Fingolimod (FTY720) Stimulates Ca2+/Calcineurin Signaling in Fission Yeast

Fingolimod hydrochloride (FTY720) is the first in class of sphingosine 1-phosphate (S1P) receptor modulator approved to treat multiple sclerosis via down-regulation of G protein-coupled S1P receptor 1 by its phosphorylated form (FTY720-P). Many studies have revealed that FTY720 exerts various biological effects, including antitumor activities, angiogenesis inhibition, Ca(2+) mobilization and apoptosis, independently of S1P receptors. However, the exact mechanisms underlying their effects or signaling pathways mediated by FTY720 have not been completely established. To gain further insights into molecular mechanisms of FTY720 action, the effect of FTY720 on Ca(2+) signaling in fission yeast was analyzed. The addition of Ca(2+) enhanced the sensitivity induced by FTY720, and mutants lacking genes required for calcium homeostasis, including calcineurin and its downstream transcription factor, Ppb1-responsive zinc finger protein (Prz1), were hypersensitive to FTY720 and CaCl2. The effect of FTY720 on calcineurin signaling was monitored by utilizing a luciferase reporter construct fused to three tandem repeats of the calcineurin-dependent response element (CDRE), which gives an accurate measure of calcineurin activity. The addition of FTY720 increased calcineurin activity as well as Ca(2+) influx in a concentration-dependent manner. Notably, the FTY720-mediated Ca(2+) influx and calcineurin activation were reduced markedly by the deletion of yam8 (+) or cch1 (+) encoding putative subunits of a Ca(2+) channel. Consistently, the deletion of Pmk1 mitogen-activated protein kinase (MAPK), which plays an important role in the activation of the Yam8/Cch1 channel, markedly decreased the intracellular Ca(2+) levels upon FTY720 treatment. These results suggest that the FTY720-stimulated Ca(2+)/calcineurin signaling activation partly involves the Yam8/Cch1 channel in fission yeast.