Ceramide 1-phosphate Research Articles

Monitoring ceramide and sphingosine-1-phosphate levels in cancer cells and macrophages from tumours treated by photodynamic therapy

Eradication of tumours by photodynamic therapy (PDT) is accompanied by marked changes in local sphingolipid (SL) engagement. Because of the heterogeneity of cellular composition, analysis of tumour tissue homogenates to quantify SL species is inadequate for evaluating their levels in parenchymal cancer cell population. By staining tumour-derived single cell suspensions with antibodies specific to ceramide and sphingosine 1-phosphate (S1P) followed by flow cytometry, we were able to document changes in the levels of these two key SLs in cancer cells and tumour-associated macrophages (TAMs) of mouse SCCVII tumours following PDT. The results confirm previously obtained indications that tumour treatment by PDT induces a marked rise in ceramide levels in cancer cells within these lesions. Cancer cells from PDT-treated SCCVII tumours undergoing apoptosis were found to have much higher ceramide levels and substantially lower S1P levels than their viable counterparts. Compared to cancer cells, considerably higher ceramide and S1P levels were consistently found in TAMs. Treatment of SCCVII tumour-bearing mice with ceramide analog LCL29 induced a rise in ceramide levels in TAMs but not in cancer cells. When combined with PDT, LCL29 treatment produced a further increase in ceramide levels in TAMs while having no evident impact on ceramide content in cancer cells within same tumours. The results highlight SLs as important participants in tumour response to PDT and potential adjuvant therapeutic targets to PDT.

Inhibition of Ceramide Metabolism Key Enzymes and its Implication in Cell Physiology and Pathology

The simple sphingolipids ceramide, sphingosine, ceramide 1-phosphate and sphingosine 1-phosphate, and their metabolizing enzymes are implicated in the regulation of vital cellular functions, including angiogenesis, cell differentiation, migration, and cell growth and death. These sphingolipids are also involved in the establishment and progression of many illnesses including cardiovascular diseases such as atherosclerosis, or neurodegenerative diseases such as Parkinson's or Alzheimer's diseases, chronic inflammation, and cancer. Therefore, the development of inhibitors of the enzymes that are responsible for the synthesis and degradation of these metabolites may be essential tools for studying normal cell metabolism as well as for establishing new therapeutic strategies for treatment of disease.

The Characterization and Identification of Ceramide‐1‐Phosphate Binding Proteins

The activation of cytosolic phospholipase A2 α (cPLA2α) has been implicated in atherosclerosis and cerebral ischemia in previous animal models. The sphingolipid metabolite ceramide‐1‐phosphate (C1P) is known to specifically regulate the activation of cPLA2α and has been shown to increase enzymatic activity 5–10 fold through increased membrane residence. We have recently determined the amino acids that are responsible for C1P binding to cPLA2α in vitro and in a cellular system. In addition to heart disease, the conversation of ceramide to C1P has been implicated in cancer and phagocytosis signaling, suggesting that there are unidentified proteins that are regulated by C1P binding in membranes. Using the novel cPLA2α consensus sequence to search the proteome, we have predicted and are currently investigating new C1P binding proteins. Through the characterization of the C1P binding site, we have constructed a novel model for C1P recognition in membranes that deviates from the traditional lipid binding mechanism. This molecular characterization provides great insight into the molecular basis of C1P recognition and selectivity in biological membranes and will significantly increase the molecular basis of C1P binding by proteins. The understanding of this binding site in particular would lay the groundwork for a promising therapy for heart disease.AHA 11PRE7640028 (KEW)NIH T32GM075762 (KEW)

Signaling and Regulatory Functions of Bioactive Sphingolipids as Therapeutic Targets in Multiple Sclerosis

Spingolipids (SLs) are an important component of central nervous system (CNS) myelin sheaths and affect the viability of brain cells (oligodendrocytes, neurons and astrocytes) that is determined by signaling mediated by bioactive sphingoids (lyso-SLs). Recent studies indicate that two lipids, ceramide and sphingosine 1-phosphate (S1P), are particularly involved in many human diseases including the autoimmune inflammatory demyelination of multiple sclerosis (MS). In this review we: (1) Discuss possible sources of ceramide in CNS; (2) Summarize the features of the metabolism of S1P and its downstream signaling through G-protein-coupled receptors; (3) Link perturbations in bioactive SLs metabolism to MS neurodegeneration and (4) Compile ceramide and S1P relationships to this process. In addition, we described recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod) as well as proposed intervention to specify critical SL levels that tilt balances of apoptotic/active ceramide versus anti-apoptotic/inactive dihydroceramide that may offer a novel and important therapeutic approach to MS.

Ceramide kinase deficiency improves diet-induced obesity and insulin resistance

Ceramide kinase (CERK) is an enzyme that phosphorylates ceramide to produce ceramide 1-phosphate. Recently, evidence has emerged that CERK has a role in inflammatory signaling of immune cells. Since obesity is accompanied by chronic, low-grade inflammation, we examined whether CERK might be involved using CERK-null mice. We determined that CERK deficiency suppresses diet-induced increases in body weight, and improves glucose intolerance. Furthermore, we demonstrated that CERK deficiency attenuates MCP-1/CCR2 signaling in macrophages infiltrating the adipose tissue, resulting in the suppression of inflammation in adipocytes, which might otherwise lead to obesity and diabetes.

Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.

Protein Phosphatase 1α Mediates Ceramide-induced ERM Protein Dephosphorylation: A NOVEL MECHANISM INDEPENDENT OF PHOSPHATIDYLINOSITOL 4, 5-BIPHOSPHATE (PIP2) AND MYOSIN/ERM PHOSPHATASE

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP(2)) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP(2) hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP(2) and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1α, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1α acted as dominant negative of the endogenous PP1α. Additional results showed that the ceramide mechanism of PP1α activation is largely independent of PIP(2) hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1α and plasma membrane ceramide.

Serum sphingolipids and inflammatory mediators in adolescents at risk for metabolic syndrome

The purpose of this study was to determine low-grade inflammation associated with obesity that is mediated partially by TNF-α, an adipocytokine which stimulates sphingomyelinase activity in adipocytes. Circulating ceramide (Cer) and sphingosine 1-phosphate (S1P) are elevated in genetically obese (ob/ob) mice. We aimed to determine whether serum sphingolipid concentrations correlate with measures of obesity, insulin resistance, and lipid profiles in overweight versus lean adolescents. This cross-sectional study recruited 30 healthy overweight (body mass index, BMI≥85%) and 15 lean (BMI 10-84%) adolescents. Anthropometric measurements and fasting blood samples were collected at one clinic visit. Serum glucose, insulin, and fasting lipid profiles were measured. Serum adipocytokine concentrations were measured by ELISA or colorimetric assay and sphingolipids were measured by HPLC-mass spectrometry. Between group differences in serum sphingolipid concentrations were assessed. Correlations between sphingolipid concentrations and (i) body mass index, (ii) calculated homeostasis model assessment of insulin resistance (HOMA-IR), (iii) adipocytokines, and (iv) lipoproteins were determined. The results showed that significant differences in HOMA-IR (4.5±3.2 vs. 1.2±0.7), free fatty acids (0.8±0.3mmol/l vs. 0.4±0.3mmol/l), and adiponectin (6.4±3.8 vs. 12.6±9.9 μg/ml) were seen between groups (overweight vs. lean). There were significant correlations between Cer and TNF-α (r=0.429), S1P and TNF-α (r=0.288), Cer and adiponectin (r=0.321), Cer:S1P and adiponectin (r=0.324), Cer and HOMA-IR (r=0.307), and Cer:S1P and LDL cholesterol (r=0.453); these associations persisted after adjustment for BMI Z-score, sex, and Tanner stage. We concluded that elevated sphingolipid concentrations correlate with TNF-α, adiponectin, lipoprotein profiles, and HOMA-IR. Ceramide is associated with atherogenic lipid profiles and the development of insulin resistance in obese adolescents, similar to adults.

Obesity-associated steatotic liver exhibits aberrant or altered sphingolipid composition and preferentially accumulates ceramide species containing long chain fatty acids

The sphingolipid (SL) signaling pathways are induced by reactive oxygen species and proin-flammatory molecules, which are shown to be upregulated in the obese state. The present work was conducted to determine if an altered SL pathway exists, and contributes to the pathogenesis of hepatic steatosis associated with obesity. Steatotic and non-steatotic livers were procured from Zucker Obese female rats and their lean counterparts in this pre-clinical study, and assessed for enzymes involved in degradation as well as in phos-phorylation of proapoptotic SLs. The expression of enzymes [sphingo-myelinase (SMase), ceramidase, and sphingosine kinase-1 (SK1)] and apoptotic proteins (Bax and Bcl-2) was quantified by ELISA and by Western Blot. Sphingomyelin (SM), ceramide, ceramide-1 phosphate (C1P), sphingosine (SPH), and sphingosine-1-phosphate (S1P) levels were quantified by high-performance liquid chroma-tography (HPLC)-tandem mass spectroscopy (MS). Obese steatotic livers exhibited significantly upregulated ceramidase and down-regulated SK1 and C1P levels (P accumulation of ceramides containing long chain fatty acids could be involved in the pathogenesis of hepatic steatosis.

Ceramide 1-Phosphate: First, Second, or Dual Messenger?

Ceramide 1-Phosphate: First, Second, or Dual Messenger?

The Expanding Family of Bone Marrow Homing Factors for Hematopoietic Stem Cells: Stromal Derived Factor 1 Is Not the Only Player in the Game

The α-chemokine stromal derived factor 1 (SDF-1), which binds to the CXCR4 and CXCR7 receptors, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) and plays a crucial role in retention of these cells in stem cell niches. However, this unique role of SDF-1 has been recently challenged by several observations supporting SDF-1-CXCR4-independent BM homing. Specifically, it has been demonstrated that HSPCs respond robustly to some bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), and migrate in response to gradients of certain extracellular nucleotides, including uridine triphosphate (UTP) and adenosine triphosphate (ATP). Moreover, the responsiveness of HSPCs to an SDF-1 gradient is enhanced by some elements of innate immunity (e.g., C3 complement cascade cleavage fragments and antimicrobial cationic peptides, such as cathelicidin/LL-37 or β2-defensin) as well as prostaglandin E2 (PGE2). Since all these factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors supporting, and in some situations even replacing, the SDF-1-CXCR4 axis.

Acetylation of sphingosine kinase 1 regulates cell growth and cell-cycle progression

Sphingosine kinase 1 (SPK1) is a key enzyme in the sphingolipid metabolic pathway. It forms an essential checkpoint to regulate the relative levels of bioactive sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate (S1P). Here, we present evidence that SPK1 is acetylated by the intrinsic acetyltransferase activity of p300/cAMP-response element-binding protein (CREB)-binding protein (CBP) at a conserved acetylation motif (the GK motif). This post-translational modification may be an important regulator of SPK1 protein, as acetylation by p300 or CBP increased its stability. Mutation of two lysine (K) residues in its GK motif to either arginine (R) or glutamine (Q) blocked SPK1 ubiquitination and prevented its degradation by the proteasome. The processes of acetylation and ubiquitination may compete for the same lysine residues and, therefore, form a switch for SPK1 protein regulation. Intriguingly, human embryonic kidney (HEK) 293 cells stably expressed the mutated form of SPK1, in which the K residue was mutated to Q (Q-SPK1), and this mutated form mimicked acetylated SPK1. These cells were larger in size and had a slower growth rate compared to cells that expressed wild-type SPK1 (W-SPK1) or the K/R-mutated SPK1 (R-SPK1). These data suggest that SPK1 acetylation plays a key role in cell growth, cell size, and cell-cycle control.

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A 2 and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA 2-α in this pathway.

Ceramide Kinase Regulates the Production of Tumor Necrosis Factor α (TNFα) via Inhibition of TNFα-converting Enzyme

Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK(-/-)) generated higher levels of TNFα than the wild-type mice (CERK(+/+)) in response to LPS. An increase in basal TNFα secretion was also observed in CERK(-/-) murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK(-/-) as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.

Exogenous Ceramide-1-phosphate Reduces Lipopolysaccharide (LPS)-mediated Cytokine Expression

Toll-like receptor 4 (TLR4) is a component of the innate immune system that recognizes a diverse group of molecular structures, such as lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 signaling ultimately leads to activation of the transcription factor, nuclear factor κB (NF-κB), and the production of cytokines. Ceramide is a bioactive sphingolipid that has been suggested to regulate TLR4-induced NF-κB signaling, although reports on the role of ceramide in TLR4 activation conflict. We investigated the possibility that ceramide metabolites, such as ceramide-1-phosphate (C-1-P), may explain these discrepancies. We now report that exogenous C-1-P, but not ceramide, reduces NF-κB-mediated gene transcription in HEK 293 cells stably transfected with human TLR4, CD14, and MD-2. We demonstrate that inhibition of NF-κB by exogenous C-1-P is dose-dependent and specific to TLR4 in a reporter assay. We further demonstrate a requirement for both the phosphate moiety and the sphingoid backbone to inhibit LPS-activated NF-κB transcription. Specifically, C-1-P prevents the degradation of IκB, the phosphorylation of the p65 subunit of NF-κB, and LPS-stimulated MAPK activation. The functional consequence of C-1-P inhibition of NF-κB is a reduction in LPS-mediated cytokine release from HEK 293 TLR4-expressing cells and human peripheral blood mononuclear cells. Taken together, these data demonstrate that C-1-P may function as an anti-inflammatory lipid mediator of immune response.

Differential Responsiveness of HSPCs Harvested From Bone Marrow (BM), Mobilized Peripheral Blood (mPB) and Umbilical Cord Blood (UCB) to the BM Homing Factors Stromal-Derived Factor-1 (SDF-1), Sphingosine-1 Phosphate (S1P) and Ceramide-1 Phosphate (C1P) Is Related to the Desensitization of mPB and UCB HSPCs by S1P and C1P Present in mPB and UCB Plasma

Abstract 2958 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches in stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis-dependent manner. While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, recent evidence confirms that due to induction of proteolytic microenvironment in BM after myeloablative radio-chemotherapy, SDF-1 level in BM decreases (Leukemia 2011, doi: 10.1038/leu.2011.185) which supports the potential role of other non-SDF-1-mediated homing mechanisms. The cumulative evidence indicates that gradients of bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are important homing factors. To support this further we demonstrated recently that S1P and C1P are upregulated in BM after conditioning for transplantation (Leukemia 2011, doi: 10.1038/leu.2011.185). Hypothesis. Based on the fact that SDF-1, S1P and C1P are present at relatively high concentrations in umbilical cord blood (UCB) and mobilized peripheral blood (mPB) plasma, they may desensitize responsiveness of HSPCs to BM homing gradients of SDF-1, S1P and C1P. Experimental approach. Based on this we i) measured concentration of SDF-1, S1P and C1P in BM aspirates, mPB and UCB, ii) evaluated chemotactic responsiveness of BM-, UCB- and mPB-derived HSPCs to homing gradients of SDF-1, S1P and C1P and iii) investigated molecular mechanisms potentially involved in this phenomenon by examining the surface expression of CXCR4 and S1P receptors 1–5 (S1PR1-5) at baseline and following exposure to appropriate ligands. Finally, we modulated the expression of S1PR1-5 on HSPCs and their responsiveness to chemotactic gradients by removal of S1PR1-5 ligands from the culture medium. Results. The highest expression of S1PR1-5 was detected on the surface of BM-derived CD34+/Lin− cells as compared to mPB and UCB counterparts. The downregulation of S1PR1-5 on mPB- and UCB-derived BM CD34+Lin− cells correlated with elevated level of circulating S1P in UCB and mPB plasma. Next, we found that BM-derived HSPCs responded more robustly to S1P and C1P as compared to SDF-1 when these chemoattractants were employed at physiologically relevant concentrations. Addition of S1P to the BM-derived HSPCs or incubation of BM HSPCs in S1P and C1P rich UCB or mPB plasma lead to downregulation of S1PR1-5 and desensitized their responsiveness to S1P and C1P, but not SDF-1 gradient. The expression of S1PR1-5 and responsiveness to S1P gradient by UCB- and mPB-derived HSPCs, however, could be re-established after incubating HSPCs in S1P-free medium. At the same time, since SDF-1 concentration in mPB and UCB is very low, the responsiveness of mPB- and UCB-derived HSPCs to SDF-1 gradient was not affected. Conclusions. We demonstrate, for the first time, significant differences in responsiveness of HSPCs from different sources to homing gradients of bioactive lipids. The relatively high concentration of S1P and C1P in mPB and UCB plasma may potentially desensitize responsiveness of HSPCs to BM homing gradients of bioactive lipids and interfere with their homing. These observations will have substantial clinical implications in HSPCs’ transplantation. Disclosures:No relevant conflicts of interest to declare.

New Evidence That the Bioactive Lipid Ceramide-1-Phosphate (C1P) Is a Potent Chemoattractant for Mesenchymal Stromal Cells (MSC), Endothelial Progenitor Cells (EPCs) and Very Small Embryonic-Like Stem Cells (VSELs), Demonstrating Its Potential Involvement in Tissue/Organ Repair and Angiogenesis

Abstract 2387 Background:Ceramide-1-phosphate (C1P), like its related derivative sphingosine-1-phosphate (S1P), is derived from sphingolipids, which are important components of cell membranes. In contrast to S1P, which is secreted as a signaling molecule from intact cells, C1P is released from cells that are damaged and “leaky”. Unlike ceramide (which is often pro-apoptotic), C1P is anti-apoptotic and, as we have demonstrated, does not impair the clonogenicity of hematopoietic progenitors (Leukemia 2011, doi: 10.1038/leu.2011.185) and promotes their migration through an unknown receptor-initiated signaling pathway that is pertussis toxin-sensitive and therefore likely to involve a Gai protein-coupled seven-transmembrane-spanning receptor. Hypothesis: Taking into consideration the anti-apoptotic effects of C1P and the fact that it is released in damaged tissues, we have hypothesized that C1P plays an important and under-appreciated role in tissue/organ regeneration. Materials and Methods: To better address the potential role of C1P in this process we i) employed the Transwell system and FACS analysis to evaluate chemotactic responsiveness of stem cells involved in tissue and organ regeneration, including CD31+CD45−Lin− mesenchymal stroma cells (MSCs), Sca-1+CD51+CD45-Lin−CD31− endothelial progenitor cells (EPCs) and Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs), ii) measured the C1P level in damaged tissues (BM conditioned for transplantation, myocardium after heart infarct, and chemically damaged liver), iii) studied the activation of intracellular signaling pathways related to cell migration/adhesion (MAPKp42/44, AKT, and p38) in bone marrow stromal cells and umbilical cord blood-derived endothelial cells (HUVECs) in response to C1P stimulation, iv) evaluated the effect of C1P on adhesion and migration of stromal fibroblasts and endothelial cells, v) evaluated the influence of C1P on tube formation by HUVECs, and v) evaluated the effect of C1P stimulation on secretion of cytokines and chemokines by bone marrow stromal cells. All these effects were studied in parallel in comparison to other bioactive lipids (S1P, lysophosphatidic acid [LPA], and lysophosphatidylocholine [LPC]), as well as directly compared to the effects of known chemottractants of stem cells, bone marrow (BM)-derived fibroblasts, and endothelial cells (SDF-1, HGF, VEGF, and FGF-2). Results: We observed that C1P is upregulated in damaged tissues and strongly chemoattracts stem cells from BM that are potentially involved in tissue/organ regeneration (VSELs, MSCs, and EPCs). In chemotactic assays performed on expanded BM-derived fibroblasts and HUVECs, C1P had a similar strong chemotactic effect as S1P, but stronger than other bioactive lipids (LPA and LPC). C1P strongly stimulated phosphorylation of MAPKp42/44 and AKT in HUVECs and MAPKp42/44 in BM-derived fibroblasts. Interestingly, compared to all bioactive lipids tested in this study, C1P most strongly stimulated tube formation by HUVECs and stimulated secretion from BM-derived fibroblasts of several cytokines and chemokines, including stromal derived factor-1 (SDF-1). Conclusions: Our data demonstrate, for the first time, that C1P is a, potent bioactive lipid released from damaged cells that plays an important novel role not only in homing of HSPCs to BM, as we demonstrated recently, (Leukemia 2011, doi: 10.1038/leu.2011.185) but also directs migration and activates stem cells that are involved in repair of other damaged organs and tissues. We also envision that modulation of C1P signaling may turn out to be a viable strategy for tissue/organ repair and this is currently tested in our laboratory. Disclosures:No relevant conflicts of interest to declare.

Novel View on Unwanted Side Effects of Radio-Chemotherapy on Bone Marrow (BM) Microenvironment - Radio-Chemotherapy Upregulates BM-Level of Bioactive Lipids, Sphingosine-1- Phosphate (S1P) and Ceramide-1-Phosphate (C1P), That Chemoattract Metastasizing Cancer Cells

Abstract 726 Background:Rhabdomyosracoma (RMS), the most common soft-tissue sarcoma of adolescents and children, frequently infiltrates the BM to such a degree that it often mimics acute lymphoblastic leukemia. The prognosis is poor, particularly for the more aggressive and metastatic alveolar RMS (ARMS) compared to embryonal RMS (ERMS). In our previous work, we demonstrated a pivotal role for two signaling axes, a-chemokine stromal-derived factor-1 (SDF-1)–CXCR4 (a seven-transmembrane-spanning G protein-coupled receptor) and hepatocyte growth factor/scatter factor (HGF/SF)–c-met, in metastasis of pediatric sarcomas to bone marrow (BM) (Blood 2002;100:2597-2606,Cancer Research 2003; 63:7926–7935, IJC 2010;127: 2554–2568). Recently, however, we observed that the bioactive lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are much more potent chemotractants for human rhabdomyosarcoma (RMS) than SDF-1 or HGF/SF. Importantly, we observed that S1P and C1P levels are highly increased in BM after radio-chemotherapy. Hypothesis: Based on these observations, we hypothesized that S1P and C1P direct chemotaxis of RMS cells to BM. This could be particularly important in patients treated with radio-chemotherapy, where upregulation of S1P and C1P levels in BM may facilitate the spread to the bones of tumor cells that survived initial treatment. Material and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of bioactive lipids in BM metastasis of RMS cells. The expression of S1P seven-transmembrane-spanning G protein-coupled receptors, chemotaxis, adhesion, proliferation, and cell signaling studies in response to S1P and C1P were performed on 8 human ARMS and 3 human ERMS cell lines. The secretion of S1P and C1P in BM and by RMS cells was measured by mass spectrometry (MS). The S1P1 receptor was downregulated by employing an shRNA strategy and S1P1-KO cells were evaluated for their ability to grow tumors in immunodeficient mice. Finally, to address the role of the S1 P–S 1P1 axis in the unwanted spread of sarcoma cells after radio-chemotherapy, we compared seeding of S1P1-KO and control RMS cells in irradiated immunodeficient mice. Results: S1P and C1P are much more potent chemoattractants than SDF-1 or HGF/SF, particularly if employed at “physiological” tissue concentrations. S1P1–5 receptors are expressed on RMS cells and stimulation by S1P induced chemotaxis, adhesion of these cells, and phosphorylation of MAPPp42/44 and AKT. However, while receptor/s for C1P have not yet been identified, C1P also exerted similar effects on human RMS cells. Finally, S1P1-KO cells grew smaller tumors in immunodefcient mice and had impaired seeding efficiency in irradiated animals compared to control RMS cells transduced with empty vector. In parallel experiments, we also observed that both bioactive lipids increase stromalization of the RMS by i) chemoattracting and activating cancer-associated fibroblasts (CAF) and ii) promoting tumor angiogenesis. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of bioactive lipids in damaged tissues and side effect of such treatment is induction of unwanted prometastatic microenvironment in different organs. By employing an RMS model, we confirmed S1P and identified C1P as novel under-appreciated factor directing metastasis of cancer cells. Since S1P and C1P become upregulated in BM after radio-chemotherapy, both bioactive lipids are involved in the unwanted spread to the bones of RMS cells that survived initial treatment. The role of S1P and C1P in metastasis of other pediatric sarcomas and other types of solid tumors and dissemination of leukemias/lymphomas is currently being investigated in our laboratory, similarly as different strategies to inhibit pro-metastatic effects of S1P and C1P. Disclosures:No relevant conflicts of interest to declare.

A Novel Perspective on Stem Cell Homing – Emerging Interplay Between Bioactive Lipids As Potent Chemoattractants and Cationic Peptides As Underappreciated Modulators of Responsiveness to SDF-1 Gradients

Abstract 2957 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1 integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been proposed that concentration differences between the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells will home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/– fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Leukemia 2011, doi: 10.1038/leu.2011.185). Aim of the study. Based on these observations that strongly suggest the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs we become interested in a role of bioactive lipids (sphingosine-1 phosphate; S1P and ceramide-1 phosphate; C1P) as well as priming phenomenon of SDF-1-CXCR4 axis by cationic antimicrobial peptides (CAMPs) in HSPCs homing. Results. We present evidence that gradients of bioactive lipids, such as S1P and C1P, which are products of membrane lipid metabolism, are involved in stem cell lodgment into BM. In addition, we present another important mechanism that enhances stem cell response to an low SDF-1 concentration gradient, namely, the “priming effect”. This mechanism is based on a phenomenon in which CAMPs such as complement cascade cleavage fragment C3a and stroma fibroblast- and granulocytes-derived antimicrobial peptide LL-37 significantly increase (up to 25 times) the chemotactic responsiveness of HSPCs to very low “tissue concentration-relevant” SDF-1 gradients (1–3 ng/ml). To support this further murine HSPCs primed ex vivo with CAMPs engrafted by 3–5 days faster in lethally irradiated littermates. At the molecular level, this sensitization of the responsiveness to SDF-1 after exposure to CAMPs depends on the incorporation of the CXCR4 receptor into membrane lipid rafts. This phenomenon is specific for SDF-CXCR4 axis as we did not observe that this phenomenon influences S1P- and C1P-mediated in vitro chemotaxis. Overall, these changes of SDF-1, S1P, and C1P chemotactic gradients and the appearance of priming factors in the BM microenvironment and PB plasma are triggered during conditioning for transplantation by radio-chemotherapy by the induction of a proteolytic microenvironment in BM and the activation of the complement cascade (CC). Conclusions. We provide in vivo evidence that C3a and LL-37, which have primarily antimicrobial functions and are harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach based on a short ex vivo incubation of HSPCs in the graft before transplantation by CAMPs (C3a and LL-37) is of particular important in umbilical cord blood transplantations, where the number of available HSPCs in single unit for transplant is limited. This strategy is currently tested in the clinic. Disclosures:No relevant conflicts of interest to declare.

A Suppressor/Enhancer Screen in Drosophila Reveals a Role for Wnt-Mediated Lipid Metabolism in Primordial Germ Cell Migration

Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration.