miR-126-Rac1/p67phox信号在淫羊藿苷促小鼠胚胎干细胞分化为心肌细胞中的关键作用研究
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摘要
胚胎干(embryonic stem, ES)细胞是能在体外自我更新并具有多分化潜能的细胞,可分化为功能性心肌细胞,应用于心脏发育生理学、心脏病理学及新药发现等研究。本课题前期研究发现淫羊藿苷(icariin, ICA)能够通过激活活性氧(reactive oxygen species, ROS)信号通路促进小鼠ES细胞分化为心肌细胞,但关于ICA如何促ROS生成的作用机制还知之甚少。同时,本课题组前期利用蛋白组学技术研究了ICA促ES细胞成心体系中整套蛋白表达谱及功能,但对ICA促心肌分化是否涉及转录后调控和翻译后修饰尚不清楚。本研究主要目的是从转录后调控和翻译后修饰层面进一步论证ICA的促分化作用,揭示其促心肌分化的启动机制,为阐明其诱导分化药理作用提供实验依据,也为发展其新的应用提供理论依据。
鉴于NADPH氧化酶(NADPH oxidases, NOXs)产生的ROS在ES细胞心肌分化中发挥关键作用,课题组前期研究了其中的一个亚型NOX4在ICA促心肌分化中的作用。研究发现除了NOX4的激活不需要胞浆亚基外,其它亚型激活都需要胞浆亚基,但其它亚型NOXs在ES细胞分化心肌中是否被激活,及激活所涉及必需胞浆亚基尚不清楚。作为NADPH氧化酶的胞浆亚基,Rac1异戊烯化修饰后锚着在细胞膜上对ROS生成至关重要。此外,Rac1与其他胞浆亚基,如p67phox结合也是NOX激活产生ROS的关键一步。据此,本研究第一部分提出了Rac1异戊烯化激活是ICA促ES细胞心肌分化的早期分子事件假说；并运用siRNA沉默Rac1(si-Racl)、Rac1CAAX残基缺失(Rac1△)质粒、牛儿基转移酶-1(geranylgeranyl transferases type-I, GGTase-I)抑制剂GGTI-298,法尼基转移酶(farnesyl transferase, FTase)抑制剂FTI-277和Rac1-GTP装载抑制剂NSC23766四步法,从翻译后修饰层面探索ICA促心肌分化的可能机理。
近年来,众多研究显示微小RNA (microRNAs, miRNAs)在ES细胞心肌分化中发挥重要调控作用。基于miRNAs芯片分析,我们发现miR-126在ICA促ES细胞心肌分化中显著上调,且其拟似物或拮抗剂可分别增强或削弱心肌分化作用,提示miR-126在心肌分化过程中发挥关键作用。文献报道,miR-126可提高血管内皮生长因子(vascular endothelial growth factor, VEGF)的促血管生成作用,且VEGF与ROS形成密切相关。另有研究报道,分化d4Flk-1+(Flk-1阳性)细胞高表达miR-126。因此,本研究第二部分提出miR-126通过调节VEGF及其受体Flk-1介导Racl-p67phox复合物在细胞膜上锚着,进而激活ROS产生这一假说；并运用miR-126拟似物,miR-126拮抗剂,siRNA沉默Flk-1和Flk-1抑制剂SU5416从转录后调控层面探索ICA促心肌分化的可能机理。
ROS-Ca2+信号作为可兴奋细胞的重要生命活动,对心肌细胞正常生理功能的维持举足轻重。有研究报道在血管细胞中ROS可以激活内质网钙调控蛋白。内质网作为胞内重要的钙储存细胞器,参与调节胞内钙平衡,调控各种信号通路。内质网中的钙离子动态平衡过程由兰尼碱受体(ryanodinereceptors,RyRs)、IP3受体(inositol1,4,5-triphosphate receptors, IP3Rs)以及肌浆(内质)网Ca2+-ATP酶(sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SERCA)协调运作控制。因此,本研究第三部分探索了miR-126-Racl/p67phox-ROS-Ca2+信号通路在ICA促ES细胞心肌分化中的作用。
1.Racl异戊烯化激活介导ICA促ES细胞心肌分化研究
为探索Racl异戊烯化激活是否介导ICA促ES细胞心肌分化过程,本研究首先考察了Racl异戊烯化激活对ICA促ROS生成的调控作用,采用ROS荧光染料DCF-DA检测了si-Racl, Racl△质粒,GGTI-298, FTI-277和NSC23766对ICA促拟胚体(embryoid bodies, EBs)内ROS水平影响。Western blot检测ICA对Rac1从胞浆向胞膜移位的影响。免疫荧光法检测Rac1△对心肌特异性肌小节蛋白α-辅肌动蛋白(a-actinin)的影响。
实验结果显示,ICA作用EBs0.5,1,2,4h能时间依赖性诱导Rac1从胞浆向细胞膜移位,当用si-Racl, Rac1△, GGTI-298或NSC23766时,ICA促ROS产生作用消失,但是FTI-277却无此作用。si-Racl, Rac1△, GGTI-298或NSC23766均能显著抑制ICA促ES细胞心肌分化作用,表现为心肌分化率降低,心肌转录因子Nkx2.5和心肌小节蛋白a-actinin表达减少,但FTI-277并不能抑制ICA促分化作用。本实验结果提示Racl CAAX端被20碳牛儿基异戊烯化激活,调节ROS生成介导ICA促ES细胞心肌分化过程。
2. miR-126参与VEGF及其受体Flk-1激活介导ICA促ES细胞心肌分化研究
第一部分研究了ROS上游靶点Rac1激活在ICA促ES细胞心肌分化中的作用,基于上述研究结果,为进一步明确ROS上游信号分子在ICA促ES细胞心肌分化中的角色,本部分着重对miR-126, VEGF及Flk-1进行了考察。采用Western blot去检测ICA对VEGF和Flk-1表达的影响。miRNAs芯片技术检测ICA介入后miRNAs差异表达谱。进一步利用Flk-1抑制剂SU5416, siRNA沉默Flk-1(si-Flk-1), miR-126拟似物和miR-126抑制剂研究miR-126介导VEGF及其受体Flk-1对ICA促ES细胞心肌分化的影响。
实验结果显示,VEGF在d5EBs时高表达,随着EBs向心肌细胞分化,VEGF显著下调(d5-d5+3),之后VEGF表达相对不变。Flk-1在整个分化过程中都有表达。ICA作用3天后能一定程度上增加VEGF表达。MTT结果显示,5μ,M,2.5μM和1μM SU5416对ES细胞均无增值抑制作用。在d5EBs时加入5μM,2.5μM或SU5416并不能抑制ES细胞自发的心肌文化,表现为心肌分化率增加,a-actinin表达上调。但在d0加入1μM SU5416就可以显著抑制ES细胞自发的心肌文化和ICA促ES细胞心肌文化,表现为心肌分化率降低,Nkx2.5和a-actinin表达减少。
miRNAs芯片结果显示,在d5+3,ICA可以诱导miR-126表达增加2.115倍。qRT-PCR结果确证了miR-126的差异表达。ES细胞转染miR-126拟似物可以促进其分化为心肌细胞,表现为心肌分化率增加,VEGF, Nkx2.5和α-actinin表达上调,Rac1-p67phox复合物形成和ROS生成增多。ES细胞转染miR-126抑制剂可以抑制ICA促ES细胞心肌文化,表现为心肌分化率降低,VEGF, Nkx2.5和α-actinin表达下调,Rac1-p67phox复合物形成和ROS生成减少。本实验结果提示miR-126通过调节VEGF及其受体Flk-1介导Rac1-p67phox复合物在细胞膜上锚着,进而激活ROS产生。
3. miR-126-Rac1/p67phox-ROS-Ca2+信号通路在ICA促ES细胞心肌分化中作用研究
在第一部分和第二部分的基础上,为进一步明确ROS下游信号分子在ICA促ES细胞心肌分化的作用,第三部分着重对Ca2+和内质网钙调控蛋白进行了考察。采用荧光光度法测定EBs内Ca2+浓度；免疫荧光双染法测定内质网钙调控蛋白RyR2, IP3R2或SERCA2与α-actinin共定位情况；Western blot法测定RyR2, IP3R2和SERCA2表达；活细胞工作站测定Ca2+瞬变。
实验结果显示,ICA能增加d5EBs内Ca2+浓度,提高心肌分化过程中RyR2, IP3R2和SERCA2表达水平；在d5+7上述内质网钙调控蛋白与α-actinin均有较好的共定位；ICA尚能提高d5+11(分化末期)心肌细胞内Ca2+瞬变幅度。miR-126抑制剂可抑制ICA促ROS生成,进而下调上述作用。
结论：
1.Rac1CAAX端被牛儿基(20碳)异戊烯化激活是ICA促ES细胞心肌分化的早期分
子事件,该作用与其锚着细胞膜并与p67phox结合激活NOXs促ROS生成相关。
2.ICA促分化早期miR-126可能经由VEGF/Flk-1信号参与Rac1-p67phox复合物在细胞膜上聚集,激活NOXs而促ROS生成。
3. ROS-Ca2+信号介导ICA促心肌分化过程,miR-126抑制剂通过抑制ROS生成导致分化早期EBs内Ca2+浓度降低,分化中期内质网钙调控蛋白表达下调和分化末期心肌细胞内Ca2+瞬变幅度减小。
Embryonic stem (ES) cells can be cultured and maintained in undifferentiated state in vitro. Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology, cardiac pathology and new drug discovery. In our previous work, Icariin (ICA) facilitates the directional differentiation of ES cells into cardiomyocytes, and reactive oxygen species (ROS) signaling plays a vital role in the process. However, the mechanisms by which ROS are activated by ICA are still not understood.
In the present studies, in order to reveal the underlying mechanisms of ICA in cardiomyocyte differentiation, the relationship between structure and inducing effect was investigated. Given that ICA is a prenylflavonid compound with a prenyl group at position8of ring A, we presume that protein prenylation may be responsible for its role in cardiomyocyte differentiation.
Racl, which belongs to the small G protein Rho GTPases family, work as molecular switches to transduce intracellular signals from growth factors or G protein coupled receptors. As the cytosolic subunit of NADPH oxidase complex, Racl protein prenylation is essential to its migration from the cytosol to the plasma membrane where its attachment favors assembly of p67phox.This section demonstrated that Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation. Small interfering RNA (siRNA) targeting Racl (si-Racl), the plasmid with Racl CAAX box deletion (Racl△), the selective GGTase-I inhibitor GGTI-298, the selective FTase inhibitor FTI-277and the Racl GTP-binding inhibitor NSC23766were used to determine whether the ultimate activation of Racl is essential to ICA induced ROS generation.
MicroRNAs (miRNAs) play an important role in regulating cardiomyocyte differentiation of ES cells by repressing the translation of selected mRNAs. With miRNAs arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. miR-126mimics enhanced cardiomyocyte differentiation, while miR-126inhibitor abolished the differentiation. Accordingly, miR-126enhances the pro-angiogenic actions of VEGF (vascular endothelial growth factor).
Based on the observations that VEGF elicits a burst of ROS that enhances angiogenesis, this section is aimed to investigate whether miR-126promotes Racl-p67phox membrane accumulation, thus activating ROS. Former studies have showed the role of Ca2+in cardiomyocyte differentiation, and Ca2+is the direct molecular target of ROS. The third section was aimed to explore whether miR-126-Racl/p67phox-ROS-Ca2+pathway involved in ICA induced cardiomyocyte differentiation.
1. Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation of ES cells
si-Racl, Racl△, GGTI-298, FTI-277and NSC23766were used to determine the role of Racl protein prenylation in ICA induced cardiomyocyte differentiation. Intracellular ROS generation was measured using the fluorescent dye2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). The differential protein fractions (the membrane and cytosol fractions) of Racl were assessed by western blot. The sarcomeric structure of cardiomyocytes derived from ES cells was assessed by immunofluorescence with a-actinin antibody.
The results demonstrated that ICA0.5,1,2,4h rapidly elevated Racl translocation to the membranes. si-Racl, Racl△, GGTI-298and NSC23766all could abolish the inducible effect of ICA, however, FTI-277couldn't disrupt the effect. These results suggested that Racl protein prenylation was involved in ICA induced cardiomyocyte differentiation.
2. miR-126/VEGF/Flk-1signaling is involved in ICA induced cardiomyocyte differentiation of ES cells
The first section has explored the role of Racl in ICA induced cardiomyocyte differentiation, to further investigate the upstream target of ROS, miR-126, VEGF and Flk-1was analyzed. The expression of VEGF and Flk-1was assessed by western blot. The different expressions of miRNAs induced by ICA were assessed by miRNAs chips. The Flk-1inhibitor SU5416, the siRNAs targeting Flk-1(si-Flk-1) miR-126mimic and miR-126inhibitor was used to investigate the miR-126/VEGF/Flk-1signaling in ICA induced cardiomyocyte differentiation.
The results demonstrated that VEGF is highly expressed in EBs on day5, when EBs initiated to differentiate into cardiomyocytes, VEGF showed a markedly decrease. Flk-1kept a relative steady expression during the differentiation. ICA slightly increased VEGF expression on day5+3. SU5416(5μM,2.5μM and1μM) all had no toxic effect on ES cells. SU5416couldn't inhibit the spontaneous cardiomyocyte differentiation, as seen by the increased beating activity and a-actinin expression. SU5416was applied to ES cells from hanging drop cultures, it did inhibit the spontaneous cardiomyocyte differentiation.
With microRNA arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. Overexpression of miR-126in ES cells promoted cardiomyocyte differentiation by activating VEGF signaling, while knockdown of miR-126blocked cardiomyocyte differentiation by inhibiting VEGF signaling. ICA2h significantly enhanced the formation of Rac1-p67phox complex, which was disrupted by miR-126inhibitor.
3. miR-126-Rac1/p67phox-ROS-Ca2+signaling participated in ICA induced cardiomyocyte differentiation of ES cells
Based on the first and second sections, in order to further verify the role of ICA in cardiomyocyte differentiation, this section is aimed to investigate the downstream target of ROS, the Ca2+. The co-locations of Ca2+handing proteins on endoplasmic reticulum (ryanodine receptors, RyRs; inositol1,4,5-triphosphate receptors, IP3Rs;(sarco) endoplasmic reticulum Ca2+-ATPase, SERCA) and α-actinin were assessed by fluorescence. The expressions of RyR2, IP3R2and SERCA2were assessed by western blot. The intracellular Ca2+transients were tested by Fluo4-AM staining.
The results demonstrated that there was a correlation between α-actinin and RyR2, IP3R2or SERCA2during differentiation. The expressions of RyR2, IP3R2and SERCA2were increased in EBs treated with ICA. The concentration of intracellular Ca2+in EBs on day5was increased by ICA. The amplitude of the intracellular Ca2+transients was elevated in the cardiomyocytes derived from ES cells with ICA treatment. miR-126inhibitor could hinder the inducible effect of ICA.
Conclusion:
1. Rac1protein prenylation is catalysed by GGTase-I, addition of geranylgeranyl diphosphate to the cysteine residue in the CAAX sequence and subsequently migrates to the membrane where its attachment favors assembly of p67phox, thus activating NOXs and generating ROS. This process takes a critical role in ICA induced cardiomyocyte differentiation.
2. At the early differentiation stage, up-regulation of miR-126induced by ICA promotes Racl-p67phox membrane accumulation may via VEGF and its receptor Flk-1, thus activating ROS.
3. Ca2+signaling is regulated by ROS in ICA induced cardiomyocyte differentiation, miR-126inhibitor decreases ROS generation, thus down-regulation [Ca2+]i in day5EBs and RyR2, IP3R2, SERCA2proein expression in day5+7EBs and the amplitude of Ca2+transient in the cardiomyocytes derived from ES cells.
鉴于NADPH氧化酶(NADPH oxidases, NOXs)产生的ROS在ES细胞心肌分化中发挥关键作用,课题组前期研究了其中的一个亚型NOX4在ICA促心肌分化中的作用。研究发现除了NOX4的激活不需要胞浆亚基外,其它亚型激活都需要胞浆亚基,但其它亚型NOXs在ES细胞分化心肌中是否被激活,及激活所涉及必需胞浆亚基尚不清楚。作为NADPH氧化酶的胞浆亚基,Rac1异戊烯化修饰后锚着在细胞膜上对ROS生成至关重要。此外,Rac1与其他胞浆亚基,如p67phox结合也是NOX激活产生ROS的关键一步。据此,本研究第一部分提出了Rac1异戊烯化激活是ICA促ES细胞心肌分化的早期分子事件假说；并运用siRNA沉默Rac1(si-Racl)、Rac1CAAX残基缺失(Rac1△)质粒、牛儿基转移酶-1(geranylgeranyl transferases type-I, GGTase-I)抑制剂GGTI-298,法尼基转移酶(farnesyl transferase, FTase)抑制剂FTI-277和Rac1-GTP装载抑制剂NSC23766四步法,从翻译后修饰层面探索ICA促心肌分化的可能机理。
近年来,众多研究显示微小RNA (microRNAs, miRNAs)在ES细胞心肌分化中发挥重要调控作用。基于miRNAs芯片分析,我们发现miR-126在ICA促ES细胞心肌分化中显著上调,且其拟似物或拮抗剂可分别增强或削弱心肌分化作用,提示miR-126在心肌分化过程中发挥关键作用。文献报道,miR-126可提高血管内皮生长因子(vascular endothelial growth factor, VEGF)的促血管生成作用,且VEGF与ROS形成密切相关。另有研究报道,分化d4Flk-1+(Flk-1阳性)细胞高表达miR-126。因此,本研究第二部分提出miR-126通过调节VEGF及其受体Flk-1介导Racl-p67phox复合物在细胞膜上锚着,进而激活ROS产生这一假说；并运用miR-126拟似物,miR-126拮抗剂,siRNA沉默Flk-1和Flk-1抑制剂SU5416从转录后调控层面探索ICA促心肌分化的可能机理。
ROS-Ca2+信号作为可兴奋细胞的重要生命活动,对心肌细胞正常生理功能的维持举足轻重。有研究报道在血管细胞中ROS可以激活内质网钙调控蛋白。内质网作为胞内重要的钙储存细胞器,参与调节胞内钙平衡,调控各种信号通路。内质网中的钙离子动态平衡过程由兰尼碱受体(ryanodinereceptors,RyRs)、IP3受体(inositol1,4,5-triphosphate receptors, IP3Rs)以及肌浆(内质)网Ca2+-ATP酶(sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SERCA)协调运作控制。因此,本研究第三部分探索了miR-126-Racl/p67phox-ROS-Ca2+信号通路在ICA促ES细胞心肌分化中的作用。
1.Racl异戊烯化激活介导ICA促ES细胞心肌分化研究
为探索Racl异戊烯化激活是否介导ICA促ES细胞心肌分化过程,本研究首先考察了Racl异戊烯化激活对ICA促ROS生成的调控作用,采用ROS荧光染料DCF-DA检测了si-Racl, Racl△质粒,GGTI-298, FTI-277和NSC23766对ICA促拟胚体(embryoid bodies, EBs)内ROS水平影响。Western blot检测ICA对Rac1从胞浆向胞膜移位的影响。免疫荧光法检测Rac1△对心肌特异性肌小节蛋白α-辅肌动蛋白(a-actinin)的影响。
实验结果显示,ICA作用EBs0.5,1,2,4h能时间依赖性诱导Rac1从胞浆向细胞膜移位,当用si-Racl, Rac1△, GGTI-298或NSC23766时,ICA促ROS产生作用消失,但是FTI-277却无此作用。si-Racl, Rac1△, GGTI-298或NSC23766均能显著抑制ICA促ES细胞心肌分化作用,表现为心肌分化率降低,心肌转录因子Nkx2.5和心肌小节蛋白a-actinin表达减少,但FTI-277并不能抑制ICA促分化作用。本实验结果提示Racl CAAX端被20碳牛儿基异戊烯化激活,调节ROS生成介导ICA促ES细胞心肌分化过程。
2. miR-126参与VEGF及其受体Flk-1激活介导ICA促ES细胞心肌分化研究
第一部分研究了ROS上游靶点Rac1激活在ICA促ES细胞心肌分化中的作用,基于上述研究结果,为进一步明确ROS上游信号分子在ICA促ES细胞心肌分化中的角色,本部分着重对miR-126, VEGF及Flk-1进行了考察。采用Western blot去检测ICA对VEGF和Flk-1表达的影响。miRNAs芯片技术检测ICA介入后miRNAs差异表达谱。进一步利用Flk-1抑制剂SU5416, siRNA沉默Flk-1(si-Flk-1), miR-126拟似物和miR-126抑制剂研究miR-126介导VEGF及其受体Flk-1对ICA促ES细胞心肌分化的影响。
实验结果显示,VEGF在d5EBs时高表达,随着EBs向心肌细胞分化,VEGF显著下调(d5-d5+3),之后VEGF表达相对不变。Flk-1在整个分化过程中都有表达。ICA作用3天后能一定程度上增加VEGF表达。MTT结果显示,5μ,M,2.5μM和1μM SU5416对ES细胞均无增值抑制作用。在d5EBs时加入5μM,2.5μM或SU5416并不能抑制ES细胞自发的心肌文化,表现为心肌分化率增加,a-actinin表达上调。但在d0加入1μM SU5416就可以显著抑制ES细胞自发的心肌文化和ICA促ES细胞心肌文化,表现为心肌分化率降低,Nkx2.5和a-actinin表达减少。
miRNAs芯片结果显示,在d5+3,ICA可以诱导miR-126表达增加2.115倍。qRT-PCR结果确证了miR-126的差异表达。ES细胞转染miR-126拟似物可以促进其分化为心肌细胞,表现为心肌分化率增加,VEGF, Nkx2.5和α-actinin表达上调,Rac1-p67phox复合物形成和ROS生成增多。ES细胞转染miR-126抑制剂可以抑制ICA促ES细胞心肌文化,表现为心肌分化率降低,VEGF, Nkx2.5和α-actinin表达下调,Rac1-p67phox复合物形成和ROS生成减少。本实验结果提示miR-126通过调节VEGF及其受体Flk-1介导Rac1-p67phox复合物在细胞膜上锚着,进而激活ROS产生。
3. miR-126-Rac1/p67phox-ROS-Ca2+信号通路在ICA促ES细胞心肌分化中作用研究
在第一部分和第二部分的基础上,为进一步明确ROS下游信号分子在ICA促ES细胞心肌分化的作用,第三部分着重对Ca2+和内质网钙调控蛋白进行了考察。采用荧光光度法测定EBs内Ca2+浓度；免疫荧光双染法测定内质网钙调控蛋白RyR2, IP3R2或SERCA2与α-actinin共定位情况；Western blot法测定RyR2, IP3R2和SERCA2表达；活细胞工作站测定Ca2+瞬变。
实验结果显示,ICA能增加d5EBs内Ca2+浓度,提高心肌分化过程中RyR2, IP3R2和SERCA2表达水平；在d5+7上述内质网钙调控蛋白与α-actinin均有较好的共定位；ICA尚能提高d5+11(分化末期)心肌细胞内Ca2+瞬变幅度。miR-126抑制剂可抑制ICA促ROS生成,进而下调上述作用。
结论：
1.Rac1CAAX端被牛儿基(20碳)异戊烯化激活是ICA促ES细胞心肌分化的早期分
子事件,该作用与其锚着细胞膜并与p67phox结合激活NOXs促ROS生成相关。
2.ICA促分化早期miR-126可能经由VEGF/Flk-1信号参与Rac1-p67phox复合物在细胞膜上聚集,激活NOXs而促ROS生成。
3. ROS-Ca2+信号介导ICA促心肌分化过程,miR-126抑制剂通过抑制ROS生成导致分化早期EBs内Ca2+浓度降低,分化中期内质网钙调控蛋白表达下调和分化末期心肌细胞内Ca2+瞬变幅度减小。
Embryonic stem (ES) cells can be cultured and maintained in undifferentiated state in vitro. Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology, cardiac pathology and new drug discovery. In our previous work, Icariin (ICA) facilitates the directional differentiation of ES cells into cardiomyocytes, and reactive oxygen species (ROS) signaling plays a vital role in the process. However, the mechanisms by which ROS are activated by ICA are still not understood.
In the present studies, in order to reveal the underlying mechanisms of ICA in cardiomyocyte differentiation, the relationship between structure and inducing effect was investigated. Given that ICA is a prenylflavonid compound with a prenyl group at position8of ring A, we presume that protein prenylation may be responsible for its role in cardiomyocyte differentiation.
Racl, which belongs to the small G protein Rho GTPases family, work as molecular switches to transduce intracellular signals from growth factors or G protein coupled receptors. As the cytosolic subunit of NADPH oxidase complex, Racl protein prenylation is essential to its migration from the cytosol to the plasma membrane where its attachment favors assembly of p67phox.This section demonstrated that Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation. Small interfering RNA (siRNA) targeting Racl (si-Racl), the plasmid with Racl CAAX box deletion (Racl△), the selective GGTase-I inhibitor GGTI-298, the selective FTase inhibitor FTI-277and the Racl GTP-binding inhibitor NSC23766were used to determine whether the ultimate activation of Racl is essential to ICA induced ROS generation.
MicroRNAs (miRNAs) play an important role in regulating cardiomyocyte differentiation of ES cells by repressing the translation of selected mRNAs. With miRNAs arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. miR-126mimics enhanced cardiomyocyte differentiation, while miR-126inhibitor abolished the differentiation. Accordingly, miR-126enhances the pro-angiogenic actions of VEGF (vascular endothelial growth factor).
Based on the observations that VEGF elicits a burst of ROS that enhances angiogenesis, this section is aimed to investigate whether miR-126promotes Racl-p67phox membrane accumulation, thus activating ROS. Former studies have showed the role of Ca2+in cardiomyocyte differentiation, and Ca2+is the direct molecular target of ROS. The third section was aimed to explore whether miR-126-Racl/p67phox-ROS-Ca2+pathway involved in ICA induced cardiomyocyte differentiation.
1. Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation of ES cells
si-Racl, Racl△, GGTI-298, FTI-277and NSC23766were used to determine the role of Racl protein prenylation in ICA induced cardiomyocyte differentiation. Intracellular ROS generation was measured using the fluorescent dye2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). The differential protein fractions (the membrane and cytosol fractions) of Racl were assessed by western blot. The sarcomeric structure of cardiomyocytes derived from ES cells was assessed by immunofluorescence with a-actinin antibody.
The results demonstrated that ICA0.5,1,2,4h rapidly elevated Racl translocation to the membranes. si-Racl, Racl△, GGTI-298and NSC23766all could abolish the inducible effect of ICA, however, FTI-277couldn't disrupt the effect. These results suggested that Racl protein prenylation was involved in ICA induced cardiomyocyte differentiation.
2. miR-126/VEGF/Flk-1signaling is involved in ICA induced cardiomyocyte differentiation of ES cells
The first section has explored the role of Racl in ICA induced cardiomyocyte differentiation, to further investigate the upstream target of ROS, miR-126, VEGF and Flk-1was analyzed. The expression of VEGF and Flk-1was assessed by western blot. The different expressions of miRNAs induced by ICA were assessed by miRNAs chips. The Flk-1inhibitor SU5416, the siRNAs targeting Flk-1(si-Flk-1) miR-126mimic and miR-126inhibitor was used to investigate the miR-126/VEGF/Flk-1signaling in ICA induced cardiomyocyte differentiation.
The results demonstrated that VEGF is highly expressed in EBs on day5, when EBs initiated to differentiate into cardiomyocytes, VEGF showed a markedly decrease. Flk-1kept a relative steady expression during the differentiation. ICA slightly increased VEGF expression on day5+3. SU5416(5μM,2.5μM and1μM) all had no toxic effect on ES cells. SU5416couldn't inhibit the spontaneous cardiomyocyte differentiation, as seen by the increased beating activity and a-actinin expression. SU5416was applied to ES cells from hanging drop cultures, it did inhibit the spontaneous cardiomyocyte differentiation.
With microRNA arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. Overexpression of miR-126in ES cells promoted cardiomyocyte differentiation by activating VEGF signaling, while knockdown of miR-126blocked cardiomyocyte differentiation by inhibiting VEGF signaling. ICA2h significantly enhanced the formation of Rac1-p67phox complex, which was disrupted by miR-126inhibitor.
3. miR-126-Rac1/p67phox-ROS-Ca2+signaling participated in ICA induced cardiomyocyte differentiation of ES cells
Based on the first and second sections, in order to further verify the role of ICA in cardiomyocyte differentiation, this section is aimed to investigate the downstream target of ROS, the Ca2+. The co-locations of Ca2+handing proteins on endoplasmic reticulum (ryanodine receptors, RyRs; inositol1,4,5-triphosphate receptors, IP3Rs;(sarco) endoplasmic reticulum Ca2+-ATPase, SERCA) and α-actinin were assessed by fluorescence. The expressions of RyR2, IP3R2and SERCA2were assessed by western blot. The intracellular Ca2+transients were tested by Fluo4-AM staining.
The results demonstrated that there was a correlation between α-actinin and RyR2, IP3R2or SERCA2during differentiation. The expressions of RyR2, IP3R2and SERCA2were increased in EBs treated with ICA. The concentration of intracellular Ca2+in EBs on day5was increased by ICA. The amplitude of the intracellular Ca2+transients was elevated in the cardiomyocytes derived from ES cells with ICA treatment. miR-126inhibitor could hinder the inducible effect of ICA.
Conclusion:
1. Rac1protein prenylation is catalysed by GGTase-I, addition of geranylgeranyl diphosphate to the cysteine residue in the CAAX sequence and subsequently migrates to the membrane where its attachment favors assembly of p67phox, thus activating NOXs and generating ROS. This process takes a critical role in ICA induced cardiomyocyte differentiation.
2. At the early differentiation stage, up-regulation of miR-126induced by ICA promotes Racl-p67phox membrane accumulation may via VEGF and its receptor Flk-1, thus activating ROS.
3. Ca2+signaling is regulated by ROS in ICA induced cardiomyocyte differentiation, miR-126inhibitor decreases ROS generation, thus down-regulation [Ca2+]i in day5EBs and RyR2, IP3R2, SERCA2proein expression in day5+7EBs and the amplitude of Ca2+transient in the cardiomyocytes derived from ES cells.
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