9Ceramide and Lipid Mediators in Apoptosis

1. INTRODUCTION

As a cellular signaling program, apoptosis is a highly controlled and complex process that depends on the orchestrated interactions of multiple soluble factors: ions (e.g., Ca2+ ), proteins (e.g., caspases, Bcl-2 family members), and nonprotein substrates (e.g., DNA). Equally important, although less well characterized, is signaling through cellular membranes and the lipids and proteins contained therein. Lipids are the primary constituents of biological membranes and thus play a structural role in defining cellular and organellar boundaries. However, lipids are not merely passive molecules serving inert, structural functions in these membranes. Many lipids are now appreciated as signaling molecules, capable of influencing diverse cellular processes and exerting powerful influence over many physiologic and pathophysiologic processes, such as programmed cell death. Sphingolipids represent one class of bioactive lipid mediators that are now recognized as key determinants of cell fate. This chapter discusses the regulated generation of bioactive sphingolipids (e.g., ceramide) and how sphingolipid signaling impacts the regulation of programmed cell death.

Lipid signaling is the control of cellular function through the modulation of membrane lipid composition. Although a full discussion of cellular lipid composition would require its own textbook, a few general concepts should be presented. In most metazoan cells, the predominant classes of lipids are the glycerolipids, sphingolipids, sterols, and eicosanoids. Major glycerolipid species include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (PS), and phosphatidylinositol, and important minor species are diacylglycerol (DAG), phosphatidic acid, lysophosphatidic acid, and the phosphatidylinositol phosphates

(PIPs). Sphingomyelin (SM) and glycosphingolipids (GSL), such as glucosylceramide (GlcCer) and gangliosides, make up the bulk of the cellular sphingolipid repertoire. Ceramide is comparatively less abundant, and sphingosine and sphingosine-1-phosphate (S1P) are found at even lower levels. Of the sterols, cholesterol is the most abundant. Finally, the highly diverse class of lipids known as eicosanoids (i.e., metabolites of arachidonic acid) are involved in the regulation of a multitude of physiologic processes – most notably, inflammation. Clearly, for cellular signaling, the eukaryotic cell has an immense array of lipids at its disposal.

Cells accomplish signaling through lipids via several mechanisms, but the simplest signaling paradigm is based on the production of a bioactive lipid from an inert, high-abundance precursor. For example, in G protein-coupled receptor signaling, phos- phatidylinositol-(4,5)-phosphate (PIP2) is hydrolyzed by phospholipase C (PLC) to form DAG and inositol-(3,4,5)- triphosphate (IP3). DAG proceeds to bind to protein kinase C (PKC), recruiting it to the membrane, allowing its activation, and promoting a signaling cascade. Other bioactive lipid/precursor pairs are included in Table 9-1. However, it must be understood that the terms bioactive and inert are relativistic and depend on the context and biology in question.

Many lipids are involved in the regulation of cell death. Lipids such as DAG, phosphoinositides, and S1P generally oppose proapoptotic pathways, whereas lipids such as ceramide and sphingosine can promote these pathways. Although tremendously important in the regulation of cell fate, lipid-mediated pathways of cell growth and survival (e.g., phosphoinositide-3-kinase [PI3K]/Akt pathways, sphingosine-1-phosphate receptor signaling) are not discussed at length in this chapter. Another topic that is not elaborated on is that of PS

Table 9-1. Signaling lipids and their precursors

exposure on the outer leaflet of the plasma membrane and its role in the recognition of apoptotic cell fragments by macrophages. Instead we focus on the lipids and pathways that have been shown to play largely proapoptotic signaling roles.

In this chapter, we mostly examine the roles of ceramide in the regulation of apoptosis. The aims of this

chapter are to (1) introduce the pertinent sphingolipids, metabolic enzymes, and basic properties and precepts essential for understanding the complex role of sphingolipids as signaling molecules; (2) review the evidence supporting a role for sphingolipids in the apoptotic program; (3) highlight studies that illustrate the vital role of ceramide in apoptosis-related disease; and (4) present a few of the many remaining unanswered questions concerning sphingolipids in cell death.

2. SPHINGOLIPID METABOLISM: CONSTITUENTS,

COMPARTMENTALIZATION, AND KEY CONCEPTS

The synthesis of all sphingolipids depends on the de novo formation of ceramide, which occurs by a series of catalytic steps (Figure 9-1). The rate-limiting step of sphingolipid synthesis occurs when serine and palmitoylCoA are condensed by the enzyme serine palmitoyltransferase (SPT) to form 3-ketosphinganine – a transient metabolite that is readily converted to dihydrosphingosine (also known as sphinganine). Dihydrosphingosine is then subject to acylation by ceramide synthases (CerSes). Fatty acyl chains are transferred from

glycosphingolipids

sphingosine sphingosine kinase

sphingosine-1-phosphate S1P lyase

O H

ceramide-1-phosphate

Plasma membrane

S1P

SK

SM

SM

GSL

Golgi

SM

L S G

GSL

Lysosomes

Cer

aSMase

SM

Cer

Sph

CerS

SK

SPP

Nucleus

M A

M s

acyl-coenzyme (acyl-CoA) onto the free amine group of dihydrosphingosine producing dihydroceramide. Dihydroceramide is subsequently reduced by dihydroceramide desaturase to form ceramide.

The cellular compartmentalization of sphingolipid metabolism is as important as the individual biochemical reactions (Figure 9-2). The early steps of sphingolipid biosynthesis are largely confined to the endoplasmic reticulum (ER). With some exceptions, most enzymes (e.g., SPT, CerSes) of the de novo synthetic pathway are found exclusively in this organelle. Ceramide formed in the ER must then be transferred to the Golgi complex via vesicular and nonvesicular trafficking for metabolism into complex sphingolipids such as SM, glucosylceramide, and gangliosides. The degradation of complex sphingolipids proceeds primarily in the endo-lysosomal compartment, yielding free sphingosine. Sphingosine can be phosphorylated to S1P, or it may be re-acylated to form ceramide – a process known as the salvage pathway.

Although a detailed assessment of the many complexities of sphingolipid metabolism is beyond the scope of this chapter, several key concepts should be emphasized. First, and most apparent from the metabolic scheme, is that ceramide occupies a central position in sphingolipid metabolism and represents a “hub” in sphingolipid synthesis, degradation, and interconversion. As a metabolic intermediate, ceramide may seem like an unlikely candidate for a signaling molecule. However, when one considers the compartmentalization of the enzymes of ceramide metabolism, it becomes apparent that ceramide is uniquely positioned to behave as a signaling lipid. Because ceramide is both substrate and product of multiple enzymes, its levels represent a balance between synthetic and degradative processes that must be highly regulated. Furthermore, multiple sphingolipid enzymes, including sphingomyelinases (SMases), CerSes, and ceramidases (CDases), exhibit unique localization and allow for ceramide levels to

Figure B9-1. Schematic representation of DGK method for ceramide quantification. Lipid extracts are prepared from any source, and samples and standards are reconstituted in detergent-containing micelles and incubated with DGK and [γ-32P]ATP. The lipids are separated by TLC. Bands corresponding to ceramide-1-phosphate (C1P) are scraped, and radioactivity is quantified by scintillation counting.

be differentially regulated in the subcompartments of the cell. Thus the concept of compartmentalization of ceramide metabolism emerges as a second and key concept in analyzing ceramide function. Accordingly, ceramide function needs to be considered in a pathwayspecific and compartment-specific manner, which is further corroborated by the molecular heterogeneity of ceramide species that may localize to distinct compartments.

The third key concept – that of metabolic flux – also relies on an understanding of the various sphingolipid enzymes and their compartmentalization. On detecting elevations in ceramide, it is insufficient to consider just one enzyme as a source of ceramide; instead, the cause(s) of ceramide accumulation must be addressed both in terms of its generation (e.g., from

SM hydrolysis) and its catabolism (e.g., by a ceramidase). Although such consideration has been previously arduous from an experimental perspective, new tools and knowledge of the multiple sphingolipid enzymes are making complex sphingolipid analyses possible (Boxes 9-1 and 9-2).

3. SPHINGOLIPIDS AS MEDIATORS OF

APOPTOTIC SIGNALING

The current synthesis of the role of sphingolipids in the regulation and execution of cell death signaling incorporates 17 years of research and more than 2,500 publications. An understanding of the role of ceramide in cellular signaling first requires an understanding of signaling paradigms. Basic cellular signaling involves the