RESUMEN
Pleomorphic rhabdomyosarcoma (PRMS) predominantly arises in adult skeletal musculature and is usually associated with poor prognosis. Thus, effective treatments must be developed. PRMS is a rare tumor; therefore, it is critical to develop an experimental system to understand the cellular and molecular mechanisms of PRMS. We previously demonstrated that PRMS develops after p53 gene deletion and oncogenic K-Ras expression in the skeletal muscle tissue. In that study, oncogenic K-Ras-expressing cells were diverse and the period until disease onset was difficult to control. In this study, we developed an experimental system to address this problem. Single cell-derived murine cell lines, designated as RMS310 and RMSg2, were established by limiting the dilution of cells from a lung metastatic tumor colony that were positive for various cancer stem cells and activated skeletal muscle-resident stem/progenitor cell marker genes by RT-PCR. All cell lines stably recapitulated the histological characteristics of human PRMS as bizarre giant cells, desmin-positive cells, and lung metastases in C57BL/6 mice. All subclones of the RMSg2 cells by the limiting dilution in vitro could seed PRMS subcutaneously, and as few as 500 RMSg2 cells were sufficient to form tumors. These results suggest that the RMSg2 cells are multipotent cancer cells that partially combine the properties of skeletal muscle-resident stem/progenitor cells and high tumorigenicity. Thus, our model system's capacity to regenerate tumor tissue in vivo and maintain stable cells in vitro makes it useful for developing therapeutics to treat PRMS.
Asunto(s)
Rabdomiosarcoma , Proteína p53 Supresora de Tumor , Adulto , Humanos , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Ratones Endogámicos C57BL , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Músculo Esquelético/metabolismo , Línea CelularRESUMEN
Tenascin-C (TNC) is an extracellular matrix glycoprotein that is expressed during embryogenesis. It is not expressed in normal adults, but is up-regulated under pathological conditions. Although TNC knockout mice do not show a distinct phenotype, analyses of disease models using TNC knockout mice combined with in vitro experiments revealed the diverse functions of TNC. Since high TNC levels often predict a poor prognosis in various clinical settings, we developed a transgenic mouse that overexpresses TNC through Cre recombinase-mediated activation. Genomic walking showed that the transgene was integrated into and truncated the Atp8a2 gene. While homozygous transgenic mice showed a severe neurological phenotype, heterozygous mice were viable, fertile, and did not exhibit any distinct abnormalities. Breeding hemizygous mice with Nkx2.5 promoter-Cre or α-myosin heavy chain promoter Cre mice induced the heart-specific overexpression of TNC in embryos and adults. TNC-overexpressing mouse hearts did not have distinct histological or functional abnormalities. However, the expression of proinflammatory cytokines/chemokines was significantly up-regulated and mortality rates during the acute stage after myocardial infarction were significantly higher than those of the controls. Our novel transgenic mouse may be applied to investigations on the role of TNC overexpression in vivo in various tissue/organ pathologies using different Cre donors.
Asunto(s)
Infarto del Miocardio/inmunología , Enfermedades Neurodegenerativas/genética , Tenascina/genética , Animales , Paseo de Cromosoma , Citocinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma , Homocigoto , Mediadores de Inflamación/metabolismo , Integrasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Regiones Promotoras Genéticas/genética , Tenascina/metabolismo , Miosinas Ventriculares/genéticaRESUMEN
Diacylglycerol kinase γ (DGKγ) is a lipid kinase to convert diacylglycerol (DG) to phosphatidic acid (PA) and indirectly regulates protein kinase C γ (PKCγ) activity. We previously reported that the basal PKCγ upregulation impairs cerebellar long-term depression (LTD) in the conventional DGKγ knockout (KO) mice. However, the precise mechanism in impaired cerebellar LTD by upregulated PKCγ has not been clearly understood. Therefore, we first produced Purkinje cell-specific DGKγ KO (tm1d) mice to investigate the specific function of DGKγ in Purkinje cells and confirmed that tm1d mice showed cerebellar motor dysfunction in the rotarod and beam tests, and the basal PKCγ upregulation but not PKCα in the cerebellum of tm1d mice. Then, the LTD-induced chemical stimulation, K-glu (50 mM KCl + 100 µM, did not induce phosphorylation of PKCα and dissociation of GluR2 and glutamate receptor interacting protein (GRIP) in the acute cerebellar slices of tm1d mice. Furthermore, treatment with the PKCγ inhibitor, scutellarin, rescued cerebellar LTD, with the phosphorylation of PKCα and the dissociation of GluR2 and GRIP. In addition, nonselective transient receptor potential cation channel type 3 (TRPC3) was negatively regulated by upregulated PKCγ. These results demonstrated that DGKγ contributes to cerebellar LTD by regulation of the basal PKCγ activity.
Asunto(s)
Cerebelo/fisiopatología , Diacilglicerol Quinasa/genética , Trastornos Motores/genética , Proteína Quinasa C/metabolismo , Regulación hacia Arriba , Animales , Apigenina/farmacología , Diacilglicerol Quinasa/metabolismo , Técnicas de Inactivación de Genes , Glucuronatos/farmacología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Ratones , Trastornos Motores/metabolismo , Trastornos Motores/fisiopatología , Fosforilación , Células de Purkinje , Receptores AMPA/metabolismo , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
GABA and glycine are inhibitory neurotransmitters. However, the mechanisms underlying the formation of GABAergic and glycinergic synapses remain unclear. The influence of GABAergic input deprivation on inhibitory terminal formation was investigated using Purkinje cell (PC)-specific vesicular GABA transporter (VGAT) knockout (L7-VGAT) mice, in which GABA release from PCs diminishes in an age-dependent manner. We compared the late development of GABAergic and glycinergic terminals in the cerebellar nucleus (CN) between control and L7-VGAT mice. In the control CN, the density of glutamate decarboxylase (GAD)-positive dots remained unchanged between postnatal 2â¯months (P2M) and 13â¯months (P13M), whereas glycine transporter 2 (GlyT2)-positive dots increased in density during this time frame. No difference in the density of GlyT2-positive dots was observed between control and L7-VGAT mice at P2M, but the density was significantly higher in the L7-VGAT fastigial nuclei (FN) than the control FN at P13M. When VGAT was absent from PC terminals, GlyT2-positive dots included GAD and VGAT and formed synapses. These results indicated that GABAergic terminals were formed by P2M, glycinergic terminals were actively formed after P2M, and more glycinergic terminals were formed in the L7-VGAT FN than in the control FN, suggesting that the increased glycinergic terminals may derive from interneurons within the FN and may also release GABA. These results suggest that the deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals derived from interneurons and that the inhibitory terminal numbers and types may be regulated by the quantity of functional GABAergic inputs.
Asunto(s)
Núcleos Cerebelosos/metabolismo , Neurotransmisores/metabolismo , Células de Purkinje/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Núcleos Cerebelosos/efectos de los fármacos , Glutamato Descarboxilasa/metabolismo , Glicina/metabolismo , Interneuronas/metabolismo , Ratones Transgénicos , Células de Purkinje/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.
Asunto(s)
Cerebelo/metabolismo , Cerebelo/ultraestructura , Aparato de Golgi/ultraestructura , Plasticidad Neuronal , Neuronas/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/patología , Modelos Animales de Enfermedad , Femenino , Aparato de Golgi/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Ratones Noqueados , Vías Nerviosas/metabolismo , Vías Nerviosas/ultraestructura , Neuronas/metabolismo , Cultivo Primario de Células , Células de Purkinje/metabolismo , Células de Purkinje/ultraestructuraRESUMEN
Outside of the neurogenic niches of the brain, postmitotic neurons have not been found to undergo efficient regeneration. We demonstrate that mouse Purkinje cells (PCs), which are born at midgestation and are crucial for development and function of cerebellar circuits, are rapidly and fully regenerated following their ablation at birth. New PCs are produced from immature FOXP2+ Purkinje cell precursors (iPCs) that are able to enter the cell cycle and support normal cerebellum development. The number of iPCs and their regenerative capacity, however, diminish soon after birth and consequently PCs are poorly replenished when ablated at postnatal day five. Nevertheless, the PC-depleted cerebella reach a normal size by increasing cell size, but scaling of neuron types is disrupted and cerebellar function is impaired. Our findings provide a new paradigm in the field of neuron regeneration by identifying a population of immature neurons that buffers against perinatal brain injury in a stage-dependent process.
Asunto(s)
Proliferación Celular , Cerebelo/crecimiento & desarrollo , Cerebelo/lesiones , Células de Purkinje/fisiología , Regeneración , Células Madre/fisiología , Factores de Edad , Animales , RatonesRESUMEN
BACKGROUND: Brain natriuretic peptide (BNP) is an important biomarker for patients with cardiovascular diseases, including heart failure, hypertension and cardiac hypertrophy. It is also known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease; however, the mechanism remains unclear. METHODS AND RESULTS: We developed a BNP reporter mouse and occasionally found that this promoter was activated specifically in the papillary tip of the kidneys, and its activation was not accompanied by BNP mRNA expression. No evidence was found to support the existence of BNP isoforms or other nucleotide expression apart from BNP and tdTomato. The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. Unexpectedly, both the expression and secretion of BNP increased in primary cultured neonatal cardiomyocytes after their treatment with an extract of the renal papillary tip. Intraperitoneal injection of the extract of the papillary tips reduced blood pressure from 210 mmHg to 165 mmHg, the decrease being accompanied by an increase in serum BNP and urinary cGMP production in stroke-prone spontaneously hypertensive (SHR-SP) rats. Furthermore the induction of BNP by the papillary extract from rats with heart failure due to myocardial infarction was increased in cardiomyocytes. CONCLUSIONS: These results suggested that the papillary tip express a substance that can stimulate BNP production and secretion from cardiomyocytes.
Asunto(s)
Enfermedades Cardiovasculares/genética , GMP Cíclico/genética , Péptido Natriurético Encefálico/genética , Insuficiencia Renal Crónica/genética , Animales , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , GMP Cíclico/metabolismo , Humanos , Médula Renal/citología , Médula Renal/metabolismo , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Péptido Natriurético Encefálico/biosíntesis , Cultivo Primario de Células , Ratas , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patologíaRESUMEN
CTLA4 is an inhibitory regulator of immune responses. Therapeutic CTLA4 blockade enhances T cell responses against cancer and provides striking clinical results against advanced melanoma. However, this therapy is associated with immune-related adverse events. Paraneoplastic neurologic disorders are immune-mediated neurological diseases that develop in the setting of malignancy. The target onconeural antigens are expressed physiologically by neurons, and aberrantly by certain tumour cells. These tumour-associated antigens can be presented to T cells, generating an antigen-specific immune response that leads to autoimmunity within the nervous system. To investigate the risk to develop paraneoplastic neurologic disorder after CTLA4 blockade, we generated a mouse model of paraneoplastic neurologic disorder that expresses a neo -self antigen both in Purkinje neurons and in implanted breast tumour cells. Immune checkpoint therapy with anti-CTLA4 monoclonal antibody in this mouse model elicited antigen-specific T cell migration into the cerebellum, and significant neuroinflammation and paraneoplastic neurologic disorder developed only after anti-CTLA4 monoclonal antibody treatment. Moreover, our data strongly suggest that CD8 + T cells play a final effector role by killing the Purkinje neurons. Taken together, we recommend heightened caution when using CTLA4 blockade in patients with gynaecological cancers, or malignancies of neuroectodermal origin, such as small cell lung cancer, as such treatment may promote paraneoplastic neurologic disorders.
Asunto(s)
Anticuerpos/toxicidad , Antígeno CTLA-4/metabolismo , Síndromes Paraneoplásicos del Sistema Nervioso/etiología , Síndromes Paraneoplásicos del Sistema Nervioso/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Neoplasias de la Mama/patología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Cerebelo/patología , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Actividad Motora/fisiología , Trastornos del Movimiento/etiología , Neuropéptidos/metabolismo , Síndromes Paraneoplásicos del Sistema Nervioso/complicaciones , Síndromes Paraneoplásicos del Sistema Nervioso/patología , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
A high incidence of oncogenic K-ras mutations is observed in lung adenocarcinoma of human cases and carcinogen-induced animal models. The process of oncogenic K-ras-mediated lung adenocarcinogenesis can be dissected into two parts: pre- and post-K-ras mutation. Adoption of transgenic lines containing a flox-K-rasG12V transgene eliminates the use of chemical carcinogens and enables us to study directly crucial events post-K-ras mutation without considering the cellular events involved with oncogenic K-ras mutation, e.g., distribution and metabolism of chemical carcinogens, DNA repair, and somatic recombination by host factors. We generated two mouse strains C57BL/6J-Ryr2(tm1Nobs) and A/J-Ryr2(tm1Nobs) in which K-rasG12V can be transcribed from the cytomegalovirus early enhancer/chicken beta actin promoter in virtually any tissue. Upon K-rasG12V induction in lung epithelial cells by an adenovirus expressing the Cre recombinase, the number of tumors in the C57BL/6J-Ryr2(tm1Nobs/+) mouse line was 12.5 times that in the A/J-Ryr2(tm1Nobs/+) mouse line. Quantitative trait locus (QTL) analysis revealed that new three modifier loci, D3Mit19, D3Mit45 and D11Mit20, were involved in the differential susceptibility between the two lines. In addition, we found that differential expression of the wild-type K-ras gene, which was genetically turn out to be anti-oncogenic activity on K-rasG12V, could not account for the different susceptibility in our two K-rasG12V-mediated lung tumor models. Thus, we provide a genetic system that enables us to explore new downstream modifiers post-K-ras mutation.
Asunto(s)
Carcinogénesis/genética , Genes ras/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas/genética , Sitios de Carácter Cuantitativo/genética , Proteínas ras/genética , Animales , Línea Celular Tumoral , Genes Modificadores/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Mutación/genéticaRESUMEN
The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1ß antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.
Asunto(s)
Amiloidosis/inmunología , Enfermedades Cardiovasculares/inmunología , Emaciación/inmunología , Interleucina-1/antagonistas & inhibidores , Interleucina-1/metabolismo , Piel/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Colesterol/sangre , Citocinas/sangre , Ensayo de Inmunoadsorción Enzimática , Interleucina-1/inmunología , Ratones , Ratones Transgénicos , Piel/inmunología , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND AND PURPOSE: Anticonvulsants have been developed according to the traditional neurotransmission imbalance hypothesis. However, the anticonvulsive pharmacotherapy currently available remains unsatisfactory. To develop new antiepileptic drugs with novel antiepileptic mechanisms, we have tested the antiepileptic actions of ONO-2506, a glial modulating agent, and its effects on tripartite synaptic transmission. EXPERIMENTAL APPROACH: Dose-dependent effects of ONO-2506 on maximal-electroshock seizure (MES), pentylenetetrazol-induced seizure (PTZ) and epileptic discharge were determined in a genetic model of absence epilepsy in mice (Cacna1a(tm2Nobs/tm2Nobs) strain). Antiepileptic mechanisms of ONO-2506 were analysed by examining the interaction between ONO-2506 and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on release of l-glutamate, d-serine, GABA and kynurenic acid in the medial-prefrontal cortex (mPFC) of freely moving rats using microdialysis and primary cultured rat astrocytes. KEY RESULTS: ONO-2506 inhibited spontaneous epileptic discharges in Cacna1a(tm2Nobs/tm2Nobs) mice without affecting MES or PTZ. Given systemically, ONO-2506 increased basal release of GABA and kynurenic acid in the mPFC through activation of both neuronal and glial exocytosis, but inhibited depolarization-induced releases of all transmitters. ONO-2506 increased basal glial release of kynurenic acid without affecting those of l-glutamate, d-serine or GABA. However, ONO-2506 inhibited AMPA-induced releases of l-glutamate, d-serine, GABA and kynurenic acid. CONCLUSIONS AND IMPLICATIONS: ONO-2506 did not affect traditional convulsive tests but markedly inhibited epileptic phenomena in the genetic epilepsy mouse model. ONO-2506 enhanced release of inhibitory neuro- and gliotransmitters during the resting stage and inhibited tripartite transmission during the hyperactive stage. The results suggest that ONO-2506 is a novel potential glial-targeting antiepileptic drug. LINKED ARTICLE: This article is commented on by Onat, pp. 1086-1087 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12050.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Caprilatos/uso terapéutico , Epilepsia/tratamiento farmacológico , Animales , Anticonvulsivantes/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Caprilatos/farmacología , Células Cultivadas , Convulsivantes , Modelos Animales de Enfermedad , Electrochoque , Epilepsia/etiología , Epilepsia/metabolismo , Epilepsia/fisiopatología , Ácido Glutámico/metabolismo , Ácido Quinurénico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pentilenotetrazol , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Serina/metabolismo , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismoRESUMEN
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the adult mammalian central nervous system and plays modulatory roles in neural development. The vesicular GABA transporter (VGAT) is an essential molecule for GABAergic neurotransmission due to its role in vesicular GABA release. Cerebellar Purkinje cells (PCs) are GABAergic projection neurons that are indispensable for cerebellar function. To elucidate the significance of VGAT in cerebellar PCs, we generated and characterized PC-specific VGAT knockout (L7-VGAT) mice. VGAT mRNAs and proteins were specifically absent in the 40-week-old L7-VGAT PCs. The morphological characteristics, such as lamination and foliation of the cerebellar cortex, of the L7-VGAT mice were similar to those of the control littermate mice. Moreover, the protein expression levels and patterns of pre- (calbindin and parvalbumin) and postsynaptic (GABA-A receptor α1 subunit and gephyrin) molecules between the L7-VGAT and control mice were similar in the deep cerebellar nuclei that receive PC projections. However, the L7-VGAT mice performed poorly in the accelerating rotarod test and displayed ataxic gait in the footprint test. The L7-VGAT mice also exhibited severer ataxia as VGAT deficits progressed. These results suggest that VGAT in cerebellar PCs is not essential for the rough maintenance of cerebellar structure, but does play an important role in motor coordination. The L7-VGAT mice are a novel model of ataxia without PC degeneration, and would also be useful for studying the role of PCs in cognition and emotion.
RESUMEN
Bone morphogenetic protein (BMP) receptor type 1A (BMPR1A) mutations are associated with facial dysmorphism, which is one of the main clinical signs in both juvenile polyposis and chromosome 10q23 deletion syndromes. Craniofacial development requires reciprocal epithelial/neural crest (NC)-derived mesenchymal interactions mediated by signaling factors, such as BMP, in both cell populations. To address the role of mesenchymal BMP signaling in craniofacial development, we generated a conditional knockdown mouse by expressing the dominant-negative Bmpr1a in NC-derived cells expressing the myelin protein zero(Mpz)-Cre transgene. At birth, 100% of the conditional mutant mice had wide-open anterior fontanelles, and 80% of them died because of cleft face and cleft palate soon after birth. The other 20% survived and developed short faces, hypertelorism and calvarial foramina. Analysis of the NC-derived craniofacial mesenchyme of mutant embryos revealed an activation of the P53 apoptosis pathway, downregulation of both c-Myc and Bcl-XL, a normal growth rate but an incomplete expansion of mesenchymal cells. These findings provide genetic evidence indicating that optimal Bmpr1a-mediated signaling is essential for NC-derived mesenchymal cell survival in both normal nasal and frontal bone development and suggest that our model is useful for studying some aspects of the molecular etiology of human craniofacial dysmorphism.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Cara/anomalías , Cresta Neural/metabolismo , Cresta Neural/patología , Transducción de Señal , Animales , Animales Recién Nacidos , Supervivencia Celular , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Cara/embriología , Cara/patología , Femenino , Hueso Frontal/anomalías , Hueso Frontal/patología , Genes Dominantes , Defectos de los Tabiques Cardíacos/embriología , Defectos de los Tabiques Cardíacos/patología , Humanos , Mesodermo/embriología , Mesodermo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Mucosa Nasal/metabolismo , Nariz/anomalías , Nariz/patología , PigmentaciónRESUMEN
Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of transforming growth factor (TGF)-ß1 as the major player in the pathogenesis of the disease. However, attempts to control its expression and to improve the outcome of pulmonary fibrosis have been disappointing. We tested the hypothesis that TGF-ß1 is the dominant factor in the acute and chronic phases of pulmonary fibrosis and developed short interfering (si)RNAs directed toward molecules implicated in the disease. This study developed novel sequences of siRNAs targeting the TGF-ß1 gene and evaluated their therapeutic efficacy in two models of pulmonary fibrosis: a model induced by bleomycin and a novel model of the disease developed spontaneously in mice overexpressing the full length of human TGF-ß1 in the lungs. Intrapulmonary delivery of aerosolized siRNAs of TGF-ß1 with sequences common to humans and rodents significantly inhibited bleomycin-induced pulmonary fibrosis in the acute and chronic phases of the disease and in a dose-dependent manner. Aerosolized human-specific siRNA also efficiently inhibited pulmonary fibrosis, improved lung function, and prolonged survival in human TGF-ß1 transgenic mice. Mice showed no off-target effects after intratracheal administration of siRNA. These results suggest the applicability of these novel siRNAs as tools for treating pulmonary fibrosis in humans.
Asunto(s)
Terapia Genética/métodos , Fibrosis Pulmonar Idiopática/terapia , Pulmón/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor de Crecimiento Transformador beta1/genética , Aerosoles , Animales , Bleomicina , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/patología , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño/administración & dosificación , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.
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Transformación Celular Neoplásica/genética , Daño del ADN/genética , Genes ras , Guanina/análogos & derivados , Estrés Oxidativo/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Guanina/metabolismo , Ratones , Mutación , Óxido Nítrico Sintasa de Tipo II/biosíntesisRESUMEN
Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε-deficient mice that PLCε plays a critical role in activation of cytokine production in non-immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL-23, in keratinocytes, and with the accumulation of CD4(+) T cells producing IL-22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL-23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL-22-producing T-cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL-23 from keratinocytes, leading to the activation of IL-22-producing T cells.
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Citocinas/inmunología , Dermatitis/inmunología , Queratinocitos/inmunología , Fosfoinositido Fosfolipasa C/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Bloqueadores/farmacología , Citocinas/metabolismo , Células Dendríticas/inmunología , Dermatitis/enzimología , Dermatitis/patología , Femenino , Humanos , Inmunosupresores/farmacología , Interleucina-23/análisis , Interleucina-23/antagonistas & inhibidores , Interleucina-23/inmunología , Interleucinas/análisis , Interleucinas/inmunología , Queratinocitos/enzimología , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoinositido Fosfolipasa C/análisis , Fosfoinositido Fosfolipasa C/metabolismo , Tacrolimus/farmacología , Regulación hacia Arriba , Interleucina-22RESUMEN
The CACNA1A gene encodes the poreforming, voltage-sensitive subunit of the voltage-dependent Ca(v)2.1 calcium channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. In mice, mutations of the homolog Cacna1a cause recessively inherited phenotypes in tottering, rolling Nagoya, rocker, and leaner mice. Here we describe two knockdown mice with 28.4+/-3.4% and 13.8+/-3.3% of the wild-type Ca(v)2.1 quantity. 28.4+/-3.4% level mutants displayed ataxia, absence-like seizures and progressive cerebellar atrophy, although they had a normal life span. Mutants with 13.8+/-3.3% level exhibited ataxia severer than the 28.4+/-3.4% level mutants, absence-like seizures and additionally paroxysmal dyskinesia, and died premature around 3 weeks of age. These results indicate that knock down of Ca(v)2.1 quantity to 13.8+/-3.3% of the wild-type level are sufficient to induce the all neurological disorders observed in natural occurring Cacna1a mutants. These knockdown animals with Ca(v)2.1 calcium channels intact can contribute to functional studies of the molecule in the disease.
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Canales de Calcio Tipo N/genética , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Ataxia Cerebelosa/genética , Animales , Ataxia Cerebelosa/patología , Ataxia Cerebelosa/fisiopatología , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones MutantesRESUMEN
To clarify the antiepileptic mechanisms of zonisamide (ZNS), we determined the interaction between ZNS and inositol-1,4,5-triphosphate receptor (IP3R) on exocytosis of GABA and glutamate in rat frontal cortex using microdialysis. ZNS increased basal GABA release, but not glutamate, concentration-dependently, and reduced concentration-dependently K(+)-evoked GABA and glutamate releases. Inhibition and activation of IP3R reduced and enhanced basal and K(+)-evoked GABA releases, respectively. The K(+)-evoked glutamate release was reduced and enhanced by IP3R antagonist and agonist, respectively, whereas basal glutamate release was increased by IP3R agonist but not affected by IP3R antagonist. Under extracellular Ca(2+) depletion, IP3R agonist increased basal GABA and glutamate releases. The latter effects of IP3R agonist were weakly enhanced by ZNS, but such stimulatory action of ZNS was abolished by extracellular Ca(2+) depletion. In contrast, ZNS inhibited the stimulatory effect of IP3R agonist on K(+)-evoked release. The stimulatory effect of IP3R agonist on basal release was regulated by N-type voltage-sensitive Ca(2+) channel (VSCC) rather than P- and L-type VSCCs, whereas the stimulatory effect of IP3R agonist on K(+)-evoked release was regulated by P- and L-type VSCCs rather than N-type VSCC. These results suggest that ZNS-activated N-type VSCC enhances IP3R-associated neurotransmitter release during resting stage, whereas ZNS-induced suppression of P- and L-type VSCCs possibly attenuates IP3R-associated neurotransmitter release during neuronal hyperexcitability. Therefore, the combination of both of these two actions of ZNS on IP3R-associated neurotransmitter release mechanism seems to be involved, at least in part, in the mechanisms of antiepileptic and neuroprotective actions of ZNS.
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Anticonvulsivantes/farmacología , Lóbulo Frontal/efectos de los fármacos , Ácido Glutámico/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/efectos de los fármacos , Isoxazoles/farmacología , Ácido gamma-Aminobutírico/efectos de los fármacos , Animales , Canales de Calcio , Relación Dosis-Respuesta a Droga , Lóbulo Frontal/metabolismo , Ácido Glutámico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Microdiálisis , Ratas , Ratas Sprague-Dawley , Zonisamida , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/-; Crx-/- retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx-/- retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/-; Crx-/- retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.
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Factores de Transcripción Otx/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Recuento de Células , Diferenciación Celular , Supervivencia Celular , Células Clonales , Electrorretinografía , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Ratones , Ratones Noqueados , Células 3T3 NIH , Especificidad de Órganos , Fenotipo , Células Fotorreceptoras/citología , Células Fotorreceptoras/metabolismo , Retina/citología , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/metabolismo , Retroviridae , Transactivadores/metabolismoRESUMEN
Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.