Ceramide 1-phosphate Research Articles

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate.

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser 225 and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A 2 and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.

Phosphorylation and inhibition of ceramide kinase by protein kinase C-β: Their changes by serine residue mutations

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P), and various roles for the CerK/C1P pathway in the regulation of cellular/biological functions have been demonstrated. CerK is constitutively phosphorylated at several serine (Ser, S) residues, however, the roles of Ser residues, including their phosphorylation, in CerK activity, have not yet been elucidated in detail. Therefore, we conducted the present study to investigate this issue. In A549 cells expressing wild-type CerK, a treatment with phorbol 12-myristate 13-acetate (PMA) decreased the formation of C1P in a protein kinase C (PKC)-βI/II-mediated manner. In the Phos-tag SDS-PAGE analysis, CerK existed in its phosphorylated form and was further phosphorylated by the PMA treatment in a PKC-βI/II-mediated manner. We examined the effects of the displacement of Ser residues (72/300/340/403/408/427) in CerK by alanine (Ala, A) on its activity and phosphorylation. Triple mutations (S340/408/427A), but not a single or double mutations (S340/408A), in CerK significantly decreased the formation of C1P. PMA-induced phosphorylation levels in S340/408A- and S340/408/427A-CerK were significantly and maximally reduced, respectively, but were similar in CerK with a single mutation and wild-type CerK. Ser residue mutations tested, including six mutations, did not affect PMA-induced decreases in C1P formation more than expected. Treatments with the protein phosphatase inhibitors, okadaic acid and cyclosporine A, decreased the formation of C1P. These results demonstrated that the activity of CerK was regulated in a phosphorylation-dependent manner in cells.

Neutral ceramidase: Advances in mechanisms, cell regulation, and roles in cancer.

Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of ceramidases, neutral ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.

Regulation of adipogenesis by ceramide 1-phosphate

We showed previously that ceramide kinase (CerK) expression increases during adipogenesis pointing to a relevant role of intracellular C1P in this process. In the present work we demonstrate that administration of exogenous C1P inhibits the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes through a mechanism involving activation of extracellularly regulated kinases (ERK) 1–2. Exogenous C1P reduced the accumulation of lipid droplets and the content of triacylglycerol in these cells, and potently inhibited the expression of the early and late adipogenic markers C/EBPβ and PPARγ, respectively. C1P also reduced the secretion of leptin, which is a crucial regulator of energy balance and appetite in the organism, and is considered to be a late marker of adipogenesis. Interestingly, all of these C1P actions were reversed by pertussis toxin, suggesting the intervention of a Gi protein-coupled receptor previously identified for C1P, in this process. Also, exogenous C1P significantly reduced CerK activity. Altogether, the data presented in this work suggest that exogenous C1P may balance adipogenesis, and that targeting CerK may be a novel way for potential applications in the treatment of obesity or other inflammation-associated diseases.

Pancreatic ductal adenocarcinoma cell secreted extracellular vesicles containing ceramide-1-phosphate promote pancreatic cancer stem cell motility

The high mortality rate associated with pancreatic ductal adenocarcinoma (PDAC) is in part due to lack of effective therapy for this highly chemoresistant tumor. Cancer stem cells, a subset of cancer cells responsible for tumor initiation and metastasis, are not targeted by conventional cytotoxic agents, which renders the identification of factors that facilitate cancer stem cell activation useful in defining targetable mechanisms. We determined that bioactive sphingolipid induced migration of pancreatic cancer stem cells (PCSC) and signaling was specific to ceramide-1-phosphate (C1P). Furthermore, PDAC cells were identified as a rich source of C1P. Importantly, PDAC cells express the C1P converting enzyme ceramide kinase (CerK), secrete C1P-containing extracellular vesicles that mediate PCSC migration, and when co-injected with PCSC reduce animal survival in a PDAC peritoneal dissemination model. Our findings suggest that PDAC secrete C1P-containing extracellular vesicles as a means of recruiting PCSC to sustain tumor growth therefore making C1P release a mechanism that could facilitate tumor progression.

Sphingosine-1-Phosphate Signaling and Metabolism Gene Signature in Pediatric Inflammatory Bowel Disease: A Matched-case Control Pilot Study.

The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population. IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD. Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS. Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls. A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

P-96 - Sphingolipidomic analysis in acne vulgaris patient serum shows decreased C24-ceramide and increased ceramide-1-phosphate levels

This study aimed to identify circulating levels of neutral sphingomyelinase activity (N-SMase), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), C16-C24 sphingomyelin (SM), C16-C24 ceramide (CER) in acne vulgaris (AV) patients and controls. Serum was collected from AV patients and age, gender matched controls. Serum levels of C16-C24 SMs and C16-C24 CERs were determined by multiple reaction monitoring (MRM) method using ultrafast-liquid chromatography coupled with tandem mass spectrometry. Serum activity of N-SMase was assayed by standard kit methods, C1P and S1P levels were determined by enzyme-linked immunosorbent assay (ELISA). A significant increase was observed in serum levels of C16 SM in patients with AV compared to controls. Very-long-chain C24 CER was significantly decreased in AV patients compared to controls. A significant positive correlation was found between serum total cholesterol levels and all measured SMs and CERs in both the control and patient groups. Patients with AV had increased circulating levels of C16 SM, C1P and lower circulating levels of C24 CER compared to healthy controls, which may provide prognostic value for the disease. Future studies are needed to understand the role of altered sphingolipid levels in the pathophysiology of AV.

P-95 - Decreased circulating sphingomyelins and ceramides in sickle cell disease patients

This study aimed to identify levels of C16-C24 sphingomyelin (CerPCho) and C16-C24 ceramide (CER) in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase activity (N-SMase), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) were also determined. Blood was collected from hemoglobin (Hb)A volunteers and homozygous HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24 CER were determined by an optimized multiple reaction monitoring method using ultrafast-liquid chromatography coupled with tandem mass spectrometry. Serum activity of N-SMase was assayed by standard kit methods, C1P and S1P levels were determined by enzyme-linked immunosorbent assay. A significant decrease was observed in serum levels of C18-C24 CerPCho and very-long-chain C22-C24 CERs in SCD patients compared to controls. A significant positive correlation was found between serum total cholesterol levels and C18-C24 CerPCho, C22-C24 CERs in SCD patients. Patients with SCD had significantly elevated serum activity of N-SMase, increased circulating levels of C1P and S1P compared to controls. Future studies are needed to understand the role of decreased CerPCho and CERs in the pathophysiology of SCD.

Knockout of Ceramide Kinase Aggravates Pathological and Lethal Responses in Mice with Experimental Colitis.

Sphingolipids and their metabolic enzymes are implicated in ulcerative colitis. Ceramide kinase (CerK) catalyzes the phosphorylation of ceramide to ceramide-1-phosphate (C1P). Previous studies showed the activation of CerK by the pro-inflammatory cytokine interleukin-1β, the C1P-induced up-regulation of prostanoids exerting protective effects against colitis, and the C1P-induced down-regulation of the pro-inflammatory cytokine tumor necrosis factor-α. In order to elucidate CerK/C1P functions in colitis, we examined the severity of dextran sodium sulfate (DSS)-induced colitis in wild-type (WT) and CerK deletion (CerK(-/-)) mice. Lethal responses were observed in C57BL/6 mice treated with DSS in dose- and time-dependent manners. The depletion of CerK enhanced DSS-induced lethal responses without affecting the onset of these responses. In colons from mice treated with 2.5% DSS for 10 d, epithelial damage was significantly enhanced by the depletion of CerK by day 5, whereas decreases in occluding and E-cadherin levels were similar in both mice. On day 5, the DSS treatment increased spleen weights and colonic levels of cyclooxygenase-2, but not cytosolic phospholipase A2α, and induced a contractile dysfunction in the colons of both mice. The DSS-induced increase in the damage activity index score between days 5 and 10 was slightly enhanced and the decrease in this score from day 10 was slower in CerK(-/-) mice than in WT mice. On day 7 after the DSS treatment, spleen weights slightly decreased and increased in WT and CerK(-/-) mice, respectively. These results indicate that the depletion of CerK enhances the pathology of colitis and lethal responses in DSS-treated mice.

Physcion 8-O-β-glucopyranoside exhibits anti-leukemic activity through targeting sphingolipid rheostat.

Acute lymphoblastic leukemia (ALL) is the most common fatal cancer in people younger than 20 years of age. This study was designed to explore the anti-leukemia activity of physcion 8-O-β-glucopyranoside (PG) in B-cell ALL. NALM6 and SupB15 cells were used as model cell lines. Cell viability, cell apoptosis, cell cycle distribution were determined by CCK-8 assay, DNA fragmentation assay and flow cytometry, and flow cytometry, respectively. Expression of proteins involved in cell apoptosis and cell cycle regulation was determined by western blot and the levels of ceramide and sphingosine 1-phosphate (S1P) were determined by ELISA. Activity of sphingosine kinase 1 (SphK1) was also determined with a Sphingosine Kinase Assay Kit. In the present study, both model cell lines were transfected with siRNA targeting SphK1 or an overexpression plasmid to examine the role of SphK1 in the anti-leukemia activity of PG. Moreover, the efficacy of PG was examined in vivo in a mouse model by measuring survival and spleen weight. Our results provided experimental evidence that PG could significantly induce apoptosis and cell cycle arrest in vitro. Mechanistically, the anti-leukemia activity of PG was mediated by its ability to repress SphK1 and thus modulate ceramide-S1P rheostat. Moreover, the anti-leukemia activity of PG was also verified in a murine model. Collectively, our results indicate that PG may be a promising agent for the treatment of B-cell leukemia.

Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure.

Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology. Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy. The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration. Animals were euthanized by cervical dislocation or cardiac puncture 2 weeks after surgery for collection of blood orovary and uterus samples, which were cleaned of adhering tissue in culture medium and used for subsequent assays. Ovaries were used for Western blotting or immunohistochemical and/or histological analyses or steroid extraction, as required (n = 5-8 per group). A set of mice (n = 3/group) was destined for oocyte recovery and IVF. Finally, another set (n = 5-6/group) was separated to study fertility parameters. The number of primordial (P < 0.01), primary (P < 0.05) and preantral follicles (P < 0.05) were decreased in Cy-treated mice compared to control animals, while atretic follicles were increased (P < 0.001). In Cy + C1P mice, the ovaries recovered control numbers of these follicular structures, in both C1P doses studied. Cy affected AMH expression, while it was at least partially recovered when C1P is administered as well. Cy caused an increase in serum FSH concentration (P < 0.01), which was prevented by C1P coadministration (P < 0.01). E2 levels in Cy-treated ovaries decreased significantly compared to control ovaries (P < 0.01), whilst C1P restored E2 levels to those of control ovaries (P < 0.01). Cy increased the expression of BAX (P < 0.01) and decreased the expression of BCLX-L compared to control ovaries (P < 0.01). The ovarian BCLX-L:BAX ratio was also lower in Cy-treated mice (P < 0.05). In the Cy + C1P group, the expression levels of BAX, BCLX-L and BCLX-L:BAX ratio were no different than those in control ovaries. In addition, acid sphingomyelinase (A-SMase) expression was higher in Cy-treated ovaries, whilst remaining similar to the control in the Cy + C1P group. Cy increased the apoptotic index (TUNEL-positive follicles/total follicles) in preantral and early antral stages, compared to control ovaries (P < 0.001 and P < 0.01, respectively). C1P protected follicles from this increase. No primordial or primary follicular cells stained for either cleaved caspase-3 or TUNEL when exposed to Cy, therefore, we have found no evidence for follicular reserve depletion in response to Cy being due to apoptosis. Cy caused evident vascular injury, especially in large cortical stromal vessels, and some neovascularization. In the Cy + C1P group, the disruptions in vascular wall continuity were less evident and the number of healthy stromal blood vessels seemed to be restored. In Cy-treated ovaries α-SMA-positive cells showed a less uniform distribution around blood vessels. C1P coadministration partially prevented this Cy-induced effect, with a higher presence of α-SMA-positive cells surrounding vessels. By H&E staining, Cy-treated mice showed endometrial alterations compared to controls, affecting both epithelial and stromal compartments. However, C1P allowed that the stromal tissue to maintain its loose quality and its glandular branches. Cy-treated animals had significantly lower pregnancy rates and smaller litter sizes compared with control mice (P = 0.013 and P < 0.05, respectively), whereas cotreatment with C1P preserved normal fertility. Furthermore, a higher (P < 0.05) proportion of abnormal oocytes was recovered from Cy-treated mice compared to the control, which was prevented by C1P administration. N/A. The results of this study were generated from an in-vivo animal experimental model, already used by several authors. Further studies on C1P functions in female reproduction in pathological conditions such as chemotherapy-induced ovarian failure and on the safety of use of this sphingolipid are required. The present findings showed that C1P administration prior to Cy might be a promising fertility preservation strategy in female patients who undergo chemotherapy. This work was supported by grants from ANPCyT (PICT 2015-1117), CONICET (PIP 380), Cancer National Institute (INC) and Roemmers Foundation, Argentina. The authors declare no conflicts of interest.

Decreased Serum Levels of Sphingomyelins and Ceramides in Sickle Cell Disease Patients.

Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle-cell disease (SCD). This study was aimed at identifying the levels of C16-C24 CerPCho and C16-C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N-SMase) activity, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16-C24 CerPCho and C16-C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N-SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme-linked immunosorbent assay. A significant decrease was observed in the serum levels of C18-C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very-long-chain C22-C24 CER were significantly decreased in SCD, while long-chain C16-C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18-C24 CerPCho and C22-C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N-SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18-C24 CerPCho in patients with SCD was accompanied by decreased levels of C22-C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation

ABSTRACTThe macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.

Lysophosphatidic Acid Signaling Axis Mediates Ceramide 1-Phosphate-Induced Proliferation of C2C12 Myoblasts.

Sphingolipids are not only crucial for membrane architecture but act as critical regulators of cell functions. The bioactive sphingolipid ceramide 1-phosphate (C1P), generated by the action of ceramide kinase, has been reported to stimulate cell proliferation, cell migration and to regulate inflammatory responses via activation of different signaling pathways. We have previously shown that skeletal muscle is a tissue target for C1P since the phosphosphingolipid plays a positive role in myoblast proliferation implying a role in muscle regeneration. Skeletal muscle displays strong capacity of regeneration thanks to the presence of quiescent adult stem cells called satellite cells that upon trauma enter into the cell cycle and start proliferating. However, at present, the exact molecular mechanism by which C1P triggers its mitogenic effect in myoblasts is lacking. Here, we report for the first time that C1P stimulates C2C12 myoblast proliferation via lysophosphatidic acid (LPA) signaling axis. Indeed, C1P subsequently to phospholipase A2 activation leads to LPA1 and LPA3 engagement, which in turn drive Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinases 1/2) activation, thus stimulating DNA synthesis. The present findings shed new light on the key role of bioactive sphingolipids in skeletal muscle and provide further support to the notion that these pleiotropic molecules might be useful therapeutic targets for skeletal muscle regeneration.

Identification of an acid sphingomyelinase ceramide kinase pathway in the regulation of the chemokine CCL5 [S

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to produce the biologically active lipid ceramide. Previous studies have implicated ASM in the induction of the chemokine CCL5 in response to TNF-α however, the lipid mediator of this effect was not established. In the present study, we identified a novel pathway connecting ASM and ceramide kinase (CERK). The results show that TNF-α induces the formation of ceramide 1-phosphate (C-1-P) in a CERK-dependent manner. Silencing of CERK blocks CCL5 production in response to TNF-α. Interestingly, cells lacking ASM have decreased C-1-P production following TNF-α treatment, suggesting that ASM may be acting upstream of CERK. Functionally, ASM and CERK induce a highly concordant program of cytokine production and both are required for migration of breast cancer cells. Taken together, these data suggest ASM can produce ceramide which is then converted to C-1-P by CERK, and that C-1-P is required for production of CCL5 and several cytokines and chemokines, with roles in cell migration. These results highlight the diversity in action of ASM through more than one bioactive sphingolipid.

The Role of Ceramide 1-Phosphate in Tumor Cell Survival and Dissemination.

Ceramide 1-phosphate (C1P) is a pleiotropic bioactive sphingolipid metabolite capable of regulating key physiologic cell functions and promoting pathologic processes. Concerning pathology, C1P or ceramide kinase (CerK), the enzyme responsible for its biosynthesis in mammalian cells, has been implicated in cancer cell growth, survival, and dissemination and is involved in inflammatory responses associated with different types of cancer cells. The mechanisms or signaling pathways mediating these C1P actions have only been partially described. This chapter reviews recent progress in identifying signal transduction pathways involved in the promotion of cancer cell growth, survival, and dissemination by CerK and C1P.

Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.

Sphingosine 1-phosphate activation of ERM contributes to vascular calcification

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.

Enhanced phosphorylation of sphingosine and ceramide sustains the exuberant proliferation of endothelial progenitors in Kaposi sarcoma.

Endothelial colony-forming cells (ECFCs), a unique endothelial stem cell population, are highly increased in the blood of Kaposi sarcoma (KS) patients. KS-derived ECFCs (KS-ECFCs) are also endowed with increased proliferative and vasculogenic potential, thus suggesting that they may be precursors of KS spindle cells. However, the mechanisms underlying the increased proliferative activity of KS-ECFCs remain poorly understood. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are metabolically interconnected sphingoid mediators crucial to cell proliferation. Here, we investigated the metabolism, release, and proliferative effects of S1P and C1P in KS-ECFCs compared with control ECFCs (Ct-ECFCs). Metabolic studies by cell labeling, chromatographic analyses, and digital autoradiography revealed that S1P and C1P biosynthesis and S1P secretion are all efficient processes in KS-ECFCs, more efficient in KS-ECFCs than Ct-ECFCs. Quantitative PCR analyses demonstrated a significantly higher ceramide kinase and sphingosine kinase-2 expression in KS-ECFCs. Notably, also the expression of S1P1 and S1P3 receptors was augmented in KS-ECFCs. Accordingly, treatment with exogenous C1P or S1P induced a significant, concentration-dependent stimulation of KS-ECFC proliferation, but was almost completely ineffective in Ct-ECFCs. Hence, we identified C1P and S1P as autocrine/paracrine proliferative signals in KS-ECFCs. A better understanding of the mechanisms that enhance S1P/C1P formation in KS-ECFCs may yield effective therapeutic modalities.

Vascular endothelial growth factor mediates ceramide 1-phosphate-stimulated macrophage proliferation

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.