Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans.

In recent years, mounting evidence has indicated that sphingolipid metabolism is an important cell signaling system. Rapid and transient changes in sphingolipid metabolism are closely associated with a wide range of cellular activities, including the stress response, apoptosis, inflammation, cell cycle regulation, and cancer development (19, 20, 33). For instance, stress signals rapidly and transiently elevate the level of cellular ceramide; conversely, growth factors stimulate the rapid, transient generation of sphingosine-1-phosphate (S-1-P). Importantly, the treatment of cells with short-chain ceramide analogs or the expression of sphingomyelinases reproduces most of the effects of endogenous ceramide, particularly in the stress response and apoptosis (19, 20, 33). Furthermore, S-1-P, as a high-affinity ligand of the edg family of G-protein-coupled receptors, promotes cell survival and proliferation by antagonizing ceramide-mediated apoptosis (52, 61). Thus, ceramide, as an important regulatory component in the stress response and apoptosis, and S-1-P, involved in cell survival and proliferation, have attracted enormous scientific interest in recent years. In particular, a large body of evidence has now accumulated to implicate an important role of ceramide in the stress response and apoptosis. It is proposed that the relative levels of cellular ceramide and S-1-P determine whether cells undergo apoptosis or continue to proliferate (61). The sphingoid long-chain bases dihydrosphingosine (DHS) and sphingosine are central components of the sphingolipid metabolic pathway (20). They are substrates for the synthesis of both ceramide and sphingoid base phosphates (S-1-P and DHS-1-phosphate [DHS-1-P]). Sphingosine is also the product of ceramide and S-1-P metabolism. It has been shown that sphingosine is also a highly bioactive molecule (20). However, compared to the situation for ceramide and S-1-P, very few studies have been directed toward characterizing the cellular functions of sphingosine. Interestingly, a recent study showed that, paralleling the change in ceramide levels, a rapid, transient elevation of sphingosine levels is also observed in the stress response and apoptosis (9). It was further shown that treatment of cells with exogenous C2-ceramide not only increases the level of endogenous ceramide but also leads to a marked increase in the level of cellular sphingosine (9). The biological significance of such an increase in the cellular sphingosine level during the stress response and apoptosis is not well understood. These studies do raise the possibility that, like ceramide, sphingosine may also play a role in the stress response and apoptosis. This possibility is further supported by the fact that exogenous sphingosine is a potent elicitor of apoptosis (9, 10, 30). Most importantly, sphingosine regulates multiple key signal transduction factors, such as protein kinase C, 3-phosphoinositide-dependent kinase 1 (PDK1), and p21-activated kinase 1 (PAK1) (4, 18, 32). The basic chemical structure, biosynthesis, and metabolism of sphingolipids are conserved in mammalian and fungal systems (15). In Saccharomyces cerevisiae, an important role for sphingolipids in the stress response was originally proposed based on the observation that SLC (suppressor of long-chain bases) mutants were unable to grow under many types of stress (55). A role for sphingolipids in the yeast stress response to heat now has been well established (28, 71). Interestingly, a detailed analysis of changes in the sphingolipid composition showed that upon heat stress, the levels of the sphingoid long-chain bases DHS and phytosphingosine (PHS) rapidly and transiently increase severalfold within 10 to 20 min (28). Although the level of cellular ceramide also increases severalfold upon heat stress, the increase occurs much more slowly, within 1 to 2 h (28). Furthermore, it was demonstrated that the rapid elevation of the levels of cellular DHS and PHS but not of ceramide is responsible for transient G1 arrest upon heat stress (27) and that heat resistance requires DHS-1-P and PHS-1-phosphate (PHS-1-P) produced by sphingoid base kinase-phosphatase activities (31, 34, 43, 44). Exogenous PHS has also been shown to inhibit yeast growth in a very specific manner that does not involve ceramide (8) but occurs in part through the regulation of ubiquitin-dependent proteolysis (7) and nutrient uptake (59). Moreover, in S. cerevisiae, PHS plays an important role in endocytosis and proper actin cytoskeleton organization by regulating Pkc1 activity via Pkh1/2 (11, 65). Together, these studies strongly suggest that sphingoid bases play a specific signaling role in growth regulation and the stress response in S. cerevisiae. We are using Aspergillus nidulans, a filamentous fungus, as a model genetic system to study the cellular functions of sphingolipids. Recently, Cheng et al. showed that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that the accumulation of cellular ceramide causes cell cycle arrest in G1 (6). In this study, we investigated the cellular functions of DHS and PHS by analyzing the physiological and cellular effects of treating A. nidulans cells with exogenous DHS and PHS. Both DHS and PHS are able to diffuse readily between cell membranes and, most importantly, are able to fully complement the lack of serine palmitoyltransferase (SPT) activity, the first committed and rate-limiting step of the sphingolipid biosynthesis pathway (6). We found that DHS and PHS have potent antifungal activity with remarkable structural and stereochemical specificity. We demonstrate that the antifungal activity of DHS and PHS does not involve ceramide and intriguingly acts through the rapid induction of metacaspase-independent apoptosis. Furthermore, we show that fungal apoptosis is remarkably similar to that of mammalian cells morphologically.