Analysis of Wnt signaling pathway during cardiomyocyte differentiation

有报道称，在鸡胚胎原肠发育过程中，Wnt-8c在原始条纹和后侧板中胚层中表达，而在心脏前区的异位表达抑制了心脏特异基因的表达。Wnt抑制剂DKK-1在表达Wnt-8c的后外侧板中胚层诱导心脏特异基因的表达，提示抑制Wnt活性可诱导非心脏前中胚层细胞心脏特异基因的异位表达和心肌细胞搏动，Wnt是心脏发育过程中的抑制信号分子。然而，Wnt调控心肌细胞分化的确切分子机制在很大程度上还不清楚。在本研究中，我们利用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。P19CL6是P19胚胎畸胎癌细胞的克隆衍生物。我们分离了两个永久性的P19CL6细胞系，CL6-XWNT-8和CL6-hDkk-1，它们分别结构性地过表达非洲爪哇Wnt-8和人DKK-1。亲代P19CL6细胞在1%二甲基亚砜(DMSO)作用下可分化为搏动心肌细胞，而CL6-Xwnt-8细胞在相同条件下不能分化为搏动心肌细胞。RT-PCR显示心脏特异性转录因子CSX/Nkx2-5在CL6-Xwnt-8细胞中的表达明显低于亲本P19CL6细胞，提示Xwnt-8抑制了调控P19CL6分化的CSx/Nkx2-5转录。CL6-hDkk-1细胞分化为搏动的心肌细胞，其表达CSx/Nkx2-5的能力与亲本P19CL6细胞相同，但在没有1%DMSO的情况下，CL6-hDkk-1细胞不能分化，提示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的分化。

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.