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Am80诱导KLF5脱乙酰化和抑制血管平滑肌细胞增殖的机制研究

Am80 Inhibits Vsmc Proliferation Through Upregulation of P21 Via Inhibiting KLF5 Activity

【作者】 舒亚南

【导师】 韩梅;

【作者基本信息】 河北医科大学 , 生物化学与分子生物学, 2010, 硕士

【摘要】 血管平滑肌细胞(vascular smooth muscle cells, VSMC)的增殖和分化受一系列调节因子构成的信号网络所调控。其中转录因子Krüppel样因子5(krüppel-like factor 5, KLF5)通过调节增殖/分化基因的表达活性而影响细胞的增殖活性。有研究显示,人工合成的维甲酸衍生物Am80通过激活其受体RARα而抑制KLF5与RARα相互作用和KLF5的活化,进而解除KLF5对p21基因表达的抑制。然而,Am80是通过何种机制抑制KLF5活性尚不清楚。乙酰化修饰在KLF5活性调节中具有重要意义,但对其在VSMC分化中的作用研究较少。本研究以组蛋白脱乙酰酶(histone deacetylases, HDACs)为切入点,以p21为靶基因,探讨KLF5乙酰化水平改变与其转录激活活性的关系,并揭示其作用机制。方法与结果:1 Am80抑制VSMC细胞周期的进程用流式细胞术检测细胞周期分布,结果显示,与对照组相比Am80刺激VSMC 24 h后,G0/G1期细胞所占比例上升了13 %,S期细胞比例下降了29 %。两组之间具有显著性差异。说明Am80可诱导细胞周期阻滞。2 Am80抑制VSMC增殖MTT分析结果显示,Am80刺激细胞12、24、48 h,VSMC增殖活性显著被抑制。与对照组相比,细胞增殖活性分别降低了44 %、35 %、26 %,该结果与细胞周期分析数据具有一致性。3 Am80诱导p21蛋白的表达为了探讨Am80诱导细胞阻滞的作用机制,我们选用不同浓度Am80刺激细胞24 h,提取总蛋白,观察细胞周期抑制蛋白p21表达的变化,Western blot结果显示,在Am80浓度为0-5μM范围内,随着Am80浓度增加,p21的表达水平呈剂量依赖性的升高,提示Am80可上调p21蛋白表达。4 Am80显著抑制球囊损伤诱导的血管内膜增生与假手术组相比,球囊内皮剥脱后14天的模型组颈总动脉中膜内的VSMC排列紊乱,内膜呈弥散性增厚,管腔变小,内膜与中膜的厚度比值(I/M)(2.10±0.26)明显高于假手术组(0.18±0.08);Am80组新生内膜的增生程度和I/M比值(0.3±0.11)明显小于模型组(P < 0.05,n=3)。结果表明,Am80可以明显抑制球囊损伤诱导的血管内膜增生。5 Am80通过抑制KLF5乙酰化上调p21表达为探讨Am80抑制p21表达的分子机制以及KLF5乙酰化水平变化是否在该过程中发挥作用,进行观察了Am80对KLF5乙酰化的影响。用不同浓度Am80(0、1、2、5和10μM)处理VSMC 2 h,提取细胞总蛋白,经抗KLF5抗体免疫沉淀后,用抗赖氨酸抗体进行Western印迹分析,以检测KLF5的乙酰化水平。结果显示,随着Am80浓度的增加,KLF5乙酰化水平逐渐降低,具有明显的剂量依赖关系。整体动物实验也证明,与模型组相比,给予Am80的大鼠血管组织中,KLF5乙酰化水平降低。在术后3 d和14 d时,Am80组血管乙酰化KLF5水平分别较模型组下降了17 %和34 %。上述结果在整体水平及细胞水平证实,Am80能抑制KLF5的乙酰化水平。6 Am80诱导KLF5与HDAC2相互作用HDAC2是调节蛋白质乙酰化修饰的关键酶。为确定HDAC2是否参与Am80诱导的KLF5脱乙酰化过程,本研究观察了KLF5与HDAC2的相互作用。免疫共沉淀分析结果显示,在Am80刺激前后的VSMC中,KLF5均可与HDAC2共沉淀;但是Am80刺激后,KLF5和HDAC2相互作用程度呈剂量依赖性增强,说明Am80能显著促进二者结合。免疫双荧光共定位分析结果进一步证实,在VSMC中KLF5与HDAC2共定位,叠加后的荧光强度随着Am80刺激而增强。给予RARα受体抑制剂Ro 41-5253预处理VSMC,可消除Am80诱导的KLF5与HDAC2相互作用。上述结果表明Am80诱导KLF5脱乙酰基与促进KLF5与HDAC2的相互作用有关。7乙酰化的KLF5促进p21启动子活性为了确定KLF5是否参与调节p21启动子活性以及与HDAC2相互作用对其转录激活作用的影响,分别将p21启动子指导的报告基因质粒(p21-luc)与KLF5和/或HDAC2表达质粒共转染293A细胞,通过双荧光素酶报告基因检测系统定量分析启动子的活性。结果显示,293A细胞共转染HDAC2和KLF5后,所测得荧光素酶的活性比单独转染KLF5升高了3倍。提示,KLF5与HDAC2对p21基因启动子的激活具有协同效应。为了确定该协同效应是否与KLF5的乙酰化有关,进而构建KLF5乙酰化位点突变(K369R)的表达质粒,并分别将其与乙酰化酶p300表达质粒和p21-luc共转染293A细胞,观察对报告基因活性的影响。结果显示,p300和KLF5表达质粒共转染细胞时,可协同抑制报告基因的表达活性。当KLF5突变体与P300表达质粒共转染时,对p21启动子的协同抑制作用消失。上述结果表明:KLF5的乙酰化抑制p21的表达;KLF5的去乙酰化促进p21的表达。结论:1 Am80阻滞VSMC细胞周期进程;2 Am80抑制VSMC增殖;3 Am80通过诱导HDAC2与KLF5的相互作用而促进KLF5的脱乙酰化;4 KLF5对p21基因的转录抑制有赖于其乙酰化修饰。

【Abstract】 Proliferation and differentiation of vascular smooth muscle cells (VSMC) are governed by a variety of regulatory factors, such as krüppel-like factor 5 (KLF5) that regulates gene expression. Recent study has shown that Am80, a synthetic RARα-specific agonist, removed the inhibitory effect of KLF5 p21 expression via inhibiting the interaction between KLF5 and RARα. Posttranslational modifications, such as acetylation, phosphorylation and ubiquitination, play a critical role in activation of KLF5. In the present studies, we investigated the effect of the interaction between KLF5 and HDAC2 induced by Am80 on p21 transcriptional activation, which may be contributes to the inhibition VSMC proliferation.Methods and results:1 Am80 blocks cell cycle progression in VSMCsThe effect of Am80 on cell cycle progression was detected by flow cytometry. The cell population of G0/G1 increased by 13 %, and S phase population displayed a 29 % decrease after treated by Am80, respectively, compared with control group. These results suggest that Am80 blocks progression of cell cycle through the G1 phase to the S phase in VSMCs.2 Am80 inhibits proliferation of VSMCsThe results of MTT assays showed that Am80 (12, 24, 48 h) treatment resulted in a significant reduction of VSMC proliferation in a time-dependent manner. The activity of VSMC proliferation decreased by 44 %, 35 %, and 26 %, respectively, compared with control group.3 Am80 induces p21 expression in VSMCsTo understand the mechanism of Am80 action, the effect of Am80 on the expression of p21 was detected by Western blot analysis. VSMCs were treated with Am80 at different doses (0-5μM) and used to examine the expression of p21. The results showed that the expression of p21 was up-regulated by Am80 in a dose-dependent manner.4 Am80 inhibits neointimal hyperplasia induced by balloon injuryThe rats injured with balloon were treatd by Am80, and then the intima to media area (I/M) ratio of carotid arteries was evaluated. The results showed that Am80 significantly reduced neointimal hyperplasia and I/M ratio (P < 0.05).5 KLF5 deacetylation induced by Am80 is associated with up-regulation p21 levelWestern blotting showed that Am80 treatment resulted in reduced acetylation of KLF5 in a dose-dependent manner in VSMCs. In vivo, the acetylated-KLF5 level in the Am80-administered group was lower than that in injured groups. These results suggest that Am80 induces the deacetylation of KLF5.6 The interaction of HDAC2 with KLF5 induced by Am80 is involved in the deacetylation of KLF5To examine whether HDAC2 mediates deacetylation of KLF5 induced by Am80, the interaction of HDAC2 with KLF5 was detected by Co-IP. The results revealed that the interaction between KLF5 and HDAC2 was significantly increased after Am80 treatment.7 KLF5 deacetylation activates the activity of p21 promoterp21 luciferase reportor analysis showed that a 3-fold increase in the p21 promoter activity was observed when the expression plasmids of KLF5 and HDAC2 were co-transfected. However, KLF5-K369R mutant that is acetylation-deficient plasmid was not able to enhance the promoter activity, suggesting that KLF5 deacetylation by HDAC2 is required for the activation of the p21 promoter.Conclusions:1 Am80 blocks the cell cycle progression in VSMCs;2 Am80 inhibits the proliferation of VSMCs; 3 Am80 induces the interaction between HDAC2 and KLF5, and subsequently leading to KLF5 deacetylation;4 The deacetylation of KLF5 is involved in activation of p21 transcription.

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