RESUMEN
Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.
Asunto(s)
Mutación con Ganancia de Función , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófisis/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Niño , Preescolar , Corticotrofos/citología , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Facies , Insuficiencia de Crecimiento/genética , Células HEK293 , Cardiopatías Congénitas/genética , Humanos , Lactante , Sistema de Señalización de MAP Quinasas/genética , Melanotrofos/citología , Melanotrofos/metabolismo , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.
Asunto(s)
Factor 2 Eucariótico de Iniciación/genética , Genes Ligados a X , Glucosa/metabolismo , Hipopituitarismo/etiología , Hipopituitarismo/metabolismo , Fenotipo , Sustitución de Aminoácidos , Apoptosis , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Línea Celular , Preescolar , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Hipopituitarismo/diagnóstico , Hibridación in Situ , Lactante , Imagen por Resonancia Magnética , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Biosíntesis de ProteínasRESUMEN
Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.
Asunto(s)
Potenciales de Acción/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hipotálamo/fisiología , Somatostatina/fisiología , Animales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-ClampRESUMEN
Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Células Ependimogliales/patología , Hipopituitarismo/etiología , Hipotálamo/patología , Neuronas/patología , Uniones Estrechas/patología , Animales , Núcleo Arqueado del Hipotálamo/inmunología , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/patología , Biomarcadores/metabolismo , Células Ependimogliales/inmunología , Células Ependimogliales/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hipopituitarismo/inmunología , Hipopituitarismo/metabolismo , Hipopituitarismo/patología , Hipotálamo/inmunología , Hipotálamo/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Eminencia Media/inmunología , Eminencia Media/metabolismo , Eminencia Media/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/inmunología , Neuronas/metabolismo , Permeabilidad , Proteínas Recombinantes de Fusión/metabolismo , Tercer Ventrículo/inmunología , Tercer Ventrículo/metabolismo , Tercer Ventrículo/patología , Uniones Estrechas/inmunología , Uniones Estrechas/metabolismoRESUMEN
There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.
Asunto(s)
Hormonas Esteroides Gonadales/metabolismo , Hormona del Crecimiento/metabolismo , Caracteres Sexuales , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Femenino , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Background/Aims. 20 Kilodalton-hGH (20 K-hGH) is the second most abundant pituitary GH variant after 22 K-hGH. In the steady state the proportion of 20 : 22 K-hGH appears constant; does this proportion change with repetitive somatotroph stimulation? Methods. Forty adult males were randomised to receive a GHRH(1-29)NH(2) bolus (0.5 mug/kg (n = 20) or 1.0 mug/kg (n = 20)), preceded or followed by a saline bolus, 1 week apart. Four to six weeks later, 10 subjects received 0.5 mug/kg GHRH(1-29)NH(2) at 0, 60, 120, and 180 minutes. Clearance rate of 22 and 20 K-hGH was measured in 10 subjects. Results. Total amount/proportion of 22 K-hGH/20 K-hGH secreted was similar for both GHRH(1-29)NH(2) doses. Repetitive stimulation reduced the amount of 22 K-hGH released whereas the amount of 20 K-hGH did not change significantly leading to an increase in the proportion of 20 K-hGH (P = .05). Half-life of 20 and 22 K-hGH were not significantly different (P = .55). Conclusions. Repetitive stimulation of the somatotroph may alter the proportion of GH variant released.
RESUMEN
Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.
Asunto(s)
Hormona del Crecimiento/metabolismo , Microcirculación , Hipófisis/irrigación sanguínea , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Hipófisis/citología , Hipófisis/metabolismoRESUMEN
BACKGROUND: Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin. PRINCIPAL FINDINGS: Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by gamma-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate. CONCLUSION: Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Ghrelina/farmacología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/fisiología , Animales , Núcleo Arqueado del Hipotálamo/citología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Estrenos/farmacología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Indoles , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Oligopéptidos/farmacología , Técnicas de Placa-Clamp , Pirrolidinonas/farmacología , Receptores de Ghrelina/agonistas , Triptófano/análogos & derivados , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Normal childhood growth is determined by ultradian and infradian variations in GH secretion, yet GH treatment of children with short stature is restricted to daily fixed doses. We have used GH-deficient dwarf rats to determine whether variable GH dose regimens promote growth more effectively than fixed doses. Animals were treated with saline or 4.2 mg of recombinant bovine GH given as 1) 700 microg/wk in 100 microg/day doses, 2) alternating weekly doses of 966 (138 microg/day) or 434 microg (62 microg/day), or 3) 700 microg/wk in randomized daily doses (5-250 microg/day). Body weight and length were measured weekly. Femur and tibia lengths and internal organ, fat pad, and muscle weights were recorded at the end of the study (6 wk); blood was collected for IGF axis measurements. GH promoted femur [F(3,60) = 14.67, P < 0.05], tibia [F(3,60) = 14.90, P < 0.05], muscle [F(3,60) = 10.37, P < 0.05], and organ growth [liver: F(3,60) = 9.30, P < 0.05; kidney: F(3,60) = 2.82, P < 0.05] and an increase in serum IGF-I [F(3,60) = 9.18, P < 0.05] and IGFBP-3 [F(3,60) = 6.70, P < 0.05] levels. IGF-I levels correlated with final weight (r = 0.45, P < 0.05) and length (r = 0.284, P < 0.05) in the whole cohort, but within each group, growth parameters correlated with serum IGF-I only in animals treated with random GH doses. The variable regimens promoted femur length (P < 0.05) and muscle (P < 0.05) and kidney (P < 0.05) weight more effectively than treatment with the fixed regimen. This study demonstrates that aspects of growth are improved following introduction of infradian variation to GH treatment in a GH-deficient model. The data suggest that varying the pattern of GH doses administered to children may enhance growth performance without increasing the overall GH dose.
Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Enanismo Hipofisario/fisiopatología , Hormona del Crecimiento/administración & dosificación , Somatomedinas/metabolismo , Animales , Tamaño Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Enanismo Hipofisario/tratamiento farmacológico , Masculino , RatasRESUMEN
Normal hypothalamopituitary development is closely related to that of the forebrain and is dependent upon a complex genetic cascade of transcription factors and signaling molecules that may be either intrinsic or extrinsic to the developing Rathke's pouch. These factors dictate organ commitment, cell differentiation, and cell proliferation within the anterior pituitary. Abnormalities in these processes are associated with congenital hypopituitarism, a spectrum of disorders that includes syndromic disorders such as septo-optic dysplasia, combined pituitary hormone deficiencies, and isolated hormone deficiencies, of which the commonest is GH deficiency. The highly variable clinical phenotypes can now in part be explained due to research performed over the last 20 yr, based mainly on naturally occurring and transgenic animal models. Mutations in genes encoding both signaling molecules and transcription factors have been implicated in the etiology of hypopituitarism, with or without other syndromic features, in mice and humans. To date, mutations in known genes account for a small proportion of cases of hypopituitarism in humans. However, these mutations have led to a greater understanding of the genetic interactions that lead to normal pituitary development. This review attempts to describe the complexity of pituitary development in the rodent, with particular emphasis on those factors that, when mutated, are associated with hypopituitarism in humans.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hipófisis/embriología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Regulación de la Expresión Génica/genética , Humanos , Hipotálamo/embriología , Hipotálamo/fisiología , Ratones , Morfogénesis/genética , Morfogénesis/fisiología , Hipófisis/fisiología , Hormonas Hipofisarias/genética , Hormonas Hipofisarias/fisiología , Factores de Transcripción/genéticaRESUMEN
GH has physiological functions in many tissues, but the cellular targets for direct effects of GH remain ill defined in complex tissues such as the growth plate in which the contribution of direct vs. indirect actions of GH remains controversial. The Janus kinase (Jak)-signal transducer and activator of transcription (STAT)-5 pathway is activated by GH, so we developed a method to visualize nuclear Stat5b and phosphorylated Stat5 in single cells in response to a pulse of GH. Hep2 cells did not show a Stat5 phosphorylation (pY-Stat5) response to GH except in cells transfected to express GH receptors. ATDC5 cells express GH receptors and showed GH-induced pY-Stat5 responses, which varied with their state of chondrocyte differentiation. In vivo, Stat5b(+ve) nuclei were seen in the resting and prehypertrophic chondrocytes of the growth plate. After a single ip pulse of human GH or mouse GH, but not prolactin, pY-Stat5 responses were visible in cells in the resting zone and groove of Ranvier, 10-45 min later. Prehypertrophic chondrocytes showed no pY-Stat5 response to GH. GH target cells were also identified in other tissues, and a marked variability in spatiotemporal pY-Stat5 responses was evident. Endogenous hepatic pY-Stat5 was detected in mice with intact GH secretion but only during a GH pulse. Fasting and chronic exposure to GH attenuated the pY-Stat5 response to an acute GH injection. In conclusion, pY-Stat5 responses to GH vary in time and space, are sensitive to nutritional status, and may be inhibited by prior GH exposure. In the growth plate, our data provide direct in vivo support for an early role of GH to regulate the fate of immature chondrocytes.
Asunto(s)
Hormona del Crecimiento/farmacología , Placa de Crecimiento/metabolismo , Hígado/metabolismo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Placa de Crecimiento/efectos de los fármacos , Humanos , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/farmacología , Hígado/efectos de los fármacos , Prolactina/farmacología , Ratas , Receptores de Somatotropina/metabolismoRESUMEN
In mammals, males and females exhibit anatomical, hormonal, and metabolic differences. A major example of such sex dimorphism in mouse involves hepatic drug metabolism, which is also a noticeable target of circadian timekeeping. However, whether the circadian clock itself contributes to sex-biased metabolism has remained unknown, although several daily output parameters differ between sexes in a number of species, including humans. Here we show that dimorphic liver metabolism is altered when the circadian regulators Cryptochromes, Cry1 and Cry2, are inactivated. Indeed, double mutant Cry1(-/-) Cry2(-/-) male mice that lack a functional circadian clock express a number of sex-specific liver products, including several cytochrome P450 enzymes, at levels close to those measured in females. In addition, body growth of Cry-deficient mice is impaired, also in a sex-biased manner, and this phenotype goes along with an altered pattern of circulating growth hormone (GH) in mutant males, specifically. It is noteworthy that hormonal injections able to mimic male GH pulses reversed the feminized gene expression profile in the liver of Cry1(-/-) Cry2(-/-) males. Altogether, our observations suggest that the 24-h clock paces the dimorphic ultradian pulsatility of GH that is responsible for sex-dependent liver activity. We thus conclude that circadian timing, sex dimorphism, and liver metabolism are finely interconnected.
Asunto(s)
Ritmo Circadiano/fisiología , Flavoproteínas/metabolismo , Hígado/metabolismo , Caracteres Sexuales , Animales , Materiales Biomiméticos/farmacología , Criptocromos , Femenino , Flavoproteínas/genética , Regulación de la Expresión Génica , Hormona del Crecimiento/análogos & derivados , Hormona del Crecimiento/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Fenotipo , Testosterona/metabolismoRESUMEN
OBJECTIVE: Somatostatin (SST) is secreted by islet delta-cells and by extraislet neuroendocrine cells. SST receptors have been identified on alpha- and beta-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating alpha- and beta-cell function. However, the specific intraislet function of delta-cell SST remains uncertain. We have used Sst(-/-) mice to investigate the role of delta-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo. RESEARCH DESIGN AND METHODS: Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst(-/-) mice in vivo and from isolated islets in vitro. RESULTS: Islet size and organization did not differ between Sst(-/-) and control islets, nor did islet glucagon or insulin content. Sst(-/-) mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst(-/-) islets compared with control islets and was inhibited by exogenous SST in Sst(-/-) but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst(-/-) islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst(-/-) islets. CONCLUSIONS: We suggest that delta-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, delta-cell SST is implicated in the nutrient-induced suppression of glucagon secretion.
Asunto(s)
Islotes Pancreáticos/metabolismo , Células Secretoras de Somatostatina/metabolismo , Somatostatina/fisiología , Animales , Femenino , Glucagón/metabolismo , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Masculino , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Radioinmunoensayo , Somatostatina/deficiencia , Somatostatina/genéticaRESUMEN
CONTEXT: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke's pouch, the developing anterior pituitary, and the eye. CONCLUSIONS: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Ojo/embriología , Proteínas HMGB/fisiología , Hipófisis/embriología , Prosencéfalo/embriología , Factores de Transcripción/fisiología , Adolescente , Adulto , Niño , Proteínas de Unión al ADN/genética , Anomalías del Ojo/etiología , Anomalías del Ojo/genética , Femenino , Proteínas HMGB/genética , Humanos , Hipopituitarismo/etiología , Hipopituitarismo/genética , Mutación , ARN Mensajero/análisis , Factores de Transcripción SOXB1 , Transducción de Señal , Factores de Transcripción/genética , beta Catenina/fisiologíaRESUMEN
The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form "pituispheres" in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathke's pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2(+) cells in the adult gland also express SOX9 and S100. We suggest that this SOX2(+)/SOX9(+) population represents transit-amplifying cells, whereas the SOX2(+)/SOX9(-) cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas HMGB/metabolismo , Hipófisis/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Ratones , Microscopía Confocal , Microscopía Fluorescente , Hipófisis/metabolismo , Factores de Transcripción SOXB1 , Células Madre/metabolismoRESUMEN
Splicing mutations in the human GH (hGH) gene (GH-1) that cause skipping of exon 3 result in a form of GH deficiency termed isolated GH deficiency type II (IGHD II). The GH-1 gene contains five exons; constitutive splicing produces the wild-type 22-kDa hormone, whereas skipping of exon 3 results in transcripts encoding a 17.5-kDa isoform that acts as a dominant-negative to block secretion of the wild-type hormone. Common characteristics of IGHD II include short stature due to impaired bone elongation, growth, and, in severe cases, anterior pituitary hypoplasia. Typically, IGHD II is treated by sc delivery of hGH, which can rescue stature but, unfortunately, does not inhibit pituitary hypoplasia. Direct destruction of transcripts encoding the dominant-negative 17.5-kDa isoform should both rescue stature and prevent hypoplasia. Here, we have used delivery of short hairpin RNAs to rescue a murine model of IGHD II by specifically targeting transcripts encoding the 17.5-kDa isoform using RNA interference. To our knowledge, this is the first example where a short hairpin RNA has been expressed to specifically degrade an incorrectly spliced transcript and rescue a dominant-negative disease phenotype in vivo.
Asunto(s)
Enanismo Hipofisario/terapia , Terapia Genética/métodos , Hormona de Crecimiento Humana/genética , Adenohipófisis/fisiología , Interferencia de ARN , Animales , Modelos Animales de Enfermedad , Enanismo Hipofisario/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Adenohipófisis/patología , Adenohipófisis/ultraestructura , Recuperación de la FunciónRESUMEN
Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.
Asunto(s)
Senescencia Celular/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/fisiología , Terminales Presinápticos/ultraestructura , Potenciales de Acción , Vías Aferentes/fisiología , Animales , Potenciales Postsinápticos Excitadores , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Hormona del Crecimiento/fisiología , Hormona Liberadora de Hormona del Crecimiento/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Terminales Presinápticos/metabolismoRESUMEN
The organization of the peptidergic neurons of the hypothalamic arcuate nucleus is not fully understood. These include growth hormone-releasing hormone (GHRH) neurons involved in growth and metabolism. We studied identified GHRH neurons of GHRH-green fluorescent protein transgenic mice using patch-clamp methods and focused on gender differences, which govern the physiological patterns of GHRH release. Both the spontaneous firing rates and the intrinsic properties of GHRH neurons were similar in males and females, although higher glutamatergic currents were noticed in females. Surprisingly, marked gender differences in GHRH neuronal activity were observed in response to the muscarinic agonist carbachol (CCh). In females, CCh enhanced action potential firing in all GHRH neurons. In males, CCh enhanced action potential firing in two-thirds of GHRH neurons, whereas it decreased firing in the remainders. M1 agonist McN-A343 (10 microM) mimicked, and M1 antagonist pirenzepine (3 microM) blocked the effects of CCh. In both genders, CCh did not change the intrinsic properties of GHRH neurons, although it strongly increased the frequency of glutamatergic currents, in the presence or absence of tetrodotoxin. In males only, CCh enhanced the frequency of GABAergic currents, and this modulation was antagonized by tetrodotoxin. Thus, the muscarinic regulation involved differential control of afferent inputs at short and long distances in male and female mice. The dual-level control could be a mechanism whereby the selective modulation of the GHRH system (short-distance control) is adjusted to the integrated regulation of arcuate nucleus activity (long-distance control).
Asunto(s)
Vías Aferentes/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Análisis de Varianza , Animales , Núcleo Arqueado del Hipotálamo/citología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Inmunohistoquímica/métodos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Factores Sexuales , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.
Asunto(s)
Apoptosis/fisiología , Trastornos del Crecimiento/genética , Hormona de Crecimiento Humana/genética , Hipófisis/citología , Sitios de Empalme de ARN/fisiología , Animales , División Celular/fisiología , Células Cultivadas , Fragmentación del ADN , Dexametasona/farmacología , Exones/genética , Genes Dominantes , Glucocorticoides/farmacología , Trastornos del Crecimiento/fisiopatología , Hormona de Crecimiento Humana/deficiencia , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Mutación/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , TransfecciónRESUMEN
In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40-44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25-22% in 8-month-old mice. TH immunoreactivity occurred in 36-35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se, may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.