Identification and functional characterization of the G-protein coupled receptor APJ and its ligand apelin in embryonic stem cells: a novel pathway regulating mammalian cardiomyogenesis
D'Aniello, Cristina (2010) Identification and functional characterization of the G-protein coupled receptor APJ and its ligand apelin in embryonic stem cells: a novel pathway regulating mammalian cardiomyogenesis. [Tesi di dottorato] (Inedito)
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The commitment of mesodermal precursors to a cardiac fate is one of the first events in embryogenesis. It results from inductive interactions occurring during gastrulation in a temporally and spatially regulated manner. Embryonic Stem Cells (ESCs) are a powerful tool to study the early events of cardiac lineage specification in mammals; in particular, several cardiac cell populations have been generated from differentiating ESCs and studies of gene expression profiles indicate that their development in culture recapitulates cardiogenesis in the early embryo. Thus, ESCs represent a valid model to elucidate the origin and the molecular identity of cardiovascular progenitor populations as well as to identify key regulators of cardiac specification in mammals for which the molecular control is largely unknown. Cripto/ALK4/Smad2 signaling is a key pathway required for the correct cardiac myogenesis, acting in the early phases of ESC differentiation. Here we report the identification of the Angiotensin II Type ReceptorLike I (AGTRL1/ APJ/msr1) and its ligand Apelin as previously unrecognized downstream targets of Cripto/Smad2 signaling both in vivo and in ESCs. Gain of function experiments show that APJ suppresses neuronal differentiation, which spontaneously occurs in cripto/ ESCs, and restore the cardiac program, activating the expression of genes pivotal for cardiac specification and terminal differentiation. Furthermore, lossoffunction experiments reveal, for the first time to our knowledge, a central role for APJ/Apelin signaling in the gene regulatory cascade promoting ESC cardiac specification and differentiation. Most remarkably, we show that Apelin promotes cardiomyogenesis via activation of pERK/p70S6 through coupling to a Go/Gi protein. Together our data point for a previously undescribed functional link between Cripto/Smad2 and APJ/Apelin in the signaling pathways that govern mesoderm patterning and cardiac specification in mammals. Finally, to further investigate the role of APJ/Apelin signaling in the control of ESC differentiation and, in particular, in the specification of cardiovascular progenitors, we have generated an inducible ESC line, which allow overexpression of APJ in a timespecific manner, by using the tetracycline (Tet)regulated transactivator system. Our data reveal that APJ overexpression, in an early time window of wildtype ESC differentiation, promotes the expression of markers of the cardiovascular lineage and markedly increases the number of cardiomyocytes.
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