ORCID Profile
0000-0001-8935-2448
Current Organisations
Universidad Autónoma de Madrid Facultad de Medicina
,
University of South Australia
,
Instituto Ramón y Cajal de Investigación Sanitaria
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Publications
Purification and characterization of an extracellular (1 → 6)- β -glucanase from the filamentous fungus Acremonium persicinum
Publisher: Portland Press Ltd.
Date: 15-06-1996
DOI: 10.1042/BJ3160841
Abstract: An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.
Dengue Virus-Induced Inflammation of the Endothelium and the Potential Roles of Sphingosine Kinase-1 and MicroRNAs
Publisher: Hindawi Limited
Date: 2015
DOI: 10.1155/2015/509306
Abstract: 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.
Sphingosine Kinase 2 Promotes Acute Lymphoblastic Leukemia by Enhancing MYC Expression
Publisher: American Association for Cancer Research (AACR)
Date: 14-05-2014
DOI: 10.1158/0008-5472.CAN-13-2732
Abstract: Sphingosine kinase 2 (SK2) may have utility as a prognostic marker in inflammatory diseases such as cancer in which it has been rationalized as a candidate therapeutic target. Here, we show that SK2 has an oncogenic role in acute lymphoblastic leukemia (ALL) by influencing expression of MYC. Genetic ablation of SK2 impaired leukemia development in a mouse model of ALL and pharmacologic inhibition extended survival in mouse xenograft models of human disease. SK2 attenuation in both the settings reduced MYC expression in leukemic cells, with reduced levels of acetylated histone H3 within the MYC gene associated with reduced levels of MYC protein and expression of MYC-regulated genes. Our results demonstrated that SK2 regulates MYC, which has a pivotal role in hematologic malignancies, providing a preclinical proof of concept for this pathway as a broad-based therapeutic target in this setting. Cancer Res 74(10) 2803–15. ©2014 AACR.
An oncogenic role for sphingosine kinase 2
Publisher: Impact Journals, LLC
Date: 30-08-2016
Identification of sphingosine kinase 1 as a therapeutic target in B-lineage acute lymphoblastic leukaemia
Publisher: Wiley
Date: 23-01-2019
DOI: 10.1111/BJH.15097
Isoflurane via TGF-ss1 release increases caveolae formation and organizes sphingosine kinase signaling in renal proximal tubules
Publisher: American Physiological Society
Date: 04-2010
DOI: 10.1152/AJPRENAL.00115.2009
Abstract: We previously showed that the inhalational anesthetic isoflurane protects against renal proximal tubule necrosis via isoflurane-mediated stimulation and translocation of sphingosine kinase-1 (SK1) with subsequent synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells (Kim M, Kim M, Kim N, D'Agati VD, Emala CW Sr, Lee HT. Am J Physiol Renal Physiol 293: F1827–F1835, 2007). We also demonstrated that the anti-necrotic and anti-inflammatory effect of isoflurane is due in part to phosphatidylserine (PS) externalization and subsequent release of transforming growth factor-β1 (TGF-β1) (Lee HT, Kim M, Kim J, Kim N, Emala CW. Am J Nephrol 27: 416–424, 2007). In this study, we tested the hypothesis that isoflurane, via TGF-β1 release, increases caveolae formation in the buoyant fraction of the cell membrane of human renal proximal tubule (HK-2) cells to organize SK1 and S1P signaling. To detect SK1 protein in the caveolae/caveolin fractions, we overexpressed human SK1 in HK-2 cells (SK1-HK-2). SK1-HK-2 cells exposed to isoflurane increased caveolae/caveolin formation in the buoyant membrane fractions which contained key signaling intermediates involved in isoflurane-mediated renal tubule protection, including S1P, SK1, ERK MAPK, and TGF-β1 receptors. Furthermore, treating SK1-HK-2 cells with recombinant TGF-β1 or PS liposome mixture increased caveolae formation, mimicking the effects of isoflurane. Conversely, TGF-β1-neutralizing antibody blocked the increase in caveolae formation induced by isoflurane in SK1-HK-2 cells. The increase in SK1 activity in the caveolae-enriched fractions from isoflurane-treated nonlentivirus-infected HK-2 cells, while smaller in magnitude, was qualitatively similar to that found in the SK1-HK-2 cell line. Finally, isoflurane also increased caveolae formation in the kidneys of TGF-β1 +/+ mice but not in TGF-β1 +/− mice (mice with reduced levels of TGF-β1). Our study demonstrates that isoflurane organizes several key cytoprotective signaling intermediates including TGF-β1 receptors, SK1 and ERK, within the caveolae fraction of the plasma membrane. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.
Sphingolipids as multifaceted mediators in ovarian cancer
Publisher: Elsevier BV
Date: 05-2021
DOI: 10.1016/J.CELLSIG.2021.109949
Abstract: Ovarian cancer is the most lethal gynaecological malignancy. It is commonly diagnosed at advanced stage when it has metastasised to the abdominal cavity and treatment becomes very challenging. While current standard therapy involving debulking surgery and platinum + taxane-based chemotherapy is associated with high response rates initially, the large majority of patients relapse and ultimately succumb to chemotherapy-resistant disease. In order to improve survival novel strategies for early detection and therapeutics against treatment-refractory disease are urgently needed. A promising new target against ovarian cancer is the sphingolipid pathway which is commonly hijacked in cancer to support cell proliferation and survival and has been shown to promote chemoresistance and metastasis in a wide range of malignant neoplasms. In particular, the sphingosine kinase 1-sphingosine 1-phosphate receptor 1 axis has been shown to be altered in ovarian cancer in multiple ways and therefore represents an attractive therapeutic target. Here we review the roles of sphingolipids in ovarian cancer progression, metastasis and chemoresistance, highlighting novel strategies to target this pathway that represent potential avenues to improve patient survival.
Tumor Necrosis Factor-Induced Neutrophil Adhesion Occurs Via Sphingosine Kinase-1-Dependent Activation of Endothelial α5β1 Integrin
Publisher: Elsevier BV
Date: 07-2010
Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1
Publisher: Portland Press Ltd.
Date: 11-11-2019
DOI: 10.1042/BCJ20190245
Abstract: Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.
The GM-CSF receptor family: Mechanism of activation and implications for disease
Publisher: Informa UK Limited
Date: 18-01-2012
DOI: 10.3109/08977194.2011.649919
Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.
Molecular targets of FTY720 (Fingolimod)
Publisher: Bentham Science Publishers Ltd.
Date: 03-2017
DOI: 10.2174/156652412803833599
Abstract: FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.
Ultrafast photochemistry produces superbright short-wave infrared dots for low-dose in vivo imaging
Publisher: Springer Science and Business Media LLC
Date: 10-06-2020
DOI: 10.1038/S41467-020-16333-2
Abstract: Optical probes operating in the second near-infrared window (NIR-II, 1,000-1,700 nm), where tissues are highly transparent, have expanded the applicability of fluorescence in the biomedical field. NIR-II fluorescence enables deep-tissue imaging with micrometric resolution in animal models, but is limited by the low brightness of NIR-II probes, which prevents imaging at low excitation intensities and fluorophore concentrations. Here, we present a new generation of probes (Ag 2 S superdots) derived from chemically synthesized Ag 2 S dots, on which a protective shell is grown by femtosecond laser irradiation. This shell reduces the structural defects, causing an 80-fold enhancement of the quantum yield. PEGylated Ag 2 S superdots enable deep-tissue in vivo imaging at low excitation intensities ( mW cm −2 ) and doses ( .5 mg kg −1 ), emerging as unrivaled contrast agents for NIR-II preclinical bioimaging. These results establish an approach for developing superbright NIR-II contrast agents based on the synergy between chemical synthesis and ultrafast laser processing.
Enhancing ER stress in myeloma.
Publisher: Impact Journals, LLC
Date: 30-07-2017
Roles, regulation and inhibitors of sphingosine kinase 2
Publisher: Wiley
Date: 07-06-2013
DOI: 10.1111/FEBS.12314
Abstract: The bioactive sphingolipids ceramide, sphingosine and sphingosine-1-phosphate (S1P) are important signalling molecules that regulate a erse array of cellular processes. Most notably, the balance of the levels of these three sphingolipids in cells, termed the 'sphingolipid rheostat', can dictate cell fate, where ceramide and sphingosine enhance apoptosis and S1P promotes cell survival and proliferation. The sphingosine kinases (SKs) catalyse the production of S1P from sphingosine and are therefore central regulators of the sphingolipid rheostat and attractive targets for cancer therapy. Two SKs exist in humans: SK1 and SK2. SK1 has been extensively studied and there is a large body of evidence to demonstrate its role in promoting cell survival, proliferation and neoplastic transformation. SK1 is also elevated in many human cancers which appears to contribute to carcinogenesis, chemotherapeutic resistance and poor patient outcome. SK2, however, has not been as well characterized, and there are contradictions in the key physiological functions that have been proposed for this isoform. Despite this, many studies are now emerging that implicate SK2 in key roles in a variety of diseases, including the development of a range of solid tumours. Here, we review the literature examining SK2, its physiological and pathophysiological functions, the current knowledge of its regulation, and recent developments in targeting this complex enzyme.
Sphingosine kinase regulates the rate of endothelial progenitor cell differentiation
Publisher: American Society of Hematology
Date: 26-02-2009
DOI: 10.1182/BLOOD-2008-07-166942
Abstract: Circulating endothelial progenitor cells (EPCs) are incorporated into foci of neovascularization where they undergo differentiation to mature endothelial cells (ECs). We show here that the enzyme sphingosine kinase-1 (SK-1) regulates the rate and direction of EPC differentiation without effect on the hematopoietic compartment. EPCs have high levels of SK-1 activity, which diminishes with differentiation and is, at least partially, responsible for maintaining their EPC phenotype. EPCs from SK-1 knockout mice form more adherent EC units and acquire a mature EC phenotype more rapidly. Conversely, EPCs from mice overexpressing SK-1 in the EC compartment are retarded in their differentiation. Exogenous regulation of SK-1 levels in normal EPCs, by genetic and pharmacologic means, including the immunomodulating drug FTY720, recapitulates these effects on EC differentiation. SK-1 knockout mice have higher levels of circulating EPCs, an exaggerated response to erythropoietin-induced EPC mobilization, and, in a mouse model of kidney ischemia reperfusion injury, exhibit a recovery similar to that of ischemic mice administered exogenous EPCs. Thus, SK-1 is a critical player in EPC differentiation into EC pointing to the potential utility of SK-1 modifying agents in the specific manipulation of endothelial development and repair.
Stereochemical Course of Hydrolysis Catalysed by α-l-Rhamnosyl and α-d-Galacturonosyl Hydrolases fromAspergillus aculeatus
Publisher: Elsevier BV
Date: 1998
Abstract: The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.
Targeting human CALR‐mutated MPN progenitors with a neoepitope‐directed monoclonal antibody
Publisher: EMBO
Date: 14-02-2022
From Sphingosine Kinase to Dihydroceramide Desaturase: A Structure–Activity Relationship (SAR) Study of the Enzyme Inhibitory and Anticancer Activity of 4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenol
Publisher: American Chemical Society (ACS)
Date: 03-02-2016
DOI: 10.1021/ACS.JMEDCHEM.5B01439
Abstract: The sphingosine kinase (SK) inhibitor, SKI-II, has been employed extensively in biological investigations of the role of SK1 and SK2 in disease and has demonstrated impressive anticancer activity in vitro and in vivo. However, interpretations of results using this pharmacological agent are complicated by several factors: poor SK1/2 selectivity, additional activity as an inducer of SK1-degradation, and off-target effects, including its recently identified capacity to inhibit dihydroceramide desaturase-1 (Des1). In this study, we have delineated the structure-activity relationship (SAR) for these different targets and correlated them to that required for anticancer activity and determined that Des1 inhibition is primarily responsible for the antiproliferative effects of SKI-II and its analogues. In the course of these efforts, a series of novel SK1, SK2, and Des1 inhibitors have been generated, including compounds with significantly greater anticancer activity.
Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma
Publisher: Springer Science and Business Media LLC
Date: 24-09-2018
Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity
Publisher: Elsevier BV
Date: 2018
Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.
Publisher: Wiley
Date: 24-07-2021
Abstract: Multiple myeloma (MM) is the second most common haematological malignancy and is an incurable disease of neoplastic plasma cells (PC). Newly diagnosed MM patients currently undergo lengthy genetic testing to match chromosomal mutations with the most potent drug/s to decelerate disease progression. With only 17% of MM patients surviving 10‐years postdiagnosis, faster detection and earlier intervention would unequivocally improve outcomes. Here, we show that the cell surface protein desmoglein‐2 (DSG2) is overexpressed in ~ 20% of bone marrow biopsies from newly diagnosed MM patients. Importantly, DSG2 expression was strongly predictive of poor clinical outcome, with patients expressing DSG2 above the 70 th percentile exhibiting an almost 3‐fold increased risk of death. As a prognostic factor, DSG2 is independent of genetic subtype as well as the routinely measured biomarkers of MM activity (e.g. paraprotein). Functional studies revealed a nonredundant role for DSG2 in adhesion of MM PC to endothelial cells. Together, our studies suggest DSG2 to be a potential cell surface biomarker that can be readily detected by flow cytometry to rapidly predict disease trajectory at the time of diagnosis.
Targeting sphingosine kinase 1 in acute myeloid leukemia: translation to clinic
Publisher: Future Medicine Ltd
Date: 11-2017
Isoform-Selective Assays for Sphingosine Kinase Activity
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-800-9_2
Abstract: Sphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form -sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both extracellularly via cell surface S1P receptors and intracellularly through a number of recently identified protein targets. The two enzymes arise from different genes, and differ in their cellular localisation, developmental expression, catalytic properties, and in at least some functional roles. Here, we describe methods for selectively detecting SK1 and SK2 activities in vitro, highlighting conditions that can discriminate between the activities of these two enzymes. The assays measure the production of (32)P-labelled S1P following the addition of exogenous sphingosine and [γ(32)P] adenosine-5'-triphosphate. The S1P product can be purified by Bligh-Dyer solvent extraction, separated by thin-layer chromatography (TLC), and the radiolabelled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 and SK2 activities in tissue, cell, and recombinant protein s les.
Regulation of Sphingosine Kinase in Hematological Malignancies and Other Cancers
Publisher: Bentham Science Publishers Ltd.
Date: 11-2011
DOI: 10.2174/187152011797655078
Abstract: The sphingolipids ceramide, sphingosine and sphingosine 1-phosphate have emerged as important signaling molecules that regulate a number of important cellular processes. Sphingosine 1-phosphate enhances cell survival and proliferation, and also regulates angiogenesis, cell invasion, and differentiation via both its cell surface G protein-coupled receptors and recently identified intracellular effectors. In contrast, ceramide and sphingosine elicit growth arrest and apoptosis through direct modulation of a number of intracellular targets. The cellular balance of these sphingolipids contributes to the determination of cell fate, and it is now clear that disruption in this 'sphingolipid rheostat' contributes to the development, progression and chemotherapeutic resistance of both hematological malignancies and solid tumors. The sphingosine kinases are central regulators of this pathway since they not only increase sphingosine 1-phosphate and assist in reduction of ceramide and sphingosine, but are also regulated at multiple levels by external stimuli. Thus, targeting the regulation of the sphingosine kinases may be a viable therapeutic strategy for a erse array of cancers. Here, we describe the current knowledge of sphingosine kinase regulation, effects of current and potential chemotherapeutic agents on this system, and discuss the implications of this for the treatment of hematological malignancies and other cancers.
Resensitising proteasome inhibitor-resistant myeloma with sphingosine kinase 2 inhibition
Publisher: Elsevier BV
Date: 2022
The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function.
Publisher: Elsevier BV
Date: 06-2006
The role of the extracellular matrix and its molecular and cellular regulators in cancer cell plasticity
Publisher: Frontiers Media SA
Date: 09-10-2018
Inhibitors of the Sphingosine Kinase Pathway as Potential Therapeutics
Publisher: Bentham Science Publishers Ltd.
Date: 06-2010
DOI: 10.2174/156800910791208599
Abstract: Sphingosine kinase (SK) 1 and 2 are lipid kinases that phosphorylate sphingosine to form sphingosine-1 phosphate, a potent signalling molecule with pleiotrophic effects. SK1 is commonly up-regulated in tumours and its inhibition or genetic ablation has been shown to slow tumour growth as well as sensitise cancer cells to other chemotherapeutics. Therefore, SK1 is of particular interest as a target therapeutic intervention in cancer. Initial SK inhibitors were sphingosine derivatives and displayed efficacy in a number of disease models, establishing a premise for SK inhibition for anti-proliferative and anti-inflammatory therapies, even though these compounds had questionable specificity. More recently, a number of new SK inhibitors have been developed that display higher affinities and greater specificity for the SKs. Here we summarise the current small molecule inhibitors and related approaches for targeting the SKs, and their in vitro and in vivo efficacy. Furthermore, we highlight findings demonstrating the success of SK inhibition in cancer and a range of other disease models that promotes the continued interest in targeting the SKs for therapeutic benefit.
Examining the Role of Sphingosine Kinase-2 in the Regulation of Endothelial Cell Barrier Integrity
Publisher: Wiley
Date: 04-2016
DOI: 10.1111/MICC.12271
Abstract: A key mediator of vascular EC barrier integrity, S1P, is derived from phosphorylation of sphingosine by the SK-1 and SK-2. While previous work indicates that SK-1 can regulate EC barrier integrity, whether SK-2 has a similar role remains to be determined. A cell impedance assay was used to assess human umbilical vein EC and bone marrow EC barrier integrity in vitro, with application of the SK inhibitors ABC294640, PF543, SKi, and MP-A08. In vivo studies were conducted using intravital microscopy to assess EC barrier integrity in SK-1 (Sphk1(-/-)) and SK-2 (Sphk2(-/-)) knock-out mice. Only ABC294640 and MP-A08, which can both inhibit SK-2, caused a decrease in EC barrier integrity in vitro in both cell types. Intravital microscopy revealed that Sphk1(-/-) mice had reduced EC barrier integrity compared to WT mice, whereas no change was evident in Sphk2(-/-) mice. Our data suggest that in vitro inhibition of SK-2, can compromise the integrity of the EC monolayer, while SK-1 exerts a more dominant control in vivo. These data may have clinical implications and could aid in the development of new treatments for disorders of vascular barrier function.
The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells
Publisher: Walter de Gruyter GmbH
Date: 2009
DOI: 10.2478/S11658-009-0008-2
Abstract: The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.
Action patterns and mapping of the substrate-binding regions of endo-(1 → 5)-α-l-arabinanases from Aspergillus niger and Aspergillus aculeatus
Publisher: Elsevier BV
Date: 09-1997
DOI: 10.1016/S0008-6215(97)00159-6
Abstract: The substrate binding sites of endo-(1-->5)-alpha-L-arabinanases (EC 3.2.1.99) from Aspergillus niger and Aspergillus aculeatus were investigated using reduced and regular (1-->5)-alpha-L-arabino-oligosaccharides and high performance anion exchange chromatographic analysis. Calculation of bond cleavage frequencies and kcat/K(m) parameters for these substrates enabled the determination of the number of arabinofuranosyl binding subsites and the estimation of the binding affinities of each subsite. The A. aculeatus endo-arabinanase has six subsites arranged symmetrically around the catalytic site, while the A. niger endo-arabinanase has five subsites two from the catalytic site towards the non-reducing end of the bound substrate and three toward the reducing end. The two subsites directly adjacent to the catalytic sites in both the A. niger and A. aculeatus endo-arabinanase have near-zero net free energy of binding. These results are unlike most glycopyranosyl endo-hydrolases studied which have net negative (unfavourable) energies of interaction at these two subsites, and may be related to the greater conformational flexibility of arabinofuranosyl residues than glycopyranosyl residues. The complete subsite maps are also rationalized with regard to the observed action patterns of these enzymes on linear (1-->5)-alpha-L-arabinan.
Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells
Publisher: Oxford University Press (OUP)
Date: 11-2005
DOI: 10.1634/STEMCELLS.2004-0338
Abstract: Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.
Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling
Publisher: Rockefeller University Press
Date: 28-12-2004
DOI: 10.1084/JEM.20040559
Abstract: Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a erse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal–regulated kinase 1/2–mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK1 is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK1 to the plasma membrane, rather than from enhanced catalytic activity of this enzyme.
Dengue Virus Infection of Primary Endothelial Cells Induces Innate Immune Responses, Changes in Endothelial Cells Function and Is Restricted by Interferon-Stimulated Responses
Publisher: Mary Ann Liebert Inc
Date: 08-2015
Abstract: Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study, we investigated DENV infection of primary EC and defined factors that influence infection in this cell type. Consistent with in vivo findings where EC infection is infrequent, only 3%-15% of EC became productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was associated with induction of innate immune responses, including increased STAT1 protein, STAT1- phosphorylation, interferon (IFN)-β, OAS-1, IFIT-1/ISG56, and viperin mRNA. Antibody blocking of IFN-β inhibited the induction of OAS1, IFIT1/ISG56, and viperin while shRNA knockdown of viperin enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and stimulated changes in barrier integrity of DENV-infected EC monolayers. Thus, DENV productively infects only a small percentage of primary EC but this has a major influence on induction of IFN-β driven innate immune responses that can restrict infection while the EC themselves are functionally altered. These changes may have important consequences for the endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in DENV disease.
Expression of a Catalytically Inactive Sphingosine Kinase Mutant Blocks Agonist-induced Sphingosine Kinase Activation
Publisher: Elsevier BV
Date: 10-2000
Potential link between the sphingosine-1-phosphate (S1P) system and defective alveolar macrophage phagocytic function in chronic obstructive pulmonary disease (COPD)
Publisher: Public Library of Science (PLoS)
Date: 20-10-2015
Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: Functional differences between sphingosine kinase 1a and 1b
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.BIOCEL.2012.05.012
Abstract: Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel 'see-saw' model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed 'see-saw' model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.
An oncogenic role of sphingosine kinase
Publisher: Elsevier BV
Date: 11-2000
DOI: 10.1016/S0960-9822(00)00834-4
Abstract: Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate erse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.
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
Publisher: Impact Journals, LLC
Date: 25-02-2016
Sphingolipids—who’s controlling who in disease?
Publisher: Wiley
Date: 04-08-2015
DOI: 10.1038/ICB.2015.70
The effect of dihydroceramide desaturase 1 inhibition on endothelial impairment induced by indoxyl sulfate.
Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.VPH.2021.106923
Abstract: Protein-bound uremic toxins (PBUTs) have adverse effects on vascular function, which is imperative in the progression of cardiovascular and renal diseases. The role of sphingolipids in PBUT-mediated vasculo-endothelial pathophysiology is unclear. This study assessed the therapeutic potential of dihydroceramide desaturase 1 (Des1) inhibition, the last enzyme involved in de novo ceramide synthesis, to mitigate the vascular effects of the PBUT indoxyl sulfate (IS). Rat aortic rings were isolated and vascular reactivity was assessed in organ bath experiments followed by immunohistochemical analyses. Furthermore, cultured human aortic endothelial cells were assessed for phenotypic and mechanistic changes. Inhibition of Des1 by a selective inhibitor CIN038 (0.1 to 0.3 μM) improved IS-induced impairment of vasorelaxation and modulated immunoreactivity of oxidative stress markers. Des1 inhibition also reversed IS-induced reduction in endothelial cell migration (1.0 μM) by promoting the expression of angiogenic cytokines and reducing inflammatory and oxidative stress markers. These effects were associated with a reduction of TIMP1 and the restoration of Akt phosphorylation. In conclusion, Des1 inhibition improved vascular relaxation and endothelial cell migration impaired by IS overload. Therefore, Des1 may be a suitable intracellular target to mitigate PBUT-induced adverse vascular effects.
Meeting Report: Barossa 2005--Signaling Networks
Publisher: American Association for the Advancement of Science (AAAS)
Date: 28-02-2006
Abstract: Cellular signal transduction involves an elaborate network of interrelated signaling pathways. Dissecting the components of these signaling pathways and the functional relationships between them is crucial to our understanding of biological processes. This was the central theme of the November 2005 Signaling Networks meeting held in the Barossa Valley, South Australia. The meeting highlighted recent exciting advances in this area, covering topics such as the initiation, integration, regulation, and architecture of signaling networks, and the importance of these pathways in normal physiological functions and pathophysiological processes.
A critical role for the protein phosphatase 2A B′α regulatory subunit in dephosphorylation of sphingosine kinase 1
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.BIOCEL.2010.11.001
Abstract: Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and erse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.
Mitochondrial translocation of TFEB regulates complex I and inflammation
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2023
DOI: 10.1101/2023.02.27.530219
Abstract: TFEB is a master regulator of autophagy, lysosome biogenesis and mitochondrial metabolism that works, and immunity, primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomic analysis revealed extensive TFEB co-precipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. Localization of TFEB in the mitochondrial matrix was confirmed by high resolution confocal microscopy and biochemistry with translocation dependent on a conserved N-terminal TOMM20-binding motif enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.
Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia
Publisher: American Society of Hematology
Date: 30-06-2022
Abstract: Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
Sphingosine kinase 2-deficiency mediated changes in spinal pain processing
Publisher: Frontiers Media SA
Date: 03-08-2015
A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties
Publisher: Impact Journals, LLC
Date: 11-03-2015
Abstract: The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.
Production of β-glucan degrading enzymes by Acremonium and Cephalosporium species
Publisher: Elsevier BV
Date: 02-1997
Sphingosine Kinases: Biochemistry, Regulation, and Roles
Publisher: Wiley
Date: 19-02-2013
Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3′ untranslated region of dengue virus type-2 RNA
Publisher: Microbiology Society
Date: 11-2013
Abstract: Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 3′ UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and co-precipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.
Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity.
Publisher: Elsevier BV
Date: 04-2008
Sphingosine kinase localization in the control of sphingolipid metabolism
Publisher: Elsevier BV
Date: 2011
Sphingosine kinase 1 is a critical component of the copper-dependent FGF1 export pathway
Publisher: Elsevier BV
Date: 09-2007
The Nucleotide-binding Site of Human Sphingosine Kinase 1
Publisher: Elsevier BV
Date: 12-2002
The tricarboxylic acid cycle of Helicobacter pylori
Publisher: Wiley
Date: 05-02-1999
DOI: 10.1046/J.1432-1327.1999.00153.X
Abstract: The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-γ that regulates neoangiogenesis
Publisher: Wiley
Date: 09-2015
DOI: 10.1096/FJ.14-261289
Characterising Distinct Migratory Profiles of Infiltrating T-Cell Subsets in Human Glioblastoma
Publisher: Frontiers Media SA
Date: 06-04-2022
DOI: 10.3389/FIMMU.2022.850226
Abstract: Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is T-cell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastoma-infiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.
Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection.
Publisher: Wiley
Date: 07-2017
DOI: 10.1038/CTI.2017.32
Sphingosine Kinase Interacts with TRAF2 and Dissects Tumor Necrosis Factor-α Signaling
Publisher: Elsevier BV
Date: 03-2002
Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion
Publisher: Elsevier BV
Date: 03-2021
DOI: 10.1016/J.CUB.2021.01.003
Abstract: Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation.
Basal and angiopoietin-1-mediated endothelial permeability is regulated by sphingosine kinase-1
Publisher: American Society of Hematology
Date: 04-2008
DOI: 10.1182/BLOOD-2007-05-092148
Abstract: Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)–regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P1, S1P2, or S1P3, did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1–induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability.
Noncellulolytic fungal β-glucanases: Their physiology and regulation
Publisher: Elsevier BV
Date: 03-1993
DOI: 10.1016/0141-0229(93)90136-P
Abstract: The occurrence, regulation, and action of fungal enzymes capable of degrading noncellulosic beta-glucans, especially 1,3-beta- and 1,6-beta-glucans, are reviewed. Special consideration is given to their roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis. Also examined are the protocols currently available for their purification, with some of the properties of purified beta-glucanases discussed in terms of their potential applications in industrial, agricultural, and medical fields.
The involvement of sphingosine kinase 1 in LPS-induced Toll-like receptor 4-mediated accumulation of HIF-1α protein, activation of ASK1 and production of the pro-inflammatory cytokine IL-6
Publisher: Wiley
Date: 27-07-2010
DOI: 10.1038/ICB.2010.91
Abstract: Toll-like receptors (TLRs) lie in the core of resistance to infectious diseases allowing host immune cells to specifically detect pathogens by recognising their specific molecular patterns. Cell membrane-associated TLR4 (recognises lipopolysaccharide (LPS) of Gram-negative bacteria) and endosomal TLR7/8 (recognise viral single-stranded RNA) are known to activate hypoxia inducible factor-1α (HIF-1α) protein (necessary for cellular adaptation to the inflammatory stress) via redox-dependent mechanism. TLR4 triggers the cross talk between HIF-1α and apoptosis signal-regulating kinase 1 (ASK1), whereas TLR7/8 activates HIF-1α in the ASK1-independent manner. Here, we report that in THP-1 and RAW264.7 macrophages, ligand-induced activation of the TLR4 but not TLR7/8 induces activation and transcriptional upregulation of sphingosine kinase 1 (SphK1) in extracellular signal-regulating kinase and phospholipase C-1γ/PI3 kinase-dependent manner. TLR4-mediated SphK1 activation was found to be critical for the redox-dependent activation of HIF-1α and ASK1, as well as for the prevention of LPS-induced activation of caspase 3 and the expression of pro-inflammatory cytokine interleukin-6.
Targeting sphingosine kinase 2 suppresses MYC expression and kills acute lymphoblastic leukemia cells
Publisher: Elsevier BV
Date: 08-2013
Sphingosine kinase 1 (SK1) is recruited to nascent phagosomes in human macrophages: inhibition of SK1 translocation by Mycobacterium tuberculosis.
Publisher: The American Association of Immunologists
Date: 15-03-2005
DOI: 10.4049/JIMMUNOL.174.6.3551
Abstract: Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.
The calmodulin-binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane.
Publisher: Elsevier BV
Date: 04-2006
Perspectives for Ag
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9NR05733A
Abstract: Research on near-infrared (NIR) bioimaging has progressed very quickly in the past few years, as fluorescence imaging is reaching a credible implementation as a preclinical technique. The applications of NIR bioimaging in theranostics have contributed to its increasing impact. This has brought about the development of novel technologies and, simultaneously, of new contrast agents capable of acting as efficient NIR optical probes. Among these probes, Ag
Sphingosine Kinase Transmits Estrogen Signaling in Human Breast Cancer Cells
Publisher: The Endocrine Society
Date: 10-2003
DOI: 10.1210/ME.2003-0119
Abstract: Current understanding of cytoplasmic signaling pathways that mediate estrogen action in human breast cancer is incomplete. Here we report that treatment with 17β-estradiol (E2) activates a novel signaling pathway via activation of sphingosine kinase (SphK) in MCF-7 breast cancer cells. We found that E2 has dual actions to stimulate SphK activity, i.e. a rapid and transient activation mediated by putative membrane G protein-coupled estrogen receptors (ER) and a delayed but prolonged activation relying on the transcriptional activity of ER. The E2-induced SphK activity consequently activates downstream signal cascades including intracellular Ca2+ mobilization and Erk1/2 activation. Enforced expression of human SphK type 1 gene in MCF-7 cells resulted in increases in SphK activity and cell growth. Moreover, the E2-dependent mitogenesis were highly promoted by SphK overexpression as determined by colony growth in soft agar and solid focus formation. In contrast, expression of SphKG82D, a dominant-negative mutant SphK, profoundly inhibited the E2-mediated Ca2+ mobilization, Erk1/2 activity and neoplastic cell growth. Thus, our data suggest that SphK activation is an important cytoplasmic signaling to transduce estrogen-dependent mitogenic and carcinogenic action in human breast cancer cells.
Enhanced expression of transferrin receptor 1 contributes to oncogenic signalling by sphingosine kinase 1
Publisher: Springer Science and Business Media LLC
Date: 25-11-2013
DOI: 10.1038/ONC.2013.502
Abstract: Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cell-surface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis.
Clinical MDR1 inhibitors enhance Smac-mimetic bioavailability to kill murine LSCs and improve survival in AML models
Publisher: American Society of Hematology
Date: 20-10-2020
DOI: 10.1182/BLOODADVANCES.2020001576
Abstract: The specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML s les to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.
Intracranial Injection of Dengue Virus Induces Interferon Stimulated Genes and CD8+ T Cell Infiltration by Sphingosine Kinase 1 Independent Pathways.
Publisher: Public Library of Science (PLoS)
Date: 17-01-2017
Sphingolipids and the unfolded protein response
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.BBALIP.2019.06.002
Abstract: The unfolded protein response (UPR) is a response by the endoplasmic reticulum to stress, classically caused by any disruption to cell homeostasis that results in an accumulation in unfolded proteins. However, there is an increasing body of research demonstrating that the UPR can also be activated by changes in lipid homeostasis, including changes in sphingolipid metabolism. Sphingolipids are a family of bioactive lipids with important roles in both the formation and integrity of cellular membranes, and regulation of key cellular processes, including cell proliferation and apoptosis. Bi-directional interactions between sphingolipids and the UPR have now been observed in a range of diseases, including cancer, diabetes and liver disease. Determining how these two key cellular components influence each other could play an important role in deciphering the causes of these diseases and potentially reveal new therapeutic approaches.
Overexpression of sphingosine kinase 1 prevents ceramide accumulation and ameliorates muscle insulin resistance in high-fat diet-fed mice
Publisher: American Diabetes Association
Date: 15-11-2012
DOI: 10.2337/DB12-0029
Abstract: The sphingolipids sphingosine-1-phosphate (S1P) and ceramide are important bioactive lipids with many cellular effects. Intracellular ceramide accumulation causes insulin resistance, but sphingosine kinase 1 (SphK1) prevents ceramide accumulation, in part, by promoting its metabolism into S1P. Despite this, the role of SphK1 in regulating insulin action has been largely overlooked. Transgenic (Tg) mice that overexpress SphK1 were fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK1 Tg mice fed an HFD displayed increased SphK activity in skeletal muscle, which was associated with an attenuated intramuscular ceramide accumulation compared with wild-type (WT) littermates. This was associated with a concomitant reduction in the phosphorylation of c-jun amino-terminal kinase, a serine threonine kinase associated with insulin resistance. Accordingly, skeletal muscle and whole-body insulin sensitivity were improved in SphK1 Tg, compared with WT mice, when fed an HFD. We have identified that the enzyme SphK1 is an important regulator of lipid partitioning and insulin action in skeletal muscle under conditions of increased lipid supply.
Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.
Publisher: Portland Press Ltd.
Date: 23-08-2000
DOI: 10.1042/BJ3500429
Abstract: Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 106-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.
Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia
Publisher: Springer Science and Business Media LLC
Date: 12-05-2022
DOI: 10.1038/S41467-022-30223-9
Abstract: The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 ( IDH1 ), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1 -mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.
Inhibition of Pol I transcription treats murine and human AML by targeting the leukemia-initiating cell population
Publisher: American Society of Hematology
Date: 25-05-2017
DOI: 10.1182/BLOOD-2016-05-718171
Abstract: Inhibition of RNA Pol I by CX-5461 treats aggressive AML and outperforms standard chemotherapy regimens. CX-5461 induces p53-dependent apoptosis, p53-independent cell-cycle defects and differentiation, and reduces LICs.
Recent advances in the development of sphingosine kinase inhibitors
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.CELLSIG.2016.06.007
Abstract: Sphingosine kinase (SK) 1 and 2 are lipid kinases that catalyse the formation of sphingosine 1-phosphate (S1P), a potent signalling molecule with a wide array of cellular effects. SK1 and 2 have been shown to be up-regulated in tumours and their genetic ablation or inhibition has been shown to slow tumour growth as well as sensitise cancer cells to chemotherapeutics. The SKs have been extensively studied, with a plethora of inhibitors developed that target the sphingosine-binding pocket of the enzyme, some with nanomolar affinities. Recently, inhibitors targeting the ATP pocket of SK have also been described. Here we discuss the development of these new small molecule SK inhibitors, summarise the recent discovery of off-targets effects of many current SK inhibitors, and provide an overview of the usefulness of these inhibitors as in vitro tools and therapeutic agents.
A point mutant of human sphingosine kinase 1 with increased catalytic activity
Publisher: Wiley
Date: 27-11-2001
DOI: 10.1016/S0014-5793(01)03162-3
Abstract: Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)-->Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)-->Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).
3D-printed microplate inserts for long term high-resolution imaging of live brain organoids.
Publisher: Springer Science and Business Media LLC
Date: 04-2021
DOI: 10.1186/S42490-021-00049-5
Abstract: Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2 mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months). Here we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates high-resolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids. This new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.
Sphingosine kinase-1 predicts overall survival outcomes in non-small cell lung cancer patients treated with carboplatin and navelbine
Publisher: Spandidos Publications
Date: 07-06-2019
Targeting sphingosine kinase 1 induces MCL1-dependent cell death in acute myeloid leukemia
Publisher: American Society of Hematology
Date: 09-02-2017
DOI: 10.1182/BLOOD-2016-06-720433
Abstract: Inhibition of SPHK1 in human AML cells induces MCL1 degradation and caspase-dependent cell death. SPHK1 inhibitors reduce leukemic burden and prolong survival in orthotopic patient-derived xenografts of AML.
Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGF beta receptor-sphingosine 1-phosphate receptor complexes in airway smooth muscle cells.
Publisher: Elsevier BV
Date: 02-2002
Purification and characterization of an extracellular β-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in β-glucan degradation
Publisher: Elsevier BV
Date: 08-1997
DOI: 10.1016/S0141-0229(96)00263-3
Abstract: A beta-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH4)2SO4 precipitation followed by anion-exchange and gel filtration chromatography. SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa. The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5. Comparison of the N-terminal amino acid sequence revealed similarities between the A. persicinum enzyme and several other extracellular fungal beta-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala. In addition to the hydrolysis of p-nitrophenyl-beta-glucoside, the enzyme was also active against several other aryl-beta-glucosides as well as a range of beta-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose. D-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by N-bromosuccinimide, N-acetylimidazole, dicyclohexyl carbodiimide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4, and some metal ions. Possible roles for this enzyme in the noncellulolytic fungus A. persicinum are discussed in light of the increase in the rate of reducing sugar release from beta-glucans by (1-->3)- and (1-->6)-beta-glucanases when the beta-glucosidase is also present in the reaction mixtures.
Sphingosine kinase 1 in viral infections
Publisher: Wiley
Date: 28-05-2012
DOI: 10.1002/RMV.1718
Abstract: Sphingosine kinase 1 (SphK1) is an enzyme that phosphorylates the lipid sphingosine to generate sphingosine-1-phosphate (S1P). S1P can act intracellularly as a signaling molecule and extracellularly as a receptor ligand. The SphK1/S1P axis has well-described roles in cell signaling, the cell death/survival decision, the production of a pro-inflammatory response, immunomodulation, and control of vascular integrity. Agents targeting the SphK1/S1P axis are being actively developed as therapeutics for cancer and immunological and inflammatory disorders. Control of cell death/survival and pro-inflammatory immune responses is central to the pathology of infectious disease, and we can capitalize on the knowledge provided by investigations of SphK1/S1P in cancer and immunology to assess its application to selected human infections. We have herein reviewed the growing literature relating viral infections to changes in SphK1 and S1P. SphK1 activity is reportedly increased following human cytomegalovirus and respiratory syncytial virus infections, and elevated SphK1 enhances influenza virus infection. In contrast, SphK1 activity is reduced in bovine viral diarrhea virus and dengue virus infections. Sphingosine analogs that modulate S1P receptors have proven useful in animal models in alleviating influenza virus infection but have shown no benefit in simian human immunodeficiency virus and lymphocytic choriomeningitis virus infections. We have rationalized a role for SphK1/S1P in dengue virus, chikungunya virus, and Ross River virus infections, on the basis of the biology and the pathology of these diseases. The increasing number of effective SphK1 and S1P modulating agents currently in development makes it timely to investigate these roles with the potential for developing modulators of SphK1 and S1P for novel anti-viral therapies.
The sphingosine 1-phosphate receptor 2/4 antagonist JTE-013 elicits off-target effects on sphingolipid metabolism
Publisher: Springer Science and Business Media LLC
Date: 10-01-2022
DOI: 10.1038/S41598-021-04009-W
Abstract: Sphingosine 1-phosphate (S1P) is a signaling lipid that has broad roles, working either intracellularly through various protein targets, or extracellularly via a family of five G-protein coupled receptors . Agents that selectively and specifically target each of the S1P receptors have been sought as both biological tools and potential therapeutics. JTE-013, a small molecule antagonist of S1P receptors 2 and 4 (S1P 2 and S1P 4 ) has been widely used in defining the roles of these receptors in various biological processes. Indeed, our previous studies showed that JTE-013 had anti-acute myeloid leukaemia (AML) activity, supporting a role for S1P 2 in the biology and therapeutic targeting of AML. Here we examined this further and describe lipidomic analysis of AML cells that revealed JTE-013 caused alterations in sphingolipid metabolism, increasing cellular ceramides, dihydroceramides, sphingosine and dihydrosphingosine. Further examination of the mechanisms behind these observations showed that JTE-013, at concentrations frequently used in the literature to target S1P 2/4 , inhibits several sphingolipid metabolic enzymes, including dihydroceramide desaturase 1 and both sphingosine kinases. Collectively, these findings demonstrate that JTE-013 can have broad off-target effects on sphingolipid metabolism and highlight that caution must be employed in interpreting the use of this reagent in defining the roles of S1P 2/4 .
The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells
Publisher: Elsevier BV
Date: 12-2010
Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence.
Publisher: F1000 Research Ltd
Date: 23-03-2016
DOI: 10.12688/F1000RESEARCH.10336.2
Abstract: Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo . Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2 -/- ) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.
Guanine nucleotides regulate sphingosine kinase 1 activation by eukaryotic elongation factor 1A and provide a mechanism for eEF1A-associated oncogenesis
Publisher: Springer Science and Business Media LLC
Date: 13-09-2010
DOI: 10.1038/ONC.2010.420
Abstract: Sphingosine kinase 1 (SK1) catalyses the formation of bioactive phospholipid sphingosine 1-phosphate (S1P). Elevated cellular SK1 activity and S1P levels enhance cell proliferation and survival, and are strongly implicated in tumourigenesis. Regulation of SK1 activity can occur through various mechanisms, including phosphorylation and protein-protein interactions. We have previously shown that eukaryotic elongation factor 1A (eEF1A) interacts with and directly activates SK1, but the mechanisms regulating this were undefined. Notably, eEF1A has GTPase activity and can exist in GTP- or GDP-bound forms, which are associated with distinct structural conformations of the protein. Here, we show that the guanine nucleotide-bound state of eEF1A regulates its ability to activate SK1, with eEF1A.GDP, but not eEF1A.GTP, enhancing SK1 activity in vitro. Furthermore, we show that enhancing cellular eEF1A.GDP levels through expression of a guanine nucleotide dissociation inhibitor of eEF1A, translationally controlled tumour protein (TCTP), increased SK1 activity in cells. We also examined a truncated isoform of eEF1A1, termed prostate tumour inducer-1 (PTI-1), which can induce neoplastic cell transformation through undefined mechanisms. PTI-1 lacks the G protein domain of eEF1A1 and is therefore unable to undergo the GTP-binding-induced conformational change. Notably, we found that PTI-1 can directly activate SK1 and that this seems to be essential for neoplastic transformation induced by PTI-1, as chemical SK1 inhibitors or overexpression of a dominant-negative SK1 blocked this process. Thus, this study defines the mechanism regulating eEF1A-mediated SK1 activation, and also establishes SK1 as being integral for PTI-1-induced oncogenesis.
Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence
Publisher: F1000 Research Ltd
Date: 06-12-2016
DOI: 10.12688/F1000RESEARCH.10336.1
Abstract: Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo . Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2 -/- ) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.
Expression profile of the sphingosine kinase signalling system in the lung of patients with chronic obstructive pulmonary disease
Publisher: Elsevier BV
Date: 11-2011
DOI: 10.1016/J.LFS.2011.08.018
Abstract: Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Despite its importance, treatment methods are limited and restricted to symptomatic care, highlighting the urgent need for new treatment options. Tissue damage in COPD is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. Lung inflammation involves the bioactive sphingolipid sphingosine 1-phosphate (S1P). We investigated lung tissue s les from 55 patients (25 with COPD) undergoing lobectomies for management of cancer. We analysed the sphingosine-kinase (SphK) mRNA expression profile, SphK enzyme activity as well as the localisation and expression of in idual proteins related to the SphK-signalling system. We show in this study for the first time a comprehensive expression profile of all synthesising enzymes, receptors and degrading enzymes of the SphK-signalling system in the human lung. Multivariate ANOVA showed that the relative mRNA expression of S1P receptor (S1PR) subtype 5 was reduced in COPD. There were strong positive correlations between the mRNA expression of S1PR5 and S1PR1 and S1PR3, and between S1PR3 and S1PR2. A significant negative correlation was found between S1PR1 and SphK protein activity. The correlations between expression levels of receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. Our findings of reduced S1PR5 in COPD and the correlation with other S1P receptors in COPD identify S1PR5 as a possible novel target for pharmacotherapy.
Purification and characterization of three extracellular (1→3)- β -d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum
Publisher: Portland Press Ltd.
Date: 15-06-1995
DOI: 10.1042/BJ3080733
Abstract: Three (1--& )-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1--& )-beta-linkages and hydrolysed a range of (1--& )-beta- and (1--& )(1--& )-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1--& )-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1--& )-beta-D-glucan glucohydrolases (EC 3.2.1.58).
Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells
Publisher: Walter de Gruyter GmbH
Date: 2009
DOI: 10.2478/S11658-009-0009-1
Abstract: Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (ECSK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. ECSK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (ECEV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in ECSK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.
Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response.
Publisher: Elsevier BV
Date: 07-2005
DOI: 10.1016/J.CELLSIG.2004.11.022
Abstract: Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.
A drug screening pipeline using 2d and 3d patient-derived in vitro models for pre-clinical analysis of therapy response in glioblastoma
Publisher: MDPI AG
Date: 21-04-2021
DOI: 10.3390/IJMS22094322
Abstract: Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microenvironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cultures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.
Reduced sphingosine kinase-1 and enhanced sphingosine 1-phosphate lyase expression demonstrate deregulated sphingosine 1-phosphate signaling in Alzheimer’s disease
Publisher: Springer Science and Business Media LLC
Date: 27-01-2014
Activation of endothelial extracellular signal-regulated kinase is essential for neutrophil transmigration: potential involvement of a soluble neutrophil factor in endothelial activation.
Publisher: The American Association of Immunologists
Date: 12-2003
DOI: 10.4049/JIMMUNOL.171.11.6097
Abstract: During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-α-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass ∼30 kDa released from neutrophils after activation.
ROCK-mediated selective activation of PERK signalling causes fibroblast reprogramming and tumour progression through a CRELD2-dependent mechanism
Publisher: Springer Science and Business Media LLC
Date: 25-05-2020
DOI: 10.1038/S41556-020-0523-Y
Abstract: It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.
Novel therapies for multiple myeloma.
Publisher: Impact Journals, LLC
Date: 28-08-2017
Isoflurane Protects Human Kidney Proximal Tubule Cells against Necrosis via Sphingosine Kinase and Sphingosine-1-Phosphate Generation
Publisher: S. Karger AG
Date: 2010
DOI: 10.1159/000298339
Abstract: i Background/Aims: /i We previously showed that the inhalational anesthetic isoflurane protects against renal ischemia reperfusion injury in part via sphingosine kinase (SK)-mediated synthesis of sphingosine-1-phosphate (S1P). In this study, we tested the hypothesis that isoflurane directly targets renal proximal tubule cells via SK activation, S1P synthesis and activation of S1P receptors to initiate cytoprotective signaling. i Methods and Results: /i Isoflurane-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt and induction of HSP70 in human kidney proximal tubule (HK-2) cells were inhibited by dimethylsphingosine (DMS), an SK inhibitor, and VPC23019, an S1P sub /3 /sub receptor selective antagonist, in HK-2 cells. A selective S1P sub /sub receptor agonist, SEW2781, mimicked isoflurane-induced phosphorylation of ERK and Akt and induction of HSP70. Moreover, isoflurane-mediated protection against H sub /sub O sub /sub -induced necrosis of HK-2 cells was significantly attenuated by an S1P sub /3 /sub receptor antagonist, VPC23019, and by SK inhibitors DMS or 4-[[4- (4-chlorophenyl)-2-thiazolyl]amino]phenol. Finally, overexpression of the SK1 enzyme in HK-2 cells protected against H sub /sub O sub /sub -induced necrosis. i Conclusions: /i Collectively, our study demonstrates that S1P released via isoflurane-mediated SK1 stimulation produces direct anti-necrotic effects probably via S1P sub /sub receptor-mediated cytoprotective signaling (ERK/Akt phosphorylation and HSP70 induction) in HK-2 cells. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of volatile anesthetics during the perioperative period.
Cellular signalling by sphingosine kinase and sphingosine 1-phosphate
Publisher: Wiley
Date: 08-2006
DOI: 10.1080/15216540600871126
Abstract: Sphingosine kinases, through the formation of the bioactive phospholipid sphingosine 1-phosphate, have been implicated in a erse range of cellular processes, including cell proliferation, apoptosis, calcium homeostasis, angiogenesis and vascular maturation. The last few years have seen a number of significant advances in understanding of the mechanisms of action, activation, cellular localisation and biological roles of these enzymes. Here we review the current understanding of the regulation of and cellular signalling by sphingosine kinase and sphingosine 1-phosphate and discuss recent findings implicating sphingosine kinase as a potential therapeutic target for the control of cancer, inflammation and a number of other diseases. We suggest that, since the activation and subcellular localization of these enzymes appear to play critical roles in their biological functions, targeting these processes may provide more specific therapeutic options than direct catalytic inhibitors.
Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics
Publisher: Impact Journals, LLC
Date: 04-05-2015
Abstract: 14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.
Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses
Publisher: Microbiology Society
Date: 2016
DOI: 10.1099/JGV.0.000334
Abstract: Sphingosine kinase (SK) 1 is a host kinase that enhances some viral infections. Here we investigated the ability of SK1 to modulate dengue virus (DENV) infection in vitro. Overexpression of SK1 did not alter DENV infection however, targeting SK1 through chemical inhibition resulted in reduced DENV RNA and infectious virus release. DENV infection of SK1⁻/ ⁻ murine embryonic fibroblasts (MEFs) resulted in inhibition of infection in an immortalized line (iMEF) but enhanced infection in primary MEFs (1°MEFs). Global cellular gene expression profiles showed expected innate immune mRNA changes in DENV-infected WT but no induction of these responses in SK1⁻/⁻ iMEFs. Reverse transciption PCR demonstrated a low-level induction of IFN-β and poor induction of mRNA for the interferon-stimulated genes (ISGs) viperin, IFIT1 and CXCL10 in DENV-infected SK1⁻/⁻ compared with WT iMEFs. Similarly, reduced induction of ISGs was observed in SK1⁻/⁻ 1°MEFs, even in the face of high-level DENV replication. In both iMEFs and 1°MEFs, DENV infection induced production of IFN-β protein. Additionally, higher basal levels of antiviral factors (IRF7, CXCL10 and OAS1) were observed in uninfected SK1⁻/⁻ iMEFs but not 1°MEFs. This suggests that, in this single iMEF line, lack of SK1 upregulates the basal levels of factors that may protect cells against DENV infection. More importantly, regardless of the levels of DENV replication, all cells that lacked SK1 produced IFN-β but were refractory to induction of ISGs such as viperin, IFIT1 and CXCL10. Based on these findings, we propose new roles for SK1 in affecting innate responses that regulate susceptibility to DENV infection.
Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1
Publisher: Elsevier BV
Date: 2009
Resistance to proteasome inhibitors and other targeted therapies in myeloma
Publisher: Wiley
Date: 20-04-2018
DOI: 10.1111/BJH.15210
Abstract: The number of novel therapies for the treatment of myeloma is rapidly increasing, as are the clinical trials evaluating them in combination with other novel and established therapies. Proteasome inhibitors, immunomodulatory agents and monoclonal antibodies are the most well known and studied classes of novel agents targeting myeloma, with histone deacetylase inhibitors, nuclear export inhibitors and several other approaches also being actively investigated. However, in parallel with the development and clinical use of these novel myeloma therapies is the emergence of novel mechanisms of resistance, many of which remain elusive, particularly for more recently developed agents. Whilst resistance mechanisms have been best studied for proteasome inhibitors, particularly bortezomib, class effects do not universally apply to all class members, and within-class differences in efficacy, toxicity and resistance mechanisms have been observed. Although immunomodulatory agents share the common cellular target cereblon and thus resistance patterns relate to cereblon expression, the unique cell surface antigens to which monoclonal antibodies are directed means these agents frequently exhibit unique within-class differences in clinical efficacy and resistance patterns. This review describes the major classes of novel therapies for myeloma, highlights the major clinical trials within each class and discusses known resistance mechanisms.
Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A
Publisher: Elsevier BV
Date: 12-2008
Production and regulation of β-glucanases in Acremonium and Cephalosporium isolates
Publisher: Elsevier BV
Date: 03-1991
Attenuation of leakiness in doxycycline-inducible expression via incorporation of 3′ AU-rich mRNA destabilizing elements
Publisher: Future Science Ltd
Date: 08-2008
DOI: 10.2144/000112896
Abstract: Tetracycline-regulated expression systems have been widely used for inducible protein expression in cultured mammalian cells. With these systems, however, leakiness in expression of the target gene in the absence of the inducing agent is a frequent problem. Here we describe a novel approach to overcome this problem that involves the incorporation of AU-rich mRNA destabilizing elements (AREs) into the 3′ untranslated regions of the tetracycline-inducible constructs. Using the inducible expression of sphingosine kinase 1 and 2 in HEK293 cells as model systems, we found this ARE approach to be remarkably successful in ablating expression of these proteins in the absence of doxycycline through decreasing stability of their mRNAs. We show that this undemanding and flexible process results in a substantial decrease in the leakiness of the tetracycline-inducible expression system while maintaining a high level of target protein expression following induction.
Lipids as central mediators of cellular signalling
Publisher: Wiley
Date: 08-2006
The Sphingolipid Rheostat: A Potential Target for Improving Pancreatic Islet Survival and Function
Publisher: Bentham Science Publishers Ltd.
Date: 12-2011
DOI: 10.2174/187153011797881201
Abstract: Beta cell apoptosis and suboptimal islet function are implicated in the development of Type I (T1D) and Type II (T2D) diabetes, as well as the failure of the only current clinical beta cell replacement therapy for T1D, islet transplantation. Sphingosine kinase (SK) is a ubiquitous lipid kinase that controls the balance between prosurvival and proapoptotic precursors (e.g. sphingosine-1-phosphate (S1P) and ceramide, respectively), the so-called 'sphingolipid rheostat', in many cell types. S1P, a potent lipid mediator, acts intracellularly through second messengers and extracellularly through five G-protein coupled receptors (S1P1-5), to promote calcium mobilization, intracellular signaling events, cytoskeleton rearrangements and mitogenesis. SK is important for revascularization responses, regulating the maturation of vascular endothelial progenitors and controlling cellular recruitment. The aim of this review is to highlight the sphingolipid rheostat in pancreatic biology as a therapeutic target for pharmacological and therapeutic intervention for diabetes and islet transplantation. SK and the sphingolipid rheostat are likely to be important for both islet function and beta cell survival and represent a common therapeutic target to protect the beta cell from diabetogenic insults and ultimately improve pancreatic islet function. A number of SK inhibitors and S1P receptor agonists/antagonists (including FTY720 (fingolimod) and its newer derivatives) have been recently described, with some now being used in the clinic. Recent developments in SK biochemistry and islet biology indicate the potential importance of the sphingolipid rheostat in determining islet survival and function. Pharmacological manipulation of this pathway represents a novel therapeutic strategy to prevent diabetes and improve islet transplantation outcomes.
Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1194/JLR.M004374
Effect of carbon source on extracellular (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium persicinum
Publisher: Canadian Science Publishing
Date: 05-1997
DOI: 10.1139/M97-061
Abstract: The effect of carbon source on the levels of three (1 → 3)-β-glucanases and a (1 → 6)-β-glucanase in the culture filtrates of the filamentous fungus Acremonium persicinum was investigated. All four enzymes were produced during growth of the fungus on (1 → 3)-, (1 → 6)-, and (1 → 3)(1 → 6)-β-glucans as well as β-linked oligoglucosides. However, only one (1 → 3)-β-glucanase and the (1 → 6)-β-glucanase were detected during growth on a range of other carbon sources including glucose, carboxymethylcellulose, and the α-glucan pullulan. The presence of glucose in the medium markedly decreased the production of all four glucanases, although the concentration required to effect complete repression of enzyme levels varied for the different enzymes. Similar repressive effects were also observed with sucrose, fructose, and galactose. The most likely explanations for these observations are that the synthesis of the (1 → 6)-β-glucanase and one of the (1 → 3)-β-glucanases is controlled by carbon catabolite repression, while the remaining two (1 → 3)-β-glucanases are inducible enzymes subject to carbon catabolite repression.Key words: (1 → 3)-β-glucanase, (1 → 6)-β-glucanase, Acremonium persicinum, regulation of synthesis, fungal β-glucanases.
Regulation of EPCs: The Gateway to Blood Vessel Formation
Publisher: Hindawi Limited
Date: 22-09-2014
DOI: 10.1155/2014/972043
Abstract: Endothelial progenitor cells (EPCs) are primitive endothelial precursors which are known to functionally contribute to the pathogenesis of disease. To date a number of distinct subtypes of these cells have been described, with differing maturation status, cellular phenotype, and function. Although there is much debate on which subtype constitutes the true EPC population, all subtypes have endothelial characteristics and contribute to neovascularisation. Vasculogenesis, the process by which EPCs contribute to blood vessel formation, can be dysregulated in disease with overabundant vasculogenesis in the context of solid tumours, leading to tumour growth and metastasis, and conversely insufficient vasculogenesis can be present in an ischemic environment. Importantly, it is widely known that transcription factors tightly regulate cellular phenotype and function by controlling the expression of particular target genes and in turn regulating specific signalling pathways. This suggests that transcriptional regulators may be potential therapeutic targets to control EPC function. Herein, we discuss the observed EPC subtypes described in the literature and review recent studies describing the role of a number of transcriptional families in the regulation of EPC phenotype and function in normal and pathological conditions.
Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism
Publisher: Elsevier BV
Date: 07-2005
Arabinans and arabinan degrading enzymes
Publisher: Elsevier
Date: 1997
Stem cell regulation by lysophospholipids
Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.PROSTAGLANDINS.2007.08.004
Abstract: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) regulate a erse range of mammalian cell processes, largely through engaging multiple G protein-coupled receptors specific for these lysophospholipids. LPA and S1P have been clearly identified to have widespread physiological and pathophysiological actions, controlling events within the reproductive, gastrointestinal, vascular, nervous and immune systems, and also having a prominent role in cancer. Here we review the recent literature showing the additional emerging role for LPA and S1P in the regulation of stem cells and their progenitors. We discuss the role of these lysophospholipids in regulating the proliferation, survival, differentiation and migration of a range of adult and embryonic stem cells and progenitors, and thus are likely to play a substantial role in the maintenance, generation, mobilisation and homing of stem cell and progenitor populations in the body.
Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases
Publisher: Wiley
Date: 25-11-1996
DOI: 10.1016/S0014-5793(96)01153-2
Abstract: The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H-NMR analysis of the action of endo-(1-->5)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of alpha-L-arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatum and Bacillus subtilis were investigated using both 1H-NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. niger we predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1-->4)-beta-D-arabinoxylan arabinohydrolases from A. awamori, Trichoderma reesei and Bifidobacterium adolescentis only enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.
Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase
Publisher: Oxford University Press (OUP)
Date: 05-1998
DOI: 10.1104/PP.117.1.153
Abstract: A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).
Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology
Publisher: Cold Spring Harbor Laboratory
Date: 07-12-2021
DOI: 10.1101/2021.12.07.471519
Abstract: Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, ision and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.
Sphingosine Kinase-1 Associates with Integrin alpha(V)beta(3) to Mediate Endothelial Cell Survival
Publisher: Elsevier BV
Date: 11-2009
Slit‐Robo signalling establishes a Sphingosine‐1‐phosphate gradient to polarise fin mesenchyme
Publisher: EMBO
Date: 09-06-2022
CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1
Publisher: Springer Science and Business Media LLC
Date: 12-12-2017
DOI: 10.1038/ONC.2016.428
Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation
Publisher: Wiley
Date: 15-10-2003
DOI: 10.1093/EMBOJ/CDG540
Sphingosine kinase 2 inhibition synergises with bortezomib to target myeloma by enhancing endoplasmic reticulum stress
Publisher: Impact Journals, LLC
Date: 14-04-2017
Sphingosine kinase-1 activity and expression in human prostate cancer resection specimens
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.EJCA.2010.07.053
Abstract: Sphingosine kinase-1 (SphK1) was shown in preclinical models and non-genitourinary cancers to be instrumental in cancer progression, adaptation to hypoxia and in tumour angiogenesis. No data were available in human prostate cancer. The present study was designed to assess SphK1 expression and activity in radical prostatectomy specimens and to research correlations with clinical features. Transverse section of fresh tissue was obtained from 30 consecutive patients undergoing laparoscopic prostatectomy. SphK1 enzymatic activities of tumour and normal counterpart were determined. Relationships with PSA, Gleason sum, pathological stage, resection margin status and treatment failure were researched. SphK1 pattern of expression was then assessed on tissue microarray. A significant 2-fold increase in SphK1 enzymatic activity(11.1 ± 8.4 versus 5.9 ± 3.2 (P<0.04)) was observed in cancer. The upper quartile of SphK1 activity was associated with higher PSA (16.7 versus 6.4 ng/ml, P = 0.04), higher tumor volumes (20.7 versus 9.8, P = 0.002), higher rates of positive margins (85.7% versus 28.6%, P = 0.01) and surgical failure (71.4% versus 9.5%, P = 0.003) than the lower three quartiles. Odds ratios (OR) for treatment failure showed a strong relationship with SphK1 activity (OR: 23.7, P = 0.001), positive resection margins (OR: 15.0, P = 0.007) and Gleason sum (≥4+3, OR: 8.0, P = 0.003). Tissue microarrays showed discrete epithelial expression that varied with Gleason sum with significant relationship between SphK1 expression and higher Gleason sum. In complement to preclinical literature, the demonstrated relationships between SphK1-increased activity in cancer and relevant clinical features confirm a central role for SphK1 in prostate cancer that herald promising avenues in risk-assessment and treatment.
The CCT/TRiC chaperonin is required for maturation of sphingosine kinase 1
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.BIOCEL.2008.08.012
Abstract: Sphingosine kinase 1 (SK1) catalyses the generation of sphingosine 1-phosphate (S1P), a bioactive phospholipid that influences a erse range of cellular processes, including proliferation, survival, adhesion, migration, morphogenesis and differentiation. SK1 is controlled by various mechanisms, including transcriptional regulation, and post-translational activation by phosphorylation and protein-protein interactions which can regulate both the activity and localisation of this enzyme. To gain a better understanding of the regulatory mechanisms controlling SK1 activity and function we performed a yeast two-hybrid screen to identify SK1-interacting proteins. Using this approach we identified that SK1 interacts with subunit 7 (eta) of cytosolic chaperonin CCT (chaperonin containing t-complex polypeptide, also called TRiC for TCP-1 ring complex), a hexadecameric chaperonin that binds unfolded polypeptides and mediates their folding and release in an ATP-dependent manner. Further analysis of the SK1-CCTeta interaction demonstrated that other CCT/TRiC subunits also associated with SK1 in HEK293T cell lysates in an ATP-sensitive manner, suggesting that the intact, functional, multimeric CCT/TRiC complex associated with SK1. Furthermore, pulse-chase studies indicated that CCT/TRiC binds specifically to newly translated SK1. Finally, depletion of functional CCT/TRiC through the use of RNA interference in HeLa cells or temperature sensitive CCT yeast mutants reduced cellular SK1 activity. Thus, combined this data suggests that SK1 is a CCT/TRiC substrate, and that this chaperonin facilitates folding of newly translated SK1 into its mature active form.
Sphingosine Kinase-1 Is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters
Publisher: MDPI AG
Date: 19-01-2022
Abstract: The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA s les (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones.
In vitro and in vivo roles of sphingosine kinase 2 during dengue virus infection
Publisher: Microbiology Society
Date: 04-2019
DOI: 10.1099/JGV.0.001245
Abstract: There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2
Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors
Publisher: Elsevier BV
Date: 05-2009
DOI: 10.1016/J.BBALIP.2009.01.019
Abstract: Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3-5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein alpha-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to G(i) and G12/13 signalling pathways.
Targeting sphingolipid metabolism as an approach for combination therapies in haematological malignancies
Publisher: Springer Science and Business Media LLC
Date: 28-06-2018
DOI: 10.1038/S41420-018-0075-0
Abstract: Conventional chemotherapy-based drug combinations have, until recently, been the backbone of most therapeutic strategies for cancer. In a time of emerging rationale drug development, targeted therapies are beginning to be added to traditional chemotherapeutics to synergistically enhance clinical responses. Of note, the importance of pro-apoptotic ceramide in mediating the anti-cancer effects of these therapies is becoming more apparent. Furthermore, reduced cellular ceramide in favour of pro-survival sphingolipids correlates with tumorigenesis and most importantly, drug resistance. Thus, agents that manipulate sphingolipid metabolism have been explored as potential anti-cancer agents and have recently demonstrated exciting potential to augment the efficacy of anti-cancer therapeutics. This review examines the biology underpinning these observations and the potential use of sphingolipid manipulating agents in the context of existing and emerging therapies for haematological malignancies.
FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells
Publisher: Elsevier BV
Date: 10-2010
TRAF2 regulates TNF and NF-kappa B signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1
Publisher: eLife Sciences Publications, Ltd
Date: 23-12-2015
DOI: 10.7554/ELIFE.10592
Abstract: TRAF2 is a component of TNF superfamily signalling complexes and plays an essential role in the regulation and homeostasis of immune cells. TRAF2 deficient mice die around birth, therefore its role in adult tissues is not well-explored. Furthermore, the role of the TRAF2 RING is controversial. It has been claimed that the atypical TRAF2 RING cannot function as a ubiquitin E3 ligase but counterclaimed that TRAF2 RING requires a co-factor, sphingosine-1-phosphate, that is generated by the enzyme sphingosine kinase 1, to function as an E3 ligase. Keratinocyte-specific deletion of Traf2, but not Sphk1 deficiency, disrupted TNF mediated NF-κB and MAP kinase signalling and caused epidermal hyperplasia and psoriatic skin inflammation. This inflammation was driven by TNF, cell death, non-canonical NF-κB and the adaptive immune system, and might therefore represent a clinically relevant model of psoriasis. TRAF2 therefore has essential tissue specific functions that do not overlap with those of Sphk1.
Rapid Histamine-Induced Neutrophil Recruitment Is Sphingosine Kinase-1 Dependent
Publisher: Elsevier BV
Date: 04-2012
DOI: 10.1016/J.AJPATH.2011.12.024
Abstract: Leukocyte recruitment to sites of inflammation is critical for the development of acute allergic responses. Rapid P-selectin up-regulation by endothelial cells is a key promoter of leukocyte infiltration in response to mediators such as histamine. However, the mechanisms underpinning this process are still incompletely understood. We examined the role of the sphingosine kinase/sphingosine-1-phosphate (SK/S1P) pathway and showed that in human umbilical vein endothelial cells, histamine rapidly activates SK in an extracellular signal-regulated kinase (ERK) 1/2-dependent manner, concurrent with the induction of P-selectin expression. Histamine activated both SK-1 and SK-2 isoforms inhibition of SK-1, but not SK-2, attenuated histamine-induced P-selectin up-regulation and neutrophil rolling in vitro. S1P receptor antagonists failed to prevent histamine-induced P-selectin expression, and exogenous S1P did not increase P-selectin expression, suggesting that S1P cell surface receptors are not involved in this process. Finally, the role of both SK-1 and SK-2 in histamine-induced leukocyte rolling in vivo was assessed using pharmacological and genetic methods. Consistent with the in vitro findings, mice pretreated with either sphingosine kinase inhibitor or fingolimod (FTY720) significantly attenuated histamine-induced leukocyte rolling in the cremaster muscle. Similarly, Sphk1(-/-) but not Sphk2(-/-) mice exhibited reduced histamine-induced leukocyte rolling. These findings demonstrate a key role for SK-1 in histamine-induced rapid P-selectin up-regulation and associated leukocyte rolling, and suggest that endothelial SK-1 is an important contributor to allergic inflammation.
The localization and activity of sphingosine kinase 1 are coordinately regulated with actin cytoskeletal dynamics in macrophages
Publisher: Elsevier BV
Date: 08-2007
Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy
Publisher: Wiley
Date: 2020
DOI: 10.1002/CTI2.1191
Author Correction: Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia
Publisher: Springer Science and Business Media LLC
Date: 15-07-2022
Sphingosine and FTY720 directly bind pro-survival 14-3-3 proteins to regulate their function
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.CELLSIG.2010.04.004
Abstract: The dimeric 14-3-3 protein family protects cells from apoptosis by regulating pro-apoptotic molecules. Conversely, the cationic lipid sphingosine is associated with physiological apoptosis and induces apoptosis in its own right by a largely undefined mechanism. We show here that sphingosine and 14-3-3 interact directly in the control of cell death. The binding of sphingosine to 14-3-3 proteins renders them phosphorylatable at the dimer interface, an event that abolishes the pro-survival signalling of 14-3-3. Sphingosine kinase 1 reduces availability of sphingosine for interaction with 14-3-3, thus inhibiting cell death and providing a new mechanistic insight into the role of this enzyme in cell survival and oncogenesis. Importantly, FTY720, a sphingosine analogue with apoptotic activity that is currently in phase III clinical trials for multiple sclerosis, acts in a similar manner to sphingosine in potentiating 14-3-3 phosphorylation. The biological significance of 14-3-3 phosphorylation was demonstrated with a non-phosphorylatable 14-3-3zeta mutant which retarded apoptosis induced by sphingosine and FTY720. These results demonstrate that direct association of sphingosine with 14-3-3 is required for 14-3-3 phosphorylation, and that this axis can control cell fate. Furthermore, these results suggest a new therapeutic activity for FTY720 as an anti-cancer agent based on this mechanism.
Integrated in silico and experimental assessment of disease relevance of PCDH19 missense variants.
Publisher: Hindawi Limited
Date: 15-06-2021
DOI: 10.1002/HUMU.24237
Abstract: PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.
Author Correction: Targeting sphingolipid metabolism as an approach for combination therapies in haematological malignancies
Publisher: Springer Science and Business Media LLC
Date: 18-06-2021
Regulation of sphingosine kinase and sphingolipid signaling
Publisher: Elsevier BV
Date: 02-2011
DOI: 10.1016/J.TIBS.2010.08.001
Abstract: Bioactive sphingolipids, including ceramide, sphingosine and sphingosine 1-phosphate are important regulators of many cellular processes, including cell survival, proliferation, differentiation, migration and immune responses. Although the levels of these bioactive sphingolipids are regulated by complex pathways subject to spatial and temporal control, the sphingosine kinases have emerged as critical central regulators of this system and, as a consequence, they have received substantial recent attention as potential therapeutic targets for cancer and a range of other conditions. Deciphering the molecular mechanisms that regulate both the activity and subcellular localization of these enzymes is vital for understanding the control of bioactive sphingolipid generation and action, and has clear implications for therapeutic strategies targeting these enzymes.
Induction and carbon source control of extracellular β-glucosidase production in Acremonium persicinum
Publisher: Elsevier BV
Date: 02-1999
14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function
Publisher: Springer Science and Business Media LLC
Date: 27-09-2016
DOI: 10.1038/NCOMMS12862
Abstract: The 14-3-3 family of adaptor proteins regulate erse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.
Science amongst the vines. Meeting on Signalling Systems
Publisher: EMBO
Date: 11-04-2008
The near‐infrared autofluorescence fingerprint of the brain
Publisher: Wiley
Date: 19-08-2020
The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-2009
DOI: 10.1161/ATVBAHA.109.194803
Abstract: Objective— The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results— In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1 S225A reduced resting and myogenic tone, resting Ca 2+ , pressure-induced Ca 2+ elevations, and Ca 2+ sensitivity. The lack of function of the Sk1 S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation almitylation motif the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca 2+ sensitivity and myogenic tone were attenuated, whereas Ca 2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca 2+ sensitivity) and intracellular effects of S1P (Ca 2+ handling). Accordingly, S1P 2 receptor inhibition (1μmol/L JTE013) attenuated myogenic tone without effect on Ca 2+ . Conclusions— Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.
Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 alpha-galactosidase.
Publisher: Springer Science and Business Media LLC
Date: 03-11-1999
Abstract: Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an alpha-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (alpha-->alpha), releasing alpha-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As alpha-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of alpha-galactosides using the transgalactosylation activity of Bi. adolescentis alpha-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 degrees C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and tetrasaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp- (1-->6)-alpha-D-Galp-(1-->6)-D-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride alpha-D-Galp-(1-->6)-alpha- D-Galp-(1-->6)-D-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.
GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma.
Publisher: BMJ
Date: 09-2022
Abstract: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited. We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.
Proteolytic inactivation of an extracellular (1 â 3)- β -glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH
Publisher: Oxford University Press (OUP)
Date: 12-1996
DOI: 10.1111/J.1574-6968.1996.TB08591.X
Abstract: The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1-->3)-beta-glucanases produced by this fungus, although the activities of the remaining two (1-->3)-beta-glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1-->3)-beta-glucanase inactivation.
Roles of lysophosphatidic acid and sphingosine-1-phosphate in stem cell biology.
Publisher: Elsevier BV
Date: 10-2018
DOI: 10.1016/J.PLIPRES.2018.09.001
Abstract: Stem cells are unique in their ability to self-renew and differentiate into various cell types. Because of these features, stem cells are key to the formation of organisms and play fundamental roles in tissue regeneration and repair. Mechanisms controlling their fate are thus fundamental to the development and homeostasis of tissues and organs. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids that play a wide range of roles in multiple cell types, during developmental and pathophysiological events. Considerable evidence now demonstrates the potent roles of LPA and S1P in the biology of pluripotent and adult stem cells, from maintenance to repair. Here we review their roles for each main category of stem cells and explore how those effects impact development and physiopathology.
Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 22-07-2003
DOI: 10.1161/01.CIR.0000080324.12530.0D
Abstract: Background— RhoA and Rho kinase are important modulators of microvascular tone. Methods and Results— We tested whether sphingosine kinase (Sphk1) that generates the endogenous sphingolipid mediator sphingosine-1-phosphate (S1P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk1 in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk1 mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk1 completely inhibited development of tone and myogenic responses. Conclusions— The tone-increasing effects of a Sphk1 overexpression suggest that Sphk1 may play an important role in the control of peripheral resistance.
Local Sphingosine Kinase 1 Activity Improves Islet Transplantation
Publisher: American Diabetes Association
Date: 07-02-2017
DOI: 10.2337/DB16-0837
Abstract: Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive β-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.
Modification of the tumour microenvironment via exosomal shedding of sphingosine 1-phosphate receptor 2 by breast cancer cells
Publisher: Impact Journals, LLC
Date: 24-07-2018
Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition
Publisher: Elsevier BV
Date: 10-2202
An Improved Isoform-Selective Assay for Sphingosine Kinase 1 Activity.
Publisher: Springer New York
Date: 2018
DOI: 10.1007/7651_2017_41
Abstract: Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell s les. The assay measures the production of
Regulation of Stem Cell Pluripotency and Neural Differentiation by Lysophospholipids
Publisher: S. Karger AG
Date: 2009
DOI: 10.1159/000231891
Abstract: Lysophospholipids are bioactive signalling molecules able to act through the binding of their specific G-protein-coupled receptors to exert pleiotropic effects on a wide range of cells. The most widely studied signalling lysophospholipids are lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). LPA and S1P have been identified to have widespread developmental, physiological and pathological actions in the central nervous system and more recently have been shown to induce biological effects on various stem cell types. This review aims to summarise the current knowledge on LPA and S1P regulation of embryonic and neural stem cell biology.
Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival
Publisher: Public Library of Science (PLoS)
Date: 19-03-2013
Targeting the integrated stress response in hematologic malignancies
Publisher: Springer Science and Business Media LLC
Date: 08-11-2022
DOI: 10.1186/S40164-022-00348-0
Abstract: While numerous targeted therapies have been recently adopted to improve the treatment of hematologic malignancies, acquired or intrinsic resistance poses a significant obstacle to their efficacy. Thus, there is increasing need to identify novel, targetable pathways to further improve therapy for these diseases. The integrated stress response is a signaling pathway activated in cancer cells in response to both dysregulated growth and metabolism, and also following exposure to many therapies that appears one such targetable pathway for improved treatment of these diseases. In this review, we discuss the role of the integrated stress response in the biology of hematologic malignancies, its critical involvement in the mechanism of action of targeted therapies, and as a target for pharmacologic modulation as a novel strategy for the treatment of hematologic malignancies.
A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.DEVCEL.2015.11.026
Abstract: ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient s les of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.
Sphingosine kinase 1 in murine dorsal root ganglia
Publisher: American Institute of Mathematical Sciences (AIMS)
Date: 2015
Targeting the sphingolipid system as a therapeutic direction for glioblastoma
Publisher: MDPI AG
Date: 2020
Abstract: Glioblastoma (GBM) is the most commonly diagnosed malignant brain tumor in adults. The prognosis for patients with GBM remains poor and largely unchanged over the last 30 years, due to the limitations of existing therapies. Thus, new therapeutic approaches are desperately required. Sphingolipids are highly enriched in the brain, forming the structural components of cell membranes, and are major lipid constituents of the myelin sheaths of nerve axons, as well as playing critical roles in cell signaling. Indeed, a number of sphingolipids elicit a variety of cellular responses involved in the development and progression of GBM. Here, we discuss the role of sphingolipids in the pathobiology of GBM, and how targeting sphingolipid metabolism has emerged as a promising approach for the treatment of GBM.
Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries
Publisher: Wiley
Date: 13-02-2006
Abstract: Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.
Post-translational regulation of sphingosine kinases
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.BBALIP.2012.07.005
Abstract: Sphingosine kinases (SKs) catalyse the conversion of sphingosine to sphingosine 1-phosphate (S1P), a signalling lipid that is involved in a plethora of cellular processes including proliferation, apoptosis, calcium homeostasis, angiogenesis, vascular and neuronal maturation, cell migration and immune responses. Over the last few years, it has become clear that SKs are subject to various forms of post-translational regulation which play important roles in the function of these enzymes. Moreover, dysregulation of SKs has been implicated in many pathological conditions, such as cancer. Here we review the various mechanisms of post-translational regulation of the SKs with the view that such knowledge may lead to the development of therapeutic strategies to modulate the activities of these enzymes in the treatment of cancer and a range of other conditions. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
CIB2 negatively regulates oncogenic signaling in ovarian cancer via sphingosine kinase 1.
Publisher: American Association for Cancer Research (AACR)
Date: 14-09-2017
DOI: 10.1158/0008-5472.CAN-17-0025
Abstract: Sphingosine kinase 1 (SK1) is a key regulator of the cellular balance between proapoptotic and prosurvival sphingolipids. Oncogenic signaling by SK1 relies on its localization to the plasma membrane, which is mediated by the calcium and integrin binding protein CIB1 via its Ca2+-myristoyl switch function. Here we show that another member of the CIB family, CIB2, plays a surprisingly opposite role to CIB1 in the regulation of SK1 signaling. CIB2 bound SK1 on the same site as CIB1, yet it lacks the Ca2+-myristoyl switch function. As a result, CIB2 blocked translocation of SK1 to the plasma membrane and inhibited its subsequent signaling, which included sensitization to TNFα-induced apoptosis and inhibition of Ras-induced neoplastic transformation. CIB2 was significantly downregulated in ovarian cancer and low CIB2 expression was associated with poor prognosis in ovarian cancer patients. Notably, reintroduction of CIB2 in ovarian cancer cells blocked plasma membrane localization of endogenous SK1, reduced in vitro neoplastic growth and tumor growth in mice, and suppressed cell motility and invasiveness both in vitro and in vivo. Consistent with the in vitro synergistic effects between the SK1-specific inhibitor SK1-I and standard chemotherapeutics, expression of CIB2 also sensitized ovarian cancer cells to carboplatin. Together, these findings identify CIB2 as a novel endogenous suppressor of SK1 signaling and potential prognostic marker and demonstrate the therapeutic potential of SK1 in this gynecologic malignancy. Cancer Res 77(18) 4823–34. ©2017 AACR.
Genome-wide transcription factor–binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs
Publisher: American Society of Hematology
Date: 26-10-2023
Abstract: Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (the Heptad, PU.1, CTCF, and STAG2) in HSPC subsets (HSC-MPP, CMP, GMP, MEP) and found that TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs such as PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of specific TF complexes was at least partially regulated by features encoded in specific DNA sequence motifs. Taken together, this study provides a comprehensive characterisation of the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying datasets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analysing aberrant regulatory networks in leukemic cells.
Over‐Expression of Sphingosine Kinase‐1 Enhances a Progenitor Phenotype in Human Endothelial Cells
Publisher: Wiley
Date: 10-2011
DOI: 10.1111/J.1549-8719.2011.00119.X
Abstract: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.
A proinflammatory role for proteolytically cleaved annexin A1 in neutrophil transendothelial migration
Publisher: The American Association of Immunologists
Date: 09-2010
Abstract: Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of AnxA1 have opposing functions in inflammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route.
Intracellular and cell wall associated β-glucanases and β-glucosidases of Acremonium persicinum
Publisher: Elsevier BV
Date: 10-1999
Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice
Publisher: American Society of Hematology
Date: 19-01-2012
DOI: 10.1182/BLOOD-2011-04-348904
Abstract: CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P1) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P1 are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P1 agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P1 agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.
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A New Target For Allergic Inflammation: The Sphingolipid Pathway
Start Date: 2012
End Date: 2014
Funder: National Health and Medical Research Council
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