Sphingosine 1-phosphate inhibits cell migration in C2C12 myoblasts Laura Becciolini a , Elisabetta Meacci a,b , Chiara Donati a,b , Francesca Cencetti a , Elena Rapizzi a,b , Paola Bruni a,b, ⁎ a Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy b Istituto Interuniversitario di Miologia (IIM), Università degli Studi di Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy Received 1 August 2005; received in revised form 11 January 2006; accepted 11 January 2006 Available online 10 February 2006 Abstract This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P 2 , being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P 2 -overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P 2 -dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P 2 -encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the antichemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair. © 2006 Elsevier B.V. All rights reserved. Keywords: Sphingosine 1-phosphate; Cell migration; S1P 2 receptor; RhoA; C2C12 myoblast 1. Introduction Cell migration plays a central role in a wide variety of biological phenomena. During embryogenesis cell directed motility is essential for the accomplishment of key morphogenic events, while in adult organism is prominent for physiological and pathological processes such as inflammatory response, wound healing, and tumour metastasis. Cell migration is also fundamental for skeletal muscle physiology: the motility of myogenic precursor cells is critical for vertebrate embryo limb development; moreover, in postnatal life, the migration of skeletal muscle satellite cells is required for regeneration of damaged skeletal muscle [1–3]. In this regard, the regulation of myoblast chemotaxis appears to be crucial to reach the optimal cell density whereupon fusion on preexisting fiber can initiate. Cell migration is regulated by expression of adhesion molecules, deposition of basement membrane or matrix proteins and soluble extracellular cues interacting with specific cell surface receptors [4]. Among the signalling factors secreted at the site of muscle lesion that favour muscle regeneration by acting on satellite cells, hepatocyte growth factor, basic fibroblast growth factor and insulin-like growth factor-1 (IGF-1) have been identified as potent stimulators of myoblast chemotaxis [5,6]. However, the picture of the extracellular cues which are recognized to be able to regulate myoblast motility is far to be completed. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated by sphingomyelin metabolism which acts almost ubiquitously, mainly via ligation to specific receptors, influ- encing many key biological parameters including cell prolifer- ation, differentiation, motility and survival [7,8]. Of note, the Biochimica et Biophysica Acta 1761 (2006) 43 – 51 http://www.elsevier.com/locate/bba Abbreviations: IGF-1, insulin-like growth factor 1; S1P, sphingosine 1-phosphate; S1PR, sphingosine 1-phosphate receptors; PBS, phosphate- buffered saline, FCS fetal calf serum; BSA, bovine serum albumin; ODN, oligodeoxyribonucleotides; siRNA, small interfering RNA; ECL, enhanced chemiluminescence ⁎ Corresponding author. Dipartimento di Scienze Biochimiche, Viale G.B. Morgagni 50, 50134 Florence, Italy. Tel.: +390554598328; fax: +390554598905. E-mail address: paola.bruni@unifi.it (P. Bruni). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.01.006