Novel signaling aspects of ceramide 1-phosphate

Novel signaling aspects of ceramide 1-phosphate Natalia Presa1, Ana Gomez-Larrauri2, Asier Dominguez-Herrera1, Miguel Trueba1, and Antonio Gomez-Muñoz1*, 1Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University ofthe Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Vizcaya), Spain. 2Department of Pneumology, Cruces University Hospital, Barakaldo (Vizcaya), Spain. *Corresponding author: Telephone: 34-94-601 2455; FAX: 34-94-601 3500 E-mail: [email protected] Abbreviations: AA, arachidonic acid, Akt (PKB), protein kinase B; BMDM, bone marrow derived macrophages; CerK, ceramide kinase; ClP, ceramide 1-phosphate; cPLA2, calcium dependent cytosolic phospholipase 2; ERK, extracellularly regulated kinases; GLUT, glucose transporter; iNOS, inducible nitric oxide synthase MCP-1, monocyte chemoattractant protein1 iNOS, inducible nitric oxide synthase; mTOR, mammalian target of rapamycin; PA, phosphatidic acid; PBK, phosphatidylinositol 3-kinase; PKC, protein kinase C; PTX, pertussis toxin; ROS, reactive oxygen species; SMase, sphingomyelinase; SphK, sphingosine kinase; SPT, serine palmitoyl transferase; SlP, sphingosinel-phosphate; TAG, triacylglycerol; VEGF, vascular endothelial growth factor. Keywords: Bioactive lipids; ceramides; ceramide kinase; ceramide 1-phosphate; sphingolipids. 1 This is the accepted manuscript of the article that appeared in final form in Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1865(4) : (2020) // Article ID 158630, which has been published in final form at https://doi.org/10.1016/j.bbalip.2020.158630. © 2020 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) ABSTRACT The bioactive sphingolipid ceramide 1-phosphate (ClP) regulates key physiologic cell functions and is implicated in a number of metabolic alterations and pathological processes. Initial studies using different types of fibroblasts and monocytes/macrophages revealed that ClP was mitogenic and that it promoted cell survival through inhibition of apoptosis. Subsequent studies implicated ClP in inflammatory responses with a specific role as pro inflammatory agent. Specifically, ClP potently stimulated cytosolic phospholipase A2 (cPLA2) resulting in elevation of arachidonic acid and pro-inflammatory eicosanoid levels. However, increasing experimental evidence suggests that ClP can also exert anti-inflammatory actions in sorne cell types and tissues. Specifically, it has been demonstrated that ClP inhibits the release of pro-inflammatory cytokines and blocks activation of the pro-inflammatory transcription factor NF-KB in sorne cell types. Moreover, ClP was shown to increase the release of anti inflammatory interleukin-1 O in macrophages, and to overcome airway inflammation and reduce lung emphysema in vivo. Noteworthy, ClP stimulated cell migration, an action that is associated with diverse physiological cell functions, as well as with inflammatory responses and tumor dissemination. More recently, ceramide kinase (CerK), the enzyme that produces C 1 P in mammalian cells, has been shown to be upregulated during differentiation of pre adipocytes into mature adipocytes, and that exogenous C 1 P, acting through a putative Gi protein-coupled receptor, negatively regulates adipogenesis. Although the latter actions seem to be contradictory, it is plausible that exogenous ClP may balance the adipogenic effects of intracellularly generated (CerK-derived) ClP in adipose tissue. The present review highlights novel signaling aspects of ClP and its impact in the regulation of cell growth and survival, inflammation and tumor dissemination. 2 Gloss Ceramide 1-phosphate is a bioactive phosphosphingolipid capable of regulating vital cell functions, including cell growth and survival, and is a key regulator of chemotaxis and tumor dissemination. ClP and ceramide kinase (CerK), the enzyme responsible for its biosynthesis in mammalian cells, are implicated in inflammatory responses by exerting either proand anti inflammatory actions depending on cell type or the metabolic context of cells. In particular, administration of ClP drives down airway inflammation, reduces pulmonary emphysema and controls adipogenesis, actions that may have potential applications in the treatment of lung disease and obesity. 3 l. Introduction Sphingolipids have been classically known as fundamental blocks of eukaryotic cell membrane architecture, however sorne ofthem play critical roles in cell biology. In particular, the simple sphingolipids, ceramide, sphingosine and their phosphorylated forms regulate key physiological functions and are implicated in many pathological processes [1-3]. Ceramide is the central hub of sphingolipid metabolism and precursor of complex sphingolipids [1]. Structurally, ceramide is composed of a sphingosine backbone and a fatty acid (FA) ofvarying carbon chain lengths that is linked to the sphingosine moiety by and amide bond. A double bond located between carbons 4 and 5 in trans configuration is of particular interest as it confers bioactivity to the molecule. The importance of this double bond is discussed below. Ceramide can be synthesized by three major pathways involving different cell compartments (Fig. 1). The de novo synthesis pathway is an anabolic pathway that takes place in the endoplasmic reticulum (ER) where serine and palmitoyl-CoA are condensed to form 3-ketosphinganine (3dehydrosphingosine) in a reaction that is catalyzed by serine palmitoyl transferase (SPT), which is the major regulatory enzyme of this pathway. 3-ketosphinganine is then converted to sphinganine ( dehydrosphingosine) by a reductase. The next step involves the incorporation of a fatty acid by sphinganine to render dihydroceramide in a reaction catalyzed by ceramide synthase activity (CerS). There are six different CerS in mammalian cells that can be distinguished by their affinities for different fatty acyl chain substrates. In other words, each CerS gives rise to specific ceramide species depending on the length of the acyl chain used as substrate (for additional information on the specificity and role of CerS in cell biology the reader is referred to elegant reviews by Futerman and co-workers [ 4-6]). The last step of the pathway is the introduction of the trans double bond between carbons 4 and 5 of dihydroceramide to render ceramide, a reaction that is catalyzed by desaturase activity. As mentioned above, this double bond confers bioactivity to ceramide, which would otherwise be 4 inert, or at least not as active, in the form of dihydroceramide. In fact, it has been recently demonstrated that the lipotoxicity of ceramides to contribute to the development of insulin resistance, type II diabetes and hepatic steatosis can be overcome by ablation of dehydroceramide desaturase 1 in mice thereby preventing the conversion of dehydroceramide to ceramide [7]. Nonetheless, a possible participation of dihydroceramide in cell biology cannot be ruled out at the present time as it has been implicated in the induction of cytotoxic autophagy in cancer cells and an increase in fibrosis markers in liver cells [8, 9]. Once synthesized, ceramides are transported to the Golgi apparatus where they serve as precursors of complex sphingolipids such as sphingomyelin (SM) or glycosphingolipids, in reactions that are catalyzed by SM synthase (SMS) and glucosylceramide synthase (GCS), respectively. Specifically, SM is synthesized from ceramides that are transported from the ER by ceramide transfer protein (CERT) in a non-vesicular transport manner, whereas synthesis of glucosylceramides requires vesicular transport of ceramide from the ER to the Golgi apparatus [10-12]. The transfer of glucosylceramides for the synthesis of more complex glucosphingolipid involves participation of the adaptor four-phosphate protein (F APP2) [3]. The second major pathway of ceramide synthesis is a catabolic pathway involving the stimulation of sphingomyelinase activity (SMase ), which generates phosphocholine and ceramides directly from the breakdown of SM. This pathway takes place in the lysosomes and in the plasma membrane of cells. There are five different types of SMases, acidic lysosomal (A-SMase), zn+2-dependent secreted A-SMase, neutral Mg2+-independent and neutral Mg2+-dependent (N-SMase), and alkaline SMase. A SMases and N-SMases are mainly involved in signal transduction processes whereas the major role of the alkaline form of this enzyme seems to be the degradation of SM that is incorporated in the diet (for additional information on the specificity and role of SMases in cell biology the reader is referred to elegant reviews by Hannun and Obeid [1, 13-15]). The third major pathway for ceramide synthesis is the salvage pathway, which takes place in the ER and mitochondria5 associated membranes (MAMs). In this pathway, sphingosine that is derived from the metabolism of complex sphingolipids is recycled back to ceramides through incorporation of a fatty acyl chain in a reaction that is catalyzed by CerS thereby bypassing dihydroceramide formation. It should be emphasized that sphingosine can only be formed after activation of catabolic pathways. A fourth pathway of ceramide synthesis was discovered in mitochondria where neutral ceramidase catalyzes the condensation of sphingosine and palmitoyl-CoA in a two-step reaction. First, the fatty acyl-CoA chain is hydrolyzed in mitochondria to free FA (palmitate) and CoA by thioesterase activity, a reaction that is followed by condensation of palmitate and sphingosine to generate ceramide in a reversed ceramidase reaction [ 16]. Ceramides contribute to the structural stability of the cell membrane and are enriched in the caveolae where they can act in cell signaling processes. These include the induction of cell cycle arrest and apoptosis, cell diff erentiation and the promotion of inflammatory responses. Noteworthy, ceramides have been implicated in a variety of pathologies including cardiovascular diseases (namely atherosclerosis), insulin resistance and type II diabetes, inflammatory respiratory illnesses such as lung emphysema, chronic obstructive pulmonary disease (COPD), asthma, and pulmonary fibrosis, the development of inflammatory bowel disease (namely Crohn's disease and ulcerative colitis), neurodegenerative disorders, multiple sclerosis, senescence, or cancer (reviewed in [6, 17, 18]). The mechanisms by which ceramides modulate signal transduction processes include activation of specific serine/threonine protein kinases [19, 20], stimulation of serine/threonine protein phosphatases (PP) [21-23], or inhibition of phospholipase D [24-26]. For example, stimulation of PP2A causes dephosphorylation and inactivation of Akt, which is a downstream target of phosphatidylinositol 3-kinase (PI3K), a key pathway involved in the promotion of cell survival. PI3K/Akt is also a major pathway by which insulin regulates glucose uptake and metabolism. In fact, inhibition of Akt largely contributes to insulin resistance and development of type 11 6 diabetes [27, 28]. It is also well established that ceramides can be metabolized further to generate different bioactive metabolites. In particular, activation of ceramidases gives rise to sphingosine, which can then be phosphorylated by two different sphingosine kinases to form sphingosine 1-phosphate (S lP). Altematively, ceramides can be phosphorylated by ceramide kinase (CerK) to generate ceramide 1-phosphate (ClP) directly. Both SlP and ClP are bioactive and can regulate a variety of physiologic cell functions. Moreover, SlP and ClP are implicated in the establishment or progression of various pathologies. The involvement of S lP in pathophysiolological processes is discussed in various elegant revisions in this special issue. The biology of ClP is discussed below. 2. Biosynthesis and transport of ceramide 1-phosphate Up to date, the only known pathway for generation of ClP in mammalian cells is the direct phosphorylation of ceramides by CerK. This kinase has been shown to reside in various cell compartments including the cytosol, the nucleus and the plasma membrane, but its major localization site is the Golgi apparatus. CerK phosphorylates ceramides that are transported from the ER to the Golgi by CERT [29]. ClP is also found in perinuclear membranes. Once synthesized, C 1 P can be transported by a specific ceramide phosphate transfer protein ( CPTP) to the plasma membrane, where it can act in signal transduction processes, and probably to other organelles [29] (Fig. 2). It is possible that ClP may also be generated by other pathways. For example, transfer of a fatty acyl-CoA to SlP, or degradation of SM by a D type phospholipase would render ClP directly. However, neither a SlP acyl transferase nor a SM specific phospholipase D have so far been detected in mammalian cells. This contrasts with the observation that ClP levels were only reduced by about 50% in cells from mice with a genetic ablation of CerK [30], suggesting the existence of pathways or enzymes other than CerK to generate ClP. However, synthesis of ClP in cells from CerK-1-animals might also be due to the existence of a different CerK isoform in mammalian cells. Of interest, unpublished 7 work frorn the Chalfant laboratory indicates that exogenous addition of [32P]S lP to cells in culture led to the production of sorne ClP, but the percentage of total cellular ClP attributed to this pathway is still unknown [31]. In addition, a SMase D capable of generating ClP frorn SM was isolated frorn the venorn of the brown recluse spider of the gender Loxosceles, and frorn the toxins that are produced by sorne bacteria including Corynebacterium tuberculosis, Archanobacterium haemoliticum, or Vibrio damsela [32]. Although a rnajor product of SMase D in the latter organisrns is ClP, this enzyrne also catalyzes a transphosphatidylation rather than a hydrolytic reaction in vivo, thereby producing cyclic-ClP (CC(l,3)P [33]. Interestingly, conversion of SM to CC(l ,3)P has been shown to disturb phospholipid integrity and rnorphology, which could affect rnernbrane asymmetry (the loss of rnernbrane asymmetry is a rnechanisrn to induce the cornplernent pathway ofthe innate irnrnune response, which can result in cell lysis) [33]. Nonetheless, the possible biological actions of cyclic-ClP await further investigation. ClP can also be synthesized and secreted into the extracellular environment by the ancient protozoan Giardia lamblia, an intestinal single-celled parasite responsible for non bacteria-associated diarrheal disease [34]. Although CerK seerns to be the only enzyme irnplicated in ClP biosynthesis in rnarnrnalian cells, recent work suggests that sphingornyelinase phosphodiesterase like 3b (SMPD3b) rnay also contribute to rnodulation ofClP levels in podocytes, which are terminally differentiated cells of the kidney filtration barrier [35]. In fact, overexpression of SMPD3b prevents the access of CerK to its cerarnide substrate leading to a decrease in the concentration of intracellular ClP, whereas gene silencing of SMPD3b with specific siRNA increased total ClP levels [35]. 8 3. Regulation of cell proliferation and survival by ceramide 1-phosphate The bioactivity of ClP was first demonstrated using short chain ClP analogs (N acetylsphingosine-1-phosphate, or C2-C1P, and N-octanoylsphingosine-1-phosphate, or C8C1P). These compounds potently stimulated DNA synthesis and proliferation in rat fibroblasts [36], an action that was subsequently confirmed using natural (long-chain) ClP also in fibroblasts [37]. The implication of ClP or CerK in the regulation of cell growth was also observed in additional cell types including primary bone marrow-derived macrophages (BMDM) [38], primary photoreceptor progenitors [39], C2C12 myoblasts [40], and different types of cancer cells such as A549 human lung adenocarcinoma [41], NCI-H358 human bronchoalveolar carcinoma [16], human neuroblastoma [12], MCF-7 breast cancer [16], RA W264.7 mouse leukemia [42], or Kaposi sarcoma [43]. Noteworthy, epidemiological studies from a cohort of 2200 breast cancer patients revealed that upregulation of CerK expression was associated with tumor recurren ce in women with this type of cancer [ 44]. It was also observed that estrogen receptor negative breast cancer patients with high expression of CerK had a worse prognosis than those with low CerK expression [ 45]. The mechanisms whereby ClP stimulates cell proliferation involve regulation of various signaling pathways (Fig. 3). Specifically, in BMDM, ClP caused phosphorylation and activation of extracellularly regulated kinases 1-2 (ERKl-2) downstream of MEK (mitogen-activated protein kinase kinase ), activation of PBK and its downstream target Akt ( also known as protein kinase B, PKB), phosphorylation of the Akt target glycogen synthase-3P (GSK-3P), c-Jun N terminal kinase (JNK) and transcription factor NF-KB [38]. ClP-stimulated BMDM proliferation also involved translocation of protein kinase C-a from the cytosol to the plasma membrane and subsequent phosphorylation and activation of this kinase, actions that were mediated by SMS derived diacylglycerol [46]. Further studies demonstrated that ClP-stimulated BMDM proliferation was also under regulation ofthe mammalian target ofrapamycin (mTOR) and its 9 hematopoietic stem/progenitor cells (HSPC), multipotent stromal cells, and human umbilical vein endothelial cells [7 6-81]. The list of chemoattractants for HSPC is rather short, so identification of ClP as novel chemotactic factor for these cells is of particular interest [82]. Moreover, ClP stimulated migration/invasion of coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells through binding to the annexin a2/protein p 11 extracellular heterotetrameric complex (A2t). Although this protein complex does not bind ClP exclusively, other structurally related lipids, including S 1 P and P A could not elicit the potent chemotactic stimulation observed with ClP [83]. By contrast, Chalfant and co-workers found that in isolated fibroblasts or in a model of mouse wound healing in vivo, ClP acts as a negative regulator of cell migration. Specifically, the interaction of ClP with cPLA2 inhibited the migration of fibroblasts into the wound environment [84, 85], and loss of the ClP-cPLA2 interaction positively affected acute wound healing [85]. It should also be pointed out that ClP-stimulated cell migration is not restricted to vertebrates as it also induced migration of primordial germ cells to the gonads in Drosophila [86]. Interestingly, exogenous ClP also stimulated glucose uptake and the subsequent generation of ATP, actions that involved activation ofthe PI3K/Akt pathway and translocation of the glucose transporter GLUT3 from the cytosol to the plasma membrane. The latter actions were inhibited by PTX, suggesting the intervention of a Gi protein-coupled receptor in these processes [28]. Since cell motility is a high energy-demanding process, ClP stimulation of glucose uptake and metabolism should not be surprising. In addition to the chemotactic properties of exogenous ClP, the generation of intracellular ClP via CerK upregulation may also be an important component of the machinery that regulates cancer cell migration. In this context, we recently showed that human pancreatic cancer cells migrate spontaneously in a CerK-dependent manner when the cells are incubated in serum-free medium, in the absence of 16 any potential chemoattractant [74]. More recently, CerK was also shown to regulate migration ofbreast cancer cells [87], and bone marrow-derived mesenchymal stem cells [88]. 5. Role of ceramide 1-phosphate in inflammation Initial work by Chalfant and co-workers showed that ClP promoted inflammation [31, 84, 89-93] (Fig. 3). This action involved translocation and activation of group IV cytosolic phospholipase A2 (cPLA2a) and subsequent generation of arachidonic acid (AA) and pro inflammatory eicosanoids [84, 89, 93-99]. Using specific siRNA to downregulate cPLA2a, Chalfant and co-workers demonstrated that the induction of ClP-stimuated AA release was strictly dependent on this phospholipase [89]. Also, the Chalfant laboratory showed that ClP bound to the C2/CaLB domains of cPLA2a with high specificity, as other structurally related lipids, including ceramides or SlP did not bind and failed to activate this enzyme [91]. Binding of CIP to C2/CaLB domains of cPLA2a increased the affinity of the enzyme to calcium and promoted translocation of the enzyme from the cytosol to the membranes, where the substrate is located [89, 100]. The relevance of intracellular ClP in the promotion of inflammation was underscored by the reduced levels of pro-inflammatory cytokines found in mice that underwent genetic ablation of CerK [30]. Also, cPLA2a may be under regulation by PKC, as downregulation of this kinase by prolonged incubation with phorbol-12-myristate-13-acetate, or pharmacological inhibition of this enzyme potently reduced ClP-stimulated AA release [94]. In addition, knockdown of CerK by specific siRNA to silence the gene encoding this kinase led to inhibition of NOX (NADPH oxidase) and depletion of eicosanoid production in neuroblastoma cells, thereby implicating NOX in brain inflammation [101]. More recently, CPTP, the protein that shuttles ClP from its site of synthesis in the Golgi to other organelles, has been involved in inflammasome activation. Specifically, knockdown of CPTP increased the release of pro17 inflammatory IL-lp and IL-18 via a NLRP3 (Nod-like receptor family pyrin domain containing 3) inflammasome-based mechanism. CPTP knockdown also induced autophagy, and treatment with exogenous ClP mimicked both stimulation of pro-inflammatory cytokine release and upregulation of autophagy [ 102]. Besides ClP, its metabolic precursor ceramide also has potent pro-inflammatory properties, independently of its conversion to ClP. For example, sorne pro-inflammatory cytokines including interleukin (IL)l-beta (IL-lP), tumor necrosis-alpha (TNFa), or platelet activating factor (PAF) [103, 104] can stimulate SMase activity leading to ceramide generation and subsequent activation of pro-inflammatory NF-KB. Activation of this transcription factor then leads to the production of additional pro-inflammatory cytokines or chemokines, such as IL-6, IL-8, RANTES (CCL5) or MCP-1 (CCL2), as well as pro-inflammatory enzymes that are involved in the synthesis of pro-inflammatory eicosanoids [105-108]. Ceramides are particularly important in inflammatory lung pathologies including chronic obstructive pulmonary disease (COPD), asthma, or lung fibrosis [109, 110] and are the molecular mediators of pulmonary edema induced by PAF [104]. Moreover, emphysema caused by exposure of human or mice lungs to cigarette smoke, was associated with increased levels of ceramides [111-115]. However, ceramides and ClP have opposing effects in cells [36], and it was demonstrated that ClP blocks many of the actions elicited by ceramides. In particular, ClP substantially reduced ceramide generation in pro-apoptotic primary bone marrow-derived macrophages through inhibition of A-SMase activity [59], or through the blockade of SPT and Aand N-SMase activities in alveolar cells [60]. The later findings suggest that ClP might have anti-inflammatory properties at least in lung tissue. This hypothesis was supported by the observation that natural ClP, or the synthetic analog C8-C1P inhibited cigarette smoke-induced airway inflammation by depleting the levels ofN-SMase-derived ceramides, and blocking pro inflammatory cytokine production, namely IL-6, IL-1 p, keratinocyte chemoattractant (KC), 18 and macrophage inflammatory protein-2 (MIP-2) [68]. Also, leukocyte infiltration in pulmonary tissue was potently reduced by ClP, and oral treatment with ClP or C8-C1P potently reduced lung emphysema. The latter action was accompanied by reduced number of macrophages and neutrophils in bronchoalveolar lavage fluid and decreased the levels of pro inflammatory cytokines [68]. The anti-inflammatory actions ofClP have also been highlighted in various reports from different groups showing that C 1 P potently inhibited the production of pro-inflammatory TNFa and NF-KB activation [116, 117]. Reduction ofTNFa levels by ClP involved inhibition of the metalloproteinase TACE (TNFa converting enzyme), the enzyme that catalyzes the conversion of pro-TNFa to its active form in macrophages [118, 119]. Also, C 1 P stimulated the secretion of anti-inflammatory IL-10 in RA W264. 7 macrophages [ 119], and ClP substantially attenuated lipopolysaccharide (LPS)-induced acute lung injury by preventing NF-KB activation in neutrophils. The latter studies revealed that intrapulmonary application of ClP before (prophylactic) or 24 h after (therapeutic) LPS instillation decreased neutrophil trafficking to the lung, reduced pro-inflammatory cytokine levels in bronchoalveolar lavage fluid, and attenuated alveolar capillary leakage [120]. Moreover, knockout ofCerK aggravated pathological or lethal responses in mice with experimental colitis [121]. Noteworthy, eicosapentenoic acid (EPA), which has shown promise as therapeutic agent in many inflammatory diseases, including skin inflammatory illnesses, potently increased the levels of ClP in the epidermis thereby contributing to its anti-inflammatory capacity in the skin. By contrast, the levels of SlP remained unchanged following EPA administration [122]. The potential of ClP as therapeutic agent in pulmonary inflammation was previously reviewed [123]. Although too early to attribute a therapeutic role to ClP, it could be envisioned that ClP analogues might be promising tools to treat lung or skin inflammation, and could perhaps be used to counteract the deleterious eff ects of ceramides in other cell types or tissues. 19 6. Regulation of adipogenesis by ceramide 1-phosphate Adipogenesis is the process by which pre-adipocytes differentiate into rnature adipocytes. In addition to its role as energy storage in the form of triacylglycerol (TAG), the adipose tissue regulates key physiological cell functions through the production of specific hormones, narnely leptin, adiponectin and resistin [ 124-126], but pre-adipocytes and rnature adipocytes can also secrete a variety of proand anti-inflammatory cytokines, sorne of which being irnplicated in insulin resistant, type II diabetes and obesity [ 127-129]. In particular, leptin has pro-inflammatory properties, and as such, it can induce the release of pro-inflammatory cytokines including TNFa, IL-6 or IL-12 [130, 131]. In turn, sorne of the pro-inflammatory cytokines can increase the levels of leptin in adipose tissue, leading to a loop that potentiates the inflarnrnatory response [131, 132]. In a recent study, we dernonstrated that CerK was upregulated during adipocyte differentiation and that knockdown of this enzyrne using specific siRNA to silence the gene encoding CerK, or pharmacological inhibition ofthis kinase resulted in a substantial decrease oflipid droplet formation and TAG levels in the adipocytes [133]. The latter findings are consistent with previous work by Claro and co-workers showing that CerK regulates lipid droplet biogenesis through activation of cPLA2a [134]. In addition, CerK knockdown caused significant reduction of leptin secretion, which is also a crucial adipokine for controlling appetite and energy horneostasis in the organisrn. Moreover, it is known that leptin levels are elevated in the obese state. The reduction of leptin secretion caused by downregulation of CerK would lead to attenuation of the pro-inflammatory response, an action that would irnpact in the context of obesity, which is a low-grade inflammatory disease. Of interest, and contrary to the adipogenic effect of CerK-generated intracellular ClP, adrninistration of exogenous ClP to pre-adipocytes inhibited their differentiation into rnature adipocytes and led to a reduction in the accurnulation of lipid droplets and depletion of T AG levels in these cells [135]. Furthermore, exogenous ClP caused a rnarked reduction in the 20 expression of the early and late adipogenic markers C/EBPP and PP ARy, and substantially reduced leptin secretion by these cells. The mechanism by which exogenous ClP inhibits adipocyte differentiation involved ERK.1-2 activation, an action that was completely blocked by PTX. Also, PTX completely restored the reduction of T AG levels and leptin secretion caused by exogenous ClP during adipogenesis, suggesting the intervention of a Gi protein-coupled receptor in this process [135]. In addition, administration of ClP during adipogenesis significantly reduced CerK activity [135] suggesting that exogenous ClP may balance the adipogenic action of CerK-derived intracellular ClP in part by reducing CerK activation. In agreement with the latter observations, exogenous ClP also negatively regulated CerK in differentiated human podocytes [35]. Therefore, it seems plausible that ClP can exert dual actions depending on whether it is generated intracellularly or acting exogenously through its putative receptor to control adipogenesis (Fig. 3). 7. Other biological actions of ClP In addition to regulating the physiologic and pathologic processes mentioned above, ClP has also been shown to participate in other biological activities. Specifically, in a murine model of hindlimb ischemia, Mena and co-workers [63] showed that local administration of ClP alone promoted blood perfusion and reduced necrosis in the ischemic muscle. The beneficia! effects of endothelial colony-forming cell infusion after ischemia induced in this model were amplified by ClP pretreatment, resulting in further enhancement ofleg reperfusion and muscle repair. Moreover, it has been recently shown that ClP can increase vasculogenesis both in vitro and in vivo [63], and that priming with ClP promotes the therapeutic effect of mesenchymal stem/stromal cells on pulmonary artery hypertension [136]. It should also be pointed out that dopamine transporter trafficking is regulated by the N-SMase-2/CerK pathway. The dopamine transporter regulates dopamine reuptake from the synaptic cleft, enabling 21 termination of dopaminergic signaling and regulating cellular dopamine recycling systems [137]. It has also been reported that ClP levels are increased in the subventricular zone (SVZ) of brains from patients with Huntington's disease (HD) (post mortem). The higher levels of ClP in HD SVZ were associated with the breakdown of SVZ myelin, where increased levels of Ca2+ stimulated CerK activity thereby enhancing ceramide phosphorylation [138]. Moreover, FTY720 ( also known as fingolimod), a drug used for the treatment of multiple sclerosis, which is capable of penetrating the blood-brain barrier [139] and to upregulate CerK, has been shown to ameliorate Alzheimer's disease [140]. In the same line of investigation, oral administration of FTY720 to a mouse model of diet induced non-alcoholic fatty liver disease improved glucose tolerance and steatosis [141], a finding that is consistent with the role of CerK in decreasing the levels of pro-inflammatory ceramides in liver, and the improvement of steatosis in phosphatidylethanolamine methyl transferase deficient mice, in which CerK was upregulated [142]. Also, ClP (specifically C22-C1P species) was elevated in Farber disease, a rare lysosomal storage disorder characterized by acid ceramidase deficiency and elevation of various pro-inflammatory cytokines and chemokines including VEGF and MCP-1 [143]. In agreement with the latter observation, we showed recently that ClP increased the secretion of VEGF [ 42] and MCP-1 [70] in macrophages, actions that were associated with the stimulation of macrophage proliferation and migration, respectively. 8. Concluding remarks The biological actions elicited by ClP are crucial for maintaining an appropriate balance between cell growth and death, and the suitable regulation of chemotaxis. Whilst CerK-derived intracellular ClP seems to be implicated in controlling cell number in a receptor-independent manner, extracellular ClP governs cell motility through interaction with a putative Gi protein coupled receptor. Any alteration in the levels of ClP, either intraor extracellular, would cause unwanted side effects and likely result in disease. Specifically, upregulation of the intracellular 22 levels of ClP would decrease ceramide levels favoring cell proliferation and reducing cell death, actions that might lead to uncontrolled cell growth and tumorigenesis. By contrast, reduction of intracellular ClP concentrations would increase ceramide levels to facilitate cell death and the development of diseases associated with apoptosis (i.e., neurodegenerative illnesses such as Alzheimer' s or Parkinson disease ). Conceming chemotaxis, migration of cells to inappropriate sites in the organism would cause alterations in tissue homeostasis, inflammation, or tumor dissemination in the case of malignant cells. Moreover, the CerK/ClP axis seems to be essential for controlling adipogenesis, which is associated with development of obesity. Whilst intracellular CerK-derived ClP levels increase during differentiation of pre adipocytes into mature adipocytes, exogenous ClP may be released to balance out adipogenesis by reducing formation of lipid droplets and decreasing TAG content in the adipocytes. Whether targeting CerK may be a way for potential applications in the treatment of cancer or obesity awaits further investigation. 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Medin, Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy, Biochim Biophys Acta Mol Basis Dis 1863 (2) (2017) 386-394. 37 Figure legends Fig. l. Biosynthesis of simple sphingolipids. Ceramide is the central hub of sphingolipid metabolism. It can be produced by three major pathways: 1) the de novo synthesis pathway is an anabolic pathway involving the coordinated actions of serine palmitoyl-CoA transferase (SPT), ceramide synthase (CerS), and desaturase (DES) activities (green area); 2) the sphingomyelinase (SMase) pathway is a catabolic pathway that generates ceramide directly from the breakdown of sphingomyelin (SM) by stimulation of different SMases. The reversed reaction is catalyzed by SM synthases to generate SM (Purple area); 3) the salvage pathway is a catabolic pathway that uses sphingosine derived from the metabolism of complex sphingolipids to form ceramide (yellow area). Once generated, ceramide can be phosphorylated to form ClP by the action of ceramide kinase (CerK). The reversed reaction is catalyzed by ClP phosphatase (CPP) or lipid phosphate phosphatases (LPP). Altematively, ceramides can be degraded by ceramidases to form sphingosine. Phosphorylation of sphingosine by sphingosine kinases (SphK) renders SlP. The reversed reaction is catalyzed by SlP phosphatases (SPP) or lipid phosphate phosphatases (LPP). S lP lyase breaksdown S lP to generate 2-trans hexadecenal and ethanolamine phosphate. Fig. 2. Biosynthesis of ceramide 1-phosphate. Ceramide 1-phosphate (ClP) is mainly synthesized in the Golgi apparatus where ceramides that are generated in the endoplasmic reticulum (ER) are transported by ceramide transfer protein (CERT). Ceramides can then be phosphorylated by ceramide kinase (CERK) to generate ClP. A ClP transfer protein (CPTP) will then transport ClP from the Golgi or from perinuclear membranes, where ClP also resides, to the plasma membrane (PM) and probably to other organelles. Fig. 3. Ceramide 1-phosphate actions in mammalian cells. ClP is generated intracellularly by the action of ceramide kinase (CerK), an enzyme that is dependent upon Ca2+ ions for activity, and that can be stimulated by interleukin 1-betta (IL-1 P), or monocyte/macrophage colony stimulating factor (M-CSF). Intracellular ClP can elicit a variety of biological effects: 1) stimulation of cell proliferation through activation of various kinases including c-Jun N terminal kinase (JNK), extracellularly regulated kinases (ERK), AKT (also known as protein kinase B, PKB), the mammalian target of rapamycin (mTOR), and protein kinase C-a (PKC38 a), as well as stimulation of VEGF release, upregulation of retinoblastoma (Rb ), activation of the RhoA/ROCK pathway and stimulation of the transcription factor NF-kB; 2) inhibition of apoptosis through blockade of acid sphingomyelinase (A-SMase) or serine palmitoyltransferase (SPT), or through upregulation of the inducible form of nitric oxide synthase (iNOS) downstream of Akt; 3) stimulation of adipogenesis; 4) stimulation or inhibition of inflammatory responses depending on cell type. Proinflammatory targets of ClP include cPLA2 whereas sorne anti-inflammatory actions of ClP occur through inhibition of A-SMase activity. ClP can be transported to various organelles including the plasma membrane by the action of a ClP transfer protein (CPTP). ClP can be released by cells and is present in plasma. Extracellular ClP stimulates cell migration through a mechanism involving interaction with a putative Gi protein-coupled receptor and subsequent activation of the mitogen activated protein kinase kinase (MEK)/ERK and phosphoinositide 3-kinase (PI3K)/Akt pathways leading to the release of macrophage chemoattractant protein-1 (MCP-1). Also, extracellular ClP promotes glucose uptake through a mechanism involving activation of the PI3K/ Akt pathway and subsequent translocation ofthe glucose transporter GLUT-3 from the cytosol to the plasma membrane. In addition, extracellular ClP can inhibit adipogenesis through a Gi protein-coupled receptor mediated mechanism. 39 Figure 1 de novo pathway Serine + Palmitoyl-CoA !SPT 3-ketosphinganine i Sphinganine i CerS Dihydroceramide Ceramide 1-phosphate SMase pathway SMase GCS Sphingomyelin E) iDES CERAMIDE ---;--------...... Glucosylceramide SMS CDase ll CerS Sphingosine ( ( � SphKl l SPPILPP Sphingosine 1-phosphate ! S1Plyase Hexadecenal+ ethanolamine phosphate Figure 1 � l Gangliosides Complex sphingolipids Salvage pathway Figure 2 NUCLEUS •• • • • • • • • • • • • • • ENDOPLASMIC RETICULUM GOLGI APPARATUS C P T P ■ ■ ■ ■ __ y__� -- ........ -........ (ff ;MMnññ;ñMint-, \\� ijijfflWij� ijijfflWijij "/' ,....,; ............ __ , .... ______ _ ■ • ■ �-----.... ,, .... , " ' ' OTHER ' I ' ,' ORGANELLES \ 1 ? 1 1 ' \ ■ I ' I I ', , ' ,; ............ ____ , Figure 2