Formation Of Sphingosine 1-phosphate Research Articles

Role of Luteolin as Potential New Therapeutic Option for Patients with Glioblastoma through Regulation of Sphingolipid Rheostat.

Glioblastoma (GBM) is the most aggressive brain tumor, still considered incurable. In this study, conducted on primary GBM stem cells (GSCs), specifically selected as the most therapy-resistant, we examined the efficacy of luteolin, a natural flavonoid, as an anti-tumoral compound. Luteolin is known to impact the sphingolipid rheostat, a pathway regulated by the proliferative sphingosine-1-phosphate (S1P) and the proapoptotic ceramide (Cer), and implicated in numerous oncopromoter biological processes. Here, we report that luteolin is able to inhibit the expression of SphK1/2, the two kinases implicated in S1P formation, and to increase the expression of both SGPL1, the lyase responsible for S1P degradation, and CERS1, the ceramide synthase 1, thus shifting the balance toward the production of ceramide. In addition, luteolin proved to decrease the expression of protumoral signaling as MAPK, RAS/MEK/ERK and PI3K/AKT/mTOR and cyclins involved in cell cycle progression. In parallel, luteolin succeeded in upregulation of proapoptotic mediators as caspases and Bcl-2 family and cell cycle controllers as p53 and p27. Furthermore, luteolin determined the shutdown of autophagy contributing to cell survival. Overall, our data support the use of luteolin as add-on therapy, having demonstrated a good ability in impairing GSC viability and survival and increasing cell sensitivity to TMZ.

Abstract 470: Sphingosine-1-Phosphate Signaling In The Human Microvascular Endothelium During Health And Disease

The loss of nitric oxide (NO)-mediated flow-induced dilation (FID) in the microvasculature, or microvascular endothelial dysfunction, is strongly linked to future cardiovascular events. Our lab has previously shown that chronic inhibition of sphingosine-1-phosphate (S1P) formation induces the change in FID mediator from the vasoprotective NO to the pro-inflammatory, pro-atherosclerotic hydrogen peroxide. The same transition is observed in arterioles from patients with coronary artery disease (CAD). Since S1P promotes NO-mediated FID, we hypothesized that formation of S1P and activation of S1P receptor 1 (S1PR1) is critical for FID in microvessels from healthy adults as opposed to arterioles from patients with CAD. Videomicroscopy was used to perform vascular function studies on human resistance arterioles (100-250μm) dissected from discarded surgical adipose tissue. The vessels were pre-constricted with endothelin-1 and changes in internal diameter in response to flow was measured. In arterioles from healthy adults, treatment with a sphingosine kinase (SpK) inhibitor for 30 minutes abolished FID (7.7±8.0% of maximal dilator capacity, n=3; mean±SEM) compared to control (73.1±8.2% n=9, p<0.05; *two-way ANOVA), whereas inhibition of S1PR1 appeared to delay the response to dilation (19.1±10.5% vs. 52.8±9.0% at 20mmHg pressure gradient, p<0.05*). In microvessels from patients with disease, both inhibition of SpK (91.8±2.8%, n=5) and S1PR1 (72.5±6.4%, n=6) did not affect FID compared to control (90.9±2.4%, n=4). Interestingly, S1PR1 expression was similar in CAD and nonCAD arterioles. Together these data suggest that formation of S1P and potentially the activation of S1PR1 is critical for FID during health, however this pathway does not contribute to FID during disease. We conclude that while arterioles can compensate for loss of this pathway in disease to maintain tissue perfusion, formation of S1P and activation of its receptors may be necessary to promote NO formation during shear and may serve as potential targets to prevent future cardiovascular disease.

Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea

AbstractSphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFβ1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor β1(TGFβ1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFβ1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFβ1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas.Injury-induced transforming growth factor β1 increases sphingosine 1-phosphate (S1P) generation by upregulating in sphingosine kinase 1 activity. The high levels of S1P formation stimulate the S1PR3-linked signaling pathway, which in turn increases vascular endothelial growth factor-A expression levels and angiogenesis in mouse corneas.

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect

To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A. Case-control laboratory study. University institute and university hospital. Patients with uterine fibroids (n = 26). Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy. Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines. The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P2, S1P3, and S1P5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P2. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells. This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.

Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate.

Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine-1-phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia-responsive master transcription factors hypoxia-inducible factor (HIF)-1α/2α, and their functions in breast cancer, with a focus on triple-negative breast cancer (TNBC). We have shown SphK2 is associated with HIF-1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF-1α. SphK2, and HIF-1α expression levels are elevated in metastatic estrogen receptor-positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K-145, that nuclear S1P, histone acetylation, HIF-1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF-1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.

Discovery of a Small Side Cavity in Sphingosine Kinase 2 that Enhances Inhibitor Potency and Selectivity.

The sphingosine-1-phosphate (S1P) signaling pathway is an attractive drug target due to its involvement in immune cell chemotaxis and vascular integrity. The formation of S1P is catalyzed by sphingosine kinase 1 or 2 (SphK1 or SphK2) from sphingosine (Sph) and ATP. Inhibition of SphK1 and SphK2 to attenuate levels of S1P has been reported to be efficacious in animal models of diseases such as cancer, sickle cell disease, and renal fibrosis. While inhibitors of both SphKs have been reported, improvements in potency and selectivity are still needed. Toward that end, we performed structure-activity relationship profiling of 8 (SLM6031434) and discovered a heretofore unrecognized side cavity that increased inhibitor potency toward SphK2. Interrogating this region revealed that relatively small hydrophobic moieties are preferred, with 10 being the most potent SphK2-selective inhibitor (Ki = 89 nM, 73-fold SphK2-selective) with validated in vivo activity.

Sphingosine-1-phosphate signaling: A novel target for simultaneous adjuvant treatment of triple negative breast cancer and chemotherapy-induced neuropathic pain.

Triple-negative breast cancer (TNBC) is very aggressive with high metastatic and mortality rates and unfortunately, except for chemotherapy, there are few therapeutic options. The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) regulates numerous processes important for cancer progression, metastasis, and neuropathic pain. The pro-drug FTY720 (fingolimod, Gilenya) used to treat multiple sclerosis is phosphorylated in the body to a S1P mimic that binds to S1PRs, except S1PR2, and also acts as a functional antagonist of S1PR1. This review highlights current findings showing that FTY720 has multiple anti-cancer activities and simultaneously prevents formation and actions of S1P. Moreover, in mouse breast cancer models, treatment with FTY720 reduces tumor growth, metastasis, and enhances sensitivity of advanced and hormonal refractory breast cancer and TNBC to conventional therapies. We discuss recent studies demonstrating that neuropathic pain induced by the chemotherapeutic bortezomib is also greatly reduced by administration of clinically relevant doses of FTY720, likely by targeting S1PR1 on astrocytes. FTY720 also shows promising anticancer potential in pre-clinical studies and is FDA approved, thus we suggest in this review that further studies are needed to pave the way for fast-tracking approval of FTY720/fingolimod for enhancing chemotherapy effectiveness and reduction of painful neuropathies.

SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.

The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.

Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis byinversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis byinducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serumS1P levels were positively associated with serumcalcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and thebone formation markerPINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Comparative analysis of the effects of a sphingosine kinase inhibitor to temozolomide and radiation treatment on glioblastoma cell lines

ABSTRACTGlioblastoma multiforme (GBM) exhibits high resistance to the standard treatment of temozolomide (TMZ) combined with radiotherapy, due to its remarkable cell heterogeneity. Accordingly, there is a need to target alternative molecules enhancing specific GBM autocrine or paracrine mechanisms and amplifying the effect of standard treatment. Sphingosine 1-phosphate (S1P) is such a lipid target molecule with an important role in cell invasion and proliferation. Sphingosine kinase inhibitors (SKI) prevent S1P formation and induce increased production of reactive oxygen species (ROS), which may potentiate radiation cytotoxicity. We analyzed the effect of SKI singular versus combined treatments with TMZ and radiation on 2 human GBM cell lines characterized by a lack of MGMT expression and low or high expression of the anti-oxidant enzyme, glutathione peroxidase 1 (GPx1). Effects were drug concentration-, cell line-dependent and partly ROS-mediated. Clonogenic survival assay demonstrates that SKI was more effective than TMZ in increasing the sensitivity of U87 cells, which express low GPx1 amount, to a 2 Gy X-ray dose. Addition of both SKI and TMZ drastically decreased U87 cells survival compared with the combination temozolomide/radiation. SKI less effectively than TMZ sensitized LN229 cells to the 2 Gy X-ray dose. Its combination to TMZ in absence of irradiation was as efficient as TMZ combination with X-ray. We provide first evidence for SKI as an alternative or complementary treatment to TMZ, and for efficient combinations of low doses of drugs and X-ray. These may help as novel bi-modal and tri-modal therapies to contend with GBM heterogeneity.

Discovery of a Potent and Selective Sphingosine Kinase 1 Inhibitor through the Molecular Combination of Chemotype-Distinct Screening Hits.

Sphingosine kinase (SphK) is the major source of the lipid mediator and G protein-coupled receptor agonist sphingosine-1-phosphate (S1P). S1P promotes cell growth, survival, and migration and is a key regulator of lymphocyte trafficking. Inhibition of S1P signaling has been proposed as a strategy for treatment of inflammatory diseases and cancer. Two different formats of an enzyme-based high-throughput screen yielded two attractive chemotypes capable of inhibiting S1P formation in cells. The molecular combination of these screening hits led to compound 22a (PF-543) with 2 orders of magnitude improved potency. Compound 22a inhibited SphK1 with an IC50 of 2 nM and was more than 100-fold selective for SphK1 over the SphK2 isoform. Through the modification of tail-region substituents, the specificity of inhibition for SphK1 and SphK2 could be modulated, yielding SphK1-selective, potent SphK1/2 dual, or SphK2-preferential inhibitors.

Abstract 3567: Metformin suppresses synthesis of pro-survival sphingolipid, sphingosine-1-phosphate, by inhibition of sphingosine kinase-1, in MCF-7 and SK-BR-3 breast cancer cell lines

Abstract The antidiabetic drug, Metformin, may possess anti-cancer properties. Metformin has been shown to suppress proliferation of breast cancer cells primarily through activation of AMP-activated protein kinase (AMPK) and its suppression of downstream signaling pathways, such as mTOR, involved in cell replication. Other mechanisms may also play a role. Sphingolipids have a role in apoptosis and survival. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, promotes cell survival, proliferation, migration, angiogenesis, lymph angiogenesis, and immune response. S1P is involved in both intracellular and extracellular functions and regulates proliferation and survival. Blocking S1P synthesis inhibits cellular proliferation. Sphingosine kinase (SphK) is a lipid kinase that catalyzes formation of S1P from the precursor sphingosine. SphK is known to be upregulated in cancer cells, promoting tumor progression. S1P has a critical role in cancer progression and is considered a viable target for cancer therapeutics. Our previous studies show that metformin has an effect on the synthesis of pro-apoptotic ceramides. We hypothesized that metformin induces cytotoxicity by reducing levels of the pro-survival sphingolipid, S1P. Firstly, MCF-7 and SK-BR-3 breast cancer cell lines were treated with increasing concentrations of metformin, and cytotoxicity was determined by MTT cell culture experiments after 24 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 2.5mM, and percentage cytotoxicity increases in a dose-dependent manner. We utilized liquid chromatography and mass spectrometry and determined that cellular S1P levels are decreased in MCF-7 cells treated with 2.5mM metformin when compared with the control group. Finally, we treated MCF-7 and SK-BR-3 breast cancer cells with metformin, SK I/II (a known SphK inhibitor), and an untreated control group for 2, 4 and 6 hours. The dose of metformin was 10mM, which was chosen from a dose-response curve using MTT assay. The dose of SK I/II was 20uM, chosen based on the IC50 given. All treatments were done using low glucose media. Using the lysates from the harvested cells, gel electrophoresis and western blots using antibodies to SphK and S1P were run. Our results showed that metformin decreased the cellular levels of SphK and S1P. Thus, metformin exhibits anticancer properties via inhibiting the production of pro-survival lipid S1P. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of synthesis of S1P in breast cancer cells. Further work is necessary to characterize the sphingolipid content of MCF-7 and SK-BR-3 cancer cells before and after metformin treatment. Citation Format: Ashley Rose, Daniel Wann, Janet Lightner, Marianne Brannon, William Stone, Victoria Palau, Koyamangalath Krishnan. Metformin suppresses synthesis of pro-survival sphingolipid, sphingosine-1-phosphate, by inhibition of sphingosine kinase-1, in MCF-7 and SK-BR-3 breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3567.

Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2.

Mast cells (MC) are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P) functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2-) mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF-) A and matrix metalloproteinase- (MMP-) 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC) or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

Increased sphingosine 1-phosphate mediates inflammation and fibrosis in tubular injury in diabetic nephropathy.

Hyperglycemia induces all isoforms of transforming growth factor β (TGFβ), which in turn play key roles in inflammation and fibrosis that characterize diabetic nephropathy. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid, derived from sphingosine by the action of sphingosine kinase (SK). S1P mediates many biological processes, which mimic TGFβ signaling. To determine the role of SK1 and S1P in inducing fibrosis and inflammation, and the interaction with TGFβ-1, 2 and 3 signalling in diabetic nephropathy, human proximal tubular cells (HK2 cells) were exposed to normal (5mmol/L) or high (30mmol/L) glucose or TGFβ-1, -2, -3 ± an SK inhibitor (SKI-II) or SK1 siRNA. Control and diabetic wild type (WT) and SK1(-/-) mice were studied. Fibrotic and inflammatory markers, and relevant downstream signalling pathways were assessed. SK1 mRNA and protein expression was increased in HK2 cells exposed to high glucose or TGFβ1,-2,-3. All TGFβ isoforms induced fibronectin, collagen IV and macrophage chemoattractant protein 1 (MCP1), which were reversed by both SKI-II and SK1 siRNA. Exposure to S1P increased phospho-p44/42 expression, AP-1 binding and NFkB phosphorylation. WT diabetic mice exhibited increased renal cortical S1P, fibronectin, collagen IV and MCP1 mRNA and protein expression compared to SK1(-/-) diabetic mice. In summary, this study demonstrates that inhibiting the formation of S1P reduces tubulointerstitial renal inflammation and fibrosis in diabetic nephropathy.

CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration.

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

Factor-Xa-induced mitogenesis and migration require sphingosine kinase activity and S1P formation in human vascular smooth muscle cells

Sphingosine-1-phosphate (S1P) is a cellular signalling lipid generated by sphingosine kinase-1 (SPHK1). The aim of the study was to investigate whether the activated coagulation factor-X (FXa) regulates SPHK1 transcription and the formation of S1P and subsequent mitogenesis and migration of human vascular smooth muscle cells (SMC). FXa induced a time- (3-6 h) and concentration-dependent (3-30 nmol/L) increase of SPHK1 mRNA and protein expression in human aortic SMC, resulting in an increased synthesis of S1P. FXa-stimulated transcription of SPHK1 was mediated by the protease-activated receptor-1 (PAR-1) and PAR-2. In human carotid artery plaques, expression of SPHK1 was observed at SMC-rich sites and was co-localized with intraplaque FX/FXa content. FXa-induced SPHK1 transcription was attenuated by inhibitors of Rho kinase (Y27632) and by protein kinase C (PKC) isoforms (GF109203X). In addition, FXa rapidly induced the activation of the small GTPase Rho A. Inhibition of signalling pathways which regulate SPHK1 expression, inhibition of its activity or siRNA-mediated SPHK1 knockdown attenuated the mitogenic and chemotactic response of human SMC to FXa. These data suggest that FXa induces SPHK1 expression and increases S1P formation independent of thrombin and that this involves the activation of Rho A and PKC signalling. In addition to its key function in coagulation, this direct effect of FXa on human SMC may increase cell proliferation and migration at sites of vessel injury and thereby contribute to the progression of vascular lesions.

Abstract 4887: FTY720 prevent tumorigenesis and suppress the progression of colitis-associated cancer.

Abstract Inflammatory bowel disease is an important risk factor for colorectal cancer. Sphingosine-1-phosphate (S1P), a pleiotropic bioactive sphingolipid metabolite formed by two sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. Enhanced tumor number and size, with a concomitant increase in tumor load was observed in mice overexpressing SphK1 treated with the colonotropic mutagen azoxymethane (AOM) followed by three rounds of oral administration of the luminal toxin dextran sodium sulfate (DSS) to induce colitis-associated cancer (CAC). There was also a large increase in adenomas with a high grade of dysplasia associated with increased SphK1 expression and formation of S1P. We demonstrated that the SphK1/S1P axis is important for production of IL-6 and is involved in persistent activation of STAT3 and consequent upregulation of one of its target genes, the S1P receptor S1PR1. Because the pro-drug FTY720 modulates the immune system by acting as a functional antagonist of S1PR1, inducing its internalization and degradation, we examined its effect on CAC progression. FTY720 drastically reduced colitis-associated tumorigenesis, when it was administered during CAC induction. Remarkably, FTY720 blocked CAC progression even when it was administered later after the last DSS treatment when tumors were already established. Moreover, it reduced SphK1 expression, production of IL-6, and persistent activation of STAT3. These findings suggest that FTY720 may be beneficial for preventing tumor formation in individuals with inflammatory bowel disease. This work was supported by grants from the National Institute of Health (R37GM043880, R01CA61774, U19AI077435 to S.S. and R01CA160688 to K.T.). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. Citation Format: Akimitsu Yamada, Jie Liang, Masayuki Nagahashi, Eugene Y. Kim, Kuzhuvelil B. Harikumar, Wei-Ching Huang, Nitai C. Hait, Jeremy C. Allegood, Megan M. Price, Dorit Avni, Tomasz Kordula, Milstien Sheldon, Kazuaki Takabe, Sarah Spiegel. FTY720 prevent tumorigenesis and suppress the progression of colitis-associated cancer. [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 4887. doi:10.1158/1538-7445.AM2013-4887