RESUMO
Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into three large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca2+-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general undestanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.
Assuntos
Inflamação/imunologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Fosfolipases A2 Secretórias/metabolismo , Receptores da Fosfolipase A2/metabolismo , Animais , Ácido Araquidônico/imunologia , Ácido Araquidônico/metabolismo , Gorduras na Dieta/metabolismo , Eicosanoides/imunologia , Eicosanoides/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Inflamação/patologia , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Lisofosfolipídeos/imunologia , Lisofosfolipídeos/metabolismo , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Independentes de Cálcio/imunologia , Fosfolipases A2 Citosólicas/genética , Fosfolipases A2 Citosólicas/imunologia , Fosfolipases A2 Secretórias/genética , Fosfolipases A2 Secretórias/imunologia , Fosfolipídeos/imunologia , Fosfolipídeos/metabolismo , Conformação Proteica , Receptores da Fosfolipase A2/imunologia , Transdução de Sinais/imunologiaRESUMO
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 , RatosRESUMO
Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity can moderate the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E2 (PGE2 ), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. Here we found that, among three PGES enzymes, only microsomal PGES-1 (mPGES-1) is induced, and its expression is associated with ß-amyloid (Aß) plaques in the cerebral cortex in human AD patients and in Tg2576 mice, a transgenic AD mouse model. Furthermore, to investigate whether mPGES-1 contributes to AD-like pathology, we bred mPGES-1-deficient mice with Tg2576 mice. We found that mPGES-1 deletion reduced the accumulation of microglia around senile plaques and attenuated learning impairments in Tg2576 mice. These results indicated that mPGES-1 is induced in the AD brain and thus plays a role in AD pathology. Blockage of mPGES-1 could form the basis for a novel therapeutic strategy for patients with AD.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Córtex Cerebral/enzimologia , Oxirredutases Intramoleculares/deficiência , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Oxirredutases Intramoleculares/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Prostaglandina-E SintasesRESUMO
Epidemiological studies have suggested that the long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity moderates the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E(2) (PGE(2)), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. To examine the involvement in AD pathology of microsomal PGES-1 (mPGES-1), a PGES enzyme, we here prepared primary cerebral neuronal cells from the cerebri of wild-type and mPGES-1-deficient mice and then treated them with ß-amyloid (Aß) fragment 31-35 (Aß(31-35)), which represents the shortest sequence of native Aß peptide required for neurotoxicity. Treatment of wild-type neuronal cells with Aß(31-35) induced mPGES-1 gene expression and PGE(2) production, followed by significant apoptotic cell death, but apoptosis was not induced in mPGES-1-deficient cells. Furthermore, the combined treatment of Aß(31-35) and PGE(2) induced apoptosis in mPGES-1-deficient neuronal cells. These results indicated that mPGES-1 is induced during Aß-mediated neuronal cell death and is involved in Aß-induced neurotoxicity associated with AD pathology.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Oxirredutases Intramoleculares/genética , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Apoptose , Células Cultivadas , Deleção de Genes , Camundongos , Microssomos/enzimologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/toxicidade , Prostaglandina-E SintasesRESUMO
We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.
Assuntos
Dinoprostona/biossíntese , Hidroxiprostaglandina Desidrogenases/metabolismo , Oxirredutases Intramoleculares/metabolismo , Animais , Células Cultivadas , Dinoprostona/antagonistas & inibidores , Dinoprostona/genética , Fibroblastos/enzimologia , Hidroxiprostaglandina Desidrogenases/genética , Oxirredutases Intramoleculares/genética , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases , RatosRESUMO
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 OxidativoRESUMO
Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3-/- mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3-/- mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3-/- mice. Moreover, the gonads of Pla2g3-/- mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.
Assuntos
Epididimo/metabolismo , Fosfolipases A2 do Grupo III/metabolismo , Espermatozoides/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Fosfatidilcolinas/metabolismo , Espermatogênese , Distribuição TecidualRESUMO
Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A(2) (sPLA(2)-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA(2)-IIA by proinflammatory cytokines was under the control of both classical cPKCalpha and atypical aPKClambda/iota pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1beta/TNFalpha-dependent induction of sPLA(2)-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA(2)-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA(2)-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA(2)-IIA expression was markedly enhanced in cPKCalpha knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKClambda/iota isoform reduced the cytokine-induced expression of sPLA(2)-IIA. These results suggest that the aPKClambda/iota pathway is required for the induction of sPLA(2)-IIA expression and that the cPKCalpha pathway acts as a negative regulator of sPLA(2)-IIA expression in cytokine-stimulated rat fibroblasts.
Assuntos
Citocinas/farmacologia , Fibroblastos/enzimologia , Fosfolipases A2 do Grupo II/metabolismo , Proteína Quinase C/metabolismo , Animais , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Isoenzimas/metabolismo , Camundongos , Ésteres de Forbol/farmacologia , Ratos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.
Assuntos
Proliferação de Células , Oxirredutases Intramoleculares/fisiologia , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/enzimologia , Neoplasias/patologia , Animais , Dinoprostona/biossíntese , Oxirredutases Intramoleculares/deficiência , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Microssomos/enzimologia , Prostaglandina-E SintasesRESUMO
Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.
Assuntos
Aterosclerose/enzimologia , Células Espumosas/enzimologia , Fosfolipases A2 do Grupo III/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Venenos de Abelha/química , Dieta Aterogênica , Células Espumosas/patologia , Fosfolipases A2 do Grupo III/química , Fosfolipases A2 do Grupo III/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas LDL/genética , Lisofosfatidilcolinas/genética , Lisofosfatidilcolinas/metabolismo , Camundongos , Camundongos Transgênicos , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genéticaRESUMO
We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.
Assuntos
Proteínas de Transporte/metabolismo , Mastócitos/metabolismo , Prostaglandina D2/biossíntese , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Mastócitos/citologia , Mastócitos/enzimologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
To understand physiological roles of tissue mast cells, we established a culture system where bone marrow-derived immature mast cells differentiate into the connective tissue-type mast cell (CTMC)-like cells through modifying the previous co-culture system with Swiss 3T3 fibroblasts. Our system was found to reproducibly mimic the differentiation of CTMCs on the basis of several criteria, such as granule maturation and sensitivity to cationic secretagogues. The gene expression profile obtained by the microarray analyses was found to reflect many aspects of the differentiation. Our system is thus helpful to gain deeper insights into terminal differentiation of CTMCs.
Assuntos
Diferenciação Celular , Mastócitos/citologia , Mastócitos/fisiologia , Modelos Biológicos , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Histamina/análise , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeo Hidrolases/metabolismo , Peritônio/citologia , Células Swiss 3T3RESUMO
OBJECTIVE: Prostaglandin E synthase (PGES) functions as the terminal enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)) and is a potent regulator of bone and cartilage metabolism. Among the 3 isozymes of PGES, microsomal PGES-1 (mPGES-1) is known to play the most critical role in the production of PGE(2) in pathophysiologic events. This study investigated the roles of mPGES-1 under normal physiologic and pathophysiologic conditions in the skeletons of mPGES-1-deficient (mPGES-1(-/-)) mice. METHODS: Skeletons of mPGES-1(-/-) mice and their wild-type littermates were compared by radiologic and histologic analyses. Four models of skeletal disorders were created: bone loss induced by ovariectomy, bone loss induced by hind limb unloading, osteoarthritis (OA) induced by instability in the knee joint, and bone fracture by osteotomy at the tibial midshaft. Expression of the PGES enzymes was examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction. The cellular mechanism of fracture healing was examined in ex vivo cultures of costal cartilage chondrocytes. RESULTS: Microsomal PGES-1(-/-) mice had unaffected skeletal phenotypes under normal physiologic conditions. In the bone fracture model, fracture healing was impaired by the mPGES-1 deficiency, with half of the mice remaining in a non-bone union state even after 21 days; normal fracture healing was restored by adenoviral reintroduction of mPGES-1. The other skeletal disorders were not affected by the mPGES-1 deficiency. In vivo and ex vivo analyses revealed an impaired proliferation of chondrocytes in cartilage with the mPGES-1 deficiency, at an early stage of fracture healing. CONCLUSION: In these mouse models of skeletal disorders, mPGES-1 was indispensable for bone repair through chondrocyte proliferation, but was not essential for the skeleton under normal physiologic conditions, nor did it play a role in the pathophysiologic conditions of bone loss due to ovariectomy, bone loss due to unloading, or stress-induced OA.
Assuntos
Doenças Ósseas Metabólicas/fisiopatologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Oxirredutases Intramoleculares/genética , Osteoartrite/fisiopatologia , Animais , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/metabolismo , Condrócitos/fisiologia , Modelos Animais de Doenças , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/metabolismo , Oxirredutases Intramoleculares/deficiência , Instabilidade Articular/diagnóstico por imagem , Instabilidade Articular/metabolismo , Instabilidade Articular/fisiopatologia , Camundongos , Camundongos Mutantes , Microssomos/enzimologia , Osteoartrite/diagnóstico por imagem , Osteoartrite/metabolismo , Ovariectomia , Fenótipo , Prostaglandina-E Sintases , Prostaglandinas/metabolismo , Radiografia , Suporte de CargaRESUMO
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 ElectrosprayRESUMO
Of 10 mammalian secreted phospholipase A(2) (sPLA(2)) enzymes identified to date, group V and X sPLA(2)s, which are two potent plasma membrane-acting sPLA(2)s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA(2), which is structurally more similar to bee venom PLA(2) than to other mammalian sPLA(2)s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA(2) hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA(2) domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA(2) depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA(2)s target distinct phospholipid molecular species in cellular membranes.
Assuntos
Infecções por Adenovirus Humanos/prevenção & controle , Fosfolipases A2 do Grupo III/fisiologia , Infecções por Adenovirus Humanos/enzimologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/patogenicidade , Linhagem Celular , Membrana Celular/metabolismo , Fosfolipases A2 do Grupo III/química , Fosfolipases A2 do Grupo III/genética , Fosfolipases A2 do Grupo V/fisiologia , Fosfolipases A2 do Grupo X/fisiologia , Humanos , Mutação , Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Several prototypes of C-C biflavones (a-f) were synthesized and evaluated their inhibitory activity against phospholipase A(2)s (PLA(2)s) activity. The synthetic C-C biflavones (a-f) showed rather different inhibitory activity against various PLA(2)s. Most synthetic C-C biflavonoids exhibited potent and broad inhibitory activity against various sPLA(2)s and cPLA(2) tested regardless of their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C-C biflavonoid (d) only showed inhibitory activity against sPLA(2) X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA(2) IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C-C biflavones which possess more selective inhibitory activity against isozymes of PLA(2).
Assuntos
Flavonas/síntese química , Flavonas/farmacologia , Isoenzimas/antagonistas & inibidores , Inibidores de Fosfolipase A2 , Ativação Enzimática/efeitos dos fármacos , Flavonas/química , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Cytosolic prostaglandin (PG) E synthase (cPGES) is constitutively expressed in various cells and regulates cyclooxygenase (COX)-1-dependent immediate PGE(2) generation. Its primary structure is identical to co-chaperone p23, a heat shock protein 90 (Hsp90)-binding protein. We have revealed that Hsp90 regulated both cPGES/p23 and its client protein kinase CK2. In this study, in order to examine the role of cPGES/p23 in vivo, we generated mice deficient in cPGES/p23 by a targeted disruption of exons 2 and 3, containing Tyr9, which is essential for catalytic activity. Heterozygotes are viable, fertile, and appear normal, despite a decrease in cPGES/p23 protein level. A generation of offsprings derived from intercrosses of cPGES/p23 homozygous mice revealed that 109, 247, and 10 pups were wild type, heterozygous, and homozygous, respectively; however, all homozygotes died at birth. The absence of viable null mutants, with heterozygotes and wild-type offspring obtained at a ratio of approximately 2:1, indicated that homozygosity for the cPGES/p23 null mutant leads to peri-natal lethality. Embryos homozygous for cPGES/p23-null had lower body weights than wild-type embryos, and abnormal morphology of skin and lungs. Moreover, the PGE(2) content in the lungs of cPGES/p23-null embryos was lower than that of the wild type. These results indicate that cPGES-derived PGES is involved in the normal development of mouse embryonic lung.
Assuntos
Dinoprostona/metabolismo , Genes Letais , Oxirredutases Intramoleculares/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Estruturas Embrionárias/anormalidades , Estruturas Embrionárias/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Coração Fetal/metabolismo , Genótipo , Oxirredutases Intramoleculares/genética , Fígado/embriologia , Fígado/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Prostaglandina-E Sintases , Pele/embriologia , Pele/metabolismoRESUMO
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 , RatosRESUMO
We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcepsilonRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.
Assuntos
Anafilaxia/genética , Anafilaxia/imunologia , Proteínas de Ciclo Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mastócitos/imunologia , Mastócitos/patologia , Anafilaxia/patologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Ciclo Celular/fisiologia , Degranulação Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/patologia , Exocitose/genética , Exocitose/imunologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Anafilaxia Cutânea Passiva/genética , Anafilaxia Cutânea Passiva/imunologiaRESUMO
Arginine vasopressin (AVP) induces immediate prostaglandin E(2) (PGE(2)) production in rat 3Y1 fibroblasts. Judging from effects of several inhibitors, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) and cyclooxygenase-1 (COX-1) were mainly involved in this reaction. The antagonist of vasopressin receptor V1a, and not that of V2, inhibited the AVP-induced PGE(2) synthesis, indicating that AVP activates cPLA(2)alpha through V1a receptor. Treatment of 3Y1 cells with AVP resulted in transient activation of p44/42 mitogen-activated protein kinase (MAPK) and cPLA(2)alpha, and phosphatidylinositol 3-kinase (PI3K) inhibitor blocked not only AVP-induced PGE(2) synthesis but also MAPK activation, suggesting that PI3K is involved in the AVP-induced MAPK and cPLA(2)alpha activation, which initiates the production of PGE(2). These results suggest that PGE(2) generated by the stimulation of AVP probably modulates the physiological effects of AVP.