Ashley J Snider

Interests

Research

sphingolipid biochemistry, diet-induced inflammation, animal models of disease, intestinal inflammation

Teaching

sphingolipid biochemistry, diet-induced inflammation, animal models of disease, intestinal inflammation

Courses

Scholarly Contributions

Chapters

• Choi, S., & Snider, A. J. (2019). Diet, lipids and colon cancer. In International review of cell and molecular biology(pp 105-144).
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  Dietary fat is digested and absorbed in the small intestine and can then be utilized as an energy source and/or as a reservoir for other bioactive lipid species. Excessive dietary fat has been implicated in the induction and/or aggravation of several diseases, including colorectal cancer (CRC). Diets with high fat content have been shown to exacerbate CRC through regulation of intestinal inflammation and proliferation, as well as alteration of bile acid pools, microbiota, and bioactive lipid species. This chapter will investigate the effects of dietary fat on CRC development and pathobiology, and possible mechanisms for specific lipid species in those processes.
• Snider, A. J., Bialkowska, A. B., Ghaleb, A. M., Yang, V. W., Obeid, L. M., & Hannun, Y. A. (2016). Murine Model for Colitis-Associated Cancer of the Colon. In Methods in molecular biology (Clifton, N.J.)(pp 245-54).
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  Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), significantly increases the risk for development of colorectal cancer. Specifically, dysplasia and cancer associated with IBD (colitis-associated cancer or CAC) develop as a result of repeated cycles of injury and healing in the intestinal epithelium. Animal models are utilized to examine the mechanisms of CAC, the role of epithelial and immune cells in this process, as well as the development of novel therapeutic targets. These models typically begin with the administration of a carcinogenic compound, and inflammation is caused by repeated cycles of colitis-inducing agents. This review describes a common CAC model that utilizes the pro-carcinogenic compound azoxymethane (AOM) followed by dextran sulfate sodium (DSS) which induces the inflammatory insult.
• Furuya, H., Choi, S., Obeid, L. M., Kawamori, T., & Snider, A. J. (2015). Colon Cancer: The Role of Sphingolipid Metabolic Enzyme. In Bioactive Sphingolipids in Cancer Biology and Therapy(pp 141-159). Springer Publishers.
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  Colorectal cancer is one of the most common tumors worldwide, with sustained incidence in developed countries and increasing incidence in developing countries. Although recent studies provide knowledge of the molecular signaling pathways that are implicated in colon carcinogenesis, treatments and outcomes still need further improvement. Bioactive sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate (S1P), are signaling molecules that regulate cellular events including cell proliferation, apoptosis, senescence, angiogenesis, and transformation in response to diverse stimuli. Ceramide and sphingosine mediate numerous cell-stress responses, including induction of apoptosis and cell senescence. In contrast, S1P plays pivotal roles in cell survival, migration, and inflammation. These sphingolipids with opposing roles can be quickly metabolized and catabolized within cells, suggesting that the balance between these potent bioactive lipids may dictate cell fate. In this chapter, we review the roles of these bioactive sphingolipids and their metabolic enzymes in colitis, colitis associated cancer, and colorectal cancer.
• Garcia-Barros, M., Coant, N., & Snider, A. J. (2015). Sphingolipids in Intestinal Inflammation and Tumorigenesis. Intestinal Tumorigenesis. In Intestinal Tumorigenesis(pp 257-286). Springer Publishers.
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  Sphingolipids, and their metabolizing enzymes, have emerged as significant players in cell signaling and biology. These bioactive lipids function not only as structural lipids in cell, but mediate significant biologic functions as well. Sphingolipid enzymes function in the intestinal lumen to degrade exogenous dietary sphingolipids and in the enterocytes themselves to generate endogenous sphingolipids. Among the most studied bioactive sphingolipids are ceramide, sphingosine, and sphingosine-1-phosphate. These sphingolipids and their synthetic enzymes have been extensively implicated in intestinal inflammation and tumorigenesis and will be the focus of this chapter.

Journals/Publications

• Chauhan, S. S., Casillas, A. L., Vizzerra, A. D., Liou, H., Clements, A. N., Flores, C. E., Prevost, C. T., Kashatus, D. F., Snider, A. J., Snider, J. M., & Warfel, N. A. (2024). PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer. Oncogene, 43(6), 406-419.
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  Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3β) via serine 9 phosphorylation. GSK3β inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3β-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.
• Richardson, W. J., Humphrey, S. B., Sears, S. M., Hoffman, N. A., Orwick, A. J., Doll, M. A., Doll, C. L., Xia, C., Hernandez-Corbacho, M., Snider, J. M., Obeid, L. M., Hannun, Y. A., Snider, A. J., & Siskind, L. J. (2024). Expression of Ceramide Synthases in Mice and Their Roles in Regulating Acyl-Chain Sphingolipids: A Framework for Baseline Levels and Future Implications in Aging and Disease. Molecular pharmacology, 105(3), 131-143.
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  Sphingolipids are an important class of lipids present in all eukaryotic cells that regulate critical cellular processes. Disturbances in sphingolipid homeostasis have been linked to several diseases in humans. Ceramides are central in sphingolipid metabolism and are largely synthesized by six ceramide synthase (CerS) isoforms (CerS1-6), each with a preference for different fatty acyl chain lengths. Although the tissue distribution of CerS mRNA expression in humans and the roles of CerS isoforms in synthesizing ceramides with different acyl chain lengths are known, it is unknown how CerS expression dictates ceramides and downstream metabolites within tissues. In this study, we analyzed sphingolipid levels and CerS mRNA expression in 3-month-old C57BL/6J mouse brain, heart, kidney, liver, lung, and skeletal muscle. The results showed that CerS expression and sphingolipid species abundance varied by tissue and that CerS expression was a predictor of ceramide species within tissues. Interestingly, although CerS expression was not predictive of complex sphingolipid species within all tissues, composite scores for CerSs contributions to total sphingolipids measured in each tissue correlated to CerS expression. Lastly, we determined that the most abundant ceramide species in mouse tissues aligned with CerS mRNA expression in corresponding human tissues (based on chain length preference), suggesting that mice are relevant preclinical models for ceramide and sphingolipid research. SIGNIFICANCE STATEMENT: The current study demonstrates that ceramide synthase (CerS) expression in specific tissues correlates not only with ceramide species but contributes to the generation of complex sphingolipids as well. As many of the CerSs and/or specific ceramide species have been implicated in disease, these studies suggest the potential for CerSs as therapeutic targets and the use of sphingolipid species as diagnostics in specific tissues.
• Espinoza, K. S., Hermanson, K. N., Beard, C. A., Schwartz, N. U., Snider, J. M., Low, B. E., Wiles, M. V., Hannun, Y. A., Obeid, L. M., & Snider, A. J. (2023). A Novel HSPB1 Mouse Model of Charcot-Marie-Tooth Disease. Prostaglandins & other lipid mediators, 106769.
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  Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old Hsp mice. Though sphingolipid levels were not altered in sciatic nerves from Hsp mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from Hsp mice. Histology was utilized to further characterize Hsp mice. Hsp mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, Hsp mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of Hsp in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between Hsp and Hsp mice. However, both coordination and grip strength were decreased in 3-month-old Hsp mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.
• Staquicini, D. I., Cardó-Vila, M., Rotolo, J. A., Staquicini, F. I., Tang, F. H., Smith, T. L., Ganju, A., Schiavone, C., Dogra, P., Wang, Z., Cristini, V., Giordano, R. J., Ozawa, M. G., Driessen, W. H., Proneth, B., Souza, G. R., Brinker, L. M., Noureddine, A., Snider, A. J., , Canals, D., et al. (2023). Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands. Proceedings of the National Academy of Sciences of the United States of America, 120(34), e2220269120.
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  The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.
• Velazquez, F. N., Stith, J. L., Zhang, L., Allam, A. M., Haley, J., Obeid, L. M., Snider, A. J., & Hannun, Y. A. (2023). Targeting sphingosine kinase 1 in p53KO thymic lymphoma. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 37(11), e23247.
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  Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.
• Sears, S. M., Dupre, T. V., Shah, P. P., Davis, D. L., Doll, M. A., Sharp, C. N., Vega, A. A., Megyesi, J., Beverly, L. J., Snider, A. J., Obeid, L. M., Hannun, Y. A., & Siskind, L. J. (2022). Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury. Journal of lipid research, 63(3), 100179.
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  Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.
• Truman, J. P., Ruiz, C. F., Montal, E., Garcia-Barros, M., Mileva, I., Snider, A. J., Hannun, Y. A., Obeid, L. M., & Mao, C. (2022). 1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase. Journal of lipid research, 63(1), 100154.
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  Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined. Here, we identify 1-deoxysphinganine (dSA), a noncanonical sphingoid base generated in the absence of serine from the alternative condensation of alanine and palmitoyl CoA by serine palmitoyl transferase, as a proximal mediator of SG deprivation in SK1 loss and Sph level elevation upon SG deprivation in cancer cells. SG starvation increased dSA levels in vitro and in vivo and in turn induced SK1 degradation through a serine palmitoyl transferase-dependent mechanism, thereby increasing Sph levels. Addition of exogenous dSA caused a moderate increase in intracellular reactive oxygen species, which in turn decreased pyruvate kinase PKM2 activity while increasing phosphoglycerate dehydrogenase levels, and thereby promoted serine synthesis. We further showed that increased dSA induces the adaptive cellular and metabolic functions in the response of cells to decreased availability of serine likely by increasing Sph levels. Thus, we conclude that dSA functions as an initial sensor of serine loss, SK1 functions as its direct target, and Sph functions as a downstream effector of cellular and metabolic adaptations. These studies define a previously unrecognized "physiological" nontoxic function for dSA.
• Al-Rashed, F., Ahmad, Z., Snider, A. J., Thomas, R., Kochumon, S., Melhem, M., Sindhu, S., Obeid, L. M., Al-Mulla, F., Hannun, Y. A., & Ahmad, R. (2021). Ceramide kinase regulates TNF-α-induced immune responses in human monocytic cells. Scientific reports, 11(1), 8259.
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  Ceramide kinase (CERK) phosphorylates ceramide to produce ceramide-1-phosphate (C1P), which is involved in the development of metabolic inflammation. TNF-α modulates inflammatory responses in monocytes associated with various inflammatory disorders; however, the underlying mechanisms remain not fully understood. Here, we investigated the role of CERK in TNF-α-induced inflammatory responses in monocytes. Our results show that disruption of CERK activity in monocytes, either by chemical inhibitor NVP-231 or by small interfering RNA (siRNA), results in the defective expression of inflammatory markers including CD11c, CD11b and HLA-DR in response to TNF-α. Our data show that TNF-α upregulates ceramide phosphorylation. Inhibition of CERK in monocytes significantly reduced the secretion of IL-1β and MCP-1. Similar results were observed in CERK-downregulated cells. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was reduced by inhibition of CERK. Additionally, NF-κB/AP-1 activity was suppressed by the inhibition of CERK. Clinically, obese individuals had higher levels of CERK expression in PBMCs compared to lean individuals, which correlated with their TNF-α levels. Taken together, these results suggest that CERK plays a key role in regulating inflammatory responses in human monocytes during TNF-α stimulation. CERK may be a relevant target for developing novel therapies for chronic inflammatory diseases.
• Selmin, O. I., Papoutsis, A. J., Hazan, S., Smith, C., Greenfield, N., Donovan, M. G., Wren, S. N., Doetschman, T. C., Snider, J. M., Snider, A. J., Chow, S. H., & Romagnolo, D. F. (2021). n-6 High Fat Diet Induces Gut Microbiome Dysbiosis and Colonic Inflammation. International journal of molecular sciences, 22(13).
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  : Concerns are emerging that a high-fat diet rich in -6 PUFA (-6HFD) may alter gut microbiome and increase the risk of intestinal disorders. Research is needed to model the relationships between consumption of an -6HFD starting at weaning and development of gut dysbiosis and colonic inflammation in adulthood. We used a C57BL/6J mouse model to compare the effects of exposure to a typical American Western diet (WD) providing 58.4%, 27.8%, and 13.7% energy (%E) from carbohydrates, fat, and protein, respectively, with those of an isocaloric and isoproteic soybean oil-rich -6HFD providing 50%E and 35.9%E from total fat and carbohydrates, respectively on gut inflammation and microbiome profile. : At weaning, male offspring were assigned to either the WD or -6HFD through 10-16 weeks of age. The WD included fat exclusively from palm oil whereas the -6HFD contained fat exclusively from soybean oil. We recorded changes in body weight, cyclooxygenase-2 (COX-2) expression, colon histopathology, and gut microbiome profile. : Compared to the WD, the -6HFD increased plasma levels of -6 fatty acids; colonic expression of COX-2; and the number of colonic inflammatory and hyperplastic lesions. At 16 weeks of age, the -6HFD caused a marked reduction in the gut presence of , , and , and induced growth of and . At the species level, the -6HFD sustains the gut growth of proinflammatory and . : An -6HFD consumed from weaning to adulthood induces a shift in gut bacterial profile associated with colonic inflammation.
• Snider, J. M., Trayssac, M., Clarke, C. J., Schwartz, N., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2021). Erratum: Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis. Journal of lipid research, 62, 100009.
• Snider, J. M., You, J. K., Wang, X., Snider, A. J., Hallmark, B., Seeds, M. C., Sergeant, S., Johnstone, L., Wang, Q., Sprissler, R., Zhang, H. H., Luberto, C., Kew, R. R., Hannun, Y. A., McCall, C. E., Yao, G., Del Poeta, M., & Chilton, F. H. (2021). Group IIA Secreted Phospholipase A Plays a Central Role in the Pathobiology of COVID-19. medRxiv : the preprint server for health sciences.
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  There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A Group IIA (sPLA -IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA -IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA -IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA -IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.
• Snider, J. M., You, J. K., Wang, X., Snider, A. J., Hallmark, B., Zec, M. M., Seeds, M. C., Sergeant, S., Johnstone, L., Wang, Q., Sprissler, R., Carr, T. F., Lutrick, K., Parthasarathy, S., Bime, C., Zhang, H. H., Luberto, C., Kew, R. R., Hannun, Y. A., , Guerra, S., et al. (2021). Group IIA secreted phospholipase A2 is associated with the pathobiology leading to COVID-19 mortality. The Journal of clinical investigation, 131(19).
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  There is an urgent need to identify the cellular and molecular mechanisms responsible for severe COVID-19 that results in death. We initially performed both untargeted and targeted lipidomics as well as focused biochemical analyses of 127 plasma samples and found elevated metabolites associated with secreted phospholipase A2 (sPLA2) activity and mitochondrial dysfunction in patients with severe COVID-19. Deceased COVID-19 patients had higher levels of circulating, catalytically active sPLA2 group IIA (sPLA2-IIA), with a median value that was 9.6-fold higher than that for patients with mild disease and 5.0-fold higher than the median value for survivors of severe COVID-19. Elevated sPLA2-IIA levels paralleled several indices of COVID-19 disease severity (e.g., kidney dysfunction, hypoxia, multiple organ dysfunction). A decision tree generated by machine learning identified sPLA2-IIA levels as a central node in the stratification of patients who died from COVID-19. Random forest analysis and least absolute shrinkage and selection operator-based (LASSO-based) regression analysis additionally identified sPLA2-IIA and blood urea nitrogen (BUN) as the key variables among 80 clinical indices in predicting COVID-19 mortality. The combined PLA-BUN index performed significantly better than did either one alone. An independent cohort (n = 154) confirmed higher plasma sPLA2-IIA levels in deceased patients compared with levels in plasma from patients with severe or mild COVID-19, with the PLA-BUN index-based decision tree satisfactorily stratifying patients with mild, severe, or fatal COVID-19. With clinically tested inhibitors available, this study identifies sPLA2-IIA as a therapeutic target to reduce COVID-19 mortality.
• Velazquez, F. N., Viscardi, V., Montemage, J., Zhang, L., Trocchia, C., Delamont, M. M., Ahmad, R., Hannun, Y. A., Obeid, L. M., & Snider, A. J. (2021). A Milk-Fat Based Diet Increases Metastasis in the MMTV-PyMT Mouse Model of Breast Cancer. Nutrients, 13(7).
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  A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.
• Velazquez, F. N., Zhang, L., Viscardi, V., Trocchia, C., Hannun, Y. A., Obeid, L. M., & Snider, A. J. (2021). Loss of sphingosine kinase 1 increases lung metastases in the MMTV-PyMT mouse model of breast cancer. PloS one, 16(5), e0252311.
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  Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.
• Al-Rashed, F., Ahmad, Z., Thomas, R., Melhem, M., Snider, A. J., Obeid, L. M., Al-Mulla, F., Hannun, Y. A., & Ahmad, R. (2020). Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α. Scientific reports, 10(1), 16802.
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  Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase-2 (nSMase2: SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1β and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1β and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.
• Choi, S., Snider, J. M., Cariello, C. P., Lambert, J. M., Anderson, A. K., Cowart, L. A., & Snider, A. J. (2020). Sphingosine kinase 1 is required for myristate-induced TNFα expression in intestinal epithelial cells. Prostaglandins & other lipid mediators, 149, 106423.
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  Saturated fatty acids (SFA) have been known to trigger inflammatory signaling in metabolic tissues; however, the effects of specific SFAs in the intestinal epithelium have not been well studied. Several previous studies have implicated disruptions in sphingolipid metabolism by oversupply of SFAs in inflammatory process. Also, our previous studies have implicated sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) as having key roles in the regulation of inflammatory processes in the intestinal epithelium. Therefore, to define the role for specific SFAs in inflammatory responses in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, significantly induced the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and it was SK1-dependent. Interestingly, myristate-induced TNFα expression was not suppressed by inhibition of S1P receptors (S1PRs), hinting at a potential novel intracellular target of S1P. Additionally, myristate regulated the expression of TNFα via JNK activation in an SK1-dependent manner, suggesting a novel S1PR-independent target as a mediator between SK1 and JNK in response to myristate. Lastly, a myristate-enriched milk fat-based diet (MFBD) increased expression of TNFα in colon tissues and elevated the S1P to sphingosine ratio, demonstrating the potential of myristate-involved pathobiologies in intestinal tissues. Taken together our studies suggest that myristate regulates the expression of TNFα in the intestinal epithelium via regulation of SK1 and JNK.
• Snider, A. J., Seeds, M. C., Johnstone, L., Snider, J. M., Hallmark, B., Dutta, R., Moraga Franco, C., Parks, J. S., Bensen, J. T., Broeckling, C. D., Mohler, J. L., Smith, G. J., Fontham, E. T., Lin, H. K., Bresette, W., Sergeant, S., & Chilton, F. H. (2020). Identification of Plasma Glycosphingolipids as Potential Biomarkers for Prostate Cancer (PCa) Status. Biomolecules, 10(10).
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  Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment naïve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids; nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids; levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and >30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.
• Snider, J. M., Trayssac, M., Clarke, C. J., Schwartz, N., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2019). Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis. Journal of lipid research, 60(4), 819-831.
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  Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.
• Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., & Snider, A. J. (2018). Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 32(10), 5724-5736.
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  Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat-based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.-Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.
• Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., & Obeid, L. M. (2018). Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 32(5), 2339-2353.
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  Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up-regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis-associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid-derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue-specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC.-Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.
• Li, F., Xu, R., Low, B. E., Lin, C. L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X. C., Li, M. S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., & Mao, C. (2018). Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 32(6), 3058-3069.
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  Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes-the major source of S1P-lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of ceramidases that are encoded by 5 distinct genes, acid ceramidase 1 ( ASAH1)/ Asah1, ASAH2/ Asah2, alkaline ceramidase 1 ( ACER1)/ Acer1, ACER2/ Acer2, and ACER3/ Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased the levels of dihydrosphingosine (dhSPH) and dihydrosphingosine-1-phosphate (dhS1P) in blood. Taken together, these results suggest that ACER2 plays a key role in the maintenance of high plasma levels of sphingoid base-1-phosphates-S1P and dhS1P-by controlling the generation of sphingoid bases-SPH and dhSPH-in hematopoietic cells.-Li, F., Xu, R., Low, B. E., Lin, C.-L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X.-C., Li, M.-S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., Mao, C. Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.
• Snider, J. M., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2018). Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry. Journal of lipid research, 59(6), 1046-1057.
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  Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.
• Dupre, T. V., Doll, M. A., Shah, P. P., Sharp, C. N., Siow, D., Megyesi, J., Shayman, J., Bielawska, A., Bielawski, J., Beverly, L. J., Hernandez-Corbacho, M., Clarke, C. J., Snider, A. J., Schnellmann, R. G., Obeid, L. M., Hannun, Y. A., & Siskind, L. J. (2017). Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. Journal of lipid research, 58(7), 1439-1452.
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  Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. 2017. 58: 1439-1452.
• Lin, C. L., Xu, R., Yi, J. K., Li, F., Chen, J., Jones, E. C., Slutsky, J. B., Huang, L., Rigas, B., Cao, J., Zhong, X., Snider, A. J., Obeid, L. M., Hannun, Y. A., & Mao, C. (2017). Alkaline Ceramidase 1 Protects Mice from Premature Hair Loss by Maintaining the Homeostasis of Hair Follicle Stem Cells. Stem cell reports, 9(5), 1488-1500.
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  Ceramides and their metabolites are important for the homeostasis of the epidermis, but much remains unknown about the roles of specific pathways of ceramide metabolism in skin biology. With a mouse model deficient in the alkaline ceramidase (Acer1) gene, we demonstrate that ACER1 plays a key role in the homeostasis of the epidermis and its appendages by controlling the metabolism of ceramides. Loss of Acer1 elevated the levels of various ceramides and sphingoid bases in the skin and caused progressive hair loss in mice. Mechanistic studies revealed that loss of Acer1 widened follicular infundibulum and caused progressive loss of hair follicle stem cells (HFSCs) due to reduced survival and stemness. These results suggest that ACER1 plays a key role in maintaining the homeostasis of HFSCs, and thereby the hair follicle structure and function, by regulating the metabolism of ceramides in the epidermis.
• McCracken, A. N., McMonigle, R. J., Tessier, J., Fransson, R., Perryman, M. S., Chen, B., Keebaugh, A., Selwan, E., Barr, S. A., Kim, S. M., Roy, S. G., Liu, G., Fallegger, D., Sernissi, L., Brandt, C., Moitessier, N., Snider, A. J., Clare, S., Müschen, M., , Huwiler, A., et al. (2017). Phosphorylation of a constrained azacyclic FTY720 analog enhances anti-leukemic activity without inducing S1P receptor activation. Leukemia, 31(3), 669-677.
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  The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.
• Pulkoski-Gross, M. J., Uys, J. D., Orr-Gandy, K. A., Coant, N., Bialkowska, A. B., Szulc, Z. M., Bai, A., Bielawska, A., Townsend, D. M., Hannun, Y. A., Obeid, L. M., & Snider, A. J. (2017). Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis. Prostaglandins & other lipid mediators, 130, 47-56.
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  Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.
• Senkal, C. E., Salama, M. F., Snider, A. J., Allopenna, J. J., Rana, N. A., Koller, A., Hannun, Y. A., & Obeid, L. M. (2017). Ceramide Is Metabolized to Acylceramide and Stored in Lipid Droplets. Cell metabolism, 25(3), 686-697.
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  In an approach aimed at defining interacting partners of ceramide synthases (CerSs), we found that fatty acyl-CoA synthase ACSL5 interacts with all CerSs. We demonstrate that ACSL5-generated FA-CoA was utilized with de novo ceramide for the generation of acylceramides, poorly studied ceramide metabolites. Functionally, inhibition of ceramide channeling to acylceramide enhanced accumulation of de novo ceramide and resulted in augmentation of ceramide-mediated apoptosis. Mechanistically, we show that acylceramide generation is catalyzed by diacylglycerol acyltransferase 2 (DGAT2) on lipid droplets. In summary, this study identifies a metabolic pathway of acylceramide generation and its sequestration in LDs in cells and in livers of mice on a high-fat diet. The study also implicates this pathway in ceramide-mediated apoptosis, and has implications in co-regulation of triglyceride and sphingolipid metabolisms.
• García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J. P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., & Hannun, Y. A. (2016). Role of neutral ceramidase in colon cancer. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 30(12), 4159-4171.
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  Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of β-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.
• Kitatani, K., Iwabuchi, K., Snider, A., & Riboni, L. (2016). Sphingolipids in Inflammation: From Bench to Bedside. Mediators of inflammation, 2016, 7602526.
• Sundaram, K., Mather, A. R., Marimuthu, S., Shah, P. P., Snider, A. J., Obeid, L. M., Hannun, Y. A., Beverly, L. J., & Siskind, L. J. (2016). Loss of neutral ceramidase protects cells from nutrient- and energy -deprivation-induced cell death. The Biochemical journal, 473(6), 743-55.
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  Sphingolipids are a family of lipids that regulate the cell cycle, differentiation and cell death. Sphingolipids are known to play a role in the induction of apoptosis, but a role for these lipids in necroptosis is largely unknown. Necroptosis is a programmed form of cell death that, unlike apoptosis, does not require ATP. Necroptosis can be induced under a variety of conditions, including nutrient deprivation and plays a major role in ischaemia/reperfusion injury to organs. Sphingolipids play a role in ischaemia/reperfusion injury in several organs. Thus, we hypothesized that sphingolipids mediate nutrient-deprivation-induced necroptosis. To address this, we utilized mouse embryonic fibroblast (MEFs) treated with 2-deoxyglucose (2DG) and antimycin A (AA) to inhibit glycolysis and mitochondrial electron transport. 2DG/AA treatment of MEFs induced necroptosis as it was receptor- interacting protein (RIP)-1/3 kinase-dependent and caspase-independent. Ceramides, sphingosine (Sph) and sphingosine 1-phosphate (S1P) were increased following 2DG/AA treatment. Cells lacking neutral ceramidase (nCDase(-/-)) were protected from 2DG/AA. Although nCDase(-/-) cells generated ceramides following 2DG/AA treatment, they did not generate Sph or S1P. This protection was stimulus-independent as nCDase(-/-) cells were also protected from endoplasmic reticulum (ER) stressors [tunicamycin (TN) or thapsigargin (TG)]. nCDase(-/-) MEFs had higher autophagic flux and mitophagy than wild-type (WT) MEFs and inhibition of autophagy sensitized them to necroptosis. These data indicate that loss of nCDase protects cells from nutrient- deprivation-induced necroptosis via autophagy, and clearance of damaged mitochondria. Results suggest that nCDase is a mediator of necroptosis and might be a novel therapeutic target for protection from ischaemic injury.
• Wang, K., Xu, R., Snider, A. J., Schrandt, J., Li, Y., Bialkowska, A. B., Li, M., Zhou, J., Hannun, Y. A., Obeid, L. M., Yang, V. W., & Mao, C. (2016). Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system. Cell death & disease, 7, e2124.
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  Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C(18:1)-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C(18:1)-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C(18:1)-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.
• Ghaleb, A. M., Bialkowska, A. B., Snider, A. J., Gnatenko, D. V., Hannun, Y. A., Yang, V. W., & Schmidt, V. A. (2015). IQ Motif-Containing GTPase-Activating Protein 2 (IQGAP2) Is a Novel Regulator of Colonic Inflammation in Mice. PloS one, 10(6), e0129314.
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  IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.
• Nowling, T. K., Mather, A. R., Thiyagarajan, T., Hernández-Corbacho, M. J., Powers, T. W., Jones, E. E., Snider, A. J., Oates, J. C., Drake, R. R., & Siskind, L. J. (2015). Renal glycosphingolipid metabolism is dysfunctional in lupus nephritis. Journal of the American Society of Nephrology : JASN, 26(6), 1402-13.
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  Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.
• Wang, K., Xu, R., Schrandt, J., Shah, P., Gong, Y. Z., Preston, C., Wang, L., Yi, J. K., Lin, C. L., Sun, W., Spyropoulos, D. D., Rhee, S., Li, M., Zhou, J., Ge, S., Zhang, G., Snider, A. J., Hannun, Y. A., Obeid, L. M., & Mao, C. (2015). Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain. PLoS genetics, 11(10), e1005591.
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  Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration.
• Janes, K., Little, J. W., Li, C., Bryant, L., Chen, C., Chen, Z., Kamocki, K., Doyle, T., Snider, A., Esposito, E., Cuzzocrea, S., Bieberich, E., Obeid, L., Petrache, I., Nicol, G., Neumann, W. L., & Salvemini, D. (2014). The development and maintenance of paclitaxel-induced neuropathic pain require activation of the sphingosine 1-phosphate receptor subtype 1. The Journal of biological chemistry, 289(30), 21082-97.
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  The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy- induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents.We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR(1))- dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/ neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration- approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.
• Snider, A. J., Ali, W. H., Sticca, J. A., Coant, N., Ghaleb, A. M., Kawamori, T., Yang, V. W., Hannun, Y. A., & Obeid, L. M. (2014). Distinct roles for hematopoietic and extra-hematopoietic sphingosine kinase-1 in inflammatory bowel disease. PloS one, 9(12), e113998.
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  Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.
• Adada, M. M., Orr-Gandy, K. A., Snider, A. J., Canals, D., Hannun, Y. A., Obeid, L. M., & Clarke, C. J. (2013). Sphingosine kinase 1 regulates tumor necrosis factor-mediated RANTES induction through p38 mitogen-activated protein kinase but independently of nuclear factor κB activation. The Journal of biological chemistry, 288(38), 27667-79.
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  Sphingosine kinase 1 (SK1) produces the pro-survival sphingolipid sphingosine 1-phosphate and has been implicated in inflammation, proliferation, and angiogenesis. Recent studies identified TRAF2 as a sphingosine 1-phosphate target, implicating SK1 in activation of the NF-κB pathway, but the functional consequences of this connection on gene expression are unknown. Here, we find that loss of SK1 potentiates induction of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5) in HeLa cells stimulated with TNF-α despite RANTES induction being highly dependent on the NF-κB pathway. Additionally, we find that SK1 is not required for TNF-induced IKK phosphorylation, IκB degradation, nuclear translocation of NF-κB subunits, and transcriptional NF-κB activity. In contrast, loss of SK1 prevented TNF-induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK, like SK1 knockdown, also potentiates RANTES induction. Finally, in addition to RANTES, loss of SK1 also potentiated the induction of multiple chemokines and cytokines in the TNF response. Taken together, these data identify a potential and novel anti-inflammatory function of SK1 in which chemokine levels are suppressed through SK1-mediated activation of p38 MAPK. Furthermore, in this system, activation of NF-κB is dissociated from SK1, suggesting that the interaction between these pathways may be more complex than currently thought.
• Gandy, K. A., Canals, D., Adada, M., Wada, M., Roddy, P., Snider, A. J., Hannun, Y. A., & Obeid, L. M. (2013). Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins. The Biochemical journal, 449(3), 661-72.
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  Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476-3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.
• Ross, J. S., Hu, W., Rosen, B., Snider, A. J., Obeid, L. M., & Cowart, L. A. (2013). Sphingosine kinase 1 is regulated by peroxisome proliferator-activated receptor α in response to free fatty acids and is essential for skeletal muscle interleukin-6 production and signaling in diet-induced obesity. The Journal of biological chemistry, 288(31), 22193-206.
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  We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3). Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1(-/-) mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1(-/-) mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.
• Snider, A. J., Ruiz, P., Obeid, L. M., & Oates, J. C. (2013). Inhibition of sphingosine kinase-2 in a murine model of lupus nephritis. PloS one, 8(1), e53521.
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  Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.
• Park, J. J., Rubio, M. V., Zhang, Z., Um, T., Xie, Y., Knoepp, S. M., Snider, A. J., Gibbs, T. C., & Meier, K. E. (2012). Effects of lysophosphatidic acid on calpain-mediated proteolysis of focal adhesion kinase in human prostate cancer cells. The Prostate, 72(15), 1595-610.
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  Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line.
• Ponnusamy, S., Selvam, S. P., Mehrotra, S., Kawamori, T., Snider, A. J., Obeid, L. M., Shao, Y., Sabbadini, R., & Ogretmen, B. (2012). Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1-phosphate signalling to regulate tumour metastasis. EMBO molecular medicine, 4(8), 761-75.
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  Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.
• Snider, A. J., Wu, B. X., Jenkins, R. W., Sticca, J. A., Kawamori, T., Hannun, Y. A., & Obeid, L. M. (2012). Loss of neutral ceramidase increases inflammation in a mouse model of inflammatory bowel disease. Prostaglandins & other lipid mediators, 99(3-4), 124-30.
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  Sphingolipids are emerging as important mediators of immune and inflammatory responses. We have previously demonstrated that sphingosine-1-phosphate (S1P) and its synthetic enzyme sphingosine kinase-1 (SK1) play an important role in inflammatory bowel disease. S1P generation is dependent on SK phosphorylation of sphingosine. Generation of sphingosine results only from the breakdown of ceramide by ceramidases (CDase). In this study, we set out to determine the role of neutral CDase (nCDase) in S1P generation and inflammatory bowel disease. To this end, we established nCDase expression is increased in patients with ulcerative colitis. Using the dextran sulfate sodium (DSS)-induced colitis model, we determined nCDase activity increased in colon epithelium, but not submucosa, in wild-type (WT) mice. Following DSS, ceramide levels were elevated in colon epithelium from WT and nCDase(-/-) mice, while S1P levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Similarly, cyclooxygenase-2 (Cox-2) levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Neutral CDase(-/-) mice also exhibited higher endotoxin levels in circulation, as well as higher circulating levels of S1P. This increase in S1P in nCDase(-/-) mice was accompanied by a marked leukocytosis, most notably circulating neutrophils and lymphocytes. Taken together these data demonstrate that loss of nCDase results in an unexpected increase in S1P generation in inflammation, and suggests that nCDase may actually protect against inflammation.
• Hernández-Corbacho, M. J., Jenkins, R. W., Clarke, C. J., Hannun, Y. A., Obeid, L. M., Snider, A. J., & Siskind, L. J. (2011). Accumulation of long-chain glycosphingolipids during aging is prevented by caloric restriction. PloS one, 6(6), e20411.
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  Chronic kidney disease and end-stage renal disease are major causes of morbidity and mortality that are seen far more commonly in the aged population. Interestingly, kidney function declines during aging even in the absence of underlying renal disease. Declining renal function has been associated with age-related cellular damage and dysfunction with reports of increased levels of apoptosis, necrosis, and inflammation in the aged kidney. Bioactive sphingolipids have been shown to regulate these same cellular processes, and have also been suggested to play a role in aging and cellular senescence.
• Jenkins, R. W., Clarke, C. J., Canals, D., Snider, A. J., Gault, C. R., Heffernan-Stroud, L., Wu, B. X., Simbari, F., Roddy, P., Kitatani, K., Obeid, L. M., & Hannun, Y. A. (2011). Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase. The Journal of biological chemistry, 286(15), 13292-303.
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  Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser(508) as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706-35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.
• Anelli, V., Gault, C. R., Snider, A. J., & Obeid, L. M. (2010). Role of sphingosine kinase-1 in paracrine/transcellular angiogenesis and lymphangiogenesis in vitro. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 24(8), 2727-38.
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  Sphingosine-1-phosphate (S1P) is an important bioactive sphingolipid involved in angiogenesis and lymphangiogenesis, 2 important processes that influence the growth, survival, and spread of tumors. S1P acts as an extracellular mediator through binding to 5 highly specific S1P receptors, S1P(1-5). Sphingosine kinase-1 (SK1), one of 2 known sphingosine kinase enzymes responsible for S1P production, appears to be overexpressed in many tumors. Although a role for S1P in angiogenesis and lymphangiogenesis has been established, it is unclear whether S1P secreted from cancer cells has a paracrine function in a tumor environment. Here we investigated whether modulation of cellular SK1 could initiate a paracrine angiogenic and lymphangiogenic switch. We found that SK1 overexpression in HEK cells or its down-regulation in glioma or breast cancer cells modulated extracellular S1P levels accordingly, which in turn increased or decreased both migration and tube formation in cocultured vascular or lymphatic endothelial cells. In contrast, down-regulation of sphingosine kinase 2 in both glioma and breast cancer cells had no appreciable effect on cellular or secreted S1P levels. In addition, vascular endothelial growth factors VEGF and VEGF-C down-regulation in cancer cells appeared insufficient to block the angiogenic and lymphangiogenic switch triggered by these cells. Moreover, S1P initiated endothelial cell sprouting in 3-dimensional collagen matrices, which is representative of a multistep angiogenic process. Our data collectively demonstrate for the first time that SK1 plays an essential role in regulating in vitro paracrine angiogenesis and lymphangiogenesis.
• Snider, A. J., Zhang, Z., Xie, Y., & Meier, K. E. (2010). Epidermal growth factor increases lysophosphatidic acid production in human ovarian cancer cells: roles for phospholipase D2 and receptor transactivation. American journal of physiology. Cell physiology, 298(1), C163-70.
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  Lysophosphatidic acid (LPA), is a lipid mediator that binds to G-protein coupled receptors. Epidermal growth factor (EGF), a polypeptide growth factor, binds to the EGF receptor (EGFR), a receptor tyrosine kinase. Both LPA and EGF induce responses in tumor cells that include proliferation, migration, metastasis, and induction of angiogenesis. LPA has the potential to act as an autocrine/paracrine factor and can transactivate the EGFR. This study explores the role of phospholipase D2 (PLD2) activation in LPA production, as well as cross-talk between EGF and LPA receptors. We demonstrate that EGF and LPA both stimulate production of LPA by OVCAR3 and SKOV3 human ovarian cancer cell lines. PD158780, an EGFR-selective tyrosine kinase inhibitor, blocks LPA production in response to both EGF and LPA in OVCAR3 and SKOV3 cells. Pertussis toxin, an inhibitor of LPA receptor signaling, inhibits LPA production in response to both EGF and LPA. Similar results were observed for the LPA receptor antagonist, Ki16425. Overexpression of PLD2 increases LPA production, while knockdown of PLD2 blocks EGF-induced LPA production. A phospholipase A2 (PLA2) inhibitor also blocks LPA- and EGF-induced LPA production. These results indicate that EGF stimulates LPA production in a manner that requires PLD2, and suggest that cross-talk can occur bidirectionally between EGF and LPA receptors.
• Snider, A. J., Kawamori, T., Bradshaw, S. G., Orr, K. A., Gilkeson, G. S., Hannun, Y. A., & Obeid, L. M. (2009). A role for sphingosine kinase 1 in dextran sulfate sodium-induced colitis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 23(1), 143-52.
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  The bioactive lipid sphingosine-1-phosphate (S1P) is emerging as an important mediator of immune and inflammatory responses. S1P formation is catalyzed by sphingosine kinase (SK), of which the SK1 isoenzyme is activated by tumor necrosis alpha (TNF-alpha). SK1 has been shown to be required for mediating TNF-alpha inflammatory responses in cells, including induction of cyclooxygenase 2 (COX-2). Because TNF-alpha and COX-2 are increased in patients with inflammatory bowel disease (IBD), we investigated the role of SK1 in a murine model of colitis. SK1(-/-) mice treated with dextran sulfate sodium (DSS) had significantly less blood loss, weight loss, colon shortening, colon histological damage, and splenomegaly than did wild-type (WT) mice. In addition, SK1(-/-) mice had no systemic inflammatory response. Moreover, WT but not SK1(-/-) mice treated with dextran sulfate sodium had significant increases in blood S1P levels, colon SK1 message and activity, and colon neutrophilic infiltrate. Unlike WT mice, SK1(-/-) mice failed to show colonic COX-2 induction despite an exaggerated TNF-alpha response; thus implicating for the first time SK1 in TNF-alpha-mediated COX-2 induction in vivo. Inhibition of SK1 may prove to be a valuable therapeutic target by inhibiting systemic and local inflammation in IBD.

Poster Presentations

• Warfel, N. A., Chauhan, S. C., Snider, J., Snider, A. J., & Cress, A. E. (2022, winter).

  PIM1 derives lipid droplet accumulation via regulation of GSK3β-PPARα signaling axis to promote proliferation and survival in prostate cancer

  . FASEB lipid droplet conferenceFASEB.

Reviews

• Clarke, C. J., & Snider, A. J. (2024. Perspective: Therapeutic Implications for Sphingolipids in Health and Disease(pp 118-120).
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  Long thought to be structural components of cell membranes, sphingolipids (SLs) have emerged as bioactive molecules whose metabolism is tightly regulated. These bioactive lipids and their metabolic enzymes have been implicated in numerous disease states, including lysosomal storage disorders, multiple sclerosis, inflammation, and cancer as well as metabolic syndrome and obesity. In addition, the indications for many of these lipids to potentially serve as biomarkers for disease continue to emerge with increasing metabolomic and lipidomic studies. The implications of these studies have, in turn, led to the examination of SL enzymes and their bioactive lipids as potential therapeutic targets and as markers for therapeutic efficacy. SIGNIFICANCE STATEMENT: Many sphingolipids (SLs) and their metabolizing enzymes have been implicated in disease. This perspective highlights the potential for SLs to serve as therapeutic targets and diagnostic markers and discusses the implications for the studies and reviews highlighted in this Special Section on Therapeutic Implications for Sphingolipids in Health and Disease.
• Doll, C. L., & Snider, A. J. (2024. The diverse roles of sphingolipids in inflammatory bowel disease(pp e23777).
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  The incidence of inflammatory bowel disease (IBD) has increased over the last 20 years. A variety of causes, both physiological and environmental, contribute to the initiation and progression of IBD, making disease management challenging. Current treatment options target various aspects of the immune response to dampen intestinal inflammation; however, their effectiveness at retaining remission, their side effects, and loss of response from patients over time warrant further investigation. Finding a common thread within the multitude causes of IBD is critical in developing robust treatment options. Sphingolipids are evolutionary conserved bioactive lipids universally generated in all cell types. This diverse lipid family is involved in a variety of fundamental, yet sometimes opposing, processes such as proliferation and apoptosis. Implicated as regulators in intestinal diseases, sphingolipids are a potential cornerstone in understanding IBD. Herein we will describe the role of host- and microbial-derived sphingolipids as they relate to the many factors of intestinal health and IBD.
• Espinoza, K. S., & Snider, A. J. (2024. Therapeutic Potential for Sphingolipids in Inflammatory Bowel Disease and Colorectal Cancer.
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  Inflammatory bowel disease (IBD), characterized by chronic inflammation in the intestinal tract, increases the risk for the development of colorectal cancer (CRC). Sphingolipids, which have been implicated in IBD and CRC, are a class of bioactive lipids that regulate cell signaling, differentiation, apoptosis, inflammation, and survival. The balance between ceramide (Cer), the central sphingolipid involved in apoptosis and differentiation, and sphingosine-1-phosphate (S1P), a potent signaling molecule involved in proliferation and inflammation, is vital for the maintenance of normal cellular function. Altered sphingolipid metabolism has been implicated in IBD and CRC, with many studies highlighting the importance of S1P in inflammatory signaling and pro-survival pathways. A myriad of sphingolipid analogues, inhibitors, and modulators have been developed to target the sphingolipid metabolic pathway. In this review, the efficacy and therapeutic potential for modulation of sphingolipid metabolism in IBD and CRC will be discussed.
• Velazquez, F. N., Hernandez-Corbacho, M., Trayssac, M., Stith, J. L., Bonica, J., Jean, B., Pulkoski-Gross, M. J., Carroll, B. L., Salama, M. F., Hannun, Y. A., & Snider, A. J. (2021. Bioactive sphingolipids: Advancements and contributions from the laboratory of Dr. Lina M. Obeid(p. 109875). Cellular Signaling.
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  Sphingolipids and their synthetic enzymes have emerged as critical mediators in numerous diseases including inflammation, aging, and cancer. One enzyme in particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P), has been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. In this review, we will discuss the contributions from the laboratory of Dr. Lina Obeid that have defined the roles for several bioactive sphingolipids in signaling and disease with an emphasis on her work defining SK1 in cellular fates and pathobiologies including proliferation, senescence, apoptosis, and inflammation.
• Choi, S., & Snider, A. J. (2015. Sphingolipids in High Fat Diet and Obesity-Related Diseases(p. 520618). Mediators of Inflammation.
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  Nutrient oversupply associated with a high fat diet (HFD) significantly alters cellular metabolism, and specifically including sphingolipid metabolism. Sphingolipids are emerging as bioactive lipids that play key roles in regulating functions, in addition to their traditional roles as membrane structure. HFD enhances de novo sphingolipid synthesis and turnover of sphingolipids via the salvage pathway, resulting in the generation of ceramide, and more specifically long chain ceramide species. Additionally, HFD elevates sphingomyelin and sphingosine-1 phosphate (S1P) levels in several tissues including liver, skeletal muscle, adipose tissue, and cardiovascular tissues. HFD-stimulated sphingolipid generation contributes to systemic insulin resistance, dysregulated lipid accumulation, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related conditions. Furthermore, altered sphingolipid levels, particularly ceramide and sphingomyelin, are involved in obesity-induced endothelial dysfunction and atherosclerosis. In this review, HFD-mediated sphingolipid metabolism and its impact on HFD-induced biology and pathobiology will be discussed.
• García-Barros, M., Coant, N., Truman, J. P., Snider, A. J., & Hannun, Y. A. (2014. Sphingolipids in colon cancer(pp 773-82).
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  Colorectal cancer is one of the major causes of death in the western world. Despite increasing knowledge of the molecular signaling pathways implicated in colon cancer, therapeutic outcomes are still only moderately successful. Sphingolipids, a family of N-acyl linked lipids, have not only structural functions but are also implicated in important biological functions. Ceramide, sphingosine and sphingosine-1-phosphate are the most important bioactive lipids, and they regulate several key cellular functions. Accumulating evidence suggests that many cancers present alterations in sphingolipids and their metabolizing enzymes. The aim of this review is to discuss the emerging roles of sphingolipids, both endogenous and dietary, in colon cancer and the interaction of sphingolipids with WNT/β-catenin pathway, one of the most important signaling cascades that regulate development and homeostasis in intestine. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
• Snider, A. J. (2013. Sphingosine kinase and sphingosine-1-phosphate: regulators in autoimmune and inflammatory disease.
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  Sphingolipids and their metabolizing enzymes are beginning to be recognized as critical mediators in biological processes, specifically in inflammation and autoimmunity. Sphingosine kinases (SKs) and their lipid product sphingosine-1-phosphate (S1P) play essential roles in inflammatory signaling processes, as well as disease development and progression. SKs can be activated by numerous growth factors and cytokines, including TNF-α and IL-1β, leading to the generation of S1P. S1P exerts its biological effects on intracellular and extracellular targets, such as S1P receptors. In addition to roles in inflammatory signaling pathways SKs, S1P and S1P receptors have been implicated in immune cell function and trafficking, specifically in lymphocytes. This review will discuss the contribution of the bioactive sphingolipid S1P, its generating enzyme SK, and its cell surface receptors in the inflammatory and autoimmune diseases systemic lupus erythematosus, arthritis and inflammatory bowel disease.
• Snider, A. J., Orr Gandy, K. A., & Obeid, L. M. (2010. Sphingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation(pp 707-15).
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  Sphingolipids and their synthetic enzymes are emerging as important mediators in inflammatory responses and as regulators of immune cell functions. In particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P) have been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. SK1 has been shown to be activated by cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin1-beta (IL1-beta). The activation of SK1 in this pathway has been shown to be, at least in part, required for mediating TNF-alpha and IL1-beta inflammatory responses in cells, including induction of cyclo-oxygenase 2 (COX2). In addition to their role in inflammatory signaling, SK and S1P have also been implicated in various immune cell functions including, mast cell degranulation, migration of neutrophils, and migration and maturation of lymphocytes. The involvement of sphingolipids and sphingolipid metabolizing enzymes in inflammatory signaling and immune cell functions has implicated these mediators in numerous inflammatory disease states as well. The contribution of these mediators, specifically SK1 and S1P, to inflammation and disease are discussed in this review.
• Snider, A. C., & Meier, K. E. (2007. Receptor transactivation cascades. Focus on "Effects of alpha1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells"(pp C1-3). American Journal of Physiology - Cell Physiology.

Others

• Wang, K., Xu, R., Schrandt, J., Shah, P., Gong, Y. Z., Preston, C., Wang, L., Yi, J. K., Lin, C. L., Sun, W., Spyropoulos, D. D., Rhee, S., Li, M., Zhou, J., Ge, S., Zhang, G., Snider, A. J., Hannun, Y. A., Obeid, L. M., & Mao, C. (2018, Jan). Correction: Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain. PLoS genetics.
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  [This corrects the article DOI: 10.1371/journal.pgen.1005591.].