Analysis of Wnt signaling pathway during cardiomyocyte differentiation

有报道称，在鸡胚原肠胚形成过程中，Wnt-8c在原始条纹和后外侧板中胚层表达，而在心前区异位表达Wnt-8c可抑制心脏特异性基因的表达。Wnt抑制剂Dkk-1可诱导Wnt-8c表达的后外侧板中胚层中心脏特异性基因表达，提示抑制Wnt活性可诱导非心前中胚层细胞中心脏特异性基因和跳动心肌细胞的异位表达，提示Wnt是心脏发育中的抑制信号分子。然而，Wnt调控心肌细胞分化的精确分子机制在很大程度上是未知的。在本研究中，我们通过P19胚胎畸胎癌细胞克隆衍生物P19CL6体外心肌细胞分化系统研究了Wnt抑制心脏分化的分子机制。我们分离了两个永久P19CL6细胞系CL6-Xwnt-8和CL6-hDkk-1，它们分别组成性过表达爪蟾Wnt-8和人Dkk-1。亲本P19CL6细胞在1%二甲亚砜（DMSO）作用下分化为搏动心肌细胞，而CL6-Xwnt-8细胞在相同条件下不分化为搏动心肌细胞。RT-PCR结果显示，与亲代P19CL6细胞相比，CL6-Xwnt-8细胞中心脏特异性转录因子Csx/Nkx2-5的表达显著降低，提示Xwnt-8抑制了Csx/Nkx2-5的转录，调节了P19CL6的分化。CL6-hDkk-1细胞分化为搏动心肌细胞，表达Csx/Nkx2-5与亲本p19cl6细胞一样多，但在没有1% DMSO的情况下不能分化，这表明可能需要额外的刺激才能分化为心肌细胞。综上所述，这些结果表明Wnt信号通过降低Csx/Nkx2-5的表达来调节心肌细胞分化。

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.