RESUMEN
A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Fenómenos Electrofisiológicos , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Intoxicación por MPTP , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genéticaRESUMEN
Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the promoter region of mu-opioid receptor (MOR) to regulate its transcriptional activity. How hnRNP K contributes to the analgesic effects of morphine, however, is largely unknown. We provide evidence that morphine increases hnRNP K protein expression via MOR activation in rat primary cortical neurons and HEK-293 cells expressing MORs, without increasing mRNA levels. Using the bicistronic reporter assay, we examined whether morphine-mediated accumulation of hnRNP K resulted from translational control. We identified potential internal ribosome entry site elements located in the 5' untranslated regions of hnRNP K transcripts that were regulated by morphine. This finding suggests that internal translation contributes to the morphine-induced accumulation of hnRNP K protein in regions of the central nervous system correlated with nociceptive and antinociceptive modulatory systems in mice. Finally, we found that down-regulation of hnRNP K mediated by siRNA attenuated morphine-induced hyperpolarization of membrane potential in AtT20 cells. Silencing hnRNP K expression in the spinal cord increased nociceptive sensitivity in wild-type mice, but not in MOR-knockout mice. Thus, our findings identify the role of translational control of hnRNP K in morphine-induced analgesia through activation of MOR.
Asunto(s)
Regiones no Traducidas 5'/efectos de los fármacos , Analgésicos Opioides/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/biosíntesis , Morfina/farmacología , Neuronas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Receptores Opioides mu/metabolismo , Animales , Secuencia de Bases , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Secuencia Conservada , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Ratones , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Neuronas/efectos de los fármacos , Nocicepción , Ratas , Ribosomas/metabolismo , Transducción de Señal , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Regulación hacia ArribaRESUMEN
Morphinan antagonists, which block opioid effects at mu-opioid receptors, have been studied for their analgesic potential. Previous studies have suggested that these antagonists elicit analgesia with fewer adverse effects in the presence of the mutant mu-opioid receptor (MOR; S196A). However, introducing a mutant receptor for medical applications represents significant challenges. We hypothesize that binding a chemical compound to the MOR may elicit a comparable effect to the S196A mutation. Through high-throughput screening and structure-activity relationship studies, we identified a modulator, 4-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-3-methylbenzoic acid (BPRMU191), which confers agonistic properties to small-molecule morphinan antagonists, which induce G protein-dependent MOR activation. Co-application of BPRMU191 and morphinan antagonists resulted in MOR-dependent analgesia with diminished side effects, including gastrointestinal dysfunction, antinociceptive tolerance, and physical and psychological dependence. Combining BPRMU191 and morphinan antagonists could serve as a potential therapeutic strategy for severe pain with reduced adverse effects and provide an avenue for studying G protein-coupled receptor modulation.
RESUMEN
Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3' untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Gastrinas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Línea Celular Tumoral , Proteínas de Unión al ADN , Gastrinas/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estabilidad del ARN/genética , ARN Mensajero/genética , Transducción de Señal , NucleolinaRESUMEN
In a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. Here, we found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Drosophila/metabolismo , Demencia Frontotemporal/metabolismo , Neuronas Motoras/metabolismo , Poro Nuclear/metabolismo , Receptores sigma/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Citosol/metabolismo , Modelos Animales de Enfermedad , Drosophila/genética , Drosophila/fisiología , Demencia Frontotemporal/genética , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Poro Nuclear/genética , Unión Proteica , ARN Interferente Pequeño , Receptores sigma/genética , Proteína de Unión al GTP ran/genética , Receptor Sigma-1RESUMEN
Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Our objective was to discover a novel enhancer of ligand-induced MOR endocytosis and determine its effects on analgesia, tolerance and dependence. We used high-throughput screening to identify convallatoxin as an enhancer of ligand-induced MOR endocytosis with high potency and efficacy. Treatment of cells with convallatoxin enhanced morphine-induced MOR endocytosis through an adaptor protein 2 (AP2)/clathrin-dependent mechanism, attenuated morphine-induced phosphorylation of MOR, and diminished desensitization of membrane hyperpolarization. Furthermore, co-treatment with chronic convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance and dependence in morphine-tolerant mice. These findings suggest convallatoxin are potentially therapeutic for morphine side effects and open a new avenue to study MOR trafficking.
Asunto(s)
Analgésicos/farmacología , Morfina/farmacología , Receptores Opioides mu/genética , Estrofantinas/farmacología , Analgesia/métodos , Analgésicos/química , Animales , Modelos Animales de Enfermedad , Endocitosis/efectos de los fármacos , Humanos , Ligandos , Ratones , Receptores Opioides mu/efectos de los fármacosRESUMEN
The accumulation of reactive oxygen species (ROS) commonly occurs during normal aging and during some acute/chronic progressive disorders. In order to avoid oxidative damage, scavenging of these radicals is important. Previously, we identified zinc finger protein 179 (Znf179) as a neuroprotector that increases antioxidant enzymes against superoxide radicals. However, the molecular mechanisms involved in the activation and regulation of Znf179 remain unresolved. Here, by performing sequence alignment, bioinformatics analysis, immunoprecipitation using two specific acetyl-lysine antibodies, and treatment with the histone deacetylase (HDAC) inhibitor SAHA, we determined the lysine-specific acetylation of Znf179. Furthermore, we investigated Znf179 interaction with HDACs and revealed that peroxide insult induced a dissociation of Znf179-HDAC1/HDAC6, causing an increase in Znf179 acetylation. Importantly, HDAC inhibition by SAHA further prompted Znf179 hyperacetylation, which promoted Znf179 to form a transcriptional complex with Sp1 and increased antioxidant gene expression against oxidative attack. In summary, the results obtained in this study showed that Znf179 was regulated by HDACs and that Znf179 acetylation was a critical mechanism in the induction of antioxidant defense systems. Additionally, HDAC inhibitors may have therapeutic potential for induction of Znf179 acetylation, strengthening the Znf179 protective functions against neurodegenerative processes.
Asunto(s)
Acetilación/efectos de los fármacos , Antioxidantes/farmacología , Proteínas de Unión al ADN/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Estrés Oxidativo/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/química , Humanos , Ratones , Alineación de SecuenciaRESUMEN
The canonical Wnt/ß-catenin pathway is constitutively activated in more than 90% of colorectal cancer (CRC) cases in which ß-catenin contributes to CRC cell growth and survival. In contrast to the Wnt/ß-catenin pathway, the non-canonical Wnt pathway can antagonize functions of the canonical Wnt/ß-catenin pathway. Wnt5a is a key factor in the non-canonical Wnt pathway, and it plays diverse roles in different types of cancers. It was shown that reintroducing Wnt5a into CRC cells resulted in inhibited cell proliferation and impaired cell motility. However, contradictory results were reported describing increased Wnt5a expression being associated with a poor prognosis of CRC patients. Recently, it was shown that the diverse roles of Wnt5a are due to two distinct roles of Wnt5a isoforms. However, the exact roles and functions of the Wnt5a isoforms in CRC remain largely unclear. The present study for the first time showed the ambiguous role of Wnt5a in CRC was due to the encoding of distinct roles of the various Wnt5a mRNA isoforms. A relatively high expression level of the Wnt5a-short (S) isoform transcript and a low expression level of the Wnt5a-long (L) isoform transcript were detected in CRC cell lines and specimens. In addition, high expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with tumor depth of CRC patients. Furthermore, knockdown of the endogenous expression of the Wnt5a-S mRNA isoform in HCT116 cells drastically inhibited their growth ability by inducing apoptosis through induction of FASLG expression and reduction of TNFRSF11B expression. Moreover, reactivation of methylation inactivation of the Wnt5a-L mRNA isoform by treatment with 5-azacytidine (5-Aza) enhanced the siWnt5a-S isoform's ability to induce apoptosis. Finally, we showed that the simultaneous reactivation of Wnt5a-L mRNA isoform and knockdown of Wnt5a-S mRNA isoform expression enhanced siWnt5a-S isoform-induced apoptosis and siWnt5a-L isoform-regulated suppression of ß-catenin expression in vitro. High expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with high mRNA levels of ß-catenin detection in vivo. Altogether, our study showed that, for the first time, different Wnt5a mRNA isoforms play distinct roles in CRC and can be used as novel prognostic markers for CRC in the future.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Proteína Wnt-5a/genética , beta Catenina/genética , Apoptosis/efectos de los fármacos , Azacitidina/administración & dosificación , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Isoformas de Proteínas/genética , ARN Mensajero/genética , Vía de Señalización Wnt/genéticaRESUMEN
After sudden traumatic brain injuries, secondary injuries may occur during the following days or weeks, which leads to the accumulation of reactive oxygen species (ROS). Since ROS exacerbate brain damage, it is important to protect neurons against their activity. Zinc finger protein 179 (Znf179) was shown to act as a neuroprotective factor, but the regulation of gene expression under oxidative stress remains unknown. In this study, we demonstrated an increase in Znf179 protein levels in both in vitro model of hydrogen peroxide (H2O2)-induced ROS accumulation and animal models of traumatic brain injury. Additionally, we examined the sub-cellular localization of Znf179, and demonstrated that oxidative stress increases Znf179 nuclear shuttling and its interaction with specificity protein 1 (Sp1). Subsequently, the positive autoregulation of Znf179 expression, which is Sp1-dependent, was further demonstrated using luciferase reporter assay and green fluorescent protein (GFP)-Znf179-expressing cells and transgenic mice. The upregulation of Sp1 transcriptional activity induced by the treatment with nerve growth factor (NGF) led to an increase in Znf179 levels, which further protected cells against H2O2-induced damage. However, Sp1 inhibitor, mithramycin A, was shown to inhibit NGF effects, leading to a decrease in Znf179 expression and lower cellular protection. In conclusion, the results obtained in this study show that Znf179 autoregulation through Sp1-dependent mechanism plays an important role in neuroprotection, and NGF-induced Sp1 signaling may help attenuate more extensive (ROS-induced) damage following brain injury.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Estrés Oxidativo/genética , Factor de Transcripción Sp1/genética , Animales , Apoptosis/genética , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Proteínas de Unión al ADN/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Plicamicina/análogos & derivados , Plicamicina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/antagonistas & inhibidores , Activación Transcripcional/genéticaRESUMEN
The accumulation of reactive oxygen species (ROS) have implicated the pathogenesis of several human diseases including neurodegenerative disorders, stroke, and traumatic brain injury, hence protecting neurons against ROS is very important. In this study, we focused on sigma-1 receptor (Sig-1R), a chaperone at endoplasmic reticulum, and investigated its protective functions. Using hydrogen peroxide (H2O2)-induced ROS accumulation model, we verified that apoptosis-signaling pathways were elicited by H2O2 treatment. However, the Sig-1R agonists, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), reduced the activation of apoptotic pathways significantly. By performing protein-protein interaction assays and shRNA knockdown of Sig-1R, we identified the brain Zinc finger protein 179 (Znf179) as a downstream target of Sig-1R regulation. The neuroprotective effect of Znf179 overexpression was similar to that of DHEAS treatment, and likely mediated by affecting the levels of antioxidant enzymes. We also quantified the levels of peroxiredoxin 3 (Prx3) and superoxide dismutase 2 (SOD2) in the hippocampi of wild-type and Znf179 knockout mice, and found both enzymes to be reduced in the knockout versus the wild-type mice. In summary, these results reveal that Znf179 plays a novel role in neuroprotection, and Sig-1R agonists may be therapeutic candidates to prevent ROS-induced damage in neurodegenerative and neurotraumatic diseases.
Asunto(s)
Apoptosis , Encéfalo/metabolismo , Proteínas de Unión al ADN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores sigma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Deshidroepiandrosterona/administración & dosificación , Sulfato de Deshidroepiandrosterona/administración & dosificación , Técnicas de Silenciamiento del Gen , Peróxido de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/administración & dosificación , Receptores sigma/agonistas , Receptores sigma/genética , Receptor Sigma-1RESUMEN
Reduction in host-activated protein C levels and resultant microvascular thrombosis highlight the important functional role of protein C anticoagulant system in the pathogenesis of sepsis and septic shock. Thrombomodulin (TM) is a critical factor to activate protein C in mediating the anticoagulation and anti-inflammation effects. However, TM protein content is decreased in inflammation and sepsis, and the mechanism is still not well defined. In this report, we identified that the TM 5' untranslated region (UTR) bearing the internal ribosome entry site (IRES) element controls TM protein expression. Using RNA probe pulldown assay, HuR was demonstrated to interact with the TM 5'UTR. Overexpression of HuR protein inhibited the activity of TM IRES, whereas on the other hand, reducing the HuR protein level reversed this effect. When cells were treated with IL-1beta, the IRES activity was suppressed and accompanied by an increased interaction between HuR and TM 5'UTR. In the animal model of sepsis, we found the TM protein expression level to be decreased while concurrently observing the increased interaction between HuR and TM mRNA in liver tissue. In summary, HuR plays an important role in suppression of TM protein synthesis in IL-1beta treatment and sepsis.