Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin. Ombretta Guardiola, Peggy Lafuste, Silvia Brunelli, Salvatore Iaconis, Thierry Touvier, Philippos Mourikis, Katrien de Bock, Enza Lonardo, Gennaro Andolfi, Ann Bouché, et al. To cite this version: Ombretta Guardiola, Peggy Lafuste, Silvia Brunelli, Salvatore Iaconis, Thierry Touvier, et al.. Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myo- statin.. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2012, 109 (47), pp.E3231-40. 10.1073/pnas.1204017109. inserm-00769969 HAL Id: inserm-00769969 https://www.hal.inserm.fr/inserm-00769969 Submitted on 5 May 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Classification: Biological Sciences, Developmental Biology; 2 3 CRIPTO REGULATES SKELETAL MUSCLE REGENERATION AND MODULATES SATELLITE 4 CELL DETERMINATION BY ANTAGONIZING MYOSTATIN 5 6 Ombretta Guardiola1,2*, Peggy Lafuste3,4,5,*, Silvia Brunelli6,7,§, Salvatore Iaconis1,2,§, 7 Thierry Touvier8, Philippos Mourikis9, Katrien De Bock3,4, Enza Lonardo1,2, Gennaro 8 Andolfi1,2, Ann Bouché3, Giovanna L. Liguori2, Michael M. Shen10, Shahragim Tajbakhsh9, 9 Giulio Cossu11, Peter Carmeliet3,4,‡ and Gabriella Minchiotti1,2‡^ 10 1. Stem Cell Fate Laboratory, Institute of Genetics and Biophysics “A. Buzzati Traverso”, 11 CNR, Naples 80131, Italy. 12 2. Institute of Genetics and Biophysics “A. Buzzati Traverso”, CNR, Naples 80131, Italy. 13 3. Vesalius Research Center, VIB, Leuven 3000, Belgium; 14 4. Vesalius Research Center, K.U.Leuven, Leuven 3000, Belgium 15 5. INSERM, U955, team 10 “Cell interactions in the neuromuscular system”, University 16 Paris Est Creteil, F-94000 Creteil, France. 17 6. Division of Regenerative Medicine, San Raffaele Scientific Institute, 20132 Milan, Italy. 18 7. Department of Experimental Medicine, University of Milano-Bicocca, 20052, Monza, 19 Italy 20 8. E Medea Scientific Institute, 23842 Bosisio Parini, Italy 21 9. Institut Pasteur, Stem Cells & Development, CNRS URA 2578, Paris, France 22 10. Columbia University College of Physicians and Surgeons Herbert Irving 23 Comprehensive Cancer Center, New York, NY 10032 1 1 11. Department of Cell and Developmental Biology, University College London. WC1E 2 6DE, United Kingdom 3 4 5 Running Title: Role of Cripto in skeletal muscle regeneration 6 7 * Equally contributed as co-first author. § Equally contributed as co-second author 8 ‡ Equally contributed as co-last author 9 ^ Corresponding author: Gabriella Minchiotti 10 Stem Cell Fate Laboratory 11 Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR 12 Via Pietro Castellino 111 13 80131 Naples, Italy 14 Tel. 0039 081 6132357; Fax 0039 081 6132595 15 [email protected] 16 2 1 SUMMARY 2 3 Skeletal muscle regeneration mainly depends on satellite cells, a population of resident 4 muscle stem cells. Yet, our understanding of the molecular mechanisms underlying 5 satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of 6 early embryogenesis, is a novel regulator of muscle regeneration and satellite cell 7 progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite 8 cells compromises skeletal muscle regeneration, whereas gain of function of Cripto 9 accelerates regeneration leading to muscle hypertrophy. Moreover, we provide evidence 10 that Cripto modulates myogenic cell determination and promotes proliferation by 11 antagonizing the TGFβ ligand Myostatin. Our data provide new insights in the molecular 12 and cellular basis of Cripto activity in skeletal muscle regeneration and raise novel 13 implications for stem cell biology and regenerative medicine. 14 15 3 1 \body 2 INTRODUCTION 3 4 It is now evident that genes and molecular mechanisms, which have key roles 5 during embryogenesis, are reactivated in the adult during tissue remodeling and 6 regeneration, and when deregulated may contribute to cancer progression (1). The cripto 7 gene has emerged as a key player in this complex scenario. Cripto is a GPI-anchored 8 protein and the founder member of a family of signaling molecules, the EGF-CFC proteins, 9 important for vertebrate development (2). Cripto is associated with the pluripotent status of 10 both human and mouse embryonic stem cells (ESCs) (3) and it acts as a key player in the 11 signaling networks orchestrating ESC differentiation (4). Intriguingly, it has been recently 12 suggested that Cripto may serve as regulator to control dormancy of hematopoietic stem 13 cells (HSCs) (5). 14 Under normal physiological conditions, Cripto is expressed during embryonic 15 development (2) and it has been shown to have activity both as a soluble factor and GPI- 16 anchored protein (6-8). Existing models indicate that Cripto can function via different 17 signaling pathways. Cripto plays distinct and opposing roles in modulating the activity of 18 several transforming growth factor-beta (TGFβ) ligands. Indeed as an obligate co-receptor, 19 Cripto binds Nodal and GDF1/GDF3 and stimulates signaling through the activin receptor 20 complex composed of type I serine-threonine ActRIB (ALK4) and type II receptor 21 (ActRII/ActRIIB) (9-11). Following receptor activation, the intracellular effectors Smad2 22 and/or Smad3 are phosphorylated and accumulate in the nucleus with Smad4 to mediate 23 transcriptional response (12). In contrast to its co-receptor function, Cripto is able to 24 antagonize signaling of other members of the TGFβ family, i.e. Activins and TGFβ. This 25 inhibitory activity of Cripto results in reduced ability to form an active ActRII/ActRIB 4 1 receptor complex (13-15). Despite the well-described role of Cripto in early development 2 and ESC differentiation, the role of this protein in post-natal life remains elusive. To date, 3 de novo expression of Cripto has been associated with several epithelial cancers (16, 17), 4 but its role in other pathological conditions such as injury or degenerative diseases has not 5 been investigated. Given the physiological activity of Cripto in the instructive events of 6 embryonic mesodermal commitment and differentiation (4), we hypothesized that Cripto 7 expression might be reactivated in response to injury in mesenchymal tissues such as 8 skeletal muscles. 9 Adult skeletal muscle has a generally low cellular turnover rate. However, in response to 10 certain pathological conditions, it undergoes robust regeneration. Regeneration is mainly 11 dependent on satellite cells, a population of resident stem cells that is in a quiescent state 12 during muscle homeostasis. Upon injury or disease, satellite cells become activated, 13 proliferate, migrate to the site of injury and either fuse to form multinucleated myotubes or 14 re-establish a self-renewing pool of quiescent satellite cells (18). Quiescent satellite cells 15 express the transcription factor Pax7, which is involved in myogenic specification (19, 20). 16 Upon injury, activated satellite cells, start proliferating and expressing MyoD, whereas 17 Pax7 expression is progressively reduced. Subsequently, expression of Myogenin and 18 MRF4 (MRF family of transcription factors) are up-regulated as cells enter their terminal 19 differentiation program. A fraction of activated cells down-regulates expression of MyoD 20 and returns to cellular quiescence to maintain a pool of satellite cells (21). A delicate 21 balance between satellite cell proliferation and exit from cell cycle, differentiation, and 22 fusion is required for the correct muscle regeneration to occur. Although some signaling 23 molecules have been found to play a crucial role in these processes (11), including 24 hepatocyte growth factor [HGF; (22)], insulin-like growth factors [IGF; (23)], Myostatin (24) 5 1 and Wnts (25), the underlying molecular mechanisms of muscle regeneration remain 2 largely undefined. 3 In the present study, we provide the first evidence that Cripto is re-expressed in 4 adult skeletal muscle in response to injury and that this response correlates with and 5 regulates muscle regeneration. We also show that Cripto is expressed in activated satellite 6 cells, and promotes myogenic cell determination and proliferation by antagonizing TGFβ 7 ligand Myostatin. 8 6 1 RESULTS 2 3 CRIPTO IS EXPRESSED DURING SKELETAL MUSCLE REGENERATION AND IN MYOGENIC CELLS IN 4 VIVO AND EX VIVO 5 To evaluate whether Cripto is activated in adult tissues under pathological conditions, we 6 performed double immunofluorescence analysis for Cripto and Laminin on normal adult 7 skeletal muscle both during homeostasis and after cardiotoxin (CTX) induced injury. Cripto 8 was undetectable in healthy uninjured muscles (Figures 1A-1C); in contrast, strong 9 expression of Cripto was observed in the regenerating area both inside and outside of the 10 basement membrane surrounding myofibers (Figures 1D-1F). Cripto expression was 11 confirmed by flow