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Previous literature suggests that sphingolipids may impact systemic coagulation and platelet aggregation, thus modulating the risks of thrombotic events. The goal of this investigation was to evaluate the role of serum sphingolipids on intrinsic platelet function to assess whether pharmacologic manipulation of sphingolipid metabolites would impact platelet aggregability. C57BL/6J mice were injected with either normal saline, 1 mg/kg FTY720 (synthetic sphingosine-1-phosphate [S1P] receptor analog), or 5 mg/kg SLM6031434 (sphingosine kinase two inhibitor). Mice were sacrificed at 6 h and whole blood (WB) was collected for impedance aggregometry assessing platelet responsiveness to arachidonic acid or adenosine diphosphate. Ex vivo studies utilized WB or platelet-rich plasma that was pretreated with S1P, FTY720, amitriptyline, or d-sphingosine then analyzed by aggregability and flow cytometry for platelet and platelet-derived microvesicle characteristics. FTY720 and SLM6031434 pretreated induced similar arachidonic acid and adenosine diphosphate–mediated platelet aggregation as controls. Ex vivo WB and platelet-rich plasma treatment with S1P, FTY720, amitriptyline and d-sphingosine did not impact platelet aggregation. The percentages of CD41+, CD62P+ and CD41+/ceramide+, CD62P+/ceramide + platelets, and platelet-derived microvesicle were not significantly different between amitriptyline-treated and normal saline-treated cohorts. Sphingolipid modulating agents, such as FTY720, SLM6031434, S1P, amitriptyline, ceramide, and d-sphingosine do not appear to independently impact platelet aggregation in murine models. [ABSTRACT FROM AUTHOR]

• Follicle-stimulating hormone and luteinizing hormone increased the synthesis of sphingosine-1-phosphate in folicular cells by activating sphingosine kinases-1. • S1P is an important mediator of FSH and LH proliferation, survival and viability effects in bovine follicular cells. • Follicle-stimulating hormone and luteinizing hormone increased the synthesis of sphingosine-1-phosphate in folicular cells via, mainly protein kinase C. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective G s protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting G q proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 2 (SphK2) inhibitors are developed for tumor therapy as considering its anti-tumor effect. Many studies also explored SphK2 modulated glucose and lipid homeostasis, which extended its potential function for metabolic diseases therapy. In this study, we discovered a significant reduction of hepatic lipid accumulation as well as recovery of liver function in ob/ob mice with intraperitoneal injection of K145. Also, db/db mice received K145 showed improvement of both NALFD and hyperglycemia. We furtherly analyzed the genes associated with lipid metabolism and found a remarkable decreased expression of lipogenic genes including FAS, ACC1 and SREBP1c whereas elevated mitochondrial fatty acid β-oxidation (FAO) related genes expression including CPT1A, MCAD, LCAD, PPAR-α, UCP2. Consistent to in vivo study, in vitro study also confirmed the role of K145 in decreasing lipid accumulation in human HL7702 cells, while inhibiting FAS, ACC1 and SREBP1c mRNA expression. It indicated a possible mechanism of K145 induced improvement of hepatic lipid accumulation partly via inhibition of lipigenesis. Our study suggested a promising role of K145 in drug development for NAFLD and diabetes therapy. • An improvement of K145 on hepatic lipid accumulation in both ob/ob and db/db mice. • A remarkable regulation of hepatic lipid metabolism related genes expression of ob/ob mice and human HL7702 cells by K145 addition. [ABSTRACT FROM AUTHOR]

Anoikis plays a crucial role in the progression, prognosis, and immune response of lung adenocarcinoma (LUAD). However, its specific impact on LUAD remains unclear. In this study, we investigated the intricate interplay of nesting apoptotic factors in LUAD. By analyzing nine key nesting apoptotic factors, we categorized LUAD patients into two distinct clusters. Further examination of immune cell profiles revealed that Cluster A exhibited greater infiltration of innate immune cells than did Cluster B. Additionally, we identified two genes closely associated with prognosis and developed a predictive model to differentiate patients based on molecular clusters. Our findings suggest that the loss of specific anoikis-related genes could significantly influence the prognosis, tumor microenvironment, and clinical features of LUAD patients. Furthermore, we validated the expression and functional roles of two pivotal prognostic genes, solute carrier family 2 member 1 (SLC2A1) and sphingosine kinase 1 (SPHK1), in regulating tumor cell viability, migration, apoptosis, and anoikis. These results offer valuable insights for future mechanistic investigations. In conclusion, this study provides new avenues for advancing our understanding of LUAD, improving prognostic assessments, and developing more effective immunotherapy strategies. [Display omitted] • The molecular characteristics associated with anoikis-related genes in lung adenocarcinoma were developed. • Anoikis-related gene prognostic model predicts lung adenocarcinoma prognosis and immunotherapy efficacy. • SLC2A1 and SPHK1 were highly expressed in lung adenocarcinoma. • SLC2A1 and SPHK1 knockdown decreased migration and increased anoikis in A549 and H1650 cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • We report a virtual screening, design, synthesis and activity of eight new inhibitors of SphK1. • Four compounds displayed anticancer activity against different tumour cell lines. • Combined techniques: docking, MD simulations and QTAIM analysis were used. • An exhaustive study of molecular interactions for the different ligands was performed. • Results provide information for the design of new inhibitors of SphK1. We report here the virtual screening design, synthesis and activity of eight new inhibitors of SphK1. For this study we used a pre-trained Graph Convolutional Network (GCN) combined with docking calculations. This exploratory analysis proposed nine compounds from which eight displayed significant inhibitory effect against sphingosine kinase 1 (SphK1) demonstrating a high level of efficacy for this approach. Four of these compounds also displayed anticancer activity against different tumor cell lines, and three of them (5), (6) and (7) have shown a wide inhibitory action against many of the cancer cell line tested, with GI 50 below 5 µM, being (5) the most promising with TGI below 10 µM for the half of cell lines. Our results suggest that the three most promising compounds reported here are the pyrimidine-quinolone hybrids (1) and (6) linked by p -aminophenylsulfanyl and o -aminophenol fragments respectively, and (8) without such aryl linker. We also performed an exhaustive study about the molecular interactions that stabilize the different ligands at the binding site of SphK1. This molecular modeling analysis was carried out by using combined techniques: docking calculations, MD simulations and QTAIM analysis. In this study we also included PF543 , as reference compound, in order to better understand the molecular behavior of these ligands at the binding site of SphK1.These results provide useful information for the design of new inhibitors of SphK1 possessing these structural scaffolds. [ABSTRACT FROM AUTHOR]

Although previous experiments have implicated sphingosine-1-phosphate (S1P) as a links between immune reactions and cancer progression, the exact mechanism of this interaction has not comprehensively studied in clinical human samples. This study sought to evaluate the S1P regulation by sphingosine kinase 1 (SPHK1), an S1P-producing enzyme, in the immunity/immuno-reactivity of clinical human breast cancer surgical specimens. S1P levels were examined in tumor, peritumoral, and normal human breast samples using mass spectrometry. Genomics Data Commons data portal of The Cancer Genome Atlas cohort was used to assess the expression of S1P-related and immune-related genes. S1P levels were significantly higher in tumor samples compared to peritumoral (P < 0.05) or normal human breast samples (P < 0.001). SPHK1 gene expression was elevated in tumoral samples compared to normal breast samples (P < 0.01). Furthermore, the elevated expression of SPHK1 in breast cancer tissue was associated with an increased expression of the different kinds of immune-related genes, such as CD68 , CD163 , CD4 , and FOXP3 (forkhead box P3), in HER2-negative breast cancer. Network analysis showed the central role of SPHK1 in the interaction of S1P signaling and expression of immune cell–related proteins. We demonstrated that S1P is mainly produced by tumor tissue, rather than peritumoral tissue, in breast cancer patients. Our data revealed the involvement of S1P signaling in the regulation of immune-related genes, suggesting the links between S1P and complicated immune–cancer interactions in breast cancer patients. [ABSTRACT FROM AUTHOR]

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P 1 –S1P 5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 μg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 μL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 μg/kg, i.p.), an antagonist for S1P 1 /S1P 3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1 , SK2 , S1P 1 , and S1P 3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling. • RvD1 attenuated IR-induced serum HMGB1 release. • RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. • RvD1 attenuated the decrease in hepatic SK1 , SK2 , S1P 1 , and S1P 3 mRNA induced by IR. • S1P 1 /S1P 3 inhibitor abolished the effects of RvD1 in serum HMGB1 release. [ABSTRACT FROM AUTHOR]

Abstract Background Lymphatic spread is the main mode of progression of esophageal squamous cell carcinoma (ESCC). Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator, which produced by sphingosine kinase 1 (SphK1) activated by phosphorylation. The SphK1-S1P axis has a crucial role in lymphangiogenesis. However, the significance of phospho-SphK1 (pSphK1) in the progression of ESCC has not been fully investigated. Materials and methods We evaluated pSphK1 expression in 92 surgically resected tumor tissues of ESCC by the immunohistochemistry. Fifty-nine (64%) patients with moderate or strong expression and 33 (36%) with negative or weak expression were classified in the pSphK1-high and pSphK1-low groups, respectively. Results Higher pathological N category (pN) was more frequently observed in the pSphK1-high group (P < 0.01). The median number of lymph node metastasis (pSphK1-high: 2 versus pSphK1-low: 0; P < 0.01), the proportion of patients with lymphatic invasion (69% versus 18%; P < 0.01) and that with intramural metastasis (27% versus 3%; P < 0.01) were significantly higher in the pSphK1-high group. The presence of lymphatic invasion (odds ratio [OR] 5.63; P < 0.01) and pN1–3 (OR 3.26; P = 0.04) were independently associated with high pSphK1 expression. The 5-y overall survival rate of the pSphK1-high group was significantly lower than that of the pSphK1-low group (50.8% versus 67.3%; P = 0.01). High pSphK1 expression was not identified as a significant independent prognostic factor. Conclusions We provide the first evidence of the association between high expression of pSphK1 and both lymphatic spread and patient outcomes in ESCC. [ABSTRACT FROM AUTHOR]

Abstract Background Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. Materials and methods S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally. Results S1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells. Conclusions S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Colitis-associated cancer (CAC) is the most serious complication of inflammatory bowel disease. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that is generated by sphingosine kinase 1 (SphK1) and is known to play an important role in inflammation and cancer progression. Moreover, SphK1 and S1P act as upstream mediators of proinflammatory cytokine interleukin 6 (IL-6) and signal transducer and activator of transcription-3 (STAT3). We hypothesized that the expression levels of phosphorylated SphK1 (pSphK1), phosphorylated STAT3 (pSTAT3), and IL-6 are universally higher in CAC patients than in sporadic colorectal cancer (CRC) patients because all of these factors are associated with inflammation. In this study, we determined the expression levels of pSphK1 in patients with sporadic CRC and CAC and clarified the importance of S1P in CAC patients. Materials and methods We randomly selected 10 sporadic CRC patients and 10 CAC patients who underwent curative resection, and we examined their surgical specimens by immunohistochemistry. We determined the expression levels of pSphK1, pSTAT3, and IL-6 in these samples. Results We found pSphK1 expression to be more prevalent in CAC patients (P = 0.019) and to have a higher immunohistochemistry score (P = 0.005) than in sporadic CRC patients. However, the expression of pSTAT3 and IL-6 did not differ between the patient groups. Conclusions To our knowledge, this is the first report comparing pSphK1 expression levels in CAC with those in sporadic CRC. The high levels of pSphK1 expression in CAC suggest an important role of S1P in the disease process of CAC. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate is synthesized by two sphingosine kinases, cytosolic SK1 and nuclear SK2 but SK2 expression was much higher than SK1in mouse skin fibroblasts. However, in SK2 −/− cells, SK1 expression was markedly increased to SK2 levels whereas in SK1 −/− cells, SK2 expression was unaffected. Ceramide, glucosylceramide and sphingosine levels were all increased in SK1 −/− but less so in SK2 −/− cells and S1P levels were not significantly reduced in either SK1 −/− or SK2 −/− cells. Greatly increased Ceramide glucosyltransferase expression was observed in SK1 −/− cells but less so in SK2 −/− cells suggested a role in drug resistance. SK2 −/− cells grew faster than control and SK1 −/− . The cell division gene PCNA was significantly overexpressed in SK2 −/− cells, suggesting a cross regulation between SKs and Ceramide glucosyltransferase. [ABSTRACT FROM AUTHOR]

The activity of ABC294640, a small-molecular sphingosine kinase 2 (SphK2) inhibitor, in human skin squamous cell carcinoma (SCC) cells was tested in this study. SphK2 mRNA and protein are expressed in established (A431 cheilocarcinoma cell line) and primary human skin SCC cells. ABC294640 dose-dependently inhibited survival, cell cycle progression and proliferation of skin SCC cells. Furthermore, ABC294640 induced caspase-3/-9 and apoptosis activation in skin SCC cells. The SphK2 inhibitor was however non-cytotoxic to SphK2-null skin melanocytes, keratinocytes and fibroblasts. ABC294640 induced ceramide accumulation, sphingosine-1-phosphate (S1P) reduction, Akt-S6K1 inhibition and JNK activation in skin SCC cells. Conversely, its cytotoxicity against SCC cells was largely attenuated by co-treatment of S1P, the Akt activator SC79, and the JNK inhibitor SP600125. In vivo , ABC294640 oral administration inhibited A431 xenograft tumor growth in nude mice. Akt-S6K1 inhibition and JNK activation were observed in ABC294640-treated tumors. Collectively, ABC294640 efficiently inhibits human skin SCC cell growth in vitro and in vivo . [ABSTRACT FROM AUTHOR]

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor β (TGF-β) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-β signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 μM sphingosine-1-phosphate (S1P), 5 μM SPHK I 2 (I 2 ; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-β1, or 0.1 ng/mL TGF-β3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-βs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-β binding proteins) expression when stimulated with S1P or I 2. However, LTBP1 was significantly upregulated via stimulation of TGF-β1 and TGF-β3, whereas LTBP2 was significantly upregulated only with TGF-β3 stimulation. Significant downregulation of TGF-β receptor II (TGF-βRII) following S1P stimulation but significant upregulation following I 2 stimulation was observed. Following TGF-β1, S1P, and I 2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I 2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I 2 only significantly downregulated FAK. Exogenous TGF-β3 caused significant upregulation of AKT. Interestingly, both I 2 and TGF-β3 caused significant downregulation of JNK expression. Lastly, TGF-β1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-β3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-β in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-β signaling modulators as novel therapeutics in corneal fibrosis. • TGF-β3 modulates LTBP1 and LTBP2 expression, where TGF-β1 only modulates LTB1 expression. • TGF-βRII is downregulated by S1P but upregulated by I 2. • TGF-β3 and I 2 both downregulated JNK expression. [ABSTRACT FROM AUTHOR]

Oxygen glucose deprivation (OGD)/re-oxygenation (OGDR) causes damages to neuronal cells. Sphingosine kinase 2 (SphK2) expression could exert neuroprotective functions. Here, we aim to induce SphK2 expression via inhibiting the anti-SphK2 microRNA: microRNA-613 (“miR-613”). In both SH-SY5Y neuronal cells and primary murine hippocampal neurons, transfection of the miR-613's specific inhibitor, antagomiR-613 (“antamiR-613”), induced miR-613 depletion and SphK2 expression. Reversely, forced over-expression of miR-613 caused SphK2 downregulation in SH-SY5Y cells. OGDR-induced cytotoxicity in neuronal cells was largely attenuated by antamiR-613. SphK2 is required for antamiR-613-induced actions in neuronal cells. SphK2 knockdown (by targeted-shRNAs) or inhibition (by its inhibitor ABC294640) almost completely abolished antamiR-613-mediated neuroprotection against OGDR. Further studies showed that OGDR-induced reactive oxygen species (ROS) production, lipid peroxidation, and DNA damages in SH-SY5Y cells were largely attenuated by antamiR-613, but were intensified by miR-613 expression. Taken together, we conclude that antamiR-613 protects neuronal cells from OGDR probably via inducing SphK2 expression. [ABSTRACT FROM AUTHOR]

Berberine (BBR) has shown neuroprotective properties. The present study aims to investigate the effects of BBR on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), and SphK1/S1P signaling, which plays a key role in MS. EAE was induced in mice, followed by treatment with BBR at 50, 100, or 300 mg/kg/d. Neurophysiological function was evaluated daily; inflammation, cell infiltration, and the severity of demyelination were also examined. The SphK1, SphK2, and S1P levels in the animals and primary astrocyte culture were measured. We found that treatment with BBR reduced the loss of neurophysiological function and the degree of demyelination. Moreover, BBR was associated with a decrease in SphK1 and S1P levels both in the animals and in culture. These results indicated that BBR suppresses demyelination and loss of neurophysiological function by inhibiting the SphK1/S1P signaling pathway. The use of BBR as a treatment of MS warrant further exploration. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related deaths globally. Although 5-Fluorouracil (5-FU) is used as the first choice treatment for advanced HCC, it exerts poor efficacy and is associated with acquired and intrinsic resistance. Sphingosine kinases (Sphk) 1 and 2 play tumour-promoting roles in different cancer types including HCC and thus represent promising pharmacological targets. In the present study, we have investigated for the first time the anticancer efficacy and underlying molecular mechanisms of combined administration of 5-FU and dual Sphk1/Sphk2 inhibitor SKI-II (4-[[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino]phenol) in HepG2 hepatocellular carcinoma cells. Here, we report that co-administration of 5-FU and SKI-II at low sub-toxic concentrations of 20 μM and 5 μM, respectively, synergistically inhibit cell proliferation, markedly reduce cell migration and the clonogenic survival, and increase apoptosis induction in HepG2 cells. Additional Western blot analyses have shown that possible mechanisms underlying enhanced sensitivity to 5-FU induced by dual Sphk 1/2 inhibition could include abrogation of FAK-regulated IGF-1R activity and down-regulation of osteopontin expression culminating in the inhibition of NF-κB activity and its downstream signalling mediated by sirtuin 1 and p38 MAPK. Our results clearly show that pharmacological blockade of both Sphk isoforms represents a promising strategy to boost the anti-tumour efficacy of 5-FU and provide a rationale for further in vivo studies into the possible use of SKI-II inhibitor as an adjunct to 5-FU treatment in HCC. [ABSTRACT FROM AUTHOR]

Necrosis- and ethylene-inducing proteins are effector molecules of microorganisms able to induce cell death in plant tissues and/or ethylene biosynthesis. The fungus Moniliophthora perniciosa , which causes the witches' broom disease in Theobroma cacao , contains five genes that encode these proteins (MpNep1-5) in its genome. Among these, MpNep2 is the most expressed Nep during disease's development, especially in the necrotic phase. Although widely studied, little is known about the mechanisms by which these proteins induce cell death. In this perspective, the present study aimed to identify potential Mp NEP2 target proteins in protein extracts of Theobroma cacao (genotype Catongo) and propose, from the results achieved, mechanisms by which Mp NEP2 can induce the process of cell death. Molecular targets captured in vitro by r Mp NEP2 immobilized on CNBr-Sepharose were identified by ms/ms. Candidate targets were identified as an Auxin Response Factor, Sphingosine Kinase and a Formin like protein. These proteins are known to participate in important processes in primary metabolism, molecular function and regulation of the plant's response. The targets: Mp NEP2 interactions were validated in silico. We discussed the different signaling pathways, membrane modulation and cell cytoskeleton, by which Mp NEP2 can act and induce responses in the plant that leads to necrosis. [Display omitted] • For the first time the possible molecular mechanisms behind cell death induced by NEP2, an elicitor secreted by M. perniciosa , are described. • NEP2 interacts with an Auxin Response Factor, a Sphingosine Kinase and a Formin-like protein during the infection process. • NEP2 interaction with target proteins modulates signaling pathways and trigger cell modifications that leads to cell death. [ABSTRACT FROM AUTHOR]

Background There are no effective treatments for pancreatic cancer peritoneal carcinomatosis (PC) or cancer dissemination in abdominal cavity. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays critical roles in cancer progression. We reported that SphK1, but not SphK2, is responsible for S1P export from breast cancer cells and recently discovered that S1P is linked to inflammation and cancer in colitis-associated cancer progression. Given the fact that inflammation is known to be essential for the establishment and progression of PC, we hypothesized that SphK1 in the host animals is involved in progression of pancreatic cancer PC. Methods Murine pancreatic adenocarcinoma panc02-luc cells were intraperitoneally injected into wildtype or SphK1 knockout (KO) mice to generate a syngeneic PC model. Cell proliferation and apoptosis were determined by Ki67 and TUNEL staining, respectively. Results All the animals developed panc02-luc PC. SphK1 KO mice developed significantly less tumor burden, less total tumor weight, and fewer number of PC nodules at 14 d after implantation. Histologically, less inflammatory cell infiltration and less cancer cell proliferation were observed in the tumors. There was no difference in apoptosis. Conclusions Our results raise an intriguing possibility that S1P generated by SphK1 in the host promotes pancreatic cancer PC progression by stimulation of proliferation of cancer cells. [ABSTRACT FROM AUTHOR]

Background Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis, and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1) in breast cancer plays important roles in progression of disease and metastasis. However, the associations between S1P and clinical parameters in human breast cancer have not been well investigated to date. Materials and methods We determined levels of S1P and other sphingolipids in breast cancer tissue by electrospray ionization-tandem mass spectrometry. Associations between S1P levels and clinicopathologic features of the tumors were analyzed. Expression of phospho-SphK1 (pSphK1) in breast cancer tissues was determined by immunohistochemical scoring. Results Levels of S1P in breast cancer tissues were significantly higher in patients with high white blood cell count in the blood than those patients without. S1P levels were lower in patients with human epidermal growth factor receptor 2 overexpression and/or amplification than those patients without. Furthermore, cancer tissues with high pSphK1 expression showed significantly higher levels of S1P than cancer tissues without. Finally, patients with lymph node metastasis showed significantly higher levels of S1P in tumor tissues than the patients with negative nodes. Conclusions To our knowledge, this is the first study to demonstrate that high expression of pSphK1 is associated with higher levels of S1P, which in turn is associated with lymphatic metastasis in breast cancer. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 2 (SphK2) is proposed as a novel oncotarget for lung cancer. Here, we studied the anti-lung cancer cell activity by ABC294640, a first-in-class SphK2 inhibitor. We showed that ABC294640 suppressed growth of primary and A549 human lung cancer cells, but sparing SphK2-low lung epithelial cells. Inhibition of SphK2 by ABC294640 increased ceramide accumulation, but decreased pro-survival sphingosine-1-phosphate (S1P) content, leading to lung cancer cell apoptosis activation. Significantly, we show that glucosylceramide synthase (GCS) might be a major resistance factor of ABC294640. The GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) or GCS shRNA/siRNA knockdown facilitated ABC294640-induced ceramide production and lung cancer cell apoptosis. Reversely, forced overexpression of GCS reduced ABC294640’s sensitivity, resulting in decreased ceramide accumulation and apoptosis induction in A549 cells. These findings provide further evidences to support that targeting SphK2 by ABC294640 may be a rational treatment option for lung cancer. Ceramide glucosylation inhibition may further sensitize lung cancer cells to ABC294640. [ABSTRACT FROM AUTHOR]

The aim of the present study is to establish the mechanism associated with the proliferation of PASMCs under ANG II stimulation. The results showed that treatment of PASMCs with ANG II induces an increase in cell proliferation and 100 nM was the optimum concentration for maximum increase in proliferation of the cells. Pretreatment of the cells with AT 1 , but not AT 2 , receptor antagonist inhibited ANG II induced cell proliferation. Pretreatment with pharmacological and genetic inhibitors of sphingomyelinase (SMase) and sphingosine kinase (SPHK) prevented ANG II-induced cell proliferation. ANG II has also been shown to induce SMase activity, SPHK phosphorylation and S1P production. In addition, ANG II caused an increase in proMMP-2 expression and activation, ERK1/2 phosphorylation and NADPH oxidase activation. Upon inhibition of MMP-2, SMase activity and S1P level were curbed leading to inhibition of cell proliferation. SPHK was phosphorylated by ERK1/2 during ET-1 stimulation of the cells. ANG II-induced ERK1/2 phosphorylation and proMMP-2 expression and activation in the cells were abrogated upon inhibition of NADPH oxidase activity. Overall, NADPH oxidase plays an important role in proMMP-2 expression and activation and that MMP-2 mediated SMC proliferation occurs through the involvement of Spm-Cer-S1P signaling axis under ANG II stimulation of PASMCs. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that hypoxic preconditioning (HPC) alleviates hypoxia/reoxygenation (H/R) injury. However, the impact and mechanism involved were not fully understood. This study aimed to evaluate the effect of HPC on H/R injury in cardiomyocytes and investigate the molecular mechanisms involved. In our study, primary neonatal rat cardiomyocytes were isolated and characterized by immunofluorescence staining. We established H/R models in vitro to mimic ischemia/reperfusion (I/R) injury in vivo. Primary cardiomyocytes were exposed to HPC and then subjected to H/R. SphK2 expression was determined by quantitative real-time PCR and Western blotting. Cell apoptosis was measured by Hoechst staining. H9c2 cells were transfected with SphK2 siRNA or pcDNA3.1-SphK2 plasmid. The transfection efficiency was evaluated 48 h post-transfection. After H/R, cell apoptosis rate was determined by Annexin V–FITC/PI and caspase-3/-9 activity was measured. The activation of FAK/AKT pathway was evaluated by Western blotting. Our results showed that HPC significantly increased SphK2 expression in primary cardiomyocytes under normal or H/R condition and protected against H/R-induced cell apoptosis, whereas SphK2 inhibitor K145 abolished the cardioprotective effect of HPC. HPC markedly reduced the cell apoptosis rate of H9c2, decreased the activities of caspase-3 and -9 and increased p-FAK and p-AKT levels, which were reversed by SphK2 knockdown. Additionally, SphK2 overexpression exerted a similar effect with HPC on cell apoptosis and FAK/AKT. Inhibition of H9c2 cell apoptosis induced by HPC and SphK2 overexpression was abolished by PI3K/AKT inhibitor LY294002. These results indicate that HPC may protect cardiomyocytes against H/R injury via SphK2 and the downstream FAK/AKT signaling pathway. Our findings provided important evidences for the protective role of HPC in ameliorating myocardial H/R injury. [ABSTRACT FROM AUTHOR]

MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3′-untranslated region (3′UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3′UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 μM S1P or 10 μM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies. • Addition of S1P and C1P enhances migration in retinal pigment epithelium cells. • S1P1 and S1P3 activation is essential for exogenous S1P stimulatory effect. • S1P and C1P require endogenous synthesis of S1P to enhance cell motility. • C1P stimulates the transcription of SphK1. • S1P and C1P stimulate IL-6, IL-8 and α-SMA transcription. [ABSTRACT FROM AUTHOR]

Sphingosine kinases (Sphks) are the rate-limiting kinases in the generation of sphingosine-1-phosphate, which is a well-established intracellular pro-survival lipid mediator. Sphk2 has been reported to be protective following experimental stroke. We investigated the role of Sphk1 in cerebral ischemia using a mouse middle cerebral artery occlusion (MCAO) model and an in vitro glucose–oxygen deprivation (OGD) model. Sphk expression and activity were assessed in the ischemic brain with quantitative PCR (qPCR), Western blot, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Pharmacological and gene knockdown approaches were utilized to investigate the effects of Sphk1 on stroke outcomes. The expression of Sphk1 but not that of Sphk2 was rapidly induced in the cortical penumbra over 96 h after MCAO, and the microglia were one of the major cellular sources of Sphk1 induction. Consistently, Sphk activity was enhanced in the cortical penumbra. In contrast to the protective role of Sphk2, pharmacological inhibition and cortical knockdown of Sphk1 reduced infarction at 24 and 96 h after reperfusion. Additionally, the Sphk1 inhibitor improved the neurological deficits at 96 h after reperfusion. Mechanistically, Sphk1 inhibition and knockdown significantly attenuated MCAO-induced expression of inflammatory mediators in the cortical penumbra. Moreover, using a conditioned medium transfer approach, we demonstrated that OGD-treated neurons induced the expression of Sphk1 and pro-inflammatory mediators in primary microglia, and the microglial induction of pro-inflammatory mediators by ischemic neurons was blunted by Sphk1 inhibition. Taken together, our results indicate that Sphk1 plays an essential role in mediating post-stroke neuroinflammation. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) dysregulation is a general feature of colorectal cancer (CRC) and other solid tumors, and is associated cancer progression. In the current study, we demonstrate that microRNA-101 (miR-101) inhibits CRC cells probably through down-regulating sphingosine kinase 1 (SphK1). Our results showed that exogenously expressing miR-101 inhibited CRC cell (HT-29 and HCT-116 lines) growth in vitro . At the molecular level, miR-101 dramatically down-regulated SphK1 mRNA and protein expression, causing pro-apoptotic ceramide production in above CRC cells. On the other hand, inhibition of miR-101 through expressing antagomiR-101 increased SphK1 expression to down-regulate ceramide level in HT-29 cells. miR-101 expression increased the in vitro anti-CRC activity of conventional chemo-agents: paclitaxel and doxorubicin. CRC cells with SphK1-shRNA knockdown showed similar phenotypes as the miR-101-expressed CRC cells, presenting with elevated level of ceramide and high sensitivity to paclitaxel or doxorubicin. In vivo, HCT-116 xenograft growth in severe combined immuno-deficient (SCID) mice was dramatically inhibited by over-expressing miR-101. Further, miR-101 enhanced paclitaxel-induced anti-HCT-116 activity in vivo . Together, these results indicate that miR-101 exerts its anti-CRC activities probably through down-regulating SphK1. [ABSTRACT FROM AUTHOR]

Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo , SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. [ABSTRACT FROM AUTHOR]

It has been reported that sphingosine kinase 1 (SPHK1), an oncogenic enzyme, was involved in the development and progression of a number of human cancers. However, the role of SPHK1 in nasopharyngeal carcinoma (NPC) is largely unknown. The present study aimed to characterize the expression of SPHK1 in human NPC and evaluate its clinical significance. Real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and Western blot analyses showed that the expression of SPHK1 mRNA and protein in NPC specimens was significantly higher than that in non-tumorous nasopharyngeal mucosa biopsies. Immunohistochemistry (IHC) was conducted to characterize the expression pattern of SPHK1 in 142 archived paraffin-embedded NPC specimens. Statistical analyses revealed that high levels of SPHK1 expression were associated with the clinical stages, locoregional recurrence and distant metastasis of NPC. NPC patients with high levels of SPHK1 expression had shorter survival time, whereas those with lower levels of SPHK1 expression survived longer. Moreover, multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic factor for NPC. Our results suggest for the first time that SPHK1 is involved in the development and progression of NPC, which can be used as a useful prognostic marker for NPC patients and may be an effective target for treating NPC. [ABSTRACT FROM AUTHOR]

Long-term glucocorticoid usage is a common cause of non-traumatic femoral head osteonecrosis. Glucocorticoids (i.e. dexamethasone (Dex)) could directly induce damages to osteoblasts. In the current study, we investigated the potential activity of K6PC-5 [ N -(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide], a novel sphingosine kinase 1 (SphK1) activator, against this process. Our data revealed that both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts were responsible to K6PC-5. K6PC-5 activated SphK1, increased sphingosine-1-phosphate (S1P) production and induced Akt phosphorylation in cultured osteoblasts. Functionally, K6PC-5 protected osteoblasts from Dex-induced apoptosis and necrosis. Such signaling and functional effects by K6PC-5 were prevented by the SphK1 inhibitor N,N-dimethylsphingosine (DMS), and by SphK1-siRNAs. On the other hand, exogenously-added S1P activated Akt and reduced Dex-induced osteoblast damages. LY294002 and MK-2206, two established Akt inhibitors, alleviated K6PC-5- or S1P-mediated osteoblast protection against Dex. Together, our results suggest that K6PC-5 alleviates Dex-induced osteoblast injuries through activating SphK1-Akt signaling. K6PC-5 might be further investigated in animal or clinical studies for its anti-glucocorticoids-associated osteonecrosis potential. [ABSTRACT FROM AUTHOR]

Sphingosine kinase 1 (SphK1) is an important signaling molecule for cell proliferation and survival. However, the role of SphK1 in acrylamide (ACR)-induced nerve injury remains unclear. The purpose of this study was to investigate the role and potential mechanism of SphK1 in ACR-induced nerve injury. Liquid chromatography triple quadrupole tandem mass spectrometry (LC-MS/MS) and reverse transcription-quantitative PCR (RT-qPCR) were used to detect sphingosine 1-phosphate (S1P) content in serum and SphK1 content in whole blood from an occupational work group exposed to ACR compared to a non-exposed group. For in vitro experiments, SphK1 in human SH-SY5Y neuroblastoma cells was activated using SphK1-specific activator phorbol 12-myristate 13-acetate (PMA). Our research also utilized cell viability assays, flow cytometry, western blots, RT-qPCR and related protein detection to assess activity of the mitogen activated protein kinase (MAPK) signaling pathway. The results of the population study showed that the contents of SphK1 and S1P in the ACR-exposed occupational contact group were lower than in the non-exposed group. The results of in vitro experiments showed that expression of SphK1 decreased with the increase in ACR concentration. Activating SphK1 improved the survival rate of SH-SY5Y cells and decreased the apoptosis rate. Activating SphK1 in SH-SY5Y cells also regulated MAPK signaling, including enhancing the phosphorylation of extracellular signal-regulated protein kinases (ERK) and inhibiting the phosphorylation of c-Jun N-terminal kinase (JNK) and p38. These results suggest that activating SphK1 can protect against nerve cell damage caused by ACR. • Scene investigation of acrylamide occupational population. • SphK1 is lowly expressed in the blood of acrylamide occupational population. • SphK1 regulates the MAPK pathway and plays a role in ACR-induced neurotoxicity. • Activation of SphK1 expression protects against the neurotoxicity produced by ACR. [ABSTRACT FROM AUTHOR]

Abstract: Measles virus (MV) manipulates host factors to facilitate virus replication. Sphingosine kinase (SK) is an enzyme catalyzing the formation of sphingosine 1-phosphate and modulates multiple cellular processes including the host defense system. Here, we determined the role of SK1 in MV replication. Overexpression of SK1 enhanced MV replication. In contrast, inhibition of SK impaired viral protein expression and infectious virus production from cells expressing MV receptor, SLAM or Nectin-4. The inhibition of virus replication was observed when the cells were infected by vaccine strain or wild type MV or V/C gene-deficient MV. Importantly, SK inhibition suppressed MV-induced activation of NF-κB. The inhibitors specific to NF-κB signal pathway repressed the synthesis of MV proteins, revealing the importance of NF-κB activation for efficient MV replication. Therefore, SK inhibition restricts MV replication and modulates the NF-κB signal pathway, demonstrating that SK is a cellular factor critical for MV replication. [Copyright &y& Elsevier]

Highlights: [•] siN-ras and U0126 enhanced the cytotoxicity of SKI II in hepatoma cells. [•] U0126 enhanced SKI II-induced apoptosis through Akt/NF-κB signaling pathway. [•] Combing U0126 and SKI II led to enhanced migratory inhibition via FAK/MLC-2 pathway. [ABSTRACT FROM AUTHOR]

Abstract: Macrophage recruitment to sites of inflammation is an essential step in host defense. However, the signals regulating the mobilization of these cells are still not fully understood. Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, is known to regulate an array of biological activities in various cell types. Here, we investigated the roles of S1P and S1P receptors (S1PRs) in macrophage migration in vitro. Furthermore, we explored the cross-talk between transforming growth factor-β1 (TGF-β1) and S1P signalling pathways in this process. We found that S1P exerted a powerful migratory action on RAW264.7 macrophages, as determined in Boyden chambers. Moreover, by employing RNA interference technology and pharmacological tools, we have demonstrated that S1PR1, but not S1PR2 and S1PR3, is required for S1P-induced macrophage migration. Importantly, we observed a pronounced increase in sphingosine kinase-1 (SphK1) mRNA expression and subsequently increase in S1P production, following transforming growth factor-β1 (TGF-β1) stimulation in RAW264.7 macrophages. The expression of S1PR1, but not S1PR2 and S1PR3, was also significantly up-regulated after TGF-β1 stimulation. Interestingly, exogenously added S1P-induced up-regulation of SphK1 and the synthesis of additional S1P, suggesting a self-amplifying loop of S1P to enhance macrophage migration. In conclusion, our results reveal that SphK1/S1PR1 signalling axis is induced by TGF-β1 and stimulates cell migration in RAW 264.7 macrophages. This study provides new clues for the molecular mechanisms of macrophage recruitment during inflammation. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH2-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-32P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K m, and the K i values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors. [Copyright &y& Elsevier]

Abstract: Acid sphingomyelinase (ASM) was identified as a gene induced by NFAT2 activation in osteoclasts. Suppression of ASM expression in bone marrow macrophages by knockdown enhanced c-Fos/NFAT2 expression, increasing the number of TRAP-positive multinucleated cells in vitro. SphK1 was upregulated during the late stage of osteoclastogenesis, while SphK2 expression remained constant. SphK1 was downregulated following ASM knockdown, while SphK2 levels were unchanged. Experiments using shRNA and catalytically-inactive form demonstrated inhibitory and stimulatory activities on osteoclast formation of SphK1 and SphK2, respectively. These results suggest that ASM regulates osteoclastogenesis by modulating the balance between SphK1 and SphK2 downstream of RANKL signaling. [Copyright &y& Elsevier]

Abstract: A Dictyostelium discoideum mutant with a disruption in the sphingosine-1-phosphate (S-1-P) lyase gene was obtained in an unbiased genetic analysis, using random insertional mutagenesis, for mutants with increased resistance to the widely used cancer chemotherapeutic drug cisplatin. This finding opened the way to extensive studies in both D. discoideum and human cells on the role and mechanism of action of the bioactive sphingolipids S-1-P and ceramide in regulating the response to chemotherapeutic drugs. These studies showed that the levels of activities of the sphingolipid metabolizing enzymes S-1-P lyase, sphingosine kinase and ceramide synthase, affect whether a cell dies or lives in the presence of specific drugs. The demonstration that multiple enzymes of this biochemical pathway were involved in regulating drug sensitivity provided new opportunities to test whether pharmacological intervention might increase sensitivity. Thus it is of considerable clinical significance that pharmacological inhibition of sphingosine kinase synergistically sensitizes cells to cisplatin, both in D. discoideum and human cells. Linkage to the p38 MAP kinase and protein kinase C (PKC) signaling pathways has been demonstrated. This work demonstrates the utility of D. discoideum as a lead genetic system to interrogate molecular mechanisms controlling the sensitivity of tumor cells to chemotherapeutic agents and for determining novel ways of increasing efficacy. The D. discoideum system could be easily adapted to a high throughput screen for novel chemotherapeutic agents. [Copyright &y& Elsevier]

Abstract: Sphingosine-1-phosphate (S1P) constricts cerebral arteries through S1P3 receptor stimulation. Because the activity of the key S1P-synthesizing enzyme, sphingosine kinase (SPK), can be stimulated by agonists of various G protein-coupled receptors, it is likely that S1P also acts as a second messenger for other vasoconstrictors. We investigated the effect of SPK inhibitors and SPK gene deletion on the contractile responses of isolated vessels to vasoactive agonists and KCl-induced depolarization. Basilar and femoral arteries of rat, mounted in a wire myograph, were incubated with dimethylsphingosine (DMS), 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (Compound 2) or FTY720, and exposed to KCl, 5-hydroxytryptamine (5-HT), S1P or phenylephrine (PE). Vasomotor responses in basilar artery were decreased by DMS, Compound 2 and FTY720, while they were not affected in femoral artery. Basilar arteries from SPK1−/− mice exhibited weaker vasoconstriction to both KCl and agonists (S1P and the prostanoid U46619) when compared to either wild type (WT) or SPK2−/−. In contrast, in mesenteric resistance arteries, neither the contraction to KCl nor the maximum contraction to PE and S1P significantly differed among WT, SPK1−/− and SPK2−/−. Quantitative analysis of SPK mRNA (reverse transcription and real time polymerase chain reaction) in mouse arteries showed 40–80-fold higher SPK1 expression in cerebral arteries than in aorta or mesenteric arteries. SPK1 critically modulates the reactivity of cerebral vasculature to vasoconstrictors. S1P plays a specific role as modulator of cerebral blood flow, potentially acting either directly outside vascular smooth muscle cells on S1P3 receptors, or indirectly after being generated inside the cell in response to vasoconstrictors. [Copyright &y& Elsevier]

Abstract: Our previous report showed that inhibition of sphingosine kinase (SphK) ameliorates eosinophilic inflammation and mucin production in a mouse asthmatic model. To clarify the role of SphK in airway mucin production, we utilized the mouse asthmatic model and found that both SphK and MUC5AC expression were increased and co-localized in airway epithelium. Next we cultured normal human bronchial epithelial cells in an air–liquid interface and treated with IL-13 to induce their differentiation into goblet cells. We found that SphK1 and MUC5AC expression was increased by IL-13 treatment at both protein and mRNA levels, whereas SphK2 expression was not changed. N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, decreased MUC5AC expression up-regulated by IL-13 treatment. Furthermore, DMS inhibited IL-13-induced ERK1/2 phosphorylation but neither p38 MAPK nor STAT6 phosphorylation. These results suggest that SphK1 is involved in MUC5AC production induced by IL-13 upstream of ERK1/2 phosphorylation, and independent of STAT6 phosphorylation. [Copyright &y& Elsevier]

Abstract: The lipid second messenger sphingosine 1-phosphate (S1P) is a critical mediator of cellular proliferation and survival signals, and is essential for vasculogenesis and neurogenesis. S1P formation is catalysed by sphingosine kinases 1 and 2 (Sphk1 and Sphk2). We have found that the endogenous glycolipid sulfatide (3-O-sulfogalactosylceramide) binds to and inhibits the activity of Sphk2 and the closely related ceramide kinase (Cerk), but not Sphk1. Using sulfatide as a probe, we mapped the lipid binding domain to the N-terminus of Sphk2 (residues 1–175), a region of sequence that is absent in Sphk1, but aligns with a pleckstrin homology domain in Cerk. Accordingly, Sphk2 bound to phosphatidylinositol monophosphates but not to abundant cellular phospholipids. Deleting the N-terminal domain reduced Sphk2 membrane localisation in cells. We have therefore identified a lipid binding domain in Sphk2 that is important for the enzyme’s sub-cellular localisation. [Copyright &y& Elsevier]

Abstract: Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-γ inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-γ production, we measured IL-18-induced IFN-γ production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-γ expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-γ production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-γ production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-γ production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-γ production via p38 MAPK. [Copyright &y& Elsevier]

Abstract: D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI–MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO3]- in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay. [Copyright &y& Elsevier]

Abstract: Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase. [Copyright &y& Elsevier]

Abstract: Ceramide kinase (CERK) and its product, ceramide-1-phosphate (Cer-1-P), are implicated in signaling processes, but the action mechanisms are not fully elucidated. When checking for intracellular effects of Cer-1-P by exposing CERK-expressing CHO cells to truncated ceramides, an unexpected decrease in CERK activity and protein level was observed. This decrease appeared dose-dependent and specific for the d-erythro-ceramide configuration and the presence of the double bond. At early time points, CERK clustered near the plasma membrane, followed later by its appearance in the culture medium. In cells expressing CERK lacking the pleckstrin domain or an inactive CERK mutant, this ceramide effect was not observed, indicating that clustering and release of CERK may be mediated by Cer-1-P. Presumably, high local Cer-1-P concentrations will increase the plasma membrane curvature and lead to release of CERK by vesicle shedding. This could be a potential regulatory mechanism in CERK/Cer-1-P signaling so far not investigated. [Copyright &y& Elsevier]

The formation of DNA methylation patterns is one of the epigenetic events that underlie mammalian development. The Sphk1 CpG island is a target for tissue-dependent DNA methylation as well as a template for generating multiple subtypes. The number of mammalian non-coding RNA genes is rapidly expanding. In this study, we found endogenous antisense transcripts, Khps1 subtypes with different sizes (600–20,000nt). A subtype, Khps1a, was a 1290-bp, non-coding, 5′-capped and 3′-polyadenylated RNA that originated from the CpG island and overlapped with a tissue-dependent differentially methylated region (T-DMR) of Sphk1. Intriguingly, overexpression of two fragments of Khps1 caused demethylation of CG sites in the T-DMR. Furthermore, this RNA-directed demethylation was associated with DNA methylation at three CC(A/T)GG sites in the T-DMR. The link between the RNA-directed CG demethylation and non-CG methylation provides a novel mechanism of epigenetic regulation and potential tool for epigenetic manipulation of mammalian cells. [Copyright &y& Elsevier]

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration. [Copyright &y& Elsevier]