Dengue Virus-Induced Inflammation of the Endothelium and the Potential Roles of Sphingosine Kinase-1 and MicroRNAs

Copyright © 2015 Amanda L. Aloia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.One of the main pathogenic effects of severe dengue virus (DENV) infection is a vascular leak syndrome. There are no available antivirals or specific DENV treatments and without hospital support severe DENV infection can be life-threatening. The cause of the vascular leakage is permeability changes in the endothelial cells lining the vasculature that are brought about by elevated vasoactive cytokine and chemokines induced following DENV infection. The source of these altered cytokine and chemokines is traditionally believed to be from DENV-infected cells such as monocyte/macrophages and dendritic cells. Herein we discuss the evidence for the endothelium as an additional contributor to inflammatory and innate responses during DENV infection which may affect endothelial cell function, in particular the ability to maintain vascular integrity. Furthermore, we hypothesise roles for two factors, sphingosine kinase-1 and microRNAs (miRNAs), with a focus on several candidate miRNAs, which are known to control normal vascular function and inflammatory responses. Both of these factors may be potential therapeutic targets to regulate inflammation of the endothelium during DENV infection

Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest

Interaction between anandamide and sphingosine-1-phosphate in mediating vasorelaxation in rat coronary artery

<b>BACKGROUND AND PURPOSE</b> Anandamide and sphingosine-1-phosphate (S1P) both regulate vascular tone in a variety of vessels. This study aimed to examine the mechanisms involved in the regulation of coronary vascular tone by anandamide and S1P, and to determine whether any functional interaction occurs between these receptor systems. <br></br> <b>EXPERIMENTAL APPROACH</b> Mechanisms used by anandamide and S1P to regulate rat coronary artery (CA) reactivity were investigated using wire myography. Interactions between S1P and the cannabinoid (CB)2 receptor were determined using human embryonic kidney 293 (HEK293) cells that stably over-express recombinant CB2 receptor. <br></br> <b>KEY RESULTS</b> Anandamide and S1P induced relaxation of the rat CA. CB2 receptor antagonists attenuated anandamide-induced relaxation, while S1P-mediated relaxation was dependent on the vascular endothelium and S1P3. Anandamide treatment resulted in an increase in the phosphorylation of sphingosine kinase-1 within the CA. Conversely, anandamide-mediated relaxation was attenuated by inhibition of sphingosine kinase. Moreover, S1P3, specifically within the vascular endothelium, was required for anandamide-mediated vasorelaxation. In addition to this, S1P-mediated relaxation was also reduced by CB2 receptor antagonists and sphingosine kinase inhibition. Further evidence that S1P functionally interacts with the CB2 receptor was also observed in HEK293 cells over-expressing the CB2 receptor. <br></br> <b>CONCLUSIONS AND IMPLICATIONS</b> In the vascular endothelium of rat CA, anandamide induces relaxation via a mechanism requiring sphingosine kinase-1 and S1P/S1P3. In addition, we report that S1P may exert some of its effects via a CB2 receptor- and sphingosine kinase-dependent mechanism, where subsequently formed S1P may have privileged access to S1P3 to induce vascular relaxation

Intracranial Injection of Dengue Virus Induces Interferon Stimulated Genes and CD8+ T Cell Infiltration by Sphingosine Kinase 1 Independent Pathways

Copyright: © 2017 Al-Shujairi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.We have previously reported that the absence of sphingosine kinase 1 (SK1) affects both dengue virus (DENV) infection and innate immune responses in vitro. Here we aimed to define SK1-dependancy of DENV-induced disease and the associated innate responses in vivo. The lack of a reliable mouse model with a fully competent interferon response for DENV infection is a challenge, and here we use an experimental model of DENV infection in the brain of immunocompetent mice. Intracranial injection of DENV-2 into C57BL/6 mice induced body weight loss and neurological symptoms which was associated with a high level of DENV RNA in the brain. Body weight loss and DENV RNA level tended to be greater in SK1-/- compared with wildtype (WT) mice. Brain infection with DENV-2 is associated with the induction of interferon-β (IFN-β) and IFN-stimulated gene (ISG) expression including viperin, Ifi27l2a, IRF7, and CXCL10 without any significant differences between WT and SK1-/- mice. The SK2 and sphingosine-1-phosphate (S1P) levels in the brain were unchanged by DENV infection or the lack of SK1. Histological analysis demonstrated the presence of a cellular infiltrate in DENV-infected brain with a significant increase in mRNA for CD8 but not CD4 suggesting this infiltrate is likely CD8+ but not CD4+ T-lymphocytes. This increase in T-cell infiltration was not affected by the lack of SK1. Overall, DENV-infection in the brain induces IFN and T-cell responses but does not influence the SK/S1P axis. In contrast to our observations in vitro, SK1 has no major influence on these responses following DENV-infection in the mouse brain

Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1 and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is over-expressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease

An oncogenic role for sphingosine kinase 2

While both human sphingosine kinases (SK1 and SK2) catalyze the generation of the pleiotropic signaling lipid sphingosine 1-phosphate, these enzymes appear to be functionally distinct. SK1 has well described roles in promoting cell survival, proliferation and neoplastic transformation. The roles of SK2, and its contribution to cancer, however, are much less clear. Some studies have suggested an antiproliferative/ pro-apoptotic function for SK2, while others indicate it has a prosurvival role and its inhibition can have anti-cancer effects. Our analysis of gene expression data revealed that SK2 is upregulated in many human cancers, but only to a small extent (up to 2.5-fold over normal tissue). Based on these findings, we examined the effect of different levels of cellular SK2 and showed that high-level overexpression reduced cell proliferation and survival, and increased cellular ceramide levels. In contrast, however, low-level SK2 overexpression promoted cell survival and proliferation, and induced neoplastic transformation in vivo. These findings coincided with decreased nuclear localization and increased plasma membrane localization of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis, supporting the use of SK2-specific inhibitors as anti-cancer agents.Heidi A. Neubauer, Duyen H. Pham, Julia R. Zebol, Paul A.B. Moretti, Amanda L. Peterson, Tamara M. Leclercq, Huasheng Chan, Jason A. Powell, Melissa R. Pitman, Michael S. Samuel, Claudine S. Bonder, Darren J. Creek, Briony L. Gliddon and Stuart M. Pitso

Sphingosine kinase 2 inhibition synergises with bortezomib to target myeloma by enhancing endoplasmic reticulum stress

Published: April 14, 2017The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.Craig T. Wallington-Beddoe, Melissa K. Bennett, Kate Vandyke, Lorena Davies, Julia R. Zebol, Paul A.B. Moretti, Melissa R. Pitman, Duncan R. Hewett, Andrew C.W. Zannettino and Stuart M. Pitso

The emerging role of FTY720 (Fingolimod) in cancer treatment

FTY720 (Fingolimod) is a clinically approved immunomodulating therapy for multiple sclerosis that sequesters T-cells to lymph nodes through functional antagonism of sphingosine-1-phosphate 1 receptor. FTY720 also demonstrates a proven efficacy in multiple in vitro and in vivo cancer models, suggesting a potential therapeutic role in cancer patients. A potential anticancer mechanism of FTY720 is through the inhibition of sphingosine kinase 1, a proto-oncogene with in vitro and clinical cancer association. In addition, FTY720's anticancer properties may be attributable to actions on several other molecular targets. This study focuses on reviewing the emerging evidence regarding the anticancer properties and molecular targets of FTY720. While the clinical transition of FTY720 is currently limited by its immune suppression effects, studies aiming at FTY720 delivery and release together with identifying its key synergetic combinations and relevant patient subsets may lead to its rapid introduction into the clinic

Introduction: Autofiction

Autofiction is a literary genre that combines the traditional genres of fiction and autobiography. The three autofictional texts in this section stem from a class on “Autofiction,” taught by Lujain Youssef in the summer term 2022, which engaged with the genre and encouraged students to produce their own creative writing

Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKC alpha, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKC alpha directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residueopen