Involvement Of Calcium In 50-Hz Magnetic Field-induced Activation Of Sphingosine Kinase1 Signaling Pathway

Since the widely using of the electricity,50 Hz power frequency magnetic fields(MF)exist profusely in the public and occupational environments.Some epidemiological studies have demonstrated the correlations between exposure to 50 Hz MF and increased risks of adverse health effects.The International Agency for Research on Cancer(IARC)has even classified ELF-MF as a `possible human carcinogenic agent’(class 2B)in 2002.However,up to now,the biological effects and mechanisms of ELF-EMFs are still ambiguous.Sphingolipid metabolites play important roles in cellular activities.Our previous study found that sphingosine kinase 1(SK1)regulates 50 Hz MF-induced human amniotic epithelial(FL)cell proliferation.However,the mechanism of SKI activation induced by MF remain unclear.Relevant studies showed that extracellular regulated protein kinase(ERK),Ca2+,protein kinase C a(PKCa),Akt(protein kinase B),and other molecules can not only respond to MF but also be closely related to SKI activation.Thus,the objective of the present study is to explore the possible roles of calcium on SKI activation and cell proliferation induced by 50 Hz MF.In this study,we found that treatment with BAPTA,the cell permeant chelator of calcium,could block 0.4 mT,50 Hz MF-induced FL cell proliferation,indicating that calcium was involved in the mechanism of MF-induced cell proliferation.Further study showed that the intracellular calcium increased after exposure to 0.4mT,50 Hz MF for 5 min and 10 min,but exposure to MF for 15 min,30 min or 60 min did not affect Ca2+concentration further,indicating that Ca2+might be an early effector in responding to MF exposure.Then,we investigated the effects of MF exposure on extracellular calcium influx with the use of Nifedipine(NIF),an inhibitor of L-type calcium channel of cellular membrane.Results showed that NIF could reduce phosphorylation/activation of ERK and PKC induced by MF,while had no effect on Akt,suggesting that MF could activate ERK and PKC through L-type calcium channel.The corresponding inhibitors were used to further investigate the relationships between protein kinases.Results showed that SKI II,an inhibitor of SKI,eliminated the phosphorylation of both ERK and PKCa induced by MF,whereas the inhibitor of PKCa,Go6976 had no effect on 50 Hz MF-induced SKI activation in FL cells.It suggests that PKCa should be a downstream signalling molecule of SKI.Interestingly,treatment with U0126,an inhibitor of ERK,also could block MF-induced SKI phosphorylation,but had no effect on PKCa phosphorylation,suggesting that SKI enhanced the phosphorylation of ERK and PKCa through different pathways and there might be a feedback mechanism between SKI and ERK activation in responding to MF exposure in FL cells.In addition,we detected the concentration of intracellular calcium in FL cells after cultured with calcium free medium,the results showed that Ca2+was significantly reduced,and 50 Hz MF exposure at 0.4mT for 10 min could weaken this reduction,suggesting that MF exposure could increase intracellular calcium through the release of calcium pool.Exposure of FL cells to MF for 60 min and cultured in calcium-free medium simultaneously enhanced the phosphorylation/activation of Akt and SKI and promoted FL cell proliferation.LY294002,the inhibitor of PI3K/Akt,eliminated the phosphorylation/activation of SKI induced by MF,suggesting that Akt participated in MF-induced SKI activation which was dependent on the release of intracellular calcium in the absence of calcium influx.However,the effects of SKI activation and cell proliferation were much lower than that of normal culture condition,and the activation of SKI failed to activate ERK and PKCa,supposing that MF could activate SKI and its downstream signaling molecules mainly through stimulating extracellular calcium influx,while intracellular calcium release might be a stress response.In summary,we concluded:MF exposure increased intracellular Ca2+and cell proliferation,which was dependent on the L-type calcium channel.Intracellular Ca2+mediated the 50 Hz MF-induced SKI activation which enhanced the phosphorylation of PKCα and ERK,and there might be a feedback mechanism between SKI and ERK activation in responding to MF exposure in FL cells.When cultured with calcium free medium,MF exposure could also increase intracellular calcium,and activate SKI through PI3K/Akt pathway.