Abstract
12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6–8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.
Highlights
Pluripotent stem (PS) cells represent a promising source of cardiomyocytes for cell-based therapies and a model for cardiac d isease[1]
To avoid potential false-positive results caused by the presence of DMSO, a solvent of TPA, an additional control was added to our experimental scheme
Treatments by both UO126 and PD184352 abolished the phosphorylation of GATA4 (Fig. 6D,E and Supplementary Fig. 8B,C; 9A’–D’, A’’–D’’). These inhibitors decreased the phosphorylation of protein kinase C (PKC) to some extent (Supplementary Fig. 9C’,C’’). Taking these findings together with the results of cardiomyocyte counting (Fig. 6A and Supplementary Fig. 7A), we suggest that the positive effect of TPA on cardiomyocyte differentiation is mediated through the activation of the extracellular signal-regulated kinase (ERK) signalling pathway by PKC
Summary
Pluripotent stem (PS) cells represent a promising source of cardiomyocytes for cell-based therapies and a model for cardiac d isease[1]. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a very potent small-molecule compound It is standardly used in biomedical research as an activator of the protein kinase C (PKC) family, a broad group of serine/threonine kinases[5,6]. PKC signalling mediates a number of cellular processes, including the regulation of cell proliferation, differentiation, and migration, etc.[7,8,9]. This treatment increased the number of cardiomyocytes by approximately 5 times compared to the standard protocol of differentiation, which is based on spontaneous cardiomyogenesis induced by the formation of embryonic bodies. Our results show that the activation of PKC by TPA and subsequent stimulation of ERK at the specific time point increases the formation of cardiomyocytes in differentiating ES cells
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