Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (SIP) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by SIP lyase. Extracellular SIP activates specific G protein-coupled receptors while intracellular SIP can mobilize Ca2+ from thapsigargin-sensitive stores. Here, we have studied Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in SIP lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca2+] was elevated and agonist-induced [Ca2+](i) increases were augmented in SIP lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+](i) increases and Ca2+ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+ storage in SI P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca2+ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca2+](i) increase and the other with a slower and prolonged [Ca2+](i) elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+](i) increases, reflecting overall Ca2+ release, was significantly increased by more than 50% in both rapidly and slowly responding SIP lyase-deficient cells. It is concluded that elevated concentrations of SIP and/or sphingosine lead to enhanced Ca2+ storage and elevated basal [Ca2+](i). SIP metabolism thus plays a role not only in acute Ca2+ mobilization but also in long-term regulation of Ca2+ homeostasis. (C) 2009 Elsevier Inc. All rights reserved.