Sphingolipids are a class of complex lipids containing a backbone of sphingoid bases, namely the organic aliphatic amino alcohol sphingosine (Sph), that are essential constituents of eukaryotic cells. They were first described as major components of cell membrane architecture, but it is now well established that some sphingolipids are bioactive and can regulate key biological functions. These include cell growth and survival, cell differentiation, angiogenesis, autophagy, cell migration, or organogenesis. Furthermore, some bioactive sphingolipids are implicated in pathological processes including inflammation-associated illnesses such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease (namely Crohn’s disease and ulcerative colitis), type II diabetes, obesity, and cancer. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism and can act as second messenger, especially when it is produced at the plasma membrane of cells. Ceramides promote cell cycle arrest and apoptosis. However, ceramide 1-phosphate (C1P), the product of ceramide kinase (CerK), and Sph 1-phosphate (S1P), which is generated by the action of Sph kinases (SphK), stimulate cell proliferation and inhibit apoptosis. Recently, C1P has been implicated in the spontaneous migration of cells from some types of cancer, and can enhance cell migration/invasion of malignant cells through interaction with a Gi protein-coupled receptor. In addition, CerK and SphK are implicated in inflammatory responses, some of which are associated with cancer progression and metastasis. Hence, targeting these sphingolipid kinases to inhibit C1P or S1P production, or blockade of their receptors might contribute to the development of novel therapeutic strategies to reduce metabolic alterations and disease.
Sphingolipids belong to a class of lipids that are major components of cell membranes. They are ubiquitous and highly conserved, and some of them play key roles in cell physiology and pathology. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism (Figure 1). It contains a sphingoid base (18-carbon amine alcohol) backbone with an N-linked fatty acid chain. Combination with one or more carbohydrates gives rise to cerebrosides or gangliosides, respectively, whereas incorporation of phosphocholine into the ceramide moiety forms sphingomyelin (SM). Ceramides can be synthesized mainly by three major pathways (Figure 1): (1) the de novo synthesis pathway takes place in the endoplasmic reticulum (ER) and is regulated primarily by serine palmitoyl transferase (SPT) and (dihydro)ceramide synthases (CerSs); (2) the SM pathway where activation of sphingomyelinases (SMase) at the plasma membrane of cells or in lysosomes, generates phosphocholine and ceramide directly from SM; (3) the salvage pathway where sphingosine (Sph) that is derived from the metabolism of complex sphingolipids is recycled back to ceramide by the action of CerSs . Ceramides can be further metabolized to generate Sph by the action of ceramidases, and in turn, Sph can be phosphorylated by Sph kinases (SphK) to form Sph 1-phosphate (S1P). In addition, ceramide can be directly phosphorylated to form ceramide 1-phosphate (C1P) by the action of ceramide kinase (CerK) (Figure 1). All of these sphingolipid metabolites, ceramide, C1P, Sph, and S1P are bioactive and can regulate vital physiological and pathological processes.
Biosynthesis of ceramide, sphingosine, S1P, and C1P
The topology of ceramide biosynthesis as well as the specificity of the enzymes implicated in ceramide production may be key factors for regulation of its biological functions. The two major regulatory enzymes of de novo synthesis of ceramides, which takes place in the ER and mitochondria, are SPT and CerS (Figure 1). SPT is a heterodimeric complex. In bacteria, the enzyme is cytoplasmic, whereas in yeast, plants, and mammals it is localized to the ER. The human enzyme is composed of two subunits encoded by separate genes. Both the SPT1 and SPT2 subunits are absolutely required for catalytic activity. A third subunit SPT3 has 68% homology to SPT2 and can also form dimers with SPT1. SPT3 is highly expressed in placenta and human trophoblasts and has preference for C14-CoAs to generate C16-sphingolipid derivatives in tissues expressing this subunit . CerSs are the second key enzymes in the pathway for de novo sphingolipid biosynthesis. These highly conserved transmembrane proteins are also known as human homologs of yeast longevity assurance gene (LASS1). Six different CerSs (CerS1–6) have been identified in vertebrates and plants . Each CerS regulates the de novo synthesis of endogenous ceramides with a high degree of fatty acid specificity. Although some CerSs are ubiquitously expressed, other isoforms present a very specific distribution among tissues, according to the need of each tissue for specific ceramide species . Importantly, CerS1 specifically generates C18 ceramide and is highly expressed in the brain and skeletal muscles but is almost undetectable in other tissues. CerS2 mainly generates C20–26 ceramides and has been demonstrated to be mainly expressed in kidney, liver, and intestine and to have the highest expression of all CerSs in oligodendrocytes and Schwann cells during myelination (reviewed in ). The selectivity of different CerS isoforms to produce different ceramide species is important since ceramides with specific acyl chain lengths might promote different responses within cells . CerS3 shows highest expression in testis, CerS4 is not specific to any particular tissue, CerS5 is the main CerS found in the lung epithelia, and CerS6 is expressed in almost all tissues, but shows a very low expression profile in each of them. CerS6 predominantly synthesizes C16 ceramides, which is a major ceramide species implicated in a variety of diseases including acute lymphoblastic leukemia, insulin resistance, or obesity, and has been associated with chemoresistance .
Ceramide generation is also topologically important at the plasma membrane of cells, an ideal location for regulating signal transduction processes. The major regulatory enzymes implicated in this pathway are the SMases. There are five different types of SMases, acidic (A-SMase), neutral Mg2+-dependent and neutral Mg2+-independent (N-SMase), alkaline SMase, and a Zn2+-dependent secreted form of A-SMase. N-SMases are membrane bound enzymes with an optimal activity at a neutral pH. Several N-SMase isoforms have been characterized. N-SMase 1 is localized to the membranes of the ER, and it is ubiquitously expressed and highly enriched in kidney . N-SMase 2 has a different domain structure than N-SMase 1 and is specifically highly expressed in brain [8–10]. A third N-SMase (N-SMase 3) is ubiquitously present in all cell types and distributed mainly in the ER and Golgi membranes . Ceramides can also be generated through the salvage pathway (reviewed in ). It should be noted that whereas sphinganine is generated by de novo sphingolipid biosynthesis (Figure 1), free Sph seems to be derived only via turnover of complex sphingolipids, more specifically by hydrolysis of ceramide. The catabolism of ceramide takes place in lysosomes from where Sph can be released. Free Sph is probably trapped at the ER-associated membranes where it undergoes reacylation to form ceramide.
Besides their crucial role as precursors of complex sphingolipids and cell membrane architecture, ceramides have also become relevant in the regulation of vital physiological processes, and are implicated in the pathogenesis of a variety of illnesses. Small changes in the molecular structure of ceramide can regulate its biological function. As shown in Figure 1, dihydroceramide is an early intermediate in the de novo ceramide biosynthesis. It differs from ceramide only by the lack of the C4–5 trans-double bond in the sphingoid backbone, which makes it biologically inert when compared with ceramide. The introduction of a trans-double bond between C4 and C5 confers bioactivity to the ceramide molecule. This reaction is catalyzed by the enzyme (dihydro)-ceramide desaturase, which is localized to the cytosolic leaflet of the ER membrane . Unsaturation in the sphingoid backbone increases the intramolecular hydration/hydrogen bonding in the polar region, which allows the close packing of the ceramide molecules and the regulation of the physical properties of membranes .
Concerning the physiological aspects of ceramide biology, it has been widely demonstrated that ceramides are major regulators of cell death, mainly by promoting apoptosis, and cell proliferation, namely by causing cell cycle arrest and inhibition of cell growth (reviewed in [12,13]). Apoptosis is an essential process for normal embryonic development and to maintain cellular homeostasis within mature tissues. A proper balance between regulation of normal cell growth and cell death is the basis of life . Moreover, long-chain ceramides (with amide linked fatty acyl chains ranging from 18 to 22 carbons in length) are a major lipid class conforming the human stratum corneum barrier , which is the outermost layer of the epidermis that protects underlying tissue from infection, dehydration, chemicals, or mechanical stress.
With regard to pathology, ceramides have been involved in numerous inflammatory processes. The intracellular levels of ceramide are fine-tuned and alteration of ceramide levels contributes to the development of age-related, neurological, and neuroinflammatory diseases. Increasing evidence points toward an important role of ceramides, or some of its metabolites, in maintaining lung cell homeostasis and proper host response to airway microbial infections . In particular, ceramides play a relevant role in asthma, chronic obstructive pulmonary disease (COPD), or pulmonary fibrosis, and have been implicated in the establishment or progression of cardiovascular diseases, including atherosclerosis [1,13]. Moreover, ceramides have been described as potent tumor suppressors [17–19]. Also, it is known that ceramides contribute to the lipotoxicity that is associated with diabetes, hepatic steatosis, and cardiovascular diseases. In this concern, Summers and co-workers have recently demonstrated the biological relevance of the 4, 5 trans-double bond that is present in the ceramide molecule . Specifically, ablation of ceramide desaturase 1 (DES1), the enzyme that introduces the 4,5 trans-double bond in the dihydroceramide moiety to form ceramide, from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance . These findings suggest that inhibition of DES1 may prove useful for treatment of metabolic disorders including hepatic steatosis, or type II diabetes.
Sph and S1P
Degradation of ceramides by ceramidase activity produces Sph, which is also bioactive. Sph was first demonstrated to inhibit PKC activity , which is a family of serine/threonine kinases that regulate vital cellular functions including cell growth and survival, differentiation, cellular transformation, motility, adhesion, or tumor suppression, and is also implicated in disease [22,23]. Subsequent studies showed that Sph was also an inhibitor of Mg2+-dependent phosphatidate phosphohydrolase activity (PAP-1, also called lipin), a key regulatory enzyme of lipid metabolism, and Mg2+-independent PAP-2 activities (also called lipid phosphate phosphatases, or LPPs) that are involved in signal transduction processes [24–27], and that it potently stimulated PLD  and DAGK activities in different cell types . In addition, Sph can modulate cell functions through stimulation of phosphatidylinositol-dependent phospholipase C activity and subsequent calcium mobilization [30,31]. Interestingly, in a drug-induced Niemann-Pick disease type C1 (NPC1) model system Sph accumulated in late endosomes and lysosomes (also called the acidic compartment) leading to calcium depletion in these organelles . The authors of the latter work suggested that Sph storage is an initiating factor in NPC1 disease pathogenesis leading to the secondary storage of cholesterol and other kind of sphingolipids. Sph (pKa 6.6) is likely to be protonated in the endolysosomes (pH 4–5) but deprotonated in the cytosol (pH 7.4)  and so it may require transport from one cell compartment to the other possibly by the NPC1 transporter protein . The accumulation of Sph is likely associated with a transport defect of Sph in NPC1 disease patient cells . A promising tool to study NPC1 biology is the cationic sterol U18666A, which can directly bind to a site that is within a section of the NPC1 protein called the sterol-sensing domain to inhibit this protein . For additional information on Sph biology the reader is referred to elegant reviews by Hannun and Obeid , and Silva and co-workers .
Sph can be phosphorylated to form S1P by the action of SphK 1 and 2. Numerous reports have demonstrated that S1P can regulate many physiologic cell functions including immune cell trafficking, vascular development, vascular tone control, cardiac function, or vascular permeability , and that is a key regulator of aldosterone and cortisol secretion [39–41]. Interestingly, the existence of a gradient between peripheral blood (PB) and bone marrow (BM) of S1P, which is a major PB chemoattractant for BM-residing hematopoietic stem/progenitor cells has been recently demonstrated in healthy volunteers . It was observed that both activation of the complement cascade and the levels of S1P undergo changes in a circadian cycle. While the complement cascade becomes highly activated during deep sleep at 2 a.m., S1P becomes activated later, with the highest level observed at 8 a.m., which precedes circadian egress of hematopoietic stem/progenitor cells from BM into PB. These observations are relevant to better understand certain circadian-associated pathologies, such as the exacerbation of inflammatory symptoms at night, the onset of hemolysis in paroxysmal nocturnal hemoglobinuria patients, and the prevalence of stroke incidence in the early morning hours . In addition, and contrary to ceramides, S1P promotes cell proliferation and survival , and has been implicated in cancer growth and dissemination. Most of the S1P actions are elicited through interaction with a family of Gi protein-coupled receptors, for which five members have been identified (S1PR1–5) [43,44]. In particular, S1PR2 facilitates lung fibrosis through a mechanism involving up-regulation of interleukin-13 expression, suggesting that this pathway may represent a novel target for treating this disease. In fact, the levels of SphK 1 and 2 in the lung are associated with disease severity and mortality of patients with idiopathic fibrosis , and S1P lyase, the enzyme that breaks down S1P into hexadecenal and ethanolamine phosphate , and ameliorates pulmonary fibrosis. S1P can also act in a receptor-independent manner through interaction with intracellular targets such as histone deacetylases to inhibit the enzymatic activity of histone acetylation and stimulate gene transcription in the nucleus , or with tumor necrosis factor receptor-associated factor 2 to inhibit apoptosis . Also of interest is the fact that S1P is increased in the plasma of patients with lung cancers  where it potently stimulates cell proliferation. In particular, S1PR3 is elevated in lung adenocarcinoma cells and is involved in up-regulation of epidermal growth factor receptor, a tyrosine kinase receptor that appears to promote solid tumor growth . Moreover, inhibition of SphK2 or silencing of the gene encoding this kinase in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), increased apoptosis in non-small cell lung cancers, thereby emphasizing the tumor promoting role of S1P in the lung. S1P was also demonstrated to play a key role in the dissemination of tumors other than lung cancers trough stimulation of cell migration and invasion. In particular, S1P promoted human leukemia cell migration [50,51] through a mechanism involving inhibition of heme oxygenase 1 and inducible nitric oxide synthase in a p38 MAPK-dependent manner . In addition, S1P has been shown to activate two critical transcription factors, nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3), which regulate the transcription of large sets of genes involved in inflammation, proliferation, and carcinogenesis (reviewed in). In a different context, S1P levels were shown to be elevated in the airways of asthmatic patients, and inhibition of SphK activity showed beneficial effects in a murine model of allergic asthma .
Another key enzyme involved in the regulation of S1P biological activities is S1P lyase, which irreversibly degrades S1P producing ethanolamine phosphate and hexadecenal . Specifically, S1P lyase has been demonstrated to regulate lymphocyte trafficking, inflammation, and other physiological and pathological processes, including different types of cancer. For additional information on S1P biology and pathology, there are numerous reports in the scientific literature, but the reader is referred to elegant reviews by Maceyka and Spiegel , Pyne and co-workers , Proia and Hla , Fyrst and Saba , and Kumar and co- workers.
Another relevant ceramide metabolite is C1P, which is produced by the action of CerK on ceramide that is transported by ceramide transfer protein (CERT) from the ER to the Golgi apparatus (Figure 2). So far, in mammalian cells, activation of CerK is the only mechanism by which C1P can be synthesized. However, some arthropods, such as spiders of the genera Loxosceles, some bacteria, including Corynebacterium tuberculosis, Archanobacterium haemolyticum, and Vibrio damsela possess sphingomyelinase D (SMase D) in their venom or toxins. SMase D is a phospholipase D-like enzyme that in addition to generating a cyclic ceramide phosphate, would directly form C1P from degradation of SM, possibly at the plasma membrane of cells  where it might act in signal transduction processes. Noteworthy, CerK-generated C1P can be transported from the Golgi apparatus to the plasma membrane by a recently identified C1P transfer protein , so that it could also contribute to the role of C1P as signaling metabolite.
Biosynthesis and transport of C1P
It was first demonstrated that C1P has mitogenic properties, and that it potently inhibits apoptosis in different cell types, thereby implicating CerK in the regulation of cell growth and survival. The mechanisms by which C1P regulates cell proliferation include activation of different signaling pathways such as mitogen-activated protein kinase kinase (MEK)/extracellularly regulated kinases (ERKs) 1/2, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, also known as Akt)/mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase (JNK), protein kinase C-α , or stimulation of vascular endothelial cell growth factor (VEGF) secretion . With regard to the mechanisms involved in C1P-promoted cell survival, these include direct inhibition of A-SMase  or SPT , which as mentioned earlier are major ceramide-producing enzymes, thereby reducing the levels of proapoptotic ceramides. Furthermore, C1P prevented cell death through stimulation of the PI3K/Akt pathway also involving up-regulation of the NF-κB transcription factor , or by stimulation of the inducible form of nitric oxide synthase . C1P was also found to be a key mediator of retina photoreceptor proliferation, survival, and differentiation [65,66]. C1P has also been extensively implicated in inflammatory responses. In numerous papers, Chalfant and co-workers demonstrated that C1P exerts pro-inflammatory actions in different cell types [67–73] and that it is a key factor in the wound healing process . A major mechanism by which C1P promotes inflammation includes activation of group IV cytosolic phospholipase A2 (cPLA2) α (cPLA2α) and subsequent generation of arachidonic acid and proinflammatory eicosanoids [68,69,73,75–81].
CerK is particularly relevant in lung cancer cell proliferation and dissemination. Down-regulation of CerK expression using specific siRNA to silence the gene encoding this kinase blocked progression of A549 human lung adenocarcinoma cells into the S and G2/M phases of the cell cycle, reduced cancer cell proliferation, and enhanced apoptosis in these cells . Likewise, inhibition of CerK activity with the selective inhibitor NVP-231, blocked NCI-H358 human epithelial lung cancer cell proliferation by inducing M-phase arrest and subsequent cell death whereas overexpression of CerK enhanced cell proliferation and protected against apoptosis. The latter action involved inhibition of initiator caspase 9 and executioner caspase 3 activation leading to blockade of DNA fragmentation in the lung cancer cells, and also in MCF-7 breast cancer cells . In addition, partial knockdown of CerK with specific siRNA-sensitized breast and lung cancer cells to drug-induced apoptosis suggesting that targeting this enzyme may improve or potentiate anticancer cell therapy. In fact, up-regulation of CerK is profoundly important for development of human breast cancer. Studies from a cohort of 2200 patients revealed that increased CerK expression is associated with an elevated risk of tumor recurrence in women with breast cancer. Also, CerK was spontaneously up-regulated during tumor recurrence of multiple mouse models of breast cancer , and down-regulation of CerK is a major mechanism by which vitamin D3 reduces proliferation of human neuroblastoma cells . However, contrary to stimulation of cell proliferation or inhibition of apoptosis, which are regulated by endogenous C1P, administration of exogenous C1P was associated with reduction of lung inflammation and emphysema in a model of mouse COPD and human lung tissue [86–88]. Also, exogenous administration of C1P protected against the deleterious or toxic effects of the anticancer drug cyclophosphamide, which induces ovarian damage and infertility in women with ovarian cancer under treatment with this drug . The actions of exogenous C1P seem to be mediated through binding of C1P to a putative Gi protein-coupled receptor and not by direct interaction with intracellular targets [90–92]. An important feature of many cancer cells is their capacity to migrate to nearby or distal tissues to form secondary tumors in the process of metastasis. This action requires up-regulation of proteolytic enzymes such as matrix metalloproteinases (MMPs), a large family of zinc endopeptidases that includes 24 different isoforms in humans. Activation of MMPs causes disruption of the extracellular matrix leading to cell detachment and invasion. In this connection, CerK-derived C1P has been recently demonstrated to up-regulate the expression of some MMPs including MMP-2 and MMP-9 , and to enhance cell migration/invasion of different cancers such as human and mouse leukemia cells [90,91], or human pancreatic cancer cells . In addition, Ratajczak and co-workers have shown that C1P regulates migration and invasion of a variety of normal and cancer cells, including multi-potent stromal cells, human umbilical vein endothelial cells, hematopoietic stem/progenitor cells, human leukemia cells, rhabdomyosarcoma and non-small and small lung cancer cells [51,94–100]. Of interest, Kester and co-workers  showed that exogenous C1P stimulated migration/invasion of coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells through binding to the annexin a2/protein p11 extracellular heterotetrameric complex (A2t). Although this protein complex does not bind C1P exclusively, other structurally related lipids, including S1P and phosphatidic acid could not elicit the potent chemotactic stimulation observed with C1P . Also, it has been demonstrated that the CerK/(endogenous) C1P axis plays a critical role in the regulation of cell migration/invasion in some types of cancer. For instance, up-regulation of CerK is a fundamental step for spontaneous migration of human pancreatic cancer cells , and this may be the case for other types of cancers. Moreover, the concerted action of A-SMase and CerK in a newly described pathway leads to depletion of ceramide levels and a concomitant increase in endogenous C1P levels resulting in tumor progression , thereby further emphasizing a possible role of endogenous C1P in controlling cell migration and invasion in cancer cells. It should also be pointed out that some bioactive phospholipids, including C1P sensitize or prime the chemotactic responsiveness of lung cancer cells to known prometastatic factors .
More recently, CerK has been implicated in adipogenesis, the process in which preadipocytes differentiate into mature adipose cells. Specifically, CerK expression was up-regulated during adipocyte differentiation, and knockdown of this enzyme using specific siRNA decreased the formation of lipid droplets and the content of triacylglycerol in the adipocytes . In addition, CerK knockdown caused significant reduction in leptin levels, an adipokine that is elevated in the obese state, pointing to a role of CerK in obesity. Noteworthy, and contrary to endogenous CerK-derived C1P, exogenous C1P inhibited adipogenesis , suggesting that extracellular C1P may balance the adipogenic action of CerK-derived intracellular C1P. Consistent with the latter observations, exogenous C1P inhibited CerK in differentiated human podocytes . Inhibition of adipogenesis by exogenous C1P involved ERK1/2 activation through a Gi protein-coupled receptor, as well as inhibition of phosphatidylethanolamine N-methyltransferase expression . At last, it should be pointed out that C1P regulates wound repair and regeneration through physical interaction with group IVA cPLA2. Genetic ablation of the C1P–cPLA2 interaction increased primary dermal fibroblast migration and accelerated wound maturation thereby pointing to a negative regulation of this lipid–protein interaction in fibroblast chemotaxis .
It can be concluded that targeting ceramide, Sph and their phosphorylated forms, or the enzymes involved in their metabolism may be relevant for controlling cell and tissue homeostasis as well as for developing novel therapeutic strategies to overcome metabolic disorders or pathology.
Sphingolipids are essential for cell architecture, and some of them regulate key physiological and pathological processes.
The simple sphingolipids: ceramide, Sph, S1P, and C1P control cell and tissue homeostasis.
While ceramides induce cell cycle arrest and apoptosis, the phosphosphingolipids, C1P and S1P are mitogenic and reduce or block cell death.
Ceramides are pro-inflammatory, whereas C1P and S1P possess pro- and anti-inflammatory properties depending on cell type.
Targeting bioactive sphingolipids, the enzymes implicated in their metabolism, or their receptors when present, may be highly relevant to overcome metabolic alterations or treat sphingolipid-associated illnesses.
The authors declare that there are no competing interests associated with the manuscript.
The work in A.G.-M. lab is supported by the ‘Departamento de Educación del Gobierno Vasco (Gasteiz-Vitoria, Basque Country, Spain)’ [grant number IT-1106-16]; and the ‘Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain)’ [grant number SAF2016-79695-R].
chronic obstructive pulmonary disease
cytosolic phospholipase A2
extracellularly regulated kinase
nuclear factor κB
Niemann-Pick disease type C1
neutral Mg2+-independent sphingomyelinase
protein kinase C
- SMase D
serine palmitoyl transferase