Nikolova-Karakashian M, Morgan E T, Alexander C, Liotta D C, Merrill A H
Department of Biochemistry, Emory University, Atlanta, Georgia 30322-3050, USA.
J Biol Chem. 1997 Jul 25;272(30):18718-24. doi: 10.1074/jbc.272.30.18718.
Interleukin 1beta (IL-1beta) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Cer has been proposed to play a role in the down-regulation of cytochrome P450 2C11 (CYP2C11) and induction of alpha1-acid glycoprotein (AGP) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (1995) J. Biol. Chem. 270, 25233-25238). Nonetheless, some of the features of the down-regulation of CYP2C11 do not fit a simple model of Cer as a second messenger as follows: N-acetylsphinganine (C2-DHCer) is as potent as N-acetylsphingosine (C2-Cer) in suppression of CYP2C11; the IL-1beta concentration dependence for SM turnover is different from that for the increase in Cer; and the increase in Cer mass is not equivalent to the amount of SM hydrolyzed nor the time course of SM hydrolysis. In this article, we report that these discrepancies are due to activation of ceramidase by the low concentrations of IL-1beta ( approximately 2.5 ng/ml) that maximally down-regulate CYP2C11 expression, whereas higher IL-1beta concentrations (that induce AGP) do not activate ceramidase and allow Cer accumulation. This bimodal concentration dependence is demonstrated both by in vitro ceramidase assays and in intact hepatocytes using a fluorescence Cer analog, 6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-Cer (NBD-Cer), and following release of the NBD-fatty acid. IL-1beta increases both acid and neutral ceramidase activities, which appear to be regulated by tyrosine phosphorylation because pretreatment of hepatocytes with sodium vanadate increases (and 25 microM genistein reduces) the basal and IL-1beta-stimulated ceramidase activities. Since these findings suggested that sphingosine (and, possibly, subsequent metabolites) is the primary mediator of the down-regulation of CYP2C11 by IL-1beta, the effects of exogenous sphingosine and C2-Cer on expression of this gene were compared. Sphingosine was more potent than C2-Cer in down-regulation of CYP2C11 when added alone or with fumonisin B1 to block acylation of the exogenous sphingosine. Furthermore, the suppression of CYP2C11 by C2-Cer (and C2-DHCer) is probably mediated by free sphingoid bases, rather than the short chain Cer directly, because both are hydrolyzed by hepatocytes and increase cellular levels of sphingosine and sphinganine. From these observations we conclude that sphingosine, possibly via sphingosine 1-phosphate, is a mediator of the regulation of CYP2C11 by IL-1beta in rat hepatocytes and that ceramidase activation provides a "switch" that determines which sphingolipids are elevated by this cytokine to produce multiple intracellular responses.
白细胞介素1β（IL-1β）可诱导大鼠原代肝细胞中鞘磷脂（SM）水解生成神经酰胺（Cer），并且有人提出Cer在该细胞因子对细胞色素P450 2C11（CYP2C11）的下调及α1-酸性糖蛋白（AGP）的诱导过程中发挥作用（Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (1995) J. Biol. Chem. 270, 25233 - 25238）。然而，CYP2C11下调的某些特征并不符合Cer作为第二信使的简单模型，具体如下：N-乙酰鞘氨醇（C2-DHCer）在抑制CYP2C11方面与N-乙酰鞘氨醇（C2-Cer）一样有效；IL-1β对SM周转的浓度依赖性与Cer增加的浓度依赖性不同；Cer量的增加既不等同于水解的SM量，也不等同于SM水解的时间进程。在本文中，我们报道这些差异是由于低浓度IL-1β（约2.5 ng/ml）激活了神经酰胺酶，该浓度可最大程度地下调CYP2C11表达，而较高浓度的IL-1β（诱导AGP）则不激活神经酰胺酶并导致Cer积累。通过体外神经酰胺酶测定以及在完整肝细胞中使用荧光Cer类似物6-((N-(7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基)氨基)-Cer（NBD-Cer）并跟踪NBD-脂肪酸的释放，证实了这种双峰浓度依赖性。IL-1β增加了酸性和中性神经酰胺酶活性，这似乎受酪氨酸磷酸化调节，因为用钒酸钠预处理肝细胞会增加（而25 μM染料木黄酮会降低）基础和IL-1β刺激的神经酰胺酶活性。由于这些发现表明鞘氨醇（以及可能随后的代谢产物）是IL-1β下调CYP2C11的主要介质，因此比较了外源性鞘氨醇和C2-Cer对该基因表达的影响。单独添加或与伏马菌素B1一起添加以阻断外源性鞘氨醇的酰化时，鞘氨醇在下调CYP2C11方面比C2-Cer更有效。此外，C2-Cer（和C2-DHCer）对CYP2C11的抑制可能是由游离鞘脂碱介导的，而不是直接由短链Cer介导，因为两者都会被肝细胞水解并增加细胞内鞘氨醇和鞘氨醇的水平。从这些观察结果我们得出结论，鞘氨醇可能通过鞘氨醇1-磷酸，是IL-1β在大鼠肝细胞中调节CYP2C11的介质，并且神经酰胺酶激活提供了一个“开关”，该开关决定了该细胞因子会升高哪些鞘脂以产生多种细胞内反应。
