The recent discovery of sphingolipid-derived second messengers that regulate fundamental cell responses such as cell growth and apoptosis has provided insight into the way cells sense and respond to stressful stimuli. This will help the understanding of the pathogenesis of stress-related diseases and eventually offer novel therapeutic approaches.
Living cells are often exposed to dramatic changes in environmental conditions and adapt to these changes by expression of inducible enzyme systems that provide an appropriate response to the environmental signals. However, the physiological systems activated by stress stimuli can not only protect but may also damage the cell. Obviously, regulation of these seemingly contradictory functions must operate on multiple pathways to ensure an adequate homeostasis. Researchers need to find out how these multiple signaling pathways are coordinated. This review focuses on ceramide, a lipid second messenger generated by sphingomyelin hydrolysis that was recently identified as a key player in stress signaling in mammalian cells (3, 12, 14, 15).
Sphingolipid-mediated signal transduction
Sphingolipids are derivatives of long-chain bases and display a great structural diversity and complexity. They have a headgroup at position 1 of the ceramide backbone, such as phosphorylcholine to form sphingomyelin and carbohydrates to form glycosphingolipids. Sphingomyelin is located predominantly in the outer leaflet of the plasma membrane. Biosynthesis of sphingolipids (Fig. 1⇓) starts with the condensation of serine and palmitoyl-CoA to yield 3-ketosphinganine, which is reduced further to sphinganine and converted to dihydroceramide by the enzyme ceramide synthase. In the next step, headgroups are added to form sphingomyelin and glycosphingolipids. This occurs mainly in the Golgi apparatus and, in some cases, in the plasma membrane.
Degradation of sphingolipids begins with the removal of headgroups. In the case of sphingomyelin, phosphorylcholine is released by acidic, neutral, and Mg2+-dependent sphingomyelinases to yield ceramide. Indeed, ceramide is a central metabolite in sphingolipid catabolism and is degraded to fatty acid and free long-chain bases by neutral or acidic ceramidases. The sphingosine thus formed can be reacylated subsequently to ceramide, phosphorylated to yield sphingosine-1-phosphate, or methylated. Several of these intermediates (ceramide, sphingosine, and sphingosine-1-phosphate) have potential roles as second messengers, with ceramide being the best characterized (14).
A number of cytokines, growth factors, and other environmental stress stimuli employ the sphingomyelin-signaling pathway to generate ceramide (Table 1⇓). The mechanism coupling the receptors of the ligands or the physical stimuli to activation of sphingomyelinases are poorly understood. It is assumed that physical stresses act directly on the membrane and, by poorly defined processes, trigger activation of an acidic sphingomyelinase and subsequent signaling through the stress-activated protein kinase (SAPK) cascade. The best-characterized membrane receptor known to activate the sphingomyelin pathway is the 55-kDa tumor necrosis factor (TNF) receptor that binds TNF-α and is able to deliver rapid signals to a neutral and an acidic sphingomyelinase. This receptor possesses two distinct cytoplasmic domains, one of which links a novel adaptor protein, designated FAN (factor associated with neutral sphingomyelinase activation) to activation of a neutral sphingomyelinase and ceramide production. The other domain, which is identical to the death domain of the 55-kDa TNF receptor, couples to the proapoptotic adapter proteins TRADD (TNF receptor-associated death domain) and FADD (Fas/Apo-1-associated death domain) and is suggested to link to the acidic sphingomyelinase (1).
Irrespective of the stimulus used and the receptor system involved, it is quite obvious that several mechanisms exist that initiate ceramide production and that each cell type may display a specific pattern of activated sphingolipid pathways.
Identification of ceramide targets
With respect to immediate targets of ceramide, suggestions for several ceramide-activated enzymes have been forwarded. These include a 97-kDa proline-directed serine/threonine protein kinase, which recently has been suggested to be identical to the kinase suppressor of Ras (KSR) (12), a ceramide-activated protein phosphatase of the okadaic acid-sensitive PP2A subgroup of protein phosphatases (3), diverse protein kinase C isoenzymes (8, 10), and the protein kinase c-Raf (6). Addition of ceramide to intact cells or the pharmacological modulation of endogenous levels of ceramide alters the activity of members of the mitogen-activated protein kinase (MAPK) cascades, the classic extracellular signal-regulated kinases (ERK) 1 and 2, and the more recently discovered SAPKs of the SAPK/c-Jun NH2-terminal kinase (JNK) subfamily or the p38/reactivating kinase (RK) subfamily. These parallel signaling pathways regulate such fundamental aspects of cell function as metabolism, secretion, and gene expression. The archetypal signaling cascade includes ERK1 and ERK2 and responds primarily to mitogenic stimulation via the small G protein Ras. In contrast, the SAPK and p38 pathways respond to cellular stresses such as inflammatory mediators, poisons, heat, and high-energy radiation. However, there are cell type-specific patterns of activation of the different MAPK cascades by a given ligand, which determine the cell-specific response to this stimulus. Despite intensive efforts in recent years, identification of the molecular targets of ceramide action has proved difficult and so far indirect. It is clear, however, that ceramide delivers signals into one or several cascades of the MAPK families in a cell type-specific manner to regulate cell responses such as proliferation, apoptosis, or gene expression and subsequent mediator synthesis.
A novel approach to identify downstream targets of ceramide is the use of a radioiodinated, photoaffinity-labeling analog of ceramide with high 125I-specific radioactivity (>2000 Ci/mmol) designated [125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine (TID)-ceramide. On irradiation with light at ~350 nm, a photolabile diazirine moiety is rapidly photolyzed to generate a carbene capable of reacting with the full range of functional groups occurring in biomolecules (2). This technique enables the cell physiologist to take snapshots of even very transient interactions between cell molecules and the photocrosslinker used. This tool has been successfully employed in identifying c-Raf as a ceramide-binding partner in renal mesangial cells (6), a cell type orchestrating inflammatory processes in the renal glomerulus and thus predestinated to process stress signals (13).
Ceramide not only bound to c-Raf but also increased protein kinase c-Raf activity. The signal was further processed along the MAPK-ERK kinase (MEK)/ERK cascade and caused an increased activity of ERK1 and ERK2 that could be inhibited by a synthetic inhibitor of MEK. Importantly, the physiological agonist interleukin (IL)-1β was found to use this signaling pathway to activate c-Raf in mesangial cells (6).
The serine/threonine kinase c-Raf is a well-studied signaling device that is responsible for phosphorylation and activation of MEK in the classic MAPK cascade. The mechanism of activation of c-Raf has been studied extensively, and it is now clear that this is a multistep event. In the first step, c-Raf translocates to the plasma membrane and associates with Ras-GTP. This association with Ras-GTP is not sufficient for c-Raf activation but is required for its recruitment to the plasma membrane. In the next step, c-Raf is activated by an unknown mechanism that may comprise a tyrosine and/or serine/threonine phosphorylation of c-Raf or the interaction with another membrane cofactor such as a lipid. The c-Raf NH2 terminus contains a highly conserved region (CR-1) that encompasses a zinc-finger motif analogous to the lipid-binding domain of protein kinase C (PKC), and it is tempting to speculate that c-Raf is activated by the binding of a lipid second messenger in a manner similar to the mechanism of activation of PKC by 1,2-diacylglycerol (DAG). It is obvious that ceramide specifically binds to c-Raf and stimulates its kinase activity, thus establishing ceramide as a lipid second messenger that acts as a major direct activator of c-Raf in IL-1β- and TNF-α-triggered signal propagation.
Interestingly, PKC-α and PKC-δ isoforms were identified as direct targets of ceramide. No binding of ceramide to PKC-ϵ and PKC-ζ, the other isoenzymes of PKC present in mesangial cells (5), could be detected. Binding of ceramide to PKC-α is accompanied by an increase in kinase activity in vitro. In vivo activation of PKC-α by ceramide was monitored by delayed translocation of the isoform from the cytosol to the membrane fraction of mesangial cells. Unexpected was the finding that PKC-ζ, in contrast to other reports (10), was not labeled with [125I]TID-ceramide in mesangial cells (6). This again suggests that ceramide may interact with potential targets in a cell type-specific manner (8).
An important question that immediately arises is concerned with the molecular mechanism of ceramide binding to its direct targets identified so far, i.e., PKC-α, PKC-δ, and c-Raf (6, 8). Is there a specific binding motif for ceramide that is common in these molecules? The conserved C1 and C2 domains in the regulatory part of PKC isoenzymes are candidates for lipid-interacting motifs. The C2 part present in the conventional cPKCs contains a Ca2+-phospholipid-binding domain (CaLB domain) that is responsible for interaction with phospholipids. The C1 part present in all PKC isoenzymes contains tandem repeats of cysteine-rich motifs, a so-called zinc butterfly, that is thought to be responsible for binding of DAG and phorbol esters (4). A list of potential further ceramide targets containing either a CaLB domain or zinc butterfly or both is shown in Table 2⇓. A homologous cysteine-rich motif is present in c-Raf. Although these lipid-binding motifs share certain common characteristics, they are not functionally equivalent, which may explain why only PKC-α and PKC-δ, but not PKC-ϵ or PKC-ζ, are able to directly bind ceramide (7).
PKC as a switching-off device of sphingolipid signaling
Nishizuka and his colleagues first suggested that DAG generated from hormone-induced phosphoinositide hydrolysis provides the physiological activator of PKC (11). PKC and cytosolic free Ca2+ were demonstrated to act independently or synergistically to initiate a myriad of receptor-controlled cell responses. An equally important function of PKC is the immediate negative feedback control of agonist-induced phosphoinositide turnover to prevent overshot of the system, thus establishing PKC as a bidirectional regulator of cellular functions (11). PKC-α isoenzyme was shown to be responsible for feedback regulation of hormone-stimulated inositol lipid hydrolysis in glomerular mesangial and endothelial cells (5, 7), which may be of importance in the mechanisms underlying homologous or heterologous desensitization (5). In this context, it is worth mentioning that ceramide also triggers PKC-α-mediated feedback inhibition of inositol lipid signaling (8). Since ceramide has a stimulatory effect on PKC-α activity (see above), it was tempting to speculate that this activation might be physiologically relevant and be followed by inhibition of inositol phosphate formation in mesangial cells. Indeed, it was observed that preincubation of cells for up to 2 h with ceramide time-dependently blocked extracellular nucleotide-evoked inositol trisphosphate generation (8).
Moreover, in analogy to feedback regulation of phosphoinositide turnover, it has been reported that IL-1β-induced ceramide formation is inhibited by stimulation of PKC and that this effect is reversed by specific PKC inhibitors or by downregulation of PKC isoenzymes in mesangial cells (9). From these data, it is tempting to speculate that endogenous ceramide, formed in mesangial cells after cytokine stimulation, activates PKC-α, thereby establishing a negative feedback loop that switches off ceramide production (Fig. 2⇓). One can envisage that cross-talk between the phosphoinositide- and sphingomyelin-signaling pathways engaging PKC-α provides an elegant and comprehensive mechanism of autoregulation.
With ceramide, the list of lipid second messengers has adopted a novel and prominent member derived from sphingomyelin turnover. By introducing photocrosslinkers to identify ceramide targets, it will be possible to trace the molecular chain that possibly constitutes one of the major signaling pathways by which messages carried by stress stimuli are transmitted from the cell membrane to the cell interior, where the appropriate response required to deal successfully with environmental changes is initiated.
We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (SFB 553), the Wilhelm Sander-Stiftung, and the Commission of the European Communities (Biomed 2, PL 90979).
- © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.