RESUMO
Stem cell-derived brain organoids replicate important stages of the prenatal human brain development and combined with the induced pluripotent stem cell (iPSC) technology offer an unprecedented model for investigating human neurological diseases including autism and microcephaly. We describe the history and birth of organoids and their application, focusing on cerebral organoids derived from embryonic stem cells and iPSCs. We discuss new insights into organoid-based model of schizophrenia and shed light on challenges and future applications of organoid-based disease model system. This review also suggests hitherto unrevealed potential applications of organoids in combining with new technologies such as nanophotonics/optogenomics for controlling brain development and atomic force microscopy for studying mechanical forces that shape the developing brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Modelos Biológicos , Organoides , Esquizofrenia/patologia , Encéfalo/citologia , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimentoRESUMO
The watershed-hypothesis of schizophrenia asserts that over 200 different mutations dysregulate distinct pathways that converge on an unspecified common mechanism(s) that controls disease ontogeny. Consistent with this hypothesis, our RNA-sequencing of neuron committed cells (NCCs) differentiated from established iPSCs of 4 schizophrenia patients and 4 control subjects uncovered a dysregulated transcriptome of 1349 mRNAs common to all patients. Data reveals a global dysregulation of developmental genome, deconstruction of coordinated mRNA networks, and the formation of aberrant, new coordinated mRNA networks indicating a concerted action of the responsible factor(s). Sequencing of miRNA transcriptomes demonstrated an overexpression of 16 miRNAs and deconstruction of interactive miRNA-mRNA networks in schizophrenia NCCs. ChiPseq revealed that the nuclear (n) form of FGFR1, a pan-ontogenic regulator, is overexpressed in schizophrenia NCCs and overtargets dysregulated mRNA and miRNA genes. The nFGFR1 targeted 54% of all human gene promoters and 84.4% of schizophrenia dysregulated genes. The upregulated genes reside within major developmental pathways that control neurogenesis and neuron formation, whereas downregulated genes are involved in oligodendrogenesis. Our results indicate (i) an early (preneuronal) genomic etiology of schizophrenia, (ii) dysregulated genes and new coordinated gene networks are common to unrelated cases of schizophrenia, (iii) gene dysregulations are accompanied by increased nFGFR1-genome interactions, and (iv) modeling of increased nFGFR1 by an overexpression of a nFGFR1 lead to up or downregulation of selected genes as observed in schizophrenia NCCs. Together our results designate nFGFR1 signaling as a potential common dysregulated mechanism in investigated patients and potential therapeutic target in schizophrenia.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Mutação/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Esquizofrenia/genética , Transdução de Sinais/genética , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Redes Reguladoras de Genes , Genômica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Receptor Notch1/metabolismo , Esquizofrenia/patologia , Transcriptoma , Adulto JovemRESUMO
Studies of induced pluripotent stem cells (iPSCs) from schizophrenia patients and control individuals revealed that the disorder is programmed at the preneuronal stage, involves a common dysregulated mRNA transcriptome, and identified Integrative Nuclear FGFR1 Signaling a common dysregulated mechanism. We used human embryonic stem cell (hESC) and iPSC-derived cerebral organoids from four controls and three schizophrenia patients to model the first trimester of in utero brain development. The schizophrenia organoids revealed an abnormal scattering of proliferating Ki67+ neural progenitor cells (NPCs) from the ventricular zone (VZ), throughout the intermediate (IZ) and cortical (CZ) zones. TBR1 pioneer neurons and reelin, which guides cortico-petal migration, were restricted from the schizophrenia cortex. The maturing neurons were abundantly developed in the subcortical regions, but were depleted from the schizophrenia cortex. The decreased intracortical connectivity was denoted by changes in the orientation and morphology of calretinin interneurons. In schizophrenia organoids, nuclear (n)FGFR1 was abundantly expressed by developing subcortical cells, but was depleted from the neuronal committed cells (NCCs) of the CZ. Transfection of dominant negative and constitutively active nFGFR1 caused widespread disruption of the neuro-ontogenic gene networks in hESC-derived NPCs and NCCs. The fgfr1 gene was the most prominent FGFR gene expressed in NPCs and NCCs, and blocking with PD173074 reproduced both the loss of nFGFR1 and cortical neuronal maturation in hESC cerebral organoids. We report for the first time, progression of the cortical malformation in schizophrenia and link it to altered FGFR1 signaling. Targeting INFS may offer a preventive treatment of schizophrenia.
Assuntos
Córtex Cerebral/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Esquizofrenia/patologia , Calbindina 2/metabolismo , Córtex Cerebral/metabolismo , Células-Tronco Embrionárias/patologia , Humanos , Interneurônios/metabolismo , Interneurônios/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteína Reelina , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMO
In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor-1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene-controlling Feed-Forward-And-Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Núcleo Celular/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Humanos , Neoplasias/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Schizophrenia is a neurodevelopmental disorder featuring complex aberrations in the structure, wiring, and chemistry of multiple neuronal systems. The abnormal developmental trajectory of the brain appears to be established during gestation, long before clinical symptoms of the disease appear in early adult life. Many genes are associated with schizophrenia, however, altered expression of no one gene has been shown to be present in a majority of schizophrenia patients. How does altered expression of such a variety of genes lead to the complex set of abnormalities observed in the schizophrenic brain? We hypothesize that the protein products of these genes converge on common neurodevelopmental pathways that affect the development of multiple neural circuits and neurotransmitter systems. One such neurodevelopmental pathway is Integrative Nuclear FGFR1 Signaling (INFS). INFS integrates diverse neurogenic signals that direct the postmitotic development of embryonic stem cells, neural progenitors and immature neurons, by direct gene reprogramming. Additionally, FGFR1 and its partner proteins link multiple upstream pathways in which schizophrenia-linked genes are known to function and interact directly with those genes. A th-fgfr1(tk-) transgenic mouse with impaired FGF receptor signaling establishes a number of important characteristics that mimic human schizophrenia - a neurodevelopmental origin, anatomical abnormalities at birth, a delayed onset of behavioral symptoms, deficits across multiple domains of the disorder and symptom improvement with typical and atypical antipsychotics, 5-HT antagonists, and nicotinic receptor agonists. Our research suggests that altered FGF receptor signaling plays a central role in the developmental abnormalities underlying schizophrenia and that nicotinic agonists are an effective class of compounds for the treatment of schizophrenia.
Assuntos
Modelos Animais de Doenças , Genômica/métodos , Esquizofrenia , Animais , Genômica/tendências , Humanos , Camundongos , Esquizofrenia/genética , Esquizofrenia/patologia , Esquizofrenia/terapiaRESUMO
Genetic and post mortem evidence has implicated the α7 neuronal nicotinic receptor (NNR) in the etiology of schizophrenia and related disorders. In schizophrenia, enhanced subcortical dopamine (DA) correlates with positive and cognitive of the disease, including impairments in sensorimotor gating. We measured the levels of extracellular DA and DA metabolites during an acoustic test session of prepulse inhibition (PPI) of the startle response, a measure of sensorimotor gating, by microdialysis and HPLC-EC in a transgenic mouse model of schizophrenia. In th-fgfr1(tk-) mice, blockade of fibroblast growth factor receptor 1 (FGFR1) signaling during development in catecholaminergic neurons results in reduced size and density of midbrain DA neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These mice displayed reduced PPI and enhanced startle response relative to control mice as well as a potentiation of DA release in the dorsal striatum during a 30 minute PPI test session. Acute administration of a partial α7 NNR agonist TC-7020 (1.0 mg/kg) normalized PPI and startle deficits and attenuated increases of DA release during acoustic PPI testing. These results provide direct evidence of elevated striatal dopaminergic transmission with impaired sensorimotor gating that may underlie cognitive and positive symptoms and motor deficits in schizophrenia and related disorders. Also, systemic targeting of alpha7 NNRs may ameliorate these deficits by functionally suppressing striatal DA activity.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Inibição Neural/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Esquizofrenia/fisiopatologia , Filtro Sensorial/efeitos dos fármacos , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Estimulação Acústica/métodos , Animais , Cromatografia Líquida de Alta Pressão , Corpo Estriado/metabolismo , Relação Dose-Resposta a Droga , Ácido Homovanílico/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microdiálise/métodos , Inibição Neural/genética , Agonistas Nicotínicos/farmacologia , Quinuclidinas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/genética , Esquizofrenia/genética , Filtro Sensorial/genética , Tiofenos/farmacologiaRESUMO
A growing body of evidence suggests that the alpha7 neuronal nicotinic receptor (NNR) subtype is an important target for the development of novel therapies to treat schizophrenia, offering the possibility to address not only the positive but also the cognitive and negative symptoms associated with the disease. In order to probe the relationship of alpha7 function to relevant behavioral correlates we employed TC-5619, a novel selective agonist for the alpha7 NNR subtype. TC-5619 binds with very high affinity to the alpha7 subtype and is a potent full agonist. TC-5619 has little or no activity at other nicotinic receptors, including the alpha4beta2, ganglionic (alpha3beta4) and muscle subtypes. The transgenic th(tk-)/th(tk-) mouse model that reflects many of the developmental, anatomical, and multi-transmitter biochemical aspects of schizophrenia was used to assess the antipsychotic effects of TC-5619. In these mice TC-5619 acted both alone and synergistically with the antipsychotic clozapine to correct impaired pre-pulse inhibition (PPI) and social behavior which model positive and negative symptoms, respectively. Antipsychotic and cognitive effects of TC-5619 were also assessed in rats. Similar to the results in the transgenic mice, TC-5619 significantly reversed apomorphine-induced PPI deficits. In a novel object recognition paradigm in rats TC-5619 demonstrated long-lasting enhancement of memory over a wide dose range. These results suggest that alpha7-selective agonists such as TC-5619, either alone or in combination with antipsychotics, could offer a new approach to treating the constellation of symptoms associated with schizophrenia, including cognitive dysfunction.
Assuntos
Comportamento Animal/efeitos dos fármacos , Benzofuranos/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Neurônios/metabolismo , Agonistas Nicotínicos/uso terapêutico , Quinuclidinas/uso terapêutico , Receptores Nicotínicos/fisiologia , Esquizofrenia/tratamento farmacológico , Psicologia do Esquizofrênico , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Benzofuranos/farmacologia , Clozapina/farmacologia , Clozapina/uso terapêutico , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/psicologia , Comportamento Exploratório/efeitos dos fármacos , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Agonistas Nicotínicos/farmacologia , Regiões Promotoras Genéticas , Quinuclidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Reflexo de Sobressalto/efeitos dos fármacos , Esquizofrenia/metabolismo , Comportamento Social , Tirosina 3-Mono-Oxigenase/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Studies have shown the presence of expanded polyQ containing proteins in brain cells related to Huntington disease (HD) and other poly-glutamine disorders. We report the use of organically modified silica (ORMOSIL) nanoparticles as an efficient non-viral gene carrier in an effort to model brain pathology associated with those disorders induced by expanded polyQ peptides. In experiment 1, plasmids expressing Hemaglutinin-tagged polypeptides with 20 glutamine repeats (Q20) or with extended 127-glutamine repeats (Q127) were complexed with ORMOSIL nanoparticles and injected twice (2 weeks apart) into the lateral ventricle of the mouse brain. Fourteen days post-injection of Q127, immunocytochemistry revealed the presence of the characteristic nuclear and cytoplasmic Q127 aggregates in numerous striatal, septal and neocortical neuronal cells as well as ubiquitin-containing aggregates indicative of the neuronal pathology. The mice receiving Q127 showed a marked increase in the reactive GFAP (+) astrocytes in striatum, septum and brain cortex, further indicating the neurodegenerative changes, accompanied by motor impairments. In experiment 2, plasmids Q20 or Q127 were complexed with ORMOSIL and were injected into the brain lateral ventricle or directly into the striatum of adult rats. In both routes of transfection, Q127 induced the appearance of reactive GFAP (+) astrocytes and activated ED1 antigen expressing microglia. An increase in the size of the lateral ventricle was also observed in rats receiving Q127. In transgenic mouse polyQ models, extensive pathologies occur outside the nervous system and the observed brain pathologies could reflect developmental effects of the toxic polyQ proteins. Our experiments show that the nervous tissue restricted expression of poly Q-extended peptides in adult brain is sufficient to evoke neuropathologies associated with HD and other polyQ disorders. Thus, nanotechnology can be employed to model pathological and behavioral aspects of genetic brain diseases in mice as well as in other species, providing a novel research tool for in vivo testing of single or multi-gene therapies.
Assuntos
Técnicas de Transferência de Genes/tendências , Vetores Genéticos/genética , Nanopartículas/química , Peptídeos/genética , Siloxanas/farmacologia , Transfecção/métodos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Ectodisplasinas/análise , Ectodisplasinas/biossíntese , Feminino , Gliose/genética , Gliose/metabolismo , Gliose/fisiopatologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/fisiopatologia , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Nanopartículas/toxicidade , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Peptídeos/metabolismo , Peptídeos/toxicidade , Plasmídeos/genética , Ratos , Ratos Wistar , Dióxido de SilícioRESUMO
Activation of cAMP signaling pathway and its transcriptional factor cyclic AMP response element binding protein (CREB) and coactivator are key determinants of neuronal differentiation and plasticity. We show that nuclear fibroblast growth factor receptor-1 (FGFR1) mediates cAMP-induced neuronal differentiation and regulates CREB and CREB binding protein (CBP) function in alpha-internexin-expressing human neuronal progenitor cells (HNPC). In proliferating HNPC, FGFR1 was associated with the cytoplasm and plasma membrane. Treatment with dB-cAMP induced nuclear accumulation of FGFR1 and caused neuronal differentiation, accompanied by outgrowth of neurites expressing MAP2 and neuron-specific neurofilament-L protein and enolase. HNPC transfected with nuclear/cytoplasmic FGFR1 or non-membrane FGFR1(SP-/NLS), engineered to accumulate exclusively in the cell nucleus, underwent neuronal differentiation in the absence of cAMP stimulation. In contrast, FGFR1/R4, with highly hydrophobic transmembrane domain of FGFR4, was membrane associated, did not enter the nucleus and failed to induce neuronal differentiation. Transfection of tyrosine kinase-deleted dominant negative receptor mutants, cytoplasmic/nuclear FGFR1(TK-) or nuclear FGFR1(SP-/NLS)(TK-), prevented cAMP-induced neurite outgrowth. Nuclear FGFR1 localized in speckle-like domains rich in phosphorylated histone 3 and splicing factors, regions known for active RNA transcription and processing, and activated the neurofilament-L gene promoter. FGFR1(SP-/NLS) transactivated CRE, up-regulated phosphorylation and transcriptional activity of CREB and stimulated the activity of CBP several-fold. Thus, cAMP-induced nuclear accumulation of FGFR1 provides a signal that triggers molecular events leading to neuronal differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Neurônios/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Bucladesina/farmacologia , Proteína de Ligação a CREB , Núcleo Celular/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Genes Dominantes , Humanos , Mutagênese Sítio-Dirigida , Neuritos/efeitos dos fármacos , Proteínas de Neurofilamentos/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transativadores/metabolismo , TransfecçãoRESUMO
Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor (TGF)-beta superfamily of signaling cytokines, induces dendritic growth in rat sympathetic neurons. In this study, we present evidence that the recently discovered integrative nuclear FGFR1 signaling (INFS) pathway is involved in dendrite outgrowth mediated by BMP-7. Immunocytochemical analysis of expressed fibroblast growth factors (FGFs) showed that little FGF-2 was detected in control neurons, but the expression of this molecule in the cytoplasm and nucleus increased within 6 h after BMP-7 treatment. In contrast, FGF-1 was constitutively present in the peripheral cytoplasm and in neurites under control conditions, and its distribution did not change with BMP-7 exposure. The high-affinity receptor FGFR1 was present in low amounts in control neurons and was associated with the cytoplasm, the plasma membrane, and the nucleus. Twenty-four hours of BMP-7 treatment elicited an increase in FGFR1 nuclear localization. Overexpressed constructs of FGFR1 that lack the tyrosine kinase domain, and have been shown to act in a dominant-negative manner on FGFR1 signaling, inhibited BMP-7 mediated initial dendrite outgrowth in transfected neurons by approximately 50%. However, targeted inhibition of extracellular FGF-2 by overexpression of a secreted receptor mutant FGFR1(TM-) lacking the transmembrane domain failed to affect BMP-7 induced dendritic growth, as did treatment with the extracellular FGFR antagonist inositol hexakisphosphate. These results suggest that the INFS, which has already been implicated in a broad range of activities in other cell types, may also be required for BMP-7 to stimulate dendritic development.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Dendritos/fisiologia , Neurônios/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/fisiologia , Sistema Nervoso Simpático/fisiologia , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 7 , Células Cultivadas , Dendritos/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Humanos , Membranas Intracelulares/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.
Assuntos
Núcleo Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Receptores de Angiotensina/metabolismo , Transdução de Sinais , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Animais , Sítios de Ligação , Bovinos , Núcleo Celular/genética , Células Cultivadas , Pegada de DNA , Desoxirribonuclease I/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Regiões Promotoras Genéticas , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta/fisiologia , Transativadores/metabolismo , Tirosina/metabolismoRESUMO
Manipulation of gene expression in developing or in mature central nervous systems (CNS) holds a promise for the resolution of many compelling neurobiological questions, including the feasibility of gene therapy to treat diseases of the brain. In this context, a number of viral vectors have been used in recent years to introduce and express genes into the CNS. This article discusses a gene transfer system based on the Herpes Simplex Virus-1 (HSV-1). We describe here the use of non-replicating, non-toxic HSV-1 vector, 8117/43, in a series of studies carried in our joint program. This vector proves further the utility of HSV-1 as a delivery vehicle to a number of distinct sites within the CNS.
Assuntos
Encéfalo/metabolismo , Técnicas de Transferência de Genes , Herpesvirus Humano 1 , beta-Galactosidase/genética , Animais , Encéfalo/citologia , Terapia Genética/métodos , Vetores Genéticos , Masculino , Ratos , Ratos Endogâmicos F344 , Técnicas Estereotáxicas , beta-Galactosidase/análiseRESUMO
Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.
Assuntos
Astrócitos/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Plasticidade Neuronal/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Astrócitos/citologia , Astrócitos/fisiologia , Sequência de Bases , Sítios de Ligação , Encéfalo/citologia , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Luciferases/biossíntese , Mitose , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
In this study we describe the presence of high affinity FGF-2 binding sites in the nuclei of U251MG glioma cells (K(d)=7 pM). Immunoprecipitation of total cell extracts with FGF receptor (FGFR) 1-4 antibodies showed that U251MG glioma cells express only FGFR1. [125I]FGF-2 cross linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 may account for the nuclear FGF-2 binding sites. Western blot analysis demonstrated the presence of 103, 118 kDa and small amounts of 145 kDa FGFR1 isoforms in the nuclei of glioma cells. All isoforms contain both the C- and N-terminal domains. Nuclear FGFR1 retains kinase activity. Immunocytochemistry using confocal microscopy showed specific FGFR1 immunoreactivity within the nuclear interior. In continuously proliferating glioma cells, nuclear FGFR1 is constitutively expressed, independent of cell density. In contrast, in nontransformed human astrocytes, nuclear FGFR1 levels fluctuate with the proliferative state of the cell. In quiescent, confluent astrocytes nuclear FGFR1 protein was depleted. An accumulation of nuclear FGFR1 was observed following the transition to a subconfluent, proliferating state. Transfection of a pcDNA3.1-FGFR1 expression vector into glioma cells that do not express FGFR1 resulted in the nuclear accumulation of FGFR1, increased cell proliferation, and stimulated transition from the G0/G1 to the S-phase of the cell cycle. The increased proliferative rate was resistant to inhibition by the cell-impermeable FGF binding antagonist, myoinositol hexakis [dihydrogen phosphate]. Our results suggest that the constitutive nuclear presence of FGFR1 contributes to the increased proliferation of glioma cells while the transient nuclear accumulation of FGFR1 in normal astrocytes may play a role in the transition to a reactive state.
Assuntos
Núcleo Celular/metabolismo , Glioma/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Astrócitos/química , Astrócitos/metabolismo , Western Blotting , Divisão Celular/fisiologia , Núcleo Celular/química , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioma/química , Humanos , Imuno-Histoquímica , Radioisótopos do Iodo , Mitógenos/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares , TransfecçãoRESUMO
FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.
Assuntos
Astrócitos/citologia , Núcleo Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioblastoma/patologia , Astrócitos/metabolismo , Comunicação Celular , Contagem de Células , Divisão Celular , Células Cultivadas , Feminino , Glioblastoma/metabolismo , Humanos , Masculino , Neuroglia/citologia , Neuroglia/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crestderived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.
Assuntos
Astrócitos/citologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Receptores Proteína Tirosina Quinases , Animais , Divisão Celular , Humanos , Neuroglia/citologia , Proteína Quinase C/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
In an effort to determine the localization of fibroblast growth factor (FGF) receptors (FGFR) that could mediate the intracellular action of FGF-2, we discovered the presence of high-affinity. FGF-2 binding sites in the nuclei of bovine adrenal medullary cells (BAMC). Western blot analysis demonstrated the presence of 103-, 118-, and 145-kDa forms of FGFR1 in nuclei isolated from BAMC. 125I-FGF-2 cross-linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 can account for the nuclear FGF-2 binding sites. Nuclear FGFR1 has kinase activity and undergoes autophosphorylation. Immunocytochemistry with the use of confocal and electron microscopes demonstrated the presence of FGFR1 within the nuclear interior. Nuclear subfractionation followed by Western blot or immunoelectron microscopic analysis showed that the nuclear FGFR1 is contained in the nuclear matrix and the nucleoplasm. Agents that induce translocation of endogenous FGF-2 to the nucleus (forskolin, carbachol, or angiotensin II) increased the intranuclear accumulation of FGFR1. This accumulation was accompanied by an overall increase in FGF-2-inducible tyrosine kinase activity. Our findings suggest a novel mode for growth factor action whereby growth factor receptors translocate to the nucleus in parallel with their ligand and act as direct mediators of nuclear responses to cell stimulation.
Assuntos
Medula Suprarrenal/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Medula Suprarrenal/citologia , Animais , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Cinética , Microscopia Imunoeletrônica , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismoRESUMO
Fractionation of human astrocytes revealed the presence of 103, 118, and 145 kDa forms of FGF receptor 1 (FGFR1) in isolated nuclei. Only trace amounts of FGFR1 proteins were detected in the cell membrane or cytoplasmic fractions. Nuclear FGFR1 is found in the nucleoplasm and nuclear matrix but not in chromatin. Immuno-confocal microscopy further demonstrates the intranuclear presence of FGFR1 and its colocalization with FGF-2. Nuclear FGFR1 binds to FGF-2 and has tyrosine kinase activity. Translocation of functional growth factor receptors into the cell nucleus offers a novel mechanism for growth factor action.
Assuntos
Astrócitos/química , Núcleo Celular/química , Fator 1 de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Membrana Celular/química , Células Cultivadas , Citoplasma/química , Humanos , Peso MolecularRESUMO
We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.
Assuntos
Medula Suprarrenal/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Receptores de Calcitriol/genética , Sistema Nervoso Simpático/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Vitamina D/farmacologia , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Análise de Variância , Animais , Bovinos , Técnicas In Vitro , Masculino , Camundongos , Neurotransmissores/genéticaRESUMO
Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaffin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated. The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA. The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.