Nuclear Sphingolipid Metabolism

Abstract

Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane–associated ganglioside GM1 plays a pivotal role in Ca²⁺ homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes.

INTRODUCTION

The nucleus is an organelle with a high capacity for lipid metabolism (1). In recent years, many studies have established that nuclear lipids play distinct roles in many cellular processes, including DNA replication, RNA processing, chromatin structure, and Ca²⁺ homeostasis (reviewed in References 2-5). Phosphatidylinositol phosphates (PIPs), the most extensively characterized nuclear lipids, have pivotal roles in chromatin remodeling, gene transcription, and mRNA export (6-8). Most lipids are localized to the nuclear envelope (NE), where in addition to providing structural support they participate in multiple signaling cascades. However, bioactive lipids are localized in other nuclear compartments, including chromatin (9-11) and the nuclear matrix (12). Significantly, the concentration of nuclear lipids can be dynamically altered by metabolic flux in response to signaling cascades that are often uncoupled from cytosolic processes. Similarly, extracellular stimuli can elicit lipid metabolism and signaling exclusively in the nucleus. For example, insulin growth factor 1 (IGF-1) induces the phosphorylation and activation of nuclear phospholipase C β1 (PLCβ1) (13), which consequently results in nuclear diacylglycerol (DAG) accumulation with a corresponding decrease in phosphatidylinositol biphosphate (14, 15). Furthermore, accumulation of nuclear DAG in response to IGF-1 stimulation promotes protein kinase C (PKC) nuclear translocation (15-17). Activation of this signaling pathway modulates various nuclear processes, including gene expression and cell proliferation (4).

In addition to PIPs, other classes of phospholipids, including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, also have varied nuclear functions (5, 18-24). Roles for diacylglycerol kinases (DGKs), a family of enzymes that convert DAG to phosphatidic acid (PA), in varied nuclear processes are well documented (25-28). For example, nuclear DGK-ζ expression regulates A172 cell growth by decreasing DAG concentrations (29). DGK-ζ is localized in nuclear speckles (30) and is also activated by α-thrombin in IIC9 fibroblast nuclei (31) and by nerve growth factor in PC12 cells (32). We have shown that PA regulates steroidogenic gene transcription by serving as an agonist for the nuclear receptor steroidogenic factor 1 (SF-1) (33). SF-1 regulates the transcription of multiple genes in the endocrine system, including most genes that are required for steroid hormone biosynthesis and endocrine development (34, 35). DGK-ζ directly interacts with SF-1, and activation of the cAMP pathway stimulates DGK activity in the nucleus of H295R adrenocortical cells (33). Consistent with these findings establishing roles for DAG/PA in nuclear processes, lipins (proteins that have phosphatidate phosphatase activity and catalyze the formation of DAG in the glycerol-3-phosphate pathway) are also emerging as regulators of gene expression (36). Lipin-1 binds to peroxisome proliferator–activated receptor (PPAR)α and serves as a coactivator in the expression of genes involved in fatty acid uptake, mitochondrial function, and lipid metabolism (37). Finally, recent studies have identified cyclic PA as a PPARγ antagonist that binds to the receptor with nanomolar activity and inhibits the expression of PPARγ target genes and adipogenesis (38).

In addition to phospholipids, the nucleus is emerging as a hub for sphingolipid metabolism. Sphingolipids comprise a large family of phospholipids and glycolipids (Figure 1) that share a common sphingoid base backbone (Figure 2). These molecules participate in many signal transduction pathways (39-41). To date, various sphingolipid species have been identified in multiple nuclear compartments, including chromatin, NE, and nuclear matrix (1, 42-52). In this review, we summarize studies that have identified a role for this class of lipids in regulating nuclear processes.

Figure 1.

The sphingolipid metabolic pathway. De novo biosynthesis begins with the condensation of serine and palmitoyl-CoA and various fatty acyl-CoAs. Ceramide can be generated through (a) de novo biosynthesis, (b) degradation of sphingomyelin (SM) or glucosyl-ceramides and galactosyl-ceramides, or (c) acylation of sphingosine (SPH). Ceramide can be phosphorylated into ceramide-1-phosphate (C1P) or hydrolyzed to form SPH, which is phosphorylated into sphingosine-1-phosphate (S1P). S1P can be either dephosphorylated to form SPH or irreversibly cleaved into phosphoethanolamine and hexadecenal. SPT denotes serine palmitoyltransferase.

Figure 2.

Nuclear sphingomyelin (SM) metabolism. SM synthase catalyzes the formation of SM from ceramide and phosphatidylcholine (PC). SM can be degraded by sphingomyelinase (SMase), which generates phosphorylcholine (PPC) as a by-product, or by reverse SM synthase, which catalyzes the transfer of the PPC group in SM to diacylglycerol (DAG). Ceramide formed by SM hydrolysis can be phosphorylated into ceramide-1-phosphate or hydrolyzed into sphingosine, which can then be converted into its phosphorylated form, sphingosine-1-phosphate.

NUCLEUS ORGANIZATION AND ENDONUCLEAR DOMAINS

The nucleus is a well-organized substructure with a dynamic framework (53). It is composed of a well-defined NE that encapsulates several endonuclear domains, including the nuclear matrix, chromatin, and nucleolus. The NE is a bilayer whose outer and inner leaflets display unique lipid compositions. Although a detailed comparison of the relative distribution of lipid species between the two leaflets of the nuclear membrane has not been reported, cholesterol has been shown to reside in the outer membrane but not in the inner membrane (54), whereas the gangliosides GM1 and GD1a were detected in both (55). The outer membrane is continuous with the endoplasmic reticulum (ER) and thus shares certain lipidomic properties. Conversely, the inner membrane is closely associated with the nuclear lamina and has distinct lipid characteristics (Figure 3) (53).

Figure 3.

The localization of different sphingolipid species in subnuclear domains. The outer membrane is continuous with the endoplasmic reticulum, whereas the inner membrane is associated with the nuclear lamina. The nuclear pore allows passive flow of small molecules between the cytosol and the nucleoplasm. Abbreviations: S1P, sphingosine-1-phosphate; SM, sphingomyelin.

Like plasma membranes, nuclear membranes have been suggested to contain many types of receptors, including inositol 1,4,5-triphosphate, PPAR, and retinoic acid (RA) receptors (56, 57). Some agonists activate signaling exclusively through nuclear membrane–localized receptors. RA, for example, activates phospholipase A2 (PLA2), PLC, and phospholipase D (PLD) only in the nucleus (58-60). Additionally, compelling new evidence suggests that nuclear membrane–associated enzymes have physicochemical properties that are different from those of their plasma membrane and/or cytosolic counterparts. For example, the kinetic parameters of nuclear PLC differ from those of cytoplasmic or plasma membrane–associated PLC (61).

The nuclear matrix is often viewed as the basic organizing structure of the nucleus that is responsible for maintaining nuclear shape. However, the nuclear matrix is also the site at which many processes, including DNA replication, gene transcription, and protein phosphorylation, occur (62-64). Many enzymes linked to PIP metabolism associate with the nuclear matrix (65), suggesting that the matrix is actively involved in nuclear lipid signaling cascades. Chromatin is closely associated with the nuclear matrix and exhibits a dynamic structure that is actively regulated by multiple interconnected mechanisms, including DNA methylation and histone modification (66, 67). Heterochromatin regions, which are transcriptionally inactive but contain many specific nuclear proteins that regulate gene transcription (68), are similarly organized by the nuclear matrix (Figure 3).

SPHINGOLIPID BIOSYNTHESIS AND METABOLISM

Sphingolipids are synthesized de novo from serine and palmitoyl-CoA to form a sphingoid base, which is further N-acylated with various fatty acyl-CoAs to make N-acylsphinganine (dihydroce-ramide) and is sequentially desaturated to form ceramide (Figure 1). Ceramide can be metabolized into more complex sphingolipids by the incorporation of O-linked head groups such as phosphoryl-choline or carbohydrate moieties to form sphingomyelin (SM) (Figure 2) or glycosphingolipids (Figure 4), respectively. Alternatively, ceramide can be phosphorylated into ceramide-1-phosphate (C1P) by ceramide kinase (CERK) or hydrolyzed into sphingosine (SPH) by ceramidases (Figure 2). Sphingosine kinases (SKs) then phosphorylate SPH to form sphingosine-1-phosphate (S1P). Irreversible sphingolipid degradation occurs by the action of S1P lyase, which cleaves S1P into phosphoethanolamine and hexadecenal. Aside from being de novo synthesized, ceramide can also be formed by the hydrolysis of SM and glycosphingolipids through the salvage pathway or by the N-acylation of SPH through the action of ceramide synthases (Figure 1).

Figure 4.

Ganglioside structure and biosynthetic pathway. Lactosyl-ceramide, formed by the addition of two carbohydrate moieties (glucose and galactose) to the terminal OH group of ceramide, is the precursor of gangliosides. GM1 has one terminal N-acetylneuraminic acid (sialic acid) group. GD1a, which has two terminal sialic acid groups, is converted into GM1 by neuraminidase.

Cellular sphingolipid concentrations are tightly regulated by the actions of multiple enzymes that act to maintain sphingolipid homeostasis. Because several sphingolipid species have unique physiological functions, these enzymes are important not only to ensure optimal sphingolipid concentrations but also to regulate the capacity of these bioactive lipids to activate cell signaling. For example, because ceramidases control the ratio between ceramide and S1P (Figure 2), the activity of this family of hydrolases dictates whether a cell undergoes apoptosis or proliferates (69-71). Similarly, the activity of SK1 is strongly correlated with cell growth (72-75). Furthermore, sphingomyelinase (SMase), the enzyme that breaks the phosphodiester bond of SM to form ceramide (Figure 2), is activated by multiple apoptotic factors, including tumor necrosis factor α (TNFα) (76), ionizing radiation (77), and Fas ligand (78). Finally, because the length of the N-linked fatty acyl chain on ceramide is a key determinant of the cellular function of these molecules (53, 79-83), ceramide synthases play a central role in regulating the composition of sphingolipid pools (84).

Sphingolipid-metabolizing enzymes display distinct subcellular localizations and membrane topologies (85-89). Enzymes linked to de novo sphingolipid synthesis [e.g., serine palmitoyltransferase, ceramide synthase, dihydroceramide desaturase] are localized primarily in the ER lumen, whereas different isoforms of ceramidase and SMase are expressed at the plasma membrane, lysosomes, or mitochondria (90). Glycosphingolipids are synthesized by a series of enzymes residing in the Golgi apparatus. Due to the hydrophobic nature of most sphingolipid species, compart-mentalization of sphingolipid metabolism and subsequently signaling are important themes in sphingolipid biology (90). In this manner, ceramide localized at the plasma membrane, for example, participates in signaling pathways distinct from those of mitochondrial ceramide (91-96). Adding to this complexity, different isoforms of the same enzyme may have distinct substrate specificities (85, 97-102).

NUCLEAR SPHINGOLIPIDS

Biochemical, analytical, and microscopic techniques have been utilized to identify sphingolipid-metabolizing enzymes in nuclei and to quantify the concentrations of sphingolipid species (97). SM is the most prominent sphingolipid in nuclei; its concentration in nuclear matrix is three times higher than in chromatin (103). SM is a major component of chromatin (10), where it plays a role in DNA replication and chromatin architecture (43, 50, 104). The catabolism of SM gives rise to ceramide and subsequently to SPH, S1P, and C1P (Figure 2), all of which have specific nuclear functions. S1P, which has recognized roles in cell proliferation, migration, and differentiation (39), regulates gene transcription by specifically binding to histone deacetylases (HDACs) 1 and 2 and inhibiting their enzymatic activity (52). Gangliosides are also prominent in the outer and inner membranes of the NE. The ganglioside GM1 (Figure 4), in particular, has been extensively studied for its key role in nuclear Ca²⁺ homeostasis (105). Collectively, studies of nuclear sphingolipids not only illustrate the multifaceted regulatory capabilities of these lipid mediators, which in most cases differ from their cytoplasmic functions, but also highlight the importance of location (i.e., chromatin versus NE versus nuclear matrix) in determining their nuclear functions.

The localization of sphingolipid enzymes in various subnuclear compartments facilitates the dynamic nuclear metabolism of sphingolipids. To date, enzymes involved in SM, ceramide, SPH, and glycosphingolipid metabolism have been identified in nuclear extracts isolated from different cell types (42-44, 46, 48, 106-108). SM was the first sphingolipid identified as a component of the nuclear matrix (109). However, the likelihood of nuclear sphingolipid metabolism became apparent only after enzymes that metabolize sphingolipids were found in the nucleus (12, 45, 110). In fact, the nuclear levels of distinct sphingolipid subspecies change under different cellular physiological states (50, 78, 111, 112), illustrating the intrinsic capacity for dynamic nuclear sphin-golipid metabolism. For example, chromatin-associated SM synthase and SMase control dynamic oscillations in SM concentrations during the cell cycle (112-114). Furthermore, ganglioside-metabolizing enzymes, including neuraminidase (sialidase) (115) and GM2/GD2 synthase (116), play pivotal roles in regulating GM1 levels in the NE.

Sphingomyelin

SM was first identified in the NE of hepatocytes (117, 118). It was subsequently shown to be present in chromatin (10) and the nuclear matrix (12) and to be associated with double-stranded RNA (dsRNA) (Figure 3) (49). As discussed above, SM is the most abundant sphingolipid in the nucleus and the major phospholipid associated with chromatin (10), although it is enriched three times higher in the nuclear matrix (103). Studies have demonstrated that nuclear SM levels are dynamic and oscillate in response to different cellular states (43, 45, 50, 106, 112, 119). Additionally, distinct cellular cues differentially affect SM concentration in distinct subnuclear compartments (e.g., nuclear matrix versus chromatin) (103). For example, SM levels in the nuclear matrix increase at the beginning of S phase of the cell cycle during hepatic regeneration (103), whereas chromatin-associated SM decreases during the same period (119). These studies not only highlight the specificity of SM metabolism in different nuclear domains but also suggest that the amount of SM associated with DNA is proportional to the state of chromatin condensation (Table 1).

Table 1.

Summary of reported nuclear functions for different sphingolipid species and their endonuclear localization^a

Sphingolipid	Nuclear localization	Function
Sphingomyelin	Nuclear envelope Nuclear matrix Chromatin	DNA synthesis Chromatin assembly Membrane structure RNA stability
Ceramide	?	Apoptosis
Ceramide-1-phosphate	Perinuclear region	cPLA2α translocation
Sphingosine	?	SF-1 antagonist ligand
Sphingosine-1-phosphate	Chromatin	Histone acetylation
GM1	Inner nuclear membrane	Ca2+ homeostasis
GD3	?	Histone H1 phosphorylation Apoptosis
GDla	Inner nuclear membrane	Reservoir for GM1

^aQuestion marks indicate that the specific endonuclear domain has not been determined. Abbreviations: cPLA2 α, α-type cytosolic phospholipase A2; SF-1, steroidogenic factor 1.

SMase, the enzyme that catalyzes the degradation of SM to form ceramide and phosphoryl-choline (Figure 2), has been detected in the nuclear matrix (42), the NE (43), and chromatin (112). SM synthase has also been identified in the NE and chromatin (113). These enzymes appear to have different physicochemical characteristics, depending on their intranuclear localization, and their activities change in response to different cellular cues, such as cell proliferation and apoptosis (50, 104, 107, 112). Chromatin-associated SM synthase differs in pH optimum and K_m from the same isoform localized in the NE (113). Moreover, plasma membrane–localized SMase may be involved in axonal growth (120), whereas nuclear SMase activation is associated with apoptosis (46, 78). Different isoforms of the same enzyme can also reside in different intranuclear compartments, as is believed to be the case for neutral SMase. This ceramide-generating enzyme (Figure 2) was proposed to reside in the NE of rat liver cells and to translocate to the nuclear matrix during DNA synthesis (43). However, a subsequent study used biochemical and immunohistochemical approaches to demonstrate that SMase possesses a nuclear export signal and is enriched in the nuclear matrix (121). It is thus believed that distinct isoforms of SMase participate in each of these processes.

SM plays a role in stabilizing DNA during the cell cycle (112), and the dynamic changes in the amount of chromatin-associated SM are due to the opposing activities of SMase and SM synthase (112, 119). Concomitant activation of SMase and inhibition of SM synthase at the beginning of S phase may lead to a decrease in SM levels, which facilitates DNA unwinding (5). Increased SM synthase activity at the end of S phase then facilitates double-helix restoration after DNA synthesis ends (5). Notably, these changes in SM concentrations occur selectively in chromatin (104).

Interestingly, reverse SM synthase, an enzyme that catalyzes the reverse reaction as that of SM synthase (Figure 2), has also been identified in chromatin (114). Similar to SMase, reverse SM synthase catalyzes the degradation of SM but with the key difference that it catalyzes the transfer of the phosphorylcholine group from SM to DAG, forming ceramide and phosphatidyl-choline (Figure 2). Therefore, this enzyme not only promotes the accumulation of ceramide but also decreases DAG levels while increasing phosphatidylcholine. This is significant because the ceramide/DAG ratio is linked to cell proliferation and apoptosis. Although ceramide’s role in cell fate has been described predominantly in whole-cell studies (70), ceramide is a well-known mediator of apoptosis (122). Nonetheless, the induction of apoptosis in rat liver occurs through the selective accumulation of nuclear ceramide due to the activation of neutral SMase at the NE (46). In contrast, numerous studies have shown that an increase in nuclear DAG mediates the recruitment of various PKC isoforms into the nucleus (123, 124). PKC and DAG may be necessary for the transitions from G1 to S phase (125) and from G2 to M phase (126, 127) of the cell cycle. Although the identification of direct nuclear and cytoplasmic targets for ceramide is still an area of active investigation, by analogy to cytosolic signaling, it has been proposed that nuclear ceramide concentrations and PKC activity are also directly related (128).

Early studies pointed to an interaction between SM and RNA by observing a significant reduction in SM levels after nuclear digestion with RNase (129). This association was strengthened by studies identifying a complex containing RNA, proteins, SM, phosphatidylcholine, SM synthase, and neutral SMase from hepatocyte nuclei that were sequentially treated with Triton X-100 and a DNAse/RNAse cocktail (45). RNAse-resistant RNA became sensitive to enzymatic hydrolysis when it was pretreated with SMase, suggesting that SM protects RNA from degradation (15). Furthermore, because RNA digestion was temperature sensitive (i.e., higher temperatures yielded more undigested RNA that was hydrolyzed by RNAse), this RNA was assumed to be dsRNA (49). Although the precise role of dsRNA-bound SM is unclear, SM may play a role in RNA maturation by associating with newly synthesized RNA and protecting it from enzymatic digestion prior to its export from the nucleus (5). SM may also stabilize dsRNA by forming a bridge between the two strands (Table 1) (49).

Ceramide and Ceramide-1-Phosphate

As depicted in Figure 1, ceramide sits at the hub of the sphingolipid metabolic pathway because it not only serves as the building block for more complex sphingolipids (e.g., SM and glycosphin-golipids) but is also an intermediate metabolite for either sphingolipid degradation or the generation of phosphorylated species such as S1P and C1P. The presence of SMase and SM synthase in the nucleus suggests that ceramide is actively produced and consumed. Ceramidase activity in liver nuclear membranes was also reported (110), suggesting that the ceramide generated can be further metabolized. The nuclear localization of these enzymes suggests the existence of a nuclear SM cycle, although the regulation of ceramide concentrations in the nucleus is poorly understood. Akin to nuclear SM, spatially localized ceramide metabolism in different subnuclear domains may participate in distinct nuclear processes.

Nuclear ceramide accumulation has been associated with apoptosis in rat hepatocytes after hepatic vein ligation (46). [Portal vein ligation is a procedure that involves the ligation of hepatic lobes, which promotes atrophy of ligated lobes while inducing hypertrophy of nonligated lobes. This procedure can be used as a model of apoptosis of the ligated lobes, whereas hypertrophic hepatocytes can be studied for cell proliferation mechanisms (46, 47).] Increased SMase and ceramidase activity occurs after portal vein branch ligation, which correlates with DNA fragmentation and cell death (Table 1) (46). Additionally, SM degradation in chromatin at the beginning of S phase during hepatic regeneration suggests an accumulation of ceramide in this subnuclear domain (104). In Jurkat T cells, Fas ligand simultaneously stimulates neutral SMase and inhibits SM synthase activities in a caspase-3-dependent manner, which results in the time- and dose-dependent accumulation of nuclear ceramide (78). Although the precise molecular mechanisms involved have yet to be defined, these studies suggest a role for nuclear ceramide in cell proliferation. Recently, ceramide accumulation in rat hepatic nuclei as a result of neutral SMase activation was reported to occur in response to a high-fat diet (130). The basis for the accumulation of nuclear ceramide and the site at which this occurs are unknown. However, given that whole-cell studies have established a role for ceramide in insulin resistance, nuclear ceramide metabolism may have implications in insulin signaling (131, 132). Ceramide concentrations also increase in RAW 264.7 macrophages that have been activated by the Toll-like receptor 4–specific ligand Kdo₂-lipid A (133). Finally, ceramide regulates nuclear protein import in smooth muscle cells by inducing p38 mitogen-activated protein kinase activation and the subsequent relocalization of two nuclear transport proteins, importin A and cellular apoptosis susceptibility gene (123). The inhibition of nuclear import by exogenously supplemented ceramide resulted in diminished expression of proliferation protein markers, including cyclin A and proliferating cell nuclear antigen, and in reduced proliferative capacity (123).

Rovina et al. (62) recently identified nuclear export and import signals in the primary sequence of CERK. This finding suggests that nuclear ceramide can be further metabolized into C1P, which may harbor unique nuclear functions. Although these functions are yet to be reported, there is compelling evidence for a regulatory role for C1P in α-type cytosolic phospholipase A2 (cPLA2α) activity and arachidonic acid release in many cell types (64). C1P binds to the Ca²⁺-binding regions in the C2 domain of cPLA2α and promotes its translocation to the perinuclear region of cells (134). Moreover, CERK activity is required for IL-1β-induced prostaglandin production (135), as does the interaction between C1P and cPLA2α (136). Nuclear C1P may represent a yet-to-be-established, key mediator of the inflammatory response.

Sphingosine and Sphingosine-1-Phosphate

The nuclear localization of ceramidase (46) enables the local hydrolysis of ceramide to SPH. We demonstrated that SPH plays an important role in steroid hormone production in the human adrenal cortex by serving as a ligand for SF-1 (137). SPH is bound to SF-1 under basal conditions and antagonizes receptor function (137). Activation of the cAMP signaling pathway, the major regulatory cascade in adrenal steroidogenesis (138, 139), reduces the amount of receptor-bound SPH and enables the transcription of genes involved in the conversion of cholesterol to steroid hormones (Figure 5) (137). Significantly, we have found that acid ceramidase directly binds to SF-1 in the nucleus of H295R adrenocortical cells (N.C. Lucki & M.B. Sewer, unpublished observations). This suggests that ligand formation and delivery are facilitated by a direct interaction between enzyme and receptor and provides support for a novel coregulatory role of this enzyme in controlling gene expression. We also found that cAMP rapidly decreases nuclear concentrations of ceramide and stimulates the nuclear localization of SK1 while concomitantly increasing SPH and S1P levels (D. Li & M.B. Sewer, unpublished observations). Collectively, these studies implicate signal-induced nuclear sphingolipid metabolism as a critical regulator of gene transcription.

Figure 5.

Model for the role of sphingosine (SPH) in controlling the transactivation potential of steroidogenic factor 1 (SF-1). Under basal conditions, SPH is bound to SF-1 and corepressory proteins. Adrenocorticotropin hormone (ACTH) signaling activates protein kinase A (PKA), which promotes the release of SPH from the receptor’s ligand-binding pocket. Concomitantly, activation of the ACTH/cAMP pathway increases nuclear diacylglycerol kinase (DGK) activity, leading to increased phosphatidic acid (PA) biosynthesis. PA binding to SF-1 activates the receptor, thereby facilitating the recruitment of coactivator proteins and enabling interaction with the promoters of target genes. Abbreviations: GCN5, general control of amino acid synthesis protein 5; HDAC, histone deacetylase; PSF, polypyrimidine tract–binding protein–associated splicing factor; Sin3A, Sin3 homolog A; SMRT, silencing mediator for retinoid of thyroid hormone receptors; SRC1, steroid receptor coactivator 1.

The amount of intracellular SPH is regulated not only by the action of ceramidases and ceramide synthases (Figure 1) but also by its phosphorylation to S1P by SK (Figure 2). The two isoforms of SK have distinct subcellular localizations and physiological functions (72, 140). SK1 is cytoplasmic and is associated primarily with cell proliferation and growth, whereas SK2 is mainly nuclear and is linked to apoptosis. Nuclear SK activity was first described in the NE and nucleoplasm of Swiss 3T3 cells (108). Platelet-derived growth factor was shown to upregulate nucleoplasmic SK activity that correlated with progression through S phase (108). This study provided an early indication that nuclear S1P production might be involved in cell cycle regulation. Of note, we found that cAMP promotes the phosphorylation and nuclear translocation of SK1 in H295R adrenocortical cells (D. Li & M.B. Sewer, unpublished observations). More recently, Hait et al. (52) reported that SK2 interacts with histone H3 in chromatin of MCF-7 breast cancer cells, establishing a role for endonuclear S1P in the epigenetic regulation of gene transcription (52). Expression of SK2 induced histone acetylation, which correlated with the formation of S1P and dihydro-S1P in the nuclei of these cells (Figure 3). Furthermore, the authors demonstrated that S1P and dihydro-S1P inhibited the activity of HDAC1 and -2 by binding to their active site (52). SK2 interacted with HDAC1 and HDAC2 and thus facilitated S1P transfer to the enzymes. Finally, SK2 associated with HDACs at the promoters of the cyclin-dependent kinase inhibitor p21 and c-fos genes, where it induced histone acetylation and gene transcription (Table 1) (52). These findings not only identified HDACs as nuclear targets of S1P but also uncovered a novel role for this multifaceted sphingolipid molecule as a regulator of histone posttranslational modification and global gene expression programs.

Gangliosides

Gangliosides are formed by a ceramide molecule linked to an oligosaccharide chain containing hexose and N-acetylneuraminic acid (sialic acid) groups (Figure 4). Many studies have established that gangliosides are intrinsic components of the nucleus and occur in both membranes of the NE (Figure 3). The first evidence for nuclear ganglioside localization came from subcellular fractionation studies of bovine mammary gland and rat liver cells (141, 142), in which nuclear ganglioside concentrations were found to be 10% of those of the plasma membrane (143). The major species identified were GM1 and GM3 in rat liver (142) and GM3, GD3, and GTb1 in bovine mammary gland cells (143). Subsequent studies in neuroblastoma and primary neuronal cells demonstrated by cytochemical analysis with cholera toxin B subunit linked to horseradish peroxidase that GM1 occurs at high concentrations in the NE of differentiating, but not quiescent, cells (44, 144). Similarly, GM1, GM3, and c-series gangliosides were observed in mature rat brain (145). However, developing rat brain comprised relatively more GM3 and GD3 (145), indicating that the synthesis of distinct ganglioside subspecies in the nucleus is differentially regulated during development.

Nuclear quantification of gangliosides from cultured neuro2A cells by two successive high-density sucrose gradient purifications found that GM1 and GD1a are the predominant gangliosides found in nuclei and are localized primarily in the NE (44). A more detailed characterization of the nuclear localization of gangliosides became possible by subjecting the isolated nuclei to mild treatment with a sodium citrate solution, which allows the separation of the inner and outer nuclear membranes (146, 147). The use of this technique revealed that GM1 and GD1a are present in both membranes of the NE in primary neurons (48). Interestingly, GD1a is converted to GM1 by neuraminidase (Figure 4), a membrane-associated enzyme that is present in both membranes of the NE (148, 149). Nuclear GD1a may serve as a storage reserve precursor for GM1.

The characterization of gangliosides in other endonuclear domains is relatively unexplored. However, some studies have determined the presence of ganglioside subspecies in heterochromatin and chromatin. GM1 may associate with heterochromatin from mouse epithelial cells (150), whereas immunocytochemical studies showed that GD3 colocalizes with chromatin in rat cortical neurons subjected to β-amyloid peptide (151). GD3 accumulation is concomitant with reduced levels of SM and increased activity of 2,8-sialyltransferase (GD3 synthase) (151), the enzyme that forms GD3 from GM3 (Figure 4). GD3 synthase knockdown by RNA interference prevented β-amyloid peptide–induced entry into S phase and apoptosis (151), supporting a role for GD3 in cell cycle activation and cell death. In addition, translocation of GD3 from the cytosol into the nucleus was observed in HUT-78 T-lymphoma cells (51). This translocation strongly correlated with histone H1 phosphorylation after activation of apoptosis (51), which suggests that GD3 may have an epigenetic role in the transcriptional regulation of specific genes (Table 1).

GM1 and Ca²⁺ homeostasis

GM1 associated with the inner NE plays a prominent role in nuclear Ca²⁺ homeostasis. This nuclear function of GM1 emerged after researchers discovered that this ganglioside is tightly associated with a Na⁺/Ca²⁺ exchanger (NCX) (48). NCX mediates countertransport of three Na⁺ ions for one Ca²⁺ ion against a Ca²⁺ gradient. On the basis of the topology of plasma membrane–associated NCX (152), it has been proposed that in the NE, negatively charged sialic acid groups on the GM1 oligosaccharide chain interact with positively charged amino acid residues on the polypeptide loop between transmembrane segments 5 and 6 of NCX at the nucleoplasm (153, 154). This interaction is thought to facilitate Ca²⁺ transport from the nucleoplasm (low [Ca²⁺]) to the NE lumen (high [Ca²⁺]).

Xie et al. (48) demonstrated the association between GM1 and NCX by immunoprecipitation of NE extracts with an antibody against NCX followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting analysis of GM1. These investigators further determined by immunoblot analysis of the separate NE membranes that this association occurs specifically at the inner membrane of the NE (48). Interestingly, plasma membrane NCX showed no association with GM1, suggesting a difference between plasma membrane–localized NCX and NE-localized NCX (48). Given that splice variants of this exchanger have been described (155, 156), differential association between nuclear and cytoplasmic NCX with GM1 may be due to alternatively spliced NCX isoforms.

GM1 association with NCX potentiates Na⁺ and Ca²⁺ exchange between the nucleoplasm and the NE lumen. This theory was first demonstrated by Ca²⁺ uptake experiments with isolated nuclei (48) and more recently using genetically encoded chameleon Ca²⁺ sensors (157). Subsequent studies employed a comparison of NE/ER Ca²⁺ elevation in GM1-expressing NG108-15 and GM1-deficient NG-CR72 cells (154, 157), which showed significantly higher NE/ER Ca²⁺ elevation in cells containing GM1. Similar results were observed in C6 cells (158), which contain NCX/GM1 in the NE but not in the plasma membrane, whereas no NE/ER Ca²⁺ elevation was observed in NCX-deficient Jurkat T cells (153). Because the NE lumen is continuous with the ER, these data support a function for the NCX/GM1 complex as an alternate mechanism for transferring cytosolic Ca²⁺ to the ER. Studies using knockout (KO) mice engineered to lack GM2/GD2 synthase, which results in deficient synthesis of GM2, GD2, and GM1 (Figure 4), have demonstrated key regulatory roles for nuclear membrane–associated GM1 in Ca²⁺ homeostasis (159, 160). These KO mice develop late-onset neurological disease (161) and display deficient Ca²⁺ regulatory capabilities in their cerebellar granule neurons (162). Supplementation of neuronal cultures from GM2/GD2 synthase KO mice with exogenous GM1 restored a normal phenotype (116), supporting a role for this ganglioside in regulating Ca²⁺ homeostasis. Moreover, most cell types studied to date express nuclear NCX/GM1, which suggests that this complex is a ubiquitous mechanism for Ca²⁺ homeostasis employed by cells.

CONCLUSIONS AND FUTURE PERSPECTIVES

Nuclear sphingolipid metabolism is an area of research undergoing significant progress. Emerging new data are paving the way toward a more comprehensive understanding of the unique roles for these bioactive lipids in nuclear processes. Analogous to their cytosolic functions, distinct sphingolipid species have unique nuclear functions and act via temporally and spatially specific mechanisms. Future studies aimed at elucidating the contributions that sphingolipid-metabolizing enzymes play in nuclear processes and at quantifying the nuclear concentrations of different sphingolipid species under different cellular conditions will allow for a more thorough understanding of how nuclear lipid metabolism coordinates global changes in cell function.

SUMMARY POINTS.

1. Distinct subsets of sphingolipid-metabolizing enzymes catalyze sphingolipid turnover in varied subnuclear domains.

2. Sphingolipid concentrations at different intranuclear compartments fluctuate in response to diverse physiological cues. Some extracellular stimuli may affect nuclear sphingolipid turnover independently of cytosolic signaling.

3. Sphingomyelin plays structural and regulatory roles in chromatin architecture, DNA synthesis, and RNA stability.

4. Sphingosine regulates gene transcription by serving as a ligand for the nuclear receptor steroidogenic factor 1.

5. Sphingosine-1-phosphate plays an epigenetic role in gene expression by controlling hi-stone acetylation.

6. The GM1 ganglioside modulates nuclear Ca²⁺ homeostasis by forming a complex with a Na⁺/Ca²⁺ exchanger in the inner membrane of the nuclear envelope.

Nuclear matrix: a filamentous protein network in the nucleus

Cyclic phosphatidic acid (PA): a naturally occurring analog of lysophosphatidic acid, cyclic PA differs from phosphatidic acid in having a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons. This structure is critical for its biological activity (163)

Steroidogenic factor 1 (SF-1): a nuclear receptor that regulates the transcription of genes involved in steroid hormone biosynthesis and endocrine development

Sphingolipids: a family of glycolipids and phospholipids that are characterized by the presence of a common sphingoid base backbone

Ganglioside: a glycosphingolipid with one or more sialic acids (n-acetylneuraminic acid) linked to the carbohydrate chain

GM1: the major ganglioside subspecies in the nuclear envelope that binds to NCX. Regulates nuclear Ca²⁺ homeostasis

Heterochromatin: a tightly packed form of DNA that is usually associated with silenced gene regions

Sphingomyelin (SM): a sphingolipid with phosphocholine attached to the terminal OH group of ceramide

Glycosphingolipid: a sphingolipid with carbohydrate groups attached to the terminal OH group of ceramide

Salvage pathway: the regeneration of ceramide from the breakdown of complex sphingolipids

Sphingomyelin (SM) cycle: the activation of sphingomyelinase by extracellular stimuli that leads to sphingomyelin turnover to form ceramide

NCX: a Na⁺/Ca²⁺ exchanger localized at the plasma and nuclear membranes