The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells Ola Hadadeh1,2, Emilie Barruet1,2, Franck Peiretti1,2, Monique Verdier1,2, Denis Bernot1,2, Yasmine Hadjal1,2, Claire El Yazidi1,2, Andre´ e Robaglia-Schlupp3,4, Andre Maues De Paula3,5, Didier Ne` gre6,7, Michelina Iacovino 8 , Michael Kyba8, Marie-Christine Alessi1,2, Bernard Bine´ truy1,2* 1 Inserm U626, Faculte´ de Me´decine, Marseille, France, 2 Aix-Marseille Universite´, Faculte´ de Me´decine, Marseille, France, 3 Inserm U910, Faculte´ de Me´decine, Marseille, France, 4 Laboratoire de Biologie Cellulaire, CHU (Centre Hospitalier Universitaire) La Timone AP-HM (Assistance Publique – Hoˆ pitaux de Marseille), Marseille, France, 5 Service d’Anatomie Pathologique et Neuropathologie, CHU (Centre Hospitalier Universitaire) La Timone AP-HM (Assistance Publique – Hoˆ pitaux de Marseille), Marseille, France, 6 INSERM, U758, Ecole Normale Supe´rieure de Lyon, Lyon, France, 7 Universite´ de Lyon, UCB-Lyon1, Ecole Normale Supe´rieure de Lyon, Lyon, France, 8 Department of Pediatrics and Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota, United States of America Abstract Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-12/2 induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms. Citation: Hadadeh O, Barruet E, Peiretti F, Verdier M, Bernot D, et al. (2012) The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells. PLoS ONE 7(11): e49065. doi:10.1371/journal.pone.0049065 Editor: Qingzhong Xiao, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom Received January 17, 2012; Accepted October 8, 2012; Published November 8, 2012 Copyright: 2012 Hadadeh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Agence Nationale pour la Recherche and the Association Franc¸aise contre les Myopathies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Bernard.Binetruy@univ-amu.fr Introduction Adipose, vascular and muscle tissue plasticity occurs in a number of normal and physiopathological processes including embryonic development, adult muscle aging, muscular dystrophies, obesity and diabetes. There is ample evidence both in vivo and in vitro that the extracellular matrix (ECM) surrounding the tissues plays an important functional biological role either in physiological or pathological conditions. Likewise its function in development is well documented, suggesting that ECM could be involved in the regulation of the commitment and differentiation of stem cells. The concept of niche in which the stem cell fate is dependent on interactions with its surrounding environment represents a para- digm of these regulations [1,2]. The PA system is one of the main suppliers of extracellular proteolytic activity, such as the fibrinolysis, thus contributing to ECM degradation and tissue remodelling. It is exerted by two distinct plasminogen activators, tPA (tissue-type plasminogen activator) and uPA (urokinase-type plasminogen activator) [3,4]. The plasminogen activator inhibitor-1 (PAI-1) directly inhibits the proteolytic activity of uPA and tPA [3]. This system is implicated in numerous fibrinolytic and non-fibrinolytic processes leading to either ECM degradation, activation of latent growth factors, or integrin-mediated cell adhesion on vitronectin (VN) [5,6]. All these processes are inhibited by PAI-1 [7,8]. In vitro, PAI-1 regulates adipocyte differentiation [9] and, by controlling endothelial cell migration and prolifereation, is a potent regulator of angiogenesis [10,11,12]. Finally, PAI-1 interferes with myogenesis: myogenic cells express the proteins of the plasminogen activation system PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49065