ATRA调节血管平滑肌细胞中KLF4表达的分子机制

发布时间：2018-05-14 15:21

本文选题：KLF4 + RARα　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:维生素A的衍生物全反式维甲酸(all-trans retinoic acid, ATRA)在多种细胞中具有调节细胞生长和诱导分化的作用。维甲酸受体(retinoic acid receptor, RAR)是一类重要的核受体,有RARα、RARβ和RARγ三种亚型。该类受体通过与其相应的配体ATRA相结合,激活或抑制多种基因的表达,从而在细胞增殖、分化及凋亡过程中发挥重要的作用。Krüppel样因子4(krüppel-like factor, GKLF/KLF4)是一类含有锌指结构的核转录因子,对血管平滑肌细胞(vascular smooth muscle cell, VSMC)具有抑制增殖、诱导分化的作用。大量的实验结果表明,在VSMC中,ATRA可以显著诱导KLF4基因的表达,但目前ATRA诱导KLF4表达的分子机制尚不清楚。本研究探讨ATRA激活KLF4基因表达的机制,研究RARα及相关的信号通路在ATRA诱导KLF4表达过程中的作用,以期明确ATRA诱导VSMC分化的分子基础。方法:用贴块法分离、培养大鼠VSMC,胰蛋白酶消化传代,取3～5代细胞进行实验。Western blot分析检测ATRA对KLF4和RARα表达的影响,应用不同信号通路抑制剂研究介导KLF4表达的信号转导途径。结果: 1 ATRA诱导KLF4和RARα表达以10μM ATRA刺激VSMC 0、6、12、24 h后,提取细胞总蛋白和RNA,分别进行Western blot分析和RT-PCR。结果显示,随着ATRA刺激时间的延长,KLF4的表达水平呈时间依赖性升高,同时RARα表达水平亦显著增高,提示RARα可能参与ATRA诱导KLF4的表达。 2 RARα介导ATRA诱导的KLF4表达应用RARα拮抗剂Ro 41-5253(20μM)预处理VSMC 1 h后,ATRA(10μM)刺激VSMC 24 h,分别提取细胞总蛋白和RNA,进行Western blot分析和RT-PCR。结果显示,应用RARα抑制剂预处理细胞后,ATRA上调KLF4表达的作用受到抑制。为了进一步验证RARα是否介导ATRA诱导的KLF4表达,将RARα的特异性小干扰RNA(RARα-siRNA)导入VSMC,阻断内源性RARα表达后,检测KLF4的表达变化。结果显示,转染RARα-siRNA的细胞与转染NS-siRNA的对照组相比,ATRA对KLF4表达的诱导作用被抑制,表明RARα介导ATRA对KLF4表达的诱导作用。 3 ATRA激活p38 MAPK、抑制ERK信号通路为了探讨ATRA通过哪些信号途径激活KLF4基因表达,应用ATRA(10μM),刺激VSMC 0、15、30、60 min后,分别检测ERK、p38 MAPK、Akt及β-catenin的磷酸化水平。结果发现,ATRA刺激15 min后,ERK的磷酸化水平降低,p38 MAPK的磷酸化水平显著升高,ATRA刺激不影响Akt和β-catenin的磷酸化水平。表明ATRA能够抑制ERK信号通路,激活p38 MAPK通路。 4 RARα通过ERK和p38 MAPK信号调节KLF4基因表达为了进一步探讨RARα在ATRA诱导KLF4基因表达中的作用,应用RARα拮抗剂Ro 41-5253预处理VSMC 1 h后, ATRA刺激VSMC 1 h。Western blot分析发现,ERK磷酸化水平与对照组相比明显升高,p38磷酸化水平显著降低。此外,应用RARα-siRNA转染VSMC,敲低内源性RARα表达后,ERK磷酸化水平与对照组相比升高,p38 MAPK磷酸化水平显著降低,表明RARα通过ERK和p38 MARK信号调节KLF4基因表达。 5 p38 MAPK信号介导ATRA诱导的KLF4表达为了进一步证明ATRA诱导KLF4表达与p38 MAPK和ERK信号通路的相关性,分别用p38 MAPK特异性抑制剂SB203580(20μM),ERK抑制剂PD98059(20μM)及Akt抑制剂LY294002(20μM)预孵育VSMC 2 h后,给予ATRA刺激24 h,收集细胞进行Western blot分析。结果显示,抑制p38 MAPK信号途径可使ATRA诱导KLF4表达的作用受到抑制,抑制ERK信号途径上调KLF4表达,抑制Akt信号途径不影响ATRA对KLF4表达的诱导作用。为了进一步验证ERK信号途径在ATRA诱导KLF4表达中的作用,应用ERK的持续活化型质粒pCMV-MEKca转染VSMC,ATRA刺激细胞24 h后,收集细胞检测KLF4的表达。Western blot结果显示,转染pCMV-MEKca的细胞与转染对照质粒(pCMV)的细胞相比,ATRA诱导KLF4表达的作用受到抑制。以上结果表明,p38 MAPK和ERK信号通路在ATRA调节KLF4基因表达中发挥重要的作用。结论: 1在VSMC中,ATRA诱导KLF4和RARα表达。 2 RARα介导ATRA对KLF4基因表达的诱导作用。 3 RARα通过ERK和p38 MAPK信号途径调节ATRA诱导的KLF4基因表达。
[Abstract]:Objective : All trans retinoic acid ( ATRA ) , a derivative of vitamin A , plays an important role in regulating cell growth and inducing differentiation in a variety of cells . The retinoic acid receptor ( RARs ) plays an important role in the process of cell proliferation , differentiation and apoptosis . The Kr眉ppel - like factor ( GKLF / KLF4 ) is a kind of nuclear transcription factor containing zinc finger structure .

A large number of experimental results show that ATRA can induce KLF4 gene expression significantly , but the molecular mechanism of ATRA - induced KLF4 expression is not clear . This study explores the mechanism of ATRA activation of KLF4 gene expression , and studies the role of RAR伪and related signal pathway in ATRA - induced KLF4 expression , with a view to clarifying the molecular basis of ATRA - induced differentiation of KLF4 .

Methods : The effects of ATRA on the expression of KLF4 and RAR伪were detected by Western blot , and the signal transduction pathway of KLF4 expression was studied by Western blot .

Results :

1 . ATRA - induced KLF4 and RAR伪expression

The total protein and RNA were extracted by Western blot and RT - PCR respectively . The results showed that the expression level of KLF4 increased in time - dependent manner with the prolongation of ATRA stimulation time , and the level of RAR伪was also significantly increased , suggesting that RAR伪might participate in ATRA - induced expression of KLF4 .

KLF4 expression induced by RAR伪mediated ATRA

After pretreatment with RAR伪 inhibitor Ro 41 - 5253 ( 20 渭M ) , the effects of ATRA ( 10 渭M ) on the expression of KLF4 by ATRA ( 10 渭M ) were inhibited .

3 ATRA activates p38 MAPK and inhibits ERK signaling pathway

The phosphorylation level of ERK , p38 MAPK , and 尾 - catenin was detected by ATRA ( 10 渭M ) . The phosphorylation level of ERK , p38 MAPK , and 尾 - catenin was significantly increased after 15 min of ATRA stimulation . The phosphorylation level of p38 MAPK was significantly increased , and ATRA stimulation did not affect the phosphorylation of ERK and 尾 - catenin , suggesting that ATRA could inhibit ERK signaling pathway and activate p38 MAPK pathway .

4 RAR伪regulates KLF4 gene expression via ERK and p38 MAPK signaling

In order to further study the role of RAR伪in ATRA - induced KLF4 gene expression , it was found that the phosphorylation of ERK increased significantly compared with the control group after 1 h treatment with RAR伪 antagonist Ro 41 - 5253 . The phosphorylation level of ERK increased significantly compared with the control group , and the phosphorylation level of ERK increased significantly , and the phosphorylation level of p38 MAPK was significantly decreased , indicating that RAR伪regulated KLF4 gene expression via ERK and p38 MARK .

5 - p38 MAPK signal - mediated induction of ATRA - induced KLF4 expression

In order to further demonstrate that ATRA - induced KLF4 expression was associated with p38 MAPK and ERK signaling pathways , the effect of ATRA on KLF4 expression was inhibited by the inhibition of p38 MAPK specific inhibitor SB203580 ( 20 渭M ) , ERK inhibitor PD98059 ( 20 渭M ) , and the expression of KLF4 by ATRA . The results showed that inhibition of ERK signaling pathway inhibited the expression of KLF4 by ATRA . The results showed that inhibition of ERK signaling pathway inhibited the expression of KLF4 . The results showed that p38 MAPK and ERK signaling pathway played an important role in the regulation of KLF4 gene expression in ATRA .

Conclusion :

1 . ATRA induced KLF4 and RAR伪expression .

2 RAR伪mediated the induction of ATRA on KLF4 gene expression .

3 RAR伪regulates the expression of KLF4 gene induced by ATRA via ERK and p38 MAPK signaling pathway .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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