Many enzymes of sphingolipid metabolism are regulated in response to extra- and intracellular stimuli and in turn serve as regulators of levels of bioactive lipids (such as sphingosine, ceramide, sphingosine 1-phosphate, and diacylglycerol), and as such, they serve a prototypical modular function in cell regulation. However, lipid metabolism is also closely interconnected in that a product of one enzyme serves as a substrate for another. Moreover, many cell stimuli regulate more than one of these enzymes, thus adding to the complexity of regulation of lipid metabolism. In this paper, we review the status of enzymes of sphingolipid metabolism in cell regulation and propose a role for these enzymes in integration of cell responses, a role that builds on the modular organization while also taking advantage of the complexity and interconnectedness of lipid metabolism, thus providing for a combinatorial mechanism of generating diversity in cell responses. This may be a general prototype for the involvement of metabolic pathways in cell regulation.

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A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC 50 of 1 M. It did not inhibit acid SMase at up to at least 150 M. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 M) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 M of the compound was protected completely from the loss of SM. The addition of 10 -20 M GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 M did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 M, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-B translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that NSMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

The pathogenic fungus Cryptococcus neoformans infects humans upon inhalation and causes the most common fungal meningoencephalitis in immunocompromised subjects worldwide. In the host, C. neoformans is found both intracellularly and extracellularly, but how these two components contribute to the development of the disease is largely unknown. Here we show that the glycosphingolipid glucosylceramide (GlcCer), which is present in C. neoformans, was essential for fungal growth in host extracellular environments, such as in alveolar spaces and in the bloodstream, which are characterized by a neutral/alkaline pH, but not in the host intracellular environment, such as in the phagolysosome of macrophages, which is characteristically acidic. Indeed, a C. neoformans mutant strain lacking GlcCer did not grow in vitro at a neutral/alkaline pH, yet it had no growth defect at an acidic pH. The mechanism by which GlcCer regulates alkali tolerance was by allowing the transition of C. neoformans through the cell cycle. This study establishes C. neoformans GlcCer as a key virulence factor of cryptococcal pathogenicity, with important implications for future development of new antifungal strategies.

Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. In this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC). We, therefore, tested the effects of D609, a purported PC-PLC-specific inhibitor on the activity of SMS. D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10 -50 g/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.