226 research outputs found
Sphingosine kinase 2 promotes acute lymphoblastic leukemia by enhancing MYC expression
No full textAbstract not availableCraig T. Wallington-Beddoe, Jason A. Powell, Daochen Tong, Stuart M. Pitson, Kenneth F. Bradstock and Linda J. Bendal
Science amongst the vines - Meeting on signalling systems
No full textCopyright © 2008 by the European Molecular Biology OrganizationStuart M Pitson, Gregory J Goodall and Mark A Guthridg
Roles of lysophosphatidic acid and sphingosine-1-phosphate in stem cell biology
No full textStem 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.Grace E. Lidgerwood, Stuart M. Pitson, Claudine Bonder, Alice Péba
Tumor necrosis factor-induced neutrophil adhesion occurs via sphingosine kinase-1-dependent activation of endothelial alpha(5)beta(1) integrin
No full textLeukocyte recruitment plays a major role in the immune response to infectious pathogens, as well as during inflammatory and autoimmune disorders. The process of leukocyte extravasation from the blood requires a complex cascade of adhesive events between the leukocytes and the endothelium, including initial leukocyte rolling, adhesion, and finally transendothelial migration. Current research in this area aims to identify the key leukocyte subsets that initiate a given disease and to identify the trafficking molecule(s) that will most specifically inhibit those cells. Herein we demonstrate that tumor necrosis factor (TNF)α activates the integrin α5β1 without altering total expression levels of β1 integrin on human umbilical vein endothelial cells. Moreover, our studies suggest that TNFα-induced β1 activation is dependent on sphingosine kinase-1, but independent of the sphingosine-1-phosphate family of G protein-coupled receptors. We also show, using a parallel plate flow chamber assay, that neutrophil adhesion to TNFα-activated endothelium can be attenuated by blocking α5β1 or its ligand angiopoietin-2. These observations add new complexities that broaden the accepted concept of cellular trafficking with neutrophil adhesion to TNFα activated endothelial cells being sphingosine kinase-1, α5β1, and angiopoietin-2 dependent. Moreover, this work supports the notion that sphingosine kinase-1 may be the single target required for an effective broad spectrum approach to combat inflammation and immune disorders.Wai Y. Sun, Stuart M. Pitson and Claudine S. Bonde
Identification of sphingosine kinase 1 as a therapeutic target in B-lineage acute lymphoblastic leukaemia
No full textLink to a related website: https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/bjh.15097, Open Access via UnpaywallAbstract not availableCraig T. Wallington‐Beddoe, Vicki Xie, Daochen Tong, Jason A. Powell, Alexander C. Lewis, Lorena Davies, Stuart M. Pitson, Kenneth F. Bradstock
Linda J. Bendal
The role of sphingosine kinases and SKAM1 in cutaneous wound healing
No full textSphingosine kinases (SKs) phosphorylate sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). SKs and S1P regulate a diverse range of cellular processes, including cell proliferation, survival, differentiation, migration, smooth muscle cell contraction, inflammation, cytoskeleton reorganisation and angiogenesis, mainly via the engagement of S1P to a family of five S1P-specific G protein-coupled receptors (GPCRs). As such, the SKs and S1P are involved in regulating a plethora of cellular processes that are known to be fundamental to wound healing. The role of SK and S1P in cancer and other diseases including asthma, hypertension, atherosclerosis and allergy are well established. Notably, however, the direct role of the SKs and S1P in wound healing has not been previously examined in any detail. My studies sought to fill this gap in knowledge. Using a well-established mouse model of incisional wound healing, I have shown that SK1⁻ʹ⁻, SK2⁻ʹ⁻, SK1⁻ʹ⁻ SK2⁺ʹ⁻ mice healed at a slower rate compared to wildtype mice. This may be attributed to a decrease in cellular proliferation in the early steps of wound repair. These studies highlight the importance of SKs in the very complex process of wound healing. My studies also examined the role of a relatively uncharacterised protein, fibroblast growth factor receptor-1 oncogenic partner 2 (FGFR1OP2), in wound healing. FGFR1OP2 is a protein that was identified from a yeast two-hybrid screen for SK1 interacting proteins. Unpublished work performed from the Pitson laboratory has shown that FGFR1OP2 can interact and activate SK1 in cells and in vitro. As such, we have more appropriately named this protein SKAM1 (Sphingosine Kinase Activating Molecule 1). SKAM1 has previously been reported to be upregulated following tooth extraction in rat oral mucosa. The Pitson laboratory has shown that overexpression of SKAM1 induced collagen matrix contraction, an in vitro model of wound contraction, was mediated by SK1 and the S1P receptors, S1P₁ ̷ ₃. Using a number of classical in vitro models of wound healing, I found that overexpression of SKAM1 in NIH3T3 fibroblasts did not affect cellular migration and proliferation. Notably, however, NIH3T3 fibroblasts overexpressing SKAM1 were resistant to serum deprivation-induced apoptosis. We also generated SKAM1 transgenic mice, where SKAM1 was ubiquitously expressed, to study the role of this protein in wound healing in vivo. We found no observable phenotypical difference between SKAM1 transgenic and wildtype mice at 12 and 48 weeks of age. Primary mouse embryonic fibroblasts (MEFs) isolated from SKAM1 transgenic embryos showed enhanced ability to contract collagen matrix compared with the wildtype. Somewhat surprisingly, the rate of wound healing following incisional wounding was similar between SKAM1A transgenic and wildtype mice. Notably, however, SKAM1A transgenic mice showed enhanced wound resolution compared with the wildtype following full-thickness excisional wounding. In addition, SKAM1 gene-trap mice with conditional potential have also been successfully generated and provide a tool for the study of the effect of SKAM1A knockout in wound healing in vivo. The Pitson laboratory previously showed that a 35 amino acid peptide of SKAM1, SKAM1⁷¹⁻¹⁰⁵, can surprisingly still activate SK1 and enhance collagen matrix contraction. Further to this I have shown that a 30 amino acid cell-permeable peptide of SKAM1, TAT-SKAM1⁷⁶⁻¹⁰⁵, was able to directly activate recombinant SK1 in vitro and when applied to cells. Notably, this effect was blocked by a mutant version of this peptide Tyr104→Phe mutation. Furthermore, NIH3T3 fibroblasts treated with TAT-SKAM1⁷⁶⁻¹⁰⁵ showed enhanced collagen contraction, and more importantly, intradermal injection of TAT-SKAM1A⁷⁶⁻¹⁰⁵ into full-thickness excisional wounds resulted in enhanced wound resolution in mice. Neither of these effects was observed with the mutant peptide. Taken together, my findings suggest a potential therapeutic use of this peptide for the enhancement of wound repair. In summary, my findings have demonstrated for the first time a novel role of SK and SKAM1 in wound healing. Knowledge gained from this study will be valuable for the development of potential new therapeutics for the improvement of wound healing.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2015
Roles and Regulation of Sphingosine Kinase 2 in Cancer
No full textThe two mammalian sphingosine kinases, SK1 and SK2, produce the bioactive signalling lipid sphingosine-1-phosphate (S1P), which generally promotes cell survival, proliferation and migration. In line with this, SK1 is often found to be upregulated in human cancers, and overexpression of SK1 leads to neoplastic transformation of cells and tumour growth. However, despite generating the same product, most evidence to date suggests that SK2 acts in an opposing manner to promote cell death. In contrast, knockout mouse models indicate that there is at least some functional redundancy between the two SK isoforms, and targeting SK2 via genetic or pharmacological approaches in cancer models results in reduced tumour growth. Clearly, the roles of SK2 are poorly understood, but it is apparent that these unique and complex functions of SK2 are largely dictated by its differential subcellular localisation. SK1 is generally a cytoplasmic protein, but it can also be translocated to the plasma membrane where it mediates cell survival, proliferation and oncogenic signalling through the production of S1P. SK2, however, has been reported to localise to the nucleus, endoplasmic reticulum and mitochondria, and at these locations it appears to possess anti-proliferative and pro-apoptotic functions. SK2 has also been reported to localise to the plasma membrane, but its specific roles here have not been well characterised. Therefore, the main aims of this study were to explore the roles of SK2 in cancer, and to characterise novel mechanisms that regulate SK2 subcellular localisation, such as interacting proteins and post translational modifications, in order to gain a better understanding of this complex enzyme and the potential benefits of targeting SK2 in cancer. To explore the roles of SK2 in cancer, we examined the expression of SK2 in various human tumour samples using publically available datasets, and found that SK2 shows statistically significant upregulation in many cancers, but only to modest levels up to 2.5- fold over normal tissues. As high-level SK2 overexpression has been previously shown to cause cell death, we explored the effects of low, close to physiological levels of SK2 overexpression. By engineering a series of human and mouse cell lines overexpressing graded levels of SK2, we found that low-level SK2 overexpression increased cell survival and proliferation, and activated oncogenic signalling pathways. Notably, low-level SK2 overexpression (5- to 10-fold over endogenous levels) was sufficient to induce neoplastic transformation of mouse fibroblasts, resulting in efficient tumour formation in vivo. These findings coincided with decreased nuclear localisation and increased plasma membrane localisation of SK2, as well as increases in extracellular S1P formation. Hence, we have shown for the first time that SK2 can have a direct role in promoting oncogenesis. Furthermore, the Pitson laboratory previously identified a novel SK2-interacting protein, cytoplasmic dynein 1 intermediate chain 2 (IC-2), through a yeast two-hybrid screen, and characterising this interaction formed another part of these studies. We confirmed that SK2 interacts physiologically with the dynein complex in cells via the IC subunit, and being a retrograde-directed transport motor complex, we found that dynein mediates the translocation of SK2 away from the plasma membrane. Interestingly, although IC-2 was identified in the yeast two-hybrid screen, SK2 interacts more robustly with the highly-related IC-1 isoform, which is abundantly expressed in the brain. Strikingly, we found that IC-1 is downregulated 17-fold in glioblastoma multiforme (GBM) patient samples, which correlated with poorer survival of patients with this form of brain tumour. In line with a role for dynein in transporting SK2, low IC-1 expression in GBM cells coincided with more SK2 localised to the plasma membrane, where we had found it to accumulate in an oncogenic setting. Re-expression of IC-1 in these cells reduced plasma membrane localised-SK2 and extracellular S1P formation, and notably, decreased tumour growth and tumour-associated angiogenesis in vivo. Thus, these findings demonstrate a novel tumour-suppressive function of dynein IC-1, and uncover new mechanistic insights into SK2 regulation. Through previous mass spectrometric analyses performed by the Pitson laboratory, it is evident that SK2 contains multiple uncharacterised phosphorylation sites that are not shared with SK1. We explored the function of one such site, Ser363, and found it to potentially regulate nuclear localisation of SK2. Furthermore, we identified SK2 as a bona fide substrate of glycogen synthase kinase 3 (GSK3) in vitro and in cells, involving residues Ser437 and Ser441, and we found that other phosphorylation events may act to regulate SK2 catalytic activity. Overall, the studies outlined here have revealed a previously unreported role for SK2 in driving oncogenesis, and have described the characterisation of novel mechanisms that regulate the subcellular localisation of SK2. Therefore, these findings support the use of SK2 inhibitors as promising anti-cancer therapeutic agents. Furthermore, as the opposing functions of SK2 are largely dictated by it subcellular localisation, these findings may also assist in the development of new strategies to target oncogenic SK2 in cancer.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201
Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition
No full textAbstract not availableFeby Saviraa, Ruth Magayea, Carmen V. Scullinoc, Bernard L. Flynn, Stuart M. Pitson, Dovile Anderson, Darren J. Creek, Yue Hua, Xin Xionga, Li Huanga, Danny Liew, Christopher Reid, David Kaye, Andrew R. Kompah, Bing Hui Wan
The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells
No full textThe 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.Vidya Limaye, Mathew A. Vadas, Stuart M. Pitson and Jennifer R. Gambl
Regulation of sphingosine kinase by interacting proteins.
No full textSphingosine kinase 1 (SK1) is responsible for phosphorylating the lipid sphingosine, generating the bio-active phospholipid, sphingosine 1-phosphate (S1P). Cells possess basal SK1 activity
which has been proposed to serve in a ‘housekeeping’ function to limit the levels of proapoptotic sphingosine and ceramide in the cell. In some circumstances, however, such as cell exposure to growth factors and cytokines this basal level of SK1 activity is increased, resulting in an increased production of S1P. As S1P is a pro-proliferative, pro-survival molecule, its increased production is associated with enhanced cell proliferation, survival and an oncogenic phenotype. The Pitson laboratory has shown previously that one mechanism by which SK1 is activated is through phosphorylation at Ser-225 by ERK1/2. Here, my studies focused on alternative mechanisms of SK1 activation that arise through its interaction with two proteins, eukaryotic elongation factor 1A (eEF1A) and a relatively uncharacterised protein, SK activator molecule 1 (SKAM). eEF1A is able to directly increase the catalytic activity of SK1 in vitro and is also able to increase endogenous SK activity when over-expressed in quiescent cells that have reduced levels of endogenous eEF1A protein. Due to the abundance of eEF1A protein within a cell, I hypothesized that the effect of eEF1A on SK activity may be dynamically regulated. eEF1A contains a ‘G protein-like’ domain that enables it to bind GDP and GTP. When bound by GTP, eEF1A undergoes a large conformational change that enables it to bind aminoacyltRNA for transport to the ribosome. Similarly, just as the nucleotide-bound state of eEF1A regulates its role in protein synthesis, I found that the nucleotide-bound state of eEF1A also regulates its ability to activate SK1. Strikingly, it is only the translationally inactive eEF1A.GDP that can activate SK1. A truncated form of eEF1A named PTI-1 has been described that lacks the ‘G protein-like’ domain and thus can not bind guanine nucleotides, rendering it structurally analogous to eEF1A.GDP. In keeping with my finding that only eEF1A.GDP activates SK1, I found that PTI-1 also activates SK1 both in vitro and in cells. Importantly, PTI-1 has been previously characterized as an oncoprotein and for the first time my studies have shown a likely mechanism by which PTI-1 induces a tumourigenic phenotype. Expression of PTI-1 in NIH 3T3 cells induces neoplastic transformation, as measured by focus formation. Notably, this PTI-1-induced transformation is blocked when cells are treated with SK inhibitors or when cells are co-transfected with PTI-1 and a dominant negative SK1, indicating that oncogenesis by PTI-1 is mediated through SK1. The current study also investigated the regulation of SK1 activity by its interaction with SKAM1. Previous studies have shown that SKAM1, like eEF1A, can directly increase the catalytic activity of SK1 in vitro and in cells. My studies have determined the minimal region of interaction of SKAM1 that is still able to interact with and activate SK1. Remarkably, a 35 amino acid SKAM1 peptide retained the ability to activate SK1. The physiological relevance of the SK1-SKAM1 interaction was also examined and I have shown that knock-down of SKAM1, and the related protein SKAM2, in HEK 293T cells resulted in decreased cell proliferation coupled with increased susceptibility to apoptosis. Results presented here, also suggest that phosphorylation of SKAM1 at Tyr-46 acts as a negative regulator for SKAM1-induced SK1 activation. In summary, the current study presents two novel SK1 interacting proteins that directly increase the catalytic activity of this enzyme, and investigates mechanisms by which their effects on SK1 activity are regulated. While the guanine nucleotide bound state of eEF1A1 determines its effects on SK1 activity, the phosphorylation status of SKAM1 appears to determine its ability to activate SK1.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201
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