RESUMEN
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Adulto , Humanos , Glioblastoma/metabolismo , Brasil , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Tubulina (Proteína)/metabolismoRESUMEN
Malignant mesotheliomas are rare types of cancers that affect the mesothelial surfaces, usually the pleura and peritoneum. They are associated with asbestos exposure, but due to a latency period of more than 30 years and difficult diagnosis, most cases are not detected until they reach advanced stages. Treatment options for this tumor type are very limited and survival ranges from 12 to 36 months. This review discusses the molecular physiopathology, current diagnosis, and latest therapeutic options for this disease.
Asunto(s)
Amianto , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurales , Amianto/toxicidad , Humanos , Mesotelioma/terapia , Pleura , Neoplasias Pleurales/diagnóstico , Neoplasias Pleurales/terapiaRESUMEN
Medulloblastoma (MB) is the most common malignant brain tumor in children. It is currently classified in four main molecular subgroups with different clinical outcomes: sonic hedgehog, wingless, group 3, and group 4 (MBSHH, MBWNT, MBGRP3, or MBGRP4). Presently, a 22-gene expression panel has been efficiently applied for molecular subgrouping using nCounter technology. In this study, formalin-fixed, paraffin-embedded samples from 164 Brazilian medulloblastomas were evaluated, applying the 22-gene panel, and subclassified into the low and high expression of nine key medulloblastoma-related genes. In addition, TP53 mutation status was assessed using TruSight Tumor 15 Panel, and its correlation with expression and prognostic impact was evaluated. Samples from 149 of 164 patients (90%) were classified into MBSHH (47.7%), MBWNT (16.1%), MBGRP3 (15.4%), and MBGRP4 (20.8%). GNAS presented the highest expression levels, with higher expression in MBSHH. TP53, MYCN, SOX2, and MET were also up-regulated in MBSHH, whereas PTEN was up-regulated in MBGRP4. GNAS, TP53, and PTEN low expression was associated with the unfavorable patient outcome only for MBSHH (P = 0.04, P = 0.01, and P = 0.02, respectively). TP53 mutations were detected in 28.57% of MBSHH cases and exhibited association with lower expression and worse clinical outcome, although not statistically significant. The 22-gene panel for molecular classification of medulloblastoma associated with the expression of GNAS, TP53, and PTEN improves the patient prognostication in MBSHH subgroup and can be easily incorporated in the 22-gene panel without any additional costs.
Asunto(s)
Neoplasias Cerebelosas/clasificación , Neoplasias Cerebelosas/genética , Cromograninas/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Proteínas Hedgehog/genética , Meduloblastoma/clasificación , Meduloblastoma/genética , Fosfohidrolasa PTEN/genética , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Adolescente , Brasil/epidemiología , Neoplasias Cerebelosas/epidemiología , Niño , Preescolar , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Masculino , Meduloblastoma/epidemiología , Mutación , Pronóstico , Adulto JovenRESUMEN
Medulloblastoma is the most frequent malignant brain tumor in children, representing 20% of all childhood brain tumors. Currently, medulloblastomas are molecularly classified in 4 subgroups that are associated with distinctive clinicopathological features. KBTBD4 mutations were recently described in a subset of MBGRP3 and MBGRP4 medulloblastomas subgroups. However, no other studies reported KBTBD4 mutations in medulloblastomas. Thus, our aim was to investigate KBTBD4 mutations in a Brazilian series of medulloblastoma. We evaluated 128 medulloblastoma patients molecularly classified from 4 Brazilian reference centers. DNA from formalin-fixed, paraffin-embedded samples was screened for KBTBD4 hotspot mutations by Sanger sequencing. Most of the patients were male, average age was 16.5 years old and average overall survival was 55.9 months. The predominant histological subtype was the classic subtype, followed by nodular/desmoplastic, and the predominant medulloblastoma molecular subtype was the MBSHH subgroup (46%), followed by MBGRP3 and MBGRP4 (19%/each), and MBWNT (16%). Among the 128 samples, 111 were successfully sequenced. No KBTBD4 mutations were identified in 111 samples. Our findings suggest that KBTBD4 mutations are uncommon in Brazilian MBGRP3 and MBGRP4 medulloblastomas subgroups. Further studies in a larger series of MBGRP3 and MBGRP4 medulloblastomas are warranted to better assess role of KBTBD4 mutations.
Asunto(s)
Proteínas Portadoras/genética , Neoplasias Cerebelosas/genética , Meduloblastoma/genética , Adolescente , Adulto , Brasil , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/patología , Niño , Preescolar , Femenino , Humanos , Masculino , Meduloblastoma/mortalidad , Meduloblastoma/patología , Persona de Mediana Edad , Mutación , Tasa de Supervivencia , Adulto JovenRESUMEN
Background: Human biological material has become an important resource for biomedical research. Tumor Biobanks are facilities that collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. mRNA-translation has been demonstrated to modulate protein levels and is considered a fundamental post-transcriptional mechanism of gene expression regulation. Thus, determining translation efficiencies of individual mRNAs in human tumors may add another layer of information that contributes to the understanding of tumorigenic pathways. To analyze the RNAs actively engaged in translation, RNAs associated with ribosomes (polysomes) are isolated, identified and compared to total RNA. However, the application of this technique in human tumors depends on the stability of the polysomal structure under Biobank storage conditions that usually consists of ultra-low temperature. Since the effect of freezing on the stability of the polysomal structure in stored tumor samples is not known, it is essential to evaluate this factor in the frozen samples, validating the use of biobank samples in studies of translational efficiency. Methods: Xenograft tumors were divided in two parts, half was subject to immediate processing, and half was frozen for posterior analysis. Both parts were subject to polysomal separation, RNA extraction and identification through RNAseq. Results: It was possible to successfully extract and identify total and polysomal RNA from both fresh and frozen tumoral tissue. The quantification of the polysome profile indicated no difference in the translational efficiency estimated in fresh versus frozen tissue. Gene expression data from the fresh versus frozen tissues were compared and the correlation between the polysome associated fresh x frozen (R = 0,89) and total fresh x frozen (0,90) mRNAs was calculated. No difference was identified between the two conditions. Conclusions: We demonstrated that tissue freezing does not affect the polysomal structure, consequently validating the viability of the use of biobank stored tissue for polysome associated RNA analysis (AU)
Asunto(s)
Humanos , Polirribosomas , ARN , Expresión Génica , Regulación de la Expresión Génica , NeoplasiasRESUMEN
The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Vesículas Secretoras/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Hipocampo/citología , Immunoblotting , Ratones , Proteínas PrPC/metabolismo , Vesículas Secretoras/ultraestructuraRESUMEN
The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuritas/metabolismo , Neurogénesis/fisiología , Biosíntesis de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Factor de Transcripción Activador 4/biosíntesis , Factor de Transcripción Activador 4/genética , Animales , Conducta Animal/fisiología , Células Cultivadas , Regulación hacia Abajo/fisiología , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Conducta Alimentaria/fisiología , Péptidos y Proteínas de Señalización Intracelular , Memoria/fisiología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismo , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.
Asunto(s)
Automatización , Proteínas Proto-Oncogénicas c-akt/análisis , Serina-Treonina Quinasas TOR/análisis , Análisis de Matrices Tisulares , Humanos , Inmunohistoquímica , Fosforilación , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/biosíntesis , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.
Asunto(s)
Señalización del Calcio/fisiología , Ganglios Espinales/fisiología , Proteínas de Choque Térmico/fisiología , Laminina/fisiología , Proteínas PrPC/fisiología , Receptor Cross-Talk/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Axones/química , Axones/fisiología , Supervivencia Celular/fisiología , Líquido Extracelular/química , Líquido Extracelular/fisiología , Ganglios Espinales/química , Proteínas de Choque Térmico/química , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Laminina/metabolismo , Ratones , Ratones Noqueados , Cultivo Primario de Células , Unión Proteica/fisiología , Células Receptoras Sensoriales/química , Regulación hacia Arriba/fisiologíaRESUMEN
Prion protein (PrP(C)) is the normal isoform of PrP(Sc), a protein involved in neurodegenerative disorders. PrP(C) participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress-inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP(C) mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP(C), STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP(C), in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP(C) ligands, their expression was not altered in PrP(C)-null mice.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Proteínas PrPC/metabolismo , Vitronectina/metabolismo , Animales , Axones/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Corazón/embriología , Riñón/embriología , Riñón/metabolismo , Hígado/embriología , Hígado/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Notocorda/embriología , Notocorda/metabolismo , Proteínas PrPC/genética , Médula Espinal/embriología , Médula Espinal/metabolismo , Factores de TiempoRESUMEN
Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, Prnp(0/0)ras/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation.
Asunto(s)
Agregación Celular , Integrina alfaV/metabolismo , Integrina alfaVbeta3/metabolismo , Neoplasias Pulmonares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Metástasis de la Neoplasia , Proteínas PrPC/metabolismo , Análisis de Varianza , Animales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Neoplasias Pulmonares/secundario , Ratones , Ratones Noqueados , Proteínas PrPC/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas ras/metabolismoRESUMEN
Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.
Asunto(s)
Astrocitos/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Proteínas de Choque Térmico/metabolismo , Proteínas PrPC/metabolismo , Animales , Astrocitos/citología , Encéfalo/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Neurológicos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Proteínas PrPC/genética , Proteína Quinasa C/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Vimentina/metabolismoRESUMEN
The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.
Asunto(s)
Encéfalo/metabolismo , Endocitosis/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Neuronas/metabolismo , Proteínas PrPC/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Activación Enzimática/fisiología , Proteínas de Choque Térmico/genética , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Técnicas de Cultivo de Órganos , Proteínas PrPC/genética , Transporte de Proteínas/fisiologíaRESUMEN
The physiological functions of the cellular prion protein, PrP(C), as a cell surface pleiotropic receptor are under debate. We report that PrP(C) interacts with vitronectin but not with fibronectin or collagen. The binding sites mediating this PrP(C)-vitronectin interaction were mapped to residues 105-119 of PrP(C) and the residues 307-320 of vitronectin. The two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrP(C) antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrP(C)-null mice. Functional assays demonstrated that relative to wild-type cells, PrP(C)-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alphavbeta3 activity. Our findings indicate that PrP(C) plays an important role in axonal growth, and this function may be rescued in PrP(C)-knockout animals by integrin compensatory mechanisms.
Asunto(s)
Axones/metabolismo , Integrinas/metabolismo , Proteínas PrPC/metabolismo , Vitronectina/metabolismo , Animales , Sitios de Unión , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/embriología , Humanos , Ratones , Proteínas PrPC/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Vitronectina/químicaRESUMEN
Cellular prion protein (PrP(C)) is a cell surface glycoprotein that interacts with several ligands such as laminin, NCAM (Neural-Cell Adhesion Molecule) and the stress-inducible protein 1 (STI1). PrP(C) association with these proteins in neurons mediates adhesion, differentiation and protection against programmed cell death. Herein, we used an aversively motivated learning paradigm in rats to investigate whether STI1 interaction with PrP(C) affects short-term memory (STM) formation and long-term memory (LTM) consolidation. Blockage of PrP(C)-STI1 interaction with intra-hippocampal infusion of antibodies against PrP(C) or STI1 immediately after training impaired both STM and LTM. Furthermore, infusion of PrP(C) peptide 106-126, which competes for PrP(C)-STI1 interaction, also inhibited both forms of memory. Remarkably, STI1 peptide 230-245, which includes the PrP(C) binding site, had a potent enhancing effect on memory performance, which could be blocked by co-treatment with the competitive PrP(C) peptide 106-126. Taken together, these results demonstrate that PrP(C)-STI1 interaction modulates both STM and LTM and suggests a potential use of ST11 peptide 230-245 as a pharmacological agent.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Memoria a Corto Plazo/fisiología , Memoria/fisiología , Proteínas PrPC/fisiología , Animales , Reacción de Prevención/efectos de los fármacos , Conducta Animal/fisiología , Western Blotting , Proteínas de Choque Térmico/antagonistas & inhibidores , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Indicadores y Reactivos , Masculino , Plasticidad Neuronal/fisiología , Proteínas PrPC/antagonistas & inhibidores , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Ratas , Ratas Wistar , Sinapsis/fisiologíaRESUMEN
Cellular prion protein (PrPc) has a pivotal role in prion diseases. PrPc is a specific receptor for laminin (LN) gamma1 peptide and several lines of evidence indicate that it is also involved in neural plasticity. Here we investigated whether the interaction between PrPc and LN plays a role in rat memory formation. We found that post-training intrahippocampal infusion of PrPc-derived peptides that contain the LN binding site (PrPc163-182 and PrPc173-192) or of anti-PrPc or anti-LN antibodies that inhibit PrPc-LN interaction impaired inhibitory avoidance memory retention. The amnesic effect of anti-PrPc antibodies and PrPc173-192 peptide was reversed by co-infusion of a LN gamma1 chain-derived peptide containing the PrPc-binding site, suggesting that PrPc-LN interaction is indeed crucial for memory consolidation. In addition, PrPc173-192 peptide and anti-PrPc or anti-LN antibodies also inhibited the activation of hippocampal cAMP-dependent protein kinase A (PKA) and extracellular regulated kinase (ERK1/2), two kinases that mediate the up-regulation of signaling pathways needed for consolidation of inhibitory avoidance memory. Our findings show that, through its interaction with LN, hippocampal PrPc plays a critical role in memory processing and suggest that this role is mediated by activation of both PKA and ERK1/2 signaling pathways.
Asunto(s)
Hipocampo/metabolismo , Laminina/metabolismo , Aprendizaje/fisiología , Memoria/fisiología , Proteínas PrPC/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Sitios de Unión/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Laminina/antagonistas & inhibidores , Laminina/inmunología , Masculino , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteínas PrPC/química , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Understanding the physiological function of the cellular prion (PrPc) depends on the investigation of PrPc-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrPc ligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrPc interaction with STI1 in hippocampal neurons. Both PrPc and STI1 are abundantly expressed and highly colocalized in the hippocampus in situ, indicating that they can interact in vivo. Recombinant STI1 (His6-STI1) added to hippocampal cultures interacts with PrPc at the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrPc-null cells. This effect was abolished by antibodies against either PrPc or STI1 and was dependent on the STI1 domain that binds PrPc. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His6-STI1, but not its counterpart lacking the PrPc binding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrPc-STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Neuritas/fisiología , Neuronas/metabolismo , Priones/metabolismo , Transducción de Señal/fisiología , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas de Choque Térmico/genética , Ratones , Ratones Noqueados , Neuritas/metabolismo , Priones/genética , Unión Proteica/fisiologíaRESUMEN
ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting -- in a manner dependent on the MEK/ERK pathway -- c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor -->cAMP/PKA --> Akt deactivation -->GSK3 activity liberation --> c-Myc Thr58 phosphorylation. We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.
Asunto(s)
Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/farmacología , Ciclo Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Corteza Suprarrenal/citología , Animales , Bovinos , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Transducción de SeñalRESUMEN
Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.