RESUMEN
Hepatocellular carcinoma (HCC) is a common malignant tumor with a complex immune evasion mechanism posing a challenge to treatment. The role of the S100A10 gene in various cancers has garnered significant attention. This study aims to elucidate the impact of S100A10 on CD8+ T cell exhaustion via the cPLA2 and 5-LOX axis, thereby elucidating its role in immune evasion in HCC. By analyzing the HCC-related data from the GEO and TCGA databases, we identified differentially expressed genes associated with lipid metabolism and developed a prognostic risk model. Subsequently, through RNA-seq and PPI analyses, we determined vital lipid metabolism genes and downstream factors S100A10, ACOT7, and SMS, which were significantly correlated with CD8+ T cell infiltration. Given the most significant expression differences, we selected S100A10 for further investigation. Both in vitro and in vivo experiments were conducted, including co-culture experiments of CD8+ T cells with MHCC97-L cells, Co-IP experiments, and validation in an HCC mouse model. S100A10 was significantly overexpressed in HCC tissues and potentially regulates CD8+ T cell exhaustion and lipid metabolism reprogramming through the cPLA2 and 5-LOX axis. Silencing S100A10 could inhibit CD8+ T cell exhaustion, further suppressing immune evasion in HCC. S100A10 may activate the cPLA2 and 5-LOX axis, initiating lipid metabolism reprogramming and upregulating LTB4 levels, thus promoting CD8+ T cell exhaustion in HCC tissues, facilitating immune evasion by HCC cells, ultimately impacting the growth and migration of HCC cells. This research highlights the critical role of S100A10 via the cPLA2 and 5-LOX axis in immune evasion in HCC, providing new theoretical foundations and potential targets for diagnosing and treating HCC.
Asunto(s)
Araquidonato 5-Lipooxigenasa , Linfocitos T CD8-positivos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Escape del Tumor , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Humanos , Animales , Ratones , Araquidonato 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/genética , Línea Celular Tumoral , Proteínas S100/metabolismo , Proteínas S100/genética , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolipasas A2 Citosólicas/genética , Masculino , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos C57BL , Agotamiento de Células TRESUMEN
OBJECTIVE: Since diabetic patients with coronary microvascular dysfunction (CMD) exhibit high cardiac mortality and women have higher prevalence of non-obstructive coronary artery disease than men, we tried to expand the limited understanding about the etiology and the sex difference of diabetic CMD. APPROACH AND RESULTS: Accumulated methylglyoxal (MGO) due to diabetes promotes vascular damage and it was used for mimicking diabetic status. Flow cytometry analysis and isometric tension measurement were performed to evaluate coronary artery endothelial injury. MGO induced apoptosis of coronary endothelial cells, accompanied by downregulation of androgen receptor (AR). Lentivirus-mediated stable expression of AR in coronary endothelial cells increased anti-apoptotic Bcl-2 expression and attenuated MGO-induced cell apoptosis. cPLA2 activation was the downstream of AR downregulation by MGO treatment. Moreover, MGO also activated cPLA2 rapidly to impair endothelium-dependent vasodilation of coronary arteries from mice. Reactive oxygen species (ROS) overproduction was demonstrated to account for MGO-mediated cPLA2 activation and endothelial dysfunction. Importantly, AR blockade increased endothelial ROS production whereas AR activation protected coronary artery endothelial vasodilatory function from the MGO-induced injury. Although galectin-3 upregulation was confirmed by siRNA knockdown in endothelial cells not to participate in MGO-induced endothelial apoptosis, pharmacological inhibitor of galectin-3 further enhanced MGO-triggered ROS generation and coronary artery endothelial impairment. CONCLUSIONS: Our data proposed the AR downregulation-ROS overproduction-cPLA2 activation pathway as one of the mechanisms underlying diabetic CMD and postulated a possible reason for the sex difference of CMD-related angina. Meanwhile, MGO-induced galectin-3 activation played a compensatory role against coronary endothelial dysfunction.
Asunto(s)
Apoptosis , Vasos Coronarios , Células Endoteliales , Piruvaldehído , Receptores Androgénicos , Transducción de Señal , Piruvaldehído/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ratones , Masculino , Transducción de Señal/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/efectos de los fármacos , Humanos , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Vasos Coronarios/patología , Vasos Coronarios/metabolismo , Femenino , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolipasas A2 Citosólicas/genética , Especies Reactivas de Oxígeno/metabolismo , Ratones Endogámicos C57BLRESUMEN
Hyperactivation of cyclin-dependent kinase 5 (Cdk5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinson's disease (PD) and Alzheimer's disease. However, the mechanism by which Cdk5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that Cdk5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes on each single-point mutation. Interestingly, double mutations also led to a severe decline in the activity of cPLA2 and to the disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of Cdk5, resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity, confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and Cdk5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis, and inflammatory pathways are significantly upregulated in the case of PD patients compared with that of healthy control subjects.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina , Enfermedad de Parkinson , Fosfolipasas A2 Citosólicas , Animales , Humanos , Ratones , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosfolipasas A2 Citosólicas/genética , Fosfolipasas A2 Citosólicas/metabolismo , FosforilaciónRESUMEN
HDAC5 is a class IIa histone deacetylase member that is downregulated in multiple solid tumors, including pancreatic cancer, and loss of HDAC5 is associated with unfavorable prognosis. In this study, assessment of The Cancer Genome Atlas pancreatic adenocarcinoma dataset revealed that expression of HDAC5 correlates negatively with arachidonic acid (AA) metabolism, which has been implicated in inflammatory responses and cancer progression. Nontargeted metabolomics analysis revealed that HDAC5 knockdown resulted in a significant increase in AA and its downstream metabolites, such as eicosanoids and prostaglandins. HDAC5 negatively regulated the expression of the gene encoding calcium-dependent phospholipase A2 (cPLA2), the key enzyme in the production of AA from phospholipids. Mechanistically, HDAC5 repressed cPLA2 expression via deacetylation of GATA1. HDAC5 knockdown in cancer cells enhanced sensitivity to genetic or pharmacologic inhibition of cPLA2 in vitro and in vivo. Fatty acid supplementation in the diet reversed the sensitivity of HDAC5-deficient tumors to cPLA2 inhibition. These data indicate that HDAC5 loss in pancreatic cancer results in the hyperacetylation of GATA1, enabling the upregulation of cPLA2, which contributes to overproduction of AA. Dietary management plus cPLA2-targeted therapy could serve as a viable strategy for treating HDAC5-deficient pancreatic cancer patients. SIGNIFICANCE: The HDAC5-GATA1-cPLA2-AA signaling axis regulates sensitivity to fat restriction plus cPLA2 inhibition in pancreatic ductal adenocarcinoma, proposing dietary management as a feasible strategy for treating a subset of patients with pancreatic cancer.
Asunto(s)
Adenocarcinoma , Ácido Araquidónico , Histona Desacetilasas , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/genética , Ácido Araquidónico/metabolismo , Citosol/metabolismo , Histona Desacetilasas/genética , Neoplasias Pancreáticas/genética , Fosfolipasas A2 Citosólicas/genética , Fosfolípidos/metabolismoRESUMEN
Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication.
Asunto(s)
Peces/virología , Iridovirus/genética , Fosfolipasas A2 Citosólicas/genética , Replicación Viral/genética , Animales , Acuicultura , Araquidonato 5-Lipooxigenasa/genética , Peces/genética , Glicerofosfolípidos/genética , Iridovirus/patogenicidad , Fosfatidilcolinas/genética , Fosfatidilserinas/genética , SingapurRESUMEN
Inflammatory Bowel Disease (IBD) is an autoimmune condition with complicated pathology and diverse clinical signs. TNFα is believed to play a crucial role in the pathogenesis of IBD. We recently identified fexofenadine, a well-known antagonist of histamine H1 receptor, as a novel inhibitor of TNFα signaling. Additionally, cytosolic phospholipase A2 (cPLA2) was isolated as a binding target of fexofenadine, and fexofenadine-mediated anti-TNF activity relied on cPLA2 in vitro. The objective of this study is to determine whether fexofenadine is therapeutic against chemically-induced murine IBD model and whether cPLA2 and/or histamine H1 receptor is important for fexofenadine's anti-inflammatory activity in vivo by leveraging various genetically modified mice and chemically induced murine IBD models. Both dextran sulfate sodium- and 2, 4, 6-trinitrobenzene sulfonic acid-induced murine IBD models revealed that orally delivered fexofenadine was therapeutic against IBD, evidenced by mitigated clinical symptoms, decreased secretions of the proinflammatory cytokine IL-6 and IL-1ß, lowered intestinal inflammation, and reduced p-p65 and p-IĸBα. Intriguingly, Fexofenadine-mediated protective effects against IBD were lost in cPLA2 deficient mice but not in histamine H1 receptor-deficient mice. Collectively, these findings demonstrate the therapeutic effects of over-the-counter drug Fexofenadine in treating DSS-induced IBD murine and provide first in vivo evidence showing that cPLA2 is required for fexofenadine's therapeutic effects in murine IBD model and probably other inflammatory and autoimmune diseases as well.
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Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Fosfolipasas A2 Citosólicas/fisiología , Terfenadina/análogos & derivados , Animales , Biomarcadores Farmacológicos , Modelos Animales de Enfermedad , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasas A2 Citosólicas/genética , Terfenadina/uso terapéuticoRESUMEN
The hypothalamus plays a central role in monitoring and regulating systemic glucose metabolism. The brain is enriched with phospholipids containing poly-unsaturated fatty acids, which are biologically active in physiological regulation. Here, we show that intraperitoneal glucose injection induces changes in hypothalamic distribution and amounts of phospholipids, especially arachidonic-acid-containing phospholipids, that are then metabolized to produce prostaglandins. Knockdown of cytosolic phospholipase A2 (cPLA2), a key enzyme for generating arachidonic acid from phospholipids, in the hypothalamic ventromedial nucleus (VMH), lowers insulin sensitivity in muscles during regular chow diet (RCD) feeding. Conversely, the down-regulation of glucose metabolism by high fat diet (HFD) feeding is improved by knockdown of cPLA2 in the VMH through changing hepatic insulin sensitivity and hypothalamic inflammation. Our data suggest that cPLA2-mediated hypothalamic phospholipid metabolism is critical for controlling systemic glucose metabolism during RCD, while continuous activation of the same pathway to produce prostaglandins during HFD deteriorates glucose metabolism.
Asunto(s)
Glucosa/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Prostaglandinas/biosíntesis , Núcleo Hipotalámico Ventromedial/metabolismo , Animales , Ácido Araquidónico/metabolismo , Vías Biosintéticas , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/genética , Fosfolipasas A2 Grupo IV/metabolismo , Hiperglucemia/metabolismo , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Fosfolipasas A2 Citosólicas/genética , Fosfolípidos/metabolismoRESUMEN
Spinal cord injury (SCI) produces chronic, pro-inflammatory macrophage activation that impairs recovery. The mechanisms driving this chronic inflammation are not well understood. Here, we detail the effects of myelin debris on macrophage physiology and demonstrate a novel, activation state-dependent role for cytosolic phospholipase-A2 (cPLA2) in myelin-mediated potentiation of pro-inflammatory macrophage activation. We hypothesized that cPLA2 and myelin debris are key mediators of persistent pro-inflammatory macrophage responses after SCI. To test this, we examined spinal cord tissue 28-days after thoracic contusion SCI in 3-month-old female mice and observed both cPLA2 activation and intracellular accumulation of lipid-rich myelin debris in macrophages. In vitro, we utilized bone marrow-derived macrophages to determine myelin's effects across a spectrum of activation states. We observed phenotype-specific responses with myelin potentiating only pro-inflammatory (LPS + INF-γ; M1) macrophage activation, whereas myelin did not induce pro-inflammatory responses in unstimulated or anti-inflammatory (IL-4; M2) macrophages. Specifically, myelin increased levels of pro-inflammatory cytokines, reactive oxygen species, and nitric oxide production in M1 macrophages as well as M1-mediated neurotoxicity. PACOCF3 (cPLA2 inhibitor) blocked myelin's detrimental effects. Collectively, we provide novel spatiotemporal evidence that myelin and cPLA2 play an important role in the pathophysiology of SCI inflammation and the phenotype-specific response to myelin implicate diverse roles of myelin in neuroinflammatory conditions.
Asunto(s)
Inflamación/genética , Vaina de Mielina/genética , Fosfolipasas A2 Citosólicas/genética , Traumatismos de la Médula Espinal/genética , Animales , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/patología , Interleucina-4/biosíntesis , Cetonas/farmacología , Lípidos/biosíntesis , Lípidos/genética , Activación de Macrófagos/genética , Ratones , Microglía/metabolismo , Microglía/patología , Óxido Nítrico/biosíntesis , Fagocitosis/genética , Fenotipo , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
Obesity is associated with metabolic endotoxemia, reactive oxygen species (ROS), chronic inflammation, and obese kidney fibrosis. Although the fat-intestine-kidney axis has been documented, the pathomechanism and therapeutic targets of obese kidney fibrosis remain unelucidated. To mimic obese humans with metabolic endotoxemia, high-fat-diet-fed mice (HF group) were injected with lipopolysaccharide (LPS) to yield the obese kidney fibrosis-metabolic endotoxemia mouse model (HL group). Therapeutic effects of ROS, cytosolic phospholipases A2 (cPLA2) and cyclooxygenase-2 (COX-2) inhibitors were analyzed with a quantitative comparison of immunohistochemistry stains and morphometric approach in the tubulointerstitium of different groups. Compared with basal and HF groups, the HL group exhibited the most prominent obese kidney fibrosis, tubular epithelial lipid vacuoles, and lymphocyte infiltration in the tubulointerstitium. Furthermore, inhibitors of nonspecific ROS, cPLA2 and COX-2 ameliorated the above renal damages. Notably, the ROS-inhibitor-treated group ameliorated not only oxidative injury but also the expression of cPLA2 and COX-2, indicating that ROS functions as the upstream signaling molecule in the inflammatory cascade of obese kidney fibrosis. ROS acts as a key messenger in the signaling transduction of obese kidney fibrosis, activating downstream cPLA2 and COX-2. The given antioxidant treatment ameliorates obese kidney fibrosis resulting from a combined high-fat diet and LPS-ROS could serve as a potential therapeutic target of obese kidney fibrosis with metabolic endotoxemia.
Asunto(s)
Ciclooxigenasa 2/genética , Endotoxemia/complicaciones , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Obesidad/complicaciones , Fosfolipasas A2 Citosólicas/genética , Especies Reactivas de Oxígeno/metabolismo , Animales , Biomarcadores , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Metabolismo de los Lípidos , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Terapia Molecular Dirigida , Estrés Oxidativo , Fosfolipasas A2 Citosólicas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.
Asunto(s)
Autofagia , Lisosomas/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Fosfolípidos/química , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Catepsina D/genética , Catepsina D/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Fosfolipasas A2 Citosólicas/genética , Fosfolípidos/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , alfa-N-Acetilgalactosaminidasa/metabolismoRESUMEN
BACKGROUND The aim of this study was to elucidate the involvement of cPLA2-AA-COX-2 pathway factors and their potential role in lung cancer early diagnosis and prognosis. MATERIAL AND METHODS We selected 80 lung cancer patients as the cancer group, and 30 normal patients were selected as the normal group. Serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 were measured, and mRNA levels of COX-2, cPLA2, COX-1, and mPGES in serum were determined. Spearman's P-test was used to analyze the correlation between expression of PGI2 and mPGES in serum and the clinical characteristics of these lung cancer patients. The factors affecting the prognosis lung cancer were analyzed by COX regression model. RESULTS The serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 in the cancer patient group were significantly higher (p<0.05) than in the normal group; after treatment, the serum contents of these factors were significantly decreased (p<0.05). However, distant metastasis had a significant effect on serum contents of mPGES and PGI2 (p<0.05), but not on the other factors. The mRNA levels of COX-2, cPLA2, COX-1, and mPGES in cancer patients were significantly higher than in normal patients. In addition, the 5-year survival rate of patients with high expression of mPGES and/or PGI2 was lower than that of the low expression group. Cox regression analysis showed that the expression of mPGES and PGI2 had statistical significance in predicting the prognosis of lung cancer. CONCLUSIONS The cPLA2-AA-COX-2 pathway is closely associated with lung cancer. These findings are important for clinical diagnosis of lung cancer.
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Ácido Araquidónico/metabolismo , Ciclooxigenasa 2/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfolipasas A2 Citosólicas/metabolismo , Adulto , Anciano , Ácido Araquidónico/sangre , Ácido Araquidónico/genética , Ciclooxigenasa 2/sangre , Ciclooxigenasa 2/genética , Citosol/metabolismo , Dinoprostona/metabolismo , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Isoenzimas , Pulmón/metabolismo , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Fosfolipasas A2 Citosólicas/sangre , Fosfolipasas A2 Citosólicas/genética , Pronóstico , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/genéticaRESUMEN
Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1⯱â¯0.3â¯g/kg/day), or control (dextrose)⯱â¯veliparib (25â¯mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.
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Trastornos del Sistema Nervioso Inducidos por Alcohol/prevención & control , Bencimidazoles/uso terapéutico , Proteínas del Tejido Nervioso/biosíntesis , Fosfolipasas A2 Citosólicas/biosíntesis , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Trastornos del Sistema Nervioso Inducidos por Alcohol/tratamiento farmacológico , Trastornos del Sistema Nervioso Inducidos por Alcohol/enzimología , Animales , Bencimidazoles/farmacología , Consumo Excesivo de Bebidas Alcohólicas , Giro Dentado/efectos de los fármacos , Giro Dentado/enzimología , Giro Dentado/patología , Modelos Animales de Enfermedad , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/enzimología , Corteza Entorrinal/patología , Inducción Enzimática/efectos de los fármacos , Masculino , Proteínas del Tejido Nervioso/genética , Fosfolipasas A2 Citosólicas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The ovarian steroid hormones, estrogen (E2) and progesterone (P4), are essential regulators of uterine biology. The imbalance of these ovarian steroid hormones leads to uterine diseases such as endometrial cancer, endometriosis, and infertility. Mitogen-inducible gene 6 (MIG-6) is an adaptor protein. MIG-6 mediates P4 signaling and acts as a tumor suppressor during endometrial tumorigenesis in both humans and mice. In previous studies, we developed the conditional knockout of Mig-6 in all uterine compartments (Pgrcre/+Mig-6f/f; Mig-6KO) and endometrial epithelial cell-specific Mig-6 knockout (Sprr2fcre/+Mig-6f/f; Mig-6Ep-KO) mice. Both mouse models developed endometrial hyperplasia and E2-dependent endometrial cancer. P4 treatment significantly decreases aberrant epithelial proliferation and AKT signaling in Mig-6Ep-KO mice but not in Mig-6KO mice. In the present study, we identified a calcium-dependent phospholipase A2 (cPla2) as one of the genes down-regulated by Mig-6 in the uterus. We performed immunohistochemistry and Western Blot analysis to investigate the regulation of cPLA2 by MIG-6 as well as determine the expression patterns of cPLA2 in the uterus. While the expression of cPLA2 was stronger at the uterine epithelial cells of Mig-6KO and Mig-6Ep-KO mice compared to control mice, P4 suppressed the expression of cPLA2 in Mig-6Ep-KO mice but not in Mig-6KO mice. To determine the ovarian steroid hormone regulation of cPLA2, we examined the expression of cPLA2 in ovariectomized control, Mig-6KO, Mig-6Ep-KO, and PRKO mice treated with P4 or E2. After P4 treatment, cPLA2 expression was remarkably reduced in Mig-6Ep-KO mice but not in Mig-6KO mice. However, the expression of cPLA2 was not changed in PRKO mice. Our results identified cPLA2 as a novel target of MIG-6 in the murine uterus and identified its important role during endometrial tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Neoplasias Endometriales/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfolipasas A2 Citosólicas/genética , Animales , Calcio/metabolismo , Carcinogénesis/patología , Neoplasias Endometriales/patología , Endometrio/metabolismo , Endometrio/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones NoqueadosRESUMEN
Sulfite is a commonly used preservative in food products, alcoholic beverages and pharmaceutical products. We investigated the effect of sulfite, on locomotor activity as well as the relationship of these effects with oxidant and antioxidant capacities, cPLA2 enzyme activity. Thirty male Wistar albino rats were randomly divided into two groups as control(C) and sulfite(S). Animals in the S group were given freshly prepared sulfite for 35 days via gastric gavage (100â¯mg/kg/day) while the C group received equal volumes of distilled water via gavage for the same period. Open-field tests were performed to all groups and animals were sacrificed. Total antioxidant capacity(TAC), TBARS levels, cPLA2 activity as well as amount of caspase-3 positive cells were analyzed on the hippocampi. In the open field test, distance and velocity values of the S group increased with respect to controls. TBARS and cPLA2 activity were also increased in the S group, while levels of TAC decreased compared to controls. Immunohistochemical analysis showed that sulfite ingestion caused an increase in the amount of hippocampal caspase-3 positive cells. In conclusion, sulfite seemed to increase locomotor activity. cPLA2 might play a role in ingested sulfite-induced oxidative stress and apoptotic cell death in the hippocampus.
Asunto(s)
Caspasa 3/metabolismo , Conservantes de Alimentos/toxicidad , Locomoción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfolipasas A2 Citosólicas/metabolismo , Sulfitos/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Caspasa 3/genética , Conservantes de Alimentos/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Masculino , Fosfolipasas A2 Citosólicas/genética , Ratas , Ratas Wistar , Sulfitos/metabolismoRESUMEN
During radiotherapy, an inflammatory response might be induced by activating various enzymes involved in membrane lipid metabolism. The eicosanoid pathway associated with cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COXs), and lipoxygenases (LOXs) can be induced by radiation, and many lipid metabolites might contribute to cancer-associated inflammation, cell proliferation, and cell survival in cancer. The lipid metabolites are also involved in the establishment of the tumor-associated microenvironment through promotion of angiogenesis and formation of vascular network. These biological activities of lipid metabolites are responsible for malignant progression with the acquisition of radioresistance, leading to unsatisfactory outcome of cancer radiotherapy. Many efforts have been made to identify the mechanisms associated with bioactive lipid metabolites and radiation signaling that lead to radioresistance and to develop potent radiosensitizers to improve therapeutic efficacy. Beneficial outcomes would be achieved by targeting the enzymes, such as cPLA2, COXs, and LOXs, responsible for arachidonic acid metabolism and cancer-associated inflammation during cancer radiotherapy. The current study demonstrated a brief review for the radioresistant effects of bioactive lipid metabolites and their enzymes in cancer and the radiosensitizing effects of inhibitors for the enzymes on cancer therapy.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Metabolismo de los Lípidos/efectos de la radiación , Neoplasias/metabolismo , Neoplasias/radioterapia , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Biomarcadores , Ensayos Clínicos como Asunto , Terapia Combinada , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Susceptibilidad a Enfermedades , Activación Enzimática/efectos de la radiación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Neoplasias/enzimología , Neoplasias/genética , Fosfolipasas A2 Citosólicas/genética , Fosfolipasas A2 Citosólicas/metabolismo , Pronóstico , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Resultado del TratamientoRESUMEN
The aim of the present study was to investigate the potential role of club cell secretory protein (CCSP), an endogenous modulator, in reducing pulmonary inflammation induced by sevoflurane following onelung ventilation (OLV). Healthy Japanese white rabbits were randomly assigned to six groups: Shamoperated group (group S); respiratory management of OLV group (group O); OLV + sevoflurane treated group (group OF), club cells exfoliated + shamoperated group (group NA), club cells exfoliated + OLV group (group NAO); and club cells exfoliated + OLV + sevoflurane treated group (group NAOF). At the end of the experimental observation, all animals in the different groups were sacrificed and lung injury was evaluated according to the lung wet/dry weight ratio and histological scoring system. Lung homogenates were harvested to detect the mRNA and protein expression of cytosolic phospholipase A2 (cPLA2) and CCSP. The content of arachidonic acid was measured using an ELISA. Following OLV treatment, cPLA2 expression was increased, CCSP expression was decreased and lung injury scores were significantly increased. Sevoflurane inhalation in the OLVtreated group induced an upregulation of CCSP and a downregulation of cPLA2 expression. In the group NAO, in which the club cells were simultaneously exfoliated, OLV caused more severe lung damage and induced higher expression of cPLA2 compared with that in group O. However, sevoflurane inhalation reduced the extent of lung injury and the expression of cPLA2, even when the endogenous modulator of lung inflammation, CCSP, was exfoliated (group NAOF). These results indicated that OLV promoted lung inflammation through the CCSP and cPLA2 pathway. However, the results from the club cells exfoliated group indicated that the CCSP may not be involved in the protective effect exerted by sevoflurane inhalation.
Asunto(s)
Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Éteres Metílicos/farmacología , Fosfolipasas A2 Citosólicas/genética , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Animales , Ácido Araquidónico/metabolismo , Biopsia , Modelos Animales de Enfermedad , Femenino , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Fosfolipasas A2 Citosólicas/metabolismo , Conejos , Sevoflurano , Uteroglobina/genética , Uteroglobina/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
A casein glycomacropeptide hydrolysate (GMPH) was found to possess inhibitory activity against lipopolysaccharide (LPS)-induced inflammatory response in our previous study. In the current study, the inhibitory effect and the underlying molecular mechanism of GMPH on inflammatory response in LPS-stimulated RAW264.7 macrophages were further investigated. Results showed that GMPH significantly suppressed LPS-induced intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production. GMPH reduced the production of prostaglandin E2 (PEG2) and the expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) in LPS-stimulated macrophages. GMPH also attenuated LPS-induced phosphorylation of MAPK (c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38) and protein kinase B (Akt). Furthermore, GMPH inhibited nuclear transcription factor kappa-B (NF-κB) activation by suppressing the nuclear translocation of NF-κB p65, which was markedly reversed by LY294002, an Akt inhibitor. These results demonstrated that GMPH exerts anti-inflammatory functions through the inactivation of MAPK and Akt in LPS-stimulated RAW264.7 macrophages, therefore may hold potential to ameliorate inflammation-related metabolic disorders.
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Caseínas/química , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Macrófagos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Caseínas/farmacología , Ciclooxigenasa 2/genética , Lipopolisacáridos/efectos adversos , Macrófagos/metabolismo , Malondialdehído/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , FN-kappa B/genética , Fragmentos de Péptidos/química , Fosfolipasas A2 Citosólicas/genética , Fosfolipasas A2 Citosólicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
RATIONALE: The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A2, which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A2 are formed after the concerted actions of cPLA2α (cytosolic phospholipase A2) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF1α (PGI-M) and 11-dehydro-TXB2 (TX-M) have been taken as biomarkers of prostacyclin and thromboxane A2 formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. OBJECTIVE: We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA2α, causing almost complete loss of prostacyclin and thromboxane A2, who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA2α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A2 in the circulation. METHODS AND RESULTS: Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F1α) and thromboxane A2 (measured as TXB2), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A2 remained negligible. CONCLUSIONS: These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromboxane A2 by platelets in the general circulation. Previous work relying on urinary metabolites of prostacyclin and thromboxane A2 as markers of whole-body endothelial and platelet function now requires reevaluation.
Asunto(s)
6-Cetoprostaglandina F1 alfa/análogos & derivados , Aloinjertos/metabolismo , Trasplante de Riñón , Riñón/metabolismo , Mutación con Pérdida de Función , Fosfolipasas A2 Citosólicas/genética , Tromboxano B2/análogos & derivados , 6-Cetoprostaglandina F1 alfa/metabolismo , 6-Cetoprostaglandina F1 alfa/orina , Biomarcadores/orina , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Fenotipo , Fosfolipasas A2 Citosólicas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Tromboxano B2/metabolismo , Tromboxano B2/orinaRESUMEN
Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR) or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Fosfolipasas A2 Citosólicas/metabolismo , Piruvaldehído/toxicidad , Antioxidantes/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A2 Citosólicas/antagonistas & inhibidores , Fosfolipasas A2 Citosólicas/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.