组蛋白脱乙酰酶2调节KLF4促进平滑肌细胞分化活性的作用机制
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摘要
血管平滑肌细胞(vascular smooth muscle cell, VSMC)在血管损伤因素刺激下,可从分化表型转化为去分化表型并获得增殖能力,这个过程称为表型转化。表型转化是VSMC增殖和迁移的关键性起始步骤,而由VSMC异常增殖和迁移所引发的血管重塑是高血压、动脉粥样硬化和血管再狭窄等血管病变发生发展的根本原因。因此,阐明调控VSMC表型转化的分子机制对防治心血管疾病具有重要的意义。
维甲酸类药物作用的经典途径是维甲酸通过与受体的配体结合区域互相结合,使受体活化后直接激活与分化相关的基因,进而发挥促分化作用。维甲酸类药物可以调节细胞中不同基因或酶的表达,使其活化或抑制,从而达到治疗目的。Krüppel样因子4(krüppel-like factor 4,GKLF/KLF4)是KLF家族中与胚胎发育、细胞分化和癌症发生密切相关的转录因子,我室以往的研究证实,全反式维甲酸(all-trans retinoic acid,ATRA)可显著诱导KLF4表达及促进去分化型VSMC再分化,但KLF4在ATRA诱导VSMC分化中的作用及机制尚不清楚。
组蛋白脱乙酰酶(histone deacetylases, HDACs)可通过触发组蛋白脱乙酰化及作为转录因子辅阻遏物在调节基因表达过程中发挥重要作用。已经证明,HDACs的酶促化学修饰,包括磷酸化、乙酰化和甲基化,既调节其与其他转录因子的结合活性,也调节其脱乙酰酶活性,但其是否调节KLF4的促分化活性目前尚未报道。
为了阐明HDAC2对KLF4促分化活性的调节作用及其机制,本研究系统观察在ATRA诱导的VSMC分化过程中KLF4和HDAC2之间的相互作用、KLF4乙酰化修饰及转录活性变化及其调节途径,旨在揭示VSMC分化的调控机制。
实验结果如下:
1 ATRA对KLF4与HDAC2相互作用的影响本部分实验研究ATRA诱导的VSMC分化与KLF4和HDAC2相互作用之间的关系。实验结果如下:
1.1 ATRA抑制KLF4与HDAC2相互作用
免疫共沉淀分析结果显示,在ATRA刺激的VSMC中,HDAC2和KLF4的相互作用受到抑制,二者形成的复合物明显减少。VSMC与10μM ATRA孵育0、30、60 min后,随着刺激时间的延长,KLF4与HDAC2的结合明显降低,交互免疫沉淀得到相似的结果。GST pull down分析结果显示,HDAC2和KLF4的体外结合活性也随着ATRA刺激时间的延长而逐渐降低。以上结果表明,ATRA能显著抑制KLF4与HDAC2的相互作用,并具有明显的时效关系。
1.2 ATRA通过激活JNK信号途径诱导HDAC2磷酸化
VSMC经ATRA(10μM)刺激0、15、30、60 min后,进行免疫沉淀分析。结果显示,随着ATRA刺激时间的延长,磷酸化型HDAC2水平逐渐升高,在60 min时,其磷酸化水平增加1倍;同时,ATRA刺激后,磷酸化型JNK(p-JNK)水平也显著升高,而ATRA刺激对磷酸化型ERK,磷酸化型Akt水平无显著影响。分别以ERK特异性抑制剂PD98059、Akt特异性抑制剂LY294002和JNK特异性抑制剂处理VSMC,特异性阻断ERK、Akt或者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预处理可显著抑制HDAC2磷酸化,而PD98059和LY294002对HDAC2磷酸化无明显抑制作用。免疫共沉淀分析结果显示,在SP600125预孵育的VSMC中,HDAC2与KLF4的相互作用较单纯用ATRA刺激的细胞明显增强。上述结果表明,ATRA通过JNK途径诱导HDAC2发生磷酸化修饰,而磷酸化的HDAC2与KLF4的结合活性明显降低。
用靶向JNK的siRNA敲低内源性JNK的表达后,ATRA对HDAC2磷酸化不再产生明显影响。证明ATRA诱导的HDAC2磷酸化依赖于JNK信号途径的激活。
2 HDAC2磷酸化修饰KLF4乙酰化及DNA结合活性的影响
第一部分研究证实,ATRA通过诱导HDAC2磷酸化而抑制KLF4与HDAC2的相互作用。为进一步证明检查HDAC2磷酸化是否对KLF4的化学修饰具有调节作用,本部分研究探讨HDAC2磷酸化修饰状态与KLF4乙酰化修饰及DNA结合活性的关系。
2.1 ATRA诱导KLF4乙酰化
免疫沉淀和Western blot分析结果显示,ATRA刺激VSMC不同时间(0、30、60 min)后,KLF4的乙酰化水平呈时间依赖性升高。用靶向HDAC2的siRNA敲低内源性HDAC2的表达后,ATRA对KLF4的乙酰化不再产生明显影响。证明ATRA诱导的KLF4乙酰化依赖于HDAC2的磷酸化活化。
2.2磷酸化型的HDAC2因与KLF4解离,不再催化KLF4脱乙酰基
HDAC2磷酸化位点定点突变实验证明,HDAC2磷酸化位点突变后再用ATRA处理,HDAC2与KLF4的相互作用较对照组(转染野生型HDAC2表达载体)增加,KLF4乙酰化水平降低。结果提示,ATRA诱导的KLF4乙酰化,可能是继发于HDAC2磷酸化后与KLF4解离,从而使HDAC2不能对KLF4脱乙酰基的结果。
2.3 ATRA通过诱导HDAC2磷酸化而使其出核
免疫组化染色和荧光定位染色实验结果证明,HDAC2主要定位于VSMC的细胞核内;ATRA刺激30 min后,HDAC2发生核输出,至40 min时,细胞核内几乎检测不到HDAC2阳性颗粒。HDAC2核输出与HDAC2磷酸化具有正相关关系,表明HDAC2的磷酸化修饰可能是导致其核输出的机制之一。
2.4 KLF4乙酰化修饰增强其与VSMC分化标志基因启动子的结合活性
为了确定VSMC分化标志基因SM22α和SMα-actin表达与KLF4乙酰化修饰的关系,用ChIP分析检查ATRA处理前后,KLF4与两种分化标志基因启动子的结合活性。结果显示,在ATRA诱导KLF4乙酰化水平达到峰值的处理条件下,KLF4与SM22α和SMα-actin启动子的结合活性也显著升高;用JNK特异性抑制剂SP600125阻断JNK通路后,KLF4与SM22α和SMα-actin启动子的结合活性显著下降。该结果提示,KLF4乙酰化修饰促进其与VSMC分化标志基因启动子的结合活性。
以上结果表明,ATRA通过诱导HDAC2磷酸化而使其出核,KLF4不再被其脱乙酰基而维持较高的乙酰化水平。乙酰化型KLF4与VSMC分化标志基因启动子结合活性升高是ATRA诱导VSMC分化的作用机制之一。
3 KLF4乙酰化修饰对VSMC分化标志基因表达的影响
前两部分证明,在ATRA短时间处理的VSMC中,乙酰化型KLF4与VSMC分化标志基因SM22α和SMα-actin启动子结合活性增加。为进一步证实ATRA长时间作用于VSMC使KLF4持续维持乙酰化修饰状态并激活VSMC表达,本部分研究观察ATRA长时间作用于VSMC对HDAC2磷酸化、KLF4乙酰化及VSMC分化标志基因表达的影响。
3.1 ATRA诱导VSMC分化标志基因表达
ATRA刺激VSMC 0、6、12、24和48 h,VSMC中分化标志基因SM22α和SMα-actin表达水平呈时间依赖性升高。表明ATRA长时间作用于VSMC分化标志基因能诱导其向分化表型转化。
3.2 ATRA诱导KLF4和HDAC2表达
Western blot结果显示,KLF4和HDAC2表达因受ATRA诱导而明显上调,其中KLF4在ATRA刺激12 h开始升高,HDAC2在48 h明显增加。表明,ATRA长时间作用于VSMC,可通过诱导KLF4表达而上调VSMC分化标志基因表达。
3.3 ATRA长时间作用于VSMC可使JNK信号途径处于持续活化状态
ATRA刺激VSMC 0、6、12、24和48 h,VSMC中JNK持续活化,其磷酸化水平在6 h达到最高水平,至48 h仍维持在较高水平上。而Akt和ERK磷酸化水平无明显变化。
3.4 ATRA长时间作用于VSMC对HDAC2磷酸化和KLF4修饰的影响及其作用机制与短时间作用时相同
以上结果表明,ATRA长时间作用于VSMC,除了能以与短时间作用相同的信号途径使HDAC2磷酸化及通过抑制HDAC2与KLF4相互作用,使KLF4保持高乙酰化水平外,还可诱导KLF4表达,从而在KLF4基因表达及表达产物化学修饰两个水平上调节其对VSMC分化标志基因的激活作用。
4 ATRA抑制主动脉球囊损伤引发的内膜增生
在前三部分揭示HDAC2调节KLF4促分化活性的作用机制的基础上,本部分实验利用球囊导管剥脱大鼠主动脉血管内皮后,观察ATRA对血管内膜增生的影响及其与HDAC2磷酸化和KLF4乙酰化修饰的关系。
4.1 ATRA显著抑制球囊损伤引发的血管内膜增生
与假手术组相比,主动脉内皮球囊剥脱后14天,模型组主动脉中膜内的VSMC排列紊乱,内膜呈弥散性增厚,管腔变小,模型组诱发内膜增生,内膜与外膜的厚度比值(I/M)明显高于假手术组;ATRA组新生内膜的增生程度和I/M比值明显小于模型组。结果表明,ATRA可以明显抑制球囊损伤诱导的血管内膜增生。免疫组织化学染色结果显示,球囊损伤血管内皮14天后,模型组新生内膜中PCNA阳性染色的细胞数量及单细胞染色强度均高于ATRA组;在ATRA组,PCNA在新生内膜中的阳性染色颗粒颜色较浅、数量较少。提示ATRA能有效抑制球囊损伤后新生内膜中VSMC的增殖。
4.2 ATRA促进血管新生内膜中VSMC向分化型转化
Western blot结果显示,主动脉内皮被球囊剥脱后14天,ATRA组的新生内膜中VSMC分化标志基因SM22α和SMα-actin的表达水平明显高于模型组。免疫组织化学染色结果亦显示,ATRA组的SM22α和SMα-actin阳性染色细胞数量及着色强度较模型组明显增强,与Western blot结果一致。
上述结果表明,ATRA可诱导VSMC分化标志基因SM22α和SMα-actin表达,提示ATRA可促进新生内膜中的VSMC向分化型转化。
4.3 ATRA通过JNK依赖的信号途径诱导HDAC2磷酸化
Western blot结果显示,与假手术组和模型组相比较,ATRA组的JNK磷酸化水平显著升高,p-Akt和p-ERK1/2水平无显著变化,而模型组与ATRA组相比,上述三种蛋白的磷酸化水平均无显著差异,与体外细胞学实验结果一致。
4.4 ATRA通过抑制HDAC2与KLF4相互作用而防止KLF4被HDAC2脱乙酰基
ATRA组HDAC2磷酸化水平显著增加,与此同时,与KLF4的相互作用减弱,结果使KLF4不再被HDAC2脱乙酰基而保持较高的乙酰化水平,与体外培养细胞结果一致。
结论
1在体外培养的VSMC中,ATRA刺激前KLF4与HDAC2以相互缔合的方式存在,即二者以组成型结合在一起。
2 ATRA通过激活JNK信号途径诱导HDAC2磷酸化,磷酸化型HDAC2与KLF4的结合活性降低;ATRA通过抑制HDAC2与KLF4相互作用而防止KLF4被HDAC2脱乙酰基,结果表现为KLF4乙酰化水平升高。
3 HDAC2磷酸化修饰导致其核输出加快。
4乙酰化型KLF4与VSMC分化标志基因启动子的结合活性升高,这是其反式激活VSMC分化标志基因表达的作用机制之一。
5 ATRA促进KLF4表达是其长时间作用于VSMC时,诱导其向分化型转化机制之一。
In response to pathogenic vascular injuries, vascular smooth muscle cells (VSMC) can revert from differentiated phenotype to dedifferentiated phenotype, and then proliferate. It is well established that phenotypic change of VSMC from the differentiated to the dedifferentiated state accompanies their migration and proliferation, which is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. Therefore, exploring the regulatory mechanisms of VSMC phenotype is helpful for therapy of these remodeling cardiovascular diseases.
All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to inhibit VSMC proliferation and induce VSMC differentiation through regulating numerous differentiation-related genes. The gut enriched krüppel-like factor (GKLF/KLF4) is a member of KLF family which plays important roles in cell growth, proliferation, differentiation, and embryogenesis as well as carcinogenesis. ATRA has been shown to induce expression of KLF4 and promote VSMC differentiation, but the action mechanisms of KLF4 in VSMC phenotype modulation induced by ATRA are unclear.
Histone deacetylases (HDACs) play an important role in the regulation of transcription via triggering deacetylation of histones and some transcription factors. HDAC2 is one of the most thoroughly studied HDACs, and can undergo different posttranslational modifications such as nitration, phosphorylation and ubiquitination. However, little is known of the role of HDAC2 in the regulation of KLF4 activity during ATRA-induced VSMC differentiation.
In the present studies, we investigated crucial mechanisms of the interaction between KLF4 and HDAC2 induced by ATRA, which will contribute to penetrate VSMC differentiation.
1 The effect of ATRA on the interaction between KLF4 and HDAC2
We studied the effect of ATRA on the interaction between KLF4 and HDAC2 by co-IP and GST pull down assay. The results were as follows:
1.1 ATRA inhibits the interaction between KLF4 and HDAC2
Co-immunoprecipitation assay showed that ATRA inhibited the interaction between KLF4 and HDAC2. Treatment of VSMC with ATRA resulted in a time-dependent (0, 30, 60 min) reduction in the interaction of KLF4 with HDAC2. GST pull down assay further identified that the interaction between KLF4 and HDAC2 was significantly inhibited in VSMC treated with ATRA in a time-dependent manner. The above findings suggested that ATRA inhibits the interaction between KLF4 and HDAC2 in a time-dependent manner.
1.2 ATRA induces HDAC2 phosphorylation via JNK-dependent signal pathway in VSMC
The lysates of VSMC treated with ATRA for 15, 30 and 60 min were immunoprecipitated with anti-phosphoserine antibody, and then phospho-HDAC2 was detected by Western blotting with anti-HDAC2 antibody. The results showed that ATRA treatment time-dependently increased the levels of phospho-HDAC2 within 60 min in VSMC. To determine which signal pathway mediates HDAC2 phosphorylation in VSMC treated with ATRA, we examined the phosphorylation of ERK1/2, JNK and Akt by Western blotting with phosphospecific antibodies. The results showed that ATRA markedly increased levels of phospho-JNK at 15 min after ATRA treatment. In contrast, ATRA did not affect the phosphorylation of ERK and Akt. To further identify whether JNK activation mediates HDAC2 phosphorylation induced by ATRA, VSMC were pretreated for 2 h with 20μM PD98059 (a specific inhibitor of ERK), 20μM LY294002 (a specific inhibitor of Akt) or 20μM SP600125 (a specific inhibitor of JNK), and then treated with 10μM ATRA for 1 h. Inhibition of JNK by SP600125 blocked the ATRA-induced phosphorylation of HDAC2, whereas PD98059 and LY294002 had no significant effect on HDAC2 phosphorylation induced by ATRA. After cells were transfected with JNK-specific siRNA, HDAC2 phosphorylation was no significant change even by ATRA stimulation. Together, these results suggest that JNK-, but not ERK- and Akt-dependent signal pathway, is involved in the ATRA-induced HDAC2 phosphorylation in VSMC, subsequently leading to the reduction in the interaction between KLF4 and HDAC2 in VSMC.
2 The effects of HDAC2 phosphorylation on the deacetylation of KLF4 and DNA binding activity
The above results suggested that ATRA reduces the interaction between KLF4 and HDAC2 by inducing HDAC2 phosphorylation. To identify whether HDAC2 phosphorylation can regulate KLF4 modification, we detected the phosphorylation of HDAC2, acetylation of KLF4 and its DNA binding activity.
2.1 ATRA induces KLF4 acetylation
Immunoprecipitation assay and Western blotting assays showed that the acetylation of KLF4 was induced by ATRA in a time-dependent (0, 30, 60 min) manner. After cells were transfected with HDAC2-siRNA, ATRA did not acetylate KLF4 in VSMC. The results suggested that KLF4 acetylation induced by ATRA is dependent on HDAC2 phosphorylation.
2.2 Phosphorylated HDAC2 dissociates from KLF4
Compared with wild HDAC2, the interaction of HDAC2S424A with KLF4 in ATRA-induced VSMC is significantly increased. Dissociation of phosphorylated HDAC2 from KLF4 made its catalytic activity lose. Sequentially, acetylation of KLF4 increased.
2.3 ATRA promotes nuclear export of phospho-HDAC2 by activating JNK-signaling pathway
Immunocytochemistry and cell immunofluorescence analysis indicated that majority of HDAC2 proteins were located in the nucleus, but located mostly in the cytoplasm in VSMC treated with ATRA for 40 min.The results suggested that HDAC2 phosphorylation may lead to HDAC2 nuclear export.
2.4 KLF4 acetylation increases the binding of KLF4 to the promoter of VSMC differentiation marker genes
To identify that the expression of VSMC differentiation marker genes SM22αand SMα-actin were related with KLF4 acetylation, ChIP assays were performed. The results showed that when the acetylation of KLF4 reached a maximum after ATRA stimulation, DNA binding activity of KLF4 increased significantly; the interaction of KLF4 with SM22αand SMα-actin promoter was significantly inhibited by SP600125 pretreatment. The results suggested that KLF4 acetylation increases its DNA binding activity.
3 The effects of KLF4 acetylation on espression of VSMC differentiation marker genes
In this part, we detected the effect of long-time stimulation of ATRA on HDAC2 phosphorylation, KLF4 acetylation and the expression of VSMC differentiation marker genes. The results were as follows:
3.1 ATRA induces VSMC marker gene expression
After treatment by ATRA from 0 to 48 h, SM22αand SMα-actin (α-SMA) which are highly expressed in differentiated VSMC increased in a time-dependent manner, suggesting that long-time stimulation of ATRA can induce VSMC phenotypic switching by promoting VSMC marker gene expression.
3.2 ATRA induces KLF4 and HDAC2 expression in VSMC
Western blot analysis showed that expression of KLF4 and HDAC2 was induced by ATRA in a time-dependent manner, especially 12 h and 48 h after ATRA treatment, suggesting that long-time stimulation of ATRA can induce VSMC differentiation marker gene expression by inducing the acetylation of KLF4.
3.4 ATRA induces HDAC2 phosphorylation and KLF4 acetylation
Consistent results were obtained in the model rats with the data in ATRA-induced VSMC.
These evidences prove that ATRA can keep JNK pathway persistent activating and long-timely induce HDAC2 phosphorylation. On the other hand, ATRA could induce KLF4 expression in VSMC. Therefore, ATRA-induced VSMC marker gene expression is involved in KLF4 gene expression and its post-translational modification.
4 ATRA inhibits neointimal formation induced by balloon injury
The animal model was established through de-endothelializing by a Fogarty’s tube. The effects of ATRA on neointimal formation, HDAC2 phosphorylation and KLF4 acetylation were evaluated in this part.
4.1 ATRA inhibits neointimal formation induced by balloon injury
To investigate the effect of ATRA on neointimal formation, the intima to media area (I/M) ratio was evaluated by morphometric analysis in each cross-section of arteries at 14 days after balloon injuries. The I/M ratio was 1.23±0.11 in ATRA group and 4.30±0.26 in injured group.
The expression of PCNA was detected by immunohistochemical staining,and the results demonstrated that the level of PCNA in the ATRA group was lower than that in the injured group. Immunohistochemical staining showed that the number of PCNA-positive cells in the ATRA group was significantly decreased compared to the injured group. The results indicated that ATRA significantly inhibits neointimal hyperplasia.
4.2 ATRA induce VSMC marker gene expression during the progress of neointimal formation
Western blot analysis showed that the level of SM22αand SMα-actin in the carotid arteries of ATRA group was much higher than that in the injured group. Immunohistochemical staining also showed that the number of SM22αand SMα-actin positive cells in ATRA group increased significantly, compared to the sham operated group. The results of immunohistochemistry staining were similar to that of Western blot, suggesting that ATRA induces the expression of differentiation marker genes in neointimal formation.
4.3 ATRA induces HDAC2 phosphorylation by JNK-dependent pathway in neointimal formation induced by balloon injury
Western blot analysis showed that compared with the sham operated group and the injured group, phospho-JNK in the ATRA group was significantly increased, whereas phospho-ERK or phospho-Akt had no valuable change. The results from animal model were similar to those from cultured VSMC in vitro.
4.4 Dissociation of HDAC2 from KLF4 inhibits KLF4 deacetylation
HDAC2 phosphorylation was induced in the ATRA group compared with the injured group. Simultaneously, the interaction between KLF4 and HDAC2 was reduced, leading to KLF4 acetylation at a relative high level.
Conclusion:
1 ATRA induces HDAC2 phosphorylation by activating JNK-dependent pathway. Dissociation of phospho-HDAC2 from KLF4 prevents KLF4 deacetylation.
2 Phosphorylation of HDAC2 at serine residue leads to its nuclear export.
3 Acetylation of KLF4 increases its binding activity to the promoter of VSMC differentiation marker genes.
4 ATRA induces KLF4 expression in VSMC.
5 ATRA inhibits neointimal formation inducing by balloon injury in rats via the JNK signaling.
维甲酸类药物作用的经典途径是维甲酸通过与受体的配体结合区域互相结合,使受体活化后直接激活与分化相关的基因,进而发挥促分化作用。维甲酸类药物可以调节细胞中不同基因或酶的表达,使其活化或抑制,从而达到治疗目的。Krüppel样因子4(krüppel-like factor 4,GKLF/KLF4)是KLF家族中与胚胎发育、细胞分化和癌症发生密切相关的转录因子,我室以往的研究证实,全反式维甲酸(all-trans retinoic acid,ATRA)可显著诱导KLF4表达及促进去分化型VSMC再分化,但KLF4在ATRA诱导VSMC分化中的作用及机制尚不清楚。
组蛋白脱乙酰酶(histone deacetylases, HDACs)可通过触发组蛋白脱乙酰化及作为转录因子辅阻遏物在调节基因表达过程中发挥重要作用。已经证明,HDACs的酶促化学修饰,包括磷酸化、乙酰化和甲基化,既调节其与其他转录因子的结合活性,也调节其脱乙酰酶活性,但其是否调节KLF4的促分化活性目前尚未报道。
为了阐明HDAC2对KLF4促分化活性的调节作用及其机制,本研究系统观察在ATRA诱导的VSMC分化过程中KLF4和HDAC2之间的相互作用、KLF4乙酰化修饰及转录活性变化及其调节途径,旨在揭示VSMC分化的调控机制。
实验结果如下:
1 ATRA对KLF4与HDAC2相互作用的影响本部分实验研究ATRA诱导的VSMC分化与KLF4和HDAC2相互作用之间的关系。实验结果如下:
1.1 ATRA抑制KLF4与HDAC2相互作用
免疫共沉淀分析结果显示,在ATRA刺激的VSMC中,HDAC2和KLF4的相互作用受到抑制,二者形成的复合物明显减少。VSMC与10μM ATRA孵育0、30、60 min后,随着刺激时间的延长,KLF4与HDAC2的结合明显降低,交互免疫沉淀得到相似的结果。GST pull down分析结果显示,HDAC2和KLF4的体外结合活性也随着ATRA刺激时间的延长而逐渐降低。以上结果表明,ATRA能显著抑制KLF4与HDAC2的相互作用,并具有明显的时效关系。
1.2 ATRA通过激活JNK信号途径诱导HDAC2磷酸化
VSMC经ATRA(10μM)刺激0、15、30、60 min后,进行免疫沉淀分析。结果显示,随着ATRA刺激时间的延长,磷酸化型HDAC2水平逐渐升高,在60 min时,其磷酸化水平增加1倍;同时,ATRA刺激后,磷酸化型JNK(p-JNK)水平也显著升高,而ATRA刺激对磷酸化型ERK,磷酸化型Akt水平无显著影响。分别以ERK特异性抑制剂PD98059、Akt特异性抑制剂LY294002和JNK特异性抑制剂处理VSMC,特异性阻断ERK、Akt或者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预处理可显著抑制HDAC2磷酸化,而PD98059和LY294002对HDAC2磷酸化无明显抑制作用。免疫共沉淀分析结果显示,在SP600125预孵育的VSMC中,HDAC2与KLF4的相互作用较单纯用ATRA刺激的细胞明显增强。上述结果表明,ATRA通过JNK途径诱导HDAC2发生磷酸化修饰,而磷酸化的HDAC2与KLF4的结合活性明显降低。
用靶向JNK的siRNA敲低内源性JNK的表达后,ATRA对HDAC2磷酸化不再产生明显影响。证明ATRA诱导的HDAC2磷酸化依赖于JNK信号途径的激活。
2 HDAC2磷酸化修饰KLF4乙酰化及DNA结合活性的影响
第一部分研究证实,ATRA通过诱导HDAC2磷酸化而抑制KLF4与HDAC2的相互作用。为进一步证明检查HDAC2磷酸化是否对KLF4的化学修饰具有调节作用,本部分研究探讨HDAC2磷酸化修饰状态与KLF4乙酰化修饰及DNA结合活性的关系。
2.1 ATRA诱导KLF4乙酰化
免疫沉淀和Western blot分析结果显示,ATRA刺激VSMC不同时间(0、30、60 min)后,KLF4的乙酰化水平呈时间依赖性升高。用靶向HDAC2的siRNA敲低内源性HDAC2的表达后,ATRA对KLF4的乙酰化不再产生明显影响。证明ATRA诱导的KLF4乙酰化依赖于HDAC2的磷酸化活化。
2.2磷酸化型的HDAC2因与KLF4解离,不再催化KLF4脱乙酰基
HDAC2磷酸化位点定点突变实验证明,HDAC2磷酸化位点突变后再用ATRA处理,HDAC2与KLF4的相互作用较对照组(转染野生型HDAC2表达载体)增加,KLF4乙酰化水平降低。结果提示,ATRA诱导的KLF4乙酰化,可能是继发于HDAC2磷酸化后与KLF4解离,从而使HDAC2不能对KLF4脱乙酰基的结果。
2.3 ATRA通过诱导HDAC2磷酸化而使其出核
免疫组化染色和荧光定位染色实验结果证明,HDAC2主要定位于VSMC的细胞核内;ATRA刺激30 min后,HDAC2发生核输出,至40 min时,细胞核内几乎检测不到HDAC2阳性颗粒。HDAC2核输出与HDAC2磷酸化具有正相关关系,表明HDAC2的磷酸化修饰可能是导致其核输出的机制之一。
2.4 KLF4乙酰化修饰增强其与VSMC分化标志基因启动子的结合活性
为了确定VSMC分化标志基因SM22α和SMα-actin表达与KLF4乙酰化修饰的关系,用ChIP分析检查ATRA处理前后,KLF4与两种分化标志基因启动子的结合活性。结果显示,在ATRA诱导KLF4乙酰化水平达到峰值的处理条件下,KLF4与SM22α和SMα-actin启动子的结合活性也显著升高;用JNK特异性抑制剂SP600125阻断JNK通路后,KLF4与SM22α和SMα-actin启动子的结合活性显著下降。该结果提示,KLF4乙酰化修饰促进其与VSMC分化标志基因启动子的结合活性。
以上结果表明,ATRA通过诱导HDAC2磷酸化而使其出核,KLF4不再被其脱乙酰基而维持较高的乙酰化水平。乙酰化型KLF4与VSMC分化标志基因启动子结合活性升高是ATRA诱导VSMC分化的作用机制之一。
3 KLF4乙酰化修饰对VSMC分化标志基因表达的影响
前两部分证明,在ATRA短时间处理的VSMC中,乙酰化型KLF4与VSMC分化标志基因SM22α和SMα-actin启动子结合活性增加。为进一步证实ATRA长时间作用于VSMC使KLF4持续维持乙酰化修饰状态并激活VSMC表达,本部分研究观察ATRA长时间作用于VSMC对HDAC2磷酸化、KLF4乙酰化及VSMC分化标志基因表达的影响。
3.1 ATRA诱导VSMC分化标志基因表达
ATRA刺激VSMC 0、6、12、24和48 h,VSMC中分化标志基因SM22α和SMα-actin表达水平呈时间依赖性升高。表明ATRA长时间作用于VSMC分化标志基因能诱导其向分化表型转化。
3.2 ATRA诱导KLF4和HDAC2表达
Western blot结果显示,KLF4和HDAC2表达因受ATRA诱导而明显上调,其中KLF4在ATRA刺激12 h开始升高,HDAC2在48 h明显增加。表明,ATRA长时间作用于VSMC,可通过诱导KLF4表达而上调VSMC分化标志基因表达。
3.3 ATRA长时间作用于VSMC可使JNK信号途径处于持续活化状态
ATRA刺激VSMC 0、6、12、24和48 h,VSMC中JNK持续活化,其磷酸化水平在6 h达到最高水平,至48 h仍维持在较高水平上。而Akt和ERK磷酸化水平无明显变化。
3.4 ATRA长时间作用于VSMC对HDAC2磷酸化和KLF4修饰的影响及其作用机制与短时间作用时相同
以上结果表明,ATRA长时间作用于VSMC,除了能以与短时间作用相同的信号途径使HDAC2磷酸化及通过抑制HDAC2与KLF4相互作用,使KLF4保持高乙酰化水平外,还可诱导KLF4表达,从而在KLF4基因表达及表达产物化学修饰两个水平上调节其对VSMC分化标志基因的激活作用。
4 ATRA抑制主动脉球囊损伤引发的内膜增生
在前三部分揭示HDAC2调节KLF4促分化活性的作用机制的基础上,本部分实验利用球囊导管剥脱大鼠主动脉血管内皮后,观察ATRA对血管内膜增生的影响及其与HDAC2磷酸化和KLF4乙酰化修饰的关系。
4.1 ATRA显著抑制球囊损伤引发的血管内膜增生
与假手术组相比,主动脉内皮球囊剥脱后14天,模型组主动脉中膜内的VSMC排列紊乱,内膜呈弥散性增厚,管腔变小,模型组诱发内膜增生,内膜与外膜的厚度比值(I/M)明显高于假手术组;ATRA组新生内膜的增生程度和I/M比值明显小于模型组。结果表明,ATRA可以明显抑制球囊损伤诱导的血管内膜增生。免疫组织化学染色结果显示,球囊损伤血管内皮14天后,模型组新生内膜中PCNA阳性染色的细胞数量及单细胞染色强度均高于ATRA组;在ATRA组,PCNA在新生内膜中的阳性染色颗粒颜色较浅、数量较少。提示ATRA能有效抑制球囊损伤后新生内膜中VSMC的增殖。
4.2 ATRA促进血管新生内膜中VSMC向分化型转化
Western blot结果显示,主动脉内皮被球囊剥脱后14天,ATRA组的新生内膜中VSMC分化标志基因SM22α和SMα-actin的表达水平明显高于模型组。免疫组织化学染色结果亦显示,ATRA组的SM22α和SMα-actin阳性染色细胞数量及着色强度较模型组明显增强,与Western blot结果一致。
上述结果表明,ATRA可诱导VSMC分化标志基因SM22α和SMα-actin表达,提示ATRA可促进新生内膜中的VSMC向分化型转化。
4.3 ATRA通过JNK依赖的信号途径诱导HDAC2磷酸化
Western blot结果显示,与假手术组和模型组相比较,ATRA组的JNK磷酸化水平显著升高,p-Akt和p-ERK1/2水平无显著变化,而模型组与ATRA组相比,上述三种蛋白的磷酸化水平均无显著差异,与体外细胞学实验结果一致。
4.4 ATRA通过抑制HDAC2与KLF4相互作用而防止KLF4被HDAC2脱乙酰基
ATRA组HDAC2磷酸化水平显著增加,与此同时,与KLF4的相互作用减弱,结果使KLF4不再被HDAC2脱乙酰基而保持较高的乙酰化水平,与体外培养细胞结果一致。
结论
1在体外培养的VSMC中,ATRA刺激前KLF4与HDAC2以相互缔合的方式存在,即二者以组成型结合在一起。
2 ATRA通过激活JNK信号途径诱导HDAC2磷酸化,磷酸化型HDAC2与KLF4的结合活性降低;ATRA通过抑制HDAC2与KLF4相互作用而防止KLF4被HDAC2脱乙酰基,结果表现为KLF4乙酰化水平升高。
3 HDAC2磷酸化修饰导致其核输出加快。
4乙酰化型KLF4与VSMC分化标志基因启动子的结合活性升高,这是其反式激活VSMC分化标志基因表达的作用机制之一。
5 ATRA促进KLF4表达是其长时间作用于VSMC时,诱导其向分化型转化机制之一。
In response to pathogenic vascular injuries, vascular smooth muscle cells (VSMC) can revert from differentiated phenotype to dedifferentiated phenotype, and then proliferate. It is well established that phenotypic change of VSMC from the differentiated to the dedifferentiated state accompanies their migration and proliferation, which is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and postangioplasty restenosis. Therefore, exploring the regulatory mechanisms of VSMC phenotype is helpful for therapy of these remodeling cardiovascular diseases.
All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to inhibit VSMC proliferation and induce VSMC differentiation through regulating numerous differentiation-related genes. The gut enriched krüppel-like factor (GKLF/KLF4) is a member of KLF family which plays important roles in cell growth, proliferation, differentiation, and embryogenesis as well as carcinogenesis. ATRA has been shown to induce expression of KLF4 and promote VSMC differentiation, but the action mechanisms of KLF4 in VSMC phenotype modulation induced by ATRA are unclear.
Histone deacetylases (HDACs) play an important role in the regulation of transcription via triggering deacetylation of histones and some transcription factors. HDAC2 is one of the most thoroughly studied HDACs, and can undergo different posttranslational modifications such as nitration, phosphorylation and ubiquitination. However, little is known of the role of HDAC2 in the regulation of KLF4 activity during ATRA-induced VSMC differentiation.
In the present studies, we investigated crucial mechanisms of the interaction between KLF4 and HDAC2 induced by ATRA, which will contribute to penetrate VSMC differentiation.
1 The effect of ATRA on the interaction between KLF4 and HDAC2
We studied the effect of ATRA on the interaction between KLF4 and HDAC2 by co-IP and GST pull down assay. The results were as follows:
1.1 ATRA inhibits the interaction between KLF4 and HDAC2
Co-immunoprecipitation assay showed that ATRA inhibited the interaction between KLF4 and HDAC2. Treatment of VSMC with ATRA resulted in a time-dependent (0, 30, 60 min) reduction in the interaction of KLF4 with HDAC2. GST pull down assay further identified that the interaction between KLF4 and HDAC2 was significantly inhibited in VSMC treated with ATRA in a time-dependent manner. The above findings suggested that ATRA inhibits the interaction between KLF4 and HDAC2 in a time-dependent manner.
1.2 ATRA induces HDAC2 phosphorylation via JNK-dependent signal pathway in VSMC
The lysates of VSMC treated with ATRA for 15, 30 and 60 min were immunoprecipitated with anti-phosphoserine antibody, and then phospho-HDAC2 was detected by Western blotting with anti-HDAC2 antibody. The results showed that ATRA treatment time-dependently increased the levels of phospho-HDAC2 within 60 min in VSMC. To determine which signal pathway mediates HDAC2 phosphorylation in VSMC treated with ATRA, we examined the phosphorylation of ERK1/2, JNK and Akt by Western blotting with phosphospecific antibodies. The results showed that ATRA markedly increased levels of phospho-JNK at 15 min after ATRA treatment. In contrast, ATRA did not affect the phosphorylation of ERK and Akt. To further identify whether JNK activation mediates HDAC2 phosphorylation induced by ATRA, VSMC were pretreated for 2 h with 20μM PD98059 (a specific inhibitor of ERK), 20μM LY294002 (a specific inhibitor of Akt) or 20μM SP600125 (a specific inhibitor of JNK), and then treated with 10μM ATRA for 1 h. Inhibition of JNK by SP600125 blocked the ATRA-induced phosphorylation of HDAC2, whereas PD98059 and LY294002 had no significant effect on HDAC2 phosphorylation induced by ATRA. After cells were transfected with JNK-specific siRNA, HDAC2 phosphorylation was no significant change even by ATRA stimulation. Together, these results suggest that JNK-, but not ERK- and Akt-dependent signal pathway, is involved in the ATRA-induced HDAC2 phosphorylation in VSMC, subsequently leading to the reduction in the interaction between KLF4 and HDAC2 in VSMC.
2 The effects of HDAC2 phosphorylation on the deacetylation of KLF4 and DNA binding activity
The above results suggested that ATRA reduces the interaction between KLF4 and HDAC2 by inducing HDAC2 phosphorylation. To identify whether HDAC2 phosphorylation can regulate KLF4 modification, we detected the phosphorylation of HDAC2, acetylation of KLF4 and its DNA binding activity.
2.1 ATRA induces KLF4 acetylation
Immunoprecipitation assay and Western blotting assays showed that the acetylation of KLF4 was induced by ATRA in a time-dependent (0, 30, 60 min) manner. After cells were transfected with HDAC2-siRNA, ATRA did not acetylate KLF4 in VSMC. The results suggested that KLF4 acetylation induced by ATRA is dependent on HDAC2 phosphorylation.
2.2 Phosphorylated HDAC2 dissociates from KLF4
Compared with wild HDAC2, the interaction of HDAC2S424A with KLF4 in ATRA-induced VSMC is significantly increased. Dissociation of phosphorylated HDAC2 from KLF4 made its catalytic activity lose. Sequentially, acetylation of KLF4 increased.
2.3 ATRA promotes nuclear export of phospho-HDAC2 by activating JNK-signaling pathway
Immunocytochemistry and cell immunofluorescence analysis indicated that majority of HDAC2 proteins were located in the nucleus, but located mostly in the cytoplasm in VSMC treated with ATRA for 40 min.The results suggested that HDAC2 phosphorylation may lead to HDAC2 nuclear export.
2.4 KLF4 acetylation increases the binding of KLF4 to the promoter of VSMC differentiation marker genes
To identify that the expression of VSMC differentiation marker genes SM22αand SMα-actin were related with KLF4 acetylation, ChIP assays were performed. The results showed that when the acetylation of KLF4 reached a maximum after ATRA stimulation, DNA binding activity of KLF4 increased significantly; the interaction of KLF4 with SM22αand SMα-actin promoter was significantly inhibited by SP600125 pretreatment. The results suggested that KLF4 acetylation increases its DNA binding activity.
3 The effects of KLF4 acetylation on espression of VSMC differentiation marker genes
In this part, we detected the effect of long-time stimulation of ATRA on HDAC2 phosphorylation, KLF4 acetylation and the expression of VSMC differentiation marker genes. The results were as follows:
3.1 ATRA induces VSMC marker gene expression
After treatment by ATRA from 0 to 48 h, SM22αand SMα-actin (α-SMA) which are highly expressed in differentiated VSMC increased in a time-dependent manner, suggesting that long-time stimulation of ATRA can induce VSMC phenotypic switching by promoting VSMC marker gene expression.
3.2 ATRA induces KLF4 and HDAC2 expression in VSMC
Western blot analysis showed that expression of KLF4 and HDAC2 was induced by ATRA in a time-dependent manner, especially 12 h and 48 h after ATRA treatment, suggesting that long-time stimulation of ATRA can induce VSMC differentiation marker gene expression by inducing the acetylation of KLF4.
3.4 ATRA induces HDAC2 phosphorylation and KLF4 acetylation
Consistent results were obtained in the model rats with the data in ATRA-induced VSMC.
These evidences prove that ATRA can keep JNK pathway persistent activating and long-timely induce HDAC2 phosphorylation. On the other hand, ATRA could induce KLF4 expression in VSMC. Therefore, ATRA-induced VSMC marker gene expression is involved in KLF4 gene expression and its post-translational modification.
4 ATRA inhibits neointimal formation induced by balloon injury
The animal model was established through de-endothelializing by a Fogarty’s tube. The effects of ATRA on neointimal formation, HDAC2 phosphorylation and KLF4 acetylation were evaluated in this part.
4.1 ATRA inhibits neointimal formation induced by balloon injury
To investigate the effect of ATRA on neointimal formation, the intima to media area (I/M) ratio was evaluated by morphometric analysis in each cross-section of arteries at 14 days after balloon injuries. The I/M ratio was 1.23±0.11 in ATRA group and 4.30±0.26 in injured group.
The expression of PCNA was detected by immunohistochemical staining,and the results demonstrated that the level of PCNA in the ATRA group was lower than that in the injured group. Immunohistochemical staining showed that the number of PCNA-positive cells in the ATRA group was significantly decreased compared to the injured group. The results indicated that ATRA significantly inhibits neointimal hyperplasia.
4.2 ATRA induce VSMC marker gene expression during the progress of neointimal formation
Western blot analysis showed that the level of SM22αand SMα-actin in the carotid arteries of ATRA group was much higher than that in the injured group. Immunohistochemical staining also showed that the number of SM22αand SMα-actin positive cells in ATRA group increased significantly, compared to the sham operated group. The results of immunohistochemistry staining were similar to that of Western blot, suggesting that ATRA induces the expression of differentiation marker genes in neointimal formation.
4.3 ATRA induces HDAC2 phosphorylation by JNK-dependent pathway in neointimal formation induced by balloon injury
Western blot analysis showed that compared with the sham operated group and the injured group, phospho-JNK in the ATRA group was significantly increased, whereas phospho-ERK or phospho-Akt had no valuable change. The results from animal model were similar to those from cultured VSMC in vitro.
4.4 Dissociation of HDAC2 from KLF4 inhibits KLF4 deacetylation
HDAC2 phosphorylation was induced in the ATRA group compared with the injured group. Simultaneously, the interaction between KLF4 and HDAC2 was reduced, leading to KLF4 acetylation at a relative high level.
Conclusion:
1 ATRA induces HDAC2 phosphorylation by activating JNK-dependent pathway. Dissociation of phospho-HDAC2 from KLF4 prevents KLF4 deacetylation.
2 Phosphorylation of HDAC2 at serine residue leads to its nuclear export.
3 Acetylation of KLF4 increases its binding activity to the promoter of VSMC differentiation marker genes.
4 ATRA induces KLF4 expression in VSMC.
5 ATRA inhibits neointimal formation inducing by balloon injury in rats via the JNK signaling.
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