RESUMEN
Parkinson disease (PD) is a devastating, largely nonfamilial, age-related disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Release of DA from these neurons into the dorsal striatum is crucial for regulating movement and their loss causes PD. Unfortunately, the mechanisms underlying SNc neurodegeneration remain unclear, and currently there is no cure for PD, only symptomatic treatments. Recently, several regulator of G protein signaling (RGS) proteins have emerged as critical modulators of PD pathogenesis and/or motor dysfunction and dyskinesia: RGSs 4, 6, 9, and 10. Striatal RGS4 has been shown to exacerbate motor symptoms of DA loss by suppressing M4-autoreceptor-Gα i/o signaling in striatal cholinergic interneurons. RGS6 and RGS9 are key regulators of D2R-Gα i/o signaling in SNc DA neurons and striatal medium spiny neurons, respectively. RGS6, expressed in human and mouse SNc DA neurons, suppresses characteristic PD hallmarks in aged mice, including SNc DA neuron loss, motor deficits, and α-synuclein accumulation. After DA depletion, RGS9 (through its inhibition of medium spiny neuron D2R signaling) suppresses motor dysfunction induced by L-DOPA or D2R-selective agonists. RGS10 is highly expressed in microglia, the brain's resident immune cells. Within the SNc, RGS10 may promote DA neuron survival through the upregulation of prosurvival genes and inhibition of microglial inflammatory factor expression. Thus, RGSs 4, 6, 9, and 10 are critical modulators of cell signaling pathways that promote SNc DA neuron survival and/or proper motor control. Accordingly, these RGS proteins represent novel therapeutic targets for the treatment of PD pathology. SIGNIFICANCE STATEMENT: Parkinson disease (PD), the most common movement disorder, is a progressive neurodegenerative disease characterized by substantia nigra pars compacta (SNc) dopamine (DA) neuron loss and subsequent motor deficits. Current PD therapies only target disease motor symptomology and are fraught with side effects. Therefore, researchers have begun to explore alternative therapeutic options. Regulator of G protein signaling (RGS) proteins, whether primarily expressed in SNc DA neurons (RGS6), striatal neurons (RGSs 4 and 9), or microglia (RGS10), modulate key signaling pathways important for SNc DA neuron survival and/or proper motor control. As such, RGS proteins represent novel therapeutic targets in PD.
Asunto(s)
Actividad Motora/fisiología , Degeneración Nerviosa/patología , Enfermedad de Parkinson/patología , Porción Compacta de la Sustancia Negra/patología , Proteínas RGS/metabolismo , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Proteínas de Unión al GTP/metabolismo , Humanos , Ratones , Microglía/metabolismo , Enfermedad de Parkinson/fisiopatología , Porción Compacta de la Sustancia Negra/fisiopatología , Transducción de Señal/fisiologíaRESUMEN
Recent studies of leukemic tumors in individual extramedullary sites showed they adopt the clinical and metastatic behavior of solid cancers originating in those sites. To elucidate features of leukemic tumors that render them resistant to agents effective against marrow leukemia, we analyzed a series of AML breast tumors by histology, immunohistochemistry, and RNA sequencing. Striking histologic similarities to solid cancers were found: a single-filing architectural pattern virtually identical to that of invasive lobular breast carcinoma and dense desmoplastic keloid-like fibrosis similar to colon, gallbladder, and pancreas carcinomas. Sequencing found 2157 genes significantly downregulated in AML breast tumors compared to normal breast. Comparison to triple-negative breast cancer found 859 genes similarly downregulated. At least 30 of these genes have been associated with poor prognosis in breast cancers. Five were reported in AML marrow studies to correlate with poor prognosis. The findings of this pilot study suggest the seed-and-soil interaction recognized in solid cancer growth may help explain how leukemic cells, in some patients, adopt solid tumor behavior in non-marrow sites. Transformed cells that metastasize from tumor to marrow can impart chemoresistance and be an unrecognized cause of treatment failure and death. Further studies comparing leukemic tumor to simultaneous marrow could potentially identify biomarkers that predict extramedullary resistance and lead to new therapeutic targets. Recognizing the potential for leukemia to adopt solid tumor phenotype, and implementation of body scanning and ablative tumor treatment, could decrease the persistently high rates of marrow resistance and treatment failure.
Asunto(s)
Mama/patología , Leucemia Mieloide Aguda/patología , Sarcoma Mieloide/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor , Mama/química , Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Carcinoma/patología , Terapia Combinada , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Especificidad de Órganos , Proyectos Piloto , Pronóstico , ARN Mensajero/análisis , ARN Neoplásico/análisis , Sarcoma Mieloide/tratamiento farmacológico , Sarcoma Mieloide/genética , Sarcoma Mieloide/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
G protein-coupled receptor signaling mechanisms are implicated in many aspects of cardiovascular control, and dysfunction of such signaling mechanisms is commonly associated with disease states. Investigators have identified a large number of regulator of G protein signaling (RGS) proteins that variously contribute to the modulation of intracellular second-messenger signaling kinetics. These many RGS proteins each interact with a specific set of second-messenger cascades and receptor types and exhibit tissue-specific expression patterns. Increasing evidence supports the contribution of RGS proteins, or their loss, in the pathogenesis of cardiovascular dysfunctions. This review summarizes the current understanding of the functional contributions of RGS proteins, particularly within the B/R4 family, in cardiovascular disorders of pregnancy including gestational hypertension, uterine artery dysfunction, and preeclampsia.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares/genética , Polimorfismo de Nucleótido Simple , Proteínas RGS/genética , Transducción de Señal/genética , Animales , Femenino , Humanos , Embarazo , Unión Proteica , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Alcohol is the most commonly abused drug worldwide, and chronic alcohol consumption is a major etiological factor in the development of multiple pathological sequelae, including alcoholic cardiomyopathy and hepatic cirrhosis. Here, we identify regulator of G protein signaling 6 (RGS6) as a critical regulator of both alcohol-seeking behaviors and the associated cardiac and hepatic morbidities through two mechanistically divergent signaling actions. RGS6(-/-) mice consume less alcohol when given free access and are less susceptible to alcohol-induced reward and withdrawal. Antagonism of GABA(B) receptors or dopamine D2 receptors partially reversed the reduction in alcohol consumption in RGS6(-/-) animals. Strikingly, dopamine transporter inhibition completely restored alcohol seeking in mice lacking RGS6. RGS6 deficiency was associated with alterations in the expression of genes controlling dopamine (DA) homeostasis and a reduction in DA levels in the striatum. Taken together, these data implicate RGS6 as an essential regulator of DA bioavailability. RGS6 deficiency also provided dramatic protection against cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia when mice were forced to consume alcohol. Although RGS proteins canonically function as G-protein regulators, RGS6-dependent, alcohol-mediated toxicity in the heart, liver, and gastrointestinal tract involves the ability of RGS6 to promote reactive oxygen species-dependent apoptosis, an action independent of its G-protein regulatory capacity. We propose that inhibition of RGS6 might represent a viable means to reduce alcohol cravings and withdrawal in human patients, while simultaneously protecting the heart and liver from further damage upon relapse.
Asunto(s)
Consumo de Bebidas Alcohólicas , Conducta Animal , Proteínas RGS/fisiología , Recompensa , Animales , Apoptosis/fisiología , Cardiomiopatías/etiología , Condicionamiento Operante , Ratones , Ratones Noqueados , Proteínas RGS/genéticaRESUMEN
Gαi-coupled receptors play important roles in protecting the heart from ischemic injury. Regulator of G protein signaling (RGS) proteins suppress Gαi signaling by accelerating the GTPase activity of Gαi subunits. However, the roles of individual RGS proteins in modulating ischemic injury are unknown. In this study, we investigated the effect of RGS6 deletion on myocardial sensitivity to ischemic injury. Hearts from RGS6 knockout (RGS6-/-) and RGS6 wild-type (RGS6+/+) mice were subjected to 30 minutes of ischemia and 2 hours of reperfusion on a Langendorff heart apparatus. Infarcts in RGS6-/- hearts were significantly larger than infarcts in RGS6+/+ hearts. RGS6-/- hearts also exhibited increased phosphorylation of ß2-adrenergic receptors and G protein-coupled receptor kinase 2 (GRK2). Mitochondrial GRK2 as well as caspase-3 cleavage were increased significantly in RGS6-/- hearts compared with RGS6+/+ hearts after ischemia. Chronic propranolol treatment of mice prevented the observed increases in ischemic injury and the GRK2 phosphorylation observed in RGS6-/- hearts. Our findings suggest that loss of RGS6 predisposes the ventricle to prodeath signaling through a ß2AR-GRK2-dependent signaling mechanism, and they provide evidence for a protective role of RGS6 in the ischemic heart. Individuals expressing genetic polymorphisms that suppress the activity of RGS6 may be at increased risk of cardiac ischemic injury. Furthermore, the development of agents that increase RGS6 expression or activity might provide a novel strategy for the treatment of ischemic heart disease.
Asunto(s)
Caspasa 3/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Mitocondrias Cardíacas/metabolismo , Infarto del Miocardio , Isquemia Miocárdica , Proteínas RGS/metabolismo , Animales , Diseño de Fármacos , Ratones , Ratones Noqueados , Infarto del Miocardio/etiología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Miocardio/patología , Sustancias Protectoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3-/- mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6-/- mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Proteínas RGS/fisiología , Sustancia Negra/patología , Animales , Proteínas de Homeodominio/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT(1A)Rs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT(1A)R-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT(1A)R-expressing cells implicated in mood disorders. RGS6(-/-) mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT(1A)R blockade. Effects of the SSRI fluvoxamine and 5-HT(1A)R agonist 8-OH-DPAT were also potentiated in RGS6(+/-) mice. The phenotype of RGS6(-/-) mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gα(i)-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6(-/-) animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents.
Asunto(s)
Adenilil Ciclasas/metabolismo , Ansiedad/fisiopatología , Depresión/fisiopatología , Proteínas RGS/fisiología , Receptor de Serotonina 5-HT1A/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Animales Recién Nacidos , Ansiedad/genética , Ansiedad/prevención & control , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Depresión/genética , Depresión/prevención & control , Femenino , Fluvoxamina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Proteínas RGS/deficiencia , Proteínas RGS/genética , Serotonina/metabolismo , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). The neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6fl/fl; DAT-iCreER mice to determine the role of RGS6 in DA neurons on EtOH consumption, reward, and relapse behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse ventral tegmental area (VTA), and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6-/- mice. Strikingly, RGS6 loss from DA neurons before or after EtOH behavioral reward is established significantly reduced (~ 50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies identify DA neurons as the locus of RGS6 action in promoting EtOH consumption, preference, reward, and relapse. RGS6 is unique among R7 RGS proteins in promoting rather than suppressing behavioral responses to drugs of abuse and to modulate EtOH behavioral reward. This is a result of RGS6's pre-synaptic actions that we hypothesize promote VTA DA transmission by suppressing GPCR-Gαi/o-DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.
RESUMEN
Breast cancer is a large global health burden and the most frequently diagnosed malignancy in women worldwide. Here, we utilize RGS6(-/-) mice to interrogate the role of regulator of G protein signaling 6 (RGS6), localized to the ductal epithelium in mouse and human breast, as a novel tumor suppressor in vivo. RGS6(-/-) mice exhibit accelerated 7,12-dimethylbenza[α]anthracene (DMBA)-induced tumor initiation and progression, as well as decreased overall survival. Analysis of carcinogenic aberrations in the mammary glands of DMBA-treated mice revealed a failure of the DNA damage response concurrent with augmented oncogenesis in RGS6(-/-) animals. Furthermore, RGS6 suppressed cell growth induced by either human epidermal growth factor receptor 2 or estrogen receptor activation in both MCF-7 breast cancer cells and mammary epithelial cells (MECs). MECs isolated from RGS6(-/-) mice also showed a deficit in DMBA-induced ATM/p53 activation, reactive oxygen species generation and apoptosis confirming that RGS6 is required for effective activation of the DNA damage response in these cells, a critical countermeasure against carcinogen-mediated genotoxic stress. The ability of RGS6 to simultaneously enhance DNA-damage-induced apoptotic signaling and suppress oncogenic cell growth likely underlie the accelerated tumorigenesis and cellular transformation observed in DMBA-treated RGS6(-/-) mice and isolated MECs, respectively. Unsurprisingly, spontaneous tumor formation was also seen in old female RGS6(-/-) but not in wild-type mice. Our finding that RGS6 is downregulated in all human breast cancer subtypes independent of their molecular classification indicates that obtaining a means to restore the growth suppressive and pro-apoptotic actions of RGS6 in breast might be a viable means to treat a large spectrum of breast tumors.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis/genética , Carcinogénesis/patología , Proteínas RGS/genética , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Línea Celular Tumoral , Daño del ADN/genética , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Proteínas RGS/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gßγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gß(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.
Asunto(s)
Cerebelo/metabolismo , Locomoción , Proteínas del Tejido Nervioso/metabolismo , Proteínas RGS/metabolismo , Receptores de GABA-B/metabolismo , Transducción de Señal , Animales , Baclofeno/farmacología , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/patología , Cerebelo/patología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Agonistas de Receptores GABA-B/farmacología , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Humanos , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas RGS/genética , Receptores de GABA-B/genéticaRESUMEN
Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gß5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gß5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gß5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gß5S is undetectable, whereas levels of the photoreceptor-specific Gß5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gß5S expression in retina, the DBC-related defects in Gß5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gß5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/biosíntesis , Proteínas RGS/metabolismo , Retina/metabolismo , Animales , Subunidades beta de la Proteína de Unión al GTP/genética , Regulación de la Expresión Génica/genética , Ratones , Ratones Noqueados , Proteínas RGS/genética , Retina/patologíaRESUMEN
Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Galpha subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.
Asunto(s)
Tipificación del Cuerpo , Señalización del Calcio , Proteínas RGS/fisiología , Somitos/embriología , Proteínas Wnt/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Calcio/análisis , Desarrollo Embrionario , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Imagen Molecular , Proteínas RGS/análisis , Transducción de Señal/fisiología , Proteínas Wnt/análisis , Proteína Wnt-5a , Pez Cebra , Proteínas de Pez Cebra/análisisRESUMEN
Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). EtOH is the most abused substance worldwide with chronic consumption often leading to the development of dependence and abuse. Unfortunately, the neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6 fl/fl ; DAT-iCreER mice to determine the role of RGS6 in VTA DA neurons on EtOH consumption and reward behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse VTA, and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in VTA DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6 -/- mice. Strikingly, RGS6 loss from VTA DA neurons before or after EtOH behavioral reward is established significantly reduced (â¼50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies illuminate a critical role of RGS6 in the mesolimbic circuit in promoting EtOH seeking, reward, and reinstatement. We propose that RGS6 functions to promote DA transmission through its function as a negative modulator of GPCR-Gα i/o -DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.
RESUMEN
Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice (Atm TgC3001L, Nes-Cre, and Syn1-Cre) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.
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Genotipo , Ratones , Animales , Alelos , Transgenes/genética , Ratones Transgénicos , Secuencia de BasesRESUMEN
The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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Bases de Datos Farmacéuticas , Farmacología , Humanos , Bases de Datos Factuales , Canales Iónicos , Ligandos , Receptores Citoplasmáticos y NuclearesRESUMEN
Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.
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Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Carcinoma Ductal/metabolismo , Mitocondrias/metabolismo , Proteínas RGS/metabolismo , Transducción de Señal/fisiología , Animales , Neoplasias de la Mama/patología , Carcinoma Ductal/patología , División Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas RGS/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
RATIONALE: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G(i/o) proteins to promote G protein-coupled inwardly rectifying K(+) (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart. OBJECTIVE: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. METHODS AND RESULTS: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I(K)(ACh)) activation and deactivation, as well as the extent of I(K)(ACh) desensitization. CONCLUSIONS: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.
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Potenciales de Acción/fisiología , Frecuencia Cardíaca/fisiología , Corazón/fisiología , Fibras Parasimpáticas Posganglionares/fisiología , Proteínas RGS/fisiología , Transducción de Señal/fisiología , Potenciales de Acción/genética , Animales , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/fisiopatología , Células Cultivadas , Frecuencia Cardíaca/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas RGS/deficiencia , Proteínas RGS/genética , Receptor Muscarínico M2/fisiología , Transducción de Señal/genética , Nodo Sinoatrial/fisiologíaRESUMEN
Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G(i/o)-dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBPß and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBPß lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.
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Factor de Unión a CCAAT/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Proteínas RGS/metabolismo , Elementos de Respuesta/fisiología , Transcripción Genética/fisiología , Acetilación/efectos de los fármacos , Animales , Factor de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Mutación , Proteínas Proto-Oncogénicas c-bcl-6 , Proteínas RGS/genética , Ratas , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15537. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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Bases de Datos Farmacéuticas , Farmacología , Humanos , Canales Iónicos , Ligandos , Transporte de Proteínas , Receptores Citoplasmáticos y NuclearesRESUMEN
Cascade-specific termination of G protein signaling is catalyzed by the RGS (regulator of G protein signaling) family members, including RGS2. Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated by these hormones. Mutations in RGS2 are associated with human hypertension and increased risk of developing preeclampsia and its sequelae. RGS family members are known to influence maternal vascular function, but the role of RGS2 within the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the development of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared with clinically matched controls and RGS2 is known to be a CREB-responsive gene, RGS2 mRNA was reduced in placentas from pregnancies affected by preeclampsia. Experimentally reducing Rgs2 expression within the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in pregnant wild-type C57BL/6J mice. Stimulation of RGS2 transcription within immortalized human HTR8/SVneo trophoblasts by cAMP/CREB signaling was discovered to be dependent on the activity of histone deacetylase activity, and more specifically, HDAC9 (histone deacetylase-9), and HDAC9 expression was reduced in placentas from human pregnancies affected by preeclampsia. We conclude that reduced expression of RGS2 within the placenta may mechanistically contribute to preeclampsia. More generally, this work identifies RGS2 as an HDAC9-dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.