Research Article 1819 Introduction The dissection of signaling pathways regulating cardiac versus vascular differentiation is of particular impact because recent evidence has shown that, during embryogenesis, cardiomyogenesis and vasculogenesis occur in a strictly time- and space-dependent manner. Using mouse embryonic stem (ES) cells, which mimic early embryonic development, it was discovered that cardiac- independent vasculogenesis arises from Flk-1 + (vascular endothelial growth factor receptor 2-positive, VEGFR2 + ) hemangioblasts, whereas at cardiogenic day 3.25 a ‘second’ Flk-1 + cell population with enriched cardiogenic as well as vasculogenic potential was identified (Yang et al., 2008). This observation raised the intriguing possibility that the cardiac lineage develops from a progenitor cell that also displays vascular potential and thus might represent the cardiac equivalent to the hemangioblast (Bu et al., 2009; Garry and Olson, 2006; Kattman et al., 2007). As a consequence, Flk-1 + cardiac as well as endothelial progenitor cells should be responsive towards VEGF stimulation, and might activate distinct signaling pathways that variegate cardiac-associated vasculogenesis versus cardiac-independent (yolk sac hemangioblast-derived) vascular differentiation (Iida et al., 2005; Kattman et al., 2006). The signal transduction pathways that are activated in different cell types in response to VEGF are well established (Cross et al., 2003; Wang et al., 2008). However, relatively little is known about the involvement of distinct phosphoinositide 3-kinase (PI3K) catalytic subunits and protein kinase C (PKC) isoforms in VEGF action (Hamada et al., 2005; Gliki et al., 2002; Gerber et al., 1998). VEGF receptors (VEGFR1 and VEGFR2) are present on vascular endothelial cells and their signaling is mediated by receptor dimerization leading to autophosphorylation of the cytosolic domains of the receptors. Phosphorylated VEGF receptors serve as docking sites for adapter molecules or signaling enzymes such as PI3K. VEGF has been shown to activate PI3K generating phosphatidylinositol (3,4,5)-trisphosphates (Bos 1995; Gerber et al., 1998). Moreover, numerous studies demonstrated that PI3K plays an important role in regulating endothelial proliferation, migration and survival (Gerber et al., 1998; Thakker et al., 1999; Jiang et al., 2000). PI3Ks are classified into classes I, II and III (Vanhaesebroeck et al., 1997). Class I PI3Ks, which include class IA and class IB (consisting of PI3K only), are a family of dual-specificity lipid and protein kinases that control many cellular functions, such as growth and proliferation, survival and apoptosis, as well as adhesion and migration of a wide range of cell types (Katso et al., 2001; Wymann and Pirola, 1998). All four class I PI3Ks are heterodimers composed of a catalytic subunit with a molecular weight of 110 kDa and a tightly associated regulatory subunit that controls activation and subcellular localization (Whitman et al., 1988; Stephens et al., 1991). The importance of PKC regarding the VEGF–PI3K pathway was suggested by several authors (Chou et al., 1998; Dutil et al., 1998; Gliki et al., 2002; Le Good et al., 1998), although a detailed VEGF- Summary VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1 + cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110a and p110 using short hairpin RNA (shRNA), or inhibition of p110a with compound 15e and of p110 with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110b, and shRNA knockdown of p110b were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCa/bII) and rottlerin (targeting PKC) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCa/bII and PKC but not PKCz. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide- dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110a and p110 are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis. Key words: Embryonic stem cells, Cardiovascular differentiation, Phosphoinositide 3-kinase, Protein kinase C Accepted 7 January 2011 Journal of Cell Science 124, 1819-1830 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.077594 VEGF-mediated PI3K class IA and PKC signaling in cardiomyogenesis and vasculogenesis of mouse embryonic stem cells Mohamed M. Bekhite 1,2 , Andreas Finkensieper 1 , Stephanie Binas 1 , Jörg Müller 3 , Reinhard Wetzker 3 , Hans-Reiner Figulla 1 , Heinrich Sauer 4 and Maria Wartenberg 1, * 1 Department of Internal Medicine I, Cardiology Division, Friedrich Schiller University, 07743 Jena, Germany 2 Zoology Department, Faculty of Science, Tanta University, Tanta 31527, Egypt 3 Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University, 07737 Jena, Germany 4 Department of Physiology, Justus Liebig University Gießen, 35392 Gießen, Germany *Author for correspondence (maria.wartenberg@med.uni-jena.de) Journal of Cell Science