血管紧张素Ⅱ诱导其前体基因表达及促进血管平滑肌细胞增殖的作用机制研究
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摘要
血管平滑肌细胞(vascular smooth muscle cells, VSMC)异常增殖是动脉粥样硬化斑块形成、高血压和血管成形术后再狭窄等血管增殖性疾病共同的细胞病理学基础。虽然多种细胞因子和血管活性物质能促进VSMC的增殖,但血管紧张素II(angiotensinII, AngII)在VSMC增殖及高血压、动脉粥样硬化、血管再狭窄等血管重塑性疾病的发生发展过程中处于关键地位。已经证明,在心血管系统中,VSMC不仅是AngII的一个重要来源,而且也是AngII调节血管功能的重要靶细胞。因此,研究AngII调节其前体基因表达及促进VSMC增殖的作用机制,对阐明血管增殖性疾病的分子机制具有重要意义。
为了阐明AngII调节血管紧张素原基因表达及促进VSMC增殖的分子机制,本研究观察AngII对转录激活蛋白-1(activator protein-1, AP-1)表达活化及其对血管紧张素原基因表达的影响;探讨AP-1与信号转导及转录激活因子5(signal transducer and activator of transcription 5, STAT5)在调节血管紧张素原基因表达中的相互关系;确定AngII诱导VSMC增殖的信号转导途径及roscovitine抗VSMC增殖的作用靶点。
1 AngII调节其前体基因在VSMC中表达的分子机制
AP-1是一种激活细胞增殖相关基因表达的转录因子,在AngII诱导其前体基因表达过程中,伴有AP-1(Fos-Jun二聚体)与血管紧张素原基因启动子结合活性的增强。为进一步探讨AngII促进AP-1与其顺式元件结合的分子机制。本部分实验用放线菌酮(cycloheximide, CHX)作为c-Jun的阻断剂,观察AngII对AP-1与血管紧张素原基因启动子相互作用及对血管紧张素原基因表达的影响。实验结果如下:
1.1不同剂量CHX对VSMC活力的影响
为了观察CHX对VSMC是否具有的毒性,用不同剂量的CHX处理VSMC后,进行MTT分析。结果显示,在15~45μmol/L浓度范围内,VSMC活力无明显变化。提示在该浓度范围内,CHX对VSMC不产生毒性作用。
1.2 AngII促进c-Jun蛋白表达与磷酸化
Western blot结果显示,AngII作用于VSMC 0.5 h后,细胞核内的c-Jun水平即显著升高并在此水平上保持至3 h。而且,免疫细胞化学染色证实,AngII诱导表达的c-Jun主要分布在细胞核内。用抗丝氨酸磷酸化抗体对核提取物进行免疫沉淀后检测磷酸化型c-Jun水平时发现,伴随着c-Jun表达增高,磷酸化型c-Jun水平也平行升高。用CHX预处理VSMC 0.5 h后,再用AngII刺激,c-Jun表达虽然不受影响,但其磷酸化水平明显降低。结果提示,AngII对其前体基因的正反馈调节是通过促进AP-1表达及诱导c-Jun磷酸化活化而实现的,CHX是一种AP-1磷酸化的抑制剂。
1.3 CHX抑制AngII诱导的血管紧张素原基因表达
用RT-PCR检测CHX抑制c-Jun磷酸化对血管紧张素原基因表达的影响。结果显示,用AngII处理VSMC 3 h,可显著提高血管紧张素原基因的表达活性。用CHX预处理VSMC可抑制AngII诱导的血管紧张素原基因表达。由此可见,CHX对c-Jun磷酸化的抑制可下调血管紧张素原基因的表达活性,提示AP-1的磷酸化活化是该基因表达所必需的。
1.4 AngII促进AP-1与血管紧张素原基因启动子结合
为寻找AngII诱导c-Jun磷酸化与其促进血管紧张素原基因表达之间的关系,用EMSA检测AngII对AP-1与其顺式元件结合活性的影响。结果显示,AngII处理VSMC 0.5 h后,核蛋白与探针的结合活性显著升高,至3 h达高峰。分别加入抗c-Jun和抗STAT5b抗体进行超迁移分析,均可出现抗体-抗原-探针形成的超迁移条带。在CHX预处理的细胞,其核蛋白与探针的结合活性明显下降。为了查明CHX抑制AP-1结合活性的机制,对同样条件下收集的核蛋白进行Western blot分析。结果表明,CHX对AP-1结合活性的抑制效应与AP-1蛋白水平无关,而是CHX抑制AP-1磷酸化的直接结果。上述结果提示,AngII诱导AP-1磷酸化是AngII对血管紧张素原基因正反馈调节的机制之一。
2 Roscovitine抑制血管平滑肌细胞增殖与c-Jun表达之间的关系
Roscovitine作为一种细胞周期蛋白依赖激酶(CDKs)的特异性抑制剂,具有诱导肿瘤细胞凋亡和抑制细胞增殖的作用。但roscovitine对VSMC增殖是否具有抑制作用,以及其对VSMC的抑制效应及作用机制与其他细胞是否相同目前尚不清楚。本部分比较roscovitine对VSMC和不同组织来源的细胞系增殖的影响及其作用机制。
2.1 Roscovitine对细胞增殖的影响
用细胞计数法检测细胞增殖活力。结果显示,被血清和AngII刺激后,VSMC细胞增殖速率显著加快,分别是对照组(无血清培养组)的2.63和1.68倍。不同浓度的roscovitine(15、30、45μmol/L)预处理VSMC 15 h,均可显著抑制AngII诱导的VSMC增殖。抑制率分别为37.3 %、46.7 %和51.8 %。Roscovitine也可显著抑制血清诱导的VSMC增殖。结果表明,roscovitine可显著抑制体外培养的VSMC增殖。
Roscovitine也显著抑制血清诱导的HeLa、COS-7、M17细胞的增殖。Roscovitine在30μmol/L时,对HeLa、COS-7、M17细胞的增殖抑制率分别为61.8 %、60.4 %和54.3 % (p<0.001),明显高于对VSMC的抑制作用。结果提示,不同细胞对roscovitine的敏感性有所不同。
2.2 Roscovitine对c-Jun蛋白表达的影响
免疫细胞化学染色结果显示,在AngII刺激的VSMC中,c-Jun表达明显增多。Roscovitine预处理VSMC可使c-Jun的表达明显下降。Western blot结果进一步证实,AngII和血清刺激VSMC可显著诱导c-Jun蛋白的表达,roscovitine预处理细胞15 h可显著抑制AngII诱导的c-Jun表达。然而,在HeLa、COS-7和M17细胞中,roscovitine预处理细胞对c-Jun蛋白表达无明显影响。在HeLa、COS-7、M17和VSMC中,roscovitine均不影响STAT5b的表达水平。结果提示,roscovitine抗VSMC增殖的作用与其抑制c-Jun表达有关;roscovitine抑制HeLa、COS-7和M17增殖与抑制c-Jun表达无关。说明roscovitine抗细胞增殖的机理在不同细胞是不同的。
2.3 Roscovitine在转录水平上抑制c-jun基因在VSMC中的表达
为进一步探讨roscovitine抑制c-Jun表达的作用环节,用roscovitine处理VSMC、HeLa与M17细胞后,用RT-PCR检测c-jun基因的转录活性。结果显示,roscovitine可下调VSMC中c-jun mRNA水平,但不影响HeLa和M17细胞对c-jun mRNA的表达,与Western blot结果一致。结果提示,roscovitine通过抑制c-jun基因转录而导致VSMC中c-Jun蛋白水平下降。
3 AngII促进VSMC增殖的信号转导途径及Roscovitine抑制VSMC增殖的作用靶点
AngII通过与VSMC上的相应受体相互作用而触发细胞增殖信号转导过程,最终引起细胞增殖相关基因表达及VSMC大量增殖。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联反应是转导细胞增殖信号进入细胞核的一条重要信号转导通路。本部分研究观察roscovitine对MAPK信号通路以及对血管紧张素原基因表达的影响,进一步阐明roscovitine抑制VSMC增殖与细胞增殖信号转导之间的关系。
3.1 Roscovitine对ERK1/2和c-Jun磷酸化的影响
AngII可诱导ERK1/2快速磷酸化,AngII刺激VSMC 5 min,ERK1/2的磷酸化水平达到高峰,30 min仍维持在较高水平上。给予roscovitine预处理VSMC 15 h后,再用AngII刺激细胞,则磷酸化型ERK1/2几乎检测不到,而各组细胞中ERK1/2的总含量没有变化。结果表明,roscovitine可以完全阻断AngII诱导的ERK1/2磷酸化。为观察roscovitine对AngII诱导c-Jun磷酸化的影响,细胞裂解液用抗丝氨酸磷酸化抗体进行免疫沉淀后,用抗c-Jun抗体检测磷酸化型c-Jun水平。结果显示,roscovitine显著降低c-Jun的磷酸化水平。上述结果表明,在VSMC中,roscovitine不仅抑制AngII诱导的c-Jun表达,而且抑制c-Jun的磷酸化。该抑制作用与其抑制AngII诱导的ERK1/2磷酸化有关。
3.2 Roscovitine抑制AP-1介导的血管紧张素原基因表达
本实验进一步探讨roscovitine对c-Jun/AP-1下游基因血管紧张素原基因表达的影响。RT-PCR结果显示,AngII刺激可显著上调血管紧张素原基因的表达。给予roscovitine预处理VSMC,可显著下调AngII刺激引起的血管紧张素原基因的表达水平。
为进一步证实c-Jun在体内的作用,用c-Jun抗体进行ChIP分析后,用PCR扩增血管紧张素原基因启动子中的AP-1结合序列。实验结果显示,AngII处理细胞可增强c-Jun与血管紧张素原基因启动子的结合活性,给予roscovitine预处理VSMC,可显著抑制c-Jun的结合活性。该结果进一步证实,roscovitine通过抑制c-Jun表达与磷酸化、以及抑制c-Jun与血管紧张素原基因启动子的结合活性,而起到抑制血管紧张素原基因表达的作用。
4 AP1与STAT5在调节血管紧张素原基因表达中的相互作用
AngII诱导其前体基因表达与AP-1和STAT5磷酸化活化有关,表明AP-1与STAT5均参与血管紧张素原基因的反式激活过程。本部分实验探讨这两种转录因子在血管紧张素原基因转录激活过程中的相互作用。
4.1 AngII诱导其前体基因表达与促进c-Jun与STAT5b相互作用有关
用抗STAT5b抗体对细胞裂解液进行免疫沉淀,抗c-Jun抗体进行Western blot分析。结果显示,AngII刺激前后,均可检测到STAT5b与c-Jun共沉淀,但AngII刺激后,STAT5b与c-Jun的相互作用明显增强。交互式免疫共沉淀进一步证实,STAT5b与c-Jun之间存在物理学上的相互作用,AngII能够诱导二者之间的相互缔合。
4.2 c-Jun和STAT5b以复合物的形式与血管紧张素原基因调控元件相互作用
EMSA结果显示,在AngII诱导下,VSMC核蛋白与血管紧张素原基因启动子区AP-1结合位点的结合活性明显增强,加入抗c-Jun或STAT5b抗体均可使核蛋白-DNA复合物发生超迁移。结果表明,核蛋白-DNA复合物中含有c-Jun和STAT5b。由此推测,STAT5b是以直接或间接方式与结合在AP-1结合位点上的AP-1发生相互作用。
ChIP分析结果显示,用抗STAT5b抗体沉淀富集的DNA-核蛋白复合物中的DNA片断为模板,可以扩增出含AP-1结合位点的血管紧张素原基因调控区片断;用抗c-Jun抗体沉淀富集的DNA-核蛋白复合物中的DNA片断为模板,可以扩增出含SATA5结合位点的血管紧张素原基因调控区片断。AngII刺激后,从抗STAT5b抗体沉淀的复合物中扩增出的含AP-1位点的片断明显增多;经AG490处理后,该基因片断的扩增产物显著减少。这些结果提示,在体内,c-Jun和STAT5b除分别与血管紧张素原基因启动子区相应的顺式元件结合外,同时c-Jun与STAT5b之间也存在相互作用。
4.3 STAT5b与c-Jun体外结合实验
GST pull-down结果显示,无论是核蛋白中的STAT5b还是细胞总蛋白中的STAT5b均不能被GST-c-Jun融合蛋白淘选出来。结果表明,c-Jun与STAT5b在体外无直接相互作用。
4.4 AP-1与STAT5协同激活血管紧张素原基因的表达
报告基因分析结果显示,c-Jun和STAT5b表达质粒共转染293A细胞时,报告基因的相对活力显著升高,表明AP-1与STAT5在促进血管紧张素原基因表达方面具有正协同作用。在HeLa细胞中,AP-1与STAT5可协同抑制报告基因的表达。提示在不同细胞中,血管紧张素原基因的表达需要不同的转录因子进行组合调控。
结论
1. AngII通过促进AP-1表达及磷酸化活化正反馈调节其前体基因表达。
2. Roscovitine通过特异性抑制c-Jun表达而发挥其对VSMC的抗增殖作用。
3. Roscovitine抑制VSMC增殖与抑制ERK1/2信号转导通路有关。
4. STAT5和c-Jun对血管紧张素原基因的转录激活具有正协同作用。
5. STAT5和AP-1是通过与相应顺式元件结合以及二者之间相互作用实现对血管紧张素原基因表达的组合调控。
The proliferation of vascular smooth muscle cells (VSMC) is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. It has been known that a number of the growth factors and vasoactive substances can induce the proliferation of VSMC, AngII play a central role in VSMC proliferation and a variety of pathological processes of vascular remodeling diseases including atherosclerosis, hypertension and restenosis after coronary angioplasty. Not only does VSMC as a source of AngII, but it also serves an important target tissue of AngII-regulating vascular function. Therefore, to investigate mechanisms of AngII-induced VSMC proliferation and regulating its precursor gene expression is more important to clarify the molecular mechanisms of proliferative vascular disease.
To elucidate the molecular mechanism of angiotensinogen gene expression and VSMC proliferation induced by AngII, we detected the effect of AngII on activator protein-1 (AP-1) expression and activation, and on angiotensinogen gene expression and VSMC proliferation. We also investigated the interaction between AP-1 and STAT5 in the transcriptional activation of angiotensinogen gene. Additionally, signal transduction pathways of AngII-induced VSMC proliferation and the potential targets of the inhibition of VSMC proliferation by roscovitine were studied.
1 Molecular mechanisms of angiotensinogen gene expression induced by AngII in VSMC
AP-1 was identified as a transcription factor involved in regulation of the expression of genes responsible for cell proliferation. It has been demonstrated that expression of angiotensinogen gene induced by AngII is related with the increase in the activity of AP-1 binding to angiotensinogen promoter in VSMC. To determine the molecular mechanism of AP-1 binding to its cis-element, we investigated the effect of AngII on expression of angiotensinogen gene and interaction between AP-1 and its cis-element by using cycloheximide (CHX) as an inhibitor of inhibiting c-Jun phosphorylation in this part. The results were as follows:
1.1 Effects of different concentrations of CHX on viability of the VSMC
To assess whether CHX has cytotoxic effect on VSMC, the cells were treated with different concentrations of CHX, and then the viability of cells was analyzed by MTT method. The results showed that there was no difference in viability of the VSMC at the range of 15~45μmol/L of CHX. The results suggested that CHX has no cytotoxic effect on VSMC at examined concentration ranges.
1.2 AngII induces c-Jun proteins expression and phosphorylation
Western blot results showed that the level of c-Jun in VSMC was significantly increased after the cells were treated with AngII for 0.5 h, and then kept to 3 h at this level. Immunocytochemistry analysis indicated that majority of c-Jun proteins were located in the nucleus. The phosphorylated c-Jun levels were assessed by immunoblotting with anti-c-Jun antibody after the nuclear extracts were immunoprecipitated by anti-phosphoserine antibody. The results confirmed that AngII could induce serine phosphorylation of c-Jun. VSMC was pretreated with CHX, and then stimulated by AngII, the levels of c-Jun protein were not altered, but the phosphorylated c-Jun levels were significantly decreased. These results indicated that the c-Jun expression and phosphorylation induced by AngII is one of the important mechanisms whereby AngII regulates its precursor gene expression in feedback manner. It is found that CHX is an inhibitor of AP-1 phosphorylation.
1.3 CHX inhibits angiotensinogen gene expression induced by AngII
The effect of CHX on angiotensinogen gene expression was detected by RT-PCR. The results showed that angiotensinogen gene expression activity was significantly increased after the cells were treated with AngII for 3 h. There was a reduction of angiotensinogen expression induced by AngII in VSMC pretreated with CHX. The results suggested that CHX downregulates the expression of angiotensinogen gene through inhibiting c-Jun phosphorylation. It indicated that the phosphorylation activation of AP-1 is necessary for angiotensinogen gene expression.
1.4 AngII promotes AP-1 binding to angiotensinogen gene promoter
To determine the relationship between the c-Jun phosphorylation and angiotensinogen gene expression induced by AngII, the effect of AngII on the binding activity of AP-1 to its cis-acting elements was detected by EMSA. The results showed that the binding activity of AP-1 to the probes was increased significantly after the cells were treated by AngII for 30 min, and reached to preak at 3 h. The supershift analysis using antibody to c-Jun or STAT5b showed that there was appearance of supershifted band. The binding activity of AP-1 to its cis-acting elements was declined significantly in VSMC pretreated with CHX. To explore the mechanism of the inhibition of AP-1 binding activity by CHX, the nuclear protein at same condition was detected by Western blot. The results showed that there was no relationship between c-Jun protein level and AP-1 binding activity. It was results of inhibition of AP-1 phosphorylation by CHX, which suggested that AngII-induced AP-1 phosphorylation was one of the mechanisms of AngII regulating angiotensinogen gene expression.
2 Roscovitine inhibits VSMC proliferation
Roscovitine is a potent and specific inhibitor of the CDKs that inhibits cell proliferation and induces apoptosis. However, it was not clear whether roscovitine has same antiproliferative effect in VSMC and other type cells. In this part, we compared the effect of roscovitine on VSMC proliferation with several cell lines from different tissues and the mechanism of inhibition of cell proliferation by roscovitine.
2.1 Effect of roscovitine on proliferation of different cells
The cell proliferation activity was determined by cell counting. After the cells were stimulated by 10 % FBS and angiotensin II (10-6 mol/L), the cell number was significantly increased by 2.63 and 1.68 times compared with control, respectively. VSMC was pretreated with different concentration of roscovitine (15, 30, 45μmol/L) for 15 h, the AngII-induced VSMC proliferation was inhibited by roscovitine. The inhibitive rate was 37.3 %, 46.7 % and 51.8 %, respectively. Roscovitine also inhibited FBS- induced VSMC proliferation. The results suggested that roscovitine significantly inhibited VSMC proliferation in vitro.
Roscovitine also inhibited HeLa, COS-7 and M17 proliferation induced by FBS. The inhibitive rate of roscovitine (30μmol/L) on HeLa, COS-7 and M17 proliferation was 61.8 %, 60.4 % and 54.8 % respectively, which was higher than that of VSMC. These data indicated that the different cell types exhibit variable sensitivity to roscovitine.
2.2 The effect of roscovitine on c-Jun protein expression
Immunocytochemistry staining results showed that stimulation with AngII markedly increased expression of c-Jun in VSMC. There was a reduction of AngII-stimulated c-Jun expression in VSMC pretreated with roscovitine. Western blot analysis also confirmed that the levels of c-Jun were significantly increased after the cells were stimulated by AngII or FBS, and the levels of c-Jun were decreased significantly by pretreatment with roscovitine for 15 h. However, in COS-7, HeLa, M17 cell lines, the c-Jun expression was not affected by roscovitine. The STAT5b expression was not affected by roscovitine in HeLa, COS-7, M17 and VSMC. These results suggested that inhibition of c-jun expression was involved in antiproliferative effect of roscovitine and was not related with the inhibition of HeLa, COS-7 and M17. It indicated that there were different mechanisms in roscovitine-inhibiting cell proliferation at different cells.
2.3 Roscovitine inhibits c-jun gene expression in VSMC at transcriptional level
The VSMC was pretreated with roscovitine, and then c-jun mRNA level was determined by RT-PCR. We demonstrated that roscovitine caused a down-regulation of c-Jun mRNA in VSMC, but it did not affect c-jun mRNA expression in HeLa and M17 cells. The results indicated that roscovitine down-regulates c-Jun protein expression by inhibiting c-jun gene transcription.
3 Signal transduction pathways of AngII-induced VSMC proliferation and the targets of the inhibition of VSMC proliferation by roscovitine
AngII has been shown to induce proliferation of VSMC and through AT1 receptor. Once AngII binds to the AT1R, it activates a series of signaling cascades and this led to the gene expression involved in cell proliferation. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to play a key role in transducting extracellular signals to nucleus. In this part, we observed the effect of roscovitine on MAPK signaling pathways and angiotensinogen gene expression.
3.1 Effect of roscovitine on AngII-induced ERK1/2 and c-Jun phosphorylation AngII can rapidly induce ERK1/2 phosphorylation. After stimulation with AngII for 5 min, the phosphorylation of ERK1/2 reached to peak and still kept high level at 30 min. Pretreating the cells with roscovitine for 15 h, the level of ERK1/2 phosphorylation did not detected. The total levels of ERK1/2 were not changed. It showed that roscovitine completely inhibited ERK1/2 phosphorylation induced by AngII. To examine the effect of roscovitine on c-Jun phosphorylation, the cell lysates were immunoprecipitated with anti-phospho-Ser mAb, and immunoblotted using anti-c-Jun antibody, the results showed that phosphorylated c-Jun levels decreased following treatment with roscovitine. The results indicated that roscovitine significantly inhibited the AngII-induced c-Jun expression and phosphorylation, which was associated with the inhibition of ERK1/2 phosphorylation.
3.2 Roscovitine inhibits AP-1-mediated angiotensinogen gene expression We further examined the effects of roscovitine on expression of
angiotensinogen, which was one of downstream gene of AP-1. RT-PCR results showed that roscovitine reduced AngII-induced angiotensinogen gene expression.
To confirm the effect of c-Jun in vivo, ChIP assay was performed. After chromatin was immunoprecipitated with anti-c-Jun antibody, DNA fragments containing AP-1 binding site were subjected to PCR. The ChIP assay demonstrated that treatment with AngII could enhance binding of c-Jun to the angiotensinogen promoter, an effect that could be suppressed by roscovitine, which further demonstrated that roscovitine can block AngII-mediated expression of angiotensinogen by suppressing c-Jun expression and phosphorylation, and then inhibiting the binding of c-Jun to the angiotensinogen gene promoter.
4 Interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene
AP-1 and STAT5 phosphorylation activation is involved in AngII-induced its precursor gene expression, which suggested that AP-1 and STAT5 participated in trans-activation of angiotensinogen gene. In the present study, we investigated the interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene.
4.1 AP-1 and STAT5 interaction is involved in AngII-induced its precursor gene expression
We carried out co-immunoprecipitation (co-IP) experiments using VSMC lysates treated with or without AngII. The results showed that IP with anti-STAT5b antibody followed by Western blot analysis with anti-c-Jun antibody clearly demonstrated the presence of c-Jun among the immunoprecipitated proteins, and the AngII treatment caused increase in obviously the interaction between c-Jun and STAT5. These results confirmed that the physical interaction between c-Jun and STAT5b occurs in vivo. AngII can induce interaction between c-Jun and STAT5b.
4.2 The complex formed by c-Jun and STAT5b interacts with cis-elements of angiotensinogen gene
EMSA results showed that AngII enhanced the binding activity of AP-1 to the promoter of angiotensinogen gene. Supershift analysis was performed with anti-c-Jun or anti-STAT5b antibody. The results indicated that the DNA-protein complex contained both c-Jun and STAT5b. These data indicated that STAT5b and c-Jun can interact directly or indirectly with each other at the AP-1 binding site.
ChIP assay results showed that DNA sequences contained AP-1 binding site could be amplified when DNA immunoprecipitated using anti-STAT5b antibody were subjected to PCR. DNA immunoprecipitated with anti-c-Jun antibody contained STAT5 binding site. The results demonstrated that there is presence of the interaction between STAT5 and c-Jun when they bind, respectively, to their cis-elements located in the angiotensinogen gene promoter.
4.3 GST pull-down analysis for interaction between STAT5 and c-Jun in vitro
GST pull-down results showed that STAT5b in whole cell lysates or nuclear extracts could not be pulled down by using GST-c-Jun fusion proteins. It indicated that c-Jun could not interact directly with STAT5b in vitro.
4.4 STAT5 synergizes with c-Jun in transactivation of the expression of angiotensinogen gene
The reporter gene assay results showed that the relative luciferase activity was significantly enhanced, when 293A cells were co-transfected with c-Jun and STAT5b, These results suggested that interaction between AP-1 and STAT5 mediates the synergistic enhancement of angiotensinogen gene expression. Co-transfected c-Jun and STAT5b into HeLa cells could synergistically inhibit angiotensinogen gene expression. These results suggested that in different cells there were distinct transcriptional factors which mediate the angiotensinogen gene expression.
Conclusion:
1. AngII positive-feedback regulates its precursor gene expression by inducing AP-1 phosphorylation activation.
2. Roscovitine exerts an antiproliferative effect on VSMC by specifically inhibiting c-Jun expression.
3. ERK1/2 signal transduction pathway is involved in inhibition of VSMC proliferation by roscovitine.
4. STAT5 and c-Jun have a synergistic effect in trans-activation of angiotensinogen gene.
为了阐明AngII调节血管紧张素原基因表达及促进VSMC增殖的分子机制,本研究观察AngII对转录激活蛋白-1(activator protein-1, AP-1)表达活化及其对血管紧张素原基因表达的影响;探讨AP-1与信号转导及转录激活因子5(signal transducer and activator of transcription 5, STAT5)在调节血管紧张素原基因表达中的相互关系;确定AngII诱导VSMC增殖的信号转导途径及roscovitine抗VSMC增殖的作用靶点。
1 AngII调节其前体基因在VSMC中表达的分子机制
AP-1是一种激活细胞增殖相关基因表达的转录因子,在AngII诱导其前体基因表达过程中,伴有AP-1(Fos-Jun二聚体)与血管紧张素原基因启动子结合活性的增强。为进一步探讨AngII促进AP-1与其顺式元件结合的分子机制。本部分实验用放线菌酮(cycloheximide, CHX)作为c-Jun的阻断剂,观察AngII对AP-1与血管紧张素原基因启动子相互作用及对血管紧张素原基因表达的影响。实验结果如下:
1.1不同剂量CHX对VSMC活力的影响
为了观察CHX对VSMC是否具有的毒性,用不同剂量的CHX处理VSMC后,进行MTT分析。结果显示,在15~45μmol/L浓度范围内,VSMC活力无明显变化。提示在该浓度范围内,CHX对VSMC不产生毒性作用。
1.2 AngII促进c-Jun蛋白表达与磷酸化
Western blot结果显示,AngII作用于VSMC 0.5 h后,细胞核内的c-Jun水平即显著升高并在此水平上保持至3 h。而且,免疫细胞化学染色证实,AngII诱导表达的c-Jun主要分布在细胞核内。用抗丝氨酸磷酸化抗体对核提取物进行免疫沉淀后检测磷酸化型c-Jun水平时发现,伴随着c-Jun表达增高,磷酸化型c-Jun水平也平行升高。用CHX预处理VSMC 0.5 h后,再用AngII刺激,c-Jun表达虽然不受影响,但其磷酸化水平明显降低。结果提示,AngII对其前体基因的正反馈调节是通过促进AP-1表达及诱导c-Jun磷酸化活化而实现的,CHX是一种AP-1磷酸化的抑制剂。
1.3 CHX抑制AngII诱导的血管紧张素原基因表达
用RT-PCR检测CHX抑制c-Jun磷酸化对血管紧张素原基因表达的影响。结果显示,用AngII处理VSMC 3 h,可显著提高血管紧张素原基因的表达活性。用CHX预处理VSMC可抑制AngII诱导的血管紧张素原基因表达。由此可见,CHX对c-Jun磷酸化的抑制可下调血管紧张素原基因的表达活性,提示AP-1的磷酸化活化是该基因表达所必需的。
1.4 AngII促进AP-1与血管紧张素原基因启动子结合
为寻找AngII诱导c-Jun磷酸化与其促进血管紧张素原基因表达之间的关系,用EMSA检测AngII对AP-1与其顺式元件结合活性的影响。结果显示,AngII处理VSMC 0.5 h后,核蛋白与探针的结合活性显著升高,至3 h达高峰。分别加入抗c-Jun和抗STAT5b抗体进行超迁移分析,均可出现抗体-抗原-探针形成的超迁移条带。在CHX预处理的细胞,其核蛋白与探针的结合活性明显下降。为了查明CHX抑制AP-1结合活性的机制,对同样条件下收集的核蛋白进行Western blot分析。结果表明,CHX对AP-1结合活性的抑制效应与AP-1蛋白水平无关,而是CHX抑制AP-1磷酸化的直接结果。上述结果提示,AngII诱导AP-1磷酸化是AngII对血管紧张素原基因正反馈调节的机制之一。
2 Roscovitine抑制血管平滑肌细胞增殖与c-Jun表达之间的关系
Roscovitine作为一种细胞周期蛋白依赖激酶(CDKs)的特异性抑制剂,具有诱导肿瘤细胞凋亡和抑制细胞增殖的作用。但roscovitine对VSMC增殖是否具有抑制作用,以及其对VSMC的抑制效应及作用机制与其他细胞是否相同目前尚不清楚。本部分比较roscovitine对VSMC和不同组织来源的细胞系增殖的影响及其作用机制。
2.1 Roscovitine对细胞增殖的影响
用细胞计数法检测细胞增殖活力。结果显示,被血清和AngII刺激后,VSMC细胞增殖速率显著加快,分别是对照组(无血清培养组)的2.63和1.68倍。不同浓度的roscovitine(15、30、45μmol/L)预处理VSMC 15 h,均可显著抑制AngII诱导的VSMC增殖。抑制率分别为37.3 %、46.7 %和51.8 %。Roscovitine也可显著抑制血清诱导的VSMC增殖。结果表明,roscovitine可显著抑制体外培养的VSMC增殖。
Roscovitine也显著抑制血清诱导的HeLa、COS-7、M17细胞的增殖。Roscovitine在30μmol/L时,对HeLa、COS-7、M17细胞的增殖抑制率分别为61.8 %、60.4 %和54.3 % (p<0.001),明显高于对VSMC的抑制作用。结果提示,不同细胞对roscovitine的敏感性有所不同。
2.2 Roscovitine对c-Jun蛋白表达的影响
免疫细胞化学染色结果显示,在AngII刺激的VSMC中,c-Jun表达明显增多。Roscovitine预处理VSMC可使c-Jun的表达明显下降。Western blot结果进一步证实,AngII和血清刺激VSMC可显著诱导c-Jun蛋白的表达,roscovitine预处理细胞15 h可显著抑制AngII诱导的c-Jun表达。然而,在HeLa、COS-7和M17细胞中,roscovitine预处理细胞对c-Jun蛋白表达无明显影响。在HeLa、COS-7、M17和VSMC中,roscovitine均不影响STAT5b的表达水平。结果提示,roscovitine抗VSMC增殖的作用与其抑制c-Jun表达有关;roscovitine抑制HeLa、COS-7和M17增殖与抑制c-Jun表达无关。说明roscovitine抗细胞增殖的机理在不同细胞是不同的。
2.3 Roscovitine在转录水平上抑制c-jun基因在VSMC中的表达
为进一步探讨roscovitine抑制c-Jun表达的作用环节,用roscovitine处理VSMC、HeLa与M17细胞后,用RT-PCR检测c-jun基因的转录活性。结果显示,roscovitine可下调VSMC中c-jun mRNA水平,但不影响HeLa和M17细胞对c-jun mRNA的表达,与Western blot结果一致。结果提示,roscovitine通过抑制c-jun基因转录而导致VSMC中c-Jun蛋白水平下降。
3 AngII促进VSMC增殖的信号转导途径及Roscovitine抑制VSMC增殖的作用靶点
AngII通过与VSMC上的相应受体相互作用而触发细胞增殖信号转导过程,最终引起细胞增殖相关基因表达及VSMC大量增殖。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联反应是转导细胞增殖信号进入细胞核的一条重要信号转导通路。本部分研究观察roscovitine对MAPK信号通路以及对血管紧张素原基因表达的影响,进一步阐明roscovitine抑制VSMC增殖与细胞增殖信号转导之间的关系。
3.1 Roscovitine对ERK1/2和c-Jun磷酸化的影响
AngII可诱导ERK1/2快速磷酸化,AngII刺激VSMC 5 min,ERK1/2的磷酸化水平达到高峰,30 min仍维持在较高水平上。给予roscovitine预处理VSMC 15 h后,再用AngII刺激细胞,则磷酸化型ERK1/2几乎检测不到,而各组细胞中ERK1/2的总含量没有变化。结果表明,roscovitine可以完全阻断AngII诱导的ERK1/2磷酸化。为观察roscovitine对AngII诱导c-Jun磷酸化的影响,细胞裂解液用抗丝氨酸磷酸化抗体进行免疫沉淀后,用抗c-Jun抗体检测磷酸化型c-Jun水平。结果显示,roscovitine显著降低c-Jun的磷酸化水平。上述结果表明,在VSMC中,roscovitine不仅抑制AngII诱导的c-Jun表达,而且抑制c-Jun的磷酸化。该抑制作用与其抑制AngII诱导的ERK1/2磷酸化有关。
3.2 Roscovitine抑制AP-1介导的血管紧张素原基因表达
本实验进一步探讨roscovitine对c-Jun/AP-1下游基因血管紧张素原基因表达的影响。RT-PCR结果显示,AngII刺激可显著上调血管紧张素原基因的表达。给予roscovitine预处理VSMC,可显著下调AngII刺激引起的血管紧张素原基因的表达水平。
为进一步证实c-Jun在体内的作用,用c-Jun抗体进行ChIP分析后,用PCR扩增血管紧张素原基因启动子中的AP-1结合序列。实验结果显示,AngII处理细胞可增强c-Jun与血管紧张素原基因启动子的结合活性,给予roscovitine预处理VSMC,可显著抑制c-Jun的结合活性。该结果进一步证实,roscovitine通过抑制c-Jun表达与磷酸化、以及抑制c-Jun与血管紧张素原基因启动子的结合活性,而起到抑制血管紧张素原基因表达的作用。
4 AP1与STAT5在调节血管紧张素原基因表达中的相互作用
AngII诱导其前体基因表达与AP-1和STAT5磷酸化活化有关,表明AP-1与STAT5均参与血管紧张素原基因的反式激活过程。本部分实验探讨这两种转录因子在血管紧张素原基因转录激活过程中的相互作用。
4.1 AngII诱导其前体基因表达与促进c-Jun与STAT5b相互作用有关
用抗STAT5b抗体对细胞裂解液进行免疫沉淀,抗c-Jun抗体进行Western blot分析。结果显示,AngII刺激前后,均可检测到STAT5b与c-Jun共沉淀,但AngII刺激后,STAT5b与c-Jun的相互作用明显增强。交互式免疫共沉淀进一步证实,STAT5b与c-Jun之间存在物理学上的相互作用,AngII能够诱导二者之间的相互缔合。
4.2 c-Jun和STAT5b以复合物的形式与血管紧张素原基因调控元件相互作用
EMSA结果显示,在AngII诱导下,VSMC核蛋白与血管紧张素原基因启动子区AP-1结合位点的结合活性明显增强,加入抗c-Jun或STAT5b抗体均可使核蛋白-DNA复合物发生超迁移。结果表明,核蛋白-DNA复合物中含有c-Jun和STAT5b。由此推测,STAT5b是以直接或间接方式与结合在AP-1结合位点上的AP-1发生相互作用。
ChIP分析结果显示,用抗STAT5b抗体沉淀富集的DNA-核蛋白复合物中的DNA片断为模板,可以扩增出含AP-1结合位点的血管紧张素原基因调控区片断;用抗c-Jun抗体沉淀富集的DNA-核蛋白复合物中的DNA片断为模板,可以扩增出含SATA5结合位点的血管紧张素原基因调控区片断。AngII刺激后,从抗STAT5b抗体沉淀的复合物中扩增出的含AP-1位点的片断明显增多;经AG490处理后,该基因片断的扩增产物显著减少。这些结果提示,在体内,c-Jun和STAT5b除分别与血管紧张素原基因启动子区相应的顺式元件结合外,同时c-Jun与STAT5b之间也存在相互作用。
4.3 STAT5b与c-Jun体外结合实验
GST pull-down结果显示,无论是核蛋白中的STAT5b还是细胞总蛋白中的STAT5b均不能被GST-c-Jun融合蛋白淘选出来。结果表明,c-Jun与STAT5b在体外无直接相互作用。
4.4 AP-1与STAT5协同激活血管紧张素原基因的表达
报告基因分析结果显示,c-Jun和STAT5b表达质粒共转染293A细胞时,报告基因的相对活力显著升高,表明AP-1与STAT5在促进血管紧张素原基因表达方面具有正协同作用。在HeLa细胞中,AP-1与STAT5可协同抑制报告基因的表达。提示在不同细胞中,血管紧张素原基因的表达需要不同的转录因子进行组合调控。
结论
1. AngII通过促进AP-1表达及磷酸化活化正反馈调节其前体基因表达。
2. Roscovitine通过特异性抑制c-Jun表达而发挥其对VSMC的抗增殖作用。
3. Roscovitine抑制VSMC增殖与抑制ERK1/2信号转导通路有关。
4. STAT5和c-Jun对血管紧张素原基因的转录激活具有正协同作用。
5. STAT5和AP-1是通过与相应顺式元件结合以及二者之间相互作用实现对血管紧张素原基因表达的组合调控。
The proliferation of vascular smooth muscle cells (VSMC) is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. It has been known that a number of the growth factors and vasoactive substances can induce the proliferation of VSMC, AngII play a central role in VSMC proliferation and a variety of pathological processes of vascular remodeling diseases including atherosclerosis, hypertension and restenosis after coronary angioplasty. Not only does VSMC as a source of AngII, but it also serves an important target tissue of AngII-regulating vascular function. Therefore, to investigate mechanisms of AngII-induced VSMC proliferation and regulating its precursor gene expression is more important to clarify the molecular mechanisms of proliferative vascular disease.
To elucidate the molecular mechanism of angiotensinogen gene expression and VSMC proliferation induced by AngII, we detected the effect of AngII on activator protein-1 (AP-1) expression and activation, and on angiotensinogen gene expression and VSMC proliferation. We also investigated the interaction between AP-1 and STAT5 in the transcriptional activation of angiotensinogen gene. Additionally, signal transduction pathways of AngII-induced VSMC proliferation and the potential targets of the inhibition of VSMC proliferation by roscovitine were studied.
1 Molecular mechanisms of angiotensinogen gene expression induced by AngII in VSMC
AP-1 was identified as a transcription factor involved in regulation of the expression of genes responsible for cell proliferation. It has been demonstrated that expression of angiotensinogen gene induced by AngII is related with the increase in the activity of AP-1 binding to angiotensinogen promoter in VSMC. To determine the molecular mechanism of AP-1 binding to its cis-element, we investigated the effect of AngII on expression of angiotensinogen gene and interaction between AP-1 and its cis-element by using cycloheximide (CHX) as an inhibitor of inhibiting c-Jun phosphorylation in this part. The results were as follows:
1.1 Effects of different concentrations of CHX on viability of the VSMC
To assess whether CHX has cytotoxic effect on VSMC, the cells were treated with different concentrations of CHX, and then the viability of cells was analyzed by MTT method. The results showed that there was no difference in viability of the VSMC at the range of 15~45μmol/L of CHX. The results suggested that CHX has no cytotoxic effect on VSMC at examined concentration ranges.
1.2 AngII induces c-Jun proteins expression and phosphorylation
Western blot results showed that the level of c-Jun in VSMC was significantly increased after the cells were treated with AngII for 0.5 h, and then kept to 3 h at this level. Immunocytochemistry analysis indicated that majority of c-Jun proteins were located in the nucleus. The phosphorylated c-Jun levels were assessed by immunoblotting with anti-c-Jun antibody after the nuclear extracts were immunoprecipitated by anti-phosphoserine antibody. The results confirmed that AngII could induce serine phosphorylation of c-Jun. VSMC was pretreated with CHX, and then stimulated by AngII, the levels of c-Jun protein were not altered, but the phosphorylated c-Jun levels were significantly decreased. These results indicated that the c-Jun expression and phosphorylation induced by AngII is one of the important mechanisms whereby AngII regulates its precursor gene expression in feedback manner. It is found that CHX is an inhibitor of AP-1 phosphorylation.
1.3 CHX inhibits angiotensinogen gene expression induced by AngII
The effect of CHX on angiotensinogen gene expression was detected by RT-PCR. The results showed that angiotensinogen gene expression activity was significantly increased after the cells were treated with AngII for 3 h. There was a reduction of angiotensinogen expression induced by AngII in VSMC pretreated with CHX. The results suggested that CHX downregulates the expression of angiotensinogen gene through inhibiting c-Jun phosphorylation. It indicated that the phosphorylation activation of AP-1 is necessary for angiotensinogen gene expression.
1.4 AngII promotes AP-1 binding to angiotensinogen gene promoter
To determine the relationship between the c-Jun phosphorylation and angiotensinogen gene expression induced by AngII, the effect of AngII on the binding activity of AP-1 to its cis-acting elements was detected by EMSA. The results showed that the binding activity of AP-1 to the probes was increased significantly after the cells were treated by AngII for 30 min, and reached to preak at 3 h. The supershift analysis using antibody to c-Jun or STAT5b showed that there was appearance of supershifted band. The binding activity of AP-1 to its cis-acting elements was declined significantly in VSMC pretreated with CHX. To explore the mechanism of the inhibition of AP-1 binding activity by CHX, the nuclear protein at same condition was detected by Western blot. The results showed that there was no relationship between c-Jun protein level and AP-1 binding activity. It was results of inhibition of AP-1 phosphorylation by CHX, which suggested that AngII-induced AP-1 phosphorylation was one of the mechanisms of AngII regulating angiotensinogen gene expression.
2 Roscovitine inhibits VSMC proliferation
Roscovitine is a potent and specific inhibitor of the CDKs that inhibits cell proliferation and induces apoptosis. However, it was not clear whether roscovitine has same antiproliferative effect in VSMC and other type cells. In this part, we compared the effect of roscovitine on VSMC proliferation with several cell lines from different tissues and the mechanism of inhibition of cell proliferation by roscovitine.
2.1 Effect of roscovitine on proliferation of different cells
The cell proliferation activity was determined by cell counting. After the cells were stimulated by 10 % FBS and angiotensin II (10-6 mol/L), the cell number was significantly increased by 2.63 and 1.68 times compared with control, respectively. VSMC was pretreated with different concentration of roscovitine (15, 30, 45μmol/L) for 15 h, the AngII-induced VSMC proliferation was inhibited by roscovitine. The inhibitive rate was 37.3 %, 46.7 % and 51.8 %, respectively. Roscovitine also inhibited FBS- induced VSMC proliferation. The results suggested that roscovitine significantly inhibited VSMC proliferation in vitro.
Roscovitine also inhibited HeLa, COS-7 and M17 proliferation induced by FBS. The inhibitive rate of roscovitine (30μmol/L) on HeLa, COS-7 and M17 proliferation was 61.8 %, 60.4 % and 54.8 % respectively, which was higher than that of VSMC. These data indicated that the different cell types exhibit variable sensitivity to roscovitine.
2.2 The effect of roscovitine on c-Jun protein expression
Immunocytochemistry staining results showed that stimulation with AngII markedly increased expression of c-Jun in VSMC. There was a reduction of AngII-stimulated c-Jun expression in VSMC pretreated with roscovitine. Western blot analysis also confirmed that the levels of c-Jun were significantly increased after the cells were stimulated by AngII or FBS, and the levels of c-Jun were decreased significantly by pretreatment with roscovitine for 15 h. However, in COS-7, HeLa, M17 cell lines, the c-Jun expression was not affected by roscovitine. The STAT5b expression was not affected by roscovitine in HeLa, COS-7, M17 and VSMC. These results suggested that inhibition of c-jun expression was involved in antiproliferative effect of roscovitine and was not related with the inhibition of HeLa, COS-7 and M17. It indicated that there were different mechanisms in roscovitine-inhibiting cell proliferation at different cells.
2.3 Roscovitine inhibits c-jun gene expression in VSMC at transcriptional level
The VSMC was pretreated with roscovitine, and then c-jun mRNA level was determined by RT-PCR. We demonstrated that roscovitine caused a down-regulation of c-Jun mRNA in VSMC, but it did not affect c-jun mRNA expression in HeLa and M17 cells. The results indicated that roscovitine down-regulates c-Jun protein expression by inhibiting c-jun gene transcription.
3 Signal transduction pathways of AngII-induced VSMC proliferation and the targets of the inhibition of VSMC proliferation by roscovitine
AngII has been shown to induce proliferation of VSMC and through AT1 receptor. Once AngII binds to the AT1R, it activates a series of signaling cascades and this led to the gene expression involved in cell proliferation. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to play a key role in transducting extracellular signals to nucleus. In this part, we observed the effect of roscovitine on MAPK signaling pathways and angiotensinogen gene expression.
3.1 Effect of roscovitine on AngII-induced ERK1/2 and c-Jun phosphorylation AngII can rapidly induce ERK1/2 phosphorylation. After stimulation with AngII for 5 min, the phosphorylation of ERK1/2 reached to peak and still kept high level at 30 min. Pretreating the cells with roscovitine for 15 h, the level of ERK1/2 phosphorylation did not detected. The total levels of ERK1/2 were not changed. It showed that roscovitine completely inhibited ERK1/2 phosphorylation induced by AngII. To examine the effect of roscovitine on c-Jun phosphorylation, the cell lysates were immunoprecipitated with anti-phospho-Ser mAb, and immunoblotted using anti-c-Jun antibody, the results showed that phosphorylated c-Jun levels decreased following treatment with roscovitine. The results indicated that roscovitine significantly inhibited the AngII-induced c-Jun expression and phosphorylation, which was associated with the inhibition of ERK1/2 phosphorylation.
3.2 Roscovitine inhibits AP-1-mediated angiotensinogen gene expression We further examined the effects of roscovitine on expression of
angiotensinogen, which was one of downstream gene of AP-1. RT-PCR results showed that roscovitine reduced AngII-induced angiotensinogen gene expression.
To confirm the effect of c-Jun in vivo, ChIP assay was performed. After chromatin was immunoprecipitated with anti-c-Jun antibody, DNA fragments containing AP-1 binding site were subjected to PCR. The ChIP assay demonstrated that treatment with AngII could enhance binding of c-Jun to the angiotensinogen promoter, an effect that could be suppressed by roscovitine, which further demonstrated that roscovitine can block AngII-mediated expression of angiotensinogen by suppressing c-Jun expression and phosphorylation, and then inhibiting the binding of c-Jun to the angiotensinogen gene promoter.
4 Interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene
AP-1 and STAT5 phosphorylation activation is involved in AngII-induced its precursor gene expression, which suggested that AP-1 and STAT5 participated in trans-activation of angiotensinogen gene. In the present study, we investigated the interaction between AP-1 and STAT5 in transcriptional activation of angiotensinogen gene.
4.1 AP-1 and STAT5 interaction is involved in AngII-induced its precursor gene expression
We carried out co-immunoprecipitation (co-IP) experiments using VSMC lysates treated with or without AngII. The results showed that IP with anti-STAT5b antibody followed by Western blot analysis with anti-c-Jun antibody clearly demonstrated the presence of c-Jun among the immunoprecipitated proteins, and the AngII treatment caused increase in obviously the interaction between c-Jun and STAT5. These results confirmed that the physical interaction between c-Jun and STAT5b occurs in vivo. AngII can induce interaction between c-Jun and STAT5b.
4.2 The complex formed by c-Jun and STAT5b interacts with cis-elements of angiotensinogen gene
EMSA results showed that AngII enhanced the binding activity of AP-1 to the promoter of angiotensinogen gene. Supershift analysis was performed with anti-c-Jun or anti-STAT5b antibody. The results indicated that the DNA-protein complex contained both c-Jun and STAT5b. These data indicated that STAT5b and c-Jun can interact directly or indirectly with each other at the AP-1 binding site.
ChIP assay results showed that DNA sequences contained AP-1 binding site could be amplified when DNA immunoprecipitated using anti-STAT5b antibody were subjected to PCR. DNA immunoprecipitated with anti-c-Jun antibody contained STAT5 binding site. The results demonstrated that there is presence of the interaction between STAT5 and c-Jun when they bind, respectively, to their cis-elements located in the angiotensinogen gene promoter.
4.3 GST pull-down analysis for interaction between STAT5 and c-Jun in vitro
GST pull-down results showed that STAT5b in whole cell lysates or nuclear extracts could not be pulled down by using GST-c-Jun fusion proteins. It indicated that c-Jun could not interact directly with STAT5b in vitro.
4.4 STAT5 synergizes with c-Jun in transactivation of the expression of angiotensinogen gene
The reporter gene assay results showed that the relative luciferase activity was significantly enhanced, when 293A cells were co-transfected with c-Jun and STAT5b, These results suggested that interaction between AP-1 and STAT5 mediates the synergistic enhancement of angiotensinogen gene expression. Co-transfected c-Jun and STAT5b into HeLa cells could synergistically inhibit angiotensinogen gene expression. These results suggested that in different cells there were distinct transcriptional factors which mediate the angiotensinogen gene expression.
Conclusion:
1. AngII positive-feedback regulates its precursor gene expression by inducing AP-1 phosphorylation activation.
2. Roscovitine exerts an antiproliferative effect on VSMC by specifically inhibiting c-Jun expression.
3. ERK1/2 signal transduction pathway is involved in inhibition of VSMC proliferation by roscovitine.
4. STAT5 and c-Jun have a synergistic effect in trans-activation of angiotensinogen gene.
引文
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