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1.
Int J Mol Sci ; 22(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502192

RESUMO

The release of exosomes can lead to cell-cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.


Assuntos
Diferenciação Celular , Ceramidas/metabolismo , Colecalciferol/farmacologia , Exossomos/metabolismo , Hipocampo/metabolismo , Vitaminas/farmacologia , Células Cultivadas , Exossomos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/embriologia , Humanos , Receptores de Calcitriol/metabolismo , Esfingomielina Fosfodiesterase/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445139

RESUMO

Recent evidence pinpoints extracellular vesicles (EVs) as key players in intercellular communication. Given the importance of cholesterol and sphingomyelin in EV biology, and the relevance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in cholesterol/sphingomyelin homeostasis, we evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EV release. EVs were isolated from the extracellular medium of BV2 microglia treated or not with ethanol (50 and 100 mM). Radioactive metabolic tracers combined with thin layer chromatography were used as quantitative methods to assay phospholipid transfer, SMase activity and cholesterol uptake/esterification. Inhibitors of SMase (desipramine and GW4869) and MAM (cyclosporin A) activities were also utilized. Our data show that ethanol increases the secretion and inflammatory molecule concentration of EVs. Ethanol also upregulates MAM activity and alters lipid metabolism by increasing cholesterol uptake, cholesterol esterification and SMase activity in microglia. Notably, the inhibition of either SMase or MAM activity prevented the ethanol-induced increase in EV secretion. Collectively, these results strongly support a lipid-driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EV secretion in glial cells.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Etanol/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Células Cultivadas , Colesterol/metabolismo , Ciclosporina/farmacologia , Retículo Endoplasmático/metabolismo , Vesículas Extracelulares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445706

RESUMO

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.


Assuntos
Fosfolipídeos/biossíntese , Esfingomielina Fosfodiesterase/biossíntese , Esfingomielina Fosfodiesterase/metabolismo , Transporte Biológico , Ceramidas/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Doença de Niemann-Pick Tipo A/metabolismo , Fosfolipídeos/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Esfingomielinas/metabolismo , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologia
4.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069814

RESUMO

Osteogenesis imperfecta (OI) is a bone fragility disorder that is usually caused by mutations affecting collagen type I. We compared the calvaria bone tissue transcriptome of male 10-week-old heterozygous Jrt (Col1a1 mutation) and homozygous oim mice (Col1a2 mutation) to their respective littermate results. We found that Jrt and oim mice shared 185 differentially expressed genes (upregulated: 106 genes; downregulated: 79 genes). A total of seven genes were upregulated by a factor of two or more in both mouse models (Cyp2e1, Slc13a5, Cgref1, Smpd3, Ifitm5, Cthrc1 and Rerg). One gene (Gypa, coding for a blood group antigen) was downregulated by a factor of two or more in both OI mouse models. Overrepresentation analyses revealed that genes involved in 'ossification' were significantly overrepresented among upregulated genes in both Jrt and oim mice, whereas hematopoietic genes were downregulated. Several genes involved in Wnt signaling and transforming growth factor beta signaling were upregulated in oim mice, but less so in Jrt mice. Thus, this study identified a set of genes that are dysregulated across various OI mouse models and are likely to play an important role in the pathophysiology of this disorder.


Assuntos
Osteogênese Imperfeita/genética , Crânio/metabolismo , Animais , Colágeno Tipo I/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Modelos Animais de Doenças , Fêmur/metabolismo , Perfilação da Expressão Gênica/métodos , Heterozigoto , Homozigoto , Masculino , Camundongos , Mutação , Osteogênese , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/fisiopatologia , Crânio/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Simportadores/metabolismo , Transcriptoma/genética
5.
Toxicology ; 458: 152831, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34097992

RESUMO

Aryl hydrocarbon receptor (AHR) activation via 2,3,7,8-tetrachlorodibenzofuran (TCDF) induces the accumulation of hepatic lipids. Here we report that AHR activation by TCDF (24  µg/kg body weight given orally for five days) induced significant elevation of hepatic lipids including ceramides in mice, was associated with increased expression of key ceramide biosynthetic genes, and increased activity of their respective enzymes. Results from chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and cell-based reporter luciferase assays indicated that AHR directly activated the serine palmitoyltransferase long chain base subunit 2 (Sptlc2, encodes serine palmitoyltransferase 2 (SPT2)) gene whose product catalyzes the initial rate-limiting step in de novo sphingolipid biosynthesis. Hepatic ceramide accumulation was further confirmed by mass spectrometry-based lipidomics. Taken together, our results revealed that AHR activation results in the up-regulation of Sptlc2, leading to ceramide accumulation, thus promoting lipogenesis, which can induce hepatic lipid accumulation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ceramidas/biossíntese , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Metabólica/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzofuranos/farmacologia , Ceramidas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipidômica , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genética , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Triglicerídeos/metabolismo
6.
J Clin Invest ; 131(9)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33938457

RESUMO

Intestinal farnesoid X receptor (FXR) signaling is involved in the development of obesity, fatty liver disease, and type 2 diabetes. However, the role of intestinal FXR in atherosclerosis and its potential as a target for clinical treatment have not been explored. The serum levels of fibroblast growth factor 19 (FGF19), which is encoded by an FXR target gene, were much higher in patients with hypercholesterolemia than in control subjects and were positively related to circulating ceramide levels, indicating a link between intestinal FXR, ceramide metabolism, and atherosclerosis. Among ApoE-/- mice fed a high-cholesterol diet (HCD), intestinal FXR deficiency (in FxrΔIE ApoE-/- mice) or direct FXR inhibition (via treatment with the FXR antagonist glycoursodeoxycholic acid [GUDCA]) decreased atherosclerosis and reduced the levels of circulating ceramides and cholesterol. Sphingomyelin phosphodiesterase 3 (SMPD3), which is involved in ceramide synthesis in the intestine, was identified as an FXR target gene. SMPD3 overexpression or C16:0 ceramide supplementation eliminated the improvements in atherosclerosis in FxrΔIE ApoE-/- mice. Administration of GUDCA or GW4869, an SMPD3 inhibitor, elicited therapeutic effects on established atherosclerosis in ApoE-/- mice by decreasing circulating ceramide levels. This study identified an intestinal FXR/SMPD3 axis that is a potential target for atherosclerosis therapy.


Assuntos
Aterosclerose , Ceramidas/biossíntese , Mucosa Intestinal/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Ácido Ursodesoxicólico/análogos & derivados , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Ceramidas/genética , Dieta Hiperlipídica/efeitos adversos , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout para ApoE , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Esfingomielina Fosfodiesterase/genética , Ácido Ursodesoxicólico/farmacologia
7.
J Biol Chem ; 297(1): 100813, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023384

RESUMO

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.


Assuntos
Autofagia , Colesterol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Lisofosfolipídeos/metabolismo , Lisossomos/metabolismo , Monoglicerídeos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Endossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Doença de Niemann-Pick Tipo C/genética , Fosfatidilgliceróis/farmacologia , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Proteína Sequestossoma-1/metabolismo , Esfingomielina Fosfodiesterase/metabolismo
8.
Toxins (Basel) ; 13(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918753

RESUMO

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.


Assuntos
Toxinas Botulínicas/metabolismo , Catepsina B/metabolismo , Membrana Celular/enzimologia , Clostridium botulinum/metabolismo , Endocitose , Lisossomos/enzimologia , Animais , Catepsina B/genética , Membrana Celular/microbiologia , Clostridium botulinum/patogenicidade , Cães , Exocitose , Interações Hospedeiro-Patógeno , Lisossomos/genética , Lisossomos/microbiologia , Células Madin Darby de Rim Canino , Esfingomielina Fosfodiesterase/metabolismo
9.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917976

RESUMO

In liposomal delivery, a big question is how to release the loaded material into the correct place. Here, we will test the targeting and release abilities of our sphingomyelin-consisting liposome. A change in release parameters can be observed when sphingomyelin-containing liposome is treated with sphingomyelinase enzyme. Sphingomyelinase is known to be endogenously released from the different cells in stress situations. We assume the effective enzyme treatment will weaken the liposome making it also leakier. To test the release abilities of the SM-liposome, we developed several fluorescence-based experiments. In in vitro studies, we used molecular quenching to study the sphingomyelinase enzyme-based release from the liposomes. We could show that the enzyme treatment releases loaded fluorescent markers from sphingomyelin-containing liposomes. Moreover, the release correlated with used enzymatic activities. We studied whether the stress-related enzyme expression is increased if the cells are treated with radiation as a stress inducer. It appeared that the radiation caused increased enzymatic activity. We studied our liposomes' biodistribution in the animal tumor model when the tumor was under radiation stress. Increased targeting of the fluorescent marker loaded to our liposomes could be found on the site of cancer. The liposomal targeting in vivo could be improved by radiation. Based on our studies, we propose sphingomyelin-containing liposomes can be used as a controlled release system sensitive to cell stress.


Assuntos
Corantes Fluorescentes , Lipossomos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Estresse Fisiológico/efeitos da radiação , Animais , Catálise , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ativação Enzimática , Corantes Fluorescentes/química , Lipossomos/química , Camundongos , Imagem Molecular , Neoplasias/radioterapia , Imagem Óptica , Esfingomielinas/química , Coloração e Rotulagem
10.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925997

RESUMO

Sphingomyelin phosphodiesterase (SMPD1) is a key enzyme in the sphingolipid metabolism. Genetic SMPD1 variants have been related to the Niemann-Pick lysosomal storage disorder, which has different degrees of phenotypic severity ranging from severe symptomatology involving the central nervous system (type A) to milder ones (type B). They have also been linked to neurodegenerative disorders such as Parkinson and Alzheimer. In this paper, we leveraged structural, evolutionary and stability information on SMPD1 to predict and analyze the impact of variants at the molecular level. We developed the SMPD1-ZooM algorithm, which is able to predict with good accuracy whether variants cause Niemann-Pick disease and its phenotypic severity; the predictor is freely available for download. We performed a large-scale analysis of all possible SMPD1 variants, which led us to identify protein regions that are either robust or fragile with respect to amino acid variations, and show the importance of aromatic-involving interactions in SMPD1 function and stability. Our study also revealed a good correlation between SMPD1-ZooM scores and in vitro loss of SMPD1 activity. The understanding of the molecular effects of SMPD1 variants is of crucial importance to improve genetic screening of SMPD1-related disorders and to develop personalized treatments that restore SMPD1 functionality.


Assuntos
Doenças de Niemann-Pick/genética , Esfingomielina Fosfodiesterase/genética , Simulação por Computador , Bases de Dados Genéticas , Éxons/genética , Variação Genética/genética , Humanos , Mutação/genética , Doenças de Niemann-Pick/metabolismo , Fenótipo , Índice de Gravidade de Doença , Esfingolipídeos/genética , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo
11.
Int J Nanomedicine ; 16: 2849-2877, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33883895

RESUMO

Background: Exosomes are endosome-derived nano-sized vesicles that have emerged as important mediators of intercellular communication and play significant roles in various diseases. However, their applications are rigorously restricted by the limited secretion competence of cells. Therefore, strategies to enhance the production and functions of exosomes are warranted. Studies have shown that nanomaterials can significantly enhance the effects of cells and exosomes in intercellular communication; however, how palladium nanoparticles (PdNPs) enhance exosome release in human leukemia monocytic cells (THP-1) remains unclear. Therefore, this study aimed to address the effect of PdNPs on exosome biogenesis and release in THP-1 cells. Methods: Exosomes were isolated by ultracentrifugation and ExoQuickTM and characterized by dynamic light scattering, nanoparticle tracking analysis system, scanning electron microscopy, transmission electron microscopy, EXOCETTM assay, and fluorescence polarization. The expression levels of exosome markers were analyzed via quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results: PdNP treatment enhanced the biogenesis and release of exosomes by inducing oxidative stress, endoplasmic reticulum stress, apoptosis, and immunomodulation. The exosomes were spherical in shape and had an average diameter of 50-80 nm. Exosome production was confirmed via total protein concentration, exosome counts, acetylcholinesterase activity, and neutral sphingomyelinase activity. The expression levels of TSG101, CD9, CD63, and CD81 were significantly higher in PdNP-treated cells than in control cells. Further, cytokine and chemokine levels were significantly higher in exosomes isolated from PdNP-treated THP-1 cells than in those isolated from control cells. THP-1 cells pre-treated with N-acetylcysteine or GW4869 showed significant decreases in PdNP-induced exosome biogenesis and release. Conclusion: To our knowledge, this is the first study showing that PdNPs stimulate exosome biogenesis and release and simultaneously increase the levels of cytokines and chemokines by modulating various physiological processes. Our findings suggest a reasonable approach to improve the production of exosomes for various therapeutic applications.


Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Exossomos/metabolismo , Imunomodulação/efeitos dos fármacos , Leucemia/patologia , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo , Paládio/toxicidade , Acetilcolinesterase/metabolismo , Acetilcisteína/farmacologia , Compostos de Anilina/farmacologia , Antioxidantes/metabolismo , Compostos de Benzilideno/farmacologia , Biomarcadores Tumorais/metabolismo , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Dano ao DNA , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia/sangue , Nanopartículas Metálicas/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Esfingomielina Fosfodiesterase/metabolismo , Células THP-1
12.
J Biol Chem ; 296: 100701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895135

RESUMO

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.


Assuntos
Ambroxol/farmacologia , Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Vesiculovirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Administração por Inalação , Animais , Transporte Biológico , Ceramidas/metabolismo , Chlorocebus aethiops , Reposicionamento de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Expectorantes , Expressão Gênica , Humanos , Cultura Primária de Células , Vírus Reordenados/efeitos dos fármacos , Vírus Reordenados/fisiologia , SARS-CoV-2/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Vesiculovirus/fisiologia
13.
J Cell Mol Med ; 25(7): 3601-3609, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33719188

RESUMO

Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha-mangostin has been reported to have anti-diabetic capacity in recent years. Here, we investigated the protective function of alpha-mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha-mangostin improved impaired endothelium-dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up-regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha-mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha-mangostin increased phosphorylation of eNOS and NO production in high glucose-treated aortas. Alpha-mangostin normalized high glucose-induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha-mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation.


Assuntos
Ceramidas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Xantonas/metabolismo , Animais , Aorta/metabolismo , Células Cultivadas , Angiopatias Diabéticas/metabolismo , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Vasodilatação/efeitos dos fármacos , Xantonas/farmacologia
14.
Science ; 371(6534)2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33707237

RESUMO

Antiphospholipid antibodies (aPLs) cause severe autoimmune disease characterized by vascular pathologies and pregnancy complications. Here, we identify endosomal lysobisphosphatidic acid (LBPA) presented by the CD1d-like endothelial protein C receptor (EPCR) as a pathogenic cell surface antigen recognized by aPLs for induction of thrombosis and endosomal inflammatory signaling. The engagement of aPLs with EPCR-LBPA expressed on innate immune cells sustains interferon- and toll-like receptor 7-dependent B1a cell expansion and autoantibody production. Specific pharmacological interruption of EPCR-LBPA signaling attenuates major aPL-elicited pathologies and the development of autoimmunity in a mouse model of systemic lupus erythematosus. Thus, aPLs recognize a single cell surface lipid-protein receptor complex to perpetuate a self-amplifying autoimmune signaling loop dependent on the cooperation with the innate immune complement and coagulation pathways.


Assuntos
Apresentação do Antígeno , Autoimunidade , Coagulação Sanguínea/imunologia , Receptor de Proteína C Endotelial/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lisofosfolipídeos/imunologia , Monoglicerídeos/imunologia , Animais , Anticorpos Antifosfolipídeos/biossíntese , Autoanticorpos/biossíntese , Modelos Animais de Doenças , Perda do Embrião/imunologia , Endossomos/imunologia , Receptor de Proteína C Endotelial/genética , Humanos , Imunidade Inata , Lúpus Eritematoso Sistêmico/sangue , Camundongos , Camundongos Mutantes , Esfingomielina Fosfodiesterase/metabolismo , Trombose/imunologia , Receptor 7 Toll-Like/imunologia
15.
Hum Exp Toxicol ; 40(2): 259-273, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527851

RESUMO

Disorders of the endoplasmic reticulum (ER) lead to cellular damage but can cause cell death if ER dysfunction is prolonged. We aimed to examine liver/kidney functions, neutral sphingomyelinase (N-SMase) activity, sphingolipid levels, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) protein expression in rats under ER stress. ER stress was induced by tunicamycin (TM) and the ER stress inhibitor taurodeoxycholic acid (TUDCA) was injected before induction of ER stress. ER stress was confirmed by increased tissue levels of GRP78. Hematological and biochemical profiles were measured by autoanalyzers while hepatic and renal injury was evaluated via microscopy and histopathological scoring. Tissue levels of C16-C24 sphingomyelins (SM), C16-C24 ceramides (CERs) and sphingosine-1-phosphate (S1P) were determined by LC-MS/MS. Tissue cPLA2 and COX-2 were measured by western blot and activity assays. Tunicamycin treatment caused kidney and liver function test abnormalities, increased hematocrit and hemoglobin levels but decreased white blood cell counts. Histopathological findings showed hepatic necroinflammation and renal tubular damage in rats treated with TM. TUDCA administration attenuated WBC abnormalities and TM- induced hepatic/renal functional impairment in ER stress, as evident by significantly restored serum ALT, AST, creatinine, and total bilirubin levels. A significant increase was observed in N-SMase activity, tissue levels of C16-C24 CERs, cPLA2 and COX-2 expression in liver and kidney tissue under ER stress. TUDCA administration decreased tissue CER levels, cPLA2 and COX-2 expression as well as prostaglandin E2 (PGE2) formation. These results signify that ER stress causes hepatic and renal toxicity as well as CER-induced PGE2 formation in liver and kidney.


Assuntos
Estresse do Retículo Endoplasmático , Inflamação/metabolismo , Rim/metabolismo , Fígado/metabolismo , Esfingolipídeos/metabolismo , Animais , Ceramidas/metabolismo , Dinoprostona/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Fosfolipases A2/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos Wistar , Esfingomielina Fosfodiesterase/metabolismo , Tunicamicina
16.
Int J Nanomedicine ; 16: 515-538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33519199

RESUMO

Background: Several studies have demonstrated various molecular mechanisms involved in the biogenesis and release of exosomes. However, how external stimuli, such as platinum nanoparticles (PtNPs), induces the biogenesis and release of exosomes remains unclear. To address this, PtNPs were synthesized using lutein to examine their effect on the biogenesis and release of exosomes in human lung epithelial adenocarcinoma cancer cells (A549). Methods: The size and concentration of isolated exosomes were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis system (NTA). Morphology and structure of exosomes were examined using scanning electron microscopy and transmission electron microscopy (TEM), respectively. Quantification of exosomes were analyzed by EXOCETTM assay and fluorescence polarization (FP). The expression of typical markers of exosomes were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: A549 cells cultured with PtNPs enhance exosome secretion by altering various physiological processes. Interestingly, A549 cells treated with PtNPs increases total protein concentration, biogenesis and release of exosomes associated with PtNPs-induced oxidative stress. GW4869 inhibits PtNPs induced biogenesis and release of exosomes and also acetylcholinesterase (AChE), neutral sphingomyelinase activity (n-SMase), and exosome counts. A549 cells pre-treated with N-acetylcysteine (NAC) significantly inhibited PtNPs induced exosome biogenesis and release. These findings confirmed that PtNPs-induced exosome release was due to the induction of oxidative stress and the ceramide pathway. These factors enhanced exosome biogenesis and release and may be useful in understanding the mechanism of exosome formation, release, and function. Conclusion: PtNPs provide a promising agent to increase exosome production in A549 cells. These findings offer novel strategies for enhancing exosome release, which can be applied in the treatment and prevention of cancer. Importantly, this is the first study, to our knowledge, showing that PtNPs stimulate exosome biogenesis by inducing oxidative stress and the ceramide pathway.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Ceramidas/metabolismo , Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , Nanopartículas Metálicas/química , Estresse Oxidativo , Platina/farmacologia , Células A549 , Acetilcolinesterase/metabolismo , Acetilcisteína/farmacologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Compostos de Anilina/farmacologia , Compostos de Benzilideno/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Exossomos/ultraestrutura , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Luteína/farmacologia , Nanopartículas Metálicas/ultraestrutura , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Soro , Esfingomielina Fosfodiesterase/metabolismo , Eletricidade Estática
17.
Cell Death Dis ; 12(1): 26, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33414455

RESUMO

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.


Assuntos
Ceramidas/metabolismo , Ferroptose/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Autofagia , Humanos , Camundongos , Transfecção
18.
J Biosci Bioeng ; 131(1): 8-12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33023861

RESUMO

Sphingomyelinase C (SMC) hydrolyzes sphingomyelin to ceramide and phosphocholine. Prokaryotic SMCs share sequence homology with mammalian SMCs that have enzymatic pH optima at neutral pH. SMC from the nonpathogenic prokaryote Streptomyces griseocarneus shows notable enzymatic features such as higher optimum pH and thermostability than other prokaryotic SMCs. Determination of the three-dimensional structure of S. griseocarneus-SMC (Sg-SMC) and comparison with other SMC structures represents a promising strategy to elucidate the unique enzymatic features of Sg-SMC on a structural basis. Therefore, we determined the crystal structure of Sg-SMC at 2.0 Å resolution by X-ray crystallography. Comparison of the Sg-SMC structure with three other structurally known SMCs from Listeria ivanovii, Bacillus cereus, and Staphylococcus aureus indicated that Sg-SMC is more diverse in sequence and that structural differences in the main chain between these SMCs are primarily located on the molecular surface distant from the active site. Comparison of the surface area of the four SMCs revealed that Sg-SMC has the most compact structure, which may contribute to the enhanced thermostability of Sg-SMC. Regarding the hydrogen bond network in the active site of Sg-SMC, a basic amino acid, Arg278, is involved, whereas the corresponding residue in other SMCs (Ser or Asn) does not form hydrogen bonds with metal-coordinating water molecules. Hydrogen bond formation between Arg278 and a Mg2+ ion-coordinating water molecule may be responsible for the higher optimal pH of Sg-SMC compared to that of other SMCs.


Assuntos
Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/metabolismo , Streptomyces/enzimologia , Temperatura , Animais , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio
19.
Methods Mol Biol ; 2187: 215-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770509

RESUMO

The prevailing mechanism of action of chemotherapeutic drugs has been challenged by the role of ceramide, a second messenger, shown to induce apoptosis, differentiation, growth arrest, senescence, and autophagy in different cells (Chabner BA, Roberts TG Jr, Nat Rev Cancer 5:65-72, 2005; Jacobi J et al, Cell Signal 29:52-61, 2017; Rotolo J et al, J Clin Invest 122:1786-1790, 2012; Truman JP et al, PLoS One 5:e12310, 2010). Certain chemotherapeutic drugs activate the acid sphingomyelinase (ASMase)/ceramide pathway, generating ceramide in the tumor endothelium and this microvascular dysfunction is crucial for the tumor response. Ceramide has fusigenic properties and as such, when generated within the plasma membrane, initiates the oligomerization of ceramide-and cholesterol-rich domains in the outer leaflet of the plasma membrane, leading to the formation of ceramide-rich microdomains/platforms (CRP) (Jacobi J et al, Cell Signal 29:52-61, 2017; Truman JP et al, PLoS One 5:e12310, 2010; van Hell AJ et al, Cell Signal 34:86-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216:115-130, 2013) known as "signaling platform." This chapter will discuss the generation, detection, and quantitation of CRP and their possible modulation in endothelial cells, in vitro and in vivo in response to certain chemotherapeutic drugs.


Assuntos
Antineoplásicos/farmacologia , Ceramidas/metabolismo , Células Endoteliais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo
20.
J Appl Microbiol ; 131(1): 182-196, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33326676

RESUMO

AIMS: The present study aims to evaluate the capability of rosuvastatin to synergize with levofloxacin against Staphylococcus aureus. METHODS AND RESULTS: Rosuvastatin inhibited the growth of S. aureus with minimum inhibitory concentration of 16 µg ml-1 . Additionally, it showed a bactericidal effect at 4x minimum inhibition concentration. Using a checkerboard method, a synergistic effect was recorded when rosuvastatin was combined with levofloxacin showing against S. aureus isolate 28 (S 28). Furthermore, this combination was also able to display a significant reduction in biofilm formation (92·8%) and suppress the production of coagulase and ß-haemolysin, and virulence factors of S. aureus isolate 28. An animal model for wound infection was used to assess the therapeutic effect of the test combination, in vivo. It was found that the test combination reduced the bacterial burden in the infected wounds by 91·3%. Pathological and histological analyses have revealed a decline in cell infiltration in the excisional wound skin tissue after treatment with rosuvastatin and levofloxacin combination. CONCLUSIONS: Rosuvastatin combined with levofloxacin can be considered as a promising solution to combat S. aureus antibiotic resistance phenomenon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study unveils the potential effect of rosuvastatin when used in combination with levofloxacin can be used as a topical antibacterial agent to treat S. aureus skin infections.


Assuntos
Biofilmes/efeitos dos fármacos , Levofloxacino/farmacologia , Rosuvastatina Cálcica/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Toxinas Bacterianas/metabolismo , Coagulase/metabolismo , Modelos Animais de Doenças , Combinação de Medicamentos , Resistência Microbiana a Medicamentos , Sinergismo Farmacológico , Proteínas Hemolisinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Esfingomielina Fosfodiesterase/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Infecção dos Ferimentos/microbiologia
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