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1.
FASEB J ; 37(12): e23330, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37983658

RESUMO

Long-chain acyl-CoA synthetase 4 (ACSL4) converts free highly unsaturated fatty acids (HUFAs) into their acyl-CoA esters and is important for HUFA utilization. HUFA-containing phospholipids produced via ACSL4-dependent reactions are involved in pathophysiological events such as inflammatory responses and ferroptosis as a source for lipid mediators and/or a target of oxidative stress, respectively. However, the in vivo role of ACSL4 in inflammatory responses is not fully understood. This study sought to define the effects of ACSL4 deficiency on lipopolysaccharide (LPS)-induced systemic inflammatory responses using global Acsl4 knockout (Acsl4 KO) mice. Intraperitoneal injection of LPS-induced more severe symptoms, including diarrhea, hypothermia, and higher mortality, in Acsl4 KO mice within 24 h compared with symptoms in wild-type (WT) mice. Intestinal permeability induced 3 h after LPS challenge was also enhanced in Acsl4 KO mice compared with that in WT mice. In addition, plasma levels of some eicosanoids in Acsl4 KO mice 6 h post-LPS injection were 2- to 9-fold higher than those in WT mice. The increased mortality observed in LPS-treated Acsl4 KO mice was significantly improved by treatment with the general cyclooxygenase inhibitor indomethacin with a partial reduction in the severity of illness index for hypothermia, diarrhea score, and intestinal permeability. These results suggest that ACSL4 deficiency enhances susceptibility to endotoxin at least partly through the overproduction of cyclooxygenase-derived eicosanoids.


Assuntos
Hipotermia , Choque Séptico , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Choque Séptico/induzido quimicamente , Eicosanoides , Diarreia , Ligases , Coenzima A Ligases/genética
2.
Prostaglandins Other Lipid Mediat ; 153: 106523, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33383181

RESUMO

Prostacyclin (PGI2) synthase (PGIS) functions downstream of inducible cyclooxygenase COX-2 in the PGI2 biosynthetic pathway. Although COX-2 and PGI2 receptor (IP) are known to be involved in adipogenesis and obesity, the involvement of PGIS has not been fully elucidated. In this study, we examined the role of PGIS in adiposity by using PGIS-deficient mice. Although PGIS deficiency did not affect in vitro adipocyte differentiation, when fed a high-fat diet (HFD), PGIS knockout (KO) mice showed reductions in both body weight gain and epididymal fat mass relative to wild-type (WT) mice. PGIS deficiency might reduce HFD-induced obesity by suppressing PGI2 production. We further found that additional gene deletion of microsomal prostaglandin (PG) E synthase-1 (mPGES-1), one of the other PG terminal synthases that also functions downstream of COX-2, emphasized the metabolic phenotypes of PGIS-deficient mice. More marked reduction in obesity and improved insulin resistance were observed in PGIS/mPGES-1 double KO (DKO) mice. Since an additive increase in PGF2α level in epididymal fat was observed in DKO mice, mPGES-1 deficiency might affect adiposity by enhancing the production of PGF2α. Our immunohistochemical analysis further revealed that in adipose tissues, PGIS was expressed in vascular and stromal cells but not in adipocytes. These results suggested that PGI2 produced from PGIS-expressed stromal tissues might enhance HFD-induced obesity by acting on IP expressed in adipocytes. The balance of expressions of PG terminal synthases and the subsequent production of prostanoids might be critical for adiposity.


Assuntos
Sistema Enzimático do Citocromo P-450 , Oxirredutases Intramoleculares , Animais , Dieta Hiperlipídica , Camundongos , Prostaglandina-E Sintases
3.
Drug Metab Dispos ; 48(4): 255-263, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980500

RESUMO

Liver X receptors (LXRs), LXRα and LXRß, are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 (hUGT1) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1-null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrß -/- , and hUGT1/Lxrαß -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXRα, with the induction pattern paralleling induction of LXRα-specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαß -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXRα, showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXRα is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXRα, and not LXRß, is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXRα is critical for the developmental expression of UGT1A1.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glucuronosiltransferase/metabolismo , Receptores X do Fígado/metabolismo , Animais , Animais Recém-Nascidos , Bilirrubina/sangue , Bilirrubina/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucuronosiltransferase/genética , Hidrocarbonetos Fluorados/administração & dosagem , Receptores X do Fígado/agonistas , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Transgênicos , Sulfonamidas/administração & dosagem , Uridina Difosfato Ácido Glucurônico/metabolismo
4.
Biol Pharm Bull ; 43(9): 1375-1381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879212

RESUMO

Adipogenic differentiation is a complex process by which fibroblast-like undifferentiated cells are converted into cells that accumulate lipid droplets. We here investigated the effect of gene deletion of calcium-independent phospholipase A2γ (iPLA2γ), a membrane-bound PLA2 enzyme, on adipogenic differentiation in mice. Since iPLA2γ knockout (KO) mice showed reduced fat volume and weight, we prepared mouse embryonic fibroblasts (MEF) from wild-type (WT) and iPLA2γ KO mice and examined the effect of iPLA2γ deletion on in vitro adipogenic differentiation. iPLA2γ increased during adipogenic differentiation in WT mouse-derived MEFs, and the differentiation was partially abolished in iPLA2γ KO-derived MEFs. In KO-derived MEFs, the inductions of peroxisome proliferator activator receptor γ (PPARγ) and CAAT/enhancer-binding protein α (C/EBPα) were also reduced during adipogenic differentiation, and the reductions in PPARγ and C/EBPα expressions and the defect in adipogenesis were restored by treatment with troglitazone, a PPARγ ligand. These results indicate that iPLA2γ might play a critical role in adipogenic differentiation by regulating PPARγ expression.


Assuntos
Adipogenia/fisiologia , Fibroblastos/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Lisofosfolipase/metabolismo , PPAR gama/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fosfolipases A2 do Grupo VI/genética , Lisofosfolipase/genética , Camundongos , Camundongos Knockout , Cultura Primária de Células , Troglitazona/farmacologia
5.
Biochim Biophys Acta ; 1841(1): 44-53, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24095834

RESUMO

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1ß-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1ß-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1ß-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1ß stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1ß stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.


Assuntos
Coenzima A Ligases/metabolismo , Fibroblastos/metabolismo , Interleucina-1beta/metabolismo , Prostaglandinas/biossíntese , Animais , Linhagem Celular , Coenzima A Ligases/genética , Fibroblastos/citologia , Técnicas de Silenciamento de Genes , Humanos , Interleucina-1beta/genética , Camundongos , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Prostaglandinas/genética , Ratos
6.
Free Radic Biol Med ; 220: 301-311, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38734266

RESUMO

Cisplatin (CDDP) is a platinum-based drug with anti-cancer activity and is widely used as a standard therapy for bladder cancer. It is well known that CDDP causes cell death by increasing the generation of reactive oxygen species (ROS) and lipid peroxidation, but the mechanism of its anti-cancer effects has not been fully elucidated. There are still some problems such as chemoresistance in CDDP therapy. In the present study, we found the expression of Ca2+-independent phospholipase A2γ (iPLA2γ), which has been reported to regulate cellular redox homeostasis by inhibiting lipid peroxide accumulation, in human bladder cancer tissues. Thus, we investigated the effect of iPLA2γ knockdown on CDDP-induced bladder cancer cell death. As a result, we found that iPLA2γ knockdown significantly enhanced CDDP-induced apoptosis, intracellular and mitochondrial ROS production, cytochrome c release and caspase activation in bladder cancer cells. Moreover, mitochondrial membrane potential was decreased and peroxidation of mitochondrial phospholipids was increased by iPLA2γ knockdown. It was also shown that co-treatment of bromoenol lactone, an iPLA2 inhibitor, increased CDDP-induced apoptosis. These results indicated that iPLA2γ plays an important role in protecting bladder cancer cells from CDDP-induced apoptosis, and that iPLA2γ inhibitors might represent a novel strategy in CDDP-based multi-drug therapy.


Assuntos
Apoptose , Cisplatino , Fosfolipases A2 do Grupo VI , Peroxidação de Lipídeos , Mitocôndrias , Fosfolipídeos , Espécies Reativas de Oxigênio , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Cisplatino/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Fosfolipases A2 do Grupo VI/metabolismo , Fosfolipases A2 do Grupo VI/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Linhagem Celular Tumoral , Fosfolipídeos/metabolismo , Antineoplásicos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Pironas/farmacologia , Naftalenos
7.
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
8.
Biosci Rep ; 42(2)2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35103282

RESUMO

Acyl-CoA synthetase long-chain family member 4 (ACSL4) activates polyunsaturated fatty acids (PUFAs) to produce PUFA-derived acyl-CoAs, which are utilised for the synthesis of various biological components, including phospholipids (PLs). Although the roles of ACSL4 in non-apoptotic programmed cell death ferroptosis are well-characterised, its role in the other types of cell death is not fully understood. In the present study, we investigated the effects of ACSL4 knockdown on the levels of acyl-CoA, PL, and ferroptosis in the human normal kidney proximal tubule epithelial (HK-2) cells. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that the knockdown of ACSL4 markedly reduced the levels of PUFA-derived acyl-CoA, but not those of other acyl-CoAs. In contrast with acyl-CoA levels, the docosahexaenoic acid (DHA)-containing PL levels were preferentially decreased in the ACSL4-knockdown cells compared with the control cells. Cell death induced by the ferroptosis inducers RSL3 and FIN56 was significantly suppressed by treatment with ferrostatin-1 or ACSL4 knockdown, and, unexpectedly, upon treating with a necroptosis inhibitor. In contrast, ACSL4 knockdown failed to suppress the other oxidative stress-induced cell deaths initiated by cadmium chloride and sodium arsenite. In conclusion, ACSL4 is involved in the biosynthesis of DHA-containing PLs in HK-2 cells and is specifically involved in the cell death induced by ferroptosis inducers.


Assuntos
Coenzima A Ligases , Espectrometria de Massas em Tandem , Morte Celular , Cromatografia Líquida , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Humanos , Fosfolipídeos/metabolismo
9.
J Lipid Res ; 51(10): 3003-15, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20625036

RESUMO

Group VIB Ca(2+)-independent phospholipase A(2)γ (iPLA(2)γ) is a membrane-bound iPLA(2) enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA(2)γ by disrupting its gene in mice. iPLA(2)γ-knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofilaments in iPLA(2)γ-KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA(2)γ-KO muscles. These results provide evidence that impairment of iPLA(2)γ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA(2)γ-KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA(2)γ may contribute to modulation of lipid mediator production in vivo.


Assuntos
Fosfolipases A2 do Grupo VI/genética , Mitocôndrias/metabolismo , Músculo Esquelético/enzimologia , Prostaglandinas/biossíntese , Animais , Feminino , Fosfolipases A2 do Grupo VI/deficiência , Fosfolipases A2 do Grupo VI/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/enzimologia , Monócitos/citologia , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo
10.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(6): 861-868, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30391710

RESUMO

Calcium-independent phospholipase A2γ (iPLA2γ)/patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is one of the iPLA2 enzymes, which do not require Ca2+ ion for their activity. iPLA2γ is a membrane-bound enzyme with unique features, including the utilization of four distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. This enzyme is preferentially distributed in the mitochondria and peroxisomes and is thought to be responsible for the maintenance of lipid homeostasis in these organelles. Thus, both the overexpression and the deletion of iPLA2γ in vivo caused mitochondrial abnormalities and dysfunction. Roles of iPLA2γ in lipid mediator production and cytoprotection against oxidative stress have also been suggested by in vitro and in vivo studies. The dysregulation of iPLA2γ can therefore be a critical factor in the development of many diseases, including metabolic diseases and cancer. In this review, we provide an overview of the biochemical properties of iPLA2γ and then summarize the current understanding of the in vivo roles of iPLA2γ revealed by knockout mouse studies.


Assuntos
Cálcio/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Animais , Humanos , Camundongos Knockout/metabolismo , Mitocôndrias/metabolismo
11.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(11): 1606-1618, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31376475

RESUMO

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.


Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Macrófagos/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Células Cultivadas , Coenzima A Ligases/genética , Feminino , Camundongos , Camundongos Knockout , Especificidade por Substrato
12.
Drug Metab Pharmacokinet ; 33(1): 9-16, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29079228

RESUMO

More than 20% of clinically used drugs are glucuronidated by a microsomal enzyme UDP-glucuronosyltransferase (UGT). Inhibition or induction of UGT can result in an increase or decrease in blood drug concentration. To avoid drug-drug interactions and adverse drug reactions in individuals, therefore, it is important to understand whether UGTs are involved in metabolism of drugs and drug candidates. While most of glucuronides are inactive metabolites, acyl-glucuronides that are formed from compounds with a carboxylic acid group can be highly toxic. Animals such as mice and rats are widely used to predict drug metabolism and drug-induced toxicity in humans. However, there are marked species differences in the expression and function of drug-metabolizing enzymes including UGTs. To overcome the species differences, mice in which certain drug-metabolizing enzymes are humanized have been recently developed. Humanized UGT1 (hUGT1) mice were created in 2010 by crossing Ugt1-null mice with human UGT1 transgenic mice in a C57BL/6 background. hUGT1 mice can be promising tools to predict human drug glucuronidation and acyl-glucuronide-associated toxicity. In this review article, studies of drug metabolism and toxicity in the hUGT1 mice are summarized. We further discuss research and strategic directions to advance the understanding of drug glucuronidation in humans.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/enzimologia , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Previsões , Glucuronídeos/genética , Glucuronosiltransferase/genética , Humanos , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Especificidade da Espécie
13.
FEBS J ; 285(11): 2056-2070, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29637744

RESUMO

Chemokines are secreted proteins that regulate cell migration and are involved in inflammatory and immune responses. Here, we sought to define the functional crosstalk between the lipid signaling and chemokine signaling. We obtained evidence that the induction of some chemokines is regulated by group VIA calcium-independent phospholipase A2 ß (iPLA2 ß) in IL-1ß-stimulated rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with IL-1ß elicited an increased release of chemotactic factor(s) for monocytic THP-1 cells into culture medium in a time-dependent manner. Inhibitor studies revealed that an intracellular PLA2 inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3 ), but not the cyclooxygenase inhibitor indomethacin, attenuated the release of chemotactic factor(s). The chemotactic activity was inactivated by treatment with either heat or proteinase K, suggesting this chemotactic factor(s) is a proteinaceous factor(s). We purified the chemotactic factor(s) from the conditioned medium of IL-1ß-stimulated 3Y1 cells using a heparin column and identified several chemokines, including CCL2 and CXCL10. The inducible expressions of CCL2 and CXCL10 were significantly attenuated by pretreatment with AACOCF3 . Gene silencing using siRNA revealed that the inductions of CCL2 and CXCL10 were attenuated by iPLA2 ß knockdown. Additionally, the transcriptional activation of nuclear factor of activated T-cell proteins (NFATs), but not nuclear factor-κB, by IL-1ß stimulation was markedly attenuated by the iPLA2 inhibitor bromoenol lactone, and NFATc4 knockdown markedly attenuated the IL-1ß-induced expression of both CCL2 and CXCL10. Collectively, these results indicated that iPLA2 ß plays roles in IL-1ß-induced chemokine expression, in part via NFATc4 signaling.


Assuntos
Quimiocina CCL2/genética , Quimiocina CXCL10/genética , Fatores de Transcrição NFATC/genética , Proteínas do Tecido Nervoso/genética , Fosfolipases A2 Independentes de Cálcio/genética , Animais , Ácidos Araquidônicos/farmacologia , Fibroblastos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica , Indometacina/farmacologia , Interleucina-1beta/genética , Monócitos/metabolismo , Fosfolipases A2 Independentes de Cálcio/antagonistas & inibidores , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
14.
Sci Rep ; 7: 46489, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28422158

RESUMO

Isothiocyanates, such as phenethyl isothiocyanate (PEITC), are formed following the consumption of cruciferous vegetables and generate reactive oxygen species (ROS) that lead to the induction of cytoprotective genes such as the UDP-glucuronosyltransferases (UGTs). The induction of ROS activates the Nrf2-Keap 1 pathway leading to the induction of genes through antioxidant response elements (AREs). UGT1A1, the sole enzyme responsible for the metabolism of bilirubin, can be induced following activation of Nrf2. When neonatal humanized UGT1 (hUGT1) mice, which exhibit severe levels of total serum bilirubin (TSB) because of a developmental delay in expression of the UGT1A1 gene, were treated with PEITC, TSB levels were reduced. Liver and intestinal UGT1A1 were induced, along with murine CYP2B10, a consensus CAR target gene. In both neonatal and adult hUGT1/Car-/- mice, PEITC was unable to induce CYP2B10. A similar result was observed following analysis of UGT1A1 expression in liver. However, TSB levels were still reduced in hUGT1/Car-/- neonatal mice because of ROS induction of intestinal UGT1A1. When oxidative stress was blocked by exposing mice to N-acetylcysteine, induction of liver UGT1A1 and CYP2B10 by PEITC was prevented. Thus, new findings in this report link an important role in CAR activation that is dependent upon oxidative stress.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/biossíntese , Isotiocianatos/farmacologia , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Bilirrubina/sangue , Receptor Constitutivo de Androstano , Glucuronosiltransferase/genética , Humanos , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Receptores Citoplasmáticos e Nucleares/genética
15.
PLoS One ; 9(10): e109409, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25313821

RESUMO

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.


Assuntos
Plaquetas/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Colágeno/metabolismo , Suscetibilidade a Doenças , Fosfolipases A2 do Grupo VI/deficiência , Fosfolipases A2 do Grupo VI/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/análise , Ativação Plaquetária , Agregação Plaquetária , Receptores Purinérgicos P2Y/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Trombose/metabolismo , Trombose/patologia , Tromboxano A2/metabolismo
16.
J Biol Chem ; 282(28): 20124-32, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17475622

RESUMO

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.


Assuntos
Cálcio/metabolismo , Fibroblastos/enzimologia , Fosfolipases A/biossíntese , Fosfolipases A/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Linhagem Celular , Citocinas/farmacologia , Eicosanoides/biossíntese , Indução Enzimática/efeitos dos fármacos , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo VI , Inflamação/genética , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Naftalenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/deficiência , Fosfolipases A/genética , Fosfolipases A2 , Pironas/farmacologia , Ratos
17.
J Biol Chem ; 280(14): 14028-41, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15695510

RESUMO

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.


Assuntos
Membrana Celular/metabolismo , Isoenzimas/metabolismo , Fosfolipases A/metabolismo , Prostaglandinas/biossíntese , Adenocarcinoma/metabolismo , Animais , Ácido Araquidônico/metabolismo , Morte Celular , Linhagem Celular , Membrana Celular/química , Neoplasias Colorretais/metabolismo , Dinoprostona/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Fosfolipases A2 do Grupo VI , Humanos , Hidrólise , Isoenzimas/genética , Fosfolipases A/genética , Fosfolipases A2 , Fosfolipídeos/química , Fosfolipídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia
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