Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts.
Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts.
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DOI：
10.1016/j.cellsig.2009.11.001
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发表时间：
2010-03
影响因子：
4.8
通讯作者：
R. Claas;M. ter Braak;B. Hegen;Verena Hardel;C. Angioni;H. Schmidt;K. Jakobs;P. V. Van Veldhoven;D. Z. zu Heringdorf
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作者：
R. Claas;M. ter Braak;B. Hegen;Verena Hardel;C. Angioni;H. Schmidt;K. Jakobs;P. V. Van Veldhoven;D. Z. zu Heringdorf
鞘氨醇-1-磷酸(S1P)在多种细胞类型中调节细胞的生长和存活、迁移和黏附。S1P由鞘氨醇激酶(SphKs)产生，被磷酸酶去磷酸化或被S1P裂解酶裂解。细胞外的S1P激活特定的G蛋白偶联受体，而细胞内的S1P可以从thapsigargin敏感的库中动员钙离子。在这里，我们研究了缺乏S1P裂解酶的小鼠胚胎成纤维细胞(MEF)中的钙信号转导。在这些细胞中，S1P和鞘氨醇的浓度分别增加了约6倍和2倍，根据液相色谱/串联质谱仪的测量。悬液中Fura-2负载细胞的测量结果显示，无论有无细胞外钙离子，S1P裂解酶缺陷的MEF静息[Ca~(2+)]i均升高，激动剂诱导的[Ca~(2+)]i升高。重要的是，SERCA抑制剂thapsigargin引起的[Ca~(2+)]i升高和Ca~(2+)动员增加，表明S1P裂解酶缺乏的MEF的Ca~(2+)储存增加。对表达基于钙调蛋白的钙离子感受器Cameleon的单个细胞的测量表明，在两个MEF细胞群中至少可以区分两种细胞类型，一种是在thapsigargin刺激下快速而短暂的[Ca~(2+)]升高，另一种是缓慢而持久的[Ca~(2+)]升高。在反应迅速和缓慢反应的S1P裂解酶缺陷细胞中，反映总钙释放的thapsigargin诱导的[Ca~(2+)]i增加的时间进程下的面积显著增加超过50%。结论：S1P和/或鞘氨醇浓度升高导致钙储存增加，基础[Ca~(2+)]i升高，因此，S1P代谢不仅在急性钙动员中起作用，而且在钙稳态的长期调节中起作用。
Sphingosine-1-phosphate (S1P) 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 S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca2+from thapsigargin-sensitive stores. Here, we have studied Ca2+signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P 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+]iwas elevated and agonist-induced [Ca2+]iincreases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+]iincreases and Ca2+mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+storage in S1P 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+]iincrease and the other with a slower and prolonged [Ca2+]ielevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+]iincreases, reflecting overall Ca2+release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca2+storage and elevated basal [Ca2+]i. S1P metabolism thus plays a role not only in acute Ca2+mobilization but also in long-term regulation of Ca2+homeostasis.