RESUMEN
NEDD9 (neural precursor cell expressed, developmentally down-regulated 9) is a member of the CAS (Crk-associated substrate) family of scaffolding proteins that regulate cell adhesion and migration. A Nedd9 knock-out/lacZ knock-in mouse (Nedd9(-/)(-)) was developed in order to study Nedd9 expression and function in the nervous system. Herein we show that NEDD9 is expressed in the adult brain and is prominently expressed in the hippocampus. Behavioral testing uncovered functional deficits in Nedd9(-)(/)(-) mice. In the Morris water maze test, Nedd9(-)(/)(-) mice showed deficits in both the ability to learn the task as well as in their ability to recall the platform location. There was no change in the gross morphology of the hippocampus, and stereological analysis of BrdU-labeled newly formed hippocampal cells suggested that this defect is not secondary to altered neurogenesis. However, analysis of the hippocampus revealed extensive loss of dendritic spine density in both the dentate gyrus (DG) and CA1 regions. Spine loss occurred equally across all branch orders and regions of the dendrite. Analysis of spine density in Nedd9(-)(/)(-) mice at 1.5, 6 and 10 months revealed an age-dependent spine loss. This work shows that NEDD9 is required for the maintenance of dendritic spines in the hippocampus, and suggests it could play a role in learning and memory.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Trastornos del Conocimiento/metabolismo , Espinas Dendríticas/metabolismo , Hipocampo/metabolismo , Animales , Trastornos del Conocimiento/patología , Espinas Dendríticas/patología , Femenino , Técnicas de Sustitución del Gen , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.
Asunto(s)
Axones/fisiología , Regulación de la Expresión Génica/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuritas/fisiología , Neuronas/citología , Animales , Animales Modificados Genéticamente , Axones/efectos de los fármacos , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/fisiología , Línea Celular , Chlorocebus aethiops , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Microfilamentos/fisiología , Mutación/fisiología , Factores de Crecimiento Nervioso/genética , Neuritas/efectos de los fármacos , Neuroblastoma , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Interferencia de ARN/fisiología , Factores de Tiempo , Transfección , Tretinoina/farmacología , Tubulina (Proteína)/metabolismoRESUMEN
We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from -1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from -475 to -445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Neuritas/fisiología , Fosfoproteínas/genética , Elementos de Respuesta , Tretinoina/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Exones , Humanos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Fosfoproteínas/fisiología , ARN Mensajero/genética , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Regulación hacia ArribaRESUMEN
INTRODUCTION: 2-Methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD) is a new analog of 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that has unique properties (distinct from 1alpha,25-dihydroxyvitamin D3) in stimulating osteoblasts to form bone in culture. This analog has now been extensively tested in aged ovariectomized female rats maintained on a diet adequate in calcium and phosphorus. METHODS: Retired female rats obtained from Sprague-Dawley were ovariectomized, and were either dosed with vehicle or 2MD at 5-7 ng/kg body weight each day. RESULTS: A marked increase in total bone mass resulted during the 28-week study. This increase in bone mass resulted from an increase in both cortical and trabecular bone, with increases to the order of 25% in the cancellous bone. Histomorphometry revealed that 2MD increased bone mass primarily by increasing bone formation. It also revealed little or no effect on bone resorption. The resulting bone is of high quality revealed by histology and biomechanical testing. CONCLUSION: Throughout the study, serum calcium remained within the normal range and thus 2MD shows great promise for the treatment of bone diseases characterized by bone loss, including osteoporosis.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Calcitriol/análogos & derivados , Osteogénesis/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Anabolizantes/uso terapéutico , Análisis de Varianza , Animales , Densidad Ósea/efectos de los fármacos , Calcitriol/uso terapéutico , Calcio/sangre , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
The vitamin A metabolite, all-trans retinoic acid (atRA), plays an important role in neuronal development, including neurite outgrowth. However, the genes that lie downstream of atRA and its receptors in neuronal cells are largely unknown. By using the human neuroblastoma cell line, SH-SY5Y, we have identified an atRA-responsive gene (RAINB1: retinoic acid inducible in neuroblastoma cells) that is induced within 4 h after exposure of SH-SY5Y cells to atRA. RAINB1 mRNA is highly expressed in the nervous system (10.5- to 11-kb transcript) in both developing embryos and adults. Its expression is perturbed in developing rat embryos exposed to excess or insufficient atRA. RAINB1 is present on chromosome 11 and is spread over 38 exons, resulting in a putative ORF of 2,429 amino acids. The RAINB1 protein shows high similarity to a gene in Caenorhabditis elegans, unc-53, that is required for axonal elongation of mechanosensory neurons, suggesting that these proteins are orthologs. Thus, RAINB1 may represent a critical downstream gene in atRA-mediated neurite outgrowth.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Cromosomas Humanos Par 11/genética , Clonación Molecular , Embrión de Mamíferos/metabolismo , Embrión no Mamífero , Exones/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Sistema Nervioso/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Mapeo Físico de Cromosoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Tretinoina/administración & dosificación , Células Tumorales CultivadasRESUMEN
Both nerve growth factor (NGF) and neurotrophin-3 (NT-3) are necessary for the survival of embryonic sympathetic neurons in vivo. All-trans retinoic acid (atRA) has been shown to promote neurite outgrowth and long-term survival of chick embryonic sympathetic neurons cultured in the presence of NGF. The present study shows that atRA can also potentiate the survival and neurite outgrowth-promoting activities of NT-3. This was accomplished by enhancing the survival of existing neurons, as cell proliferation was unaffected by exposure to atRA. atRA also enhanced neurite outgrowth of the NT-3-treated cells; however, the neurites appeared thicker and less branched than cells treated with atRA in combination with NGF. Using a quantitative PCR assay, trkA and p75(NTR) mRNAs, but not trkC mRNA, were increased ( approximately 1.5- to 2-fold) after 72 and 48 hr of exposure of the cultures to atRA, respectively. The atRA-induced increase in trkA mRNA may play a role in the enhanced survival of neurons cultured in the presence of either NGF or NT-3, as both neurotrophins have been shown to signal through this receptor. The time course of these mRNA changes would indicate that atRA does not regulate the neurotrophin receptor mRNA directly, rather, intervening gene transcription is required. Thus, during development, atRA may play a role in fine-tuning embryonic responsiveness to both NT-3 and NGF.
Asunto(s)
Neuronas/citología , Neuronas/metabolismo , Neurotrofina 3/uso terapéutico , Tretinoina/uso terapéutico , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Interacciones Farmacológicas , Queratolíticos/uso terapéutico , Factor de Crecimiento Nervioso/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/biosíntesis , Receptor trkC/biosíntesis , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Factores de TiempoRESUMEN
The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic effects.
Asunto(s)
Antineoplásicos/farmacocinética , Fenretinida/análogos & derivados , Fenretinida/farmacocinética , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Biotransformación , Femenino , Fenretinida/síntesis química , Fenretinida/farmacología , Hidrólisis , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratas , Vitamina A/sangreRESUMEN
Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.
Asunto(s)
Proteínas de Drosophila , Epitelio/efectos de los fármacos , Riñón/embriología , Mesodermo/efectos de los fármacos , Vitamina A/farmacología , Animales , Epitelio/metabolismo , Femenino , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Hibridación in Situ , Riñón/anomalías , Riñón/efectos de los fármacos , Riñón/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Morfogénesis/efectos de los fármacos , Mutación , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras , Transducción de Señal/efectos de los fármacos , Vitamina A/administración & dosificación , Vitamina A/genética , Deficiencia de Vitamina A/genética , Deficiencia de Vitamina A/fisiopatologíaRESUMEN
The antitumor effects of N-(4-hydroxyphenyl)retinamide (4-HPR), and its stable C-linked analog, 4-hydroxybenzylretinone (4-HBR) on the regression of established 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors were compared. 4-HBR is a stable and nonhydroyzable derivative which cannot be converted in vivo to retinoic acid (RA). The results indicate that 4-HBR decreased mammary tumor volumes to the same extent as equimolar concentration (2 mmol/kg diet) of 4-HPR (-45% for 4-HBR vs. -42% for 4-HPR, p<0.01). Both 4-HPR and 4-HBR bind very poorly to nuclear retinoid receptors RARs and RXRs. The similarity of physicochemical properties of 4-HPR and 4-HBR as well as their equal antitumor potency suggests that 4-HPR like 4-HBR, is acting directly rather than through hydrolysis to free RA. Treatment with 4-HPR caused an almost 65% decrease in serum retinol levels. These results suggest that 4-HBR may have a significant chemotherapeutic advantage over 4-HPR, as the nonhydrolyzable analog may not cause night blindness which occurs as a significant side effect of 4-HPR usage.
Asunto(s)
Antineoplásicos/farmacología , Fenretinida/análogos & derivados , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Vitamina A/farmacología , 9,10-Dimetil-1,2-benzantraceno , Animales , Anticarcinógenos/metabolismo , Anticarcinógenos/farmacología , Antineoplásicos/metabolismo , Carcinógenos , Femenino , Fenretinida/metabolismo , Fenretinida/farmacología , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/metabolismo , Vitamina A/análogos & derivados , Vitamina A/sangre , Vitamina A/metabolismoRESUMEN
BACKGROUND: Normal embryonic development and survival in utero is dependent on an adequate supply of vitamin A. Embryos from vitamin A-deficient (VAD) pregnant rats fed an inadequate amount of all-trans retinoic acid (atRA; 12 microg per g of diet or approximately 230 microg per rat per day) exhibit severe developmental abnormalities of the anterior cardinal vein and hindbrain by embryonic day (E) 12.5 and die shortly thereafter. METHODS: In the present study, we sought to determine whether supplementation of VAD-RA supported (12 microg per g of diet) pregnant rats with retinol (ROL) at the late-gastrula (presomite or rat E9.5) or early somite stages (E10.5), or provision of higher levels of atRA throughout this period could prevent abnormalities in the developing cardiovascular and nervous systems. RESULTS: A newly described defect in the sinuatrial venus valve along with enlarged anterior cardinal veins and nervous system abnormalities and the later death of embryos are prevented by supplementing pregnant animals with ROL on the morning of E9.5. If ROL supplementation is delayed by 1 day (E10.5), most embryos are abnormal and die by E18.5. Supplementation of VAD rats with atRA (250 microg per g of diet) between E8.5 and E10.5 also prevents the cardiovascular and nervous system abnormalities and a significant number of these embryos survive to parturition. Thus, high levels of atRA can obviate the need for ROL between E9.5 and E10.5. CONCLUSIONS: These results support an essential role for retinoid signaling between the late gastrula and early somite stages in the rat embryo for normal morphogenesis of the primitive heart tube and the posterior hindbrain. Further, these results suggest that embryonic death occurring at midgestation in the VAD rat may be linked to the abnormal development of one or both of these embryonic structures.
Asunto(s)
Anomalías Múltiples/etiología , Corazón Fetal/efectos de los fármacos , Reabsorción del Feto/etiología , Complicaciones del Embarazo/fisiopatología , Rombencéfalo/anomalías , Tretinoina/uso terapéutico , Venas/anomalías , Deficiencia de Vitamina A/fisiopatología , Vitamina A/análogos & derivados , Anomalías Múltiples/prevención & control , Alimentación Animal , Animales , Nervios Craneales/anomalías , Nervios Craneales/embriología , Diterpenos , Relación Dosis-Respuesta a Droga , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Muerte Fetal/etiología , Muerte Fetal/prevención & control , Reabsorción del Feto/prevención & control , Gástrula/efectos de los fármacos , Genes Homeobox , Edad Gestacional , Morfogénesis/efectos de los fármacos , Embarazo , Complicaciones del Embarazo/sangre , Ratas , Ésteres de Retinilo , Rombencéfalo/embriología , Factores de Transcripción/genética , Tretinoina/administración & dosificación , Venas/embriología , Vitamina A/administración & dosificación , Vitamina A/uso terapéutico , Deficiencia de Vitamina A/sangreRESUMEN
The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5-like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal.
Asunto(s)
Rombencéfalo/embriología , Deficiencia de Vitamina A/embriología , Animales , Biomarcadores , Nervios Craneales/embriología , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Oído/embriología , Proteína 2 de la Respuesta de Crecimiento Precoz , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Rombencéfalo/anomalías , Rombencéfalo/efectos de los fármacos , Factores de Tiempo , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Vitamina A/farmacología , Vitamina A/fisiologíaRESUMEN
Interactions between vitamin A and vitamin D have been suggested for several decades but have not been established. In particular, vitamin A has been proposed to intensify the severity of the bone mineralization disease, rickets and inhibit the ability of vitamin D to cure this disease. To investigate this hypothesis, weanling Holtzman rats were fed a 1.2% calcium, 0.1% phosphorus diet and 15.5 ng ergocalciferol (vitamin D(2)) every 3 d for 21 d in the presence of increasing amounts of retinyl acetate (0 microg to 8621 microg/d). The increasing amounts of retinyl acetate produced a progressive and significant decrease in total bone ash (P < 0.001) and an increase in epiphyseal plate width (P < 0.001). The same experiment conducted with increasing amounts of vitamin D(2) (0 to 645 ng/d) indicated that the antagonism by retinyl acetate could be demonstrated at all vitamin D(2) dosages. To further investigate this antagonistic relationship, weanling Holtzman rats were fed a 0. 47% calcium, 0.3% phosphorus diet and 15.5 ng vitamin D(2) every 3 d for 33 d in the presence of increasing retinyl acetate (0 to 3448 microg/d). In the absence of retinyl acetate, these rats maintained a normal serum calcium level (2.34 mmol/L). Increasing retinyl acetate, however, eliminated the ability of vitamin D(2) to elevate the level of serum calcium (1.35 mmol/L). These results illustrated in vivo antagonism of vitamin D(2) action on intestine and bone by retinyl acetate.
Asunto(s)
Vitamina A/farmacología , Vitamina D/antagonistas & inhibidores , Animales , Calcio/sangre , Diterpenos , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/crecimiento & desarrollo , Masculino , Minerales/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Ésteres de Retinilo , Vitamina A/análogos & derivadosRESUMEN
The long term chemopreventive effects of the N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG), and its stable C-linked benzyl glucuronide analog, retinamidobenzyl glucuronide (4-HPRCG) on the growth and development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors were compared. The retinamidobenzyl glucuronide is stable toward acid hydrolysis and resists the actions of beta-glucuronidase. The results indicate that the C-linked glucuronide analog, 4-HPRCG has a greater chemopreventive potency than an equimolar concentration of 4-HPROG. Tumor latency was 15% longer in rats fed 2 mmol/kg diet of 4-HPRCG as compared to 4-HPROG. At 80 days post DMBA-intubation, tumor incidence was 57% and 27% in the 4-HPROG and 4-HPRCG treated rats, respectively. Tumor multiplicity was also markedly decreased in the 4-HPRCG treated rats. At 80 days post DMBA intubation the control rats had an average of 1.43 tumors/rat compared to 0.71 and 0.36 tumors/rat in the 4-HPROG and 4-HPRCG respectively. The higher potency and low toxicity of 4-HPRCG suggest that this stable analog may have an in vivo chemopreventive advantage over its analog, 4-HPROG. The results also demonstrated that these glucuronide analogs do not bind effectively in vitro either to the nuclear retinoid receptors or to the cellular retinoid binding proteins. Regardless of the mode of action of these retinoids, they are clearly effective chemopreventive agents.
Asunto(s)
Anticarcinógenos/uso terapéutico , Fenretinida/análogos & derivados , Glucuronatos/uso terapéutico , Neoplasias Mamarias Experimentales/prevención & control , Retinoides/uso terapéutico , 9,10-Dimetil-1,2-benzantraceno , Animales , Femenino , Fenretinida/uso terapéutico , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.
Asunto(s)
Reabsorción del Feto/prevención & control , Queratolíticos/farmacología , Rombencéfalo/embriología , Tretinoina/farmacología , Deficiencia de Vitamina A/complicaciones , Animales , Dieta , Femenino , Reabsorción del Feto/etiología , Reabsorción del Feto/metabolismo , Intercambio Materno-Fetal , Embarazo , Ratas , Ratas Sprague-Dawley , Rombencéfalo/anomalías , Rombencéfalo/metabolismoRESUMEN
Retinoic acid receptors (RAR) are members of the steroid/thyroid hormone receptor superfamily and serve as ligand-activated transcription factors. In order to facilitate studies of receptor protein, we have generated a monoclonal antibody to the human RAR gamma, and have developed a procedure to purify the full-length receptor expressed in insect cells. The monoclonal antibody (A10) was developed using as antigen a carboxy-terminal fragment of the human RAR gamma expressed as a bacterial fusion protein. The A10 monoclonal antibody binds to both native and denatured forms of the human RAR gamma. This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RAR gamma to near homogeneity. The immunoaffinity-purified receptor is > 90-95% pure as revealed by silver-stained gels. The identity of the single protein band as RAR gamma was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RAR gamma is functional with respect to both ligand and DNA binding. Scatchard analysis of 3H-labeled all-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a Kd of approximately 2 nM. Binding of the pure RAR gamma to a DR5-type retinoic acid response element was also studied. Response element binding by RAR gamma required the presence of the retinoid X receptor, but did not require the presence of additional proteins. Human RAR gamma protein purified in this fashion will be useful in future structural and functional studies.
Asunto(s)
Receptores de Ácido Retinoico/aislamiento & purificación , Animales , Anticuerpos Monoclonales , Baculoviridae/genética , Sitios de Unión , Cromatografía de Afinidad , Escherichia coli/genética , Humanos , Cinética , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Ratas , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera , Receptor de Ácido Retinoico gammaAsunto(s)
Receptores de Ácido Retinoico/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Línea Celular , Clonación Molecular/métodos , Escherichia coli , Vectores Genéticos , Humanos , Cinética , Ratones , Receptores de Ácido Retinoico/aislamiento & purificación , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide , Retinoides/farmacología , Spodoptera , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Transfección/métodos , Tretinoina/metabolismoRESUMEN
The discovery and development of information surrounding the retinoic acid receptors (RAR and RXR) has ushered in a new era in understanding the molecular mechanism of action of vitamin A in embryonic development and cellular differentiation. The mechanisms involved in the regulation of gene expression by the retinoids is at least partially known and involves binding of the RAR and RXR to retinoic acid response elements. Additional factors, including coregulatory proteins, associated regulatory elements, and cell-specific factors, may also be involved in determining the specificity of retinoid-regulation of gene expression during development. During embryogenesis, retinoids are required for the development of the posterior hindbrain and its associated structures, as well as for the survival and differentiation of certain classes of neurons and neural crest cell derivatives. At least some of the effects of retinoid on hindbrain development are related to the regulation of Hox gene expression. Additional retinoid-regulated genes have been implicated in nervous system development, and the manner in which they lead to phenotypic changes during embryogenesis remains to be determined.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Retinoides/farmacología , Animales , Humanos , Sistema Nervioso/efectos de los fármacos , Retinoides/metabolismoRESUMEN
All-trans 3,4-didehydroretinoic acid (at-ddRA) has been identified as a biologically important retinoid in avian, but not mammalian, embryonic development. In this report, we show that at-ddRA, like all-trans retinoic acid (atRA), supports the survival and differentiation of sympathetic neurons of the embryonic chick. Furthermore, the expression of the retinoid-responsive gene RARbeta2 is increased in neurons exposed to either at-ddRA or atRA. The mechanism whereby at-ddRA exerts its effects in chick neurons may involve binding to and activation of nuclear retinoid receptors. For this reason, the binding of recombinant chick RARbeta2 to at-ddRA and to receptor-specific DNA response elements was examined and compared with the binding characteristics of recombinant murine RARbeta2. The chick RARbeta2, like the mammalian RAR, binds to [3H]atRA with high affinity (Kd=0.7-2 nM). Furthermore, both chick and murine RARbeta2 bind equally well to at-ddRA, atRA, and 9-cis RA, but neither receptor shows appreciable binding to 13-cis RA. The chick RARbeta2 recognizes previously described retinoic acid response elements of mammalian gene promoters and, like mammalian RARbeta2, shows enhanced binding in the presence of RXR. This study provides evidence that at-ddRA, like atRA, supports neuronal development in the chick by its interaction with nuclear retinoid receptors.
Asunto(s)
Ganglios Simpáticos/efectos de los fármacos , Neuronas/efectos de los fármacos , Tretinoina/análogos & derivados , Tretinoina/farmacología , Animales , Baculoviridae/genética , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Ganglios Simpáticos/embriología , Ligandos , Ratones , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , ARN Mensajero/análisis , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad de la EspecieRESUMEN
The O-glucuronide analog of N-(4-hydroxyphenyl)retinamide (4-HPROG) has shown a greater chemopreventive activity than the parent N-(4-hydroxyphenyl)retinamide (4-HPR). However, this compound is relatively unstable. In order to improve stability and efficacy, we have prepared a number of stable C-linked analogs of 4-HPROG (C-phenyl and C-benzyl glucuronosyl, glucosyl, and xylosyl analogs). These analogs are stable toward acid hydrolysis and the glucuronosyl analogs resist the actions of beta-glucuronidase. The analogs were prescreened for their antiproliferative potential in vitro using cultured human MCF-7 breast cancer cells. Selected analogs were then evaluated for their ability to inhibit the development and growth of tumors in the 7,12-dimethylbenzanthracene-induced rat mammary tumor model. Although the stable C-linked analogs bound poorly to the nuclear retinoic acid receptors, many showed more potency than the less stable 4-HPROG in inhibiting tumor incidence and multiplicity in vivo. The glucuronide/glucoside analogs are more potent than the xylosides, and the C-benzyl more effective than the C-phenyl analogs. The higher potency of at least two C-linked analogs (retinamidobenzyl glucuronide and retinamidobenzyl glucose) suggests that these analogs may have a chemopreventive advantage over the parent retinamide and its natural O-glucuronide.
Asunto(s)
Anticarcinógenos/uso terapéutico , Fenretinida/análogos & derivados , Glucuronatos/uso terapéutico , 9,10-Dimetil-1,2-benzantraceno , Animales , Anticarcinógenos/química , Neoplasias de la Mama/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Fenretinida/química , Fenretinida/uso terapéutico , Glucuronatos/química , Humanos , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/prevención & control , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
In explanted embryonic chick sympathetic neurons, all-trans retinoic acid (RA) as well as nerve growth factor (NGF) were found to be required for neuronal survival and neurite outgrowth at early stages of development (day 6.5-7) in agreement with previous work (Rodriguez-Tébar and Rohrer [1991] Development 112:813-820). The dependence of neurons on all-trans RA for survival diminished at later stages of development. However, all-trans RA was found to be needed at all stages of development in order to maximize neurite outgrowth. Further, removal of all-trans RA from the cultures led to a rapid degeneration of the formed neurites, demonstrating the essentiality of all-trans RA for both the development of neurites, and for the maintenance of existing neurites in cultured embryonic sympathetic neurons. The mechanism whereby all-trans RA exerts its effects on embryonic sympathetic neurons may involve activation of the nuclear retinoic acid and retinoid-X receptor (RAR and RXR) families. The results of Northern blot analyses and/or reverse transcriptase-polymerase chain reaction (RT-PCR) studies show that embryonic sympathetic ganglia express RAR beta, RAR gamma and RXR gamma mRNAs. RXR gamma mRNA is expressed at highest levels in immature neurons that are not yet responsive to NGF (day 6.5-7) and message levels decline with increasing developmental age. In contrast, RAR beta transcript levels are barely detectable at day 6.5-7, and increase approximately 4-fold in ganglia from embryos at day 8.5-9 and decline thereafter. RT-PCR studies show that RAR gamma mRNA is expressed both early (day 6.5-7) and late (day 15) in ganglionic development. Transcripts for the NGF receptors, p75NGFR and p140trk were also examined. The appearance of a single 2.7 kb p140trk transcript coincides with the time when RAR beta mRNA is maximally expressed, raising the possibility that NGF receptors may be targets of retinoid action. Evidence is also presented that all-trans RA may enhance neurite outgrowth by mechanisms other than simply inducing NGF-responsiveness of neurons.