RESUMEN
Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (â¼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.
Asunto(s)
Aciltransferasas/genética , Neoplasias de las Glándulas Suprarrenales/genética , Mutación de Línea Germinal , Paraganglioma/genética , Feocromocitoma/genética , Adulto , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinogénesis , Dominio Catalítico , Ciclo del Ácido Cítrico , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO(·)) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ET(B)), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species generation and decreasing NO(·) levels. We hypothesized that aldosterone modulates PAH by disrupting ET(B)-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. METHODS AND RESULTS: In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO(·) metabolites in the absence of left-sided heart failure. In human pulmonary artery endothelial cells, endothelin-1 increased aldosterone levels via peroxisome proliferator-activated receptor gamma coactivator-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased reactive oxygen species production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ET(B) to decrease endothelin-1-stimulated eNOS activity. Substitution of ET(B)-Cys405 with alanine improved ET(B)-dependent NO(·) synthesis under conditions of oxidant stress, confirming that Cys405 is a redox-sensitive thiol that is necessary for ET(B)-eNOS signaling. In human pulmonary artery endothelial cells, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated reactive oxygen species generation and restored ET(B)-dependent NO(·) production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in 2 animal models of PAH in vivo. CONCLUSIONS: Our findings demonstrate that aldosterone modulates an ET(B) cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO(·) and promote PAH.
Asunto(s)
Aldosterona/metabolismo , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptor de Endotelina B/metabolismo , Animales , Células Cultivadas , Cisteína/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelina-1/metabolismo , Endotelina-1/farmacología , Hipertensión Pulmonar Primaria Familiar , Humanos , Hipertensión Pulmonar/patología , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacología , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Arteria Pulmonar/citología , Presión Esfenoidal Pulmonar/efectos de los fármacos , Presión Esfenoidal Pulmonar/fisiología , Ratas , Ratas Sprague-Dawley , Espironolactona/farmacología , Compuestos de Sulfhidrilo/metabolismoRESUMEN
BACKGROUND: Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk. METHODS AND RESULTS: We recently developed a genetic mouse model to assess platelet function and thrombosis in the setting of GPx-3 deficiency. The GPx-3((-/-)) mice showed an attenuated bleeding time and an enhanced aggregation response to the agonist ADP compared with wild-type mice. GPx-3((-/-)) mice displayed increased plasma levels of soluble P-selectin and decreased plasma cyclic cGMP compared with wild-type mice. ADP infusion-induced platelet aggregation in the pulmonary vasculature produced a more robust platelet activation response in the GPx-3((-/-)) than wild-type mice; histological sections from the pulmonary vasculature of GPx-3((-/-)) compared with wild-type mice showed increased platelet-rich thrombi and a higher percentage of occluded vessels. Cremaster muscle preparations revealed endothelial dysfunction in the GPx-3((-/-)) compared with wild-type mice. With a no-flow ischemia-reperfusion stroke model, GPx-3((-/-)) mice had significantly larger cerebral infarctions compared with wild-type mice and platelet-dependent strokes. To assess the neuroprotective role of antioxidants in this model, we found that manganese(III) meso-tetrakis(4-benzoic acid)porphyrin treatment reduced stroke size in GPx-3((-/-)) mice compared with vehicle-treated controls. CONCLUSIONS: These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.
Asunto(s)
Plaquetas/fisiología , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Trombosis/metabolismo , Adenosina Difosfato/farmacología , Animales , Antioxidantes/farmacología , Tiempo de Sangría , Plaquetas/efectos de los fármacos , GMP Cíclico/sangre , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Genotipo , Glutatión/sangre , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ratones , Ratones Noqueados , Selectina-P/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Trombosis/tratamiento farmacológico , Trombosis/epidemiologíaRESUMEN
CD14 contributes to LPS signaling in leukocytes through formation of toll-like receptor 4/CD14 receptor complexes; however, a specific role for endogenous cell-surface CD14 in endothelial cells is unclear. We have found that suppression of glutathione peroxidase-1 (GPx-1) in human microvascular endothelial cells increases CD14 gene expression compared to untreated or siControl (siCtrl)-treated conditions. Following LPS treatment, GPx-1 deficiency augmented LPS-induced intracellular reactive oxygen species accumulation, CD14 expression, and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression compared to LPS-treated control cells. GPx-1 deficiency also transiently augmented LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression. Adenoviral overexpression of GPx-1 significantly diminished LPS-mediated responses in adhesion molecule expression. Consistent with these findings, LPS responses were also greater in endothelial cells derived from GPx-1-knockout mice, whereas adhesion molecule expression was decreased in cells from GPx-1-overexpressing transgenic mice. Knockdown of CD14 attenuated LPS-mediated up-regulation of ICAM-1 and VCAM-1 mRNA and protein, and it mitigated the effects of GPx-1 deficiency on LPS-induced adhesion molecule expression. Taken together, these data suggest that GPx-1 modulates the endothelial cell response to LPS, in part, by altering CD14-mediated effects.
Asunto(s)
Moléculas de Adhesión Celular/genética , Células Endoteliales/metabolismo , Glutatión Peroxidasa/fisiología , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/farmacología , Activación Transcripcional/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Endotelio Vascular/citología , Glutatión Peroxidasa/deficiencia , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/genética , ARN Mensajero/análisis , Especies Reactivas de Oxígeno/metabolismo , Molécula 1 de Adhesión Celular Vascular/análisis , Molécula 1 de Adhesión Celular Vascular/genética , Glutatión Peroxidasa GPX1RESUMEN
Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3(S)-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.
Asunto(s)
Escualeno-Monooxigenasa/química , Escualeno-Monooxigenasa/metabolismo , Animales , Dominio Catalítico , Línea Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Insectos , Conformación Proteica , Dominios Proteicos , Escualeno/metabolismo , Escualeno-Monooxigenasa/antagonistas & inhibidoresRESUMEN
Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Escualeno-Monooxigenasa/antagonistas & inhibidores , Escualeno-Monooxigenasa/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Colesterol/biosíntesis , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.
Asunto(s)
Ácido Aspártico/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Neoplasias/metabolismo , Animales , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Vascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast-like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR). METHODS AND RESULTS: Human coronary artery SMCs (CASMCs) treated with bone morphogenetic protein-2 (BMP-2; 100 ng/mL) demonstrated a 1.7-fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR-30b and miR-30c (miR-30b-c) as miRs that regulate Runx2 expression. Real-time-polymerase chain reaction confirmed that BMP-2 decreased miR-30b and miR-30c expression. A luciferase reporter assay verified that both miR-30b and miR-30c bind to the 3'-untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR-30b-c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR-30b-c by transfection with pre-miR-30b-c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP-2 and lower levels of miR-30b than did noncalcified donor coronary arteries. CONCLUSIONS: BMP-2 downregulates miR-30b and miR-30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR-30b and miR-30c may influence vascular calcification.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , MicroARNs/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Calcificación Vascular/etiología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/metabolismo , Regulación hacia Abajo , Humanos , MicroARNs/efectos de los fármacos , MicroARNs/genética , MicroARNs/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Calcificación Vascular/metabolismoRESUMEN
Repression of mitochondrial respiration represents an evolutionarily ancient cellular adaptation to hypoxia and profoundly influences cell survival and function; however, the underlying molecular mechanisms are incompletely understood. Primarily utilizing pulmonary arterial endothelial cells as a representative hypoxic cell type, we identify the iron-sulfur cluster assembly proteins (ISCU1/2) as direct targets for repression by the hypoxia-induced microRNA-210 (miR-210). ISCU1/2 facilitate the assembly of iron-sulfur clusters, prosthetic groups that are critical for electron transport and mitochondrial oxidation-reduction reactions. Under in vivo conditions of upregulating miR-210 and repressing ISCU1/2, the integrity of iron-sulfur clusters is disrupted. In turn, by repressing ISCU1/2 during hypoxia, miR-210 decreases the activity of prototypical iron-sulfur proteins controlling mitochondrial metabolism, including Complex I and aconitase. Consequently, miR-210 represses mitochondrial respiration and associated downstream functions. These results identify important mechanistic connections among microRNA, iron-sulfur cluster biology, hypoxia, and mitochondrial function, with broad implications for cellular metabolism and adaptation to cellular stress.