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 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.
Monzen
K
K
Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan.
Shiojima
I
I
Hiroi
Y
Y
Kudoh
S
S
Oka
T
T
Takimoto
E
E
Hayashi
D
D
Hosoda
T
T
Habara-Ohkubo
A
A
Nakaoka
T
T
Fujita
T
T
Yazaki
Y
Y
Komuro
I
I
D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002352:Carrier Proteins; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000378:metabolism; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins; D000099176:Noggin Protein; D000099151:MAP Kinase Kinase Kinase 7
D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D020930:MAP Kinase Kinase Kinases; D011506:Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D000099176:Noggin Protein; D000099151:MAP Kinase Kinase Kinase 7; C095552:NKX2-5 protein, human