Sphingosine 1-phosphate receptors modulate intracellular Ca 2+ homeostasis Elena Rapizzi a , Chiara Donati a , Francesca Cencetti a , Paolo Pinton b , Rosario Rizzuto b , Paola Bruni a, * a Department of Biochemical Sciences, Istituto Interuniversitario di Miologia (IIM), University of Firenze, 44100 Firenze, Italy b Department of Experimental and Diagnostic Medicine, Section of General Pathology, Telethon Center for Cell Imaging (TCCI) and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, 44100 Ferrara, Italy Received 24 November 2006 Available online 11 December 2006 Abstract Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca 2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca 2+ homeostasis in HeLa cells. Over- expression of S1P 2 , or S1P 3 , but not S1P 1, leads to a significant increase in cytosolic and mitochondrial [Ca 2+ ] in response to S1P chal- lenge. Moreover, cells ectopically expressing S1P 2 , or S1P 3 exhibited an appreciable decrease of the free Ca 2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompa- nied by a reduced susceptibility to C 2 -ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca 2+ homeostasis and its possible implication in the regulation of cell survival. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Sphingosine 1-phosphate; Calcium; Apoptosis; Ceramide; Sphingosine 1-phosphate receptors Within the last decade the importance of some sphingolip- id metabolites, such as ceramide and sphingosine 1-phos- phate (S1P), as regulators of many cellular functions has become clear, and have lead to an explosion of interest for these molecules [1]. It is now widely accepted that most if not all the effects of S1P are exerted from outside the cells via ligation to G-protein-coupled receptors, which have been named S1P receptors (S1PRs). To date, five closely related S1PRs (S1P 1 –S1P 5 ) have been identified [2]. S1P appears to be able to modulate the intracellular Ca 2+ signaling through the action of S1P 2 and S1P 3 , which mobilize the cation from intracellular stores (namely endoplasmic reticulum (ER)) [3,4], while the ability of S1P 1 to mobilize intracellular Ca 2+ is still matter of debate. In native C2C12 cells Ca 2+ mobilization induced by S1P was found to be a receptor- mediated process that involves S1P 2 and S1P 3 , but not S1P 1 [5]. Since Ca 2+ is considered one of the most important intracellular messengers, Ca 2+ signals need to be flexible and precisely regulated. In this process, mitochondria and ER have a pivotal role because they are not only involved as modulators of the variations in Ca 2+ concentration, but also as Ca 2+ intracellular targets. Although it has been extensive- ly demonstrated that S1P is able to induce an increase in cytosolic Ca 2+ concentration in various cell types [2,6], the kinetics of Ca 2+ signaling in mitochondria and ER, and the role of the different S1PRs in modulating Ca 2+ homeo- stasis in these organelles, have yet to be investigated. 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.010 Abbreviations: S1P, sphingosine 1-phosphate; ER, endoplasmic retic- ulum; S1PRs, sphingosine 1-phosphate receptors; [Ca 2+ ] cyt , cytosolic Ca 2+ concentration; [Ca 2+ ] m , mitochondrial Ca 2+ concentration; [Ca 2+ ] er , endoplasmic reticulum Ca 2+ concentration; GFP, green fluorescent protein; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; BSA, bovine serum albumin; PTx, pertussis toxin; KRB, Krebs– Ringer modified buffer; SDS, sodium dodecylsulfate. * Corresponding author. Fax: +39 055 4598905. E-mail address: paola.bruni@unifi.it (P. Bruni). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 353 (2007) 268–274