Analysis for Molecular Mechanism of Cardiomyocyte Differentiation by Using a Cell Line Which Efficiently Differentiates into Cardiomyocytes

了解心肌细胞分化和心脏发育的分子机制的基础研究目标包括建立诱导心肌细胞分化的遗传技术，这可能有助于组织再生和随后的心力衰竭基因治疗的实际治疗应用。从这方面来看，我们研究了心肌细胞分化的分子机制，通过使用心肌细胞系P19CL6，它可以有效地分化为跳动的心肌细胞。我们之前已经证明骨形态发生蛋白（BMPs）通过MAPKKK TAK1诱导心肌细胞分化。据报道，转录因子Smad在BMP信号传导中起关键作用，ATF/CREB家族转录因子ATF-2最近被证明是Smad和TAK1途径的共同核靶点。我们利用小鼠P19细胞的克隆衍生物P19CL6研究Smads和ATF-2是否参与心肌细胞分化。虽然P19CL6在二甲亚砜处理后能有效分化为心肌细胞，但P19CL6noggin（一种组成型过表达BMP拮抗剂noggin的P19CL6细胞系）不能分化为心肌细胞。Smadl（配体特异性Smad）和Smad4（常见介质Smad）的共同过表达恢复了P19CL6noggin向心肌细胞分化的能力，而Smad6（抑制性Smad）的稳定过表达完全阻断了P19CL6的分化，这表明Smad途径是心肌细胞分化所必需的。ATF-2通过与Smad1/4和TAK1的协同方式刺激βMHC启动子活性，而ATF-2显性阴性形式的过表达降低了几个心脏特异性基因的启动子活性，抑制了P19CL6的心肌细胞分化。这些结果表明Smads、TAK1和它们的共同靶点ATF-2协同在心肌细胞分化中起关键作用。少

The aims of basic research for understanding the molecular mechanism of cardiomyocyte differentiation and cardiac development include the establishment of the genetic technique by which cardiomyocyte differentiation can be induced, which may contribute to the practical therapeutic application for tissue regeneration and the subsequent gene-therapy for heart failure. From this respect, we investigated the molecular mechanism by which cardiomyocyte differentiation is regulated by using a cardiomyogenic cell line, P19CL6, which can differentiate efficiently into beating cardiomyocytes.We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiornyocyte differentiation through the MAPKKK TAK1. Transcription factors Smads have been reported to play a critical role in BMP signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to be a common nuclear target of the Smad and the TAK1 pathways. We here examined whether Smads and ATF-2 are involved … More in cardiomyocyte differentiation using P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfbxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Co-overexpression of Smadl, a ligand-specific Smad, and Smad4, a common mediator Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated βMHC promoter acitvity by the synergistic manner with Smad1/4 and TAK1, while overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited cardiomyocyte differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation. Less