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

Abstract

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.

IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis

“…Cell culture experiments. P19CL6 cells and ES cells were cultured and induced to differentiate into cardiomyocytes essentially as described 6,10 . P19CL6 cells (2,000 cells per 35-mm dish) were treated with various conditioned media for screening of their cardiogenic activities.…”

“…P19CL6 cells differentiate into cardiomyocytes with high efficiency in the presence of 1% dimethylsulphoxide (DMSO) 6 . We cultured P19CL6 cells with culture media conditioned by various cell types in the absence of DMSO, and screened the cardiogenic activity of the conditioned media.…”

Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2 (BMP-2)-induced Myocyte Enhancer Factor 2A-dependent Transcription of BMP-2 Gene in Cardiomyocyte Precursor Cells

“…I. Komuro, Department of Cardiovascular Medicine, University of Tokyo, Japan. CL6 cells undergo differentiation in the presence of 1% Me 2 SO into beating cardiomyocyte and express cardiac-specific genes (16). The cells were grown in ␣-minimal essential medium with 10% fetal bovine serum.…”

“…Stimulates PI 3-kinase Activity-CL6 cells, a clonal derivative of embryonic P19 teratocarcinoma cells, undergo differentiation into beating cardiomyocytes when treated with Me 2 SO in a BMP-2-dependent manner (16). To investigate the role of PI 3-kinase in response to BMP-2, CL6 cells were incubated with 100 ng/ml BMP-2 for different periods of time.…”

“…BMP-2 also induces expression of cardiac transcription factors in embryonic teratocarcinoma cells (16). The mechanism by which BMP-2 regulates cardiac development and cardiac gene expression is largely unknown.…”