Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expres sion of cardiac transcription factors and beating cardiomyocytes in nonprec ardiac mesodermal cells in chicks, suggesting that BMPs are inductive signa ling molecules that participate in the development of the heart. However, t he precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown, In the present study, we examined the molecular mechan isms by which BMPs induce cardiac differentiation by using the P19CL6 in vi tro cardiomyocyte differentiation system, a clonal derivative of P19 embryo nic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19 CL6noggin, which constitutively overexpresses the BMP antagonist noggin, Al though almost all parental P19CL6 cells differentiate into beating cardiomy ocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac tran scription factors or contractile protein genes. The failure of differentiat ion was rescued by overexpression of BMP-2 or addition of BMP protein to th e culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system, Overexpression of TAK1, a member of the mit ogen-activated protein kinase kinase kinase superfamily which transduces BM P signaling, restored the ability of P19CL6noggin cells to differentiate in to cardiomyocytes and concomitantly express cardiac genes, whereas overexpr ession of the dominant negative form of TAK1 in parental P19CL6 cells inhib ited cardiomyocyte differentiation. Overexpression of both cardiac transcri ption 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. Th ese results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.