RESUMEN
BACKGROUND: GNAS encodes the Gαs (stimulatory G-protein alpha subunit) protein, which mediates G protein-coupled receptor (GPCR) signaling. GNAS mutations cause developmental delay, short stature, and skeletal abnormalities in a syndrome called Albright's hereditary osteodystrophy. Because of imprinting, mutations on the maternal allele also cause obesity and hormone resistance (pseudohypoparathyroidism). METHODS: We performed exome sequencing and targeted resequencing in 2548 children who presented with severe obesity, and we unexpectedly identified 22 GNAS mutation carriers. We investigated whether the effect of GNAS mutations on melanocortin 4 receptor (MC4R) signaling explains the obesity and whether the variable clinical spectrum in patients might be explained by the results of molecular assays. RESULTS: Almost all GNAS mutations impaired MC4R signaling. A total of 6 of 11 patients who were 12 to 18 years of age had reduced growth. In these patients, mutations disrupted growth hormone-releasing hormone receptor signaling, but growth was unaffected in carriers of mutations that did not affect this signaling pathway (mean standard-deviation score for height, -0.90 vs. 0.75, respectively; P = 0.02). Only 1 of 10 patients who reached final height before or during the study had short stature. GNAS mutations that impaired thyrotropin receptor signaling were associated with developmental delay and with higher thyrotropin levels (mean [±SD], 8.4±4.7 mIU per liter) than those in 340 severely obese children who did not have GNAS mutations (3.9±2.6 mIU per liter; P = 0.004). CONCLUSIONS: Because pathogenic mutations may manifest with obesity alone, screening of children with severe obesity for GNAS deficiency may allow early diagnosis, improving clinical outcomes, and melanocortin agonists may aid in weight loss. GNAS mutations that are identified by means of unbiased genetic testing differentially affect GPCR signaling pathways that contribute to clinical heterogeneity. Monogenic diseases are clinically more variable than their classic descriptions suggest. (Funded by Wellcome and others.).
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Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Mutación , Obesidad Infantil/genética , Receptor de Melanocortina Tipo 4/metabolismo , Adolescente , Estatura , Niño , Cromograninas/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Humanos , Masculino , Mutación Missense , Receptores de Tirotropina/metabolismo , Transducción de Señal , Secuenciación del ExomaRESUMEN
OBJECTIVE: Early postnatal life is a critical period for the establishment of the functional ß-cell mass that will sustain whole-body glucose homeostasis during the lifetime. ß cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult ß cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal ß-cell development. METHODS: To study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from ß-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. ß-cell mass was studied using histomorphometric analysis and optical projection tomography. ß-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted ß cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. RESULTS: Elimination of Gsα in ß cells led to reduced ß-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key ß-cell identity and maturation genes in postnatal ß-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. CONCLUSION: We conclude that Gsα is required for ß-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal ß cells, which may have implications for using cAMP-raising agents to promote ß-cell regeneration and maturation in diabetes.
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Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Ratones Noqueados , Ratones Transgénicos , Transducción de SeñalRESUMEN
Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
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AMP Cíclico/fisiología , Dexametasona/farmacología , Dinoprostona/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Sistemas de Mensajero Secundario/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Niño , Cromograninas/antagonistas & inhibidores , Colforsina/farmacología , AMP Cíclico/farmacología , Dexametasona/administración & dosificación , Dinoprostona/administración & dosificación , Dinoprostona/antagonistas & inhibidores , Dinoprostona/fisiología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Modelos Animales , Terapia Molecular Dirigida , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Quimera por Radiación , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/biosíntesis , Subtipo EP4 de Receptores de Prostaglandina E/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The stimulatory subunit of G-protein, Gsα, acts as a secondary messenger of G-protein coupled receptors (GPCRs) that primarily activates cAMP-induced signaling. GPCRs, such as the parathyroid hormone receptor (PTHR), are critical regulators of bone formation as shown by number of genetic manipulation studies targeting early osteoblast lineage cells. In this study, we have examined the role of Gsα in osteocytes, the terminally differentiated and most abundant cells of the osteoblast lineage. Mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes (DMP1-GsαKO) have significant decrease of both trabecular and cortical bone, as assessed by µCT. Histomorphometric analysis showed that the osteopenia was mostly driven by more than 90% decrease in osteoblast numbers and activity whereas osteoclasts were only slightly decreased. The decrease in osteoblast number was associated with a striking lack of endocortical osteoblasts. We have previously shown that loss of the stimulatory subunit of G-proteins (Gsα) in osteocytes in vitro or in vivo induces high expression of sclerostin. To determine if the increased sclerostin levels contributed to the decreased endosteal bone lining cells and osteopenia, we treated wild-type mice with recombinant sclerostin and the DMP1-GsαKO mice with anti-sclerostin antibody. Treatment of wild-type mice with 100⯵g/kg sclerostin for 3-weeks significantly reduced the numbers of bone lining cells and led to osteopenia. Next, the DMP1-GsαKO and control littermates were treated with the anti-sclerostin antibody (25â¯mg/kg, 2 times per week) for 4-weeks. Upon the antibody treatment, the endocortical osteoblasts reappeared in the DMP1-GsαKO mice to a comparable level to that of the vehicle treated control littermates. In control mice, E11/gp38 positive osteocytes were observed in parallel with the endocortical osteoblasts with higher dendrite density towards the endocortical osteoblasts. In DMP1-GsαKO mice, E11/gp38 positive osteocytes were lacking dendrites and were randomly scattered throughout the bone matrix. After treatment with anti-sclerostin antibody, DMP1-GsαKO mice showed increased E11/gp38 positive osteocytes near the endosteal bone surface and endosteal osteoblasts. The anti-sclerostin antibody treatment proportionally increased the bone volume but it could not completely rescue the osteopenia in the DMP1-GsαKO mice. Taken together, this data suggests that Gsα signaling in osteocytes leads to osteopenia driven, at least in part, by increased secretion of sclerostin.
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Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Neutralizantes/metabolismo , Enfermedades Óseas Metabólicas/diagnóstico por imagen , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/metabolismo , Hueso Cortical/diagnóstico por imagen , Hueso Cortical/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fémur/diagnóstico por imagen , Fémur/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Microtomografía por Rayos XRESUMEN
CONTEXT: The term pseudohypoparathyroidism (PHP) was coined to describe the clinical condition resulting from end-organ resistance to parathormone (rPTH), caused by genetic and/or epigenetic alterations within or upstream of GNAS. Although knowledge about PHP is growing, there are few data on the prevalence of underlying molecular defects. OBJECTIVE: The purpose of our study was to ascertain the relative prevalence of PHP-associated molecular defects. DESIGN: With a specially designed questionnaire, we collected data from all patients (n = 407) clinically and molecularly characterized to date by expert referral centers in France, Italy, and Spain. RESULTS: Isolated rPTH (126/407, 31%) was caused only by epigenetic defects, 70% of patients showing loss of imprinting affecting all four GNAS differentially methylated regions and 30% loss of methylation restricted to the GNAS A/B:TSS-DMR. Multihormone resistance with no Albright's hereditary osteodystrophy (AHO) signs (61/407, 15%) was essentially due to epigenetic defects, although 10% of patients had point mutations. In patients with rPTH and AHO (40/407, 10%), the rate of point mutations was higher (28%) and methylation defects lower (about 70%). In patients with multihormone resistance and AHO (155/407, 38%), all types of molecular defects appeared with different frequencies. Finally, isolated AHO (18/407, 4%) and progressive osseous heteroplasia (7/407, 2%) were exclusively caused by point mutations. CONCLUSION: With European data, we have established the prevalence of various genetic and epigenetic lesions in PHP-affected patients. Using these findings, we will develop objective criteria to guide cost-effective strategies for genetic testing and explore the implications for management and prognosis.
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Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Hormona Paratiroidea , Seudohipoparatiroidismo/genética , Adolescente , Adulto , Niño , Cromograninas , Epigénesis Genética , Femenino , Francia/epidemiología , Humanos , Italia/epidemiología , Masculino , Mutación , Prevalencia , Seudohipoparatiroidismo/epidemiología , España/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. RESULTS: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while ß-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. CONCLUSION: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections.
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Células Ependimogliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipotálamo/metabolismo , Neuroglía/metabolismo , Delgadez/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Recuento de Células , Cromograninas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Sitios Genéticos , Ratones , Nestina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMEN
The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.
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Huesos Faciales/embriología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Morfogénesis , Cráneo/embriología , Animales , Diferenciación Celular , Fisura del Paladar/embriología , Fisura del Paladar/genética , Huesos Faciales/anomalías , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Ganglios Simpáticos/embriología , Técnicas de Inactivación de Genes , Ratones , Mutación , Cresta Neural/citología , Cresta Neural/metabolismo , Cráneo/anomalías , Raíces Nerviosas Espinales/embriologíaRESUMEN
Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-Gsα(OsxKO) mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 µg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-Gsα(OsxKO) mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-Gsα(OsxKO) mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.
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Anabolizantes/administración & dosificación , Desarrollo Óseo/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Terapia de Reemplazo de Hormonas , Osteoporosis/tratamiento farmacológico , Osteoporosis/enzimología , Hormona Paratiroidea/administración & dosificación , Animales , Huesos/efectos de los fármacos , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Humanos , Masculino , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismoRESUMEN
Gsα, the G protein that transduces receptor-stimulated cAMP generation, mediates sympathetic nervous system stimulation of brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT), which are both potential targets for treating obesity, as well as lipolysis. We generated a mouse line with Gsα deficiency in mature BAT and WAT adipocytes (Ad-GsKO). Ad-GsKO mice had impaired BAT function, absent browning of WAT, and reduced lipolysis, and were therefore cold-intolerant. Despite the presence of these abnormalities, Ad-GsKO mice maintained normal energy balance on both standard and high-fat diets, associated with decreases in both lipolysis and lipid synthesis. In addition, Ad-GsKO mice maintained at thermoneutrality on a standard diet also had normal energy balance. Ad-GsKO mice had improved insulin sensitivity and glucose metabolism, possibly secondary to the effects of reduced lipolysis and lower circulating fatty acid binding protein 4 levels. Gsα signaling in adipose tissues may therefore affect whole-body glucose metabolism in the absence of an effect on body weight.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Peso Corporal/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Glucosa/metabolismo , Insulina/farmacología , Adenoviridae/metabolismo , Adenilato Quinasa/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Lipólisis/efectos de los fármacos , Ratones Noqueados , Actividad Motora , Músculos/metabolismo , Especificidad de Órganos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Termogénesis/efectos de los fármacos , Triglicéridos/metabolismoRESUMEN
In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here, we show that targeted ablation of Gs α in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage.
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Adipocitos/metabolismo , Adipogénesis , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Adipocitos/citología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Osteoblastos/citologíaAsunto(s)
Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Proteínas Hedgehog/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Enfermedades Cutáneas Genéticas/genética , Enfermedades Cutáneas Genéticas/metabolismo , Animales , Femenino , Humanos , MasculinoRESUMEN
Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gαs restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS, which encodes Gαs, Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT-ß-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gαs as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt-ß-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.
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Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Proteínas Hedgehog/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Enfermedades Cutáneas Genéticas/genética , Enfermedades Cutáneas Genéticas/metabolismo , Animales , Enfermedades Óseas Metabólicas/patología , Diferenciación Celular , Cromograninas , Femenino , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Osificación Heterotópica/patología , Osteoblastos/metabolismo , Osteoblastos/patología , Transducción de Señal , Enfermedades Cutáneas Genéticas/patología , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Imbalances of energy homeostasis are often associated with cardiovascular complications. Previous work has shown that Gnasxl-deficient mice have a lean and hypermetabolic phenotype, with increased sympathetic stimulation of adipose tissue. The Gnasxl transcript from the imprinted Gnas locus encodes the trimeric G-protein subunit XLαs, which is expressed in brain regions that regulate energy homeostasis and sympathetic nervous system (SNS) activity. To determine whether Gnasxl knock-out (KO) mice display additional SNS-related phenotypes, we have now investigated the cardiovascular system. The Gnasxl KO mice were â¼20 mmHg hypertensive in comparison to wild-type (WT) littermates (P ≤ 0.05) and hypersensitive to the sympatholytic drug reserpine. Using telemetry, we detected an increased waking heart rate in conscious KOs (630 ± 10 versus 584 ± 12 beats min(-1), KO versus WT, P ≤ 0.05). Body temperature was also elevated (38.1 ± 0.3 versus 36.9 ± 0.4°C, KO versus WT, P ≤ 0.05). To investigate autonomic nervous system influences, we used heart rate variability analyses. We empirically defined frequency power bands using atropine and reserpine and verified high-frequency (HF) power and low-frequency (LF) LF/HF power ratio to be indicators of parasympathetic and sympathetic activity, respectively. The LF/HF power ratio was greater in KOs and more sensitive to reserpine than in WTs, consistent with elevated SNS activity. In contrast, atropine and exendin-4, a centrally acting agonist of the glucagon-like peptide-1 receptor, which influences cardiovascular physiology and metabolism, reduced HF power equally in both genotypes. This was associated with a greater increase in heart rate in KOs. Mild stress had a blunted effect on the LF/HF ratio in KOs consistent with elevated basal sympathetic activity. We conclude that XLαs is required for the inhibition of sympathetic outflow towards cardiovascular and metabolically relevant tissues.
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Presión Sanguínea/fisiología , Temperatura Corporal , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Frecuencia Cardíaca/fisiología , Animales , Atropina/farmacología , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Cromograninas , Exenatida , Receptor del Péptido 1 Similar al Glucagón , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Glucagón/metabolismo , Reserpina/farmacología , Estrés Psicológico , Ponzoñas/farmacologíaRESUMEN
Reciprocal physiological modulation of heart rate is controlled by the sympathetic and parasympathetic systems acting on the sinoatrial (SA) node. However, there is little direct in vivo work examining the role of stimulatory and inhibitory G protein signaling in the SA node. Thus, we designed a study to examine the role of the stimulatory (Gαs) and inhibitory G protein (Gαi2) in in vivo heart rate regulation in the SA node in the mouse. We studied mice with conditional deletion of Gαs and Gαi2 in the conduction system using cre-loxP technology. We crossed mice in which cre recombinase expression was driven by a tamoxifen-inducible conduction system-specific construct with "Gαs floxed" and "Gαi2 floxed" mice. We studied the heart rate responses of adult mice compared with littermate controls by using radiotelemetry before and after administration of tamoxifen. The mice with conditional deletion of Gαs and Gαi2 had a loss of diurnal variation and were bradycardic or tachycardic, respectively, in the daytime. In mice with conditional deletion of Gαs, there was a selective loss of low-frequency power, while with deletion of Gαi2, there was a loss of high-frequency power in power spectral analysis of heart rate variability. There was no evidence of pathological arrhythmia. Pharmacological modulation of heart rate by isoprenaline was impaired in the Gαs mice, but a muscarinic agonist was still able to slow the heart rate in Gαi2 mice. We conclude that Gαs- and Gαi2-mediated signaling in the sinoatrial node is important in the reciprocal regulation of heart rate through the autonomic nervous system.
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Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Frecuencia Cardíaca , Nodo Sinoatrial/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Sistema Nervioso Autónomo/metabolismo , Sistema Nervioso Autónomo/fisiopatología , Bradicardia/metabolismo , Bradicardia/fisiopatología , Cromograninas , Ritmo Circadiano , Electrocardiografía Ambulatoria/métodos , Subunidad alfa de la Proteína de Unión al GTP Gi2/deficiencia , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Frecuencia Cardíaca/efectos de los fármacos , Integrasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Agonistas Muscarínicos/farmacología , Transducción de Señal , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/inervación , Nodo Sinoatrial/fisiopatología , Taquicardia/metabolismo , Taquicardia/fisiopatología , Telemetría , Factores de TiempoRESUMEN
Abnormalities in the cAMP/PKA signaling pathway have been linked to the formation of benign adrenal tumors, as well as a possible predisposition to adrenocortical cancer. Mutations in the G-protein coupled receptor are associated with McCune-Albright syndrome and ACTH-independent macronodular adrenal hyperplasia, while defects in cAMP-dependent protein kinase A can lead to the development of Carney's complex, as well as primary pigmented nodular adrenocortical disease (PPNAD), and micronodular adrenocortical hyperplasia (MAH). Defects in phosphodiesterases, which regulate cAMP levels, have also been demonstrated in PPNAD and MAH. The Wnt signaling pathway, which is involved in oncogenesis in a variety of tumors, has also been implicated in adrenocortical tumorigenesis. MicroRNA profiling has added to our understanding of the signaling pathways involved in tumor formation in the adrenal cortex. Will this all lead to the development of specific targets for pharmacologic therapies? In this article, we review the molecular genetics of adrenocortical tumors and refer to potential targets for pharmacologic therapy.
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Neoplasias de la Corteza Suprarrenal/genética , Carcinoma/genética , Transducción de Señal/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Hormona Adrenocorticotrópica/fisiología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma/tratamiento farmacológico , Cromograninas , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Humanos , MicroARNs/fisiología , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Receptores de Corticotropina/deficiencia , Receptores de Corticotropina/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Wnt/fisiologíaRESUMEN
The G protein α-subunit G(s)α mediates receptor-stimulated cAMP generation. Heterozygous inactivating G(s)α mutations on the maternal allele result in obesity primarily due to reduced energy expenditure in Albright hereditary osteodystrophy patients and in mice. We previously showed that mice with central nervous system (CNS)-specific G(s)α deletion on the maternal allele (mBrGs KO) also develop severe obesity with reduced energy expenditure and that G(s)α is primarily expressed from the maternal allele in the paraventricular nucleus (PVN) of the hypothalamus, an important site of energy balance regulation. We now generated mice with PVN-specific G(s)α deficiency by mating Single-minded 1-cre and G(s)α-floxed mice. Homozygous G(s)α deletion produced early lethality. Heterozygotes with maternal G(s)α deletion (mPVNGsKO) also developed obesity and had small reductions in energy expenditure. However, this effect was much milder than that found in mBrGsKO mice and was more prominent in males. We previously showed mBrGsKO mice to have significant reductions in melanocortin receptor agonist-stimulated energy expenditure and now show that mBrGsKO mice have impaired cold-induced brown adipose tissue stimulation. In contrast, these effects were absent in mPVNGsKO mice. mPVNGsKO mice also had minimal effects on glucose metabolism as compared with mBrGsKO mice. Consistent with the presence of G(s)α imprinting, paternal heterozygotes showed no changes in energy or glucose metabolism. These results indicate that although G(s)α deficiency in PVN partially contributes to the metabolic phenotype resulting from maternal G(s)α mutations, G(s)α imprinting in other CNS regions is also important in mediating the CNS effects of G(s)α mutations on energy and glucose metabolism.
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Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Hipotálamo/metabolismo , Obesidad/genética , Núcleo Hipotalámico Paraventricular/metabolismo , Animales , Composición Corporal/genética , Composición Corporal/fisiología , Ingestión de Alimentos/genética , Ingestión de Alimentos/fisiología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Masculino , Ratones , Ratones Noqueados , MutaciónRESUMEN
The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues.
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Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica/genética , Transducción de Señal/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Biomarcadores/metabolismo , Encéfalo/efectos de los fármacos , Cromograninas , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Marcación de Gen , Sitios Genéticos/genética , Impresión Genómica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Insulina/farmacología , Leptina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Músculos/efectos de los fármacos , Músculos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neuropéptidos/metabolismo , Fenotipo , Transducción de Señal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Control of the renin system by physiological mechanisms such as the baroreceptor or the macula densa (MD) is characterized by asymmetry in that the capacity for renin secretion and expression to increase is much larger than the magnitude of the inhibitory response. The large stimulatory reserve of the renin-angiotensin system may be one of the causes for the remarkable salt-conserving power of the mammalian kidney. Physiological stimulation of renin secretion and expression relies on the activation of regulatory pathways that converge on the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. Mice with selective Gs-alpha (Gsα) deficiency in juxtaglomerular granular cells show a marked reduction of basal renin secretion, and an almost complete unresponsiveness of renin release to furosemide, hydralazine, or isoproterenol. Cyclooxygenase-2 generating prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) in MD and thick ascending limb cells is one of the main effector systems utilizing Gsα-coupled receptors to stimulate the renin-angiotensin system. In addition, ß-adrenergic receptors are critical for the expression of high basal levels of renin and for its release response to lowering blood pressure or MD sodium chloride concentration. Nitric oxide generated by nitric oxide synthases in the MD and in endothelial cells enhances cAMP-dependent signaling by stabilizing cAMP through cyclic guanosine monophosphate-dependent inhibition of phosphodiesterase 3. The stimulation of renin secretion by drugs that inhibit angiotensin II formation or action results from the convergent activation of cAMP probably through indirect augmentation of the activity of PGE(2) and PGI(2) receptors, ß-adrenergic receptors, and nitric oxide.
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AMP Cíclico/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/fisiología , Renina/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Ciclooxigenasa 2/fisiología , Diuréticos/farmacología , Furosemida/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Aparato Yuxtaglomerular/citología , Aparato Yuxtaglomerular/metabolismo , Túbulos Renales Distales/metabolismo , Ratones , Óxido Nítrico Sintasa/metabolismo , Prostaglandinas/fisiología , Receptores Adrenérgicos beta/metabolismo , Sistema Renina-Angiotensina/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Albright hereditary osteodystrophy (AHO) is characterized by short stature, brachydactyly, and often heterotopic ossifications that are typically subcutaneous. Subcutaneous ossifications (SCO) cause considerable morbidity in AHO with no effective treatment. AHO is caused by heterozygous inactivating mutations in those GNAS exons encoding the α-subunit of the stimulatory G protein (Gα(s)). When inherited maternally, these mutations are associated with obesity, cognitive impairment, and resistance to certain hormones that mediate their actions through G protein-coupled receptors, a condition termed pseudohypoparathyroidism type 1a (PHP1a). When inherited paternally, GNAS mutations cause only AHO but not hormonal resistance, termed pseudopseudohypoparathyroidism (PPHP). Mice with targeted disruption of exon 1 of Gnas (Gnas(E1-/+)) replicate human PHP1a or PPHP phenotypically and hormonally. However, SCO have not yet been reported in Gnas(E1+/-) mice, at least not those that had been analyzed by us up to 3 months of age. Here we now show that Gnas(E1-/+) animals develop SCO over time. The ossified lesions increase in number and size and are uniformly detected in adult mice by one year of age. They are located in both the dermis, often in perifollicular areas, and the subcutis. These lesions are particularly prominent in skin prone to injury or pressure. The SCO comprise mature bone with evidence of mineral deposition and bone marrow elements. Superficial localization was confirmed by radiographic and computerized tomographic imaging. In situ hybridization of SCO lesions were positive for both osteonectin and osteopontin. Notably, the ossifications were much more extensive in males than females. Because Gnas(E1-/+) mice develop SCO features that are similar to those observed in AHO patients, these animals provide a model system suitable for investigating pathogenic mechanisms involved in SCO formation and for developing novel therapeutics for heterotopic bone formation. Moreover, these mice provide a model with which to investigate the regulatory mechanisms of bone formation.
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Displasia Fibrosa Poliostótica/complicaciones , Osificación Heterotópica/complicaciones , Animales , Biomarcadores/metabolismo , Cromograninas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Minerales/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Osificación Heterotópica/patología , Osteoblastos/metabolismo , Seudohipoparatiroidismo , Piel/patología , Factores de TiempoRESUMEN
Genetically modified mice with deficiency of the G protein α-subunit (G(s)α) in skeletal muscle showed metabolic abnormality with reduced glucose tolerance, low muscle mass, and low contractile force, along with a fast-to-slow-fiber-type switch (Chen M, Feng HZ, Gupta D, Kelleher J, Dickerson KE, Wang J, Hunt D, Jou W, Gavrilova O, Jin JP, Weinstein LS. Am J Physiol Cell Physiol 296: C930-C940, 2009). Here we investigated a hypothesis that the switching to more slow fibers is an adaptive response with specific benefit. The results showed that, corresponding to the switch of myosin isoforms, the thin-filament regulatory proteins troponin T and troponin I both switched to their slow isoforms in the atrophic soleus muscle of 3-mo-old G(s)α-deficient mice. This fiber-type switch involving coordinated changes of both thick- and thin-myofilament proteins progressed in the G(s)α-deficient soleus muscles of 18- to 24-mo-old mice, as reflected by the expression of solely slow isoforms of myosin and troponin. Compared with age-matched controls, G(s)α-deficient soleus muscles with higher proportion of slow fibers exhibited slower contractile and relaxation kinetics and lower developed force, but significantly increased resistance to fatigue, followed by a better recovery. G(s)α-deficient soleus muscles of neonatal and 3-wk-old mice did not show the increase in slow fibers. Therefore, the fast-to-slow-fiber-type switch in G(s)α deficiency at older ages was likely an adaptive response. The benefit of higher fatigue resistance in adaption to metabolic deficiency and aging provides a mechanism to sustain skeletal muscle function in diabetic patients and elderly individuals.