Analysis of Wnt signaling pathway during cardiomyocyte differentiation

点击翻译按钮获取中文摘要

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.