Stage-Specific Role of Endogenous Smad2 Activation in Cardiomyogenesis of Embryonic Stem Cells

Abstract

Abstract-The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-␤ and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB-431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-␤, and inhibition of transforming growth factor-␤-mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-␤ negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes. to study development of cardiac myocytes but also proposes the potential use of ES cell-derived cardiac myocytes for many medical applications such as cell transplantation therapy against various heart diseases and pharmacological testing on cardiac myocytes. 1 However, the molecular mechanisms governing differentiation of ES cells into specific lineages are poorly understood, and their comprehension would improve the efficiency of differentiation into specific cell types.Members of transforming growth factor (TGF)-␤ superfamily are pleiotropic cytokines involved in many biological processes and signaling via heteromeric complexes of type I and type II serine/threonine kinase receptors. 2 On ligand binding and heterodimerization, the constitutively active type II receptor kinase phosphorylates the type I receptor, which in turn activates downstream signal transduction cascades including Smad pathways. According to the usage of different sets of type I receptors and receptor-regulated Smad (RSmad), the superfamily members can be classified into 2 major branches: (1) the TGF-␤/activin/Nodal branch and (2) the bone morphogenetic protein (BMP)/growth and differentiation factor (GDF) branch. 2 The TGF-␤/activin/Nodal branch activates activin receptor-like kinase (ALK)-4, -5, and -7, which phosphorylate Smad2 and -3, whereas Smad1, -5, and -8 are substrates for the BMP/GDF branch through ALK-1, -2, -3, and -6. 2,3 The role of Smad1/5/8-activating BMP in cardiogenesis is relatively well documented. In chick embryo, BMP2 is able to induce expression of myocardial lineage markers in ectopic locations in vivo, and anterior lateral mesoderm explant cultures in vitro, 4 and Noggin, one of soluble BMP antagonists, prevents myocard...

G Protein-Coupled Receptor APJ and Its Ligand Apelin Act Downstream of Cripto to Specify Embryonic Stem Cells Toward the Cardiac Lineage Through Extracellular Signal-Regulated Kinase/p70S6 Kinase Signaling Pathway

“…By contrast, as previously described, expression of neuroectodermal marker Neurod1 was upregulated (Figure 1D). 18 Comparable results were obtained with Smad2-silenced ESCs (Online Results and Online Figures I and II).Together, our data indicate that that early activation of Smad2 is critical for induction of Cripto, Apj, and apelin expression and the consequent inhibition of the neuronal fate. …”

“…18 We also previously showed that Cripto acts via the Nodal/ALK-4/Smad2 pathway to induce ESC cardiomyogenesis. 17 We thus asked whether early activation of Smad2 induced Apj and/or apelin expression.…”

“…17 Accordingly, recent data pointed for a key role of Nodal/Cripto-dependent early activation of Smad2, which was indispensable for mesendodermal induction and the subsequent cardiac differentiation of ESCs. 18 However, little is yet known about the mechanisms of action and the identity of the factors downstream of Cripto/Smad2 in mammalian cardiomyogenesis.…”

Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-β/activin/nodal signaling using SB-431542

“…(30) Activin signaling is known to induce meso/endoderm and to further enhance definitive endoderm in monolayer culture of hESCs. (31) Moreover, a recent study by Kitamura and colleagues (32) showed that inhibition of TGF-b/activin/nodal signaling by a small molecule inhibitor SB-431542 enhanced expression of mesodermal lineage marker genes and promoted cardiomyogenesis in mouse embryoid body outgrowth cultures. (32) In the same study, the authors demonstrated that the TGF-b superfamily ligands, such as activin A/B, nodal, and TGF-b1, activated TGF-b signaling and inhibited cardiomyogensis, suggesting that TGF-b signaling is important for skeletal cell lineage specification of ex vivo expanded ESCs.…”

“…(31) Moreover, a recent study by Kitamura and colleagues (32) showed that inhibition of TGF-b/activin/nodal signaling by a small molecule inhibitor SB-431542 enhanced expression of mesodermal lineage marker genes and promoted cardiomyogenesis in mouse embryoid body outgrowth cultures. (32) In the same study, the authors demonstrated that the TGF-b superfamily ligands, such as activin A/B, nodal, and TGF-b1, activated TGF-b signaling and inhibited cardiomyogensis, suggesting that TGF-b signaling is important for skeletal cell lineage specification of ex vivo expanded ESCs. Similarly, we have reported recently that activation of TGF-b signaling by activin B induced endodermal differentiation of ex vivo cultured hESCs and promoted pancreatic and liver lineage specification.…”