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1.
Nat Immunol ; 15(3): 231-8, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24464131

RESUMEN

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.


Asunto(s)
Quimiocina CCL5/biosíntesis , Quimiocina CXCL10/biosíntesis , Factor 1 Regulador del Interferón/metabolismo , Interleucina-1/metabolismo , Transducción de Señal/inmunología , Animales , Quimiocina CCL5/inmunología , Quimiocina CXCL10/inmunología , Quimiotaxis de Leucocito/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Inmunoprecipitación , Inflamación/inmunología , Inflamación/metabolismo , Factor 1 Regulador del Interferón/inmunología , Interleucina-1/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lisina , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Ubiquitinación
2.
PLoS Pathog ; 19(11): e1011842, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38033162

RESUMEN

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy.


Asunto(s)
Células Endoteliales , Neisseria meningitidis , Receptores de Esfingosina-1-Fosfato/metabolismo , Células Endoteliales/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/metabolismo , Encéfalo/metabolismo , Lisofosfolípidos/metabolismo
3.
FASEB J ; 38(15): e23872, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39126272

RESUMEN

Paclitaxel is among the most active chemotherapy drugs for the aggressive triple negative breast cancer (TNBC). Unfortunately, it often induces painful peripheral neuropathy (CIPN), a major debilitating side effect. Here we demonstrate that in naive and breast tumor-bearing immunocompetent mice, a clinically relevant dose of FTY720/Fingolimod that targets sphingosine-1-phosphate receptor 1 (S1PR1), alleviated paclitaxel-induced neuropathic pain. FTY720 also significantly attenuated paclitaxel-stimulated glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and expression of the astrocyte-secreted synaptogenic protein Sparcl1/Hevin, a key regulator of synapse formation. Notably, the formation of excitatory synapses containing VGluT2 in the spinal cord dorsal horn induced by paclitaxel was also inhibited by FTY720 treatment, supporting the involvement of astrocytes and Sparcl1 in CIPN. Furthermore, in this TNBC mouse model that mimics human breast cancer, FTY720 administration also enhanced the anti-tumor effects of paclitaxel, leading to reduced tumor progression and lung metastasis. Taken together, our findings suggest that targeting the S1P/S1PR1 axis with FTY720 is a multipronged approach that holds promise as a therapeutic strategy for alleviating both CIPN and enhancing the efficacy of chemotherapy in TNBC treatment.


Asunto(s)
Clorhidrato de Fingolimod , Neuralgia , Paclitaxel , Animales , Clorhidrato de Fingolimod/farmacología , Paclitaxel/farmacología , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Neuralgia/patología , Ratones , Femenino , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Línea Celular Tumoral , Receptores de Esfingosina-1-Fosfato/metabolismo , Humanos , Progresión de la Enfermedad , Antineoplásicos Fitogénicos/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética
4.
Proc Natl Acad Sci U S A ; 119(39): e2204396119, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-36122218

RESUMEN

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.


Asunto(s)
Fosfatidilserinas , Esfingosina , Ceramidas/metabolismo , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Isoenzimas/metabolismo , Liposomas/metabolismo , Lisofosfolípidos , Fosfatidilserinas/metabolismo , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
5.
J Lipid Res ; 65(6): 100556, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38719150

RESUMEN

Niemann-Pick type C1 (NPC1) disease is a rare neurodegenerative cholesterol and sphingolipid storage disorder primarily due to mutations in the cholesterol-trafficking protein NPC1. In addition to catabolic-derived sphingolipids, NPC1 dysfunction also leads to an increase in de novo sphingolipid biosynthesis, yet little is known about the cellular mechanism involved. Although deletion of NPC1 or inhibition of the NPC1 sterol binding domain enhanced de novo sphingolipid biosynthesis, surprisingly levels of the ORMDLs, the regulatory subunits of serine palmitoyltransferase (SPT), the rate-limiting step in sphingolipid biosynthesis, were also greatly increased. Nevertheless, less ORMDL was bound in the SPT-ORMDL complex despite elevated ceramide levels. Instead, ORMDL colocalized with p62, the selective autophagy receptor, and accumulated in stalled autophagosomes due to defective autophagy in NPC1 disease cells. Restoration of autophagic flux with N-acetyl-L-leucine in NPC1 deleted cells decreased ORMDL accumulation in autophagosomes and reduced de novo sphingolipid biosynthesis and their accumulation. This study revealed a previously unknown link between de novo sphingolipid biosynthesis, ORMDL, and autophagic defects present in NCP1 disease. In addition, we provide further evidence and mechanistic insight for the beneficial role of N-acetyl-L-leucine treatment for NPC1 disease which is presently awaiting approval from the Food and Drug Administration and the European Medicines Agency.


Asunto(s)
Autofagia , Enfermedad de Niemann-Pick Tipo C , Esfingolípidos , Esfingolípidos/metabolismo , Esfingolípidos/biosíntesis , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Enfermedad de Niemann-Pick Tipo C/genética , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Animales , Proteína Niemann-Pick C1 , Serina C-Palmitoiltransferasa/metabolismo , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/antagonistas & inhibidores
6.
J Biol Chem ; 299(6): 104775, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37142226

RESUMEN

The vascular and lymphatic systems both comprise a series of structurally distinct vessels lined with an inner layer of endothelial cells that function to provide a semipermeable barrier to blood and lymph. Regulation of the endothelial barrier is critical for maintaining vascular and lymphatic barrier homeostasis. One of the regulators of endothelial barrier function and integrity is sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite secreted into the blood by erythrocytes, platelets, and endothelial cells and into the lymph by lymph endothelial cells. Binding of S1P to its G protein-coupled receptors, known as S1PR1-5, regulates its pleiotropic functions. This review outlines the structural and functional differences between vascular and lymphatic endothelium and describes current understanding of the importance of S1P/S1PR signaling in regulation of barrier functions. Most studies thus far have been primarily focused on the role of the S1P/S1PR1 axis in vasculature and have been summarized in several excellent reviews, and thus, we will only discuss new perspectives on the molecular mechanisms of action of S1P and its receptors. Much less is known about the responses of the lymphatic endothelium to S1P and the functions of S1PRs in lymph endothelial cells, and this is the major focus of this review. We also discuss current knowledge related to signaling pathways and factors regulated by the S1P/S1PR axis that control lymphatic endothelial cell junctional integrity. Gaps and limitations in current knowledge are highlighted together with the need to further understand the role of S1P receptors in the lymphatic system.


Asunto(s)
Endotelio Vascular , Vasos Linfáticos , Lisofosfolípidos , Receptores de Lisoesfingolípidos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Humanos , Animales , Uniones Intercelulares , Transducción de Señal , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo
7.
FASEB J ; 37(3): e22799, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36753412

RESUMEN

Genome-wide association studies have linked the ORM (yeast)-like protein isoform 3 (ORMDL3) to asthma severity. Although ORMDL3 is a member of a family that negatively regulates serine palmitoyltransferase (SPT) and thus biosynthesis of sphingolipids, it is still unclear whether ORMDL3 and altered sphingolipid synthesis are causally related to non-Th2 severe asthma associated with a predominant neutrophil inflammation and high interleukin-17 (IL-17) levels. Here, we examined the effects of ORMDL3 overexpression in a preclinical mouse model of allergic lung inflammation that is predominantly neutrophilic and recapitulates many of the clinical features of severe human asthma. ORMDL3 overexpression reduced lung and circulating levels of dihydrosphingosine, the product of SPT. However, the most prominent effect on sphingolipid levels was reduction of circulating S1P. The LPS/OVA challenge increased markers of Th17 inflammation with a predominant infiltration of neutrophils into the lung. A significant decrease of neutrophil infiltration was observed in the Ormdl3 transgenic mice challenged with LPS/OVA compared to the wild type and concomitant decrease in IL-17, that plays a key role in the pathogenesis of neutrophilic asthma. LPS decreased survival of murine neutrophils, which was prevented by co-treatment with S1P. Moreover, S1P potentiated LPS-induced chemotaxis of neutrophil, suggesting that S1P can regulate neutrophil survival and recruitment following LPS airway inflammation. Our findings reveal a novel connection between ORMDL3 overexpression, circulating levels of S1P, IL-17 and neutrophil recruitment into the lung, and questions the potential involvement of ORMDL3 in the pathology, leading to development of severe neutrophilic asthma.


Asunto(s)
Asma , Interleucina-17 , Proteínas de la Membrana , Animales , Humanos , Ratones , Asma/metabolismo , Estudio de Asociación del Genoma Completo , Inflamación/metabolismo , Interleucina-17/genética , Interleucina-17/uso terapéutico , Lipopolisacáridos , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Esfingolípidos/metabolismo
8.
FASEB J ; 36(7): e22372, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35639028

RESUMEN

Non-alcoholic steatohepatitis (NASH) results from the accumulation of excessive liver lipids leading to hepatocellular injury, inflammation, and fibrosis that greatly increase the risk for hepatocellular carcinoma (HCC). Despite the well-characterized clinical and histological pathology for NASH-driven HCC in humans, its etiology remains unclear and there is a deficiency in pre-clinical models that recapitulate the progression of the human disease. Therefore, we developed a new mouse model amenable to genetic manipulations and gene targeting that mimics the gradual NASH to HCC progression observed in humans. C57BL/6NJ mice were fed a Western high-fat diet and sugar water (HFD/SW) and monitored for effects on metabolism, liver histology, tumor development, and liver transcriptome for up to 54 weeks. Chronic HFD/SW feeding led to significantly increased weight gain, serum and liver lipid levels, liver injury, and glucose intolerance. Hepatic pathology progressed and mice developed hepatocellular ballooning, inflammation, and worse fibrosis was apparent at 16 weeks, greatly increased through 32 weeks, and remained elevated at 54 weeks. Importantly, hepatocellular cancer spontaneously developed in 75% of mice on HFD/SW, half of which were HCC, whereas none of the mice on the chow diet developed HCC. Chronic HFD/SW induced molecular markers of de novo lipogenesis, endoplasmic reticulum stress, inflammation, and accumulation of p62, all of which also participate in the human pathology. Moreover, transcriptome analysis revealed activation of HCC-related genes and signatures associated with poor prognosis of human HCC. Overall, we have identified a new preclinical model that recapitulates known hallmarks of NASH-driven HCC that can be utilized for future molecular mechanistic studies of this disease.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Animales , Carcinoma Hepatocelular/metabolismo , Dieta Occidental/efectos adversos , Fibrosis , Inflamación/complicaciones , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo
9.
Nature ; 541(7636): 233-236, 2017 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-28052056

RESUMEN

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.


Asunto(s)
Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Genoma/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Animales , Proteínas de Transporte de Anión/deficiencia , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Genómica , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Linfopenia/genética , Linfopenia/patología , Lisofosfolípidos/metabolismo , Masculino , Ratones , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Microambiente Tumoral
10.
Glia ; 70(4): 712-727, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34958493

RESUMEN

Astrocytes, the most abundant glial cells in the mammalian brain, directly associate with and regulate neuronal processes and synapses and are important regulators of brain development. Yet little is known of the molecular mechanisms that control the establishment of astrocyte morphology and the bi-directional communication between astrocytes and neurons. Here we show that neuronal contact stimulates expression of S1PR1, the receptor for the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P), on perisynaptic astrocyte processes and that S1PR1 drives astrocyte morphological complexity and morphogenesis. Moreover, the S1P/S1PR1 axis increases neuronal contact-induced expression of astrocyte secreted synaptogenic factors SPARCL1 and thrombospondin 4 that are involved in neural circuit assembly. Our findings have uncovered new functions for astrocytic S1PR1 signaling in regulation of bi-directional astrocyte-neuron crosstalk at the nexus of astrocyte morphogenesis and synaptogenesis.


Asunto(s)
Astrocitos , Neuronas , Animales , Astrocitos/metabolismo , Neuronas/metabolismo , Transducción de Señal , Receptores de Esfingosina-1-Fosfato , Sinapsis/metabolismo
11.
FASEB J ; 35(3): e21415, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33566377

RESUMEN

Acute liver failure (ALF) causes severe liver dysfunction that can lead to multi-organ failure and death. Previous studies suggest that sphingosine kinase 1 (SphK1) protects against hepatocyte injury, yet not much is still known about its involvement in ALF. This study examines the role of SphK1 in D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF, which is a well-established experimental mouse model that mimics the fulminant hepatitis. Here we report that deletion of SphK1, but not SphK2, dramatically decreased GalN/LPS-induced liver damage, hepatic apoptosis, serum alanine aminotransferase levels, and mortality rate compared to wild-type mice. Whereas GalN/LPS treatment-induced hepatic activation of NF-κB and JNK in wild-type and SphK2-/- mice, these signaling pathways were reduced in SphK1-/- mice. Moreover, repression of ALF in SphK1-/- mice correlated with decreased expression of the pro-inflammatory cytokine TNFα. Adoptive transfer experiments indicated that SphK1 in bone marrow-derived infiltrating immune cells but not in host liver-resident cells, contribute to the development of ALF. Interestingly, LPS-induced TNFα production was drastically suppressed in SphK1-deleted macrophages, whereas IL-10 expression was markedly enhanced, suggesting a switch to the anti-inflammatory phenotype. Finally, treatment with a specific SphK1 inhibitor ameliorated inflammation and protected mice from ALF. Our findings suggest that SphK1 regulates TNFα secretion from macrophages and inhibition or deletion of SphK1 mitigated ALF. Thus, a potent inhibitor of SphK1 could potentially be a therapeutic agent for fulminant hepatitis.


Asunto(s)
Apoptosis/genética , Inflamación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Factor de Necrosis Tumoral alfa/metabolismo , Alanina Transaminasa/metabolismo , Animales , Antiinflamatorios/farmacología , Apoptosis/fisiología , Modelos Animales de Enfermedad , Galactosamina/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Fallo Hepático Agudo/tratamiento farmacológico , Fallo Hepático Agudo/metabolismo , Ratones Noqueados
12.
Proc Natl Acad Sci U S A ; 116(21): 10557-10562, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-31068460

RESUMEN

Neuropathic pain afflicts millions of individuals and represents a major health problem for which there is limited effective and safe therapy. Emerging literature links altered sphingolipid metabolism to nociceptive processing. However, the neuropharmacology of sphingolipid signaling in the central nervous system in the context of chronic pain remains largely unexplored and controversial. We now provide evidence that sphingosine-1-phosphate (S1P) generated in the dorsal horn of the spinal cord in response to nerve injury drives neuropathic pain by selectively activating the S1P receptor subtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharmacological inhibition of S1PR1 with multiple antagonists in distinct chemical classes, but not agonists, attenuated and even reversed neuropathic pain in rodents of both sexes and in two models of traumatic nerve injury. These S1PR1 antagonists retained their ability to inhibit neuropathic pain during sustained drug administration, and their effects were independent of endogenous opioid circuits. Moreover, mice with astrocyte-specific knockout of S1pr1 did not develop neuropathic pain following nerve injury, thereby identifying astrocytes as the primary cellular substrate of S1PR1 activity. On a molecular level, the beneficial reductions in neuropathic pain resulting from S1PR1 inhibition were driven by interleukin 10 (IL-10), a potent neuroprotective and anti-inflammatory cytokine. Collectively, our results provide fundamental neurobiological insights that identify the cellular and molecular mechanisms engaged by the S1PR1 axis in neuropathic pain and establish S1PR1 as a target for therapeutic intervention with S1PR1 antagonists as a class of nonnarcotic analgesics.


Asunto(s)
Astrocitos/metabolismo , Neuralgia/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Sulfonas/uso terapéutico , Triazoles/uso terapéutico , Animales , Evaluación Preclínica de Medicamentos , Femenino , Interleucina-10/metabolismo , Masculino , Ratones , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Ratas Sprague-Dawley , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Sulfonas/farmacología , Triazoles/farmacología
13.
J Allergy Clin Immunol ; 147(5): 1936-1948.e9, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33130063

RESUMEN

BACKGROUND: Nothing is known about the mechanisms by which increased ceramide levels in the lung contribute to allergic responses and asthma severity. OBJECTIVE: We sought to investigate the functional role of ceramide in mouse models of allergic airway disease that recapitulate the cardinal clinical features of human allergic asthma. METHODS: Allergic airway disease was induced in mice by repeated intranasal administration of house dust mite or the fungal allergen Alternaria alternata. Processes that can be regulated by ceramide and are important for severity of allergic asthma were correlated with ceramide levels measured by mass spectrometry. RESULTS: Both allergens induced massive pulmonary apoptosis and also significantly increased reactive oxygen species in the lung. Prevention of increases in lung ceramide levels mitigated allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. In contrast, dietary supplementation of the antioxidant α-tocopherol decreased reactive oxygen species but had no significant effects on elevation of ceramide level or apoptosis, indicating that the increases in lung ceramide levels in allergen-challenged mice are not mediated by oxidative stress. Moreover, specific ceramide species were altered in bronchoalveolar lavage fluid from patients with severe asthma compared with in bronchoalveolar lavage fluid from individuals without asthma. CONCLUSION: Our data suggest that elevation of ceramide level after allergen challenge contributes to the apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide might be the trigger of formation of Creola bodies found in the sputum of patients with severe asthma and could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.


Asunto(s)
Asma/inmunología , Ceramidas/inmunología , Pulmón/inmunología , Estrés Oxidativo , Adulto , Alérgenos/inmunología , Alternaria/inmunología , Animales , Apoptosis , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/inmunología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Pyroglyphidae/inmunología , Especies Reactivas de Oxígeno/inmunología , Adulto Joven
14.
J Lipid Res ; 62: 100082, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33939982

RESUMEN

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.


Asunto(s)
Sistemas CRISPR-Cas
15.
J Biol Chem ; 295(10): 3371-3384, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32144147

RESUMEN

It is a great honor to be asked to write a "Reflections" article by one of the true icons of biochemistry, Herb Tabor. I felt humbled, especially since it follows many written by biochemists I admire and whose contributions have shaped major advances in biochemistry and molecular biology in the last century. Here I present my personal reflections on my adventure with the bioactive sphingolipid metabolite sphingosine-1-phosphate intertwined with those of my family life as a wife, mother, and grandmother. These reflections brought back many memories of events in my early career that played significant roles in determining the path I have taken for more than 40 years and that brought much fun and satisfaction into my life. It has been an exciting journey so far, with many surprises along the way, that still continues.


Asunto(s)
Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Autobiografías como Asunto , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Esfingolípidos/metabolismo , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/química , Receptores de Esfingosina-1-Fosfato/metabolismo
16.
J Biol Chem ; 295(27): 9121-9133, 2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32385114

RESUMEN

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.


Asunto(s)
Enfermedad de Niemann-Pick Tipo C/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Endosomas/metabolismo , Fibroblastos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Proteína Niemann-Pick C1/genética , Proteína Niemann-Pick C1/metabolismo , Enfermedad de Niemann-Pick Tipo C/fisiopatología , Cultivo Primario de Células , Transporte de Proteínas , Esfingolípidos/metabolismo , Esfingosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
17.
FASEB J ; 34(3): 4329-4347, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31971297

RESUMEN

Plasmacytoid dendritic cells (pDCs) express Toll like receptors (TLRs) that modulate the immune response by production of type I interferons. Here, we report that sphingosine kinase 1 (SphK1) which produces the bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), plays a critical role in the pDC functions and interferon production. Although dispensable for the pDC development, SphK1 is essential for the pDC activation and production of type I IFN and pro-inflammatory cytokines stimulated by TLR7/9 ligands. SphK1 interacts with TLRs and specific inhibition or deletion of SphK1 in pDCs mitigates uptake of CpG oligonucleotide ligands by TLR9 ligand. In the pristane-induced murine lupus model, pharmacological inhibition of SphK1 or its genetic deletion markedly decreased the IFN signature, pDC activation, and glomerulonephritis. Moreover, increases in the SphK1 expression and S1P levels were observed in human lupus patients. Taken together, our results indicate a pivotal regulatory role for the SphK1/S1P axis in maintaining the balance between immunosurveillance and immunopathology and suggest that specific SphK1 inhibitors might be a new therapeutic avenue for the treatment of type I IFN-linked autoimmune disorders.


Asunto(s)
Autoinmunidad/fisiología , Interferón Tipo I/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Autoinmunidad/genética , Western Blotting , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Receptor Toll-Like 7/genética , Receptor Toll-Like 9/genética
18.
Med Res Rev ; 40(1): 385-412, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31318078

RESUMEN

Exosomes are abundantly secreted extracellular vesicles that accumulate in the circulation and are of great interest for disease diagnosis and evaluation since their contents reflects the phenotype of their cell of origin. Tumor-derived exosomes (TDEs) are of particular interest for cancer diagnosis and therapy, since most tumor demonstrate highly elevated exosome secretion rates and provide specific information about the genotype of a tumor and its response to treatment. TDEs also contain regulatory factors that can alter the phenotypes of local and distant tissue sites and alter immune cell functions to promote tumor progression. The abundance, information content, regulatory potential, in vivo half-life, and physical durability of exosomes suggest that TDEs may represent a superior source of diagnostic biomarkers and treatment targets than other materials currently under investigation. This review will summarize current information on mechanisms that may differentially regulate TDE biogenesis, TDE effects on the immune system that promote tumor survival, growth, and metastasis, and new approaches understudy to counteract or utilize TDE properties in cancer therapies.


Asunto(s)
Exosomas/metabolismo , Neoplasias/metabolismo , Animales , Transporte Biológico , Vacunas contra el Cáncer/inmunología , Humanos , Evasión Inmune/inmunología , Neoplasias/inmunología , Microambiente Tumoral/inmunología
19.
J Lipid Res ; 61(6): 953-967, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32341007

RESUMEN

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist's toolbox.


Asunto(s)
Lipidómica/métodos , Esfingolípidos/metabolismo , Temperatura , Cromatografía Liquida , Humanos , Pulmón/citología , Pulmón/metabolismo , Espectrometría de Masas en Tándem
20.
J Neuroinflammation ; 17(1): 314, 2020 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-33092620

RESUMEN

Opioid therapies for chronic pain are undermined by many adverse side effects that reduce their efficacy and lead to dependence, abuse, reduced quality of life, and even death. We have recently reported that sphingosine-1-phosphate (S1P) 1 receptor (S1PR1) antagonists block the development of morphine-induced hyperalgesia and analgesic tolerance. However, the impact of S1PR1 antagonists on other undesirable side effects of opioids, such as opioid-induced dependence, remains unknown. Here, we demonstrate that naloxone-precipitated morphine withdrawal in mice altered de novo sphingolipid metabolism in the dorsal horn of the spinal cord and increased S1P that accompanied the manifestation of several withdrawal behaviors. Blocking de novo sphingolipid metabolism with intrathecal administration of myriocin, an inhibitor of serine palmitoyltransferase, blocked naloxone-precipitated withdrawal. Noteworthy, we found that competitive (NIBR-15) and functional (FTY720) S1PR1 antagonists attenuated withdrawal behaviors in mice. Mechanistically, at the level of the spinal cord, naloxone-precipitated withdrawal was associated with increased glial activity and formation of the potent inflammatory/neuroexcitatory cytokine interleukin-1ß (IL-1ß); these events were attenuated by S1PR1 antagonists. These results provide the first molecular insight for the role of the S1P/S1PR1 axis during opioid withdrawal. Our data identify S1PR1 antagonists as potential therapeutics to mitigate opioid-induced dependence and support repurposing the S1PR1 functional antagonist FTY720, which is FDA-approved for multiple sclerosis, as an opioid adjunct.


Asunto(s)
Analgésicos Opioides/efectos adversos , Sistema Nervioso Central/metabolismo , Morfina/efectos adversos , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Receptores de Esfingosina-1-Fosfato/metabolismo , Síndrome de Abstinencia a Sustancias/metabolismo , Animales , Sistema Nervioso Central/efectos de los fármacos , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Roedores , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico
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