNA表达的变化。
结果
1.ox-LDL对巨噬细胞CHOP、ATF4mRNA的影响
分别以0、2.5、5、10、25、50的ox-LDL、LDL孵育巨噬细胞24h后发现,ox-LDL能有效刺激巨噬细胞CHOP mRNA表达上调,而LDL作用不明显。ox-LDL刺激巨噬细胞CHOP mRNA表达随ox-LDL剂量增加而逐渐增加,其中50μg/ml ox-LDL刺激作用最强,提示ox-LDL对巨噬细胞中CHOP mRNA表达的上调呈剂量依赖性。与LDL处理组相比,25μg/ml ox-LDL处理组CHOP mRNA表达显著升高(P0.01),50μg/ml ox-LDL处理组增加更明显(P<0.01)。50μg/ml ox-LDL和50p.g／ml LDL分别处理巨噬细胞0h、4h、8h、16h、24h、48h后,发现ox-LDL刺激巨噬细胞CHOP mRNA表达上调,并呈时间依赖性。与LDL处理组相比,ox-LDL处理细胞24h后CHOP mRNA开始明显增加(P<0.05),48h后CHOP mRNA增加更明显(P＜0.01)。
50μg/ml ox-LDL亦能促进巨噬细胞ATF4 mRNA的表达,并呈时间依赖性,24h时达到高峰,为LDL对照组的1.6倍(P＜0.01),但峰值表达量低于CHOP(分别为对照组的1.6倍和3倍)。ox-LDL促进巨噬细胞ATF4、CHOP、TRB3 mRNA表达增加时,TRB3 mRNA变化要晚于ATF4和CHOP。
2. ER stress对巨噬细胞合成TRB3的影响
衣霉素能明显刺激巨噬细胞TRB3 mRNA表达上调,并呈时间依赖性。与对照组相比,衣霉素处理巨噬细胞6h后TRB3 mRNA开始明显增加(P<0.05),8h后TRB3 mRNA增加更明显(P<0.01),24h后TRB3 mRNA表达达到高峰。
3.CHOP过表达对巨噬细胞TRB3表达的影响
CHOP表达质粒转染巨噬细胞使CHOP过表达后检测TRB3 mRNA,与对照组相比,巨噬细胞过表达CHOP后其TRB3 mRNA明显增加(P＜0.01)。过表达CHOP基因的巨噬细胞经50μg／ml ox-LDL处理后,与对照组相比,ox-LDL刺激组TRB3 mRNA表达水平明显增加(P<0.01)。
4.TRB3对巨噬细胞CHOP表达的影响
TRB3重组腺病毒转染巨噬细胞使TRB3过表达后,与对照组相比,其CHOP mRNA的表达明显降低(P＜0.05)。
采用TRB3靶向siRNA转染巨噬细胞抑制TRB3表达后,与对照组相比较,siRNA抑制巨噬细胞TRB3表达后CHOP mRNA的表达虽然增加,但是差异无统计学意义。抑制TRB3表达的巨噬细胞经ox-LDL处理后,与对照组相比,CHOPmRNA表达明显增加(P<0.01)。
结论
1. ox-LDL呈剂量和时间依赖性促进巨噬细胞CHOP、ATF4 mRNA表达；
2. ox-LDL可诱导巨噬细胞发生ER stress;
3.巨噬细胞发生ER stress时以CHOP、ATF4的表达增加为主；
4. ER stress促进巨噬细胞TRB3 mRNA的合成；
5.CHOP可促进ox-LDL诱导的TRB3表达,TRB3负反馈调节CHOP表达,ATF4-CHOP-TRB3参与ox-LDL导致巨噬细胞凋亡的过程。
Background
Atherosclerosis (AS) and its complications caused by acute cardiovascular disease event have become the number one killer of mankind, its prevention and treatment have been a worldwide public health priorities and challenges. Coronary artery atherosclerosis is the principal cause of acute coronary syndrome (ACS) and is the single largest killer of both men and women. ACS is the consequence of rupture or erosion of preexisting atherosclerotic plaque, with subsequent formation of local thrombus leading to critical occlusion of coronary arteries. The principal pathological basis of ACS is vulnerable plaques.
Macrophage apoptosis contributes much to the development of vulnerable atherosclerotic plaques. A possible mechanism linking macrophage apoptosis to vulnerable plaque progression is the reduced level of macrophages failing to clear the apoptotic smooth muscle cells and macrophages, which leads to secondary necrosis of these cells and facilitates the formation of an atheromatous core within plaques. As well, apoptotic macrophages can release cholesterol, which results in accumulation of acicular cholesterol crystal in the lipid core, thus injuring the fibrous cap of plaques. In addition, apoptotic macrophages can be a source of tissue factor, a procoagulant molecule considered to play an important role in coagulation and thrombosis associated with advanced plaques.
Signal transduction of apoptosis in macrophages is a complex network system. Various risk factors such as increased level of oxidized low-density lipoprotein (ox-LDL) can induce macrophage apoptosis through multiple apoptotic signaling pathways, such as Akt, NF-kB and MAPKs. However, the mechanism of macrophage apoptosis remains to be elucidated.
Tribbles (TRBs), a Drosophila protein, slows progression through the G2 stage of the cell cycle. Three mammalian orthologs, TRB1, TRB2 and TRB3, all contain a consensus serine/threonine kinase catalytic core but lack an ATP-binding pocket and so do not possess kinase activity. TRB3, also named neuronal cell death-inducible putative protein kinase (NIPK), is expressed in liver, thymus, prostate, pancreas and heart and may have wide biological activity. Recently, TRB3 has been shown to be a very important regulatory protein involved in signal pathways and works at least through CDC25/String, Akt and MAPKs. The activation of MAPKs and inhibition of Akt kinase activity result in macrophage apoptosis, which is implied in the development of vulnerable atherosclerotic plaques. TRB3 could be involved in macrophage apoptosis induced by ox-LDL and could play an essential role in the progression of vulnerable plaques.
Therefore, we hypothesized that TRB3 may be implicated in ox-LDL-induced apoptosis by stimulating human monocyte-derived macrophages with ox-LDL, through MAPKs, Akt and NF-kB signaling pathways.
Objective
1. To investigate the expression of TRBs in human monocyte-derived macrophages.
2. To observe the effect of ox-LDL on TRB3 in macrophages.
3. To explore whether TRB3 affects macrophage apoptosis induced by ox-LDL and to study the possible signal transduction mechanism.
Methods
We took primary human monocyte-derived macrophages as the object of this research, then quantitative real time PCR, western blot, ELISA, etc. were used to observe:
1. Monocytes were first allowed to differentiate into macrophages naturally. Quantitative real-time PCR performed on days 1,3,5,7,9 and 11 detected the mRNA expression of TRB1, TRB2, and TRB3 in monocyte-derived macrophages.
2. Macrophages were treated with various concentrations of ox-LDL and LDL (0,5,10,25 and 50μg/ml) or various times (0,4,8,12,24, and 48h), then quantitative real time PCR and western blot analysis were used to detect TRB3 mRNA and protein level.
3. Cloning of human TRB3 and constructing of TRB3 adenovirus vector
Based on human TRB3 sequence in Genebank, we designed one pair of primers. Using total DNA extracted from human whole blood and reverse transcript polymerase chain reaction, we cloned the CDS sequence of human TRB3. BD Adeno-XTM Expression System 2 was used to construct adenovirus containing human TRB3.293 cells were infected with the TRB3 plasmid. Recombinant adenovirus from a single plaque was expanded and purified, viral titer was determined by plaque assay.
4. Design, synthesis and screening of TRB3 targeting small interfering RNA (siRNA).
5. Macrophages were transfected with recombinant adeno viral TRB3 or TRB3 siRNA, and cell viability was examined by MTT assay, apoptosis by ELISA measurement of ssDNA absorbance, and caspase-3 level by western blot.
6. After overexpressing or silencing TRB3 expression, the change of Akt, MAPKs and NF-kB in macrophages were measured.
Results
1. TRBs mRNA expression in human macrophages
All three genes were expressed in monocyte-derived macrophages. Macrophages predominantly expressed TRB3; the TRB3 mRNA level was significantly increased on day 3 and peaked on day 7.
2. TRB3 expression upregulated by ox-LDL
Our data showed compared with LDL treatment, TRB3 mRNA and protein level in macrophages increased with increasing ox-LDL concentration and treatment time.
3. Cloning of human TRB3 and constructing TRB3 adenovirus
We have successfully cloned human TRB3, and its sequence was identical to the human TRB3 sequence in Genebank. Using BD Adeno-XTM Expression System 2, we constructed human TRB3 adenovirus.
4. TRB3 mediates macrophage apoptosis induced by ox-LDL
Compared with control cells, cell viability of TRB3-overexpressing macrophages was decreased (P<0.01), but apoptosis was increased (P<0.05) and caspase-3 level increased. With ox-LDL treatment, TRB3-overexpressing macrophages showed increased apoptosis, as deduced by decreased cell viability and increased apoptosis (P<0.01) and caspase-3 level compared with controls. Factorial analyses revealed no significant interaction between overexpression of TRB3 and stimulation with ox-LDL on macrophage apoptosis.
TRB3-silenced macrophages showed decreased apoptosis, as inferred by increased cell viability (P<0.01), and decreased apoptosis (P<0.05) and caspase-3 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly reduced cell viability (P<0.01), accompanied by increased apoptosis (P<0.01) and caspase-3 level compared with controls. Silenced TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis (P=0.048, P=0.001), which suggests that TRB3 knockdown in part resisted macrophage apoptosis induced by ox-LDL.
5. Effect of TRB3 on Akt in macrophages
Compared with control cells, the level of p-Akt of TRB3-overexpressing macrophages was decreased. With ox-LDL treatment, TRB3-overexpressing macrophages showed decreased p-Akt level compared with controls.
TRB3-silenced macrophages showed increased p-Akt level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-Akt level compared with controls.
6. Effect of TRB3 on MAPKs in macrophages
The activity of c-Fos and c-Jun in TRB3-overexpressing macrophages with ox-LDL treatment were lower than control cells(P<0.05, P<0.05). TRB3-silenced cells treated with ox-LDL showed significantly increased AP-1 activity. c-Fos activity increased 1.3 folds and c-Jun activity increased 1.3 folds compared to control cells.
TRB3-silenced macrophages showed increased p-p38 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-p38 level compared with controls.
7. Effect of TRB3 on NF-kB in macrophages
ox-LDL decreased the activity of NF-kB in macrophages in a time-dependent manner compared to LDL treatment. After treatment with ox-LDL for 24h, the activity of p50 and p65 was significantly lower than that following treatment with LDL for 24h and was further decreased after 48h ox-LDL treatmet. However, overexpression of TRB3 has no effect on the activity of p50 and p65 reduced by ox-LDL.
Conclusions
1. Human monocyte-derived macrophages expressed TRB1, TRB2 and especially TRB3.
2. TRB3 expression was upregulated in macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.
3. TRB3 in part mediates macrophage apoptosis induced by ox-LDL through Akt and MAPKs signaling pathway.
Background
The endoplasmic reticulum (ER) near the nucleus region in the cytoplasm is a central organelle of eukaryotic cells as the place of calcium storage, lipid synthesis, protein folding and protein maturation. A variety of factors such as hypoxia, hyperglycemia, chemical poisons and so on leading to depletion of endoplasmic reticulum Ca2+, inhibition of endoplasmic reticulum protein glycosylation, mismatched disulfide, accumulation of unfolded protein (unfolded protein response, UPR), excessive protein traffics and so on, can make the endoplasmic reticulum function change, collectively referred to endoplasmic reticulum stress (ER stress).Recently, ER stress has been shown to be involved in several diseases with ER stress-dependent cell death, such as diabetes and Alzheimer disease, especially ER stress plays an essential role in the progression of atherosclerosis.
CCAAT/enhancer binding proteins homologus protein (CHOP, also known as growth arrest and DNA damage-inducible protein 153), consisting of a transcriptional activation domain and a leucine zipper for DNA binging, is a member of the C/EBP family transcription factor family that can regulate the expression of a large number of target genes during cell stress response. The expression level of CHOP is low under normal conditions but induced by ER stress. CHOP is considered to be a main mediating factor in ER stress and acts as an inducer of cell cycle arrest and apoptosis. CHOP-mediated apoptosis during ER stress has been shown to be involved in several diseases with ER stress-dependent cell death. High expression of CHOP was observed at all stages of atherosclerotic lesion development in apolipoprotein E-deficicent mice, and both in early lesion and late advanced lesion CHOP expression increaseed ER stress promoted atherosclerosis progression via acculation of free cholesterol. However, whether lipoprotein ox-LDL mediates atherogenesis by CHOP-dependent ER stress pathway remains unclear.
Tribble3 (TRB3), a mammalian homolog of Drosophila Tribbles, is a recently recognized atypical inactibe kinase that inhibits mitosis and be implicated in cell cycle regulation and aopotosis. TRB3 is reported to participate in the signal pathway of ER stress, and induced by various forms ER stress, such as tunicamycin, cannabinoids and thapsigargin. Recent studies show that TRB3 acts as a target gene of CHOP during ER stress. CHOP with a novel dimerizing partner ATF4 activates TRB3 promoter[19]. We previously found that TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosis.
Taken together, we hypothesized that CHOP regulates ox-LDL-induced TRB3 expression and is involved in macrophages apoptosis through TRB3 pathway and
Objective
1. To investigate the mRNA expression of CHOP and ATF4 in human monocyte-derived macrophages.
2. To observe the effect of ER stress on TRB3 expression in macrophages.
3. To explore the interaction between TRB3 and CHOP.
Methods
1. Human monocyte-derived macrophages were treated with various concentrations of ox-LDL and LDL (0,2.5,5,10,25 and 50μg/ml))for various times (0,4,8,16,24, and 48h), then quantitative real time PCR and western blot analysis was used to detect CHOP and ATF4 mRNA and protein level.
2. Macrophages treated with 2μg/ml tunicamycin for 0,2,4,6,8,24h, the mRNA level of TRB3 was measured by quantitative real time PCR.
3. After overexpression CHOP by CHOP expression plasmid, macrophages were incubated with ox-LDL. The mRNA expression of TRB3 was measured.
4. Macrophages were transfected with adenoviral TRB3 or TRB3 siRNA, and the mRNA expression of CHOP was dectected.
Results
1. CHOP and ATF4 mRNA expression upregulated by ox-LDL
Our data showed that the expression of CHOP mRNA was significantly upregulated by ox-LDL but not LDL. This expression increased with increasing ox-LDL concentration. Furthermore, the expression of CHOP mRNA was significantly upregulated by ox-LDL at the concentration of 25 or 50μg/ml as compared to LDL treatment.
Macrophages treated with 50μg/ml ox-LDL or LDL for various times showed increased expression of CHOP mRNA with increasing time. After treatment with 50μg/ml ox-LDL for 24 hr, the expression of CHOP mRNA was significantly higher than that with LDL treatment (P<0.05) and was further increased with 48-hr ox-LDL treatment (P<0.01).
Also,50μg/ml ox-LDL increased the expression of ATF4 mRNA in a time-dependent manner. ATF4 mRNA expression peaked at 24 hr, but the peak level was less than CHOP (1.6 folds vs 3 folds). Ox-LDL induced TRB3 expression in a time-dependent manner, too. The induction of TRB3 mRNA was later than that of CHOP and ATF4 mRNA.
2. ER stress induces mRNA expression of TRB3, CHOP and ATF4
Macrophages treated with 2μg/ml tunicamycin for various times showed augmented TRB3 mRNA expression in a time-dependent manner. Compared with control cells, the significantly increase occurred at 6 hr and the peak level at 24 hr.
3. CHOP overexpression promotes TRB3 expression
Macrophages were transfected with CHOP expression plasmid and incubated for an additional 24 hr with or without ox-LDL. TRB3 mRNA expression of CHOP-overexpressing macrophages was also found significantly increased as compared with controls. Moreover, treatment of CHOP-overexpressing macrophages with ox-LDL markedly increased TRB3 expression as compared with controls.
4. Effect of TRB3 on CHOP expression
To better characterize the interaction between CHOP and TRB3, adenoviral TRB3 were transfected into macrophages to overexpress TRB3. CHOP mRNA expression of TRB3-overexpressing macrophages was significantly lower than that of control cells.
The TRB3-targeting siRNA (siTRB3) was transfected into macrophages to silence TRB3 gene expression. CHOP mRNA expression of TRB3-silenced macrophages was higher than that of control cells but not significantly, whereas with ox-LDL treatment, CHOP mRNA expression of treated cells was significantly higher.
Conclusion
1. CHOP and ATF4 mRNA expression was upregulated in human monocyte-derived macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.
2. ox-LDL can induce ER stress in human macrophage.
3. TRB3 mRNA expression was increased during ER stress.
4. CHOP promoted TRB3 expression and TRB3 exerted a feedback inhibition on the expression of CHOP in macrophages, so ATF4/CHOP-TRB3 may be involved in ox-LDL-dependent macrophage apoptosis.
动脉粥样硬化(AS)及其并发症引起的急性心血管病事件已成为人类的头号杀手,其防治一直是世界性公共卫生的重点和难题。急性冠状动脉综合征(acute coronary syndrome, ACS)是冠状动脉粥样硬化性心脏病最重要的死因。ACS主要是由于冠状动脉内粥样斑块的破裂及其引起的血栓形成,导致冠状动脉急性管腔狭窄或闭塞所致。从病理生理角度而言,ACS最主要的深层原因是易损斑块,即易于发生血栓或可能迅速进展成为罪犯病变的斑块。
近年来的研究表明,动脉粥样斑块内的巨噬细胞凋亡是易损斑块形成的重要原因。尽管巨噬细胞减少导致其分泌基质金属蛋白酶下降,斑块细胞外胶原降解减少而具有稳定斑块的作用,但大量研究证实,巨噬细胞凋亡可通过以下的机制促进易损斑块的形成：1.巨噬细胞数量减少,凋亡的平滑肌细胞和巨噬细胞不能被有效吞噬,发生继发性坏死,胞浆内脂质内容物释放,堆积形成斑块的脂质核心,促进粥样斑块脂质核心的形成；2.巨噬细胞凋亡后释放出胆固醇,导致斑块脂质核心针形胆固醇结晶增多,损伤纤维帽；3.巨噬细胞凋亡后释放促血栓形成物质,尤其是组织因子,促进斑块血栓形成。
巨噬细胞凋亡的信号调控是一个复杂的网络体系,在各种动脉粥样硬化危险因子如氧化低密度脂蛋白(ox-LDL)等的作用下,Akt、核因子-KB(NF-κB)、促分裂原活化蛋白激酶(MAPKs)等信号分子均参与巨噬细胞凋亡调控过程,但其具体的调节机制还不清楚。
Tribbles (TRBs)基因是Groβhans等在果蝇体内发现的一类丝氨酸／苏氨酸蛋白激酶样蛋白基因家族,是抑制有丝分裂、诱导细胞凋亡的核基因,也被称为神经细胞死亡诱导蛋白激酶。人类TRBs基因家族包括TRB1、TRB2、TRB3,果蝇TRB3是哺乳动物的同系物,因此目前有关TRBs基因的研究主要集中在TRB3。TRB3可能具有广泛的生物活性,在肝脏、脾、胸腺、前列腺、胰腺和心脏均有表达。最近的研究认为,TRB3基因是一类重要的信号通路调节蛋白,至少可以通过CDC25/String、Akt和MAPKs信号途径通路发挥作用,并能调节NF-κB信号通路。
因此,本研究提出如下假说：TRB3可能参与了ox-LDL诱导的巨噬细胞的凋亡过程,并通过Akt、MAPKs、NF-kB信号转导通路介导巨噬细胞凋亡,从而在动脉粥样硬化易损斑块的发生发展中发挥重要的调节作用。
目的
1.人单核源性巨噬细胞中TRBs的表达情况；
2. ox-LDL对巨噬细胞TRB3的影响；
3.TRB3在ox-LDL诱导的巨噬细胞凋亡中的作用及其信号转导机制。
方法
本研究以原代培养的人单核源性巨噬细胞为研究对象,采用实时定量逆转录聚合酶链反应(real time RT-PCR)、蛋白免疫印迹(western blot)、酶联免疫吸附(ELISA)等实验方法,分别观察：
1.在巨噬细胞自然分化第1、3、5、7、9、11天检测TRB1、TRB2、TRB3 mRNA表达水平；
2.不同浓度(0、5、10、25、50μg／m1)的ox-LDL、LDL处理巨噬细胞不同时间(0、4、8、12、24、48h)后,检测TRB3 mRNA和蛋白含量；
3.TRB3基因克隆及腺病毒表达载体的构建：根据Genebank人TRB3基因序列,设计引物,提取人全血DNA, PCR扩增TRB3基因,回收目的基因并亚克隆至表达载体,经测序鉴定正确后,应用BD Adeno-XTM Expression System 2腺病毒载体构建系统,构建含人TRB3基因的腺病毒质粒。经转染293细胞行病毒包装、扩增及纯化,得到人TRB3腺病毒载体；
4.设计、合成、筛选TRB3 siRNA;
5.TRB3腺病毒及siRNA分别转染巨噬细胞,观察ox-LDL刺激后巨噬细胞凋亡的变化(MTT检测细胞存活率,ELISA法检测凋亡细胞ssDNA含量,western blot检测caspase-3);
6.TRB3腺病毒及siRNA分别转染巨噬细胞,观察Akt、MAPKs及NF-κB信号通路的变化。
结果
1.人单核源性巨噬细胞中TRBs的表达
三种TRBs均能在巨噬细胞中表达,其中以TRB3表达为主,而TRB1、TRB2仅微量表达。在分化的第3天,TRB3 mRNA表达水平即明显上升,第7天表达量达到最高。
2. ox-LDL对巨噬细胞TRB3的影响
分别以0μg／ml、5μg／ml、10μg／ml、25μg／ml、50μg／ml的ox-LDL、LDL孵育巨噬细胞24h后发现,ox-LDL能有效刺激巨噬细胞TRB3 mRNA表达上调,而LDL作用不明显。ox-LDL刺激巨噬细胞TRB3 mRNA表达的上调呈剂量依赖性。在50μg／ml浓度下,与LDL处理组相比,ox-LDL处理组TRB3 mRNA表达显著升高(P<0.01)。
50μg/ml ox-LDL、LDL分别处理巨噬细胞0h、4h、8h、12h、24h、48h后,发现ox-LDL刺激巨噬细胞TRB3 mRNA表达上调还呈时间依赖性。与LDL处理组相比,ox-LDL处理细胞24h后TRB3 mRNA开始明显增加(P<0.05),48h后TRB3 mRNA增加更明显(P＜0.01)。Western Blot进一步证实,50μg/m1L ox-LDL处理巨噬细胞48h后,TRB3蛋白表达量亦显著增加。
3.TRB3基因克隆及腺病毒载体构建
根据人TRB3基因序列,自行设计引物,成功克隆出了人TRB3基因。经测序分析与已知人TRB3序列完全相符。利用BD Adeno-XTM Expression System 2腺病毒构建系统,成功构建了含人TRB3基因的腺病毒载体(pLP-Adeno/TRB3)。
4.TRB3对ox-LDL诱导的巨噬细胞凋亡的影响
与对照组相比,巨噬细胞过表达TRB3后,细胞存活率降低(P<0.01),凋亡增加(P<0.05),caspase-3表达量增加,即细胞凋亡增加；过表达TRB3的巨噬细胞经ox-LDL处理后细胞凋亡也增加,即细胞存活率明显下降(P<0.01),凋亡增加(P<0.01),caspase-3表达量增加；但TRB3过表达和ox-LDL处理之间无交互作用。
TRB3 siRNA转染巨噬细胞后,与对照组相比,TRB3基因沉默的巨噬细胞凋亡减少,TRB3 RNAi组巨噬细胞存活率升高(P0.01),凋亡减少(P<0.05)；经ox-LDL处理后凋亡进一步增加,TRB3 RNAi/ox-LDL组巨噬细胞存活率亦明显降低(P<0.01),凋亡增加(P<0.01),TRB3基因沉默与ox-LDL处理之间有交互作用(P=0.048,P=0.001),为拮抗作用。
5.TRB3对Akt的影响
与对照组相比,巨噬细胞过表达TRB3后,p-Akt表达量降低。过表达TRB3的巨噬细胞经ox-LDL处理后细胞p-Akt表达量比单纯ox-LDL刺激组降低。
TRB3 siRNA转染巨噬细胞后,与对照组相比,TRB3基因沉默的巨噬细胞p-Akt表达量增加；再经ox-LDL孵育后,与单纯ox-LDL处理组相比其p-Akt表达量也增加,但与TRB3基因沉默组相比p-Akt表达量降低。
6.TRB3对MAPKs的影响
巨噬细胞过表达TRB3后,与对照组相比,p38的磷酸化水平明显减低；与ox-LDL处理的对照组相比,过表达TRB3的巨噬细胞经ox-LDL处理后p-p38表达量降低。ox-LDL刺激过表达TRB3的巨噬细胞24h后,与空载体对照组相比,c-Fos的活性明显降低约22%,c-Jun的活性也明显降低。
与对照组相比,siTRB3组的p38的磷酸化水平表达量明显增加；与ox-LDL处理的对照组相比,转染siTRB3的巨噬细胞经ox-LDL处理后p38磷酸化水平明显增加。ELLISA显示与对照组相比,TRB3基因沉默后c-Fos的活性增加1.3倍,c-Jun的活性增加1.3倍。
7.TRB3对NF-κB的影响
ox-LDL呈时间依赖性降低NF-κB的活性。Ox-LDL处理细胞24h时NF-κB的活性开始降低,与LDL对照组相比,p50、p65的活性分别降低40%(P<0.05)和21%(P<0.05)。48h时p50、p65的活性分别降低40%(P<0.01)和21%(P<0.01)。但是巨噬细胞过表达TRIB3后,与对照组相比,p50、p65的活性变化差异无统计学意义。
结论
1.人单核源性巨噬细胞中TRBs的表达以TRB3为主,TRB3主要分布于细胞核内；
2. ox-LDL呈剂量和时间依赖性刺激巨噬细胞合成TRB3,；
3.TRB3部分参与ox-LDL诱导巨噬细胞凋亡过程；
4.TRB3主要是通过抑制Akt、p38 MAPK/AP-1通路参与ox-LDL诱导的巨噬细胞凋亡；
5. NF-κB参与ox-LDL诱导的巨噬细胞凋亡,但不参与TRB3介导的巨噬细胞凋亡过程
背景
内质网位于细胞核附近的胞质区域,是哺乳动物细胞重要的Ca2+贮存器,也是蛋白质合成与翻译后修饰、多肽链正确折叠与装配的重要场所。多种因素如低氧、高血糖、化学毒物等均可使内质网腔内Ca2+耗竭、内质网蛋白质糖基化抑制、二硫键错配、内质网蛋白质向高尔基体转运减少,导致未折叠或错误折叠的蛋白质在内质网腔内积蓄等,均可使内质网功能发生改变,统称内质网应激(endoplasmic reticulum stress, ER stress)。内质网应激与多种疾病相关,最近的研究显示,内质网应激参与了动脉粥样硬化的发生和发展。
C/EBP同源蛋白(CCAAT/enhancer binding proteins homologus protein, CHOP)是C/EBPs家族成员之一,具有一个转录激活区和一个与DNA结合的亮氨酸拉链结构,可以调控细胞应激反应过程中众多基因的表达。在非应激的基础状态下,CHOP的表达水平很低,而在细胞外和内质网应激中,其表达量大大增加。在内质网应激中,PERK、ATF6以及IRE-1都能够诱导CHOP的转录,然而PERK-eIF2a-ATF4是CHOP蛋白表达所必需的。通过CHOP基因过表达和CHOP基因敲除的小鼠,CHOP已被证实在内质网应激中作为细胞周期停滞和凋亡的一个诱导物。
TRB3是晚近在果蝇体内发现一种抑制有丝分裂的蛋白激酶,TRB3可能不仅是糖尿病糖耐量异常的原因,而且作用于MAPKs及NF-KB等信号通路,在细胞生长、发育、分裂、凋亡等生理反应过程具有相当重要的作用。最近的研究认为,TRB3参与内质网应激的信号转导过程。衣霉素、大麻素、毒胡萝卜素等内质网应激诱导剂,能够刺激TRB3基因的表达,并可以同时上调ATF4和CHOP的表达,但TRB3的mRNA变化要晚于ATF4和CHOP。有研究表明,在内质网应激通路中,TRB3属于下游基因,应激反应时ATF4和CHOP所形成的复合物,通过与TRB3启动子区域的一段33bp碱基重复序列结合后促进TRB3基因的表达。Ox-LDL被认为是AS发生发展过程中的一个危险因素,本研究前期实验发现,ox-LDL呈剂量和时间依赖性刺激人单核源性巨噬细胞中TRB3的表达,并且TRB3部分参与了ox-LDL诱导巨噬细胞凋亡的过程。
因此,本研究提出如下假说：ATF4-CHOP可能参与了ox-LDL诱导的巨噬细胞的TRB3表达增加的过程,并通过TRB3参与巨噬细胞凋亡,从而在动脉粥样硬化易损斑块的发生发展中发挥重要的调节作用。
目的
1.探讨ox-LDL对人单核源性巨噬细胞CHOP、ATF4的调控作用；
2.观察ER stress时巨噬细胞TRB3的表达变化；
3.探讨巨噬细胞中TRB3与ATF4-CHOP的相互调节作用。
方法
1.不同浓度(0、2.5、5、10、25、50μg／m1)的ox-LDL、LDL处理巨噬细胞不同时间(0、4、8、12、24、48h)后,检测CHOP、ATF4 mRNA含量；
2.2μg／ml衣霉素刺激人单核源性巨噬细胞不同时间(0h、4h、8h、12h、24h、48h)后,检测TRB3 mRNA的表达；
3.CHOP表达质粒转染巨噬细胞后,观察ox-LDL刺激后TRB3 mRNA表达的变化；
4.构建TRB3重组腺病毒和TRB3siRNA,转染巨噬细胞后,观察TRB3过表达或抑制表达后CHOP mRNA表达的变化。
结果
1.ox-LDL对巨噬细胞CHOP、ATF4mRNA的影响
分别以0、2.5、5、10、25、50的ox-LDL、LDL孵育巨噬细胞24h后发现,ox-LDL能有效刺激巨噬细胞CHOP mRNA表达上调,而LDL作用不明显。ox-LDL刺激巨噬细胞CHOP mRNA表达随ox-LDL剂量增加而逐渐增加,其中50μg/ml ox-LDL刺激作用最强,提示ox-LDL对巨噬细胞中CHOP mRNA表达的上调呈剂量依赖性。与LDL处理组相比,25μg/ml ox-LDL处理组CHOP mRNA表达显著升高(P0.01),50μg/ml ox-LDL处理组增加更明显(P<0.01)。50μg/ml ox-LDL和50p.g／ml LDL分别处理巨噬细胞0h、4h、8h、16h、24h、48h后,发现ox-LDL刺激巨噬细胞CHOP mRNA表达上调,并呈时间依赖性。与LDL处理组相比,ox-LDL处理细胞24h后CHOP mRNA开始明显增加(P<0.05),48h后CHOP mRNA增加更明显(P＜0.01)。
50μg/ml ox-LDL亦能促进巨噬细胞ATF4 mRNA的表达,并呈时间依赖性,24h时达到高峰,为LDL对照组的1.6倍(P＜0.01),但峰值表达量低于CHOP(分别为对照组的1.6倍和3倍)。ox-LDL促进巨噬细胞ATF4、CHOP、TRB3 mRNA表达增加时,TRB3 mRNA变化要晚于ATF4和CHOP。
2. ER stress对巨噬细胞合成TRB3的影响
衣霉素能明显刺激巨噬细胞TRB3 mRNA表达上调,并呈时间依赖性。与对照组相比,衣霉素处理巨噬细胞6h后TRB3 mRNA开始明显增加(P<0.05),8h后TRB3 mRNA增加更明显(P<0.01),24h后TRB3 mRNA表达达到高峰。
3.CHOP过表达对巨噬细胞TRB3表达的影响
CHOP表达质粒转染巨噬细胞使CHOP过表达后检测TRB3 mRNA,与对照组相比,巨噬细胞过表达CHOP后其TRB3 mRNA明显增加(P＜0.01)。过表达CHOP基因的巨噬细胞经50μg／ml ox-LDL处理后,与对照组相比,ox-LDL刺激组TRB3 mRNA表达水平明显增加(P<0.01)。
4.TRB3对巨噬细胞CHOP表达的影响
TRB3重组腺病毒转染巨噬细胞使TRB3过表达后,与对照组相比,其CHOP mRNA的表达明显降低(P＜0.05)。
采用TRB3靶向siRNA转染巨噬细胞抑制TRB3表达后,与对照组相比较,siRNA抑制巨噬细胞TRB3表达后CHOP mRNA的表达虽然增加,但是差异无统计学意义。抑制TRB3表达的巨噬细胞经ox-LDL处理后,与对照组相比,CHOPmRNA表达明显增加(P<0.01)。
结论
1. ox-LDL呈剂量和时间依赖性促进巨噬细胞CHOP、ATF4 mRNA表达；
2. ox-LDL可诱导巨噬细胞发生ER stress;
3.巨噬细胞发生ER stress时以CHOP、ATF4的表达增加为主；
4. ER stress促进巨噬细胞TRB3 mRNA的合成；
5.CHOP可促进ox-LDL诱导的TRB3表达,TRB3负反馈调节CHOP表达,ATF4-CHOP-TRB3参与ox-LDL导致巨噬细胞凋亡的过程。
Background
Atherosclerosis (AS) and its complications caused by acute cardiovascular disease event have become the number one killer of mankind, its prevention and treatment have been a worldwide public health priorities and challenges. Coronary artery atherosclerosis is the principal cause of acute coronary syndrome (ACS) and is the single largest killer of both men and women. ACS is the consequence of rupture or erosion of preexisting atherosclerotic plaque, with subsequent formation of local thrombus leading to critical occlusion of coronary arteries. The principal pathological basis of ACS is vulnerable plaques.
Macrophage apoptosis contributes much to the development of vulnerable atherosclerotic plaques. A possible mechanism linking macrophage apoptosis to vulnerable plaque progression is the reduced level of macrophages failing to clear the apoptotic smooth muscle cells and macrophages, which leads to secondary necrosis of these cells and facilitates the formation of an atheromatous core within plaques. As well, apoptotic macrophages can release cholesterol, which results in accumulation of acicular cholesterol crystal in the lipid core, thus injuring the fibrous cap of plaques. In addition, apoptotic macrophages can be a source of tissue factor, a procoagulant molecule considered to play an important role in coagulation and thrombosis associated with advanced plaques.
Signal transduction of apoptosis in macrophages is a complex network system. Various risk factors such as increased level of oxidized low-density lipoprotein (ox-LDL) can induce macrophage apoptosis through multiple apoptotic signaling pathways, such as Akt, NF-kB and MAPKs. However, the mechanism of macrophage apoptosis remains to be elucidated.
Tribbles (TRBs), a Drosophila protein, slows progression through the G2 stage of the cell cycle. Three mammalian orthologs, TRB1, TRB2 and TRB3, all contain a consensus serine/threonine kinase catalytic core but lack an ATP-binding pocket and so do not possess kinase activity. TRB3, also named neuronal cell death-inducible putative protein kinase (NIPK), is expressed in liver, thymus, prostate, pancreas and heart and may have wide biological activity. Recently, TRB3 has been shown to be a very important regulatory protein involved in signal pathways and works at least through CDC25/String, Akt and MAPKs. The activation of MAPKs and inhibition of Akt kinase activity result in macrophage apoptosis, which is implied in the development of vulnerable atherosclerotic plaques. TRB3 could be involved in macrophage apoptosis induced by ox-LDL and could play an essential role in the progression of vulnerable plaques.
Therefore, we hypothesized that TRB3 may be implicated in ox-LDL-induced apoptosis by stimulating human monocyte-derived macrophages with ox-LDL, through MAPKs, Akt and NF-kB signaling pathways.
Objective
1. To investigate the expression of TRBs in human monocyte-derived macrophages.
2. To observe the effect of ox-LDL on TRB3 in macrophages.
3. To explore whether TRB3 affects macrophage apoptosis induced by ox-LDL and to study the possible signal transduction mechanism.
Methods
We took primary human monocyte-derived macrophages as the object of this research, then quantitative real time PCR, western blot, ELISA, etc. were used to observe:
1. Monocytes were first allowed to differentiate into macrophages naturally. Quantitative real-time PCR performed on days 1,3,5,7,9 and 11 detected the mRNA expression of TRB1, TRB2, and TRB3 in monocyte-derived macrophages.
2. Macrophages were treated with various concentrations of ox-LDL and LDL (0,5,10,25 and 50μg/ml) or various times (0,4,8,12,24, and 48h), then quantitative real time PCR and western blot analysis were used to detect TRB3 mRNA and protein level.
3. Cloning of human TRB3 and constructing of TRB3 adenovirus vector
Based on human TRB3 sequence in Genebank, we designed one pair of primers. Using total DNA extracted from human whole blood and reverse transcript polymerase chain reaction, we cloned the CDS sequence of human TRB3. BD Adeno-XTM Expression System 2 was used to construct adenovirus containing human TRB3.293 cells were infected with the TRB3 plasmid. Recombinant adenovirus from a single plaque was expanded and purified, viral titer was determined by plaque assay.
4. Design, synthesis and screening of TRB3 targeting small interfering RNA (siRNA).
5. Macrophages were transfected with recombinant adeno viral TRB3 or TRB3 siRNA, and cell viability was examined by MTT assay, apoptosis by ELISA measurement of ssDNA absorbance, and caspase-3 level by western blot.
6. After overexpressing or silencing TRB3 expression, the change of Akt, MAPKs and NF-kB in macrophages were measured.
Results
1. TRBs mRNA expression in human macrophages
All three genes were expressed in monocyte-derived macrophages. Macrophages predominantly expressed TRB3; the TRB3 mRNA level was significantly increased on day 3 and peaked on day 7.
2. TRB3 expression upregulated by ox-LDL
Our data showed compared with LDL treatment, TRB3 mRNA and protein level in macrophages increased with increasing ox-LDL concentration and treatment time.
3. Cloning of human TRB3 and constructing TRB3 adenovirus
We have successfully cloned human TRB3, and its sequence was identical to the human TRB3 sequence in Genebank. Using BD Adeno-XTM Expression System 2, we constructed human TRB3 adenovirus.
4. TRB3 mediates macrophage apoptosis induced by ox-LDL
Compared with control cells, cell viability of TRB3-overexpressing macrophages was decreased (P<0.01), but apoptosis was increased (P<0.05) and caspase-3 level increased. With ox-LDL treatment, TRB3-overexpressing macrophages showed increased apoptosis, as deduced by decreased cell viability and increased apoptosis (P<0.01) and caspase-3 level compared with controls. Factorial analyses revealed no significant interaction between overexpression of TRB3 and stimulation with ox-LDL on macrophage apoptosis.
TRB3-silenced macrophages showed decreased apoptosis, as inferred by increased cell viability (P<0.01), and decreased apoptosis (P<0.05) and caspase-3 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly reduced cell viability (P<0.01), accompanied by increased apoptosis (P<0.01) and caspase-3 level compared with controls. Silenced TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis (P=0.048, P=0.001), which suggests that TRB3 knockdown in part resisted macrophage apoptosis induced by ox-LDL.
5. Effect of TRB3 on Akt in macrophages
Compared with control cells, the level of p-Akt of TRB3-overexpressing macrophages was decreased. With ox-LDL treatment, TRB3-overexpressing macrophages showed decreased p-Akt level compared with controls.
TRB3-silenced macrophages showed increased p-Akt level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-Akt level compared with controls.
6. Effect of TRB3 on MAPKs in macrophages
The activity of c-Fos and c-Jun in TRB3-overexpressing macrophages with ox-LDL treatment were lower than control cells(P<0.05, P<0.05). TRB3-silenced cells treated with ox-LDL showed significantly increased AP-1 activity. c-Fos activity increased 1.3 folds and c-Jun activity increased 1.3 folds compared to control cells.
TRB3-silenced macrophages showed increased p-p38 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-p38 level compared with controls.
7. Effect of TRB3 on NF-kB in macrophages
ox-LDL decreased the activity of NF-kB in macrophages in a time-dependent manner compared to LDL treatment. After treatment with ox-LDL for 24h, the activity of p50 and p65 was significantly lower than that following treatment with LDL for 24h and was further decreased after 48h ox-LDL treatmet. However, overexpression of TRB3 has no effect on the activity of p50 and p65 reduced by ox-LDL.
Conclusions
1. Human monocyte-derived macrophages expressed TRB1, TRB2 and especially TRB3.
2. TRB3 expression was upregulated in macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.
3. TRB3 in part mediates macrophage apoptosis induced by ox-LDL through Akt and MAPKs signaling pathway.
Background
The endoplasmic reticulum (ER) near the nucleus region in the cytoplasm is a central organelle of eukaryotic cells as the place of calcium storage, lipid synthesis, protein folding and protein maturation. A variety of factors such as hypoxia, hyperglycemia, chemical poisons and so on leading to depletion of endoplasmic reticulum Ca2+, inhibition of endoplasmic reticulum protein glycosylation, mismatched disulfide, accumulation of unfolded protein (unfolded protein response, UPR), excessive protein traffics and so on, can make the endoplasmic reticulum function change, collectively referred to endoplasmic reticulum stress (ER stress).Recently, ER stress has been shown to be involved in several diseases with ER stress-dependent cell death, such as diabetes and Alzheimer disease, especially ER stress plays an essential role in the progression of atherosclerosis.
CCAAT/enhancer binding proteins homologus protein (CHOP, also known as growth arrest and DNA damage-inducible protein 153), consisting of a transcriptional activation domain and a leucine zipper for DNA binging, is a member of the C/EBP family transcription factor family that can regulate the expression of a large number of target genes during cell stress response. The expression level of CHOP is low under normal conditions but induced by ER stress. CHOP is considered to be a main mediating factor in ER stress and acts as an inducer of cell cycle arrest and apoptosis. CHOP-mediated apoptosis during ER stress has been shown to be involved in several diseases with ER stress-dependent cell death. High expression of CHOP was observed at all stages of atherosclerotic lesion development in apolipoprotein E-deficicent mice, and both in early lesion and late advanced lesion CHOP expression increaseed ER stress promoted atherosclerosis progression via acculation of free cholesterol. However, whether lipoprotein ox-LDL mediates atherogenesis by CHOP-dependent ER stress pathway remains unclear.
Tribble3 (TRB3), a mammalian homolog of Drosophila Tribbles, is a recently recognized atypical inactibe kinase that inhibits mitosis and be implicated in cell cycle regulation and aopotosis. TRB3 is reported to participate in the signal pathway of ER stress, and induced by various forms ER stress, such as tunicamycin, cannabinoids and thapsigargin. Recent studies show that TRB3 acts as a target gene of CHOP during ER stress. CHOP with a novel dimerizing partner ATF4 activates TRB3 promoter[19]. We previously found that TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosis.
Taken together, we hypothesized that CHOP regulates ox-LDL-induced TRB3 expression and is involved in macrophages apoptosis through TRB3 pathway and
Objective
1. To investigate the mRNA expression of CHOP and ATF4 in human monocyte-derived macrophages.
2. To observe the effect of ER stress on TRB3 expression in macrophages.
3. To explore the interaction between TRB3 and CHOP.
Methods
1. Human monocyte-derived macrophages were treated with various concentrations of ox-LDL and LDL (0,2.5,5,10,25 and 50μg/ml))for various times (0,4,8,16,24, and 48h), then quantitative real time PCR and western blot analysis was used to detect CHOP and ATF4 mRNA and protein level.
2. Macrophages treated with 2μg/ml tunicamycin for 0,2,4,6,8,24h, the mRNA level of TRB3 was measured by quantitative real time PCR.
3. After overexpression CHOP by CHOP expression plasmid, macrophages were incubated with ox-LDL. The mRNA expression of TRB3 was measured.
4. Macrophages were transfected with adenoviral TRB3 or TRB3 siRNA, and the mRNA expression of CHOP was dectected.
Results
1. CHOP and ATF4 mRNA expression upregulated by ox-LDL
Our data showed that the expression of CHOP mRNA was significantly upregulated by ox-LDL but not LDL. This expression increased with increasing ox-LDL concentration. Furthermore, the expression of CHOP mRNA was significantly upregulated by ox-LDL at the concentration of 25 or 50μg/ml as compared to LDL treatment.
Macrophages treated with 50μg/ml ox-LDL or LDL for various times showed increased expression of CHOP mRNA with increasing time. After treatment with 50μg/ml ox-LDL for 24 hr, the expression of CHOP mRNA was significantly higher than that with LDL treatment (P<0.05) and was further increased with 48-hr ox-LDL treatment (P<0.01).
Also,50μg/ml ox-LDL increased the expression of ATF4 mRNA in a time-dependent manner. ATF4 mRNA expression peaked at 24 hr, but the peak level was less than CHOP (1.6 folds vs 3 folds). Ox-LDL induced TRB3 expression in a time-dependent manner, too. The induction of TRB3 mRNA was later than that of CHOP and ATF4 mRNA.
2. ER stress induces mRNA expression of TRB3, CHOP and ATF4
Macrophages treated with 2μg/ml tunicamycin for various times showed augmented TRB3 mRNA expression in a time-dependent manner. Compared with control cells, the significantly increase occurred at 6 hr and the peak level at 24 hr.
3. CHOP overexpression promotes TRB3 expression
Macrophages were transfected with CHOP expression plasmid and incubated for an additional 24 hr with or without ox-LDL. TRB3 mRNA expression of CHOP-overexpressing macrophages was also found significantly increased as compared with controls. Moreover, treatment of CHOP-overexpressing macrophages with ox-LDL markedly increased TRB3 expression as compared with controls.
4. Effect of TRB3 on CHOP expression
To better characterize the interaction between CHOP and TRB3, adenoviral TRB3 were transfected into macrophages to overexpress TRB3. CHOP mRNA expression of TRB3-overexpressing macrophages was significantly lower than that of control cells.
The TRB3-targeting siRNA (siTRB3) was transfected into macrophages to silence TRB3 gene expression. CHOP mRNA expression of TRB3-silenced macrophages was higher than that of control cells but not significantly, whereas with ox-LDL treatment, CHOP mRNA expression of treated cells was significantly higher.
Conclusion
1. CHOP and ATF4 mRNA expression was upregulated in human monocyte-derived macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.
2. ox-LDL can induce ER stress in human macrophage.
3. TRB3 mRNA expression was increased during ER stress.
4. CHOP promoted TRB3 expression and TRB3 exerted a feedback inhibition on the expression of CHOP in macrophages, so ATF4/CHOP-TRB3 may be involved in ox-LDL-dependent macrophage apoptosis.
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