Monzen K, Shiojima I, Hiroi Y, Kudoh S, Oka T, Takimoto E, Hayashi D, Hosoda T, Habara-Ohkubo A, Nakaoka T, Fujita T, Yazaki Y, Komuro I
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan.
Mol Cell Biol. 1999 Oct;19(10):7096-105. doi: 10.1128/MCB.19.10.7096.
Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.
骨形态发生蛋白（BMPs）已被证明能在鸡的非心脏中胚层细胞中诱导心脏转录因子的异位表达和跳动的心肌细胞，这表明BMPs是参与心脏发育的诱导信号分子。然而，BMPs调节心脏发育的精确分子机制在很大程度上尚不清楚。在本研究中，我们利用P19CL6体外心肌细胞分化系统（P19胚胎性癌细胞的克隆衍生物）研究了BMPs诱导心脏分化的分子机制。我们建立了一个永久性的P19CL6细胞系，即P19CL6noggin，它组成性地过表达BMP拮抗剂头蛋白。尽管几乎所有亲代P19CL6细胞在用1%二甲亚砜处理时都会分化为跳动的心肌细胞，但P19CL6noggin细胞既没有分化为跳动的心肌细胞，也没有表达心脏转录因子或收缩蛋白基因。通过过表达BMP-2或向培养基中添加BMP蛋白挽救了分化失败的情况，这表明BMPs在该系统中对心肌细胞分化是不可或缺的。TAK1是丝裂原活化蛋白激酶激酶激酶超家族的成员，可转导BMP信号，过表达TAK1恢复了P19CL6noggin细胞分化为心肌细胞并同时表达心脏基因的能力，而在亲代P19CL6细胞中过表达TAK1的显性负性形式则抑制了心肌细胞分化。心脏转录因子Csx/Nkx-2.5和GATA-4同时过表达，但单独过表达Csx/Nkx-2.5或GATA-4则不能诱导P19CL6noggin细胞分化为心肌细胞。这些结果表明，TAK1、Csx/Nkx-2.5和GATA-4在心脏发生的BMP信号通路中起关键作用。
