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1.
J Invest Dermatol ; 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38522571

RESUMO

Although subtle barrier defects may facilitate allergen penetration, thereby enabling allergic sensitization, the relationship between sweating disturbance and skin barrier function is unknown. However, many studies on contact hypersensitivity in mice examined ear skin, which does not sweat, instead of the footpad, where sweating is uniquely present. In this study, we assessed whether sweat suppression in the footpad before hapten application provoked a skin barrier abnormality and reduced inflammatory thresholds to topical haptens. Mice without any genetic skin barrier dysfunction displayed markedly reduced inflammatory thresholds to haptens under transient sweat suppression before hapten application. Epicutaneously applied haptens penetrated the skin more robustly in the presence of sweat suppression compared with that in its absence, although this increase was abolished by exposure to high-humidity conditions. These mice displayed a subtle atopic dermatitis-like inflammation mediated by type 2 response-dominant inflammation and increased IgE responses, mimicking some events occurring in nonlesional atopic dermatitis skin in humans and in murine models. These lesions were dramatically attenuated by exposure to high-humidity conditions. In our model, hapten sensitization does not require mechanical injury, explaining why sensitization occurs through nonlesional atopic dermatitis skin. Awareness of the importance of preserving sweating responses is essential to prevent occupational contact dermatitis and atopic dermatitis.

2.
J Neurochem ; 166(4): 720-746, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37337846

RESUMO

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.


Assuntos
Leucodistrofia de Células Globoides , Camundongos , Animais , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Saposinas/genética , Psicosina/metabolismo , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Modelos Animais de Doenças
3.
Commun Biol ; 6(1): 524, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-37193762

RESUMO

Cyclic phosphatidic acid (cPA) is a lipid mediator, which regulates adipogenic differentiation and glucose homeostasis by suppressing nuclear peroxisome proliferator-activated receptor γ (PPARγ). Glycerophosphodiesterase 7 (GDE7) is a Ca2+-dependent lysophospholipase D that localizes in the endoplasmic reticulum. Although mouse GDE7 catalyzes cPA production in a cell-free system, it is unknown whether GDE7 generates cPA in living cells. Here, we demonstrate that human GDE7 possesses cPA-producing activity in living cells as well as in a cell-free system. Furthermore, the active site of human GDE7 is directed towards the luminal side of the endoplasmic reticulum. Mutagenesis revealed that amino acid residues F227 and Y238 are important for catalytic activity. GDE7 suppresses the PPARγ pathway in human mammary MCF-7 and mouse preadipocyte 3T3-L1 cells, suggesting that cPA functions as an intracellular lipid mediator. These findings lead to a better understanding of the biological role of GDE7 and its product, cPA.


Assuntos
PPAR gama , Ácidos Fosfatídicos , Camundongos , Animais , Humanos , Ácidos Fosfatídicos/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Lisofosfolipídeos/metabolismo , Retículo Endoplasmático/metabolismo , Diester Fosfórico Hidrolases/genética
4.
J Lipid Res ; 62: 100141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34673020

RESUMO

Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.


Assuntos
Ensaios Enzimáticos , Fluorescência , Diester Fosfórico Hidrolases/metabolismo , Anilidas/farmacologia , Células HEK293 , Humanos , Células MCF-7 , Naftalenos/farmacologia , Organofosfonatos/farmacologia , Ácidos Fosfatídicos/farmacologia
5.
Cell Signal ; 88: 110156, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34592416

RESUMO

Previous reports have demonstrated that sphingosine 1-phosphate receptor type 2 (S1P2) is involved in the activation of signal transducer and activator of transcription (STAT) 6. Additionally, the major signaling pathway of S1P2 is the Rho-Rho kinase pathway. In this study, we examined the role of S1P2 in STAT6 activation in a macrophage (Mφ) model using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). We established S1P2knockout THP-1 cells using the CRISPR-Cas9 gene editing system. The PMA-treated S1P2knockout THP-1 Mφs showed decreases in IL-4/IL-13-induced phosphorylation of Janus-activated kinase (JAK) 1, JAK2, and STAT6 as well as mRNA expression of the M2 marker ARG1 compared with wild-type THP-1 Mφs. Pretreatment of PMA-treated THP-1 Mφs with the S1P2 antagonist JTE-013, the Rho inhibitor Rhosin or the Rho kinase inhibitor Y27632 inhibited the IL-4/IL-13-induced increase in STAT6 phosphorylation. The expressions of suppressor of cytokine signaling 3 in the S1P2knockout THP-1 Mφs were higher than those in wild-type THP-1 Mφs. In addition, the protein tyrosine phosphatase inhibitor vanadate enhanced IL-4-induced STAT6 phosphorylation in the S1P2knockout THP-1 Mφs, suggesting that S1P2-Rho-Rho kinase inhibited the negative regulation of STAT6. These results suggest that the S1P2-Rho-Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in Mφs.


Assuntos
Interleucina-13 , Transdução de Sinais , Interleucina-13/metabolismo , Fosforilação , Fator de Transcrição STAT6/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato
6.
Mol Metab ; 54: 101338, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34547510

RESUMO

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Células Secretoras de Insulina/metabolismo , Fator 4 Ativador da Transcrição/deficiência , Animais , Estresse do Retículo Endoplasmático , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
7.
Cancer Sci ; 112(11): 4570-4579, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34459070

RESUMO

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.


Assuntos
Ceramidase Ácida/antagonistas & inibidores , Terapia Genética/métodos , Doenças Mitocondriais/etiologia , Estresse Oxidativo , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/uso terapêutico , Ceramidase Ácida/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Ceramidas/metabolismo , Dependovirus , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Artigo em Inglês | MEDLINE | ID: mdl-34033896

RESUMO

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.


Assuntos
Ceramidase Ácida/metabolismo , Etanolaminas/metabolismo , Animais , Células HEK293 , Humanos , Hidrólise , Masculino , Camundongos
9.
Exp Cell Res ; 391(2): 112010, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32305327

RESUMO

Lung fibrosis is a devastating disease characterized by fibroblast accumulation and extracellular matrix deposition in lungs. However, its molecular and cellular pathogenesis is not fully understood and the current therapeutic strategies are ineffective. Bleomycin-induced lung fibrosis is the most widely used experimental model for research aimed at in-depth analysis of lung fibrosis mechanisms. The present study aimed to analyse the effects of growth differentiation factor 15 (GDF15), which is associated with many diseases, in lung fibrosis. GDF15 mRNA expression was elevated in the lungs of bleomycin-treated mice, revealed by comprehensive gene analysis. Its protein levels were also increased in the lungs, bronchoalveolar lavage fluid, and plasma obtained from bleomycin-treated mice as compared to those in saline-treated mice. Bleomycin administration in mice resulted in a marked increase in senescence-associated ß-galactosidase-positive and p16INK4a-positive lung structural cells including alveolar epithelial cells and macrophages. Immunohistochemical staining using anti-GDF15 antibody and increased mRNA expression of GDF15 in bleomycin-induced senescent A549 cells indicated that GDF15 is produced from alveolar epithelial cells undergoing bleomycin-induced cellular senescence. GDF15 was also implicated in the augmentation of interleukin-4/interleukin-13-induced mRNA expression of M2 markers including arginase 1 and chitinase-3-like protein and was also responsible for increased α-smooth muscle actin expression through the ALK5-Smad2/3 pathway in WI-38 lung fibroblasts. Therefore, GDF15 secreted from senescent alveolar epithelial cells might act as a profibrotic factor through activation of M2 macrophages and fibroblasts. This implies that GDF15 could be a potential therapeutic target and a predictor of lung fibrosis progression.


Assuntos
Bleomicina/toxicidade , Transição Epitelial-Mesenquimal , Fibroblastos/patologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Macrófagos/patologia , Fibrose Pulmonar/patologia , Células A549 , Animais , Antibióticos Antineoplásicos/toxicidade , Senescência Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Transdução de Sinais
10.
Sci Rep ; 9(1): 18329, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31797978

RESUMO

Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Células da Medula Óssea/metabolismo , Colesterol/metabolismo , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/genética , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Esfingolipídeos/genética , Esfingolipídeos/metabolismo , Esfingosina/metabolismo
11.
Biol Pharm Bull ; 42(9): 1456-1463, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474707

RESUMO

α-Lipoic acid (ALA) is used as a dietary supplement and known as an anti-oxidant. The present study aimed to examine whether ALA improves endothelial dysfunction in high-fat diet-fed obese mice. After feeding a high-fat diet to Institute of Cancer Research (ICR) mice for 4 weeks, the mice were maintained with a high-fat diet (group HF) or a high-fat diet containing ALA (25 mg/d, group HF + ALA) for an additional 20 weeks. Age-matched normal diet-fed mice were also used (group Normal). Chronic oral treatment with ALA did not affect various plasma parameters or body weights. As compared with the aortas of Normal mice, those from HF mice showed impaired endothelium-dependent relaxation in response to clonidine. However, such an impairment was not observed in the aortas from HF + ALA mice. The plasma levels of thiobarbituric acid reactive substances, an indicator of oxidative stress, were significantly decreased in HF + ALA mice compared with HF mice, confirming the anti-oxidative effects of ALA. In addition, when the impaired clonidine-induced vasorelaxation of aortas from normal mice under high glucose conditions was used as a model of acute oxidative stress, the vasorelaxation responses were improved in the presence of ALA at 100 µM. Our results suggested that the chronic oral administration of ALA improves endothelial dysfunction in high-fat diet-fed obese mice possibly through the reduction in oxidative stress in vivo.


Assuntos
Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Dieta Hiperlipídica , Endotélio Vascular/efeitos dos fármacos , Obesidade/tratamento farmacológico , Ácido Tióctico/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Aorta/fisiopatologia , Glicemia/análise , Peso Corporal/efeitos dos fármacos , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiopatologia , Lipídeos/sangue , Masculino , Camundongos Endogâmicos ICR , Obesidade/sangue , Obesidade/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Tióctico/administração & dosagem
12.
Endocrinology ; 160(1): 235-248, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30476019

RESUMO

Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2ß, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2ß are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2ß. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2ß-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2ß are required for normal USM contraction and parturition mainly through their involvement in Rho activation.


Assuntos
Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Músculo Liso Vascular/enzimologia , Parto , Fosfatidilinositol 3-Quinases/metabolismo , Contração Uterina , Útero/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Classe II de Fosfatidilinositol 3-Quinases/genética , Feminino , Camundongos , Camundongos Knockout , Contração Muscular , Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Útero/fisiologia , Proteína rhoA de Ligação ao GTP/genética
13.
Inflamm Regen ; 38: 28, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30288203

RESUMO

The plant Cannabis sativa contains cannabinoids represented by Δ9-tetrahydrocannabinol, which exert psychoactivity and immunomodulation through cannabinoid CB1 and CB2 receptors, respectively, in animal tissues. Arachidonoylethanolamide (also referred to as anandamide) and 2-arachidonoylglycerol (2-AG) are well known as two major endogenous agonists of these receptors (termed "endocannabinoids") and show various cannabimimetic bioactivities. However, only 2-AG is a full agonist for CB1 and CB2 and mediates retrograde signals at the synapse, strongly suggesting that 2-AG is physiologically more important than anandamide. The metabolic pathways of these two endocannabinoids are completely different. 2-AG is mostly produced from inositol phospholipids via diacylglycerol by phospholipase C and diacylglycerol lipase and then degraded by monoacylglycerol lipase. On the other hand, anandamide is concomitantly produced with larger amounts of other N-acylethanolamines via N-acyl-phosphatidylethanolamines (NAPEs). Although this pathway consists of calcium-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D, recent studies revealed the involvement of several new enzymes. Quantitatively major N-acylethanolamines include palmitoylethanolamide and oleoylethanolamide, which do not bind to cannabinoid receptors but exert anti-inflammatory, analgesic, and anorexic effects through receptors such as peroxisome proliferator-activated receptor α. The biosynthesis of these non-endocannabinoid N-acylethanolamines rather than anandamide may be the primary significance of this pathway. Here, we provide an overview of the biological activities and metabolisms of endocannabinoids (2-AG and anandamide) and non-endocannabinoid N-acylethanolamines.

14.
PLoS One ; 13(5): e0197604, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29782549

RESUMO

Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.


Assuntos
Fibrose Pulmonar Idiopática/etiologia , Interleucina-13/metabolismo , Macrófagos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Bleomicina/toxicidade , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Interleucina-13/genética , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato , Quimeras de Transplante/genética , Quimeras de Transplante/metabolismo , Regulação para Cima
15.
PLoS One ; 12(8): e0182329, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28771545

RESUMO

BACKGROUND: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1-S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II-AT1 and IL-6 are involved.


Assuntos
Angiotensina II/metabolismo , Interleucina-6/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Angiotensina II/análise , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Compostos de Bifenilo , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/patologia , Cardiomegalia/prevenção & controle , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ventrículos do Coração/diagnóstico por imagem , Humanos , Interleucina-6/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosforilação/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores de Lisoesfingolipídeo/genética , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Tetrazóis/uso terapêutico
16.
J Biol Chem ; 290(10): 6086-105, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25614622

RESUMO

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P1 and thereby their signaling on endosomes. TGFß, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFß1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFß-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFß receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFß receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFß receptor internalization and Smad2/3 phosphorylation. TGFß1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFß1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFß receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFß.


Assuntos
Endocitose/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfatidilinositol 3-Quinases/genética , Serina Endopeptidases/genética , Fator de Crescimento Transformador beta1/genética , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Camundongos , Camundongos Knockout , Serina Endopeptidases/biossíntese , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Diabetologia ; 57(9): 1968-76, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24989996

RESUMO

AIMS/HYPOTHESIS: Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. METHODS: We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. RESULTS: Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP (-/-)mice. SeP (+/-)mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. CONCLUSIONS/INTERPRETATION: The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Selenoproteína P/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Hepatócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Camundongos , Camundongos Knockout , Camundongos Mutantes , Regiões Promotoras Genéticas/genética , Selenoproteína P/genética , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/genética , Cicatrização/fisiologia
19.
J Allergy Clin Immunol ; 132(5): 1205-1214.e9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24021572

RESUMO

BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. OBJECTIVE: We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. METHODS: Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. RESULTS: S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of ß-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. CONCLUSION: S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.


Assuntos
Anafilaxia/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Junções Aderentes/metabolismo , Anafilaxia/genética , Anafilaxia/mortalidade , Animais , Aorta/imunologia , Aorta/metabolismo , Permeabilidade Capilar/genética , Permeabilidade Capilar/imunologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática , Deleção de Genes , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/genética , Transdução de Sinais , beta Catenina/metabolismo
20.
Biochim Biophys Acta ; 1831(1): 185-92, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22735357

RESUMO

Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-ß (TGFß), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFß signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFß, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.


Assuntos
Progressão da Doença , Fibrose/metabolismo , Fibrose/patologia , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Humanos , Modelos Biológicos , Especificidade de Órgãos , Esfingosina/metabolismo
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