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Analysis of Wnt signaling pathway during cardiomyocyte differentiation

心肌细胞分化过程中Wnt信号通路分析

基本信息

批准号：
14570648
负责人：
IMAMURA Hiroshi
金额：
$ 2.18万
依托单位：
SHINSHU UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

It has been reported that Wnt-8c was expressed in the primitive streak and posterior lateral plate mesoderm during gastrulation in chick embryo and that ectopic expression of Wnt-8c in the precardiac region represses cardiac-specific gene expressions. Dkk-1, a Wnt inhibitor, induces cardiac-specific gene expressions in posterior lateral plate mesoderm where Wnt-8c was expressed, suggesting that inhibition of Wnt activity can induce ectopic expression of cardiac-specific genes and beating cardiomyocytes in nonprecardiac mesodermal cells, and that Wnt is an inhibitory signaling molecule in cardiac development. However, the precise molecular mechanisms by which Wnt regulates cardiac cell differentiation are largely unknown. In the present study, we examined the molecular mechanisms by which Wnt inhibits cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonal teratocarcinoma cells. We isolated two permanent P19CL6 cell lines, CL6-Xwnt-8 and CL6-hDkk-1, which constitutively overexpress Xenopus Wnt-8 and human Dkk-1, respectively. Parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide (DMSO), however, CL6-Xwnt-8 cells did not differentiate into beating cardiomyocytes under the same condition. RT-PCR showed that expression of Csx/Nkx2-5, a cardiac-specific transcription factor, was significantly reduced in CL6-Xwnt-8 cells compared with parental P19CL6 cells, suggesting that Xwnt-8 suppressed Csx/Nkx2-5 transcription regulating P19CL6 differentiation. CL6-hDkk-1 cells differentiated into beating cardiomyocytes and expressed Csx/Nkx2-5 as much as parental P 19CL6 cells, however, did not differentiate without 1% DMSO, indicating the possibility that additional stimuli are necessary for the differentiation into cardiomyocytes. Together, these results suggest that Wnt signal regulates cardiomyocyte differentiation by reducing the expression of Csx/Nkx2-5.

据报道，Wnt-8 c在鸡胚原肠胚形成过程中的原条和后侧板中胚层表达，并且Wnt-8 c在心前区的异位表达抑制心脏特异性基因的表达。Wnt抑制剂Dkk-1在表达Wnt-8 c的后侧板中胚层中诱导心脏特异性基因表达，提示抑制Wnt活性可诱导非心前区中胚层细胞中心脏特异性基因的异位表达和搏动心肌细胞，Wnt是心脏发育的抑制性信号分子。然而，Wnt调控心肌细胞分化的确切分子机制在很大程度上是未知的。在本研究中，我们研究了Wnt抑制心脏分化的分子机制，通过使用P19 CL 6在体外心肌细胞分化系统，P19胚胎畸胎瘤细胞的克隆衍生物。我们分离了两个永久性P19 CL 6细胞系，CL 6-Xwnt-8和CL 6-hDkk-1，它们分别组成型过表达非洲爪蟾Wnt-8和人Dkk-1。亲代P19 CL 6细胞经1%二甲基亚砜（DMSO）处理后可分化为有搏动的心肌细胞，而CL 6-Xwnt-8细胞在同样条件下不能分化为有搏动的心肌细胞。RT-PCR结果显示，与亲代P19 CL 6细胞相比，Csx/Nkx 2 -5在CL 6-Xwnt-8细胞中的表达明显降低，提示Xwnt-8抑制了Csx/Nkx 2 -5的转录调控P19 CL 6的分化。CL 6-hDkk-1细胞分化为搏动的心肌细胞，并表达与亲本P19 CL 6细胞一样多的Csx/Nkx 2 -5，然而，在没有1%DMSO的情况下不分化，表明分化为心肌细胞需要额外的刺激的可能性。总之，这些结果表明，Wnt信号通过减少Csx/Nkx 2 -5的表达来调节心肌细胞分化。