BIMODAL REGULATION OF CERAMIDASE BY INTERLEUKIN-1-BETA - IMPLICATIONSFOR THE REGULATION OF CYTOCHROME-P450 2C11 (CYP2C11)

Interleukin 1 beta (IL-1 beta) induces the hydrolysis of sphingomyelin (SM) to ceramide (Cer) in primary cultures of rat hepatocytes, and Ce r has been proposed to play a role in the down-regulation of cytochrom e P450 2C11 (CYP2C11) and induction of alpha(1)-acid glycoprotein (AGP ) by this cytokine (Chen, J., Nikolova-Karakashian, M., Merrill, A. H. & Morgan, E. T. (1995) J. Biol. Chem. 270, 25235-25238). Nonetheless, some of the features of the down regulation of CYPSC11 do not fit a s imple model of Cer as a second messenger as follows: N-acetylsphingani ne (C2-DHCer) is as potent as RT-acetylsphingosine (C2-Cer) in suppres sion of CYP2C11; the IL-1 beta concentration dependence for SM turnove r 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 discre pancies are due to activation of ceramidase by the low concentrations of IL-1 beta (similar to 2.5 ng/ml) that maximally down-regulate CYP2C 11 expression, whereas higher IL-1 beta concentrations (that induce AG P) 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, -((N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-Cer (NBD-Cer), and followin g release of the NBD-fatty acid, IL-1 beta increases both acid and neu tral ceramidase activities, which appear to be regulated by tyrosine p hosphorylation because pretreatment of hepatocytes with sodium vanadat e increases (and 25 mu M genistein reduces) the basal and IL-1 beta-st imulated ceramidase activities, Since these findings suggested that sp hingosine (and, possibly, subsequent metabolites) is the primary media tor of the down regulation of CYP2C11 by IL-1 beta, the effects of exo genous sphingosine and Ca-Cer on expression of this gene were compared , Sphingosine was more potent than C2-Cer in down-regulation of CYP2C1 1 when added alone or with fumonisin B-1 to block acylation of the exo genous sphingosine. Furthermore, the suppression of CYP2C11 by C2-Cer (and C2-DHCer) is probably mediated by free sphingoid bases, rather th an the short chain Cer directly, because both are hydrolyzed by hepato cytes and increase cellular levels of sphingosine and sphinganine. Fro m these observations we conclude that sphingosine, possibly via sphing osine 1-phosphate, is a mediator of the regulation of CYP2C11 by IL-1 beta in rat hepatocytes and that ceramidase activation provides a ''sw itch'' that determines which sphingolipids are elevated by this cytoki ne to produce multiple intracellular responses.