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

Abstract

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, Ca2+ 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 Ca2+ signaling in fission yeast was analyzed. The addition of Ca2+ 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 Ca2+ influx in a concentration-dependent manner. Notably, the FTY720-mediated Ca2+ influx and calcineurin activation were reduced markedly by the deletion of yam8 + or cch1 + encoding putative subunits of a Ca2+ 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 Ca2+ levels upon FTY720 treatment. These results suggest that the FTY720-stimulated Ca2+/calcineurin signaling activation partly involves the Yam8/Cch1 channel in fission yeast.

FTY720 stimulated ROS generation and the Sty1/Atf1 signaling pathway in the fission yeast Schizosaccharomyces pombe

“…FTY720 inhibits growth of S. pombe cells in a dose‐dependent manner (Hagihara et al . ), and as shown here and previously, the wild‐type (wt) cells failed to grow in the presence of 30 μ m FTY720 (Fig. A; Hagihara et al .…”

“…A; Hagihara et al . ). Notably, although FTY720 was shown to stimulate Ca 2+ influx and the subsequent activation of the calcineurin signaling pathway, the addition of EGTA, a calcium chelator, failed to rescue the growth inhibition of wt cells by FTY720 (Fig.…”

“…We have previously analyzed the signaling pathways mediated by FTY720 in fission yeast and demonstrated that FTY720 stimulated the Ca 2+ /calcineurin pathway in fission yeast (Hagihara et al . ). Notably, however, our data also suggested the possibility that FTY720 may have a broader impact on cell proliferation other than inducing Ca 2+ /calcineurin signaling and suggested the existence of the additional signaling pathway(s) involving FTY720 action.…”

The antifungal pipeline: a reality check

“…29), of calcium signalling. The sphingosine-1-phosphate receptor modulator, fingolimod hydrochloride (also known as FTY720), also has broad-spectrum antifungal activity 52 . Finally, even altering gene expression through the manipulation of transcription factors has emerged as an attractive antifungal approach: a prime example of this strategy has been the successful identification of compounds that block the transcription factor sterol uptake control protein 2 (Upc2) 53 .…”

Evaluation of a novel method for measurement of intracellular calcium ion concentration in fission yeast

“…The real-time intracellular Ca 2+ concentration-monitoring assay was performed according to the method described in Hagihara et al (2013). The light emission levels expressed as relative light units (RLU) were measured using a microplate luminometer (CentroXS³ LB960, Berthold Japan K.K., Tokyo, Japan) at 1 min intervals.…”