RESUMEN
PURPOSE: The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. MATERIAL AND METHODS: Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. RESULTS: In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. CONCLUSIONS: In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.
RESUMEN
Intravitreal NMDA injection has been shown to induce the excitotoxic loss of retinal cells. The retinal ganglion cell apoptosis induced by NMDA is thought to play an important role in retinal ischemia injury and NMDA-injected rat has been used as a model of neuronal loss in diseases such as glaucoma. In this experimental model, we studied the early effects of NMDA leading to the degeneration of retinal ganglion cells. PKCzeta regulates the NF-kappaB pathway in cellular responses to various stresses and we have shown that aspirin inhibits purified human PKCzeta. We therefore investigated the molecular mechanism by which retinal cells limit ocular injury following NMDA treatment. We found that the NMDA-induced apoptosis of ganglion cells was mediated, at least partly, by PKCzeta. This enzyme was activated early in the cellular response to NMDA. Prolonged activation was followed by PKCzeta cleavage, and nuclear translocation of the C-terminal region of this protein-a critical event for the survival of retinal cells. We also found that pretreatment with aspirin or the coinjection of NMDA with a specific PKCzeta inhibitor counteracted the effects of NMDA. These findings provide new insight into the role played by PKCzeta in neuronal loss in glaucoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Aspirina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Masculino , N-Metilaspartato/farmacología , Ratas , Ratas WistarRESUMEN
We previously reported the involvement of QN1 (quail neuroretina 1) protein in cell cycle control during retinal development. We show here that QN1 is an ATPase conserved through evolution, from fugu to humans. We show that chicken/quail QN1 protein is orthologous to the KIAA1009 protein in humans, the function of which was not known. We demonstrate here for the first time that QN1/KIAA1009 protein is located at the spindle poles of the mitotic apparatus and at centrosomes during mitosis. The siRNA-mediated depletion of KIAA1009 led to abnormal mitosis with chromosome segregation defects and abnormal centrosome separation leading to the death of PC12 and MCF7 cells. Thus, QN1/KIAA1009 is a new microtubule-associated ATPase involved in cell division.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Segregación Cromosómica , Proteínas del Ojo/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Huso Acromático/fisiología , Adenosina Trifosfatasas/metabolismo , Animales , Secuencia de Bases , Neoplasias de la Mama/patología , Muerte Celular , Proteínas del Ojo/análisis , Femenino , Humanos , Mitosis/genética , Mitosis/fisiología , Datos de Secuencia Molecular , Feocromocitoma/patología , ARN Interferente Pequeño , Ratas , Células Tumorales CultivadasRESUMEN
Abstract Aspirin has been shown to protect against glutamate neurotoxicity via the nuclear factor kappaB pathway. Some studies have implicated the atypical protein kinase C (PKC) zeta (zeta) isoform in cell protection, but the mechanism involved remains unclear. We show here that aspirin exerts at least some of its effects through PKCzeta, decreasing the NMDA-induced activation, cleavage and nuclear translocation of this molecule. Aspirin (acetylsalicylic acid) directly inhibited the protein kinase activity of PKCzeta, whereas salicylic acid did not. This direct effect of aspirin on purified human PKCzeta is consistent with PKCzeta inhibition preventing the NMDA-induced death of cortical neurones. Caspase-3 inhibition blocked the cleavage and nuclear translocation of PKCzeta, whereas caspase-1-inhibition did not. Thus, PKCzeta (protein kinase Mzeta) regulates nuclear events essential for the initiation of the apoptotic pathway. Aspirin protects cells against NMDA-induced apoptosis by means of a novel mechanism targeting PKCzeta, a key molecule in inflammatory responses and neurodegeneration.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Aspirina/farmacología , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Animales , Caspasa 3 , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Agonistas de Aminoácidos Excitadores/toxicidad , Neuronas/citología , Neuronas/enzimología , Fármacos Neuroprotectores/farmacología , Células PC12 , Proteína Quinasa C/metabolismo , RatasRESUMEN
Cell cycle withdrawal involves several transcription factors such as E2Fs members that play a key role in cell growth control. Here we describe a novel putative bZIP transcription factor isolated from the retina and involved in neuronal proliferation arrest at the terminal differentiation: PATF (Proliferation Arrest Transcription Factor). We show that PATF associates with E2F4 protein and interacts with the E2F consensus site. PATF expression increases with establishment of quiescent state. Furthermore, the nuclear PATF localization like E2F4, depends on cell growth arrest. The decrease of PATF amount, using a retroviral antisense strategy, results in pursued neuroretina cell mitosis. Our results indicate that PATF could be a new molecular signal implicated in the final neuronal cell cycle withdrawal.
Asunto(s)
Proteínas Aviares , Neuronas/citología , Retina/embriología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , División Celular , Núcleo Celular/metabolismo , Embrión de Pollo , Clonación Molecular , Secuencia de Consenso , ADN sin Sentido/farmacología , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F4 , Proteínas del Ojo/genética , Proteínas del Ojo/fisiología , Humanos , Datos de Secuencia Molecular , Neuronas/metabolismo , Células PC12 , ARN Mensajero/biosíntesis , Conejos , Ratas , Retina/citología , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
In this report, we describe the involvement of the quail neuroretina 1 (QN1) protein in retinal development. The Qn1 cDNA was isolated as a gene specifically expressed at the onset of neuronal cell cycle withdrawal (Bidou et al., Mech. Dev. 43 (1993) 159). Qn1 is located in the cytoplasm in proliferating cells during the early stages of the development. Its distribution changes, becoming predominantly nuclear, in neurons during establishment of the quiescent state upon the differentiation. We decreased the amount of QN1 protein by an antisense strategy in vitro or in vivo. This decrease of the amount of QN1 protein results in additional mitosis and in severe abnormalities such as retinal dysplasia. Our results suggest that QN1 plays a key role at the onset of neuronal cell cycle withdrawal.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/fisiología , Retina/embriología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Western Blotting , Bromodesoxiuridina/metabolismo , Diferenciación Celular , División Celular , Núcleo Celular/metabolismo , Embrión de Pollo , Proteínas Cromosómicas no Histona/metabolismo , Citoplasma/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas del Ojo/biosíntesis , Biblioteca de Genes , Inmunohistoquímica , Cinética , Microscopía Confocal , Microscopía Fluorescente , Mitosis , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/metabolismo , Estructura Terciaria de Proteína , Codorniz , Retina/anomalías , Retina/metabolismo , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The human and murine MOK2 orthologue genes encode Krüppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3'-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Ojo/genética , Regulación de la Expresión Génica/fisiología , Proteínas de Unión al Retinol/genética , Dedos de Zinc , Animales , Secuencia de Bases , Sitios de Unión , ADN , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Células HeLa , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Células Fotorreceptoras de Vertebrados/metabolismo , Regiones Promotoras GenéticasRESUMEN
The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.
Asunto(s)
Encéfalo/metabolismo , Receptores de Tirotropina/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/citología , Células Cultivadas , ADN Complementario/genética , Epéndimo/citología , Epéndimo/metabolismo , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Tirotropina/genética , Distribución TisularRESUMEN
The protein expression and the enzyme activity of the catalytic subunit (C) of the cAMP-dependent protein kinases were studied in porcine thyroid cell primary cultures stimulated with two doses of TSH (0.1 mU/ml and 1 mU/ml) for 1 to 3 days. In TSH-stimulated cells the desensitization of the catalytic subunit activity was accompanied by a simultaneous and parallel decrease of its immunoreactivity. The loss of catalytic subunit was rapid and reached its maximum after 1 day of culture. It is similar in the two subcellular compartments: cytosol and particulate extracts. Contrary to the observed loss of the C subunit protein molecules in TSH-stimulated cells, the expression of the Cbeta subunit mRNA in these cells was increased fivefold compared to controls, while no significant change was observed on the Calpha subunit mRNA. These results suggest that TSH controls the Cbeta subunits of PKA at two levels: at the transcriptional level it increases Cbeta mRNA expression, and at the translational or posttranslational level TSH decreases the amount and the activity of the Cbeta protein molecules.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glándula Tiroides/enzimología , Tirotropina/farmacología , Animales , Western Blotting , Dominio Catalítico/inmunología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Citosol/efectos de los fármacos , Citosol/enzimología , Relación Dosis-Respuesta a Droga , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Cinética , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacosRESUMEN
Identification of the K+ transporters located in the lateral wall of the cochlea is essential for a better understanding of the mechanisms by which a positive endocochlear potential and a high K+ concentration are achieved in endolymph. In this study, we have determined the distribution of the K+ channel rat ether à go-go (eag) mRNA in the cochlea. After reverse transcription of adult rat cochlear tissues, cDNA was amplified with primers specific to eag channel. The eag mRNA was localized in cochlear tissues by in situ hybridization using specific oligonucleotide probes tailed with digoxigenin conjugated UTP. Eag mRNA was detected in the organ of Corti but mainly in the fibrocytes of the spiral ligament but not in spiral prominence or in stria vascularis. The expression pattern of rat eag transcript in spiral ligament is complementary to the Na+,K+-ATPase distribution in the cochlear lateral wall. The localization of eag mRNA suggests that eag potassium channel may be produced in the corresponding cells. Considering the importance of the K+ gradient in the cochlea, the result reported here suggests that eag channel may play a role in the control of K+ fluxes in the spiral ligament.
Asunto(s)
Cóclea/metabolismo , Ligamentos/metabolismo , Canales de Potasio/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Cóclea/anatomía & histología , Cartilla de ADN/genética , Canales de Potasio Éter-A-Go-Go , Expresión Génica , Hibridación in Situ , Órgano Espiral/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
The product of the proto-oncogene p56lck is a non-receptor tyrosine kinase member of the Src family. It is found in T cells (Marth et al., 1985, 1988) and in the mouse brain (Omri et al., 1996; Van Tan et al., 1996). In this report, we describe experiments showing that Lck is present in the mouse retina neurons. Lck gene expression was identified after isolating and sequencing the specific 5' and 3' part of the cDNA obtained by RT-PCR. In adult retina Lck immunoreactivity was most abundant in photoreceptor cells and within the outer plexiform layers. Staining was also observed in the inner nuclear and plexiform layers. In transgenic mice, the disruption of the Lck gene had serious consequences on the organization of the retina causing retinal dysplasia. These mice have partial retinal detachment with infolding and rosette formation in the photoreceptor sheet. These retinal abnormalities observed in Lck deficient mice lead to the loss of normal architecture of the photoreceptor and the inner nuclear layers, and provide an important role of Lck protein in the retina development. The lack of the Lck protein produces a spectrum of retinal pathology that resembles human retinopathy of prematurity (ROP).
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/deficiencia , Mutación , Displasia Retiniana/genética , Animales , Expresión Génica , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Ratones , Ratones Mutantes , Células Fotorreceptoras/patología , Proto-Oncogenes Mas , ARN Mensajero/análisis , Distribución TisularRESUMEN
The PR264/SC35 splicing factor belongs to the family of SR proteins which function as essential and alternative splicing factors. Here, we report that the human PR264/SC35 locus is bidirectionally transcribed. Double in situ hybridization experiments have allowed simultaneous detection of sense and antisense RNA in human CCRF-CEM cells, suggesting that expression of the corresponding genes is not mutually exclusive. We have characterized three main classes of ET RNAs encoded by the opposite strand of the PR264/SC35 gene and containing PR264/SC35-overlapping sequences, PR264/SC35-non overlapping sequences or a combination of both. We show that their expression results from the use of alternative promoters, exons and polyadenylation signals. PR264/SC35-non overlapping ET mRNA species potentially encode two protein isoforms (449 and 397 amino acids) and are expressed from the PR264/SC35 promoting region. Northern blots and RNase protection analyses indicate that ET polyadenylated RNAs are differentially expressed in several human cell lines. Similar studies performed in the mouse have revealed that the bidirectional transcription of the PR264/SC35 locus is a conserved mechanism and that the open reading frame identified in a subset of human ET mRNAs is highly conserved (93% homology). Northern blot analyses performed with several murine tissues confirmed the differential expression of the ET gene and revealed that it is predominantly expressed in the testis.
Asunto(s)
Empalme Alternativo/genética , Proteínas Nucleares/genética , ARN sin Sentido/genética , Ribonucleoproteínas , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , ADN Complementario , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Factores de Empalme Serina-Arginina , Linfocitos T/citología , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Colágeno/química , Colágeno/metabolismo , Secuencia Conservada , Coturnix , ADN Complementario , Heparina/química , Heparina/metabolismo , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , ARN Mensajero/metabolismo , Conejos , Retina/metabolismo , Homología de Secuencia de Aminoácido , Somatomedinas/química , Somatomedinas/metabolismo , Distribución Tisular , Vitronectina/químicaRESUMEN
The lck gene product, p56lck, is a member of the src-related family of protein tyrosine kinases. It is known as lymphocyte specific and involved in thymocyte development and in the immune response mediated by the T cell receptor. We report that the lck gene is also expressed in adult mouse CNS and that brain p56lck is similar to the thymus protein. In situ hybridization and immunohistochemistry show that the lck gene is expressed in neurons throughout the brain in distinct regions, including hippocampus and cerebellum. In primary cultures from fetal mouse brain, neuronal cells are immunoreactive to Lck antiserum. This suggests that the lck gene product might be involved in a new signal transduction pathway in mouse brain.
Asunto(s)
Encéfalo/enzimología , Neuronas/enzimología , Familia-src Quinasas/biosíntesis , Animales , Células Cultivadas , Cerebelo/enzimología , Embrión de Mamíferos , Exones , Expresión Génica , Hipocampo/enzimología , Hibridación in Situ , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Linfocitos T/enzimología , Linfocitos T/inmunología , Familia-src Quinasas/análisisAsunto(s)
Transformación Celular Neoplásica , Neuronas/citología , Neuronas/fisiología , Oncogenes , Retina/citología , Retina/fisiología , Retroviridae/genética , Animales , Virus del Sarcoma Aviar/genética , Diferenciación Celular , División Celular , Embrión de Pollo , Pollos , Técnicas de Cultivo/métodos , Embrión no Mamífero , Regulación Viral de la Expresión Génica , Hibridación in Situ/métodos , Microscopía Electrónica/métodos , Células Fotorreceptoras/citología , Células Fotorreceptoras/fisiología , Codorniz , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructura , Transfección/métodosRESUMEN
CD4 is a member of the Ig gene super family expressed on the surface of many thymocytes and of a subset of T lymphocytes. Human CD4 is the receptor for HIV envelope glycoprotein gp120. Human and mouse CD4 transcripts are expressed in human and mouse central nervous system (CNS), but no corresponding proteins have been reported yet. We have analyzed mRNA expression and carried out immunological experiments on adult mouse brain with probes specific for the long and short CD4 transcripts and with antibodies monospecific for mouse CD4. The main result of these experiments is that the full length CD4 transcript and the CD4 protein are expressed coordinately in neurons throughout the adult mouse brain. CD4 immunoreactivity is also present in brain small vessel walls, ependymal cells, and choroid plexus. The brain mouse CD4 protein is indistinguishable from the thymus protein. In addition, we show that neuronal cells in primary cultures from human fetal CNS are immunoreactive to human CD4 mAbs.
Asunto(s)
Encéfalo/citología , Encéfalo/inmunología , Antígenos CD4/biosíntesis , Neuronas/inmunología , Animales , Secuencia de Bases , Southern Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Antígenos CD4/análisis , Células Cultivadas , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
The long term objective of this study is to isolate genes specifically expressed at the onset of neuronal cell cycle withdrawal. As an experimental paradigm we have used a quail neuroretinal cell clone (clone K2) immortalized by a thermosensitive mutant of Rous Sarcoma Virus. K2 cells proliferate at 36 degrees C but stop synthesizing DNA after a shift to 41.5 degrees C. We have constructed a cDNA library from K2 cells transferred to 41.5 degrees C and autosubtracted with RNAs from K2 cells maintained at 36 degrees C. This strategy has led to the isolation of cDNAs which recognize mRNAs expressed in quail neuroretina (NR) during development. We report here one of these cDNAs, cDNA QN1, that hybridizes with transcripts expressed in retina neurons, in parallel with their withdrawal from the cell cycle. QN1 ORF codes for a 138 kDa polypeptide corresponding to the protein observed in Western blot analysis. A role of QN1 product(s) on neuronal quiescence is suggested by the positive effect of an antisense oligonucleotide on DNA synthesis of K2 cells.
Asunto(s)
ADN Complementario/genética , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Retina/metabolismo , Animales , Secuencia de Bases , Ciclo Celular/genética , Células Cultivadas , Células Clonales , Coturnix , Hibridación in Situ , Cinética , Mitosis , Datos de Secuencia Molecular , ARN Mensajero/genética , Retina/citologíaRESUMEN
Quail neuroretinal cells (QNR cells) from 7-day-old embryos do not proliferate even in the presence of 8% fetal calf serum. After infection by the Rous sarcoma virus (RSV) they proliferate actively and exhibit a transformed phenotype; this effect is mediated by the oncoprotein pp60v-src. Secondary cultures infected by the thermosensitive strain tsNY68 of RSV are blocked in G0 either by thermal inactivation of pp60v-src at 41.5 degrees C or by serum deprivation at the permissive temperature (36.5 degrees C). Cell division is reinduced either by pp60v-src thermal renaturation or by subsequent serum addition. Our results indicate that v-src and serum control two synergic pathways leading to G0/G1 transition in QNR cells. In order to characterize genes related to the mitogenic and transforming effects of v-src in nerve cells, we have constructed a cDNA library from QNR cells transformed by tsNY68. We report the properties of five molecular clones isolated by differential screening of this library. Unlike immediate-early genes like c-fos, they are induced in mid and late G1. Four of them correspond to unknown mRNAs and the last one codes for nucleolin. This set of v-src-regulated genes is likely to code for functions deficient in terminally differentiated QNR cells and necessary for the progression in G1.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , División Celular , Genes src , Animales , Transformación Celular Neoplásica , Células Cultivadas , Fase G1 , Regulación de la Expresión Génica , Codorniz , ARN Mensajero/análisis , Retina/embriologíaRESUMEN
The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.