Analysis of molecular mechanism in cardiomyocyte differentiation and its application for regeneration medicine using embryonic stem cells

Csx/Nkx 2 -5是心脏发育的重要调节因子。据报道，许多不同的人类CSX/NKX 2 -5突变导致先天性心脏病。我们使用P19 CL 6细胞检测了三种代表性CSX/NKX 2 -5突变体对心肌细胞分化和死亡的影响，P19 CL 6细胞有效地分化为心肌细胞。野生型CSX/NKX 2 -5的稳定过表达增强了心脏特异性基因的表达和P19 CL 6向心肌细胞的终末分化，而所有CSX/NKX 2 -5突变体都减弱了它们。当暴露于H_2O_2刺激等应激时，许多过表达突变体的细胞死亡，而过表达野生型的细胞存活。因此，我们发现Csx/Nkx 2 -5不仅在调节心脏特异性基因的表达中起关键作用，而且在保护心肌细胞免受应激中起关键作用。（Biochem Biophys Res Commun 298：493-500,高迁移率族蛋白A（HMGA）家族的成员在胎儿中大量表达，并作为转录辅因子在多种生物学过程中发挥关键作用。因子通过差异显示方法，我们分离出HMGA 2，HMGA家族的成员，其表达在P19 CL 6分化过程中显著增强。有趣的是，野生型HMGA 2的稳定过表达强烈地促进了P19 CL 6的心肌细胞分化，而HMGA 2的突变体的过表达抑制了它。我们还发现，Csx/Nkx 2 -5启动子被Smad 1反式激活，Smad 1是骨形态发生蛋白（BMP）信号的重要介导者，Smad 1和HMGA 2形成复合物，并显示协同的启动子反式激活。结合我们先前的报告，证明BMP信号传导对心肌细胞分化至关重要，我们表明HMGA 2通过加速BMP-Smad-Csx/Nkx 2 -5级联反应在心肌细胞分化中起关键作用（提交论文）。

Csx/Nkx2-5 is an important regulator of cardiac development. Many different human CSX/NKX2-5 mutations have been reported to cause congenital heart disease. We examined the effects of three representative CSX/NKX2-5 mutants on cardiomyocyte differentiation and death using P19CL6 cells, which efficiently differentiate into cardiomyocytes. Stable overexpression of wild-type CSX/NKX2-5 enhanced expression of cardiac-specific genes and the terminal differentiation of P19CL6 into cardiomyocytes, while all CSX/NKX2-5 mutants attenuated them. When exposed to stress like H_2O_2 stimulation, many cells overexpressing the mutants were dead, whereas the cells overexpressing the wild-type survived. Thus, we showed that Csx/Nkx2-5 plays a critical role not only in regulating expression of cardiac-specific genes but in protecting cardiomyocytes from stresses (Biochem Biophys Res Commun 298: 493-500, 2002).Members of the high-mobility-group proteins A(HMGA) family have been reported to be expressed abundantly in fetus and play a critical role in various biological processes as a transcriptional co-factor. By the differential display method, we isolated HMGA2, a member of the HMGA family, whose expression was markedly enhanced during differentiation of P19CL6. Interestingly, stable overexpression of wild-type HMGA2 strongly enhanced cardipmyocyte differentiation of P19CL6, while overexpression of a mutant of HMGA2 inhibited it. We also found that the Csx/Nkx2-5 promoter was transactivated by Smad1, an important mediator of signals from bone morphogenetic proteins (BMPs), and that Smadi and HMGA2 formed complexes and displayed synergistic transacriptional activation of the promoter. Together with our previous reports demonstrating that BMP signaling is essential for cardiomoycyte differentiation, we showed that HMGA2 plays a key role in cardiomyocyte differentiation through accelerating the BMP-Smad-Csx/Nkx2-5 cascade (paper in submission).