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1.
FASEB J ; 36(5): e22301, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35478358

RESUMO

Psoriasis is an inflammatory disorder characterized by keratinocyte hyper-proliferation and Th17-type immune responses. However, the roles of bioactive lipids and the regulation of their biosynthesis in this chronic skin disease are not fully understood. Herein, we show that group IVE cytosolic phospholipase A2 (cPLA2 ε/PLA2G4E) plays a counterregulatory role against psoriatic inflammation by producing the anti-inflammatory lipid N-acylethanolamine (NAE). Lipidomics analysis of mouse skin revealed that NAE species and their precursors (N-acyl-phosphatidylethanolamine and glycerophospho-N-acylethanolamine) were robustly increased in parallel with the ongoing process of imiquimod (IMQ)-induced psoriasis, accompanied by a marked upregulation of cPLA2 ε in epidermal keratinocytes. Genetic deletion of cPLA2 ε exacerbated IMQ-induced ear swelling and psoriatic marker expression, with a dramatic reduction of NAE-related lipids in IMQ-treated, and even normal, skin. Stimulation of cultured human keratinocytes with psoriatic cytokines concomitantly increased PLA2G4E expression and NAE production, and supplementation with NAEs significantly attenuated the cytokine-induced upregulation of the psoriatic marker S100A9. Increased expression of cPLA2 ε was also evident in the epidermis of psoriatic patients. These findings reveal for the first time the in vivo role of cPLA2 ε, which is highly induced in the keratinocytes of the psoriatic skin, promotes the biosynthesis of NAE-related lipids, and contributes to limiting psoriatic inflammation.


Assuntos
Psoríase , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos , Citocinas/metabolismo , Etanolaminas , Humanos , Imiquimode , Inflamação , Lipídeos/efeitos adversos , Camundongos , Fosfolipases/uso terapêutico , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico
2.
Int J Mol Sci ; 24(6)2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36982638

RESUMO

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.


Assuntos
Glicoesfingolipídeos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Glicoesfingolipídeos/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Cerebelo/metabolismo , Microdomínios da Membrana/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(41): 20689-20699, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548400

RESUMO

Mutations in the iPLA2-VIA/PLA2G6 gene are responsible for PARK14-linked Parkinson's disease (PD) with α-synucleinopathy. However, it is unclear how iPLA2-VIA mutations lead to α-synuclein (α-Syn) aggregation and dopaminergic (DA) neurodegeneration. Here, we report that iPLA2-VIA-deficient Drosophila exhibits defects in neurotransmission during early developmental stages and progressive cell loss throughout the brain, including degeneration of the DA neurons. Lipid analysis of brain tissues reveals that the acyl-chain length of phospholipids is shortened by iPLA2-VIA loss, which causes endoplasmic reticulum (ER) stress through membrane lipid disequilibrium. The introduction of wild-type human iPLA2-VIA or the mitochondria-ER contact site-resident protein C19orf12 in iPLA2-VIA-deficient flies rescues the phenotypes associated with altered lipid composition, ER stress, and DA neurodegeneration, whereas the introduction of a disease-associated missense mutant, iPLA2-VIA A80T, fails to suppress these phenotypes. The acceleration of α-Syn aggregation by iPLA2-VIA loss is suppressed by the administration of linoleic acid, correcting the brain lipid composition. Our findings suggest that membrane remodeling by iPLA2-VIA is required for the survival of DA neurons and α-Syn stability.


Assuntos
Encéfalo/patologia , Membrana Celular/patologia , Neurônios Dopaminérgicos/patologia , Proteínas de Drosophila/metabolismo , Fosfolipases A2 do Grupo X/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/patologia , alfa-Sinucleína/química , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Membrana Celular/metabolismo , Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Estresse do Retículo Endoplasmático , Feminino , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/metabolismo , Fosfolipases A2 do Grupo X/genética , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Degeneração Neural/metabolismo , Doença de Parkinson/metabolismo , Fosfolipídeos/metabolismo , Transmissão Sináptica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
4.
Front Immunol ; 15: 1401294, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38720899

RESUMO

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.


Assuntos
Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fagocitose , Fagócitos/imunologia , Fagócitos/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membrana Celular/metabolismo , Ligação Proteica
5.
J Allergy Clin Immunol ; 129(2): 536-43, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22206772

RESUMO

BACKGROUND: Eosinophilic pustular folliculitis (EPF) is a chronic intractable pruritic dermatosis characterized by massive eosinophil infiltrates involving the pilosebaceous units. Recently, EPF has been regarded as an important clinical marker of HIV infection, and its prevalence is increasing in number. The precise mechanism by which eosinophils infiltrate into the pilosebaceous units remains largely unknown. Given that indomethacin, a COX inhibitor, can be successfully used to treat patients with EPF, we can assume that COX metabolites such as prostaglandins (PGs) are involved in the etiology of EPF. OBJECTIVE: To determine the involvement of PGs in the pathogenesis of EPF. METHODS: We performed immunostaining for PG synthases in EPF skin lesions. We examined the effect of PGD(2) on induction of eotaxin, a chemoattractant for eosinophils, in human keratinocytes, fibroblasts, and sebocytes and sought to identify its responsible receptor. RESULTS: Hematopoietic PGD synthase was detected mainly in infiltrating inflammatory cells in EPF lesions, implying that PGD(2) was produced in the lesions. In addition, PGD(2) and its immediate metabolite 15-deoxy-Δ 12,14-PGJ(2) (15d-PGJ(2)) induced sebocytes to produce eotaxin-3 via peroxisome proliferator-activated receptor gamma. Consistent with the above findings, eotaxin-3 expression was immunohistochemically intensified in sebaceous glands of the EPF lesions. CONCLUSION: The PGD(2)/PGJ(2)-peroxisome proliferator-activated receptor gamma pathway induces eotaxin production from sebocytes, which may explain the massive eosinophil infiltrates observed around pilosebaceous units in EPF.


Assuntos
Quimiocinas CC/imunologia , Eosinofilia/imunologia , Foliculite/imunologia , PPAR gama/imunologia , Prostaglandina D2/imunologia , Glândulas Sebáceas/imunologia , Dermatopatias Vesiculobolhosas/imunologia , Anilidas/farmacologia , Carbazóis/farmacologia , Linhagem Celular , Células Cultivadas , Quimiocina CCL26 , Quimiocinas CC/genética , Eosinofilia/patologia , Eosinófilos/imunologia , Fibroblastos/imunologia , Foliculite/patologia , Humanos , Hidantoínas/farmacologia , Queratinócitos/imunologia , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/imunologia , Glândulas Sebáceas/citologia , Dermatopatias Vesiculobolhosas/patologia , Sulfonamidas/farmacologia , Transfecção
6.
Biomedicines ; 12(1)2023 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-38255176

RESUMO

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbß3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbß3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

7.
Biomolecules ; 13(3)2023 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-36979406

RESUMO

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.


Assuntos
Colina , Lisofosfolipase , Animais , Humanos , Camundongos , Colina/metabolismo , Glicerilfosforilcolina/metabolismo , Fígado/metabolismo , Lisofosfolipase/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/metabolismo
8.
Cell Rep ; 42(2): 111940, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36719796

RESUMO

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.


Assuntos
Fígado , Lisofosfolipase , Metionina , Fosfatidilcolinas , Animais , Camundongos , Colina/metabolismo , Glicerilfosforilcolina/metabolismo , Fígado/metabolismo , Metionina/metabolismo , Racemetionina/metabolismo , S-Adenosilmetionina/metabolismo , Triglicerídeos/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Fosfatidilcolinas/metabolismo
9.
J Biol Chem ; 286(43): 37249-63, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-21880721

RESUMO

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)ß), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)ß (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)ß, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)ß is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.


Assuntos
Células da Medula Óssea/enzimologia , Fibroblastos/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Mastócitos/enzimologia , Fosfolipídeos/metabolismo , Anafilaxia/enzimologia , Anafilaxia/genética , Animais , Ácido Araquidônico/genética , Ácido Araquidônico/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Técnicas de Cocultura , Dinoprostona/genética , Dinoprostona/metabolismo , Fibroblastos/citologia , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo VI/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Knockout , Fosfolipídeos/genética , Prostaglandina-E Sintases
10.
J Clin Invest ; 130(2): 890-903, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31671075

RESUMO

The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7-KO model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7-/- epidermis, quantitative liquid chromatography-mass spectometry (LC-MS) assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its nonenzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.


Assuntos
Ceramidas/metabolismo , Epiderme/enzimologia , Oxirredutases/metabolismo , Animais , Catálise , Ceramidas/genética , Modelos Animais de Doenças , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Humanos , Ictiose/enzimologia , Ictiose/genética , Camundongos , Camundongos Knockout , Oxirredutases/genética
11.
Cell Signal ; 20(5): 815-24, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18280113

RESUMO

Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type alpha of cytosolic phospholipase A(2) (cPLA(2)alpha), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA(2)alpha has not been well established. We investigated the effects induced by transfection with mutant cPLA(2)alpha and inhibitors for CaM and CaM kinase on the Ca(2+)-stimulated release of AA and translocation of cPLA(2)alpha. The mutation of Ser515 to Ala (S515A) did not change cPLA(2)alpha activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H(2)O(2), 1 mM) and A23187 (10 microM) markedly released AA in C12 cells expressing S515A and wild-type cPLA(2)alpha, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA(2)alpha, A23187 caused the translocation of the wild-type, the every mutants, cPLA(2)alpha-C2 domain, and cPLA(2)alpha-Delta397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 microM significantly decreased the release of AA in C12-cPLA(2)alpha cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA(2)alpha-Delta397-749, but not cPLA(2)alpha-C2 domain. Our findings show a possible effect of CaM kinase on cPLA(2)alpha in a catalytic domain A-dependent and Ser515-independent manner.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fosfolipases A2 do Grupo IV/química , Fosfolipases A2 do Grupo IV/metabolismo , Substituição de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Sequência de Bases , Benzilaminas/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Domínio Catalítico , Linhagem Celular , Citosol/enzimologia , Primers do DNA/genética , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV/genética , Humanos , Ionóforos/farmacologia , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Transdução de Sinais , Sulfonamidas/farmacologia
12.
Biochem J ; 409(2): 429-38, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17868035

RESUMO

Human sPLA2-III [group III secreted PLA2 (phospholipase A2)] is an atypical sPLA2 isoenzyme that consists of a central group III sPLA2 domain flanked by unique N- and C-terminal domains. In the present study, we found that sPLA2-III is expressed in neuronal cells, such as peripheral neuronal fibres, spinal DRG (dorsal root ganglia) neurons and cerebellar Purkinje cells. Adenoviral expression of sPLA2-III in PC12 cells (pheochromocytoma cells) or DRG explants facilitated neurite outgrowth, whereas expression of a catalytically inactive sPLA2-III mutant or use of sPLA2-III-directed siRNA (small interfering RNA) reduced NGF (nerve growth factor)-induced neuritogenesis. sPLA2-III also suppressed neuronal death induced by NGF deprivation. Lipid MS revealed that sPLA2-III overexpression increased the cellular level of lysophosphatidylcholine, a PLA2 reaction product with neuritogenic and neurotropic activities, whereas siRNA knockdown reduced the level of lysophosphatidylcholine. These observations suggest the potential contribution of sPLA2-III to neuronal differentiation and its function under certain conditions.


Assuntos
Fosfolipases A2 do Grupo III/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Animais , Diferenciação Celular , Sobrevivência Celular/efeitos da radiação , DNA Complementar/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Neuritos/fisiologia , Células PC12 , RNA Interferente Pequeno/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray
13.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(6): 869-879, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30290227

RESUMO

The human genome encodes nine enzymes belonging to the patatin-like phospholipase domain-containing lipase (PNPLA)/Ca2+-independent phospholipase A2 (iPLA2) family. Although most PNPLA/iPLA2 enzymes are widely distributed and act on phospholipids or neutral lipids as (phospho)lipases to play homeostatic roles in lipid metabolism, the function of PNPLA1 remained a mystery until a few years ago. However, the recent finding that mutations in the human PNPLA1 gene are linked to autosomal recessive congenital ichthyosis (ARCI), as well as evidence obtained from biochemical and gene knockout studies, has shed light on the function of this enzyme in skin-specific sphingolipid metabolism rather than glycerophospholipid metabolism. PNPLA1 is specifically expressed in differentiated keratinocytes and plays a crucial role in the biosynthesis of ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide. In this review, we overview the biosynthetic route and biological role of epidermal ω-O-acylceramide, highlight the function of PNPLA1 as a bona fide acylceramide synthase required for proper skin barrier function and keratinocyte differentiation, and summarize the mutations of PNPLA1 currently identified in ARCI patients. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.


Assuntos
Ceramidas/metabolismo , Lipase/metabolismo , Pele/metabolismo , Animais , Diferenciação Celular/fisiologia , Epiderme/metabolismo , Humanos , Ictiose Lamelar/metabolismo , Mutação/fisiologia
15.
Eur J Pharmacol ; 590(1-3): 1-11, 2008 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-18539271

RESUMO

The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.


Assuntos
Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/farmacologia , Dronabinol/análogos & derivados , Glicerídeos/farmacologia , Fosfolipases A2 do Grupo IV/fisiologia , Fosfolipases Tipo C/fisiologia , Quinases da Família src/fisiologia , Animais , AMP Cíclico/biossíntese , Citosol/enzimologia , Dronabinol/farmacologia , Endocanabinoides , Camundongos , Camundongos Endogâmicos ICR , Células PC12 , Fosforilação , Piperidinas/farmacologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-fyn/fisiologia , Pirazóis/farmacologia , Ratos , Receptor CB1 de Canabinoide/fisiologia , Transdução de Sinais/fisiologia
16.
Biochem Pharmacol ; 73(6): 854-62, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17188653

RESUMO

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Fosfolipases A/metabolismo , Proteína Quinase C/fisiologia , Vanadatos/farmacologia , Quinases da Família src/fisiologia , Animais , Ácido Araquidônico/metabolismo , Cálcio/fisiologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/fisiologia , Peróxido de Hidrogênio/metabolismo , Camundongos , Fosfolipases A2 , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais
17.
Hum Cell ; 20(2): 52-61, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17547719

RESUMO

We examined a 32-year-old Japanese man who was clinically diagnosed with gastric cancer, type 4, and histopathologically diagnosed with mucinous and poorly differentiated adenocarcinoma (mucinous > poorly) of the stomach. We successfully established and characterized a cell line (designated as IGSK-2) derived from the ascitic fluid of the patient with recurrent and cisplatin-resistant carcinoma. The IGSK-2 cells grew in multi-layered culture in culture dishes. The cells secreted 18 pg/mL somatostatin, 9.1 mIU/mL human chorionic gonadotrophin (hCG), 8000 U/mL carbohydrate antigen 19-9 and 410 ng/mL carcinoembryonic antigen over 4 days of culture. The population doubling time was approximately 83 h. The susceptibility test of anticancer drugs revealed that IGSK-2 cells were sensitive to Taxol, but were not sensitive to cisplatin, 5-fluorouracil and irinotecan. Immunohistochemical staining revealed that the IGSK-2 cells were positive against antihCG antibody and antiserotonin antibody, and negative against antisomatostatin antibody and antigastrin antibody.


Assuntos
Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Líquido Ascítico/citologia , Linhagem Celular Tumoral , Gonadotropina Coriônica/metabolismo , Somatostatina/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Adulto , Animais , Antígenos Glicosídicos Associados a Tumores/análise , Biomarcadores Tumorais/análise , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Antígeno Carcinoembrionário/análise , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Fluoruracila/farmacologia , Humanos , Imuno-Histoquímica , Irinotecano , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Paclitaxel/farmacologia
18.
Hum Cell ; 30(4): 319-326, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28260147

RESUMO

The hZK-1 cell line was successfully established from the metastatic foci of a lymph node of an 82-year-old Japanese woman with squamous cell carcinoma of the tongue. The pathological diagnosis of the tumor was moderately to well-differentiated squamous cell carcinoma. The hZK-1 cells were angular in shape, and had neoplastic and pleomorphic features. Adjacent hZK-1 cells were joined by desmosomes and well-developed microvilli, and many free ribosomes were observed in the cytoplasm. The doubling time of the hZK-1 cells was approximately 36, 33, and 29 h at the 10th, 20th, and 30th passages, respectively. The cell line was shown to be triploid, with a chromosomal distribution of 75-80. Immunocytochemical staining of the hZK-1 cells revealed cytokeratin (CK) 17-, Ki67-, and p53-positive staining, and negative staining for CK13. The hZK-1 cells were negative for human papillomavirus (HPV)-16 or-18 infection. Grafting was not successful when the hZK-1 cells were transplanted into the subcutis of SCID mice. The hZK-1 cells (2 × 106 cells/3 ml of growth medium) secreted vascular endothelial growth factor (VEGF) that reached a concentration of 2.6 ng/ml media after 3 days of culture. Hypoxia enhanced cellular HIF-1α expression and VEGF secretion in hZK-1 cells. The HIF-1α inhibitor YC-1 partially inhibited hypoxia-induced VEGF secretion in ZK-1 cells. The reverse transcription-polymerase chain reaction (RT-PCR) results revealed that the expression of CK17, Ki67, and p53 was elevated in the hZK-1 cells. hZK-1 cells were not sensitive to CDDP, TXT, 5-FU, or a mixture of these three anti-tumor agents.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias da Língua/patologia , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indazóis/farmacologia , Queratina-13/metabolismo , Queratina-17/metabolismo , Antígeno Ki-67/metabolismo , Metástase Linfática , Camundongos SCID , Neoplasias da Língua/genética , Neoplasias da Língua/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Nat Commun ; 8: 14609, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28248300

RESUMO

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1-/- epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1-/- keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.


Assuntos
Ceramidas/biossíntese , Lipase/metabolismo , Pele/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/deficiência , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Fenótipo , Pele/ultraestrutura
20.
Biochem Pharmacol ; 71(6): 850-7, 2006 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-16443193

RESUMO

Ceramide-1-phosphate (C1P), a novel bioactive sphingolipid, is implicated in the vital cellular processes such as cell proliferation and inflammation. The role of C1P on activity of cytosolic phospholipase A2alpha (cPLA2alpha), a key enzyme for the release of arachidonic acid (AA) and prostanoids, has not been well elucidated. In this study, we investigated the effect of C1P on the release of AA from L929 cells and a variant, which lacks cPLA2alpha expression, C12 cells. C1P at 30 microM alone induced AA release from L929 cells without an increase in intracellular Ca2+ concentration. C1P-induced AA release was marginal in C12 cells, and treatment with an intracellular Ca2+ chelator (BAPTA-AM) or an inhibitor of cPLA2alpha (2 microM pyrrophenone) decreased C1P-induced AA release in L929 cells. C1P increased the enzymatic activity of cPLA2alpha over two-fold in the presence of Ca2+. C1P triggered the translocation of cPLA2alpha and its C2 domain from the cytosol to the perinuclear region in CHO-K1 cells. Interestingly, C1P at 10 microM synergistically enhanced ionomycin-induced AA release from L929 cells. The AA release induced by C1P with and without ionomycin decreased by treatment with protein kinase C (PKC) inhibitor (10 microM GF109203X) and in the PKC-depleted cells. C1P at 10 microM stimulated the translocation of PKC (alpha and delta) from the soluble to the membrane fractions. We propose that C1P stimulates AA release via two mechanisms; direct activation of cPLA2alpha, and the PKC-dependent pathway.


Assuntos
Ceramidas/farmacologia , Citosol/efeitos dos fármacos , Fosfolipases A/biossíntese , Proteína Quinase C/biossíntese , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Citosol/enzimologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fosfolipases A2 do Grupo IV , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos
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