RESUMEN
Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Supervivencia Celular , Ganglios Espinales , Compuestos de Metilmercurio , Células de Schwann , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nervios Periféricos/metabolismo , Nervios Periféricos/efectos de los fármacos , Masculino , Ratas , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
Methylmercury (MeHg), the causal substrate in Minamata disease, can lead to severe and chronic neurological disorders. The main symptom of Minamata disease is sensory impairment in the four extremities; however, the sensitivity of individual sensory modalities to MeHg has not been investigated extensively. In the present study, we performed stimulus-response behavioral experiments in MeHg-exposed rats to compare the sensitivities to pain, heat, cold, and mechanical sensations. MeHg (6.7 mg/kg/day) was orally administered to 9-week-old Wistar rats for 5 days and discontinued for 2 days, then administered daily for another 5 days. The four behavioral experiments were performed daily on each rat from the beginning of MeHg treatment for 68 days. The pain sensation decreased significantly from day 11 onwards, but recovered to control levels on day 48. Other sensory modalities were not affected by MeHg exposure. These findings suggest that the pain sensation is the sensory modality most susceptive to MeHg toxicity and that this sensitivity is reversible following discontinuation of the exposure.
Asunto(s)
Hipoestesia/etiología , Hipoestesia/fisiopatología , Intoxicación del Sistema Nervioso por Mercurio/etiología , Intoxicación del Sistema Nervioso por Mercurio/fisiopatología , Compuestos de Metilmercurio/toxicidad , Dolor/fisiopatología , Animales , Masculino , Compuestos de Metilmercurio/administración & dosificación , Ratas WistarRESUMEN
Objective We assessed the effect of canagliflozin, an sodium-glucose co-transporter type-2 inhibitor, on hepatic steatosis using three imaging modalities: magnetic resonance imaging (MRI), computed tomography, and transient elastography. We further determined factors associated with improving hepatic steatosis by canagliflozin among patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Methods We conducted a six-month prospective single-arm study between August 2015 and June 2017. The primary outcome was the change in hepatic steatosis assessed using the hepatic proton density fat fraction (PDFF) on MRI before and after treatment with canagliflozin. The secondary outcomes were changes in measures of glucose metabolism, including the hepatic glucose uptake on fluorodeoxyglucose-positron emission tomography, and the inflammation and volumes of visceral and subcutaneous adipose tissue and skeletal muscle. Patients Nine patients with type 2 diabetes and NAFLD completed this study. All participants received canagliflozin at a dose of 100 mg daily. Results Canagliflozin caused a significant reduction in hepatic PDFF from baseline [median 20.6% (interquartile range 11.7%, 29.8%)] after 6 months [10.6% (5.4%, 22.6%), p=0.008]. Canagliflozin also significantly reduced the body weight, glycated hemoglobin, homeostasis model assessment of insulin resistance (HOMA-IR), high sensitivity C-reactive protein (hs-CRP), and volumes of adipose tissue and skeletal muscle (all p<0.05). The reduction in hepatic PDFF was not correlated with changes in the body weight, HOMA-IR, hs-CRP, or volume of adipose tissue and skeletal muscle from baseline after six months. Conclusion Among patients with type 2 diabetes and NAFLD, canagliflozin improved hepatic steatosis. The effect may be independent of reducing adiposity, insulin resistance, inflammation, and skeletal muscle volume.
Asunto(s)
Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Canagliflozina/uso terapéutico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hígado/diagnóstico por imagen , Imagen por Resonancia Magnética , Músculo Esquelético/diagnóstico por imagen , Enfermedad del Hígado Graso no Alcohólico/diagnóstico por imagen , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Estudios ProspectivosRESUMEN
Chronic arsenic exposure is known to be related to the progression of atherosclerosis. However, the pathogenic mechanisms of arsenic-induced atherosclerosis have not been fully elucidated. Because disruption of the blood coagulation/fibrinolytic system is involved in the development of arteriosclerosis, we investigated the effect of arsenite on fibrinolytic activity in human vascular endothelial EA.hy926 cells in the present study. Fibrinolysis depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) secreted from vascular endothelial cells. We found that arsenite reduced fibrinolytic t-PA activity by inhibiting its synthesis without affecting PAI-1 production. The inhibitory effect of arsenite on t-PA expression was partially recovered by the reactive oxygen species (ROS) scavenger Trolox. The nuclear factor erythroid 2 related factor 2 (NRF2) pathway is known to be activated by arsenite via ROS production. We confirmed that arsenite activated the NRF2 pathway, and arsenite-induced inhibition of fibrinolytic t-PA activity was abrogated in NRF2-knockdown EA.hy926 cells. These results suggest that arsenite inhibits the fibrinolytic activity of t-PA by selectively suppressing its synthesis via activation of the NRF2 pathway in vascular endothelial cells.
Asunto(s)
Arsenitos/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/genética , Biomarcadores , Coagulación Sanguínea/efectos de los fármacos , Línea Celular , Supervivencia Celular , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Fibrinólisis/efectos de los fármacos , Expresión Génica , Humanos , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive sulfur species (RSS), such as hydropersulfides and hydropolysulfides with high nucleophilicity, contain mobilized sulfur that readily captures xenobiotic electrophiles, leading to their sulfur adducts. We have previously reported that RSS produced by cystathionine γ-lyase (CSE) captures the electrophilic metal methylmercury (MeHg) to form inert sulfur adducts, which in turn play a critical role in the protection against MeHg-induced motor impairment in mice. However, the mechanism underlying this neuroprotective effect is not fully understood. Here, we addressed this using CSE-knockout mice. The cerebellum of CSE-knockout mice was more susceptible to MeHg than that of wild type mice. Moreover, these CSE-deficient mice exhibited a higher level of mercury accumulation in the brain. However, co-treatment with sodium tetrasulfide, an RSS able to capture MeHg, leading to the formation of its sulfur adducts, blocked the increased accumulation of mercury, motor dysfunction and mortality caused by CSE deficiency. Our findings suggest that capturing MeHg by RSS in association with its sulfur adduct formation is involved in the repression of the brain distribution and deleterious effects of MeHg.
RESUMEN
Blood coagulation and the fibrinolytic system contribute to vascular lesions. Fibrinolysis in normal circulating blood strongly depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) secreted from vascular endothelial cells; however, the mechanisms by which endothelial fibrinolysis is regulated remain to be fully understood. In the present study, human vascular endothelial EA.hy926 cells were transfected with small interfering RNA for nuclear factor erythroid 2-related factor 2 (NRF2) and the expression of t-PA and PAI-1 and fibrinolytic activity in the conditioned medium were examined. EA.hy926 cells were also treated with sulforaphane, an NRF2 activator, and fibrinolytic activity was examined to confirm the NRF2 signaling pathway's effect. Enhanced fibrinolytic activity in the conditioned medium was observed in association with increased expression and secretion levels of t-PA in NRF2 knockdown EA.hy926 cells. However, sulforaphane inhibited fibrinolytic activity and t-PA synthesis in EA.hy926 cells without any cell damage. The expression level of PAI-1 did not change in either NRF2 knockdown or sulforaphane treated cells. These results suggest that transcription factor NRF2 may play a role in down-regulating endothelial t-PA synthesis and fibrinolytic activity.
Asunto(s)
Regulación hacia Abajo/genética , Células Endoteliales/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Activador de Tejido Plasminógeno/metabolismo , Células Cultivadas , Fibrinólisis/genética , Expresión Génica/genética , Humanos , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/genéticaRESUMEN
Vascular endothelial cells cover the luminal surface of blood vessels in a monolayer. Proliferation of these cells is crucial for the repair of damaged endothelial monolayers. In the present study, we identified a zinc complex, Zn(ii)2,9-dimethyl-1,10-phenanthroline (Zn-12), that stimulates the proliferation of bovine aortic endothelial cells in a culture system. No such stimulatory activity was observed for the ligand alone or in combination with other metals; however, the ligand combined with iron weakly stimulated the proliferation, as evidenced by the [3H]thymidine incorporation assay. Inorganic zinc weakly but significantly stimulated proliferation, and intracellular accumulation of zinc was similar between inorganic zinc and Zn-12 treatment, suggesting that the mechanisms by which Zn-12 stimulates vascular endothelial cell proliferation contain processes that differ from those by which inorganic zinc stimulates proliferation. Although expression of endogenous fibroblast growth factor-2 (FGF-2) and its receptor FGFR-1 was unchanged by Zn-12, both siRNA-mediated knockdown of FGF-2 and FGFR inhibition partly but significantly suppressed the stimulation of vascular endothelial cell proliferation by Zn-12, indicating that the zinc complex activates the FGF-2 pathway to stimulate proliferation. Phosphorylation of ERK1/2 and MAPKs was induced by Zn-12, and PD98059, a MEK1 inhibitor, significantly suppressed the stimulatory effect of Zn-12 on vascular endothelial cell proliferation. Therefore, it is suggested that Zn-12 activates the FGF-2 pathway via activation of ERK1/2 signaling to stimulate vascular endothelial cell proliferation, although FGF-2-independent mechanisms are also involved in the stimulation. Zn-12 and related compounds may be promising molecular probes to analyze biological systems of vascular endothelial cells.
RESUMEN
BACKGROUND: Adenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. METHODS: We investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. RESULTS: A2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. CONCLUSION: Activation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. GENERAL SIGNIFICANCE: Our results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.
Asunto(s)
Neoplasias Pulmonares/radioterapia , Tolerancia a Radiación/genética , Receptor de Adenosina A2B/genética , Células A549 , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Histonas/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosforilación/efectos de la radiación , Radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Translocación Genética/efectos de los fármacos , Translocación Genética/efectos de la radiación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Familia-src Quinasas/genéticaRESUMEN
Methylmercury (MeHg) exposure is known to induce neurodegeneration in both the central nervous system (CNS) and peripheral nervous system (PNS). Molecular mechanisms of MeHg-induced neurotoxicity have been well investigated in the CNS, however, it remains unclear in the PNS. In the present study, comprehensive gene expression analysis was performed by analyzing MeHg-exposed adult rat dorsal root ganglion (DRG) by DNA microarray. Methylmercuric chloride (6.7 mg/kg/day) was administered to nine-week-old male Wistar rats for five days, followed by two days without administration; this cycle was repeated once. Rats were anesthetized at 7 or 14 days after commencement of MeHg exposure, and their DRGs were removed and homogenized to make total RNA samples. DNA microarray data from Day 7 samples identified 100 out of 18,513 detected genes as annotated genes with more than two-fold upregulated or downregulated expression compared with controls. Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested strong involvement of immune activation and inflammation pathways in rat DRG exposed to MeHg, and some genes overlapped with previously reported genes affected by MeHg exposure in the cerebellum. The present results suggest that MeHg-induced neurotoxicity is associated with immune activation and inflammatory responses in rat DRG.
Asunto(s)
Ganglios Espinales/inmunología , Compuestos de Metilmercurio/toxicidad , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Administración Oral , Animales , Ganglios Espinales/metabolismo , Inflamación/genética , Masculino , Compuestos de Metilmercurio/administración & dosificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas WistarRESUMEN
Metallothionein (MT) is a low-molecular-weight, cysteine-rich, and metal-binding protein that protects cells from the cytotoxic effects of heavy metals and reactive oxygen species. Previously, we found that transcriptional induction of endothelial MT-1A was mediated by not only the metal-regulatory transcription factor 1 (MTF-1)-metal responsive element (MRE) pathway but also the nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile responsive element (ARE) pathway, whereas that of MT-2A was mediated only by the MTF-1-MRE pathway, using the organopnictogen compounds tris(pentafluorophenyl)stibane, tris(pentafluorophenyl)arsane, and tris(pentafluorophenyl)phosphane as molecular probes in vascular endothelial cells. In the present study, we investigated the binding sites of MTF-1 and Nrf2 in the promoter regions of MTs in cultured bovine aortic endothelial cells treated with these organopnictogen compounds. We propose potential mechanisms underlying transcriptional induction of endothelial MT isoforms. Specifically, both MRE activation by MTF-1 and that of ARE in the promoter region of the MT-2A gene by Nrf2 are involved in transcriptional induction of MT-1A, whereas only MRE activation by MTF-1 or other transcriptional factor(s) is required for transcriptional induction of MT-2A in vascular endothelial cells.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Metalotioneína/genética , Fosfinas/toxicidad , Animales , Aorta/citología , Bovinos , Células Cultivadas , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Factor 2 Relacionado con NF-E2/genética , Isoformas de Proteínas/genética , Factores de Transcripción/genética , Transcripción Genética , Factor de Transcripción MTF-1RESUMEN
Exposure to organic mercury, especially methylmercury (MeHg), causes Minamata disease, a severe chronic neurological disorder. Minamata disease predominantly affects the central nervous system, and therefore, studies on the mechanisms of MeHg neurotoxicity have focused primarily on the brain. Although the peripheral nervous system is also affected by the organometallic compound and shows signs of neural degeneration, the mechanisms of peripheral MeHg neurotoxicity remain unclear. In the present study, we performed quantitative immunohistochemical analyses of the dorsal root ganglion (DRG) and associated sensory and motor fibers to clarify the mechanisms of MeHg-induced peripheral neurotoxicity in Wistar rats. Methylmercury chloride (6.7 mg/kg/day) was orally administrated for 5 days, followed by 2 days without administration, and this cycle was repeated once again. Seven and 14 days after the beginning of MeHg exposure, rats were anesthetized, and their DRGs and sensory and motor nerve fibers were removed and processed for immunohistochemical analyses. The frozen sections were immunostained for neuronal, Schwann cell, microglial and macrophage markers. DRG sensory neuron somata and axons showed significant degeneration on day 14. At the same time, an accumulation of microglia and the infiltration of macrophages were observed in the DRGs and sensory nerve fibers. In addition, MeHg caused significant Schwann cell proliferation in the sensory nerve fibers. In comparison, there was no noticeable change in the motor fibers. Our findings suggest that in the peripheral nervous system, MeHg toxicity is associated with neurodegenerative changes to DRG sensory neurons and the induction of a neuroprotective and/or enhancement of neurodegenerative host response.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Macrófagos/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Microglía/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Células de Schwann/efectos de los fármacos , Animales , Proliferación Celular , Masculino , Ratas WistarRESUMEN
Organic-inorganic hybrid molecules, which are composed of organic-ligand(s) and metal(s), are indispensable as synthetic reagents in chemistry, but they have made very little in the way of contributions to biological research. Previously, we reported that the cytotoxicity of organic-inorganic hybrid molecules in vascular endothelial cells depends on interactions between the intramolecular metal and ligand, but remains independent of the hydrophobicity of the intramolecular metal(s). Herein, we show a synergistic cytotoxicity produced by forming a complex of copper and 2,9-dimethyl-1,10-phenanthroline in vascular endothelial cells that depends on the intracellular accumulation of copper.
Asunto(s)
Complejos de Coordinación/toxicidad , Cobre/toxicidad , Células Endoteliales/efectos de los fármacos , Ligandos , Compuestos Organometálicos/toxicidad , Fenantrolinas/toxicidad , Animales , Bovinos , Células Cultivadas , Complejos de Coordinación/metabolismo , Cobre/metabolismo , Sinergismo Farmacológico , Células Endoteliales/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Organometálicos/metabolismo , Fenantrolinas/metabolismoRESUMEN
Methylmercury is an environmental pollutant that exhibits neurotoxicity when ingested, primarily in the form of neuropathological lesions that localize along deep sulci and fissures, in addition to edematous and inflammatory changes in patient cerebrums. These conditions been known to give rise to a variety of ailments that have come to be collectively termed Minamata disease. Since prostaglandins I2 and E2 (PGI2 and PGE2) increase vascular permeability and contribute to the progression of inflammatory changes, we hypothesize that methylmercury induces the synthesis of these prostaglandins in brain microvascular endothelial cells and pericytes. To test this theory, human brain microvascular endothelial cells and pericytes were cultured and treated with methylmercury, after which the PGI2 and PGE2 released from endothelial cells and/or pericytes were quantified by enzyme-linked immunosorbent assay while protein and mRNA expressions in endothelial cells were analyzed by western blot analysis and real-time reverse transcription polymerase chain reaction, respectively. Experimental results indicate that methylmercury inhibits the activity of protein tyrosine phosphatase 1B, which in turn activates the epidermal growth factor receptor-p38 mitogen-activated protein kinase pathway that induces cyclooxygenase-2 expression. It was also found that the cyclic adenosine 3',5'-monophosphate pathway, which can be activated by PGI2 and PGE2, is involved in methylmercury-induced cyclooxygenase-2 expression. Since it appears that protein tyrosine phosphatase 1 B serves as a sensor protein for methylmercury in these mechanisms, it is our belief that the results of the present study may provide additional insights into the molecular mechanisms responsible for edematous and inflammatory changes in the cerebrum of patients with Minamata disease.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Células Endoteliales/efectos de los fármacos , Epoprostenol/metabolismo , Compuestos de Metilmercurio/toxicidad , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Encéfalo/citología , Encéfalo/efectos de los fármacos , Línea Celular , Ciclooxigenasa 2/genética , Células Endoteliales/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Intoxicación del Sistema Nervioso por Mercurio/etiología , Intoxicación del Sistema Nervioso por Mercurio/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Recent developments have shown that organic-inorganic hybrid molecules have the potential to provide useful tools for analyzing biological systems. In the case of fibrinolysis, which is the phenomenon whereby fibrin is degraded by plasmin that has been converted from plasminogen via tissue plasminogen activator (t-PA) secreted from vascular endothelial cells, we hypothesized that there may be organic-inorganic hybrid molecules that could be used to analyze the mechanisms by which endothelial fibrinolysis is regulated. In our present study, we found that a copper complex - copper diethyldithiocarbamate (Cu10) - reduces t-PA activity in a conditioned medium of cultured human coronary endothelial cells by inhibiting the t-PA synthesis without changing the synthesis of plasminogen activator inhibitor type 1, which is a t-PA inhibitor. Copper sulfate, the Cu10 ligand, and zinc/iron complexes with the same Cu10 ligand, did not exhibit such biological activity. These results indicate that Cu10 has the potential to provide a useful tool for finding alternative pathways that downregulate endothelial t-PA synthesis.
Asunto(s)
Carbamatos/farmacología , Vasos Coronarios/citología , Células Endoteliales/metabolismo , Activador de Tejido Plasminógeno/biosíntesis , Carbamatos/toxicidad , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Fibrinólisis/efectos de los fármacos , Humanos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.
Asunto(s)
Compuestos de Metilmercurio/química , Sulfuros/química , Animales , Anticuerpos/inmunología , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , GMP Cíclico/análogos & derivados , GMP Cíclico/química , GMP Cíclico/inmunología , GMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunohistoquímica , Masculino , Compuestos de Metilmercurio/análisis , Compuestos de Metilmercurio/toxicidad , Microscopía Fluorescente , Naftoquinonas/química , Naftoquinonas/toxicidad , Óxido Nítrico/análisis , Oxidación-Reducción , Células PC12 , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Sulfuros/farmacología , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
In a cerebrum damaged by methylmercury, where neuropathological lesions tend to localize along deep sulci and fissures, edematous changes in white matter have been proposed as the cause of such localization. Since hyaluronan has a high water-retention capability and can contribute to the progression of edematous changes, we hypothesize that methylmercury increases hyaluronan in brain microvascular cells. Our experimental results indicate that methylmercury induces the expression of hyaluronan in cultured human microvascular endothelial cells and pericytes through the induction of expressed UDP-glucose dehydrogenase and hyaluronan synthase 2, respectively. After exposure to methylmercury, hyaluronan largely accumulates in perivascular space, where it contributes to the progression of edematous changes.
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Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Ácido Hialurónico/biosíntesis , Compuestos de Metilmercurio/toxicidad , Pericitos/metabolismo , Células Cultivadas , Edema , Células Endoteliales/enzimología , Células Endoteliales/patología , Inducción Enzimática/efectos de los fármacos , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Pericitos/enzimología , Pericitos/patología , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
Organic-inorganic hybrid molecules constitute analytical tools used in biological systems. Vascular endothelial cells synthesize and secrete proteoglycans, which are macromolecules consisting of a core protein and glycosaminoglycan side chains. Although the expression of endothelial proteoglycans is regulated by several cytokines/growth factors, there may be alternative pathways for proteoglycan synthesis aside from downstream pathways activated by these cytokines/growth factors. Here, we investigated organic-inorganic hybrid molecules to determine a variant capable of analyzing the expression of syndecan-4, a transmembrane heparan-sulfate proteoglycan, and identified 1,10-phenanthroline (o-Phen) with or without zinc (Zn-Phen) or rhodium (Rh-Phen). Bovine aortic endothelial cells in culture were treated with these compounds, and the expression of syndecan-4 mRNA and core proteins was determined by real-time reverse transcription polymerase chain reaction and Western blot analysis, respectively. Our findings indicated that o-Phen and Zn-Phen specifically and strongly induced syndecan-4 expression in cultured vascular endothelial cells through activation of the hypoxia-inducible factor-1α/ß pathway via inhibition of prolyl hydroxylase-domain-containing protein 2. These results demonstrated an alternative pathway involved in mediating induction of endothelial syndecan-4 expression and revealed organic-inorganic hybrid molecules as effective tools for analyzing biological systems.
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Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fenantrolinas/farmacología , Sindecano-4/biosíntesis , Animales , Aorta , Bovinos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Fenantrolinas/química , Proteoglicanos/biosíntesis , Transducción de Señal/efectos de los fármacosRESUMEN
Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. A small leucine-rich dermatan sulfate proteoglycan, biglycan, is one of the predominant types of proteoglycans synthesized by vascular endothelial cells; however, the physiological functions of biglycan are not completely understood. In the present study, bovine aortic endothelial cells in culture were transfected with small interfering RNAs for biglycan, and the expression of other proteoglycans was examined. Transforming growth factor-ß1 signaling was also investigated, because the interaction of biglycan with cytokines has been reported. Biglycan was found to form a complex with either transforming growth factor-ß1 or the transforming growth factor-ß1 type I receptor, ALK5, and to intensify the phosphorylation of Smad2/3, resulting in a lower expression of the transmembrane heparan sulfate proteoglycan, syndecan-4. This is the first report to clarify the function of biglycan as a regulatory molecule of the ALK5-Smad2/3 TGF-ß1 signaling pathway that mediates the suppression of syndecan-4 expression in vascular endothelial cells. J. Cell. Biochem. 118: 1087-1096, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Biglicano/genética , Regulación hacia Abajo , Células Endoteliales/citología , Sindecano-4/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Bovinos , Células Cultivadas , Células Endoteliales/metabolismo , Silenciador del Gen , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismoRESUMEN
Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor-ß1 (TGF-ß1 ) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine-rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF-ß1 first upregulates and then downregulates the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, via the TGF-ß receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF-ß1 . Involvement of the downstream signaling pathways of ALK5-the Smad and MAPK pathways-in syndecan-4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3-p38 MAPK pathway mediates the early upregulation of syndecan-4 by TGF-ß1 , whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF-ß1 . J. Cell. Biochem. 118: 2009-2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Proteína Smad2/genética , Proteína smad3/genética , Sindecano-4/genética , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Bovinos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad2/antagonistas & inhibidores , Proteína Smad2/metabolismo , Proteína smad3/antagonistas & inhibidores , Proteína smad3/metabolismo , Sindecano-4/antagonistas & inhibidores , Sindecano-4/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1-MRE and Nrf2-ARE pathways, whereas MT-2A expression requires only activation of the MTF-1-MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism.