KLF4在TGF-β1诱导血管平滑肌细胞分化中的作用及机制研究
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摘要
血管平滑肌细胞(vascular smooth muscle cell, VSMC)异常增殖是许多血管增殖性疾病,如动脉粥样硬化、血管再狭窄、高血压等发生发展的共同病理学基础。既往研究已经证明,转化生长因子β1(transforming growth factor, TGF-β1)作为多肽生长因子家族中的一员,通过与受体TβRI/TβRII(TGF-βtype I receptor,TβRI;TGF-βtype II receptor,TβRII)相互作用而发挥对细胞生长、分化和凋亡的调节作用。阐明TGF-β在VSMC生物学行为调节中的作用及其机制,对防治血管重塑和逆转增殖性血管病变具有重要的意义。
Krüppel样因子(krüppel-like factor,KLF)是一类含有锌指结构的转录因子,其典型结构特征是在羧基端具有3个C2H2锌指结构。KLF广泛参与细胞增殖、凋亡、分化以及胚胎发育等生命活动的调控。近年来的研究结果显示,KLF4(krüppel-like factor 4)作为转录因子既可直接结合DNA,又能通过与其它转录因子相互作用而调控基因的表达。以往研究证实,TGF-β1可诱导KLF4表达并促进体外培养的去分化型VSMC进行分化,但KLF4在TGF-β诱导VSMC分化中的作用及机制尚不清楚。
为了阐明KLF4在TGF-β诱导VSMC分化中的作用及其分子机制,本文在系统研究TGF-β1对VSMC增殖、分化、迁移、细胞周期等生物学行为影响的基础上,进一步探讨KLF4在TGF-β1促进VSMC分化中的作用和分子机制。旨在为揭示动脉粥样硬化、高血压和血管再狭窄等心血管疾病的发病机制提供新的实验和理论依据,为心血管疾病的防治提供新的靶点。
1. TGF-β1诱导VSMC分化与上调KLF4和TβRI表达有关
本部分实验以VSMC分化和去分化标志基因表达水平作为判断VSMC表型的指标,检测TGF-β1对VSMC表型及相关生物学行为的影响。为了考察TβRI是否参与TGF-β1诱导VSMC分化的过程,一方面,观察敲减TβRI基因表达对VSMC分化和去分化标志基因表达的影响;另一方面,观察KLF4过表达和敲减对TβRI表达以及对VSMC分化和去分化标志基因表达的影响。实验结果如下:
1.1 TGF-β1抑制VSMC增殖、迁移和细胞周期进程
MTT分析、细胞计数、伤口愈合实验结果显示,TGF-β1能以浓度(0.5、1、2、4 ng/ml)和时间(24、48 h)依赖的方式抑制VSMC增殖和迁移,以TGF-β1浓度为2 ng/ml作用于VSMC 48 h对细胞增殖和迁移的抑制作用最强。
流式细胞术检测结果显示,TGF-β1能以时间(6、12、24、48 h)依赖的方式抑制VSMC细胞周期的进程。TGF-β1(2 ng/ml)刺激VSMC 48 h后,停滞于G0/G1期的细胞数显著增加,同时处于S期和G2/M期的细胞数明显减少。
1.2 TGF-β1诱导VSMC分化标志基因SM22α和SMα-actin表达
SM22α和SMα-actin表达是VSMC处于分化状态的重要分子标志, PCNA表达是VSMC处于增殖状态的重要标志。采用Western blot方法,检查TGF-β1对各种标志基因表达的影响。结果显示,在TGF-β1处理的VSMC中,分化标志基因SM22α和SMα-actin表达明显升高,而增殖标志物PCNA表达显著降低,这些基因的表达变化依赖于TGF-β1作用的时间和浓度。其中,浓度为2 ng/ml和作用时间为48 h时变化最为显著。
1.3敲减TβRI表达对VSMC分化和去分化标志基因表达的影响
将靶向TβRI的小干扰RNA(small interfering RNA,siRNA)导入VSMC,阻断内源性TβRI表达后,研究TβRI在TGF-β1诱导VSMC分化中的作用。Western blot结果显示,转染TβRI-siRNA可使TβRI mRNA和蛋白表达水平降低约75~80%,表明该基因的表达被有效敲低。在TGF-β1刺激下,转染TβRI-siRNA的VSMC中SM22α和SMα-actin的表达明显降低,相反,增殖标志物PCNA的表达显著增加。
1.4 KLF4在TGF-β1诱导TβRI表达及VSMC分化中的作用
虽然已经发现KLF4是参与细胞生长、分化和凋亡的调控,但KLF4是否通过TβRI介导TGF-β1的作用目前尚不清楚。
RT-PCR和Western blot结果表明,TGF-β1显著诱导KLF4和TβRI基因的转录和蛋白表达,并具有明显的时-效(6、12、24、48 h)关系。2 ng/ml TGF-β1作用于VSMC 6 h后,KLF4和TβRI的mRNA和蛋白表达水平均显著升高,两者的表达变化呈正相关关系。
为了阐明KLF4与TβRI表达之间的关系,将KLF4-siRNA导入VSMC,阻断内源性KLF4表达后,明确KLF4在TGF-β1诱导TβRI及VSMC分化标志基因表达中的作用。RT-PCR和Western blot分析结果显示,转染KLF4-siRNA可敲减KLF4表达60%以上。在敲低KLF4表达的VSMC中,TGF-β1诱导TβRI表达的作用被取消,分化标志基因SM22α和SMα-actin的表达也明显下降,表明KLF4介导TGF-β1对TβRI表达的诱导作用。
另外,将KLF4腺病毒表达载体Ad-KLF4感染VSMC,观察强制表达KLF4对TβRI和VSMC分化标志基因表达的影响。RT-PCR和Western blot分析证实,在KLF4过表达的VSMC中,TβRI表达明显高于空载体对照组,与此同时,分化标志基因SM22α和SMα-actin的表达也显著升高。
2. TGF-β1通过激活Smad和p38 MAPK信号通路诱导KLF4磷酸化和KLF4与Smad2/3相互作用
TGF-β1通过与VSMC上的相应受体相互作用而触发细胞信号的跨膜转导,进而活化Smad信号通路而激活相关基因表达。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联反应是胞内信号进行交互作用的重要信号转导通路。在上述阐明KLF4通过TβRI介导TGF-β诱导VSMC分化的基础上,本部分实验探讨TGF-β1对Smad和MAPK信号途径的影响,以期阐明TGF-β1诱导KLF4活化和促进细胞分化的信号转导机制。
2.1 TGF-β1通过激活Smad和p38 MAPK信号途径诱导KLF4磷酸化
VSMC经TGF-β1(2 ng/ml)刺激5、10、20、40 min,用磷酸化丝氨酸抗体对细胞裂解液进行免疫沉淀后检测磷酸化KLF4的水平。结果显示,随着TGF-β1刺激时间的延长,磷酸化型KLF4水平逐渐升高,在TGF-β1刺激20~40 min内,其磷酸化水平增加2倍;同时,TGF-β1刺激后,磷酸化型Smad2(p-Smad2)、Smad3(p-Smad3)和p38 MAPK(p-p38)水平也显著升高。
分别以TβRI特异性抑制剂SB431542、p38特异性抑制剂SB203580和ERK特异性抑制剂PD98059处理VSMC,特异性阻断Smad、p38或ERK信号通路的活化后,再以2 ng/ml的TGF-β1刺激细胞40 min。Western blot结果显示,SB431542或SB203580预处理细胞可显著抑制KLF4磷酸化,而PD98059对KLF4磷酸化无明显影响。用靶向Smad2、Smad3或p38 MAPK的siRNA敲低内源性Smad2、Smad3或p38 MAPK表达后,TGF-β1对KLF4磷酸化不再产生明显影响。证明TGF-β1诱导的KLF4磷酸化依赖于Smad和p38 MAPK信号途径的激活。
2.2 TGF-β1通过诱导KLF4磷酸化而促进KLF4与Smad2/3相互作用
免疫共沉淀分析结果显示,VSMC被TGF-β1处理后,KLF4与Smad2/3相互作用形成的复合物明显增加。VSMC在TGF-β1(2 ng/ml)刺激5、10、20、40 min后,随着刺激时间的延长,KLF4与Smad2/3的结合明显增加,交互免疫沉淀也得到相似的结果。表明,TGF-β1能显著促进KLF4与Smad2/3的相互作用,并具有明显的时效关系。
为了检查KLF4磷酸化是否影响其与Smad2/3的相互作用,以SB431542、SB203580和PD98059处理VSMC,特异性阻断Smad、p38或ERK信号通路的活化后,再以2 ng/ml TGF-β1刺激细胞40 min。免疫共沉淀分析结果显示,在SB431542和SB203580预孵育的VSMC中,KLF4与Smad2/3的相互作用明显降低,交互免疫沉淀得到相似的结果。用靶向Smad2、Smad3或p38 MAPK的siRNA敲低内源性Smad2、Smad3或p38 MAPK表达后,TGF-β1对KLF4与Smad2/3的相互作用不再产生明显影响。上述结果表明,TGF-β1通过Smad和p38 MAPK信号途径诱导KLF4发生磷酸化修饰,而磷酸化型KLF4与Smad2/3的结合活性明显增加。
KLF4磷酸化位点定点突变实验证明,转染KLF4磷酸化位点(S470A)突变体的细胞再用TGF-β1处理时,KLF4的磷酸化水平明显降低,KLF4与Smad2/3的相互作用较对照组(转染野生型KLF4表达载体)降低。这些结果进一步提示,TGF-β1通过诱导KLF4磷酸化而促进KLF4与Smad2/3之间的相互作用。
2.3 KLF4与Smad2协同激活TβRI基因表达
上述实验证实,TGF-β1促进KLF4与Smad2/3相互作用。为了检查KLF4与Smad2/3相互作用在调节TβRI表达中的意义,我们首先进行了报告基因分析。将TβRI启动子指导的报告基因分别与KLF4或Smad2表达质粒共转染293A细胞时,KLF4和Smad2均能促进TβRI启动子的转录活性。KLF4和Smad2共转染使TβRI启动子活性比转染其中一种表达质粒升高1倍左右。表明KLF4与Smad2协同激活TβRI基因表达。
为了进一步检查TGF-β1对KLF4与Smad2相互作用的影响,用KLF4和Smad2表达质粒共转染293A细胞后,再以2 ng/ml TGF-β1刺激细胞40 min。报告基因分析结果显示,TGF-β1处理可增加KLF4与Smad2相互作用及其对TβRI启动子的激活作用。Smad和p38 MAPK抑制剂能有效阻断TGF-β1对TβRI启动子的激活。这些结果表明,TGF-β1通过激活Smad和p38 MAPK信号通路而促进KLF4与Smad2/3的相互作用及其对TβRI启动子的反式激活功能。
3. KLF4与Smad2协同激活TβRI启动子的作用机制
对TβRI基因启动子区域的核苷酸序列进行计算机分析的结果表明,TβRI启动子区存在KLF4(CACCC)和Smad(CAGAC)结合位点。本部分实验研究KLF4和Smad2与其结合位点的相互作用,进一步揭示KLF4和Smad2对TβRI基因表达的调控方式。
3.1 KLF4直接结合于TβRI启动子区的CACCC元件上并激活TβRI基因转录
TβRI启动子指导的报告基因分析结果显示,KLF4以剂量依赖的方式增强TβRI启动子的活性。染色质免疫沉淀实验(ChIP)结果显示,内源性和外源性的KLF4均可结合到TβRI启动子上。缺失TβRI启动子区的KLF4结合位点及突变KLF4结合位点后进行报告基因分析的结果表明,KLF4通过与TβRI启动子近端的两个KLF4结合位点:即KLF4结合位点2(-402~-398,CACCC)和位点3(-343~-339,CACCC)相互作用而发挥转录激活作用。Oligo pull-down分析结果进一步证实,KLF4直接结合于TβRI启动子区的KLF4结合位点2和3上。
3.2 Smad2在TGF-β1诱导TβRI表达中的作用
Smad是TGF-β1信号转导途径中的重要成员。上述研究结果表明,KLF4可直接激活TβRI基因的表达,但与KLF4相互作用的Smad2和Smad3是否影响TβRI基因的表达,目前尚不清楚。
本部分实验将靶向Smad2或Smad3的siRNA导入VSMC,阻断内源性Smad2或Smad3的表达后,检查Smad2或Smad3在TGF-β1诱导TβRI表达中的作用。Western blot分析结果显示,转染Smad2或Smad3的siRNA在敲低Smad2或Smad3表达的同时,也显著降低TβRI基因的表达。以Smad2表达载体pcDNA3.0-Smad2转染VSMC,证实Smad2在VSMC中的过表达能显著上调TβRI的表达。这些结果提示,Smad2和Smad3与TβRI的表达呈正相关。
3.3 Smad2通过与TβRI启动子区的Smad结合元件相互作用而激活基因表达
TβRI启动子指导的报告基因分析结果表明,Smad2也能以剂量依赖性方式增强TβRI启动子的活性。ChIP分析结果显示,在用Smad2抗体沉淀的VSMC染色质片段中可以扩增得到TβRI基因启动子区的Smad(-806~-561)结合区域,TGF-β1处理使Smad2与DNA的结合显著增加。为了定位TβRI启动子区的Smad2结合元件,进一步对截短的TβRI启动子进行报告基因分析。结果表明,Smad2只能激活含TβRI转录起始点上游-993bp的TβRI启动子,不能激活截短的、缺失-806~-561核苷酸序列的TβRI启动子。结果提示,Smad结合元件位于TβRI启动子区的-806~-561区域。
3.4 KLF4与预先结合在Smad结合位点上的Smad2协同激活TβRI基因转录
交互ChIP实验结果显示,在用KLF4抗体沉淀的VSMC染色质片段中既能扩增得到TβRI基因启动子区的KLF4结合序列(-483~-241),又能扩增得到Smad结合序列(-806~-561),而在Smad2抗体沉淀的VSMC染色质片段中,只能扩增到TβRI基因启动子区的Smad结合序列,不能扩增得到KLF4的结合序列。这些结果表明,KLF4-Smad2复合物位于Smad结合区。连续ChIP分析进一步显示,在用Smad2抗体再次沉淀KLF4抗体沉淀得到的染色质片段时,二次沉淀物中仍能扩增得到Smad结合序列。以上结果再次表明,KLF4与Smad2在TβRI基因启动子的Smad结合区形成复合物。
为了进一步证实在Smad结合区形成的KLF4-Smad2复合物能协同激活TβRI基因的转录,对缺失KLF4或Smad2结合位点的TβRI启动子进行报告基因分析。结果表明,缺失Smad结合区域(-806~-561)后,Smad2的转录激活作用以及Smad2和KLF4的协同激活作用消失。用Smad2-siRNA靶向敲低Smad2表达后进行报告基因分析的结果表明,敲低Smad2的表达也消弱了KLF4的转录激活作用,说明KLF4的激活作用部分依赖于与Smad结合区结合的Smad2。以上结果表明,KLF4与Smad2在Smad结合区形成复合物协同激活TβRI基因的转录。
结论
1. TGF-β1通过KLF4介导的TβRI表达抑制血管平滑肌细胞周期的进程并促进分化。
2. TGF-β1通过激活Smad和p38 MAPK信号途径诱导KLF4的磷酸化,磷酸化型KLF4与Smad2/3的相互作用增强。
3. KLF4直接结合于TβRI启动子区KLF4结合位点2(-402~-398,CACCC)和结合位点3(-343~-339,CACCC)并转录激活TβRI基因的表达,Smad2通过与Smad结合元件相互作用激活TβRI基因的表达。
4. KLF4与预先结合在Smad结合元件上的Smad2形成复合物,协同激活TβRI基因的表达。
Proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of a variety of proliferative vascular diseases, such as atherosclerosis, restenosis and hypertension. Several lines of evidence have shown that the factors controlling VSMC proliferation and growth inhibition, including various growth factors, signal transduction molecules and transcription factors constitute a complex functional network. Krüppel-like factors (KLFs), retinoic acid receptor-alpha (RARα), platelet-derived growth factor receptor (PDGFR), and transforming growth factor-βtype I receptor (TβRI) and type II receptor (TβRII) are expressed in VSMCs and are components of such a network. Transforming growth factor-β1 (TGF-β1) signaling is involved in the regulation of cell growth, differentiation, apoptosis, cellular homeostasis and other cellular functions. Despite numerous signaling studies, however, the specific contribution of TGF-βsignaling to VSMC phenotypic modulation has not been fully elucidated.
Krüppel-like factor 4 (KLF4, GKLF) is a member of Sp1 transcription factor family characterized by three C2H2 zinc finger motifs, mainly involved in regulating cell growth, differentiation, proliferation and apoptosis, by controlling the expression of a large number of genes with GC/GT-rich promoters. It has been demonstrated that KLF4 might function as a pleiotropic factor depending on the interaction partner(s) and the cellular context in VSMCs. However, the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition is not fully understood
To elucidate whether and how TGF-β-mediated KLF4 expression regulates TGF-βsignaling in VSMCs, we detected the effects of TGF-β1 on VSMC proliferation, growth inhibition and expression of marker genes. We further detected the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition.
1. KLF4 mediates TGF-β1-induced VSMC differentiation via TβRI
TGF-β1 treatment of VSMCs either stimulates proliferation or induces differentiation, depending on the experimental conditions. Thus, using cultured rat VSMCs, we examined the effect of TGF-β1 on the proliferation, cell cycle and differentiated marker genes of VSMCs and the role of KLF4 in TGF-β-induced VSMC differentiation.
1.1 TGF-β1 inhibits proliferation, migration and cell cycle of VSMCs
The results of MTT and migration assays showed that treatment of VSMCs with TGF-β1 resulted in a significant reduction of cell proliferation and migration in a dose-dependent (0.5, 1, 2, 4 ng/ml) and time-dependent (24, 48 h) manner. A significant inhibiting effect was observed by treating VSMCs with 2 ng/ml of TGF-β1 for 48 h.
G1-S switch is a key point in cell proliferation, so we examined the alteration of VSMC cell cycle by flow cytometry. Cell cycle analysis showed that the G0/G1 cell population was increased to 42% after TGF-β1 treatment for 48 h, with only 15% of untreated cells being arrested in the G0/G1 phase of the cell cycle. In addition, there was a reduction in cell proportion in S phase and G2/M phase after TGF-β1 treatment.
1.2 TGF-β1 enhances the expression of differentiation marker genes, and simultaneously suppresses the expression of dedifferentiation marker gene
SM22αand SMα-actin highly expressed in differentiated VSMCs are the differentiated marker genes, while PCNA (proliferating cell nuclear antigen) is the VSMC-dedifferentiated marker. Western blot showed that treated with 2 ng/ml of TGF-β1 from 0 to 48 h increased expression of SM22αand SMα-actin, while decreasing the PCNA levels in a time-dependent manner. In addition, when treatment with a range of TGF-β1 concentrations from 0 to 4 ng/ml for 48 h resulted in increased the level of SM22αand SMα-actin in a dose-dependent manner, and decreased the level of PCNA in VSMCs.
1.3 TβRI expression is closely linked to VSMC differentiation by TGF-β1
RNA interference was used to silence the expression of TβRI and to assess its effects on TGF-β1-induced VSMC differentiation. Measurements of protein expression showed that the targeted TβRI siRNA knocked down between 75% and 80% of cellular TβRI that was induced by TGF-β1. Transfection with TβRI siRNA reduced SM22αand SMα-actin protein levels while upregulating PCNA expression in TGF-β1-treated cells.
1.4 KLF4 mediates TGF-β1-induced TβRI expression and VSMC differentiation
To confirm that KLF4 is involved in the regulation of VSMC differentiation we examined the effect of TGF-β1 on KLF4 protein expression. The results of Western blot analysis showed that TGF-β1 (2 ng/ml) time-dependently induced KLF4 levels, reaching a maximum after 6 h before returning to basal levels by 48 h of treatment with TGF-β1. A similar pattern of TβRI expression following TGF-β1 stimulation was observed. Measurements of mRNA levels by semi-quantitative RT-PCR showed comparable changes in KLF4 and TβRI expression in response to TGF-β1.
To assess that KLF4 mediates TGF-β1-induced TβRI expression in VSMCs, KLF4 siRNA or Ad-KLF4 was transfected into VSMCs. Transfection of VSMCs with KLF4 siRNA resulted in the suppression of TβRI mRNA and protein levels. Correspondingly, a reduction was observed in TGF-β1-induced expression of SM22αand SMα-actin proteins in response to KLF4 silencing. Conversely, overexpression of KLF4 markedly induced expression of TβRI, SM22αand SMα-actin.
2. TGF-β1 induces KLF4 phosphorylation and its interaction with Smad2/3 via Smad and p38 MAPK signaling in VSMCs
The role of KLF family in the regulation of TGF-βsignaling has been the subject of considerable investigation. However, the precise mechanism whereby KLF4 regulates TGF-βsignaling in VSMCs is still poorly understood. Having identifed TGF-βregulates KLF4, we investigated whether there exists an interesting possibility that one of the TGF-β-activated pathways regulates KLF4.
2.1 TGF-β1 induces KLF4 phosphorylation via Smad and p38 MAPK signaling in VSMCs
The potential role of KLF4 in TGF-β1 signaling and function was investigated by determining KLF4 phosphorylation in VSMCs in response to TGF-β1. This experiment showed that TGF-β1 increased phosphorylation of KLF4 within 5 min, reaching a maximum between 20 and 40 min. Under these experimental conditions, the phosphorylation of TβRI, Smad2, Smad3 and p38 MAPK increased concurrently in a time-dependent manner, whereas ERK phosphorylation was negatively affected by TGF-β1.
To assess the importance of Smad and p38 MAPK signaling in promoting KLF4 phosphorylation by TGF-β1, the effects of SB431542 and SB203580 on KLF4 phosphorylation induced by TGF-β1 were detected. Pharmacological inhibition of TβRI or p38 MAPK blocked TGF-β1-induced KLF4 phosphorylation. Moreover, silencing Smad2, Smad3 or p38αMAPK blocked TGF-β1-induced KLF4 phosphorylation.
2.2 KLF4 phosphorylation by TGF-β1 increases the interaction between KLF4 and Smad2/3
Because TGF-β1 induced KLF4 phosphorylation, experiments were conducted to determine whether KLF4 phosphorylation affected its ability to interact with Smad2 or Smad3 in VSMCs. Co-immunoprecipitation experiments confirmed that TGF-β1 time-dependently increased the interaction of Smad2 and Smad3 with KLF4. SB431542 or SB203580 suppressed TGF-β1-dependent recruitment of KLF4 with Smad2 and Smad3. Correspondingly, siRNA-mediated knockdown of p38αMAPK, Smad2 or Smad3 also decreased the interaction of KLF4 with Smad2/3 in TGF-β1-treated cells.
To verify that the KLF4-Smad interaction is elicited by KLF4 phosphorylation, point-mutation experiments were conducted. Mutation within the DNA-binding domain at 470 showed that the Ser470Ala substitution markedly reduced TGF-β1-dependent KLF4 phosphorylation; this was accompanied by impaired co-sedimentation with Smad2/3.
2.3 KLF4 and Smad2 cooperatively activate the TβRI promoter
After having demonstrated that KLF4 mediates TGF-β1-induced TβRI expression through interaction with Smad2/3, we then examined whether KLF4-Smad2 association plays a role in the activation of the TβRI promoter. Gene reporter assay showed that co-expression KLF4 and Smad2 induced an ~19-fold increase in luciferase activity.
A further induction in TβRI promoter activity was observed when the cells cotransfected with KLF4 and Smad2 expression plasmids were stimulated with TGF-β1. While, SB431542, SB203580 or a combination of both, significantly reduced the reporter gene activity in the TGF-β1-treated 293A cotransfected cells.
3. Synergistic cooperation of KLF4 with Smad2 in TGF-β1-induced TβRI gene activation in VSMCs
Since KLF4 mediates TβRI expression, we hypothesized that KLF4 enhances TGF-βsignaling via TβRI. Since it has been demonstrated that KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains KLF4-binding sites (CACCC) and the -806/-561 bp region contains Smad-responsive region (CAGAC).
3.1 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter
HEK293A cells were transfected with a TβRI promoter-reporter plasmid and increasing amounts of KLF4 expression plasmids. Measurements of luciferase activity showed that overexpression of KLF4 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.
Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains two typical KLF4-binding sites (CACCC) and one with a reverse orientation sequence (GGGTG). Chromatin immunoprecipitation studies showed that KLF4 specifically interacts with TGF-β1-responsive sites of TβRI promoter. To determine which DNA elements are involved in the KLF4 induction of TβRI, a series of 5'-deletion mutants and KLF4-binding site mutants were tested in 293A cells co-transfected with pEGFP-KLF4. Measurements of luciferase activity suggested that the KLF4-responsive elements are located in the -453/+21 region in the TβRI promoter.
To test the role of these putative KLF4-binding sites in TβRI induction by KLF4, reporter plasmids containing TβRI promoter with site-specific mutations were analyzed. Disruption of KLF4-binding site 1 (-472/-468 bp region) did not affect the transcriptional induction by KLF4, whereas disruption of site 2 (-402/-398 bp region) or 3 (-343/-339 bp region) reduced significantly the KLF4’s transcriptional response. To verify the binding activities of the KLF4-binding sites 2 and 3, Oligonucleotide pull-down assays were performed. The same pattern of KLF4 protein bands was observed with both oligonucleotide probes. Extracts from cells treated with TGF-β1 showed stronger KLF4 binding to the two probes by ~2-fold.
3.2 Effect of Smad2 on TGF-β1-induced TβRI expression
Smads are central mediators of TβR signals, so we determined whether TGF-β1-dependent induction of TβRI levels is influenced by inhibition of Smad2 and/or Smad3. Silencing of either Smad2 or Smad3 markedly suppressed TβRI up-regulation by TGF-β1. Conversely, overexpression of Smad2 markedly induced expression of TβRI.
3.3 The Smad-responsive element is responsible for Smad2 transactivation on the TβRI promoter
We examined whether Smad2 plays a role in the activation of the TβRI promoter by luciferase reporter gene assay. Measurements of luciferase activity showed that overexpression of Smad2 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.
Because Smad2 could activate the TβRI promoter, we want to determine whether Smad2 binds directly to this promoter. ChIP assays showed that constitutive Smad2 binds to the TβRI promoter. The TGF-β1 treatment increased Smad2 binding by ~2.2-fold to the putative Smad-binding region as compared to untreated cells.
To define which DNA elements are involved in the Smad2 induction of TβRI, a series of 5'-deletion mutants were tested in 293 cells co-transfected with pcDNA-Smad2. pTβRI/-993 elicited a strong transcriptional response by Smad2, whereas pTβRI/-453 and pTβRI/-386 were unresponsive to Smad2.
3.4 Interaction of KLF4 with Smad2 bound to the Smad-binding element provides cooperative activation of the TβRI promoter
Since KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. ChIP and two-step ChIP assay showed that KLF4 forms a stable complex with Smad2 that is bound to the Smad-binding region of the TβRI promoter.
To further determine the role of the Smad-binding region in the cooperative transcriptional induction of TβRI by KLF4 and Smad2, 293A cells were cotransfected with pTβRI/-993, pTβRI/-993(?-483/-241) or pTβRI/-993(?-806/-561) together with empty vector or the expression vectors encoding KLF4 and Smad2. Measurements of luciferase showed that deletion of the -483/-241 KLF4-binding region abolished KLF4’s transcriptional response while maintaining Smad2 responsiveness and the cooperative activation by Smad2 and KLF4. In contrast, deletion of the -806/-561 Smad2-binding motif had no inhibitory effect on the transcriptional induction of TβRI promoter by KLF4; however, both the Smad2 responsiveness and cooperative activation by Smad2 and KLF4 were lost. Moreover, Knockdown of Smad2 with siRNA abolished the additive effect in cells cotransfected with Smad2 and KLF4 expression plasmids.
CONCLUSIONS
1 TGF-β1 inhibits cell cycle progression and induces differentiation in cultured rat VSMCs. This activity of TGF-β1 is accompanied by upregulation of KLF4, with concomitant increase in TβRI expression.
2 KLF4 is found to transduce TGF-β1 signals via phosphorylation-mediated activation of Smad2, Smad3 and p38 MAP kinase. The activation of both pathways, in turn, increases the phosphorylation of KLF4, which enable the formation of KLF4-Smad2 complex in response to TGF-β1.
3 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter and Smad2 recruits to the Smad-responsive region to activate TβRI promoter.
4 Formation of a stable KLF4-Smad2 complex to the promoter’s Smad-responsive region mediates cooperative TβRI promoter transcription in response to TGF-β1.
Krüppel样因子(krüppel-like factor,KLF)是一类含有锌指结构的转录因子,其典型结构特征是在羧基端具有3个C2H2锌指结构。KLF广泛参与细胞增殖、凋亡、分化以及胚胎发育等生命活动的调控。近年来的研究结果显示,KLF4(krüppel-like factor 4)作为转录因子既可直接结合DNA,又能通过与其它转录因子相互作用而调控基因的表达。以往研究证实,TGF-β1可诱导KLF4表达并促进体外培养的去分化型VSMC进行分化,但KLF4在TGF-β诱导VSMC分化中的作用及机制尚不清楚。
为了阐明KLF4在TGF-β诱导VSMC分化中的作用及其分子机制,本文在系统研究TGF-β1对VSMC增殖、分化、迁移、细胞周期等生物学行为影响的基础上,进一步探讨KLF4在TGF-β1促进VSMC分化中的作用和分子机制。旨在为揭示动脉粥样硬化、高血压和血管再狭窄等心血管疾病的发病机制提供新的实验和理论依据,为心血管疾病的防治提供新的靶点。
1. TGF-β1诱导VSMC分化与上调KLF4和TβRI表达有关
本部分实验以VSMC分化和去分化标志基因表达水平作为判断VSMC表型的指标,检测TGF-β1对VSMC表型及相关生物学行为的影响。为了考察TβRI是否参与TGF-β1诱导VSMC分化的过程,一方面,观察敲减TβRI基因表达对VSMC分化和去分化标志基因表达的影响;另一方面,观察KLF4过表达和敲减对TβRI表达以及对VSMC分化和去分化标志基因表达的影响。实验结果如下:
1.1 TGF-β1抑制VSMC增殖、迁移和细胞周期进程
MTT分析、细胞计数、伤口愈合实验结果显示,TGF-β1能以浓度(0.5、1、2、4 ng/ml)和时间(24、48 h)依赖的方式抑制VSMC增殖和迁移,以TGF-β1浓度为2 ng/ml作用于VSMC 48 h对细胞增殖和迁移的抑制作用最强。
流式细胞术检测结果显示,TGF-β1能以时间(6、12、24、48 h)依赖的方式抑制VSMC细胞周期的进程。TGF-β1(2 ng/ml)刺激VSMC 48 h后,停滞于G0/G1期的细胞数显著增加,同时处于S期和G2/M期的细胞数明显减少。
1.2 TGF-β1诱导VSMC分化标志基因SM22α和SMα-actin表达
SM22α和SMα-actin表达是VSMC处于分化状态的重要分子标志, PCNA表达是VSMC处于增殖状态的重要标志。采用Western blot方法,检查TGF-β1对各种标志基因表达的影响。结果显示,在TGF-β1处理的VSMC中,分化标志基因SM22α和SMα-actin表达明显升高,而增殖标志物PCNA表达显著降低,这些基因的表达变化依赖于TGF-β1作用的时间和浓度。其中,浓度为2 ng/ml和作用时间为48 h时变化最为显著。
1.3敲减TβRI表达对VSMC分化和去分化标志基因表达的影响
将靶向TβRI的小干扰RNA(small interfering RNA,siRNA)导入VSMC,阻断内源性TβRI表达后,研究TβRI在TGF-β1诱导VSMC分化中的作用。Western blot结果显示,转染TβRI-siRNA可使TβRI mRNA和蛋白表达水平降低约75~80%,表明该基因的表达被有效敲低。在TGF-β1刺激下,转染TβRI-siRNA的VSMC中SM22α和SMα-actin的表达明显降低,相反,增殖标志物PCNA的表达显著增加。
1.4 KLF4在TGF-β1诱导TβRI表达及VSMC分化中的作用
虽然已经发现KLF4是参与细胞生长、分化和凋亡的调控,但KLF4是否通过TβRI介导TGF-β1的作用目前尚不清楚。
RT-PCR和Western blot结果表明,TGF-β1显著诱导KLF4和TβRI基因的转录和蛋白表达,并具有明显的时-效(6、12、24、48 h)关系。2 ng/ml TGF-β1作用于VSMC 6 h后,KLF4和TβRI的mRNA和蛋白表达水平均显著升高,两者的表达变化呈正相关关系。
为了阐明KLF4与TβRI表达之间的关系,将KLF4-siRNA导入VSMC,阻断内源性KLF4表达后,明确KLF4在TGF-β1诱导TβRI及VSMC分化标志基因表达中的作用。RT-PCR和Western blot分析结果显示,转染KLF4-siRNA可敲减KLF4表达60%以上。在敲低KLF4表达的VSMC中,TGF-β1诱导TβRI表达的作用被取消,分化标志基因SM22α和SMα-actin的表达也明显下降,表明KLF4介导TGF-β1对TβRI表达的诱导作用。
另外,将KLF4腺病毒表达载体Ad-KLF4感染VSMC,观察强制表达KLF4对TβRI和VSMC分化标志基因表达的影响。RT-PCR和Western blot分析证实,在KLF4过表达的VSMC中,TβRI表达明显高于空载体对照组,与此同时,分化标志基因SM22α和SMα-actin的表达也显著升高。
2. TGF-β1通过激活Smad和p38 MAPK信号通路诱导KLF4磷酸化和KLF4与Smad2/3相互作用
TGF-β1通过与VSMC上的相应受体相互作用而触发细胞信号的跨膜转导,进而活化Smad信号通路而激活相关基因表达。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联反应是胞内信号进行交互作用的重要信号转导通路。在上述阐明KLF4通过TβRI介导TGF-β诱导VSMC分化的基础上,本部分实验探讨TGF-β1对Smad和MAPK信号途径的影响,以期阐明TGF-β1诱导KLF4活化和促进细胞分化的信号转导机制。
2.1 TGF-β1通过激活Smad和p38 MAPK信号途径诱导KLF4磷酸化
VSMC经TGF-β1(2 ng/ml)刺激5、10、20、40 min,用磷酸化丝氨酸抗体对细胞裂解液进行免疫沉淀后检测磷酸化KLF4的水平。结果显示,随着TGF-β1刺激时间的延长,磷酸化型KLF4水平逐渐升高,在TGF-β1刺激20~40 min内,其磷酸化水平增加2倍;同时,TGF-β1刺激后,磷酸化型Smad2(p-Smad2)、Smad3(p-Smad3)和p38 MAPK(p-p38)水平也显著升高。
分别以TβRI特异性抑制剂SB431542、p38特异性抑制剂SB203580和ERK特异性抑制剂PD98059处理VSMC,特异性阻断Smad、p38或ERK信号通路的活化后,再以2 ng/ml的TGF-β1刺激细胞40 min。Western blot结果显示,SB431542或SB203580预处理细胞可显著抑制KLF4磷酸化,而PD98059对KLF4磷酸化无明显影响。用靶向Smad2、Smad3或p38 MAPK的siRNA敲低内源性Smad2、Smad3或p38 MAPK表达后,TGF-β1对KLF4磷酸化不再产生明显影响。证明TGF-β1诱导的KLF4磷酸化依赖于Smad和p38 MAPK信号途径的激活。
2.2 TGF-β1通过诱导KLF4磷酸化而促进KLF4与Smad2/3相互作用
免疫共沉淀分析结果显示,VSMC被TGF-β1处理后,KLF4与Smad2/3相互作用形成的复合物明显增加。VSMC在TGF-β1(2 ng/ml)刺激5、10、20、40 min后,随着刺激时间的延长,KLF4与Smad2/3的结合明显增加,交互免疫沉淀也得到相似的结果。表明,TGF-β1能显著促进KLF4与Smad2/3的相互作用,并具有明显的时效关系。
为了检查KLF4磷酸化是否影响其与Smad2/3的相互作用,以SB431542、SB203580和PD98059处理VSMC,特异性阻断Smad、p38或ERK信号通路的活化后,再以2 ng/ml TGF-β1刺激细胞40 min。免疫共沉淀分析结果显示,在SB431542和SB203580预孵育的VSMC中,KLF4与Smad2/3的相互作用明显降低,交互免疫沉淀得到相似的结果。用靶向Smad2、Smad3或p38 MAPK的siRNA敲低内源性Smad2、Smad3或p38 MAPK表达后,TGF-β1对KLF4与Smad2/3的相互作用不再产生明显影响。上述结果表明,TGF-β1通过Smad和p38 MAPK信号途径诱导KLF4发生磷酸化修饰,而磷酸化型KLF4与Smad2/3的结合活性明显增加。
KLF4磷酸化位点定点突变实验证明,转染KLF4磷酸化位点(S470A)突变体的细胞再用TGF-β1处理时,KLF4的磷酸化水平明显降低,KLF4与Smad2/3的相互作用较对照组(转染野生型KLF4表达载体)降低。这些结果进一步提示,TGF-β1通过诱导KLF4磷酸化而促进KLF4与Smad2/3之间的相互作用。
2.3 KLF4与Smad2协同激活TβRI基因表达
上述实验证实,TGF-β1促进KLF4与Smad2/3相互作用。为了检查KLF4与Smad2/3相互作用在调节TβRI表达中的意义,我们首先进行了报告基因分析。将TβRI启动子指导的报告基因分别与KLF4或Smad2表达质粒共转染293A细胞时,KLF4和Smad2均能促进TβRI启动子的转录活性。KLF4和Smad2共转染使TβRI启动子活性比转染其中一种表达质粒升高1倍左右。表明KLF4与Smad2协同激活TβRI基因表达。
为了进一步检查TGF-β1对KLF4与Smad2相互作用的影响,用KLF4和Smad2表达质粒共转染293A细胞后,再以2 ng/ml TGF-β1刺激细胞40 min。报告基因分析结果显示,TGF-β1处理可增加KLF4与Smad2相互作用及其对TβRI启动子的激活作用。Smad和p38 MAPK抑制剂能有效阻断TGF-β1对TβRI启动子的激活。这些结果表明,TGF-β1通过激活Smad和p38 MAPK信号通路而促进KLF4与Smad2/3的相互作用及其对TβRI启动子的反式激活功能。
3. KLF4与Smad2协同激活TβRI启动子的作用机制
对TβRI基因启动子区域的核苷酸序列进行计算机分析的结果表明,TβRI启动子区存在KLF4(CACCC)和Smad(CAGAC)结合位点。本部分实验研究KLF4和Smad2与其结合位点的相互作用,进一步揭示KLF4和Smad2对TβRI基因表达的调控方式。
3.1 KLF4直接结合于TβRI启动子区的CACCC元件上并激活TβRI基因转录
TβRI启动子指导的报告基因分析结果显示,KLF4以剂量依赖的方式增强TβRI启动子的活性。染色质免疫沉淀实验(ChIP)结果显示,内源性和外源性的KLF4均可结合到TβRI启动子上。缺失TβRI启动子区的KLF4结合位点及突变KLF4结合位点后进行报告基因分析的结果表明,KLF4通过与TβRI启动子近端的两个KLF4结合位点:即KLF4结合位点2(-402~-398,CACCC)和位点3(-343~-339,CACCC)相互作用而发挥转录激活作用。Oligo pull-down分析结果进一步证实,KLF4直接结合于TβRI启动子区的KLF4结合位点2和3上。
3.2 Smad2在TGF-β1诱导TβRI表达中的作用
Smad是TGF-β1信号转导途径中的重要成员。上述研究结果表明,KLF4可直接激活TβRI基因的表达,但与KLF4相互作用的Smad2和Smad3是否影响TβRI基因的表达,目前尚不清楚。
本部分实验将靶向Smad2或Smad3的siRNA导入VSMC,阻断内源性Smad2或Smad3的表达后,检查Smad2或Smad3在TGF-β1诱导TβRI表达中的作用。Western blot分析结果显示,转染Smad2或Smad3的siRNA在敲低Smad2或Smad3表达的同时,也显著降低TβRI基因的表达。以Smad2表达载体pcDNA3.0-Smad2转染VSMC,证实Smad2在VSMC中的过表达能显著上调TβRI的表达。这些结果提示,Smad2和Smad3与TβRI的表达呈正相关。
3.3 Smad2通过与TβRI启动子区的Smad结合元件相互作用而激活基因表达
TβRI启动子指导的报告基因分析结果表明,Smad2也能以剂量依赖性方式增强TβRI启动子的活性。ChIP分析结果显示,在用Smad2抗体沉淀的VSMC染色质片段中可以扩增得到TβRI基因启动子区的Smad(-806~-561)结合区域,TGF-β1处理使Smad2与DNA的结合显著增加。为了定位TβRI启动子区的Smad2结合元件,进一步对截短的TβRI启动子进行报告基因分析。结果表明,Smad2只能激活含TβRI转录起始点上游-993bp的TβRI启动子,不能激活截短的、缺失-806~-561核苷酸序列的TβRI启动子。结果提示,Smad结合元件位于TβRI启动子区的-806~-561区域。
3.4 KLF4与预先结合在Smad结合位点上的Smad2协同激活TβRI基因转录
交互ChIP实验结果显示,在用KLF4抗体沉淀的VSMC染色质片段中既能扩增得到TβRI基因启动子区的KLF4结合序列(-483~-241),又能扩增得到Smad结合序列(-806~-561),而在Smad2抗体沉淀的VSMC染色质片段中,只能扩增到TβRI基因启动子区的Smad结合序列,不能扩增得到KLF4的结合序列。这些结果表明,KLF4-Smad2复合物位于Smad结合区。连续ChIP分析进一步显示,在用Smad2抗体再次沉淀KLF4抗体沉淀得到的染色质片段时,二次沉淀物中仍能扩增得到Smad结合序列。以上结果再次表明,KLF4与Smad2在TβRI基因启动子的Smad结合区形成复合物。
为了进一步证实在Smad结合区形成的KLF4-Smad2复合物能协同激活TβRI基因的转录,对缺失KLF4或Smad2结合位点的TβRI启动子进行报告基因分析。结果表明,缺失Smad结合区域(-806~-561)后,Smad2的转录激活作用以及Smad2和KLF4的协同激活作用消失。用Smad2-siRNA靶向敲低Smad2表达后进行报告基因分析的结果表明,敲低Smad2的表达也消弱了KLF4的转录激活作用,说明KLF4的激活作用部分依赖于与Smad结合区结合的Smad2。以上结果表明,KLF4与Smad2在Smad结合区形成复合物协同激活TβRI基因的转录。
结论
1. TGF-β1通过KLF4介导的TβRI表达抑制血管平滑肌细胞周期的进程并促进分化。
2. TGF-β1通过激活Smad和p38 MAPK信号途径诱导KLF4的磷酸化,磷酸化型KLF4与Smad2/3的相互作用增强。
3. KLF4直接结合于TβRI启动子区KLF4结合位点2(-402~-398,CACCC)和结合位点3(-343~-339,CACCC)并转录激活TβRI基因的表达,Smad2通过与Smad结合元件相互作用激活TβRI基因的表达。
4. KLF4与预先结合在Smad结合元件上的Smad2形成复合物,协同激活TβRI基因的表达。
Proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of a variety of proliferative vascular diseases, such as atherosclerosis, restenosis and hypertension. Several lines of evidence have shown that the factors controlling VSMC proliferation and growth inhibition, including various growth factors, signal transduction molecules and transcription factors constitute a complex functional network. Krüppel-like factors (KLFs), retinoic acid receptor-alpha (RARα), platelet-derived growth factor receptor (PDGFR), and transforming growth factor-βtype I receptor (TβRI) and type II receptor (TβRII) are expressed in VSMCs and are components of such a network. Transforming growth factor-β1 (TGF-β1) signaling is involved in the regulation of cell growth, differentiation, apoptosis, cellular homeostasis and other cellular functions. Despite numerous signaling studies, however, the specific contribution of TGF-βsignaling to VSMC phenotypic modulation has not been fully elucidated.
Krüppel-like factor 4 (KLF4, GKLF) is a member of Sp1 transcription factor family characterized by three C2H2 zinc finger motifs, mainly involved in regulating cell growth, differentiation, proliferation and apoptosis, by controlling the expression of a large number of genes with GC/GT-rich promoters. It has been demonstrated that KLF4 might function as a pleiotropic factor depending on the interaction partner(s) and the cellular context in VSMCs. However, the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition is not fully understood
To elucidate whether and how TGF-β-mediated KLF4 expression regulates TGF-βsignaling in VSMCs, we detected the effects of TGF-β1 on VSMC proliferation, growth inhibition and expression of marker genes. We further detected the actual relationship between KLF4 and TβR in the regulation of VSMC proliferation and growth inhibition.
1. KLF4 mediates TGF-β1-induced VSMC differentiation via TβRI
TGF-β1 treatment of VSMCs either stimulates proliferation or induces differentiation, depending on the experimental conditions. Thus, using cultured rat VSMCs, we examined the effect of TGF-β1 on the proliferation, cell cycle and differentiated marker genes of VSMCs and the role of KLF4 in TGF-β-induced VSMC differentiation.
1.1 TGF-β1 inhibits proliferation, migration and cell cycle of VSMCs
The results of MTT and migration assays showed that treatment of VSMCs with TGF-β1 resulted in a significant reduction of cell proliferation and migration in a dose-dependent (0.5, 1, 2, 4 ng/ml) and time-dependent (24, 48 h) manner. A significant inhibiting effect was observed by treating VSMCs with 2 ng/ml of TGF-β1 for 48 h.
G1-S switch is a key point in cell proliferation, so we examined the alteration of VSMC cell cycle by flow cytometry. Cell cycle analysis showed that the G0/G1 cell population was increased to 42% after TGF-β1 treatment for 48 h, with only 15% of untreated cells being arrested in the G0/G1 phase of the cell cycle. In addition, there was a reduction in cell proportion in S phase and G2/M phase after TGF-β1 treatment.
1.2 TGF-β1 enhances the expression of differentiation marker genes, and simultaneously suppresses the expression of dedifferentiation marker gene
SM22αand SMα-actin highly expressed in differentiated VSMCs are the differentiated marker genes, while PCNA (proliferating cell nuclear antigen) is the VSMC-dedifferentiated marker. Western blot showed that treated with 2 ng/ml of TGF-β1 from 0 to 48 h increased expression of SM22αand SMα-actin, while decreasing the PCNA levels in a time-dependent manner. In addition, when treatment with a range of TGF-β1 concentrations from 0 to 4 ng/ml for 48 h resulted in increased the level of SM22αand SMα-actin in a dose-dependent manner, and decreased the level of PCNA in VSMCs.
1.3 TβRI expression is closely linked to VSMC differentiation by TGF-β1
RNA interference was used to silence the expression of TβRI and to assess its effects on TGF-β1-induced VSMC differentiation. Measurements of protein expression showed that the targeted TβRI siRNA knocked down between 75% and 80% of cellular TβRI that was induced by TGF-β1. Transfection with TβRI siRNA reduced SM22αand SMα-actin protein levels while upregulating PCNA expression in TGF-β1-treated cells.
1.4 KLF4 mediates TGF-β1-induced TβRI expression and VSMC differentiation
To confirm that KLF4 is involved in the regulation of VSMC differentiation we examined the effect of TGF-β1 on KLF4 protein expression. The results of Western blot analysis showed that TGF-β1 (2 ng/ml) time-dependently induced KLF4 levels, reaching a maximum after 6 h before returning to basal levels by 48 h of treatment with TGF-β1. A similar pattern of TβRI expression following TGF-β1 stimulation was observed. Measurements of mRNA levels by semi-quantitative RT-PCR showed comparable changes in KLF4 and TβRI expression in response to TGF-β1.
To assess that KLF4 mediates TGF-β1-induced TβRI expression in VSMCs, KLF4 siRNA or Ad-KLF4 was transfected into VSMCs. Transfection of VSMCs with KLF4 siRNA resulted in the suppression of TβRI mRNA and protein levels. Correspondingly, a reduction was observed in TGF-β1-induced expression of SM22αand SMα-actin proteins in response to KLF4 silencing. Conversely, overexpression of KLF4 markedly induced expression of TβRI, SM22αand SMα-actin.
2. TGF-β1 induces KLF4 phosphorylation and its interaction with Smad2/3 via Smad and p38 MAPK signaling in VSMCs
The role of KLF family in the regulation of TGF-βsignaling has been the subject of considerable investigation. However, the precise mechanism whereby KLF4 regulates TGF-βsignaling in VSMCs is still poorly understood. Having identifed TGF-βregulates KLF4, we investigated whether there exists an interesting possibility that one of the TGF-β-activated pathways regulates KLF4.
2.1 TGF-β1 induces KLF4 phosphorylation via Smad and p38 MAPK signaling in VSMCs
The potential role of KLF4 in TGF-β1 signaling and function was investigated by determining KLF4 phosphorylation in VSMCs in response to TGF-β1. This experiment showed that TGF-β1 increased phosphorylation of KLF4 within 5 min, reaching a maximum between 20 and 40 min. Under these experimental conditions, the phosphorylation of TβRI, Smad2, Smad3 and p38 MAPK increased concurrently in a time-dependent manner, whereas ERK phosphorylation was negatively affected by TGF-β1.
To assess the importance of Smad and p38 MAPK signaling in promoting KLF4 phosphorylation by TGF-β1, the effects of SB431542 and SB203580 on KLF4 phosphorylation induced by TGF-β1 were detected. Pharmacological inhibition of TβRI or p38 MAPK blocked TGF-β1-induced KLF4 phosphorylation. Moreover, silencing Smad2, Smad3 or p38αMAPK blocked TGF-β1-induced KLF4 phosphorylation.
2.2 KLF4 phosphorylation by TGF-β1 increases the interaction between KLF4 and Smad2/3
Because TGF-β1 induced KLF4 phosphorylation, experiments were conducted to determine whether KLF4 phosphorylation affected its ability to interact with Smad2 or Smad3 in VSMCs. Co-immunoprecipitation experiments confirmed that TGF-β1 time-dependently increased the interaction of Smad2 and Smad3 with KLF4. SB431542 or SB203580 suppressed TGF-β1-dependent recruitment of KLF4 with Smad2 and Smad3. Correspondingly, siRNA-mediated knockdown of p38αMAPK, Smad2 or Smad3 also decreased the interaction of KLF4 with Smad2/3 in TGF-β1-treated cells.
To verify that the KLF4-Smad interaction is elicited by KLF4 phosphorylation, point-mutation experiments were conducted. Mutation within the DNA-binding domain at 470 showed that the Ser470Ala substitution markedly reduced TGF-β1-dependent KLF4 phosphorylation; this was accompanied by impaired co-sedimentation with Smad2/3.
2.3 KLF4 and Smad2 cooperatively activate the TβRI promoter
After having demonstrated that KLF4 mediates TGF-β1-induced TβRI expression through interaction with Smad2/3, we then examined whether KLF4-Smad2 association plays a role in the activation of the TβRI promoter. Gene reporter assay showed that co-expression KLF4 and Smad2 induced an ~19-fold increase in luciferase activity.
A further induction in TβRI promoter activity was observed when the cells cotransfected with KLF4 and Smad2 expression plasmids were stimulated with TGF-β1. While, SB431542, SB203580 or a combination of both, significantly reduced the reporter gene activity in the TGF-β1-treated 293A cotransfected cells.
3. Synergistic cooperation of KLF4 with Smad2 in TGF-β1-induced TβRI gene activation in VSMCs
Since KLF4 mediates TβRI expression, we hypothesized that KLF4 enhances TGF-βsignaling via TβRI. Since it has been demonstrated that KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains KLF4-binding sites (CACCC) and the -806/-561 bp region contains Smad-responsive region (CAGAC).
3.1 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter
HEK293A cells were transfected with a TβRI promoter-reporter plasmid and increasing amounts of KLF4 expression plasmids. Measurements of luciferase activity showed that overexpression of KLF4 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.
Using the TESS computational program, we established that the -472/-339 bp region of the rat TβRI promoter contains two typical KLF4-binding sites (CACCC) and one with a reverse orientation sequence (GGGTG). Chromatin immunoprecipitation studies showed that KLF4 specifically interacts with TGF-β1-responsive sites of TβRI promoter. To determine which DNA elements are involved in the KLF4 induction of TβRI, a series of 5'-deletion mutants and KLF4-binding site mutants were tested in 293A cells co-transfected with pEGFP-KLF4. Measurements of luciferase activity suggested that the KLF4-responsive elements are located in the -453/+21 region in the TβRI promoter.
To test the role of these putative KLF4-binding sites in TβRI induction by KLF4, reporter plasmids containing TβRI promoter with site-specific mutations were analyzed. Disruption of KLF4-binding site 1 (-472/-468 bp region) did not affect the transcriptional induction by KLF4, whereas disruption of site 2 (-402/-398 bp region) or 3 (-343/-339 bp region) reduced significantly the KLF4’s transcriptional response. To verify the binding activities of the KLF4-binding sites 2 and 3, Oligonucleotide pull-down assays were performed. The same pattern of KLF4 protein bands was observed with both oligonucleotide probes. Extracts from cells treated with TGF-β1 showed stronger KLF4 binding to the two probes by ~2-fold.
3.2 Effect of Smad2 on TGF-β1-induced TβRI expression
Smads are central mediators of TβR signals, so we determined whether TGF-β1-dependent induction of TβRI levels is influenced by inhibition of Smad2 and/or Smad3. Silencing of either Smad2 or Smad3 markedly suppressed TβRI up-regulation by TGF-β1. Conversely, overexpression of Smad2 markedly induced expression of TβRI.
3.3 The Smad-responsive element is responsible for Smad2 transactivation on the TβRI promoter
We examined whether Smad2 plays a role in the activation of the TβRI promoter by luciferase reporter gene assay. Measurements of luciferase activity showed that overexpression of Smad2 dose-dependently increased by several fold the transcriptional activity of TβRI when compared with control plasmids.
Because Smad2 could activate the TβRI promoter, we want to determine whether Smad2 binds directly to this promoter. ChIP assays showed that constitutive Smad2 binds to the TβRI promoter. The TGF-β1 treatment increased Smad2 binding by ~2.2-fold to the putative Smad-binding region as compared to untreated cells.
To define which DNA elements are involved in the Smad2 induction of TβRI, a series of 5'-deletion mutants were tested in 293 cells co-transfected with pcDNA-Smad2. pTβRI/-993 elicited a strong transcriptional response by Smad2, whereas pTβRI/-453 and pTβRI/-386 were unresponsive to Smad2.
3.4 Interaction of KLF4 with Smad2 bound to the Smad-binding element provides cooperative activation of the TβRI promoter
Since KLF4 interaction with Smad2 cooperatively activates TβRI promoter, we hypothesized that KLF4 could form a stable complex with Smad2 either at the KLF4-binding region, Smad-responsive element or both of these two regions. ChIP and two-step ChIP assay showed that KLF4 forms a stable complex with Smad2 that is bound to the Smad-binding region of the TβRI promoter.
To further determine the role of the Smad-binding region in the cooperative transcriptional induction of TβRI by KLF4 and Smad2, 293A cells were cotransfected with pTβRI/-993, pTβRI/-993(?-483/-241) or pTβRI/-993(?-806/-561) together with empty vector or the expression vectors encoding KLF4 and Smad2. Measurements of luciferase showed that deletion of the -483/-241 KLF4-binding region abolished KLF4’s transcriptional response while maintaining Smad2 responsiveness and the cooperative activation by Smad2 and KLF4. In contrast, deletion of the -806/-561 Smad2-binding motif had no inhibitory effect on the transcriptional induction of TβRI promoter by KLF4; however, both the Smad2 responsiveness and cooperative activation by Smad2 and KLF4 were lost. Moreover, Knockdown of Smad2 with siRNA abolished the additive effect in cells cotransfected with Smad2 and KLF4 expression plasmids.
CONCLUSIONS
1 TGF-β1 inhibits cell cycle progression and induces differentiation in cultured rat VSMCs. This activity of TGF-β1 is accompanied by upregulation of KLF4, with concomitant increase in TβRI expression.
2 KLF4 is found to transduce TGF-β1 signals via phosphorylation-mediated activation of Smad2, Smad3 and p38 MAP kinase. The activation of both pathways, in turn, increases the phosphorylation of KLF4, which enable the formation of KLF4-Smad2 complex in response to TGF-β1.
3 KLF4 binds directly to the KLF4-binding sites 2 and 3 of the TβRI promoter and Smad2 recruits to the Smad-responsive region to activate TβRI promoter.
4 Formation of a stable KLF4-Smad2 complex to the promoter’s Smad-responsive region mediates cooperative TβRI promoter transcription in response to TGF-β1.
引文
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