S1P Levels Research Articles

Abstract 5000: Spinster 2 exports S1P, an important player in lymph node metastasis.

Abstract Sphingosine-1-phosphate (S1P), a ligand for five specific receptors, is a potent lipid mediator that plays important roles in cancer cell proliferation, migration, and cancer-induced angiogenesis/lymphangiogenesis. S1P is produced inside cells and therefore must be secreted in order to exert its effects through these receptors. We have reported that E2-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E2 important for breast cancer pathophysiology. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S1P, thus it is important to elucidate its physiological role. Although lymphatic endothelial cells contribute to regulation of S1P levels in lymph, little is known how S1P levels are maintained in the lymphatic system. Spinster 2 knockout mice were used, and the lymphatic system was examined by flow cytometry and immunofluorescent staining. Sphingolipids in the blood, lymph, and organs were determined by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We found that S1P secreted by breast cancer cells promotes tumor progression in a syngeneic model by stimulating angiogenesis and lymphangiogenesis. Interestingly, suppression of tumor S1P production resulted in less lymph node metastasis. Further, we found that serum S1P levels are elevated in breast cancer patients with lymph node metastasis. We confirmed that Spns2 can export endogenous S1P from cells and also dihydro-S1P, which is active at all cell surface S1P receptors. Moreover, Spns2-/- mice have decreased levels of both of these phosphorylated sphingoid bases in blood, accompanied by increases in very long chain ceramide species, and have defective lymphocyte trafficking. Surprisingly, levels of S1P and dihydro-S1P were increased in lymph from Spns2-/- mice as well as in specific tissues, including lymph nodes, and interstitial fluid. Moreover, lymph nodes from Spns2-/- mice have aberrant lymphatic sinuses, which appear collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is a S1P transporter in vivo that regulate not only of blood S1P but also lymph node and lymph S1P levels, which may implicate a role in lymph node metastasis. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. S.S. is supported by NIH R37GM043880, and K.T. by NIH R01CA160688 and Susan G. Komen for the Cure Investigator Initiated Research Grant. Citation Format: Masayuki Nagahashi, Eugene Y. Kim, Akimitsu Yamada, Subramaniam Ramachandran, Jeremy C. Allegood, Nitai Hait, Michael Maceyka, Sheldon Milstien, Sarah Spiegel, Kazuaki Takabe. Spinster 2 exports S1P, an important player in lymph node metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2013-5000

Biological Characterization of 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) as a Selective Sphingosine Kinase-2 Inhibitor and Anticancer Agent

In our effort to develop selective sphingosine kinase-2 (SphK2) inhibitors as pharmacological tools, a thiazolidine-2,4-dione analogue, 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145), was synthesized and biologically characterized. Biochemical assay results indicate that K145 is a selective SphK2 inhibitor. Molecular modeling studies also support this notion. In vitro studies using human leukemia U937 cells demonstrated that K145 accumulates in U937 cells, suppresses the S1P level, and inhibits SphK2. K145 also exhibited inhibitory effects on the growth of U937 cells as well as apoptotic effects in U937 cells, and that these effects may be through the inhibition of down-stream ERK and Akt signaling pathways. K145 also significantly inhibited the growth of U937 tumors in nude mice by both intraperitoneal and oral administration, thus demonstrating its in vivo efficacy as a potential lead anticancer agent. The antitumor activity of K145 was also confirmed in a syngeneic mouse model by implanting murine breast cancer JC cells in BALB/c mice. Collectively, these results strongly encourage further optimization of K145 as a novel lead compound for development of more potent and selective SphK2 inhibitors.

Identification of Sphingosine Kinases As Therapeutic Targets in B-Lineage Acute Lymphoblastic Leukemia

AbstractAbstract 1499Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SphK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SphK1. Little is known about the interaction of SphKs with intracellular signalling pathways. Here we assess the relevance of SphKs in B-lineage acute lymphoblastic leukemia (B-ALL).Gene expression signatures indicative of activation of SphK1 or SphK2 were not publicly available. Therefore we treated ALL cell lines with the SphK1 or SphK2 specific inhibitors SK1-I and ABC294640 respectively, and analysed gene expression by microarray. Although a signature for SphK1 was not obtained, two independent methods of analysis generated SphK2 gene signatures that segregated control and drug treated cell lines. The signatures consisted of 11 and 35 genes and included reduced expression of the NF-κB inhibitor TRIB3 (NF-κB activity is commonly up regulated in ALL), the C/EBP family transcription factor DDIT3, the protein phosphatase 1 regulatory subunit PPP1R15A and the sphingolipid/cholesterol transporter ABCA1, all validated by quantitative RT-PCR. These gene signatures were used to interrogate a large publicly available gene expression dataset (GSE28497) obtained from pediatric ALL patients at the time of diagnosis. SphK2 activity signatures were more highly expressed in ALL samples (p=0.001 and p=0.027 for the smaller and larger signatures respectively) than normal B-cell progenitors. Although SPHK1 or SPHK2 genes were not over expressed in this dataset, SphK1 protein levels were increased in ALL cell lines and in patient samples.The importance of SphK1 and SphK2 in the development of ALL was examined by transducing B-cell progenitors isolated from WT, SphK1−/− or SphK2−/− mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated wild type C57BL/6 mice. Twenty-two of 29 mice receiving cells from WT animals developed ALL, with a mean survival of 52.6 days (95% CI 42.3–62.8 days). Mice receiving cells lacking Sphk1 or Sphk2 had a significantly reduced incidence of ALL development with 14 of 30 (mean survival 71.6 days, 95% CI 60.5–82.8 days) and 16 of 29 animals (mean survival of 68.7 days, 95% CI 57.9–79.5 days) respectively (p=0.001). Lymphoblasts with a B-cell progenitor phenotype (B220+, CD19+, IgM−, CD11b−) were present in blood films and livers from leukemic mice and cells recovered from these animals produced a rapidly fatal ALL in secondary recipients. The presence of BCR/ABL and the expected deletion of Sphk1 or Sphk2 were confirmed by PCR in all murine leukemias examined.We have previously reported that inhibitors of SphK1 and/or SphK2 inhibit proliferation and induce cell death in ALL cell lines, and that these agents can synergize with imatinib in Ph+ ALL cell lines. We have furthered these studies to demonstrate that patient derived ALL cells similarly respond to SphK1 and SphK2 inhibition in vitro. The SphK2 inhibitor ABC294640 (100mg/kg) reduced plasma S1P levels in mice consistent with our previous reports of reduced ALL burden following ABC294640 treatment, and using a NOD/SCID γc−/− xenograft model of human ALL we have now shown that ABC294640 extends survival of ALL-bearing mice (p=0.0012 for Ph− xenograft) and further extends the survival when combined with imatinib treatment in mice engrafted with a human Ph+ ALL patient sample (p=0.044).This is the first report to suggest that sphingosine kinase 2 activity is increased in ALL, providing support for targeting SphK2 as a therapeutic strategy. Loss of SphKs reduces the incidence of ALL in a murine model of BCR/ABL-driven disease and ABC294640 reduces disease and extends survival in a human xenograft model of ALL. This compound synergizes with a number of potential therapeutic agents and further extends survival in a xenograft model of Ph+disease when combined with imatinib. This has potential to translate into a useful anti-leukemic strategy. Disclosures:No relevant conflicts of interest to declare.

Characterization of Isoenzyme-Selective Inhibitors of Human Sphingosine Kinases

Sphingosine kinases (SKs) are promising new therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate. The functions of the two SK isoenzymes, SK1 and SK2, are not redundant, with genetic ablation of SK2 having more pronounced anticancer effects than removal of SK1. Although several small molecule inhibitors of SKs have been described in the literature, detailed characterization of their molecular and cellular pharmacology, particularly their activities against human SK1 and SK2, have not been completed. Computational modeling of the putative active sites of SK1 and SK2 suggests structural differences that might allow isozyme-selective inhibitors. Therefore, we characterized several SK-inhibitory compounds which revealed differential inhibitory effects on SK1 and SK2 as follows: SKI-II and ABC294735 are SK1/2-dual inhibitors; CB5468139 is a SK1-selective inhibitor; and ABC294640 is a SK2-selective inhibitor. We examined the effects of the SK inhibitors on several biochemical and phenotypic processes in A498 kidney adenocarcinoma cells. The SK2-selective inhibitor ABC294640 demonstrated the most pronounced effects on SK1 and SK2 mRNA expression, decrease of S1P levels, elevation of ceramide levels, cell cycle arrest, and inhibition of proliferation, migration and invasion. ABC294640 also down-regulated the expression or activation of several signaling proteins, including STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to responses to the SK1/2-dual inhibitors. Overall, these results suggest that inhibition of SK2 results in stronger anticancer effects than does inhibition of SK1 or both SK1 and SK2.

Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: Functional differences between sphingosine kinase 1a and 1b

Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel ‘see-saw’ model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed ‘see-saw’ model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.

Abstract 4768: Design and development of selective sphingosine kinase 2 inhibitors

Abstract Sphingosine-1-phosphate (S1P) has been recently implicated to play a vital role in regulating a variety of physiological and pathological processes including proliferation, differentiation, migration, and cell survival. S1P is produced endogenously by sphingosine kinases (SKs) and the balance of the levels of S1P, its precursor sphingosine, and ceramide, referred to as the “sphingosine rheostat,” is important for normal cellular functions. To date, two isoforms of SPKs have been reported, sphingosine kinase 1 (SK1) and sphingosine kinase 2 (SK2). Many efforts have been invested to study the roles of SK1 and SK1 has been indicated in the regulation of cancer progression and development. However, much is still remain unknown for SK2 and current results of the function of SK2 are complex and controversial. This is also reflected in the development of SK inhibitors. Several potent selective SK1 inhibitors have been reported and tested as potential cancer treatment agents. However, no potent and selective SK2 inhibitors have been developed to date. Therefore, there is an urgent need to develop SK2 selective inhibitors as pharmacological tools to help unravel the roles of SK2. Recently, we discovered that a series of thiazolidine-2,4-dione analogs selectively inhibit SK2 and one lead compound was identified to inhibit SK2 with sub-µM potency but no activity to SK1 with a concentration up to 10 µM. Herein, we will report the structure activity relationship (SAR) studies of this newly identified lead SK2 inhibitor to optimize its potency and selectivity towards SK2. Furthermore, these analogs will be biologically characterized in human leukemia U937 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4768. doi:1538-7445.AM2012-4768

Sphingosine Kinase And Sphingosine-1-Phosphate Receptors: Novel Therapeutic Targets Of Rheumatoid Arthritis?

Rheumatoid arthritis (RA) is a chronic, destructive, autoimmune joint disease characterized by elevated levels of proinflammatory cytokine production. Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate. Synovial fluid of RA patients exhibits significantly higher levels of S1P than their non-inflammatory osteoarthritis counterparts. SphK blockade suppresses cytokines and MMP-9 release in RA peripheral blood mononuclear cells. In addition, downregulation of SphK1 either through a specific siRNA approach or transgenic human TNF-α SphK1-deficient mice (hTNF-α/SphK1(-/-)) exhibit significantly less synovial inflammation and joint pathology. By contrast, SphK2 modulation leads to disease exacerbation. These results clearly demonstrate that such anti- and proinflammatory potential of SphK1/2 modulation may alter the outcome in RA synovitis and raises the possibility that drugs that specifically target SphK1 activity may play a beneficial role in the treatment of RA and other autoimmune rheumatic diseases.

Expression of sphingosine 1-phosphate receptor 4 and sphingosine kinase 1 is associated with outcome in oestrogen receptor-negative breast cancer

Background:We previously reported that sphingosine 1-phosphate receptor 4 (S1P4) is expressed and stimulates the ERK-1/2 pathway via a human epidermal growth factor receptor 2 (HER2)-dependent mechanism in oestrogen receptor-negative (ER−) MDA-MB-453 breast cancer cells.Methods:Clinical relevance of S1P4 and sphingosine kinase 1 (SK1, which catalyses the formation of S1P) was assessed in a cohort of 140 ER− breast tumours by immunohistochemistry (IHC) and the weighted histoscore method. Additional evidence for a functional interaction between S1P4 and SK1 and between HER2 and SK1 was obtained using MDA-MB-453 cells.Results:High S1P4 expression is associated with shorter disease-free (P=0.014) and disease-specific survival (P=0.004), and was independent on multivariate analysis. In addition, patients with tumours that contain high and low levels of SK1 and S1P4, respectively, have a significantly shorter disease-free survival (P=0.043) and disease-specific survival (P=0.033) compared with patients whose tumours contain both low S1P4 and SK1 levels. In addition, high tumour expression of SK1 was significantly associated with shorter disease-specific survival (P=0.0001) in patients with HER2-positive tumours. Treatment of MDA-MB-453 cells with the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) reduced the basal and S1P/S1P4-induced activation of ERK-1/2 and altered HER2 trafficking in these cells.Conclusion:These findings highlight an important role for S1P4 and SK1 in ER− breast cancer progression.

Sphingosine-1-Phosphate Produced by Sphingosine Kinase 1 Promotes Breast Cancer Progression by Stimulating Angiogenesis and Lymphangiogenesis

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings show that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.

Discovery and Evaluation of Inhibitors of Human Ceramidase

The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analogue (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analogue, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell-cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development and provide the first nonlipid inhibitors of human ceramidase activity.

The Sphingosine-1-phospate receptor 1 mediates S1P action during cardiac development

BackgroundSphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P1-5R, with S1P1R having the highest expression level in the developing heart. S1P1R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT).ResultsWe determined, by real-time PCR analysis, that S1P1R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P1R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P1,3,4,5R agonist) or KRP203 (an S1P1R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P1R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P1R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P1R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis.ConclusionsThese data indicate that S1P1R is the primary mediator of S1P action in AV canal cultures and that loss of S1P1R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.

Simultaneous determination of sphingosine and sphingosine 1-phosphate in biological samples by liquid chromatography–tandem mass spectrometry

d-erythro-sphingosine (Sph) and its phosphorylated product, d-erythro-sphingosine 1-phosphate (S1P) are sphingolipids mediating numerous cellular processes. Imbalance of Sph/S1P levels contributes to many diseases. Given the interconversion of these two opposing signaling molecules, it is essential to examine their levels simultaneously. In the present study, we developed a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to simultaneously quantify the levels of Sph and S1P in biological samples using C17-Sph and C17-S1P as internal standards. With one step of methanol-induced protein precipitation, each sample was subjected to LC–MS/MS analysis using positive electrospray ionization under selected reaction monitoring mode. The running time was within 4 min with a simple mobile phase consisting of methanol–0.1% formic acid (95:5, v/v) at a flow rate of 0.2 mL/min. Standard curves were linear over ranges of 1–100 ng/mL for Sph and 0.1–10 ng/mL for S1P with correlation coefficient ( r 2) greater than 0.997. The lower limit of quantifications (LLOQs) were 1 ng/mL for Sph and 0.1 ng/mL for S1P. The intra-batch and inter-batch precision was less than 15% for all quality control samples. The recoveries of the method were found to be 76.36–89.84%. The method was applied to simultaneously determine the Sph and S1P levels in mouse kidney, human plasma, and HEK 293 cells treated with tumor necrosis factor-α (TNF-α) and N,N-dimethylsphingosine (DMS). The S1P levels increased in cells treated with TNF-α whereas decreased in cells treated with DMS. These results indicated that this new LC–MS/MS method was rapid, sensitive, specific and reliable to quantify Sph and S1P levels in biological samples simultaneously.

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C(14)-C(26) N-acyl part) of "normal" sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C(n)-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C(16)-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C(16)-LacCer (10.0 µM) and the abundant HexCer was C(24)-HexCer (2.5 µM). The abundant Cer, C(24)-Cer (4.0 µM), was not influenced by fasting; however, levels of C(16)-C(20) Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL(3) was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.

Novel Mechanistic Insight Into Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs): Complement Cascade and Membrane Attack Complex Activated in Bone Marrow Sinusoids During Mobilization Release From Erythrocytes Sphingosine-1 Phosphate – An Underappreciated Chemoattractant Executing Egress of HSPCs.

Abstract 31We reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) and that CC clevage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40). We also reported that C5 cleavage fragments play a crucial role in the mobilization process by: i) inducing proteolytic activity in the BM environment; ii) directing BM egress of granulocytes that “pave a road” for HSPCs; and iii) inducing secretion of cationic peptides from activated granulocytes that prime HSPC egress (Leukemia 2009; in press). In this study, we sought to determine which major chemottractant is present in PB that is responsible for egress of HSPCs and whether activation of CC plays some role in its level/expression. We noticed that plasma derived from normal and mobilized PB strongly chemoattracts murine and human HSPCs. This chemotactic effect was not dependent on plasma SDF-1 levels because: i) it occurs unaffectedly in the presence of CXCR4 antagonist AMD3100; ii) it was still robust to heat-inactivated sera; and iii) ELISA studies revealed negligible concentrations of SDF-1, which did not correlate with good or poor mobilizer status. However, to our surprise, we noticed that plasma isolated from G-CSF-mobilized mice and patients contains traces of free hemoglobin, which suggests some level of hemolysis occurs in mobilized PB. As such, we performed chemotactic assays in the presence of different concentrations of lysed erythrocytes and noticed that such diluted lysates are potent chemoattractants for HSPCs. The chemotactic activities of these lysates were resistant to heat inactivation similarly as patient sera. Based on this, we focused on S1P, a thermo-resistant lipid that, as reported, chemoattracts HSPCs and whose major reservoirs are erythrocytes (FASEB J 2007:21;1202). In fact we found by ELISA that S1P level increases during mobilization in PB and that SP1 is the most potent chemoattractant for BM-residing HSPCs, much stronger than SDF-1 - if both compounds are compared in physiologically relevant concentrations. Furthermore, activation of S1P receptors on BM-derived HSPCs augmented responsiveness to SDF-1 gradient up to 50%. However, these chemotactic effects of S1P were not visible for previously mobilized PB or umbilical cord blood HSPCs, which we explain by a fact that these mobilized cells are already desensitized to S1P gradient. Therefore, we propose the following scenario. First, a mobilizing agent (e.g., G-CSF) induces activation of CC in BM that subsequently contributes to the release of protelolytic enzymes from granulocytes that perturb SDF-1-CXCR4/VLA-4-VCAM1 interactions and stimulate egress of activated granulocytes from BM that “pave a road” for egress of HSPCs. Simultaneously, the final product of CC activation (C5b-C9), the membrane attack complex (MAC), induces in BM sinusoids the release of S1P from erythrocytes. S1P accumulating in BM sinusoids and cationic peptides released from activated granulocytes, but not changes in plasma SDF-1 levels, are crucial executors of HSPCs egress from BM into PB. Thus, our results provide novel evidence that CC activation/membrane attack complex (MAC)-induced elevated plasma S1P level is essential for egress/mobilization of HSPCs. Disclosures:No relevant conflicts of interest to declare.

The Enigma of Sphingosine 1-Phosphate Synthesis

See related article, pages 669–676 Sphingosine 1-phosphate (S1P) is well known as a key sphingolipid messenger molecule in the cardiovascular system, yet many fundamental features of its synthesis, transport, and action remain enigmatic. Indeed, the very name “sphingolipid” was coined to reflect the mysterious characteristics of this class of lipids. It is now well established (through the work of Timothy Hla and of many others subsequently) that S1P binds to and activates a family of G protein–coupled S1P receptors located in vascular endothelial cells, cardiac myocytes, blood platelets, and vascular smooth muscle cells (among other cell types) and elicits a broad range of physiological responses (reviewed elsewhere1). In vascular endothelial cells, S1P elicits such diverse responses as cell survival, proliferation, angiogenesis, cell migration, permeability, and endothelial NO synthase activation. The intracellular signaling pathways stimulated by S1P have been extensively characterized, yet the cellular origins of S1P and the pathways for its transport and action in cardiovascular tissues remain incompletely understood. The concentration of S1P in normal human plasma is within the range of several hundred nanomolar, and this amphipathic lipid is highly protein bound, mostly to HDL and albumin.2 Importantly, S1P receptors bind their ligand with an affinity in the nanomolar range; therefore, many important questions remain. What controls S1P binding to plasma proteins? Where does all this plasma S1P come from? Important clues to the latter question are provided in a recent report from the laboratory of Timothy Hla, published in this issue of Circulation Research 3 and reporting the novel and important discovery: the vascular endothelium is an important source of plasma S1P through the actions of sphingosine kinases (SphKs) in endothelial cells. For many years, conventional wisdom held that blood platelets represent the principal source for S1P in the plasma. Elegant studies by Igarashi …

Upregulation of the Human Alkaline Ceramidase 1 and Acid Ceramidase Mediates Calcium-Induced Differentiation of Epidermal Keratinocytes

Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs. haCER1 is the human ortholog of mouse alkaline ceramidase 1 that we previously identified. haCER1 catalyzed the hydrolysis of very long-chain ceramides to generate sphingosine (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER1 in HEKs decreased the levels of D-e-C(24:1)-ceramide and D-e-C(24:0)-ceramide but elevated the levels of both SPH and its phosphate (S1P), whereas RNA interference-mediated knockdown of haCER1 caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER1 overexpression, Ca2+(o) increased the levels of SPH and S1P, and this was attenuated by haCER1 knockdown. haCER1 knockdown also inhibited the Ca2+(o)-induced growth arrest of HEKs and the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Ca2+(o); and its knockdown attenuated the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. These results strongly suggest that upregulation of haCER1 and AC mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs by generating SPH and S1P.

Shp-2 tyrosine phosphatase is required for hepatocyte growth factor-induced activation of sphingosine kinase and migration in embryonic fibroblasts

Shp-2, a ubiquitously expressed protein tyrosine phosphatase containing two Src homology 2 domains, plays an important role in integrating signaling from the cell surface receptors to intracellular signaling mechanisms. Previous studies have demonstrated that the Shp-2 is involved in hepatocyte growth factor (HGF)-induced cell scattering. Here we report that Shp-2 is required for the HGF-induced activation of sphingosine kinase-1 (SPK1), a highly conserved lipid kinase that plays an important role in cell migration. Loss-of-function mutation of Shp-2 did not affect the expression of SPK1, but resulted in its inactivation and the blockage of HGF-induced migration in embryonic fibroblasts. Reintroduction of functional wild type (WT) Shp-2 into the mutant cells partially restored SPK1 activation, and overexpression of SPK1 in these mutant cells enhanced HGF-induced cell migration. Inhibition of expression or activity of SPK1 in WT cells markedly decreased intracellular S1P levels and HGF-induced cell migration. Furthermore, we found that Shp-2 co-immunoprecipitated with SPK1 and c-Met in embryonic fibroblasts. These studies suggest that Shp-2 is an SPK1-interacting protein and that it plays an indispensable role in HGF-induced SPK1 activation.