Karimian Golnar, Buist-Homan Manon, Schmidt Martina, Tietge Uwe J F, de Boer Jan Freark, Klappe Karin, Kok Jan Willem, Combettes Laurent, Tordjmann Thierry, Faber Klaas Nico, Moshage Han
Dept. of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Biochim Biophys Acta. 2013 Dec;1832(12):1922-9. doi: 10.1016/j.bbadis.2013.06.011. Epub 2013 Jun 28.
Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (-70%, P<0.05) without inducing necrosis. GCDCA increased the S1P levels in hepatocytes (P<0.05). GCDCA induced [Ca(2+)] oscillations in hepatocytes and co-treatment with the [Ca(2+)] chelator BAPTA repressed GCDCA-induced apoptosis. Ski-II inhibited the GCDCA-induced intracellular [Ca(2+)] oscillations. Transcripts of all five S1P receptors were detected in hepatocytes, of which S1PR1 and S1PR2 appear most dominant. Inhibition of S1PR1, but not S1PR2, reduced GCDCA-induced apoptosis by 20%. Exogenous S1P also significantly reduced GCDCA-induced apoptosis (-50%, P<0.05), however, in contrast to the GCDCA-induced (intracellular) SphK1 pathway, this was dependent on S1PR2 and not S1PR1. Our results indicate that SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca(2+)] signaling and activation of S1PR1.
鞘氨醇激酶（SphKs）及其产物鞘氨醇-1-磷酸（S1P）已被报道可调节肝细胞的凋亡和存活。胆汁淤积性肝病的特征是细胞毒性水平的胆汁盐诱导肝损伤。目前尚不清楚SphKs和/或S1P是否在这一致病过程中发挥作用。在此，我们研究了SphK1和S1P在胆汁盐诱导的肝细胞死亡中的潜在作用。原代大鼠肝细胞暴露于甘氨鹅去氧胆酸（GCDCA）以诱导凋亡。将暴露于GCDCA的肝细胞与S1P、SphK1抑制剂Ski-II和/或S1P受体（S1PR1和S1PR2）的特异性拮抗剂共同处理。对凋亡和坏死进行定量分析。Ski-II显著降低了GCDCA诱导的肝细胞凋亡（-70%，P<0.05），且未诱导坏死。GCDCA增加了肝细胞中的S1P水平（P<0.05）。GCDCA诱导肝细胞中的[Ca(2+)]振荡，而与[Ca(2+)]螯合剂BAPTA共同处理可抑制GCDCA诱导的凋亡。Ski-II抑制了GCDCA诱导的细胞内[Ca(2+)]振荡。在肝细胞中检测到所有五种S1P受体的转录本，其中S1PR1和S1PR2最为主要。抑制S1PR1而非S1PR2可使GCDCA诱导的凋亡减少20%。外源性S1P也显著降低了GCDCA诱导的凋亡（-50%，P<0.05），然而，与GCDCA诱导的（细胞内）SphK1途径不同，这依赖于S1PR2而非S1PR1。我们的结果表明，SphK1在介导胆汁盐诱导的肝细胞凋亡中起关键作用，部分是通过干扰细胞内[Ca(2+)]信号传导和S1PR1的激活。
