RESUMO
OBJECTIVES: Sepsis is a systemic inflammatory disorder characterized by life-threateningorgan dysfunction resulting from a dysregulated host response to infection. Prostacyclin (PGI2) is a bioactive lipid produced by PGI synthase (PGIS) and is known to play important roles in inflammatory reactions as well as cardiovascular regulation. However, little is known about the roles of PGIS and PGI2 in systemic inflammatory responses such as septic shock. METHODOLOGY: Systemic inflammation was induced by intraperitoneal injection of 5 mg/kg lipopolysaccharide (LPS) in wild type (WT) or PGIS knockout (KO) mice. Selexipag, a selective PGI2 receptor (IP) agonist, was administered 2 h before LPS injection and again given every 12 h for 3 days. RESULTS: Intraperitoneal injection of LPS induced diarrhea, shivering and hypothermia. These symptoms were more severe in PGIS KO mice than in WT micqe. The expression of Tnf and Il6 genes was notably increased in PGIS KO mice. In contrast, over 95% of WT mice survived 72 h after the administration of LPS, whereas all of the PGIS KO mice had succumbed by that time. The mortality rate of LPS-administrated PGIS KO mice was improved by selexipag administration. CONCLUSION: Our study suggests that PGIS-derived PGI2 negatively regulates LPS-induced symptoms via the IP receptor. PGIS-derived PGI2-IP signaling axis may be a new drug target for systemic inflammation in septic shock.
Assuntos
Sistema Enzimático do Citocromo P-450 , Oxirredutases Intramoleculares , Lipopolissacarídeos , Choque Séptico , Animais , Masculino , Camundongos , Acetamidas/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Citocinas/metabolismo , Epoprostenol , Inflamação/induzido quimicamente , Interleucina-6/genética , Interleucina-6/metabolismo , Oxirredutases Intramoleculares/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirazinas/farmacologia , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
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/genéticaRESUMO
Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing membrane phospholipids. Here we demonstrated decreases in various kinds of PUFA-containing phospholipid species in ACSL4-deficient murine lung. We then examined the effects of ACSL4 gene deletion on lung injury by treating mice with two pulmonary toxic chemicals: paraquat (PQ) and methotrexate (MTX). The results showed that ACSL4 deficiency attenuated PQ-induced acute lung lesion and decreased mortality. PQ-induced lung inflammation and neutrophil migration were also suppressed in ACSL4-deficient mice. PQ administration increased the levels of phospholipid hydroperoxides in the lung, but ACSL4 gene deletion suppressed their increment. We further found that ACSL4 deficiency attenuated MTX-induced pulmonary fibrosis. These results suggested that ACSL4 gene deletion might confer protection against pulmonary toxic chemical-induced lung injury by reducing PUFA-containing membrane phospholipids, leading to the suppression of lipid peroxidation. Inhibition of ACSL4 may be promising for the prevention and treatment of chemical-induced lung injury.
Assuntos
Lesão Pulmonar , Camundongos , Animais , Peroxidação de Lipídeos , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/genética , Xenobióticos , Deleção de Genes , Fosfolipídeos , Ácidos Graxos Insaturados , Pulmão , LigasesRESUMO
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/metabolismoRESUMO
Long-chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their active form, acyl-CoAs. Recent knock-out mouse studies revealed that among ACSL isoenzymes, ACSL6 plays an important role in the maintenance of docosahexaenoic acid (DHA)-containing glycerophospholipids. Several transcript variants of the human ACSL6 gene have been found; the two major ACSL6 variants, ACSL6V1 and V2, encode slightly different short motifs that both contain a conserved structural domain, the fatty acid Gate domain. In the present study, we expressed recombinant human ACSL6V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, and then, using our novel ACSL assay system with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined the substrate specificities of the recombinant human ACSL6V1 and V2 proteins. The results showed that both ACSL6V1 and V2 could convert various kinds of long-chain fatty acids into their acyl-CoAs. Oleic acid was a good common substrate and eicosapolyenoic acids were poor common substrates for both variants. However, ACSL6V1 and V2 differed considerably in their preferences for octadecapolyenoic acids, such as linoleic acid, and docosapolyenoic acids, such as DHA and docosapentaenoic acid (DPA): ACSL6V1 preferred octadecapolyenoic acids, whereas V2 strongly preferred docosapolyenoic acids. Moreover, our kinetic studies revealed that ACSL6V2 had a much higher affinity for DHA than ACSL6V1. Our results suggested that ACSL6V1 and V2 might exert different physiological functions and indicated that ACSL6V2 might be critical for the maintenance of membrane phospholipids bearing docosapolyenoic acids such as DHA.
Assuntos
Coenzima A Ligases/metabolismo , Fosfolipídeos/metabolismo , Animais , Coenzima A Ligases/genética , Ácidos Docosa-Hexaenoicos/química , Ácidos Docosa-Hexaenoicos/metabolismo , Ensaios Enzimáticos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ácido Linoleico/metabolismo , Fosfolipídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ácidos Esteáricos/metabolismo , Especificidade por Substrato/genética , Espectrometria de Massas em TandemRESUMO
Cyclophosphamide (CP) has been widely used in the treatment of various malignancies and autoimmune diseases, but acrolein, a byproduct of CP, causes severe hemorrhagic cystitis as the major side effect of CP. On the other hand, a large amount of prostacyclin (PGI2 ) is produced in bladder tissues, and PGI2 has been shown to play a critical role in bladder homeostasis. PGI2 is biosynthesized from prostaglandin (PG) H2 , the common precursor of PGs, by PGI2 synthase (PTGIS) and is known to also be involved in inflammatory responses. However, little is known about the roles of PTGIS-derived PGI2 in bladder inflammation including CP-induced hemorrhagic cystitis. Using both genetic and pharmacological approaches, we here revealed that PTGIS-derived PGI2 -IP (PGI2 receptor) signaling exacerbated CP-induced bladder inflammatory reactions. Ptgis deficiency attenuated CP-induced vascular permeability and chemokine-mediated neutrophil migration into bladder tissues and then suppressed hemorrhagic cystitis. Treatment with RO1138452, an IP selective antagonist, also suppressed CP-induced cystitis. We further found that cystitis-related nociceptive behavior was also relieved in both Ptgis-/- mice and RO1138452-treated mice. Our findings may provide new drug targets for bladder inflammation and inflammatory pain in CP-induced hemorrhagic cystitis.
Assuntos
Ciclofosfamida/efeitos adversos , Cistite/induzido quimicamente , Cistite/prevenção & controle , Epoprostenol/deficiência , Dor/prevenção & controle , Bexiga Urinária , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito , Cistite/complicações , Sistema Enzimático do Citocromo P-450/deficiência , Progressão da Doença , Epoprostenol/metabolismo , Feminino , Hemorragia/complicações , Hemorragia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Tamanho do Órgão/efeitos dos fármacos , Dor/induzido quimicamente , Dor/complicações , Prostaglandina-E Sintases , Bexiga Urinária/efeitos dos fármacosRESUMO
BACKGROUND/AIM: Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) is a terminal enzyme in PGE2 synthesis and highly expressed in several cancers. In this study, to reveal the involvement of mPGES-1 in skin carcinogenesis, the effect of mPGES-1 deficiency on two-stage skin carcinogenesis in mice was investigated. MATERIALS AND METHODS: A two-stage skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter was applied on mPGES-1 knockout (KO) mice and littermate wild-type mice of a Balb/c genetic background. RESULTS: DMBA/TPA-induced skin carcinogenesis was suppressed in mPGES-1 KO mice. The induction of IL-17 and other inflammatory cytokines by TPA was also suppressed by mPGES-1 deficiency, although DMBA-induced apoptosis was not affected. CONCLUSION: mPGES-1 promotes chemically induced skin carcinogenesis and might play an important role in the TPA-induced promotion phase of the two-stage skin carcinogenesis model. mPGES-1 inhibition may be a therapeutic target for skin cancer.
Assuntos
Prostaglandina-E Sintases/fisiologia , Neoplasias Cutâneas/prevenção & controle , 9,10-Dimetil-1,2-benzantraceno , Animais , Apoptose/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Citocinas/biossíntese , Dinoprostona/análise , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Neoplasias Cutâneas/induzido quimicamente , Acetato de TetradecanoilforbolRESUMO
Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and prostacyclin (PGI2) synthase (PGIS) are PG terminal synthases that work downstream of cyclooxygenase and synthesize PGE2 and PGI2, respectively. Although the involvement of PG receptors in acquired cutaneous immune responses was recently shown, the roles of these PG terminal synthases remain unclear. To identify the pathophysiological roles of mPGES-1 and PGIS in cutaneous immune systems, we applied contact hypersensitivity (CHS) to mPGES-1 and PGIS knockout (KO) mice as a model of acquired immune responses. Mice were treated with 1-fluoro-2,4-dinitrobenzene (DNFB) and evaluated for ear thickness and histopathological features. The results showed that the severity of ear swelling in both gene-deficient mice was much lower than that in wild-type (WT) mice. Histological examination of DNFB-treated ears showed that inflammatory cell infiltration and edema in the dermis were also less apparent in both genotypic mice. LC-MS analysis further showed that the increment in PGE2 levels in DNFB-treated ear tissue was reduced in mPGES-1 KO mice, and that 6-keto PGF1α (a stable metabolite of PGI2) was not detected in PGIS KO mice. Furthermore, we made bone marrow (BM) chimera and found that transplantation of WT mouse-derived BM cells restored the impaired CHS response in mPGES-1 KO mice but did not restore the response in PGIS KO mice. These results indicated that mPGES-1 in BM-derived cells and PGIS in non-BM-derived cells might play critical roles in DNFB-induced CHS. mPGES-1-derived PGE2 and PGIS-derived PGI2 might coordinately promote acquired cutaneous immune responses.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dermatite de Contato/enzimologia , Oxirredutases Intramoleculares/metabolismo , Prostaglandina-E Sintases/metabolismo , Transferência Adotiva , Animais , Células da Medula Óssea , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Dermatite de Contato/etiologia , Dermatite de Contato/genética , Dinitrofluorbenzeno/efeitos adversos , Orelha/patologia , Feminino , Interferon gama/metabolismo , Interleucinas/metabolismo , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Prostaglandinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina 22RESUMO
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 SintasesRESUMO
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/farmacologiaRESUMO
Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.
Assuntos
Ácidos Graxos Ômega-6/metabolismo , Ferroptose , Hepatócitos/metabolismo , Peroxidação de Lipídeos , Falência Hepática Aguda/metabolismo , Fígado/metabolismo , Acetaminofen , Animais , Antioxidantes/farmacologia , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Cicloexilaminas/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Desferroxamina/farmacologia , Modelos Animais de Doenças , Ferroptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Quelantes de Ferro/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Falência Hepática Aguda/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Fenilenodiaminas/farmacologia , alfa-Tocoferol/farmacologiaRESUMO
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 SubstratoRESUMO
The activation of long-chain free fatty acids is the first step reaction of their usage in the cells and tissues, which are catalyzed by a family of enzymes called acyl-coenzyme A synthetases long-chain isoform (ACSL). The five ACSL enzymes identified in mammals are thought to have specific and differing functions. Among them, ACSL4 is a unique isozyme that preferentially catalyzes several polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), and ACSL4 is thought to be an important isozyme for PUFA metabolism. Recent studies revealed that ACSL4 is involved in biological responses including inflammation, steroidogenesis, cell death, female fertility, and cancer. ACSL4 and its substrate PUFAs are thus likely to contribute to these responses. However, the roles of ACSL4 in PUFA metabolism are not fully understood. In this review, we describe the recent progress in ACSL4 research including the involvement of this enzyme in AA metabolism.
Assuntos
Ácido Araquidônico/metabolismo , Coenzima A Ligases/metabolismo , Animais , Encéfalo/metabolismo , Morte Celular , Humanos , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismoRESUMO
Acyl-CoA synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs. ACSL4 is an ACSL isozyme with a strong preference for arachidonic acid (AA) and has been hypothesized to modulate the metabolic fates of AA. There are two ACSL4 splice variants: ACSL4V1, which is the more abundant transcript, and ACSL4V2, which is believed to be restricted to the brain. In the present study, we expressed recombinant human ACSL4V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system and then partially purified both variants by cobalt affinity column chromatography. We then established a novel ACSL assay system with LC-MS/MS, which is highly sensitive and applicable to various kinds of fatty acids, and used it to investigate the substrate specificity of recombinant human ACSL4V1 and V2. The results showed that both ACSL4 variants preferred various kinds of highly unsaturated fatty acids (HUFAs), including docosahexaenoic acid (DHA), adrenic acid (docosatetraenoic acid) and eicosapentaenoic acid (EPA), as well as AA as a substrate. Moreover, our kinetic studies revealed that the two variants had similar relative affinities for AA, EPA and DHA but different reaction rates for each HUFA. These results confirmed the importance of both of ACSL4 variants in the maintenance of membrane phospholipids bearing HUFAs. Structural analysis of these variants might reveal the molecular mechanism by which they maintain membrane phospholipids bearing HUFAs.
Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Cromatografia Líquida , Coenzima A Ligases/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Spodoptera , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
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/metabolismoRESUMO
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 fármacosRESUMO
Amyloid-ß (Aß) deposition and oxidative stress observed in the brains of patients with Alzheimer's disease (AD) are important targets for therapeutic intervention. In this study, we conjugated the antioxidants caffeic acid (CA) and dihydrocaffeic acid (DHCA) to Aß1-42 C-terminal motifs (Aßx-42: x=38, 40) to synthesize CA-Aßx-42 and DHCA-Aßx-42, respectively. Among the compounds, CA-Aß38-42 exhibited potent inhibitory activity against Aß1-42 aggregation and scavenged Aß1-42-induced intracellular oxidative stress. Moreover, CA-Aß38-42 significantly protected human neuroblastoma SH-SY5Y cells against Aß1-42-induced cytotoxicity, with an IC50 of 4µM. These results suggest that CA-Aß38-42 might be a potential lead for the treatment of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Amiloide/antagonistas & inibidores , Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Antioxidantes/química , Ácidos Cafeicos/química , Linhagem Celular Tumoral , Humanos , Fármacos Neuroprotetores/química , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/químicaRESUMO
Two hallmarks of Alzheimer's disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-ß (Aß) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aß variants, TxAßx-n (x=34, 36, 38, 40; n=40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAß36-42 significantly inhibited Aß1-42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aß1-42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Antioxidantes/farmacologia , Cromanos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Antioxidantes/química , Cromanos/química , Desenho de Fármacos , Humanos , Fragmentos de Peptídeos/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.
Assuntos
Neoplasias do Colo/genética , Pólipos do Colo/genética , Ciclo-Oxigenase 2/genética , Sistema Enzimático do Citocromo P-450/genética , Oxirredutases Intramoleculares/genética , Dor/genética , Peritonite/genética , Ácido Acético , Animais , Azoximetano , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Pólipos do Colo/induzido quimicamente , Pólipos do Colo/metabolismo , Pólipos do Colo/patologia , Ciclo-Oxigenase 2/metabolismo , Sistema Enzimático do Citocromo P-450/deficiência , Dinoprostona/biossíntese , Epoprostenol/biossíntese , Oxirredutases Intramoleculares/deficiência , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptividade/efeitos dos fármacos , Dor/induzido quimicamente , Dor/metabolismo , Dor/patologia , Peritonite/induzido quimicamente , Peritonite/metabolismo , Peritonite/patologia , Prostaglandina-E Sintases , TioglicolatosRESUMO
Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1ß (IL-1ß)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1ß-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1ß-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1ß stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2.