Sphingolipids in mammalian cell signalling

Sphingolipids and their metabolites, ceramide, sphingosine and sphingosine-1-phosphate, are involved in a variety of cellular processes including differentiation, cellular senescence, apoptosis and proliferation. Ceramide is the main second messenger, and is produced by sphingomyelinase-induced hydrolysis of sphingomyelin and by de novo synthesis. Many stimuli, e.g. growth factors, cytokines, G protein-coupled receptor agonists and stress (UV irradiation) increase cellular ceramide levels. Sphingomyelin in the plasma membrane is located primarily in the outer (extracellular) leaflet of the bilayer, whilst sphingomyelinases are found at the inner (cytosolic) face and within lysosomes/endosomes. Such cellular compartmentalisation restricts the site of ceramide production and subsequent interaction with target proteins. Glycosphingolipids and sphingomyelin together with cholesterol are major components of specialised membrane microdomains known as lipid rafts, which are involved in receptor aggregation and immune responses. Many signalling molecules, for example Src family tyrosine kinases and glycosylinositolphosphate-anchored proteins, are associated with rafts, and disruption of these domains affects cellular responses such as apoptosis. Sphingosine and sphingosine-1-phosphate derived from ceramide are also signalling molecules. In particular, sphingosine-1-phosphate is involved in proliferation, differentiation and apoptosis. Sphingosine-1-phosphate can act both extracellularly through endothelial-differentiating gene (EDG) family G protein-coupled receptors and intracellularly through direct interactions with target proteins. The importance of sphingolipid signalling in cardiovascular development has been reinforced by recent reports implicating EDG receptors in the regulation of embryonic cardiac and vascular morphogenesis. Received 16 May 2001; received after revision 29 June 2001; accepted 3 July 2001     

The GPI-anchor and protein sorting

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a diverse class of proteins that are anchored to the membrane solely via means of a posttranslational lipid modification, the GPI-moiety. Since their discovery in the late 1970s, years of research have provided significant insight into the functions of this ubiquitous modification. In addition to the structure and biosynthesis of the GPI-moiety, perhaps the best-studied feature of this glycolipid is its ability to impart characteristic membrane-trafficking properties to the proteins that it anchors. Study of the mechanism of sorting of GPI-APs has brought to light the importance of lateral heterogeneities in cell membranes, termed rafts, in biological sorting processes. The focus of this review is to examine the emerging role of the GPI-anchor and mechanisms involved in GPI-AP sorting in the context of intracellular trafficking pathways.     

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P₂ relies on mechanisms ensuring transient appearance of PI(4,5)P₂ clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P₂ by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P₂ participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P₂ in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P₂ can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.     

Cytoskeleton-mediated templating of complex cellulose-scaffolded extracellular structure and its association with oikosins in the urochordate <Emphasis Type="Italic">Oikopleura</Emphasis>

Oriented cellulose deposition is critical to plant patterning and models suggest microtubules constrain cellulose synthase movements through the plasma membrane. Though widespread in plants, urochordates are the only animals that synthesize cellulose. We characterized the distinctive cellulose microfibril scaffold of the larvacean house and its interaction with house structural proteins (oikosins). Targeted disruption of cytoskeletal elements, secretory pathways, and plasma membrane organization, suggested a working model for templating extracellular cellulose microfibrils from animal cells that shows both convergence and differences to plant models. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain cellulose synthase complexes to specific cell membrane sites rather than orienting their movement through the membrane. Oikosins are incorporated into house compartments directly above their corresponding cellular field of expression and interact with the cellulose scaffold to a variable extent.     

Lipid sensing and lipid sensors

Specialized lipid microdomains in the cell plasma membrane, referred to as 'lipid rafts', are enriched in sphingolipids and cholesterol and have drawn considerable interest as platforms for the recruitment of signaling molecules. Despite numerous biochemical and cellular studies, debate persists on the size, lifetime and even the existence of lipid rafts, emphasizing the need for reliable lipid probes to study in situ membrane lipid organization. In this review, we summarize our recent data on living cells using two specific probes of raft components: lysenin, a sphingomyelin- binding protein and the fluorescein ester of poly(ethyleneglycol)cholesteryl ether that labels cholesterol-rich domains. Sphingomyelin-rich domains that are spatially and functionally distinct from the GM1 ganglioside-rich domains were found at the plasma membrane of Jurkat T cells. In addition, the dynamics of cholesterol-rich domains could be monitored at the cell surface as well as inside the cells.     

Scaffolding microdomains and beyond: the function of reggie/flotillin proteins

Reggie/flotillin proteins are considered to be components of lipid rafts and are commonly used as marker proteins for lipid microdomains. Yet almost a decade after their discovery, the function of reggies/ flotillins is still enigmatic. In this review we summarize the present state of knowledge on reggie/flotillin structure, localization and function, and discuss the role of the proteins in development and disease. Based on insights into reggie/flotillin function and by comparison with related proteins of the so-called SPFH (Stomatin/Prohibitin/Flotillin/HflK/C) protein family, including stomatin, podocin and prohibitin, we propose the existence of specific types of protein-defined microdomains which are sculpt by the clustering of individual SPFH proteins. As 'specialized rafts' similar to caveolae, these membrane domains provide platforms for the recruitment of multiprotein complexes. Since, under certain circumstances, reggie-2/flotillin-1 translocates to the nucleus, reggie/ flotillin microdomains are not only stable scaffolds but also dynamic units with their own regulatory functions.     

P-glycoprotein and ‘lipid rafts’: some ambiguous mutual relationships (floating on them, building them or meeting them by chance?)

P-glycoprotein (P-gp) is an active membrane transporter responsible for cell detoxification against numerous amphiphilic compounds, leading to multidrug resistance in tumor cells. It displays entangled connections with its membrane environment since it recognizes its substrates within the cytosolic leaflet and it also translocates some endogenous lipids to the exoplasmic leaflet. Regarding its relationships with membrane microdomains, ‘lipid rafts’, a literature analysis concludes that (i) P-gp also exists in rafts and non-raft membrane domains, depending on the cell considered, the experimental conditions and the method used to test it; (ii) cholesterol has a positive influence on P-gp function, and this may be a direct effect of the free cholesterol present in membrane or an indirect effect mediated by the cholesterol-enriched microdomains; (iii) when present in rafts, P-gp interacts with protein partners regulating its activity; (iv) P-gp is a lipid translocase that handles the raft-constituting lipids with particular efficiency, and it also influences membrane trafficking in the cell. Received 18 November 2005; received after revision 23 December 2005; accepted 12 January 2006     

Cytoskeleton–membrane interactions in membrane raft structure

Cell membranes are structurally heterogeneous, composed of discrete domains with unique physical and biological properties. Membrane domains can form through a number of mechanisms involving lipid–lipid and protein–lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft. How rafts form remains a current topic in membrane biology. We review here evidence of structuring of rafts by the cortical actin cytoskeleton. This includes evidence that the actin cytoskeleton associates with rafts, and that many of the structural and functional properties of rafts require an intact actin cytoskeleton. We discuss the mechanisms of the actin-dependent raft organization, and the properties of the actin cytoskeleton in regulating raft-associated signaling events. We end with a discussion of membrane rafts and the actin cytoskeleton in T cell activation, which function synergistically to initiate the adaptive immune response.     

Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling

Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose- capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine production.Received 9 September 2004; received after revision 14 October 2004; accepted 11 November 2004A. K. Shabaana and K. Kulangara made equal contributions to this work.     

Protein kinase C and phospholipase D: intimate interactions in intracellular signaling

Diacylglycerol (DAG) was discovered as a potent lipid second messenger with protein kinase C (PKC) as its major cellular target more than 25 years ago. There is increasing evidence of significant complexity within lipid signaling, and the classical DAG-PKC model no longer stands alone but is part of a larger bioactive lipid universe involving glycerolipids and sphingolipids. Multiple layers of regulation exist among PKC- and DAG-metabolizing enzymes such as phosphatidylcholine (PC)-specific phospholipase D, and cross-talk exists between the glycerolipid and sphingolipid pathways, with PKC at the center. Currently, there is intense interest in the question of whether DAG derived from PC can function as a lipid second messenger and regulate PKC analogous to DAG derived from phosphatidylinositol-4,5-bisphosphate (PIP₂). To address these issues and incorporate DAG-PKC and other signaling pathways into an expanded view of cell biology, it will be necessary to go beyond the classical approaches and concepts.Received 29 November 2004; received after revision 18 January 2005; accepted 4 March 2005This work is dedicated to the memory of Dr. Yasutomi Nishizuka, the discoverer of protein kinase C, who was both a gentleman and a scientist.     

Lipid sensing and lipid sensors

Translation of nutrient stimuli through intracellular signaling is important for adaptation and regulation of metabolic processes, while deregulation by either genetic or environmental factors predisposes towards the development of metabolic disorders. Besides providing energy, fatty acids act as prominent signaling molecules by altering cell membrane structures, affecting the lipid modification status of proteins, and by modulating ligand-activated nuclear receptor activity. Given their highly hydrophobic nature, fatty acids in the aqueous intracellular compartment are bound to small intracellular lipid binding proteins which function as intracellular carriers of these hydrophobic components. This review describes recent advances in identifying intracellular pathways for cytosolic fatty acid signaling through ligand activated receptors by means of small intracellular lipid binding proteins. The mechanism behind intracellular fatty acid transport and subsequent nuclear receptor activation is an emerging concept, and advances in understanding this process provide new potential therapeutic targets towards the treatment of metabolic disorders.     

Ceramide function in the brain: when a slight tilt is enough

Ceramide, the precursor of all complex sphingolipids, is a potent signaling molecule that mediates key events of cellular pathophysiology. In the nervous system, the sphingolipid metabolism has an important impact. Neurons are polarized cells and their normal functions, such as neuronal connectivity and synaptic transmission, rely on selective trafficking of molecules across plasma membrane. Sphingolipids are abundant on neural cellular membranes and represent potent regulators of brain homeostasis. Ceramide intracellular levels are fine-tuned and alteration of the sphingolipid–ceramide profile contributes to the development of age-related, neurological and neuroinflammatory diseases. The purpose of this review is to guide the reader towards a better understanding of the sphingolipid–ceramide pathway system. First, ceramide biology is presented including structure, physical properties and metabolism. Second, we describe the function of ceramide as a lipid second messenger in cell physiology. Finally, we highlight the relevance of sphingolipids and ceramide in the progression of different neurodegenerative diseases.     

OCIAD2 activates γ-secretase to enhance amyloid β production by interacting with nicastrin

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aβ), a key molecule that causes major neurotoxicity during the early stage of Alzheimer’s disease (AD). However, despite its important role in Aβ production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aβ production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aβ production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aβ generation.     

Sphingolipid metabolism and its role in the skeletal tissues

The regulators affecting skeletal tissue formation and its maintenance include a wide array of molecules with very diverse functions. More recently, sphingolipids have been added to this growing list of regulatory molecules in the skeletal tissues. Sphingolipids are integral parts of various lipid membranes present in the cells and organelles. For a long time, these macromolecules were considered as inert structural elements. This view, however, has radically changed in recent years as sphingolipids are now recognized as important second messengers for signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death. In the current review, we discuss the available data showing the roles of various sphingolipids in three different skeletal cell types—chondrocytes in cartilage and osteoblasts and osteoclasts in bone. We provide an overview of the biology of sphingomyelin phosphodiesterase 3 (SMPD3), an important regulator of sphingolipid metabolism in the skeleton. SMPD3 is localized in the plasma membrane and has been shown to cleave sphingomyelin to generate ceramide, a bioactive lipid second messenger, and phosphocholine, an essential nutrient. SMPD3 deficiency in mice impairs the mineralization in both cartilage and bone extracellular matrices leading to severe skeletal deformities. A detailed understanding of SMPD3 function may provide a novel insight on the role of sphingolipids in the skeletal tissues.     

Activation of CD47 receptors causes histamine secretion from mast cells

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcɛRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the βγ dimer of a G_i protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G_i protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G_i protein complex. Received 8 December 2008; received after revision 12 January 2009; accepted 29 January 2009     

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ₂) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.     

ABCA2: a candidate regulator of neural transmembrane lipid transport

Studies in the past years have implicated multispan transmembrane transport molecules of the ATP binding cassette (ABC) transporter family in cellular lipid export processes. The prototypic ABC transporter ABCA1 has recently been demonstrated to act as a major facilitator of cellular cholesterol and phospholipid export. Moreover, the transporter ABCA4 (ABCR) plays a pivotal role in retinaldehyde processing, and ABCA3 has recently implicated in lung surfactant processing. These pioneering observations have directed considerable attention to the A subfamily of ABC proteins. ABCA2 is the codefining member of the ABC A-transporter subclass. Although known for some time, it was not until recently that its complete molecular structure was established. Unlike other ABC A-subfamily members, ABCA2 is predominantly expressed in the brain and neural tissues. The unique expression profile together with available structural data suggest roles for this largest known ABC protein in neural transmembrane lipid export. Received 31 January 2002; received after revision 11 March 2002; accepted 11 March 2002     

Implication of phosphoinositide phosphatases in genetic diseases: the case of myotubularin

Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on myotubularin, the protein mutated in the X-linked myotubular myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family.Received 14 February 2003; received after revision 10 April 2003; accepted 14 April 2003     

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.