Sphingosine kinase-1/S1P 1 signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts Paola Nincheri a , Caterina Bernacchioni a,b , Francesca Cencetti a,b , Chiara Donati a,b,1 , Paola Bruni a,b, ⁎ ,1 a Dipartimento di Scienze Biochimiche, Universita` di Firenze, 50134, Firenze, Italy b Istituto Interuniversitario di Miologia, Firenze, Italy abstract article info Article history: Received 2 April 2010 Received in revised form 17 June 2010 Accepted 17 June 2010 Available online 1 July 2010 Keywords: PDGF Sphingosine 1-phosphate S1P 1 Sphingosine kinase-1 C2C12 myoblasts Skeletal muscle PDGF is known to be critically implicated in skeletal muscle repair; however its molecular mechanism of action has been only marginally investigated. In this study we show that in mouse myoblasts PDGF transactivates S1P 1 receptor via sphingosine kinase (SK)-1 activation and that this molecular event exerts a negative regulation of the mitogenic effect elicited by this growth factor. Indeed, pharmacological inhibition of S1P 1 , or its specific silencing increased PDGF-dependent cell proliferation, whereas S1P 1 overexpression diminished the biological effect. Moreover, the mitogenic response to PDGF was enhanced by pharmacological inhibition of SK activity as well as specific silencing of SK1 but not SK2. Furthermore, ERK1/2 signalling pathway was found to be upstream of the observed attenuation of PDGF-induced cell proliferation. Interestingly, PDGF-directed engagement of S1P 1 exerted also a positive modulatory action of the growth factor-dependent cell motility. The here highlighted dual role of S1P 1 -mediated signalling in response to myoblast challenge with PDGF is likely important to guarantee the fine control of the biological response to this growth factor, finalized to efficient repopulation of skeletal muscle after damage, where a tight balance between proliferation and migration of tissue progenitor cells is required. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid gener- ated by sphingomyelin metabolism which regulates key biological processes crucially implicated in cell homeostasis [1,2]. S1P is present at submicromolar concentration in plasma, being released by various cell types including platelets, erythrocytes and endothelial cells [3–6]. Additionally, S1P can be autonomously produced in many cell types, being endogenous levels of the bioactive lipid strictly regulated by the balance between biosynthetic and catabolic enzymatic activities such as sphingosine kinase (SK)-1 and SK2 on one side [7], and S1P lyase and specific phosphatases on the other [8,9]. The molecular mechan- isms by which S1P elicits specific cellular responses appear to be highly complex and only partially identified. It is commonly accepted that the extracellular action of S1P as ligand of a family of five specific G-protein coupled receptors named S1P receptors (S1PRs), accounts for the majority of its biological effects. Singularly, since all S1PRs, apart from S1P 1 , are coupled to a specific set of G proteins they can trigger a wide variety of signalling pathways determining distinct and even contrasting final cellular effects. Thus, the S1PR expression pattern exhibited by a given cell type appears to be critical for the final biological outcome elicited by S1P. A further layer of complexity in S1P action mode is added by the occurrence of strong cross- communication between S1P signalling and a wide variety of extracellular cues. Indeed, in many instances the biological effects exerted by S1P are superimposable to those elicited by specific growth factors and cytokines due to their ability to transactivate S1P signalling cascades as well as the ability of S1P to transactivate growth factor and cytokine signalling cascades [10,11]. In this respect it is important to note that S1P was originally identified as an intracellular messenger generated in response to PDGF with a role in the elicited mitogenic response [12]. Subsequently, it has been established that S1P and PDGF cross-communicate employing multiple mechanisms including the occurrence of a functional complex comprising PDGF receptor-beta (PDGFR-β) and S1P 1 , [13,14] as well as the sequential activation of S1P 1 driven by PDGF challenge via SK1 activation and S1P formation [15], both critically implicated in the regulation of cell motility. Moreover, the occurrence of S1P-directed transactivation of PDGFR-β via enhance- ment of its tyrosine phosphorylation state in vascular smooth muscle cells [16], as well as the S1P-mediated induction of PDGF-BB mRNA expression reported to occur via S1P 1 in vascular smooth Cellular Signalling 22 (2010) 1688–1699 Abbreviations: S1P, sphingosine 1-phoshate; SK, sphingosine kinase; S1PR, S1P receptor; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; FCS, fetal calf serum; siRNA, short interfering RNA; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; DMS, dimethylsphingosine; PTx, pertussis toxin; PDGFR-β, PDGF receptor-beta. ⁎ Corresponding author. Dipartimento di Scienze Biochimiche, University of Florence, Viale GB Morgagni 50, 50134 Firenze, Italy. Tel.: +39 0554598328; fax: +39 0554598905. E-mail address: paola.bruni@unifi.it (P. Bruni). 1 These authors contributed equally to this work. 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.06.009 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig