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1.
Endocr Regul ; 53(4): 250-262, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734650

RESUMO

OBJECTIVE: The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia. METHODS: The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction. RESULTS: It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells. CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.


Assuntos
Neoplasias Encefálicas/genética , Endorribonucleases/genética , Glioma/genética , Proteínas Serina-Treonina Quinases/genética , Hipóxia Tumoral/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Hipóxia Celular/genética , Linhagem Celular Tumoral , Endorribonucleases/deficiência , Endotelina-1/genética , Enzimas Conversoras de Endotelina/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/metabolismo , Glioma/patologia , Humanos , Hipóxia/genética , Hipóxia/patologia , Proteínas Serina-Treonina Quinases/deficiência , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética
2.
Cancer Lett ; 452: 152-157, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-30926432

RESUMO

The endothelin-1 (ET-1) axis contributes to the pathophysiology of several cancers by promoting tumor development and progression. This peptide is activated from its precursor, big ET-1, by endothelin-converting enzyme-1 (ECE-1). Active ET-1 binds to its cognate G-coupled receptor, ETAR, which transduces the signal to the inside of the cell. ET-1 has a short half-life of about 90 s, so its biological effects are completely dependent on its enzymatic activation by ECE-1. Expression of ECE-1 is elevated in several tumors and cancer cell lines. There are four ECE-1 isoforms -ECE-1a, -1b, -1c, and -1d- which vary in terms of their subcellular localization and, in some cases, their effects on cancer-related properties such as proliferation and invasiveness. In this article, we review findings on the role of ECE-1, particularly isoform ECE-1c, in oncogenesis and malignant progression. We also review evidence regarding ECE-1 expression in several types of tumors and cancer cell lines. Recent findings from our laboratory and others allow us to speculate on the mechanism by which ECE-1c promotes cancer aggressiveness. Finally, we evaluate potential post-translational modifications of ECE-1c, highlighting phosphorylation by several kinases, as well as evidence pointing to a putative, non-canonical, ET-1-independent mechanism for promoting invasiveness. Taken together, current evidence suggests that ECE-1c contributes to cancer aggressiveness and plays a putative role as a key regulator of cancer progression. Therefore, we propose that this protein is a promising target for prognostic and therapeutic purposes.


Assuntos
Enzimas Conversoras de Endotelina/metabolismo , Neoplasias/patologia , Biomarcadores Tumorais/genética , Domínio Catalítico/genética , Humanos , Invasividade Neoplásica/patologia , Neoplasias/genética , Fosforilação , Prognóstico , Isoformas de Proteínas/metabolismo
3.
Neurochem Res ; 44(6): 1289-1296, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30806879

RESUMO

The accumulation of amyloid beta (Aß) in the brain is believed to play a central role in the development and progression of Alzheimer's disease. Revisions to the amyloid cascade hypothesis now acknowledge the dynamic equilibrium in which Aß exists and the importance of enzymes involved in the production and breakdown of Aß in maintaining healthy Aß levels. However, while a wealth of pharmacological and immunological therapies are being generated to inhibit the Aß-producing enzymes, ß-site APP cleavage enzyme 1 and γ-secretase, the therapeutic potential of stimulating Aß-degrading enzymes such as neprilysin, endothelin-converting enzyme-1 and insulin-degrading enzyme remains relatively unexplored. Recent evidence indicates that increasing Aß degradation as opposed to inhibiting synthesis is a more effective strategy to prevent Aß build-up. Therefore Aß degrading enzymes have become valuable targets of therapy. In this review, we discuss the pathway of Aß synthesis and clearance along with the opportunities they present for therapeutic intervention, the benefits of increasing the expression/activity of Aß-degrading enzymes, and the untapped therapeutic potential of enzyme activation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Ativadores de Enzimas/farmacologia , Insulisina/metabolismo , Neprilisina/metabolismo , Proteólise/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/química , Animais , Terapia Genética , Humanos
4.
J Agric Food Chem ; 67(4): 1115-1126, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30648392

RESUMO

The interest in using food byproducts as a source of bioactive peptides has increased significantly in the recent years. The goal of this work was to determine the presence and stability of peptides showing angiotensin I-converting enzyme (ACE-I), endothelin-converting enzyme (ECE), dipeptidyl peptidase-IV (DPP-IV), and platelet-activating factor-acetylhydrolase (PAF-AH) inhibitory activity derived from dry-cured ham bones, which could exert cardiovascular health benefits. ACE-I and DPP-IV inhibitory peptides were stable against heating typically used in Mediterranean household cooking methods and also to in vitro digestion. PAF-AH inhibitory activity significantly increased following simulated gastrointestinal digestion whereas ECE inhibitory significantly decreased ( P < 0.05). The mass spectrometry analysis revealed a notable degradation of hemoglobin-derived peptides after simulated digestion, and the release of a large number of dipeptides that may have contributed to the observed bioactivities. These results suggest that natural peptides from Spanish dry-cured ham bones could contribute to a positive impact on cardiovascular health.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Cardiotônicos/química , Inibidores da Dipeptidil Peptidase IV/química , Produtos da Carne/análise , Peptídeos/química , Resíduos/análise , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Animais , Osso e Ossos/química , Cardiotônicos/isolamento & purificação , Culinária , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/isolamento & purificação , Enzimas Conversoras de Endotelina/química , Cinética , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/isolamento & purificação , Peptidil Dipeptidase A/química , Suínos , Espectrometria de Massas em Tandem
5.
Biochem J ; 476(3): 513-533, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626614

RESUMO

Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) is secreted from intestinal L-cells and mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic ß-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving ß-cell mass. These effects are mediated by the GLP-1 receptor (GLP-1R), which is a therapeutic target in type 2 diabetes. Although agonism at the GLP-1R has been well studied, desensitisation and resensitisation are perhaps less well explored. An understanding of these events is important, particularly in the design and use of novel receptor ligands. Here, using either HEK293 cells expressing the recombinant human GLP-1R or the pancreatic ß-cell line, INS-1E with endogenous expressesion of the GLP-1R, we demonstrate GLP-1R desensitisation and subsequent resensitisation following removal of extracellular GLP-1 7-36 amide. Resensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Resensitisation is also regulated by endothelin-converting enzyme-1 (ECE-1) activity, most likely through proteolysis of GLP-1 in endosomes and the facilitation of GLP-1R dephosphorylation and recycling. Inhibition of ECE-1 activity also increases GLP-1-induced activation of extracellular signal-regulated kinase and generation of cAMP, suggesting processes dependent upon the lifetime of the internalised ligand-receptor complex.


Assuntos
Endossomos/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Proteólise , Transdução de Sinais , AMP Cíclico/genética , AMP Cíclico/metabolismo , Endossomos/genética , Enzimas Conversoras de Endotelina/genética , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células HEK293 , Humanos , Fragmentos de Peptídeos/farmacologia , Transporte Proteico
6.
FASEB J ; 33(3): 3758-3771, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30481490

RESUMO

Accumulating evidence suggests that the abnormal aggregation of amyloid-ß (Αß) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aß is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aß. In this study, we determined whether disruption of Aß catabolism could trigger Aß aggregation within neurons and increase the amount of Aß associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aß levels and promoted the intracellular formation of Aß oligomers, a process that did not require internalization of secreted Aß. In vivo, the accumulation of intraneuronal Aß aggregates was accompanied by increased levels of both extracellular and exosome-associated Aß, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of Aß degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aß aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Exossomos/metabolismo , Metaloendopeptidases/metabolismo , Agregação Patológica de Proteínas/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Endossomos/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Espaço Extracelular/metabolismo , Feminino , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Corpos Multivesiculares/metabolismo , Neurônios/metabolismo , Proteólise
7.
Oral Dis ; 25(1): 44-53, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29978582

RESUMO

BACKGROUND: Endothelin-converting enzyme-1 (ECE-1) primarily converts big endothelins (ETs) into active endothelin-1 (ET-1). However, the expression pattern and prognostication status of ECE-1 in head and neck cancer (HNC) are enigmatic. In this study, we investigated ECE-1 expression and assessed the roles of ECE-1 as a predictor for HNC differentiation and prognosis. MATERIALS AND METHODS: ECE-1 expressions were evaluated by immunohistochemical analysis using a tissue microarray (TMA) composed of 100 cases of head and neck squamous cell carcinoma. The correlation of ECE-1 expression with clinicopathologic variables and patient outcomes was analyzed. RESULTS: ECE-1 may be overexpressed in HNC carcinoma cells. Higher ECE-1 level was detected more frequently in moderately to poorly differentiated tumors and showed a lower differentiation category compared to the G1 cases (p = 0.015); this finding was further confirmed by an adjusted odds ratio (OR) of 4.071 (p = 0.042). Moreover, Kaplan-Meier survival analyses showed that a higher ECE-1 expression was associated with a poorer survival in patients with HNC (p < 0.0001). On multivariate Cox proportional hazards models analysis, ECE-1 of high expression proved to be an independent prognostic factor with a hazard ratio (HR) of 3.985 (p < 0.001). CONCLUSION: Our data provide the first evidence that overexpression of ECE-1 in HNC is a predictor of poor tumor differentiation and prognosis.


Assuntos
Enzimas Conversoras de Endotelina/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais
8.
Cell Mol Neurobiol ; 38(6): 1293-1303, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29948551

RESUMO

Ischemia-reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood-spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.


Assuntos
Regulação para Baixo , Enzimas Conversoras de Endotelina/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão/genética , Regulação para Cima , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Neurônios/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Ativação Transcricional/genética
9.
Mol Pharmacol ; 94(1): 674-688, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29724789

RESUMO

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.


Assuntos
Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Enzimas Conversoras de Endotelina/metabolismo , Metabolismo Energético , Células HEK293 , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/fisiologia , Neuropeptídeos/metabolismo , Obesidade/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Neurobiol Dis ; 114: 153-163, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29524599

RESUMO

Parkinson's disease (PD) is a progressive motor neurodegenerative disorder, characterized by a selective loss of dopaminergic neurons in the substantia nigra. The complexity of disease etiology includes both genetic and environmental factors. No effective drug that can modify disease progression and protect dopamine neurons from degeneration is presently available. Human α-Synuclein A30P (A30P) is a mutant gene identified in early onset PD and showed to result selective dopamine neuron loss in transgenic A30P flies and mice. Paraquat (PQ) is an herbicide and an oxidative stress generator, linked to sporadic PD. We hypothesized that vital PD modifier genes are conserved across species and would show unique transcriptional changes to oxidative stress in animals expressing a PD-associated gene, such as A30P. We also hypothesized that manipulation of PD modifier genes would provide neuroprotection across species. To identify disease modifier genes, we performed two independently-duplicated experiments of microarray, capturing genome-wide transcriptional changes in A30P flies, chronically fed with PQ-contaminated food. We hypothesized that the best time point of identifying a disease modifier gene is at time when flies showed maximal combined toxicity of A30P transgene and PQ treatment during an early stage of disease and that effective disease modifiers gene are those showing transcriptional changes to oxidative stress in A30P expressing and not in wild type animals. Fly Neprilysin3 (Nep3) is one identified gene that is highly conserved. Its mouse and human homolog is endothelin-converting enzyme-1 (Ece1). To investigate the neuroprotective effect of Ece1, we used NS1 cells and mouse midbrain neurons expressing A30P, treated with or without PQ. We found that ECE1 expression protected against A30P toxicity on cell viability, on neurite outgrowth and ameliorated A30P accumulation in vitro. Expression of ECE1 in vivo suppressed dopamine neuron loss and alleviated the corresponding motor deficits in mice with A30P-expression. Our study leverages a new approach to identify disease modifier genes using a stress-enhanced PD animal model.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Enzimas Conversoras de Endotelina/biossíntese , Estresse Oxidativo/fisiologia , alfa-Sinucleína/biossíntese , alfa-Sinucleína/toxicidade , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Células Cultivadas , Neurônios Dopaminérgicos/efeitos dos fármacos , Método Duplo-Cego , Drosophila , Enzimas Conversoras de Endotelina/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , alfa-Sinucleína/genética
11.
Acta Physiol (Oxf) ; 223(1): e13033, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29330945

RESUMO

AIM: Cyclosporine A (CsA) induces renal vasoconstriction and hypoxia and enhances the expression of endothelin-1 (ET-1) pro-hormone (pre-pro-ET-1), plausibly leading to a feed-forward loop of renal vasoconstriction, hypoxia and enhanced synthesis of the potent vasoconstrictor ET-1. Endothelin-converting enzyme (ECE)-1 cleaves big endothelin to generate endothelin (ET)-1 and is upregulated by hypoxia via hypoxia-inducible factor (HIF). We hypothesized that in addition to the direct induction of ET-1 synthesis, CsA might also intensify renal ECE-1 expression, thus contributing to enhanced ET-1 synthesis following CsA. METHODS: CsA was administered to Sprague Dawley rats (120 mg/kg/SC) for 4 days, and renal HIF and ECE-1 expression were assessed with Western blots and immunostaining. Human umbilical vein endothelial cells (HUVEC) and proximal tubular cell line (HK-2) were subjected to CsA, and ECE-1 induction was evaluated using real-time mRNA PCR and Western blots. RESULTS: Cyclosporine A intensified renal parenchymal ECE-1 expression in the rat kidney, particularly in distal nephron segments, along with renal hypoxia (detected by pimonidazole adducts) and HIF expression, in line with our recent observations showing episodic hypoxia in mice subjected to CsA. Furthermore, in cultured normoxic HUVEC and HK-2 cells, CsA dose-dependently induced both pre-pro-ET-1 and ECE-1 mRNA and protein expression, with enhanced ET-1 generation. CONCLUSION: CsA induces ECE-1 via both hypoxic and non-hypoxic pathways. ECE-1 may contribute to increased renal ET-1 generation following CsA, participating in a feed-forward loop of renal parenchymal hypoxia and ET synthesis.


Assuntos
Ciclosporina/farmacologia , Enzimas Conversoras de Endotelina/biossíntese , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Rim/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Endotelina-1/genética , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina/sangue , Enzimas Conversoras de Endotelina/genética , Indução Enzimática , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/enzimologia , Masculino , Ratos Sprague-Dawley , Regulação para Cima
12.
Artigo em Inglês | MEDLINE | ID: mdl-28971608

RESUMO

Endothelin-1 (ET-1) and nitric oxide (NO) are two highly potent vasoactive molecules with opposing effects on the vasculature. Endothelin-converting enzyme (ECE) and nitric oxide synthase (NOS) catalyse the production of ET-1 and NO, respectively. It is well established that these molecules play a crucial role in the initiation and progression of cardiovascular diseases and have therefore become targets of therapy. Many studies have examined the mechanism(s) by which NO regulates ET-1 production. Expression and localization of ECE-1 is a key factor that determines the rate of ET-1 production. ECE-1 can either be membrane bound or be released from the cell surface to produce a soluble form. NO has been shown to reduce the expression of both membrane-bound and soluble ECE-1. Several studies have examined the mechanism(s) behind NO-mediated inhibition of ECE expression on the cell membrane. However, the precise mechanism(s) behind NO-mediated inhibition of soluble ECE production are unknown. We hypothesize that both exogenous and endogenous NO, inhibits the production of soluble ECE-1 by preventing its release via extracellular vesicles (e.g., exosomes), and/or by inhibiting the activity of A Disintegrin and Metalloprotease-17 (ADAM17). If this hypothesis is proven correct in future studies, these pathways represent targets for the therapeutic manipulation of soluble ECE-1 production.


Assuntos
Proteína ADAM17/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Óxido Nítrico/farmacologia , Animais , Membrana Celular/metabolismo , Regulação para Baixo , Endotelina-1/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Humanos
13.
CNS Neurosci Ther ; 23(11): 855-865, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28941188

RESUMO

AIMS: Lower androgen level in elderly men is a risk factor of Alzheimer's disease (AD). It has been reported that androgen reduces amyloid peptides (Aß) production and increases Aß degradation by neurons. Activated microglia are involved in AD by either clearing Aß deposits through uptake of Aß or releasing cytotoxic substances and pro-inflammatory cytokines. Here, we investigated the effect of androgen on Aß uptake and clearance and Aß-induced inflammatory response in microglia, on neuronal death induced by Aß-activated microglia, and explored underlying mechanisms. METHODS: Intracellular and extracellular Aß were examined by immunofluorescence staining and Western blot. Amyloid peptides (Aß) receptors, Aß degrading enzymes, and pro-inflammatory cytokines were detected by RT-PCR, real-time PCR, and ELISA. Phosphorylation of MAP kinases and NF-κB was examined by Western blot. RESULTS: We found that physiological concentrations of androgen enhanced Aß42 uptake and clearance, suppressed Aß42 -induced IL-1ß and TNFα expression by murine microglia cell line N9 and primary microglia, and alleviated neuronal death induced by Aß42 -activated microglia. Androgen administration also reduced Aß42 -induced IL-1ß expression and neuronal death in murine hippocampus. Mechanistic studies revealed that androgen promoted microglia to phagocytose and degrade Aß42 through upregulating formyl peptide receptor 2 and endothelin-converting enzyme 1c expression, and inhibited Aß42 -induced pro-inflammatory cytokines expression via suppressing MAPK p38 and NF-κB activation by Aß42 , in an androgen receptor independent manner. CONCLUSION: Our study demonstrates that androgen promotes microglia to phagocytose and clear Aß42 and inhibits Aß42 -induced inflammatory response, which may play an important role in reducing the neurotoxicity of Aß.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Androgênios/farmacologia , Anti-Inflamatórios/farmacologia , Encéfalo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/toxicidade , Peptídeos beta-Amiloides/metabolismo , Androgênios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Di-Hidrotestosterona/metabolismo , Di-Hidrotestosterona/farmacologia , Enzimas Conversoras de Endotelina/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Tumour Biol ; 39(9): 1010428317725922, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28945168

RESUMO

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Enzimas Conversoras de Endotelina/biossíntese , Neoplasias Esofágicas/patologia , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Enzimas Conversoras de Endotelina/análise , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais
15.
Autoimmunity ; 50(4): 223-231, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28557628

RESUMO

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.


Assuntos
Autoantígenos/imunologia , Enzimas Conversoras de Endotelina/imunologia , Poliendocrinopatias Autoimunes/imunologia , Adolescente , Processamento Alternativo , Autoanticorpos/imunologia , Autoantígenos/genética , Autoimunidade , Criança , Enzimas Conversoras de Endotelina/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Imuno-Histoquímica , Masculino , Fenótipo , Hipófise/imunologia , Hipófise/metabolismo , Poliendocrinopatias Autoimunes/diagnóstico , Poliendocrinopatias Autoimunes/genética
16.
Transgenic Res ; 26(3): 411-417, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28409408

RESUMO

Inducible Cre-ERT recombinase technology is widely used for gene targeting studies. The second generation of inducible Cre-ERT recombinase, hemizygous B6.129S-Tg(UBC-cre/ERT2)1Ejb/J (hereafter abbreviated as Cre-ERT2), a fusion of a mutated estrogen receptor and Cre recombinase, was engineered to be more efficient and specific than the original Cre-ERT. The putative mechanism of selective Cre-mediated recombination is Cre sequestration in the cytoplasm in the basal state with translocation to the nucleus only in the presence of tamoxifen. We utilized both a reporter mouse (B6.129 (Cg)-Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J) and endothelin converting enzyme-1 floxed transgenic mouse line to evaluate Cre-ERT2 activity. We observed spontaneous Cre activity in both settings. Unintended Cre activity is a confounding factor that has a potentially large impact on data interpretation. Thus, it is important to consider background Cre activity in experimental design.


Assuntos
Integrases/genética , Integrases/metabolismo , Camundongos Transgênicos , Transgenes , Animais , Enzimas Conversoras de Endotelina/genética , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamoxifeno/farmacologia
17.
Hypertens Res ; 40(8): 738-745, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28298655

RESUMO

Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 µM l-NAME), blockade of ETA- and ETB-receptors (10 µM bosentan) and stimulation of the endothelium with 0.001-10 µM acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. l-NAME (1) increased arterial contractile responses to K+ (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 µM) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of l-NAME during contractions induced by either 10 µM PHE or 40 mM K+ were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.


Assuntos
Enzimas Conversoras de Endotelina/antagonistas & inibidores , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/uso terapêutico , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Neprilisina/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Benzazepinas/uso terapêutico , Bosentana , Endotélio Vascular/efeitos dos fármacos , Hidralazina/uso terapêutico , Losartan/uso terapêutico , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/uso terapêutico , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ratos , Ratos Endogâmicos SHR , Sulfonamidas/uso terapêutico , Resistência Vascular/efeitos dos fármacos
18.
J Neurochem ; 141(2): 275-286, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28171705

RESUMO

We have examined the roles of the endothelin-converting enzyme-1 and -2 (ECE-1 and ECE-2) in the homeostasis of α-synuclein (α-syn) and pathogenesis of Lewy body disease. The ECEs are named for their ability to convert inactive big endothelin to the vasoactive peptide endothelin-1 (EDN1). We have found that ECE-1 and ECE-2 cleave and degrade α-syn in vitro and siRNA-mediated knockdown of ECE-1 and ECE-2 in SH-SY5Y neuroblastoma cells significantly increased α-syn both intracellularly (within the cell lysate) (p < 0.05 for both ECE-1 and -2) and extracellularly (in the surrounding medium) (p < 0.05 for ECE-1 and p = 0.07 for ECE-2). Double immunofluorescent labelling showed co-localization of ECE-1 and ECE-2 with α-syn within the endolysosomal system (confirmed by a proximity ligation assay). To assess the possible relevance of these findings to human Lewy body disease, we measured ECE-1 and ECE-2 levels by sandwich ELISA in post-mortem samples of cingulate cortex (a region with a predilection for Lewy body pathology) in dementia with Lewy bodies (DLB) and age-matched controls. ECE-1 (p < 0.001) and ECE-2 (p < 0.01) levels were significantly reduced in DLB and both enzymes correlated inversely with the severity of Lewy body pathology as indicated by the level of α-syn phosphorylated at Ser129 (r = -0.54, p < 0.01 for ECE-1 and r = -0.49, p < 0.05 for ECE-2). Our novel findings suggest a role for ECEs in the metabolism of α-syn that could contribute to the development and progression of DLB.


Assuntos
Encéfalo/enzimologia , Enzimas Conversoras de Endotelina/metabolismo , Doença por Corpos de Lewy/enzimologia , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Linhagem Celular Tumoral , Enzimas Conversoras de Endotelina/antagonistas & inibidores , Feminino , Humanos , Doença por Corpos de Lewy/patologia , Masculino
19.
Genesis ; 55(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28109039

RESUMO

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediate this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.


Assuntos
Enzimas Conversoras de Endotelina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5/genética , Crista Neural/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Enzimas Conversoras de Endotelina/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos , Crista Neural/embriologia , Faringe/embriologia , Faringe/metabolismo , Regulação para Cima , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
20.
Cardiovasc Res ; 113(2): 207-221, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28025386

RESUMO

AIM: To analyse the ability of TWEAK to modify the endothelin system, particularly endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1), studying the intracellular mechanisms implied. TNF-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily; it has different biological functions such as inflammation, angiogenesis, proliferation, and apoptosis. TWEAK and fibroblast growth-factor-inducible 14 are expressed in different cell types, including endothelial and smooth muscle cells. Despite their presence in endothelial cells, the effect of TWEAK on endothelial function is incompletely defined. METHODS AND RESULTS: In cells, TWEAK induced protein (Western blot) and mRNA (quantitative polymerase chain reaction) expression of ECE-1. Results were related to transcriptional changes, as ECE-1 promoter activity (transfection assays) was also increased. Transfections with serial deletions of ECE-1 promoter suggest a potential role for AP-1 and NFkB, which were confirmed by electrophoretic mobility shift assays. When AP-1 or NFkB activations were inhibited by specific inhibitors of AP-1, PD-98059 (Erk1/2 inhibitor), or SP-600125 (JNK inhibitor), and also with an inhibitor of NFKB and PDTC, TWEAK effect was partially blocked in both cases, suggesting that both transcription factors are implied in ECE-1 regulation. Moreover, the endothelial changes induced by TWEAK were also tested in vivo, using 3-month-old male CD-1 mice treated with TWEAK 10 µg/kg body weight for 24 h, finding similar effects, a rise in ET-1 production (enzyme-linked immunosorbent assay), and ECE-1 expression in aorta and lung tissues. Mice showed slight hypertension after 4 h of treatment, which disappeared at 24 h. CONCLUSIONS: In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina/metabolismo , Fatores de Necrose Tumoral/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/enzimologia , Endotelina-1/genética , Enzimas Conversoras de Endotelina/genética , Humanos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Camundongos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Transcrição Genética , Transfecção , Fatores de Necrose Tumoral/toxicidade , Regulação para Cima
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