【作者】 张睿；

【导师】 温进坤； 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， 博士

【摘要】 已经证明,血管活性物质及其受体、细胞因子及其受体和细胞信号转导分子所构成的复杂网络调节血管平滑肌细胞(vascular smooth muscle cell, VSMC)的功能。近年发现,全反式维甲酸(all-trans retinoic acid, ATRA)及其受体以直接或间接的方式参与VSMC生物学行为的调节。Krüppel-like factor 4 (KLF4)是一类具有锌指结构的转录因子,广泛参与多种细胞的生长、增殖、分化及凋亡的调节。在VSMC中,KLF4的功能取决于与之相互作用的辅助因子。我室近期证实,ATRA可诱导VSMC表达KLF4和线粒体融合蛋白2(mitofusin-2, mfn-2),然而,ATRA诱导的KLF4和mfn-2表达之间是否存在内在联系,目前尚不清楚。Mfn-2(亦称为增殖抑制基因,HSG)是一种线粒体融合蛋白,主要在高能量需求的组织,如骨骼肌和心肌中表达。多种证据表明,mfn-2通过改变线粒体膜电位、脂肪氧化以及氧化磷酸化系统来调节线粒体代谢。另外,最近的研究证实,mfn-2具有很强的抑制VSMC增殖和促进VSMC凋亡的作用。有研究表明,mfn-2抑制肿瘤细胞增殖的活性和增强肿瘤放化疗敏感性的效应甚至强于p53。因此,阐明调节mfn-2表达的分子机制有助于更好地理解其生物学功能。本研究利用现代分子生物学和细胞生物学实验技术,研究KLF4在ATRA诱导mfn-2表达中的作用及作用机制,发现在VSMC中,KLF4通过直接与mfn-2启动子结合激活mfn-2表达,ATRA通过激活JNK和p38 MAPK信号通路使KLF4磷酸化,磷酸化型KLF4与p300结合并被其乙酰化。KLF4乙酰化后与mfn-2启动子的结合活性增强。另外,在ATRA处理的VSMC中,RARα与KLF4共同激活mfn-2启动子。1. KLF4介导ATRA诱导的mfn-2表达ATRA可同时上调KLF4和mfn-2表达,但KLF4在ATRA诱导mfn-2表达中的作用尚不清楚。本部分旨在探讨KLF4是否介导ATRA诱导的mfn-2表达。实验结果如下:1.1 KLF4介导ATRA对mfn-2表达的诱导作用RT-PCR和Western blot结果显示,ATRA可显著诱导KLF4和mfn-2表达,并具有明显的时效关系。其中,ATRA浓度为10μM和作用时间为24 h时两者的表达变化最明显。为了阐明ATRA诱导mfn-2表达是否依赖于KLF4,以KLF4腺病毒表达载体Ad-KLF4感染VSMC或将靶向KLF4的小干扰RNA(KLF4-siRNA)导入VSMC,观察其对mfn-2表达的影响。Western blot和RT-PCR结果显示,在过表达KLF4的VSMC中,mfn-2 mRNA和蛋白水平均以时间依赖的方式增加。相反,将KLF4 siRNA导入VSMC,阻断ATRA诱导的内源性KLF4表达后,ATRA对mfn-2表达的诱导作用受到抑制。这些结果表明,KLF4介导ATRA对mfn-2表达的诱导作用。1.2 KLF4直接结合到mfn-2启动子上并调节其转录为了明确KLF4是否直接激活mfn-2启动子,用mfn-2报告基因(pGL3-mfn-2-luc)和KLF4表达质粒共转染A293细胞,荧光素酶活性分析结果显示,过表达KLF4以剂量依赖的方式增加mfn-2启动子活性。用TESS软件对mfn-2启动子区核苷酸序列进行分析,发现在mfn-2启动子区存在4个典型的KLF4结合位点(CACCC/GTGGG)。为了明确哪个位点与KLF4相互作用,我们构建5’端进行性缺失的mfn-2启动子报告质粒,与KLF4表达质粒共转染A293细胞。报告基因分析结果显示,KLF4对mfn-2启动子的激活依赖于-441到-273之间的核苷酸序列。进一步将位于该区域的4个KLF4结合位点进行定点突变后,检查KLF4对启动子的影响。结果显示,第3个KLF4结合位点突变后,mfn-2启动子完全失去对KLF4的应答,表明KLF4结合位点3在KLF4介导的mfn-2转录激活中起关键作用。ChIP结果也证明,在KLF4过表达的VSMC中,KLF4与mfn-2启动子的结合活性增强。1.3 KLF4介导ATRA诱导的线粒体成簇和线粒体去极化为了分析mfn-2表达上调对VSMC线粒体动力学的影响,分别用荧光染色和电镜观察ATRA处理和KLF4过表达所引起的线粒体形态变化。结果显示,在KLF4过表达和被ATRA处理的VSMC中都出现线粒体聚集现象,将KLF4 siRNA导入VSMC,阻断ATRA诱导的内源性KLF4表达后,ATRA诱导的线粒体成簇明显减少。我们也用JC-1染色和流式细胞术检测线粒体膜电位的变化,结果显示,KLF4过表达和ATRA处理均使线粒体膜电位下降,且这种效应依赖于mfn-2的表达。以上结果表明,mfn-2是受KLF4调控的靶基因,在VSMC中,KLF4介导ATRA诱导的mfn-2表达,提示KLF4在ATRA信号通路中发挥重要作用。2. KLF4对mfn-2的转录激活与ATRA诱导KLF4与p300相互作用及KLF4乙酰化相关联KLF4的转录活性受与之相互作用的辅助因子以及翻译后修饰如磷酸化、乙酰化等多种方式的影响。本部分实验探讨ATRA对KLF4磷酸化和乙酰化修饰的影响,并研究这些修饰在KLF4调节mfn-2表达中的意义。2.1 ATRA诱导KLF4乙酰化为了阐明ATRA促进KLF4激活mfn-2转录的分子机制,本部分实验首先检测ATRA对KLF4乙酰化的影响。实验结果表明,随着VSMC被ATRA刺激时间的延长,KLF4乙酰化水平逐渐升高。细胞免疫荧光染色可见,在KLF4乙酰化过程中伴随着转乙酰基酶p300的入核,在ATRA刺激VSMC 1 h,p300发生明显的核转位,提示ATRA诱导KLF4乙酰化与促进KLF4与p300相互作用有关。免疫共沉淀结果显示,ATRA促进KLF4与p300的结合。报告基因分析KLF4乙酰化对mfn-2转录激活的影响时发现,与单独过表达KLF4相比,共表达KLF4和p300显著增强KLF4对mfn-2的转录激活作用。这些结果表明,KLF4对mfn-2的转录激活与ATRA诱导KLF4与p300相互作用及KLF4乙酰化有关。2.2 ATRA通过激活JNK和p38信号通路诱导KLF4磷酸化以及KLF4与p300的相互作用为了明确KLF4与p300相互作用是否依赖于KLF4磷酸化,我们检测ATRA对KLF4磷酸化的影响。实验结果表明,ATRA刺激VSMC 0.5 h,KLF4的磷酸化水平开始升高,到1 h达到高峰,并维持在此水平至2 h。在相同的实验条件下,JNK和p38 MAPK的磷酸化水平也显著升高,但ATRA刺激不影响Akt和ERK的磷酸化水平。分别以ERK特异性抑制剂PD98059、Akt特异性抑制剂LY294002、p38 MAPK特异性抑制剂SB203580和JNK特异性抑制剂SP600125处理VSMC,阻断ERK、Akt、p38 MAPK或JNK信号通路的活化后,再以10μM ATRA刺激细胞1 h。Western blot结果显示,SP600125和SB203580预处理可显著抑制ATRA诱导的KLF4磷酸化,而PD98059和LY294002对KLF4磷酸化无明显影响。VSMC被KLF4磷酸化位点突变体转染后,再以10μM ATRA刺激细胞1 h,与对照组(转染野生型KLF4表达载体)相比,KLF4磷酸化水平明显下降,同时,KLF4与p300的结合明显减少。为了阐明KLF4乙酰化是否依赖于它的磷酸化及其与p300的相互作用,用SB203580和SP600125处理VSMC或者用KLF4磷酸化位点突变体转染VSMC后检测KLF4的磷酸化水平。结果显示,这些抑制剂或KLF4磷酸化位点突变体在抑制KLF4磷酸化的同时,也使KLF4乙酰化水平显著降低。报告基因分析结果显示,将KLF4磷酸化位点突变体和p300共转染A293后,KLF4激活mfn-2启动子的作用消失。另外,用SP600125和SB203580处理A293细胞或者用KLF4磷酸化位点突变体转染A293细胞,在KLF4磷酸化受到抑制后,ATRA丧失对mfn-2启动子的激活作用。以上结果表明,ATRA通过诱导KLF4磷酸化促进其与p300相互作用,以及KLF4乙酰化,乙酰化型KLF4对mfn-2的转录激活作用增强。3. RARα通过与KLF4相互作用促进KLF4对mfn-2启动子的激活ATRA既可通过其受体介导的信号转导途径调节转录因子的活性,也可通过调节受体与相关转录因子的相互作用而调节转录因子的转录激活作用。第二部分实验已经证实,KLF4通过JNK和p38 MAPK信号途径介导ATRA对mfn-2表达的诱导作用,本部分实验进一步探讨维甲酸受体(RAR)在KLF4激活mfn-2表达中的作用。3.1 ATRA诱导RARα和RARβ的表达ATRA能否诱导VSMC表达RAR目前尚不清楚。为了阐明RAR在KLF4介导mfn-2基因表达中的作用,首先检查ATRA对RAR表达的影响。Western blot和RT-PCR结果显示,ATRA以时间和剂量依赖的方式显著诱导RARα表达,轻微上调RARβ表达,对RARγ的表达没有影响。3.2 RARα通过与KLF4结合参与ATRA诱导KLF4乙酰化的过程因为在mfn-2启动子区不存在RAR结合元件,因此,我们推测,RAR可能通过与KLF4相互作用或通过影响KLF4修饰间接调节mfn-2的表达。我们首先用RARα拮抗剂(Ro 41-5253)和RARβ拮抗剂(LE135)处理VSMC或将靶向RAR的小干扰RNA(siRNA)导入VSMC,抑制内源性RAR表达后,进一步研究RAR对KLF4修饰的影响。Western blot结果显示, RARα和RARβ拮抗剂不影响ATRA诱导的KLF4磷酸化。用RARα-siRNA敲低RARα表达显著抑制KLF4乙酰化,敲低RARβ和RARγ对KLF4乙酰化没有影响。为了阐明RAR促进KLF4乙酰化的机制,我们检测KLF4与RAR的相互作用。免疫共沉淀分析结果证实,ATRA仅促进KLF4与RARα的结合。3.3 RARα促进KLF4对mfn-2启动子的激活报告基因分析结果表明,与单独过表达KLF4相比,共表达RARα和KLF4显著增强KLF4对mfn-2启动子的激活作用,共转染RARα和KLF4表达载体后,再以ATRA刺激细胞可使mfn-2启动子活性进一步升高。然而,在单独过表达RARα条件下,无论ATRA刺激与否,均不能增强mfn-2启动子活性。3.4 RARα与KLF4相互作用依赖于KLF4磷酸化上述实验证实,KLF4与p300相互作用依赖于KLF4磷酸化。本部分实验进一步检查KLF4磷酸化是否影响其与RARα结合。免疫共沉淀分析结果证实,用SP600125和SB203580处理VSMC或者用KLF4磷酸化位点突变体转染VSMC后,ATRA诱导的RARα与KLF4的相互作用显著降低。结果表明,RARα与KLF4相互作用依赖于KLF4的磷酸化。3.5敲低RARα表达抑制KLF4与p300结合为了进一步明确RARα促进KLF4乙酰化的分子机制,将靶向RARα的小干扰RNA导入VSMC,阻断ATRA诱导的内源性RARα表达后,检查RARα对KLF4与p300相互作用的影响。免疫共沉淀分析结果显示,敲低RARα表达显著抑制KLF4与p300的结合。以上结果显示ATRA通过诱导KLF4磷酸化而促进KLF4与RARα的结合,二者的结合有利于KLF4与p300的相互作用及KLF4被p300乙酰化,乙酰化型KLF4对mfn-2启动子的激活作用显著增强。结论1. KLF4介导ATRA诱导的mfn-2表达是通过与mfn-2启动子区的KLF4结合位点相互作用而实现的。同时,KLF4介导ATRA对核周线粒体成簇及线粒体去极化的诱导作用。2. ATRA通过激活JNK和p38 MAPK信号途径而诱导KLF4磷酸化,磷酸化型KLF4与p300相互作用增强并被p300乙酰化。KLF4乙酰化是ATRA激活mfn-2启动子的重要条件。3. ATRA通过诱导KLF4磷酸化而促进KLF4与RARα的结合,二者的结合有利于KLF4与p300相互作用,进而促进KLF4乙酰化及其对mfn-2启动子的激活作用。4.Mfn-2是受KLF4调控的靶基因,KLF4是介导ATRA信号的重要转录调控因子。更多还原

【Abstract】 Proliferative vascular diseases are multiple gene disease caused by interaction of environmental and genetic factors. Vasoactive peptides and their receptors, growth factors and cytokines as well as their receptors, cell signal transduction proteins and cyclins are all involved in the occurrence of these diseases. All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to regulate growth induce differentiation in multiple cells in direct or indirect manner.The Krüppel-like factor 4 (KLF4/GKLF) is a pleiotropic zinc finger transcription factor that regulates genes being involved in both the regulation of cell growth, proliferation, differentiation and apoptosis in several tissues. In vascular smooth muscle cells (VSMCs), KLF4 can function as an anti-proliferation factor or a prodifferentiation factor depending on the interaction partner. We have recently demonstrated that ATRA induced the expression of KLF4 and mitofusin 2 (mfn-2), a member of the mitofusin family, in VSMCs. However, the role of KLF4 in the regulation of mfn-2 expression has not been well characterized.Mfn-2 (also named hyperplasia suppressor gene [HSG]) is a mitochondrial fusion protein that is expressed mainly in tissues with high energetic requirements, such as skeletal muscle and heart. Several lines of evidence indicate that mfn-2 modulates mitochondrial metabolism by regulating mitochondrial membrane potential, fuel oxidation, and the oxidative phosphorylation system. In addition, recent study has shown that mfn-2 exhibits a profound anti-proliferative effect on VSMCs in vitro and in vivo, and promotes VSMC and cardiomyocyte apoptosis. Recent evidence also shows that mfn-2 inhibits tumor cell proliferation and increases their sensitivity to chemotherapy and radiotherapy even above p53. Thus, it is important to identify the molecular mechanisms underlying the activation or repression of mfn-2 expression in order to have a better understanding of its biological functions. Based on the experimental facts that mfn-2 and KLF4 showed a similar expression pattern and that KLF4 is a pleiotropic transcription factor that participates in a wide range of biological functions, we speculated that KLF4 might be a key effector of ATRA-induced mfn-2 expression in VSMCs.Here, we studied the mechanism by which KLF4 mediates ATRA-induced mfn-2 expression in VSMCs. We show that KLF4 binds directly to the mfn-2 promoter and activates its transcription. Further, we demonstrate that ATRA increases the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, and that the activation of JNK and p38 MAPK signaling by ATRA is essential for KLF4 phosphorylation and its interaction with p300. KLF4 acetylation by p300 increases its activity to transactivate the mfn-2 promoter. In further study, we also demonstrate that RARαis involved in KLF4-mediated transactivation of mfn-2 promoter by binding to KLF4 in ATRA-induced VSMCs.1. KLF4 mediates ATRA-induced mfn-2 gene expression in VSMCsATRA is known to induce the expression of KLF4 and mfn-2, however, direct relationship between KLF4 and mfn-2 has not yet been characterized. We sought to determine whether KLF4 is responsible for the mfn-2 expression and function in VSMCs treated by ATRA. The results were as follows:1.1 KLF4 mediates ATRA-induced mfn-2 expression in VSMCsThe results of Western blot and RT-PCR assays showed that treatment of VSMCs with ATRA resulted in upregulation of KLF4 and mfn-2 in a time-dependent manner. A significant change was observed at concentration of 10μM for 24 h.To further determine whether ATRA-induced mfn-2 gene expression was due to the increased expression of KLF4, we transfected VSMCs with KLF4 expression vector (pAd-KLF4) and KLF4-siRNA, the result of Western blotting and RT-PCR assays showed that overexpression of KLF4 resulted in a time-dependent increase in protein and mRNA levels of mfn-2. In contrast, when VSMCs were transfected with rat KLF4-specific siRNA (KLF4-siRNA) to block the endogenous KLF4 expression induced by ATRA, ATRA-elicited increase in mfn-2 expression significantly reduced. 1.2 KLF4 binds directly to the mfn-2 promoter and regulates its transcriptionTo identify whether KLF4 activates the mfn-2 promoter, A293 cells were transiently co-transfected with the mfn-2 promoter (pGL3-mfn-2-luc) and KLF4 expression plasmid (GFP-KLF4). Reporter gene assays showed that KLF4 overexpression significantly increased the mfn-2 promoter activity in a dose-dependent fashion.Using the TESS computational program, we established that the -1333/+15 bp region of the mfn-2 promoter contains four typical KLF4-binding sites (CACCC/GTGGG). To identify the KLF4-responsive elements involved in KLF4-induced mfn-2 expression, progressive 5’ deletion constructs of the mfn-2 promoter fused to the luciferase reporter gene were generated and transiently transfected into A293 cells with KLF4. Measurements of luciferase activity suggested that the region between -441 and -273 is critical for conferring the capacity to respond to KLF4.To further evaluate which site binds KLF4 and activates the promoter, the four boxes (–1152GTGGG–1148,–840CACCC–836,–356CACCC–352, and–122GTGGG–118) were mutated, respectively, and functional analysis was performed in A293 cells. The result showed that the inactivation of the box 3 completely blocked the response to KLF4, indicating that box 3 element plays a critical role in the KLF4-mediated transcriptional activation of mfn-2.To investigate whether KLF4 binds to the mfn-2 promoter within intact chromatin, ChIP assays were performed. An increased binding of KLF4 to the mfn-2 promoter was observed in KLF4-overexpressing VSMCs.1.3 KLF4 mediates ATRA-induced perinuclear mitochondrial clustering and mitochondrial depolarizationTo examine the effect of upregulation of KLF4 and mfn-2 expression induced by ATRA on mitochondrial dynamics in VSMCs, fluorescent microscopy and electron microscopy were done. The perinuclear mitochondrial clustering was observed in KLF4-overexpressing and ATRA-treated VSMCs. In fluorescent microscopy, when VSMCs were transfected with KLF4-specific siRNA (siRNA-KLF4) to block the endogenous KLF4 expression induced by ATRA, we could not observe perinuclear mitochondrial clustering anymore. To further evaluate whether mitochondrial membrane potential (MMP) was also altered in KLF4-overexpressing cells or in ATRA-treated cells and whether some of the effects of KLF4 on the membrane potential depended on mfn-2, we stained the VSMCs with JC-1. The results suggested that KLF4 overexpression induced mitochondrial perinuclear clustering, and decreased the mitochondrial membrane potential, while transfecting cells with siRNA-mfn-2 abrogated the effect of KLF4 on mitochondrial membrane potential. As expected, these changes were also reflected in the MMP as measured by flow cytometer. Thus, we speculate that KLF4-induced decrease in the mitochondrial membrane potential of VSMCs is at least in part through the up-regulation of mfn-2.These results suggest that the mfn-2 promoter is a direct transcriptional target for KLF4, while KLF4 mediates ATRA-induced mfn-2 expression and plays a role in ATRA signaling and function in VSMCs.2. ATRA promotes the activation of the mfn-2 promoter by KLF4 via inducing KLF4 acetylationLike other zinc finger-containing proteins, the KLF activity may be regulated not only by de novo synthesis but also by protein-protein interactions and posttranslational modification such as phosphorylation, acetylation, ubiquitination or SUMOylation. In this part, we investigated the modification of KLF4 in ATRA-induced VSMCs and the roles of KLF4 modification in activation of the mfn-2 promoter.2.1 ATRA induces acetylation of KLF4Because KLF4 overexpression and ATRA treatment induce the mfn-2 expression, we sought to determine the mechanism whereby KLF4 regulates mfn-2 gene expression in ATRA-treated VSMCs. Western blot assays showed that KLF4 overexpression increased mfn-2 expression, while ATRA treatment increased the inductive effect of KLF4 on mfn-2 expression. To test whether KLF4 binds to the mfn-2 promoter after ATRA stimulation, we performed ChIP assays using primers specific for the mfn-2 promoter containing the putative KLF4-binding site 3. Upon ATRA stimulation, KLF4 binding to DNA was increased.To further demonstrate the transcriptional activation of mfn-2 by KLF4, we overexpressed or knocked down KLF4 and detected the effect of ATRA on the mfn-2 promoter. Reporter gene assays showed that knockdown of KLF4 abrogated the effect of ATRA on the promoter, while ATRA further induced the mfn-2 promoter activity by KLF4 overexpression in A293 cells.To explore the molecular mechanism of ATRA-increased inductive effect of KLF4 on mfn-2 gene expression, the level of acetylated-KLF4 was detected firstly. CoIP assay showed that ATRA time-dependently enhanced the acetylation level of KLF4 within 24 h. To further understand whether acetylation of KLF4 is related to p300, effect of ATRA on p300 subcellular localization in VSMCs was observed. p300 translocated from the cytoplasm into the nucleus after ATRA stimulation for 1 hour. Effects on the interaction between KLF4 and p300 after p300 nuclear import were further investigated by CoIP assay, suggesting that ATRA promoted the interaction of KLF4 with p300.To further determine the effect of KLF4 acetylation on the mfn-2 promoter activation, the luciferase activity was detected. The results showed that the coexpression of KLF4 with p300 increased the mfn-2 promoter activity than overexpression of KLF4 alone. When A293 cells were co-transfected with KLF4 and HDAC2, the mfn-2 promoter activity was reduced to 10% of that transfected with KLF4 alone. Meanwhile, when acetylation-deficient mutant of KLF4 (K225/229R) and p300 were co-transfected, the promoter activity could not be enhanced, indicating that KLF4 acetylation by p300 is required for the activation of the mfn-2 promoter.2.2 ATRA promotes the interaction between KLF4 and p300 by inducing KLF4 phosphorylation via JNK and p38 signalingTo understand whether ATRA-induced interaction between KLF4 and p300 depends on KLF4 phosphorylation, we first detected the levels of phospho-KLF4 in VSMCs treated with ATRA. CoIP assay showed that ATRA stimulation rapidly induced the phosphorylation of KLF4 within 0.5 h. KLF4 phosphorylation reached a maximum at 1 h and remained constant for at least 2 h. Under these experimental conditions, the phosphorylation of JNK and p38 MAPK increased concurrently in a time-dependent manner, and the phosphorylation of Akt and ERK was not changed following ATRA treatment.To further determine whether JNK and p38 MAPK signaling mediate phosphorylation of KLF4 induced by ATRA, we performed the inhibitor studies on KLF4 phosphorylation. VSMCs treated with either JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 blocked ATRA-induced KLF4 phosphorylation, whereas pretreatment with ERK inhibitor PD98059 and Akt inhibitor LY294002, did not affect the KLF4 phosphorylation.Next, we investigated whether KLF4 phosphorylation affects its interaction with p300. The effects of JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 on ATRA-induced KLF4 phosphorylation and ATRA-increased interaction between KLF4 and p300 were detected. The results suggest that ATRA-increased interaction between KLF4 and p300 was reduced to the level of the control by treating VSMCs with SP600125 or SB203580 rather than PD98059 or LY294002. To further demonstrate that the phosphorylation of KLF4 is important for its interaction with p300, we mutated three phosphorylation sites (S415A, S444A, and S470A), and found that both KLF4 phosphorylation and the interaction of KLF4 with p300 induced by ATRA was reduced when the Ser470 was mutated to alanine.To further understand whether KLF4 acetylation depends on its phosphorylation and its interaction with p300, ATRA-induced KLF4 acetylation in VSMCs treated with SP600125 and SB203580 or in VSMCs transfected with the mutant of phosphorylation site (Ser470) was detected. The results showed that inhibiting JNK and p38 activation by their respective inhibitors or transfecting with the mutant of phosphorylation site (Ser470) abrogated ATRA-induced KLF4 acetylation.To evaluate the effect of the KLF4 phosphorylation on ATRA-induced mfn-2 promoter activity, luciferase activity was detected. The results showed that the coexpression of the S470A mutant of KLF4 with p300 could not activate the mfn-2 promoter as compared with the coexpression of the wild-type KLF4 with p300. Similarly, ATRA-elicited increase in the mfn-2 promoter activity was abrogated by treatment with SP600125 and SB203580 or by transfecting the S470A mutant.These results further suggest that ATRA stimulates the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, which in turn increases KLF4 acetylation and its transactivation to the mfn-2 promoter.3. RARαenhances activation of the mfn-2 promoter by KLF4 via binding to KLF4 in ATRA-induced VSMCsThe above study shows that KLF4 mediates mfn-2 expression in ATRA-stimulated VSMCs. However, it remains unclear whether RARs are involved in KLF4-mediated mfn-2 expression in ATRA-stimulated VSMCs. In this part, we determined the role of RARs in activation of the mfn-2 promoter by KLF4.3.1 ATRA induces the expression of RARαand RARβin VSMCsIt is not yet clear whether retinoid receptors (RARs) are expressed in VSMCs. Thus, as a first step toward understanding the potential role of RARs in KLF4-mediated mfn-2 expression, we examined the effect of ATRA on expression of RARs. Western blot and RT-PCR analysis showed that ATRA time- and dose- dependently induced the expression of RARα, and slightly up-regulated the expression of RARβ, and had little or no effect on RARγexpression.3.2 RARαis involved in ATRA-induced KLF4 acetylationThere is no RAR-binding site in the mfn-2 promoter region. Thus, we think that mfn-2 expression is indirectly regulated by RARs. To dissect the contribution of RARs to KLF4 activity regulation in VSMCs, we treated VSMCs with RARαantagonist (Ro 41-5253), RARβantagonist (LE135) or transfected VSMCs with RARs-specific siRNA and determined the effects of RARs on KLF4 phosphorylation in ATRA-stimulated VSMCs. The results indicated that knockdown of RARs did not affect the KLF4 phosphorylation. To further understand whether the levels of acetylated-KLF4 were changed when RARs were knocked down with their siRNA, Western blot was performed, and the result revealed that silencing of RARα, but not RARβand RARγ, inhibited the KLF4 acetylation.To further investigate whether RARs affect KLF4 acetylation in ATRA-stimulated VSMCs, we performed a co-immunoprecipitation (Co-IP) assay. The results showed that the precipitates pulled down with KLF4 antibody, RARαwas increased, RARβwas unchanged, and RARγwas undetectable.3.3 RARαpromotes activation of the mfn-2 promoter by KLF4The functional interaction between KLF4 and RARαin the activation of the mfn-2 promoter by KLF4 was further determined by reporter gene assays. The results showed that the mfn-2 promoter activity induced by KLF4 overexpression was further enhanced after cotransfection of KLF4 with RARα. Upon ATRA treatment, the mfn-2 promoter activity was further induced. However, the mfn-2 promoter activity kept at the baseline when cells were transfected with RARαalone, regardless of with or without ATRA treatment.3.4 Interaction of KLF4 with RARαis dependent on KLF4 phosphorylationBecause we have found that the phosphorylation of KLF4 induced by ATRA was independent on RARαexpression, thus we wanted to demonstrate whether KLF4 phosphorylation affects its interaction with RARα. Crossing Co-IP assay indicated that ATRA-increased interaction between KLF4 and RARαwas reduced to the level of the control by treating VSMCs with SP600125 or SB203580 and by transfecting the mutant of KLF4 phosphorylation site (Ser470).3.5 Knockdown of RARαdecreases the interaction of KLF4 with p300Since ATRA increased KLF4 acetylation by increasing the interaction of KLF4 with p300, the role of RARαin the interaction of KLF4 with p300 needs to be further detected. The result showed that the knockdown of RARαresulted in an obvious reduction in the interaction of KLF4 with p300.These results showed that the acetylation rather than the phosphorylation of KLF4 is dependent on the RARαexpression. Moreover, ATRA increases the interaction of phospho-KLF4 with RARαand their interaction promotes the binding of KLF4 to the mfn-2 promoter.CONCLUSIONS1 KLF4 mediates not only ATRA-induced mfn-2 expression in VSMCs by binding directly to the mfn-2 promoter but also ATRA-induced perinuclear mitochondrial clustering and mitochondrial depolarization.2 ATRA promotes the activation of the mfn-2 promoter by KLF4 via inducing acetylation of KLF4 by p300. KLF4 acetylation by p300 increases its activity to transactivate the mfn-2 promoter.3 ATRA increases the interaction of KLF4 with p300 by inducing KLF4 phosphorylation, and the activation of JNK and p38 MAPK signaling by ATRA is essential for KLF4 phosphorylation and its interaction with p300.4 RARαpromotes the activation of the mfn-2 promoter by KLF4 via increasing the interaction of phospho-KLF4 with p300.更多还原