RESUMEN
PURPOSE: Lung cancer is a leading cause of cancer deaths and efforts are underway to identify novel therapies to treat these tumors. Diacylglycerol kinase η (DGKη), an enzyme that phosphorylates diacylglycerol to form phosphatidic acid, has been shown to modulate MAPK signaling downstream of EGFR, which is an oncogenic driver in some lung cancers. Since mutations in EGFR and K-Ras are common in lung cancer, we hypothesized that limiting the function of DGKη would attenuate oncogenic properties of lung cancer cells. METHODS: We determined the expression levels of DGKη in a mouse models of mutant EGFR and K-Ras lung cancer and in human lung cancer cell lines with activating mutations in either EGFR or K-Ras. We also tested the effects of shRNA-mediated depletion of DGKη in lung cancer cells and tested if DGKη depletion augmented the effects of afatinib, a new generation EGFR inhibitor. RESULTS: DGKη was expressed in malignant epithelium from mice with mutant EGFR or K-Ras lung cancer. It was also expressed in human lung cancer cell lines with EGFR or K-Ras mutations. Depleting DGKη in lung cancer cell lines, harboring mutant EGFR, reduced their growth on plastic and in soft agar and also augmented the effects of afatinib, an EGFR inhibitor. DGKη depletion also reduced growth of one of two lung cancer cell lines that harbored mutant K-Ras. CONCLUSIONS: Our data indicate that DGKη is a potential therapeutic target in lung cancers, especially those harboring EGFR mutations. Our findings warrant further studies to examine the effects of limiting its function in vivo.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Neoplasias Pulmonares/enzimología , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Genes erbB-1 , Genes ras , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Transgénicos , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have cloned a cDNA for human UMP-CMP kinase from a macrophage cDNA library. Sequence analysis showed that this cDNA is derived from the same gene as a previously reported EST-derived cDNA. Here we show that a conspicuous difference between these two clones, 73 additional 5' nucleotides in the EST clone, including a putative translational start site, is not functionally significant. This work shows that the additional 5'sequence in the EST clone was unnecessary for enzymatic activity and nonfunctional in the initiation of translation. Specifically, we found that protein expressed by both the macrophage-derived cDNA and the extended cDNA had the same relative molecular mass, consistent with use of an ATG internal to the macrophage-derived clone as the functional start site. In addition, this work more precisely defines the catalytic activity of UMP-CMP kinase. Here, we show a 3-fold greater substrate preference for CMP relative to UMP, identify ATP and UTP as the preferred phosphate donors for the reaction, and demonstrate that the reaction is Mg2+-dependent. In addition, investigation of UMP-CMP-kinase expression revealed two mRNA products in immune tissues and cancer cell lines. The smaller RNA product was previously undescribed.
Asunto(s)
Regiones no Traducidas 5'/genética , Nucleósido-Fosfato Quinasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células COS/citología , Células COS/enzimología , Clonación Molecular , Citidina Monofosfato/metabolismo , ADN Complementario/análisis , Biblioteca de Genes , Humanos , Riñón/citología , Riñón/enzimología , Macrófagos/enzimología , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/metabolismo , ARN Mensajero/genética , Especificidad por Sustrato , Transfección , Uridina Monofosfato/metabolismoRESUMEN
This review describes the current understanding of the contributions of genetic alterations in platelet-activating factor (PAF) acetylhydrolase to the pathogenesis of asthma. A variety of in vitro and in vivo studies, performed by multiple laboratories, suggest that the lipid substrates of this enzyme, PAF and oxidised derivatives of phosphatidylcholines, play important roles as causative factors in many diseases including asthma. PAF acetylhydrolase inactivates PAF and oxidatively-fragmented lipids thus providing a mechanism to prevent their pro-inflammatory effects. Since it is a most unusual protein, the biochemical, structural and functional characteristics of PAF acetylhydrolase continue to be unravelled. First, the ability of this enzyme to inactivate pro-inflammatory lipid mediators is modulated by its association with lipoproteins and by its susceptibility to oxidative inactivation. Second, mediators of inflammation, such as the substrates for PAF acetylhydrolase, alter expression of the protein at the transcriptional level. Third, naturally-occurring variants of PAF acetylhydrolase have catalytic properties different from those exhibited by the most common form of this protein. Thus, a variety of factors, including genetics, contribute to determine the biological level of lipid substrates known to act as mediators of asthma and other diseases. Here, I summarise key studies that implicate PAF and related molecules as important mediators in the pathogenesis of asthma. Next, I describe clinical findings that are consistent with a role of PAF acetylhydrolase as a modulator of asthma. Third, I focus on the biochemical effects associated with naturally-occurring mutations and polymorphisms in the PAF acetylhydrolase gene and the incidence of these genetic variations in populations of asthmatic subjects. Finally, I present my views on the future of this emerging field and the potential utility of performing additional studies aimed at further characterising the contribution of PAF acetylhydrolase to the pathogenesis of a complex syndrome generally recognised as a multifactorial and heterogeneous disease.
Asunto(s)
Asma/genética , Fosfolipasas A/genética , Polimorfismo Genético , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Asma/enzimología , Asma/etiología , Ligamiento Genético , Humanos , Fosfolipasas A/deficiencia , Factor de Activación Plaquetaria/análisis , Factor de Activación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/fisiologíaRESUMEN
The platelet-activating factor (PAF) acetylhydrolases catalyze hydrolysis of the sn-2 ester bond of PAF and related pro-inflammatory phospholipids and thus attenuate their bioactivity. One secreted (plasma) and four intracellular isozymes have been described. The intracellular isozymes are distinguished by differences in primary sequence, tissue localization, subunit composition, and substrate preferences. The most thoroughly characterized intracellular isoform, Ib, is a G-protein-like complex with two catalytic subunits (alpha1 and alpha2) and a regulatory beta subunit. The beta subunit is a product of the LIS1 gene, mutations of which cause Miller-Dieker lissencephaly. Isoform II is a single polypeptide that is homologous to the plasma PAF acetylhydrolase and has antioxidant activity in several systems. Plasma PAF acetylhydrolase is also a single polypeptide with a catalytic triad of amino acids that is characteristic of the alpha/beta hydrolases. Deficiency of this enzyme has been associated with a number of pathologies. The most common inactivating mutation, V279F, is found in >30% of randomly surveyed Japanese subjects (4% homozygous, 27% heterozygous). The prevalence of the mutant allele is significantly greater in patients with asthma, stroke, myocardial infarction, brain hemorrhage, and nonfamilial cardiomyopathy. Preclinical studies have demonstrated that recombinant plasma PAF acetylhydrolase can prevent or attenuate pathologic inflammation in a number of animal models. In addition, preliminary clinical results suggest that the recombinant enzyme may have pharmacologic potential in human inflammatory disease as well. These observations underscore the physiological importance of the PAF acetylhydrolases and point toward new approaches for controlling pathologic inflammation.
Asunto(s)
Inflamación/metabolismo , Fosfolipasas A/metabolismo , Factor de Activación Plaquetaria/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Antiinflamatorios/metabolismo , Biomarcadores/análisis , Enfermedades Cardiovasculares/enzimología , Línea Celular , Células Cultivadas , Clonación Molecular , Eritrocitos/enzimología , Europa (Continente) , Regulación de la Expresión Génica , Humanos , Isoenzimas/metabolismo , Japón , Mutación , Fosfolipasas A/análisis , Fosfolipasas A/genética , Fosfolípidos/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Platelet-activating factor (PAF) is a phospholipid with potent, diverse physiological actions, particularly as a mediator of inflammation. The synthesis, transport, and degradation of PAF are tightly regulated, and the biochemical basis for many of these processes has been elucidated in recent years. Many of the actions of PAF can be mimicked by structurally related phospholipids that are derived from nonenzymatic oxidation, because such compounds can bind to the PAF receptor. This process circumvents much of the biochemical control and presumably is regulated primarily by the rate of degradation, which is catalyzed by PAF acetylhydrolase. The isolation of cDNA clones encoding most of the key proteins involved in regulating PAF has allowed substantial recent progress and will facilitate studies to determine the structural basis for substrate specificity and the precise role of PAF in physiological events.
Asunto(s)
Fosfolípidos/fisiología , Factor de Activación Plaquetaria/fisiología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Humanos , Inflamación/etiología , Inflamación/fisiopatología , Leucocitos/inmunología , Leucocitos/fisiología , Oxidación-Reducción , Fosfolipasas A/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/fisiología , Transducción de SeñalRESUMEN
Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma.
Asunto(s)
Asma/genética , Fosfolipasas A/deficiencia , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Asma/epidemiología , Asma/fisiopatología , Secuencia de Bases , Sitios de Unión , Niño , Femenino , Genotipo , Homocigoto , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Fosfolipasas A/sangre , Polimorfismo GenéticoRESUMEN
The platelet-activating factor acetylhydrolases are enzymes that were initially characterized by their ability to hydrolyze platelet-activating factor (PAF). In human plasma, PAF acetylhydrolase (EC 3.1.1.47) circulates in a complex with low density lipoproteins (LDL) and high density lipoproteins (HDL). This association defines the physical state of PAF acetylhydrolase, confers a long half-life, and is a major determinant of its catalytic efficiency in vivo. The lipoprotein-associated enzyme accounts for all of the PAF hydrolysis in plasma but only two-thirds of the protein mass. To characterize the enzyme-lipoprotein interaction, we employed site-directed mutagenesis techniques. Two domains within the primary sequence of human PAF acetylhydrolase, tyrosine 205 and residues 115 and 116, were important for its binding to LDL. Mutation or deletion of those sequences prevented the association of the enzyme with lipoproteins. When residues 115 and 116 from human PAF acetylhydrolase were introduced into mouse PAF acetylhydrolase (which normally does not associate with LDL), the mutant mouse PAF acetylhydrolase associated with lipoproteins. To analyze the role of apolipoprotein (apo) B100 in the formation of the PAF acetylhydrolase-LDL complex, we tested the ability of PAF acetylhydrolase to bind to lipoproteins containing truncated forms of apoB. These studies indicated that the carboxyl terminus of apoB plays a key role in the association of PAF acetylhydrolase with LDL. These data on the molecular basis of the PAF acetylhydrolase-LDL association provide a new level of understanding regarding the pathway for the catabolism of PAF in human blood.
Asunto(s)
Lipoproteínas LDL/metabolismo , Fosfolipasas A/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Humanos , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Fosfolipasas A/genética , Unión Proteica , Proteínas Recombinantes/metabolismo , Tirosina/metabolismoAsunto(s)
Cromatografía Liquida/métodos , Fosfolipasas A/sangre , 1-Alquil-2-acetilglicerofosfocolina Esterasa , ADN Complementario/genética , Escherichia coli/química , Vectores Genéticos/genética , Humanos , Fosfolipasas A/genética , Fosfolipasas A/aislamiento & purificación , Factor de Activación Plaquetaria/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , SefarosaRESUMEN
Platelet-activating factor (PAF) is a potent phospholipid with diverse physiological and pathological actions, and it is inactivated by PAF acetylhydrolase. In this study, we analyzed the tissue distribution of the plasma PAF acetylhydrolase mRNA in humans. We isolated a 3.5-kilobase fragment containing the 5' genomic sequence of the plasma PAF acetylhydrolase gene and further characterized the promoter activity. We determined the transcriptional initiation site by primer extension. We then prepared constructs containing various lengths of 5' genomic fragments fused to a luciferase reporter gene and transfected these constructs into COS-7 cells. We found that there is more than one region in the 1.3-kilobase 5' genomic sequence conferring promoter activity and that a very short 5'-flanking region (72 base pairs) is sufficient for more than 65% of the basal activity. In parallel, we examined the regulation of expression of the PAF acetylhydrolase gene. We found that interferon-gamma (IFNgamma) and lipopolysaccharide (LPS) significantly inhibited synthesis of PAF acetylhydrolase, whereas other cytokines, including IFNalpha, interleukin (IL) 1alpha, IL4, IL6, tumor necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor, had a smaller or no effect in human monocyte-derived macrophages. Furthermore, transfection of the promoter/reporter construct into macrophage RAW264.7 cells revealed that IFNgamma and LPS decreased the promoter activity by 35% and 50%, respectively, whereas PAF stimulated it by 52% via its receptor. The promoter activity was much lower in monocytic U937 cells compared with the basal level in COS-7 cells, while the activities in P388D1 and RAW264.7 macrophagic cells were considerably higher than the basal level in COS-7 cells. There are multiple regions in the PAF acetylhydrolase promoter that contain responsive elements for signal transducer and activators of transcription-related proteins, and also for myeloid-specific transcription factors. Our data indicate that the opposite of mRNA expression in monocytes versus macrophages is due to inhibition of the promoter activity in the former and activation in the latter cells.
Asunto(s)
Regulación de la Expresión Génica/genética , Fosfolipasas A/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Secuencia de Bases , Diferenciación Celular/genética , Línea Celular , Clonación Molecular , Citocinas/farmacología , Genes Reporteros/genética , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Fosfolipasas A/análisis , Factor de Activación Plaquetaria/farmacología , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transfección/genéticaAsunto(s)
Fosfolipasas A , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Regulación de la Expresión Génica , Homeostasis , Humanos , Inflamación/enzimología , Cinética , Fosfolipasas A/sangre , Fosfolipasas A/química , Fosfolipasas A/deficiencia , Fosfolipasas A/genética , Fosfolipasas A/fisiología , Fosfolípidos/metabolismo , Transducción de SeñalRESUMEN
Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.
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Asma/etiología , Fosfolipasas A/genética , Mutación Puntual , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Humanos , Japón , Datos de Secuencia Molecular , Fosfolipasas/genética , Fosfolipasas A/deficienciaAsunto(s)
Arteriosclerosis/etiología , Selectina-P/fisiología , Factor de Activación Plaquetaria/fisiología , Vasculitis/complicaciones , Arteriosclerosis/fisiopatología , Células Cultivadas , Endotelio Vascular/fisiología , Humanos , Leucocitos/fisiología , Músculo Liso Vascular/fisiología , Factores de Tiempo , Vasculitis/fisiopatologíaRESUMEN
We previously reported that high density lipoprotein (HDL) protects against the oxidative modification of low density lipoprotein (LDL) induced by artery wall cells causing these cells to produce pro-inflammatory molecules. We also reported that enzyme systems associated with HDL were responsible for this anti-inflammatory property of HDL. We now report studies comparing HDL before and during an acute phase response (APR) in both humans and a croton oil rabbit model. In rabbits, from the onset of APR the protective effect of HDL progressively decreased and was completely lost by day three. As serum amyloid A (SAA) levels in acute phase HDL (AP-HDL) increased, apo A-I levels decreased 73%. Concomitantly, paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-AH) levels in HDL declined 71 and 90%, respectively, from days one to three. After day three, there was some recovery of the protective effect of HDL. AP-HDL from human patients and rabbits but not normal or control HDL (C-HDL) exhibited increases in ceruloplasmin (CP). This increase in CP was not seen in acute phase VLDL or LDL. C-HDL incubated with purified CP and re-isolated (CP-HDL), lost its ability to inhibit LDL oxidation. Northern blot analyses demonstrated enhanced expression of MCP-1 in coculture cells treated with AP-HDL and CP-HDL compared to C-HDL. Enrichment of human AP-HDL with purified PON or PAF-AH rendered AP-HDL protective against LDL modification. We conclude that under basal conditions HDL serves an anti-inflammatory role but during APR displacement and/or exchange of proteins associated with HDL results in a pro-inflammatory molecule.
Asunto(s)
Endotelio Vascular/fisiología , Inflamación/fisiopatología , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Músculo Liso Vascular/fisiología , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Antiinflamatorios no Esteroideos/farmacología , Aorta/citología , Aorta/fisiología , Arildialquilfosfatasa , Secuencia de Bases , Adhesión Celular , Células Cultivadas , Ceruloplasmina/biosíntesis , Quimiocina CCL2/biosíntesis , Técnicas de Cocultivo , Aceite de Crotón , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Esterasas/metabolismo , Expresión Génica , Humanos , Lipoproteínas HDL/aislamiento & purificación , Masculino , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oxidación-Reducción , Fosfolipasas A/metabolismo , ConejosRESUMEN
Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.
Asunto(s)
Lipoproteínas LDL/farmacología , Músculo Liso Vascular/citología , Factor de Activación Plaquetaria , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Animales , Células CHO , División Celular/efectos de los fármacos , Línea Celular , Cricetinae , Peroxidación de Lípido , Lipoproteínas LDL/metabolismo , Factor de Activación Plaquetaria/metabolismo , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , RatasRESUMEN
Platelet-activating factor (PAF) is a potent pro-inflammatory autacoid with diverse physiological and pathological actions. These actions are modulated by PAF acetylhydrolase, which hydrolyzes the sn-2 ester bond to yield the biologically inactive lyso-PAF. In contrast to most secreted phospholipase A2s, plasma PAF acetylhydrolase is calcium-dependent and contains a GXSXG motif that is characteristic of the neutral lipases and serine esterases. In this study we tested whether the serine in this motif is part of the active site of plasma PAF acetylhydrolase and, if so, what the other components of the active site are. Using site-directed mutagenesis, we demonstrated that Ser-273 (of the GXSXG motif), Asp-296, and His-351 are essential for catalysis. These residues were conserved in PAF acetylhydrolase sequences isolated from bovine, dog, mouse, and chicken. The linear orientation and spacing of these catalytic residues are consistent with the alpha/beta hydrolase conformation of other lipases and esterases. In support of this model, analysis of systematic truncations of PAF acetylhydrolase revealed that deletions beyond 54 amino acids from the NH2 terminus and 21 from the COOH terminus resulted in a loss of enzyme activity. These observations demonstrate that although plasma PAF acetylhydrolase is a phospholipase A2 it has structural properties characteristic of the neutral lipases and esterases.