Sphingolipids in inflammation: roles and implications.

Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1.

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.