Regulation of primary cilia formation by ceramide

The primary cilium is an important sensory organelle, the regulation of which is not fully understood. We found that in polarized Madin-Darby Canine Kidney cells, the sphingolipid ceramide is specifically distributed to a cis-Golgi compartment at the base of the primary cilium. This compartment immunostained for the centrosome marker γ-tubulin, the Rho type GTPase cell division cycle 42 (Cdc42), and atypical protein kinase Cζ/λ (aPKC), a kinase activated by ceramide and associated with a polarity protein complex consisting of partitioning defective (Par)6 and Cdc42. Inhibition of ceramide biosynthesis with Fumonisin B1 prevented codistribution of aPKC and Cdc42 in the centrosomal/pericentriolar compartment and severely impaired ciliogenesis. Cilium formation and codistribution of aPKC and Cdc42 were restored by incubation with N-acetyl or N-palmitoyl sphingosine (C2 or C16 ceramide), or the ceramide analog N-oleoyl serinol (S18). Cilium formation was also restored by the glycogen synthase kinase-3β (GSK-3β) inhibitor indirubin-3-monoxime, suggesting that regulation of ciliogenesis depends on the inhibition of GSK-3β by ceramide-activated aPKC. Consistently, inhibition of aPKC with a pseudosubstrate inhibitor prevented restoration of ciliogenesis by C2 ceramide or S18. Our data show for the first time that ceramide is required for primary cilium formation.—Wang, G., K. Krishnamurthy, and E. Bieberich. Regulation of primary cilia formation by ceramide.     

Ceramide regulates atypical PKCzeta/lambda-mediated cell polarity in primitive ectoderm cells. A novel function of sphingolipids in morphogenesis

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCzeta/lambda, its interaction with Cdc42, and phosphorylation of GSK-3beta, a substrate of PKCzeta/lambda. Recombinant PKCzeta formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCzeta/lambda regulates primitive ectoderm cell polarity and morphogenesis.     

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

We show here that human embryonic stem (ES) and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide—in particular, saturated, long-chain C_16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C_24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C_24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.     

A Metabolic Shift Favoring Sphingosine 1-Phosphate at the Expense of Ceramide Controls Glioblastoma Angiogenesis

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.     

Sphingolipid-mediated Signalling in Plants

A plethora of biological effects, ranging from cellular survivalto apoptosis, has been assigned to sphingolipids and, in particular,to the sphingolipid metabolites ceramide, sphingosine and sphingosine-1-phosphate.One aspect of sphingolipid biology that is currently attractinga great deal of interest in animals and yeast is their rolein cell signalling. In contrast, much less is known about sphingolipidsin plants, although available information suggests that thesecompounds may also fulfil important signalling roles. Thereare suggestions that sphingolipid metabolites may be involvedin diverse processes including pathogenesis, membrane stabilityand the response to drought. Here, we review current informationon the role of sphingolipid metabolites and highlight theiremerging roles in plant signalling. Copyright 2001 Annals ofBotany Company Sphingolipid, cerebrosides, glucosylceramides, sphingosine-1-phosphate, pathogenesis, stomata, guard cells, calcium, signal transduction, cell signalling     

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.     

Par6B and atypical PKC regulate mitotic spindle orientation during epithelial morphogenesis

Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells, significantly reducing both cyst size and number. Cell survival and apical positioning depend upon different thresholds of aPKC expression, suggesting that they are controlled by distinct downstream pathways. We conclude that Par6B and aPKC control mitotic spindle orientation in polarized epithelia and, furthermore, that aPKC coordinately regulates multiple processes to promote morphogenesis.     

Cancer and sphingolipid storage disease therapy using novel synthetic analogs of sphingolipids

Sphingolipid metabolites have become recognized for their participation in cell functions and signaling events that control a wide array of cellular activities. Two main sphingolipids, ceramide and sphingosine-1-phosphate, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, angiogenesis, stress responses, protein synthesis, carbohydrate metabolism, and intracellular trafficking. Ceramide and S1P often exert opposing effects on cell survival, ceramide being pro-apoptotic and S1P generally promoting cell survival. Therefore, the conversion of one of these metabolites to the other by sphingolipid enzymes provides a vast network of regulation and provides a useful therapeutic target. Here we provide a survey of the current knowledge of the roles of sphingolipid metabolites in cancer and in lipid storage disease. We review our attempts to interfere with this network of regulation and so provide new treatments for a range of diseases. We synthesized novel analogs of sphingolipids which inhibit the hydrolysis of ceramide or its conversion to more complex sphingolipids. These analogs caused elevation of ceramide levels, leading to apoptosis of a variety of cancer cells. Administration of a synthetic analog to tumor-bearing mice resulted in reduction and even disappearance of the tumors. Therapies for sphingolipid storage diseases, such as Niemann-Pick and Gaucher diseases were achieved by two different strategies: inhibition of the biosynthesis of the substrate (substrate reduction therapy) and protection of the mutated enzyme (chaperone therapy). Sphingolipid metabolism was monitored by the use of novel fluorescent sphingolipid analogs. The results described in this review indicate that our synthetic analogs could be developed both as anticancer drugs and for the treatment of sphingolipid storage diseases.     

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. We show here that polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell.     

Autophagy regulates sphingolipid levels in the liver

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.     

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.     

A simple,highly sensitive,and facile method to quantify ceramide at the plasma membrane

The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function.     

A polarity complex of mPar-6 and atypical PKC binds,phosphorylates and regulates mammalian Lgl

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.     

Phosphoinositides specify polarity during epithelial organ development

The molecular mechanisms that integrate cellular polarity with tissue architecture during epithelial morphogenesis are poorly understood. Using a three-dimensional model of epithelial morphogenesis, report that the phosphatase PTEN and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] regulate the GTPase Cdc42 and the kinase aPKC to generate the apical plasma membrane domain and maintain apical-basolateral polarity.     

Altered sphingolipid metabolism in human endometrial cancer

There is a growing body of evidence indicating that bioactive sphingolipids play a key role in cancer development, progression and metastasis. However, sphingolipid metabolism in malignant tumors is poorly investigated. Therefore, the aim of the present study was to examine the content of selected intermediates of ceramide metabolism and the activity of key enzymes of ceramide de novo synthesis and sphingosine-1-phosphate (S1P) production in the endometrial cancer. The specimens of cancer tissue and healthy endometrium were obtained from women undergoing surgery because of the cancer (n = 23) and because of myomas (n = 18), respectively. The content of sphinganine, dihydroceramide, ceramide, sphingosine and S1P was measured using high pressure liquid chromatography. The activity of the enzymes was determined using radioactive substrates. It has been found that the content of each examined sphingolipid was markedly elevated in the cancer tissue compared with the healthy endometrium. Namely, sphinganine, sphingosine and dihydroceramide by 3–4.6-fold, ceramide and S1P by 1.9- and 1.6-fold, respectively. Interestingly, the ratio of S1P to ceramide remained stable. The activity of serine palmitoyltransferase and sphingosine kinase 1 was increased by 2.3- and 2.6-fold, respectively. We conclude that endometrial carcinoma is characterized by profound changes in sphingolipid metabolism that likely contribute to its progression and chemoresistance.     

Quantitative Analysis of Membrane Trafficking in Regulation of Cdc42 Polarity

Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post‐Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP‐tag placed on the N‐terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post‐Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.      

Tracking Sphingosine Metabolism and Transport in Sphingolipidoses: NPC1 Deficiency as a Test Case

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3) H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3) H]-sphingomyelin-derived [(3) H]-ceramide and depletion of [(3) H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3) H]-sphingosine-derived [(3) H]-ceramide and attenuation of [(3) H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3) H]-sphingosine derived from [(3) H]-sphingomyelin/LDL or [(3) H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3) H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.     

PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1

A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification. The small GTPase Cdc42 binds to PAR-6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42-PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3-aPKC-PAR-6-Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42-PAR-6 or PAR-3-STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR-3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42-PAR-6-PAR-3-STEF/Tiam1-Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6-PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity.