RESUMO
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.
Assuntos
Mutação com Ganho de Função , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófise/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Corticotrofos/citologia , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Fácies , Insuficiência de Crescimento/genética , Células HEK293 , Cardiopatias Congênitas/genética , Humanos , Lactente , Sistema de Sinalização das MAP Quinases/genética , Melanotrofos/citologia , Melanotrofos/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sequenciamento do Exoma/métodosRESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hipotálamo/fisiologia , Somatostatina/fisiologia , Animais , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Células Ependimogliais/patologia , Hipopituitarismo/etiologia , Hipotálamo/patologia , Neurônios/patologia , Junções Íntimas/patologia , Animais , Núcleo Arqueado do Hipotálamo/imunologia , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Biomarcadores/metabolismo , Células Ependimogliais/imunologia , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hipopituitarismo/imunologia , Hipopituitarismo/metabolismo , Hipopituitarismo/patologia , Hipotálamo/imunologia , Hipotálamo/metabolismo , Imunoglobulina G/metabolismo , Masculino , Eminência Mediana/imunologia , Eminência Mediana/metabolismo , Eminência Mediana/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/imunologia , Neurônios/metabolismo , Permeabilidade , Proteínas Recombinantes de Fusão/metabolismo , Terceiro Ventrículo/imunologia , Terceiro Ventrículo/metabolismo , Terceiro Ventrículo/patologia , Junções Íntimas/imunologia , Junções Íntimas/metabolismoRESUMO
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.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hipófise/embriologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Hipotálamo/embriologia , Hipotálamo/fisiologia , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Hipófise/fisiologia , Hormônios Hipofisários/genética , Hormônios Hipofisários/fisiologia , Fatores de Transcrição/genéticaRESUMO
The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.
Assuntos
Proteínas de Ligação a DNA/genética , Anormalidades do Olho/genética , Proteínas HMGB/genética , Hipotálamo/anormalidades , Mutação , Hipófise/anormalidades , Transativadores/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/genética , Adulto , Animais , Criança , Feminino , Humanos , Lactente , Masculino , Camundongos , Fatores de Transcrição SOXB1RESUMO
The pulsatile pattern of GH secretion exhibits sexual dimorphism that is likely to depend on somatostatin (SRIH) effects on somatoliberin (GHRH) neurons in the hypothalamus. Using transgenic GHRH-enhanced green fluorescent protein (eGFP) mice, no difference in the total number of GHRH-eGFP neurons or change in somatostatin receptor sst2 and SRIH mRNA levels in ventromedial hypothalamic nucleus-arcuate nucleus and periventricular nucleus regions and GHRH mRNA levels in the ventromedial hypothalamic-arcuate region were observed between male and female mice. However, the percentage of GHRH-eGFP neurons bearing sst2A receptors reached 78% in female vs. 26% in male GHRH-eGFP mice (P < 0.02). This sex difference in sst2A distribution on GHRH neurons may play an important role in the sexually differentiated pattern of GH secretion.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/análise , Hipotálamo/química , Receptores de Somatostatina/análise , Caracteres Sexuais , Animais , Feminino , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Somatostatina/genéticaRESUMO
Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/deficiência , Hipotálamo/metabolismo , Neurônios/metabolismo , Proteínas da Matriz Viral/genética , Animais , Antivirais/farmacologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Feminino , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Doenças da Hipófise/metabolismo , Adeno-Hipófise/metabolismo , Rimantadina/farmacologia , Fatores de Tempo , Proteínas da Matriz Viral/metabolismoRESUMO
Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.