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1.
Proc Natl Acad Sci U S A ; 119(39): e2204396119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122218

RESUMO

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.


Assuntos
Fosfatidilserinas , Esfingosina , Ceramidas/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Isoenzimas/metabolismo , Lipossomos/metabolismo , Lisofosfolipídeos , Fosfatidilserinas/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
2.
FASEB J ; 37(3): e22799, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36753412

RESUMO

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.


Assuntos
Asma , Interleucina-17 , Proteínas de Membrana , Animais , Humanos , Camundongos , Asma/metabolismo , Estudo de Associação Genômica Ampla , Inflamação/metabolismo , Interleucina-17/genética , Interleucina-17/uso terapêutico , Lipopolissacarídeos , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Esfingolipídeos/metabolismo
3.
J Allergy Clin Immunol ; 147(5): 1936-1948.e9, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33130063

RESUMO

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.


Assuntos
Asma/imunologia , Ceramidas/imunologia , Pulmão/imunologia , Estresse Oxidativo , Adulto , Alérgenos/imunologia , Alternaria/imunologia , Animais , Apoptose , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pyroglyphidae/imunologia , Espécies Reativas de Oxigênio/imunologia , Adulto Jovem
4.
J Biol Chem ; 295(27): 9121-9133, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32385114

RESUMO

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.


Assuntos
Doença de Niemann-Pick Tipo C/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Endossomos/metabolismo , Fibroblastos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Doença de Niemann-Pick Tipo C/fisiopatologia , Cultura Primária de Células , Transporte Proteico , Esfingolipídeos/metabolismo , Esfingosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
J Lipid Res ; 59(8): 1424-1432, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739865

RESUMO

Neutrophils form neutrophil extracellular traps (NETs), which have been implicated in microcirculatory plugging. NET formation (NETosis) involves the fusion of granule and nuclear contents, which are then released in the extracellular space. Myeloperoxidase (MPO) plays a major role in NETosis leading to the dissociation of DNA from histones. During neutrophil activation, MPO is released and activated to convert hydrogen peroxide and chloride to hypochlorous acid (HOCl). HOCl targets plasmalogens leading to the production of the chlorinated lipids, 2-chlorofatty aldehyde and 2-chlorofatty acid (2-ClFA). Here, we tested the hypothesis that 2-ClFAs are important lipid mediators of NETosis. Human neutrophils treated with physiological levels of 2-ClFAs formed NETs, characterized by MPO association with DNA and neutrophil elastase (NE) redistribution to the perinuclear area. 2-ClFA-induced NETs reduced Escerichia coli colony forming units. 2-ClFA-induced NETosis is calcium- and protein arginine deiminase 4-dependent. Interestingly, unlike PMA, 2-ClFA initiates the NETosis process without neutrophil activation and degranulation. Furthermore, 2-ClFA elicits NETosis in bone-marrow derived neutrophils from MPO-deficient mice. Taken together, these findings suggest 2-ClFA as an MPO product that triggers the NETosis pathway following neutrophil activation.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Ácidos Graxos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Neutrófilos/citologia , Cálcio/metabolismo , DNA/metabolismo , Ácidos Graxos/química , Voluntários Saudáveis , Humanos , Neutrófilos/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/metabolismo
6.
J Lipid Res ; 59(4): 696-705, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29444934

RESUMO

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.


Assuntos
Aldeídos/sangue , Bromo/sangue , Peroxidase de Eosinófilo/sangue , Glutationa Transferase/sangue , Peroxidase/sangue , Animais , Bromo/administração & dosagem , Química Click , Peroxidase de Eosinófilo/metabolismo , Glutationa Transferase/metabolismo , Voluntários Saudáveis , Humanos , Camundongos , Peroxidase/metabolismo , Células RAW 264.7
7.
Arch Biochem Biophys ; 641: 31-38, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29378164

RESUMO

Myeloperoxidase produces the two-electron oxidant HOCl, which targets plasmalogen phospholipids liberating 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde has four known fates: 1) oxidation to 2-chlorofatty acid; 2) reduction to 2-chlorofatty alcohol; 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon. 2-Chlorofatty acid does not undergo conventional fatty acid ß-oxidation due to the presence of the α-chlorinated carbon; however, 2-chlorofatty acid does undergo sequential ω-oxidation and ß-oxidation from the ω-end, ultimately resulting in 2-chloroadipic acid urinary excretion. Recent studies have demonstrated that 2-chlorofatty acid clearance is increased by treatment with the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-chlorofatty acid has been shown to be a PPAR-α agonist, and thus accelerates its own clearance. The roles of 2-chlorofatty aldehyde and 2-chlorofatty acid on leukocyte and endothelial function have been explored by several groups, suggesting that chlorinated lipids induce endothelial cell dysfunction, neutrophil chemotaxis, monocyte apoptosis, and alterations in vascular tone. Thus, the chlorinated lipidome, produced in response to leukocyte activation, is a potential biomarker and therapeutic target to modulate host response in inflammatory diseases.


Assuntos
Cloro/metabolismo , Ácido Hipocloroso/metabolismo , Metabolismo dos Lipídeos , Peroxidase/metabolismo , Plasmalogênios/metabolismo , Aldeídos/metabolismo , Animais , Biomarcadores/metabolismo , Doença , Esterificação , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos não Esterificados/urina , Halogenação , Humanos , Neutrófilos/metabolismo , Oxirredução , PPAR alfa/metabolismo
8.
J Lipid Res ; 58(2): 317-324, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28007964

RESUMO

α-Chlorofatty aldehydes are generated from myeloperoxidase-derived HOCl targeting plasmalogens, and are subsequently oxidized to α-chlorofatty acids (α-ClFAs). The catabolic pathway for α-ClFA is initiated by ω-oxidation. Here, we examine PPAR-α activation as a mechanism to increase α-ClFA catabolism. Pretreating both HepG2 cells and primary mouse hepatocytes with the PPAR-α agonist, pirinixic acid (Wy 14643), increased the production of α-chlorodicarboxylic acids (α-ClDCAs) in cells treated with exogenous α-ClFA. Additionally, α-ClDCA production in Wy 14643-pretreated wild-type mouse hepatocytes was accompanied by a reduction in cellular free α-ClFA. The dependence of PPAR-α-accelerated α-ClFA catabolism was further demonstrated by both impaired metabolism in mouse PPAR-α-/- hepatocytes and decreased clearance of plasma α-ClFA in PPAR-α-/- mice. Furthermore, Wy 14643 treatments decreased plasma 2-chlorohexadecanoic acid levels in wild-type mice. Additional studies showed that α-ClFA increases PPAR-α, PPAR-δ, and PPAR-γ activities, as well as mRNA expression of the PPAR-α target genes, CD36, CPT1a, Cyp4a10, and CIDEC. Collectively, these results indicate that PPAR-α accelerates important pathways for the clearance of α-ClFA, and α-ClFA may, in part, accelerate its catabolism by serving as a ligand for PPAR-α.


Assuntos
Hepatócitos/metabolismo , PPAR alfa/genética , Ácidos Palmíticos/metabolismo , Animais , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Metabolismo/genética , Camundongos , Camundongos Knockout , Oxirredução , PPAR alfa/metabolismo , PPAR delta/biossíntese , RNA Mensageiro/biossíntese
9.
J Lipid Res ; 58(5): 941-954, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28264879

RESUMO

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.


Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Surfactantes Pulmonares/metabolismo , Células A549 , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Animais , Colesterol/biossíntese , Colesterol/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Homeostase , Humanos , Imunoglobulinas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteinose Alveolar Pulmonar/metabolismo , Proteinose Alveolar Pulmonar/patologia
10.
Cancer Res ; 83(4): 553-567, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36541910

RESUMO

Reciprocal interactions between breast cancer cells and the tumor microenvironment (TME) are important for cancer progression and metastasis. We report here that the deletion or inhibition of sphingosine kinase 2 (SphK2), which produces sphingosine-1-phosphate (S1P), markedly suppresses syngeneic breast tumor growth and lung metastasis in mice by creating a hostile microenvironment for tumor growth and invasion. SphK2 deficiency decreased S1P and concomitantly increased ceramides, including C16-ceramide, in stromal fibroblasts. Ceramide accumulation suppressed activation of cancer-associated fibroblasts (CAF) by upregulating stromal p53, which restrained production of tumor-promoting factors to reprogram the TME and to restrict breast cancer establishment. Ablation of p53 in SphK2-deficient fibroblasts reversed these effects, enabled CAF activation and promoted tumor growth and invasion. These data uncovered a novel role of SphK2 in regulating non-cell-autonomous functions of p53 in stromal fibroblasts and their transition to tumor-promoting CAFs, paving the way for the development of a strategy to target the TME and to enhance therapeutic efficacy. SIGNIFICANCE: Sphingosine kinase 2 (SphK2) facilitates the activation of stromal fibroblasts to tumor-promoting cancer-associated fibroblasts by suppressing host p53 activity, revealing SphK2 as a potential target to reprogram the TME.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Mamárias Animais , Fosfotransferases (Aceptor do Grupo Álcool) , Microambiente Tumoral , Animais , Camundongos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Microambiente Tumoral/fisiologia , Proteína Supressora de Tumor p53/genética
11.
Cell Stem Cell ; 22(2): 206-220.e4, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395055

RESUMO

Adequate availability of cellular building blocks, including lipids, is a prerequisite for cellular proliferation, but excess dietary lipids are linked to increased cancer risk. Despite these connections, specific regulatory relationships between membrane composition, intestinal stem cell (ISC) proliferation, and tumorigenesis are unclear. We reveal an unexpected link between membrane phospholipid remodeling and cholesterol biosynthesis and demonstrate that cholesterol itself acts as a mitogen for ISCs. Inhibition of the phospholipid-remodeling enzyme Lpcat3 increases membrane saturation and stimulates cholesterol biosynthesis, thereby driving ISC proliferation. Pharmacologic inhibition of cholesterol synthesis normalizes crypt hyperproliferation in Lpcat3-deficient organoids and mice. Conversely, increasing cellular cholesterol content stimulates crypt organoid growth, and providing excess dietary cholesterol or driving endogenous cholesterol synthesis through SREBP-2 expression promotes ISC proliferation in vivo. Finally, disruption of Lpcat3-dependent phospholipid and cholesterol homeostasis dramatically enhances tumor formation in Apcmin mice. These findings identify a critical dietary-responsive phospholipid-cholesterol axis regulating ISC proliferation and tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Colesterol/metabolismo , Intestinos/patologia , Fosfolipídeos/metabolismo , Células-Tronco/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/deficiência , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Animais , Vias Biossintéticas , Carcinogênese/patologia , Proliferação de Células , Camundongos , Organoides/metabolismo
12.
Lipids ; 51(12): 1421-1425, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27757707

RESUMO

Platelet-activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre-analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography-mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally-activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn-2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed-phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.


Assuntos
Cromatografia de Fase Reversa/métodos , Monócitos/metabolismo , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/análise , Acetatos/química , Cromatografia Líquida de Alta Pressão , Humanos , Fator de Ativação de Plaquetas/química , Espectrometria de Massas por Ionização por Electrospray
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