RESUMEN
All-trans retinoic acid (ATRA), a metabolite of vitamin A, binds to retinoic acid receptors (RARs) to mediate gene transcription in target cells. We previously found that an ATRA supplement enhanced olfactory recovery rate in adult mice after olfactory bulb deafferentation. In this study, we examined the cellular localization of RARalpha, RARbeta, and RARgamma and the effects of surgery and ATRA treatment using immunocytochemistry. Mice received a left olfactory nerve transection with the right side serving as internal control. One day after surgery, the mice were given either ATRA mixed with sesame oil or just sesame oil. In the unoperated olfactory bulb, only RARalpha immunoreactivity (ir) was observed. In the unoperated right olfactory epithelium, RARalpha-ir was found in flask-shaped cells located in the supporting cell layer, in cell clusters above the basal cell layer, in cells in the lamina propria, in some respiratory cells and in the olfactory bulb. The flask-shaped cells did not immunostain for either neurons or sustentacular cells. RARbeta-ir was localized only in the respiratory cells while no RARgamma-ir was observed in the olfactory epithelium. The density of RARalpha-ir cells was higher in the operated left olfactory epithelium and highest after ATRA treatment. This study demonstrates the presence of RARs in the olfactory system, provides additional support that the ATRA-signaling pathway may be involved in the recovery of the olfactory epithelium after injury, and suggests a role for an unstudied cell type in that process.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Nervio Olfatorio/fisiología , Vías Olfatorias/metabolismo , Receptores de Ácido Retinoico/metabolismo , Animales , Antígenos de Diferenciación/metabolismo , Western Blotting/métodos , Recuento de Células/métodos , Desnervación/métodos , Diagnóstico por Imagen/métodos , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica/métodos , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteína Marcadora Olfativa , Vías Olfatorias/citología , Vías Olfatorias/efectos de los fármacos , Receptores de Ácido Retinoico/clasificación , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.
Asunto(s)
Transformación Celular Neoplásica/genética , Genes myb/fisiología , Receptores de Ácido Retinoico/fisiología , Factores de Transcripción/fisiología , Animales , Virus de la Mieloblastosis Aviar/genética , Diferenciación Celular/genética , División Celular/genética , Línea Celular Transformada , Pollos , ADN Complementario/biosíntesis , ADN Complementario/genética , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiología , Regulación de la Expresión Génica , Humanos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/fisiología , Codorniz , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/clasificación , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Supresión Genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Activación Transcripcional , TransfecciónRESUMEN
PURPOSE: In the mature retina retinoic acid (RA) biosynthesis was reported to be regulated by light. RA is of vital importance for proper function of the retina. RA activity is mediated by interaction with nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The purpose of this study was to determine if and which RARs and RXRs are present in the mature retina, and to determine their location within the retina. METHODS: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify cDNA fragments encoding RARalpha, RARbeta, RARgamma, RXRalpha, RXRbeta, and RXRgamma from human retinal RNA. RT-PCR products were cloned, sequenced, and used in Northern blot experiments. Antibodies directed against each receptor subtype were used for immunocytochemical analysis. RESULTS: RT-PCR and Northern blot analysis indicated that all RAR and RXR subtypes are present in the mature retina. Western blot analysis, using a cytoplasmic protein fraction isolated from the bovine and human neural retina, showed the presence of RXRalpha. Immunocytochemical analysis of the human, bovine, and rat retina showed that RARalpha is highly expressed in the outer segments of cone photoreceptor cells. RXRalpha expression was observed in the rod inner segment layer. RXRgamma was detected in the nuclei and outer segments of cone photoreceptor cells. CONCLUSIONS: The retinal expression pattern of RARs and RXRs is heterogeneous. The observation that RXRalpha is present in rods whereas RARalpha is present in cones, suggests a mechanism by which RA that is produced upon bleaching, could generate different responses in the two photoreceptor cell subtypes.
Asunto(s)
Proteínas Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retina/metabolismo , Factores de Transcripción/metabolismo , Animales , Northern Blotting , Western Blotting , Bovinos , Cartilla de ADN/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Persona de Mediana Edad , ARN/aislamiento & purificación , Ratas , Receptores de Ácido Retinoico/clasificación , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Factores de Transcripción/clasificación , Factores de Transcripción/genéticaRESUMEN
Subtype-specific antipeptide antibodies have been developed against each of the retinoic acid receptors (RARs alpha, beta, and gamma) and each of the retinoid X receptors (RXRs alpha, beta, and gamma). Each antibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by both immunoblot and immunoprecipitation technology. We describe a sensitive and specific assay that combines the binding of cultured cell and tumor extracts to [3H]all-trans-retinoic acid or [3H]9-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by the subtype-specific antibodies to determine the levels of functional retinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compounds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six retinoid receptor proteins ranging in concentration from 9 fmol/mg total protein for RAR beta and RXR gamma to 50 fmol/mg for RXR alpha. Hep G2 and HL60 cells express RAR alpha and RXR alpha proteins at approximately 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (approximately 5 fmol/mg) in Hep G2 cells. MCF-7 cells in culture express RAR alpha (approximately 32 fmol/mg), RAR gamma (approximately 35 fmol/mg), and RXR alpha (approximately 60 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)