RESUMEN
Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Müller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca2+-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.
Asunto(s)
Apoptosis/fisiología , Efrina-A3/biosíntesis , Efrina-A4/biosíntesis , Glaucoma/metabolismo , Células Ganglionares de la Retina/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Glaucoma/inducido químicamente , Inyecciones Intravítreas , Masculino , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Espermina/administración & dosificación , Espermina/análogos & derivados , Espermina/toxicidadRESUMEN
MicroRNA (miR) plays an important role in tumorigenesis including malignant peripheral nerve sheath tumor (MPNST). miR-210 downregulation is frequently observed in a variety of tumors. In this study, miR-210 was identified as downregulated in MPNST cells, and its potential target ephrin-A3 (EFNA3) was upregulated in them compared with neurofibroma cells using quantitative real-time (qRT)-PCR. Luciferase reporter assay further demonstrates that EFNA3 is a target of miR-210. Then it is confirmed that miR-210 can regulate EFNA3 mRNA and protein expression in MPNST ST88-14 (NF1 wild-type) and sNF96.2 (NF1 mutant type) cell lines. The functions of miR-210 in MPNST cells were investigated, and the results showed that overexpression of miR-210 increased cellular viability, colony formation, S phase percentage, and invasiveness of MPNST cells. Inversely, inhibition of miR-210 expression induced suppression of proliferation and invasion of MPNST cells. These results suggest that miR-210-mediated EFNA3 promotion of proliferation and invasion of MPNST cells plays an important role in MPNST tumorigenesis and progression. miR-210 and EFNA3 may be candidate novel therapeutic targets for MPNST.
Asunto(s)
Efrina-A3/genética , MicroARNs/genética , Neurofibroma/genética , Neurofibroma/patología , Procesos de Crecimiento Celular/genética , Regulación hacia Abajo , Efrina-A3/biosíntesis , Efrina-A3/metabolismo , Células HEK293 , Humanos , MicroARNs/biosíntesis , MicroARNs/metabolismo , Invasividad Neoplásica , Neurofibroma/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Fase S/genética , TransfecciónRESUMEN
OBJECTIVE: To investigate the relationship among mossy fiber axon sprouting(MFS), synaptic reorganization, and the alteration of expression of Eph A5 and ephrin A3 in the dentate gyrus in rats with pilocarpine-induced chronic temporal lobe epilepsy. METHODS: Mossy fiber sprouting and synaptic formation in rats were observed by Neo-Timm staining, after the acute status epilepticus and chronic spontaneous temporal lobe epilepsy induced by lithium-chloride and pilocarpine. In situ hybridization was used to detect ephrin A3 mRNA and an immunohistochemical staining was applied to determine Eph A5 protein. RESULTS: In entorhinal cortex, only Eph A5 mRNA and protein expressed, which significantly decreased on Day 7 after pilocarpine induced status epilepticus(P<0.01),and resumed to normal levels on Day 30 (P>0.05). In the dentate granule cells, ephrin A3 mRNA reduced obviously on Day 7 after pilocarpine-induced status epilepticus (P<0.01), and returned to normal levels on Day 30 (P>0.05). CONCLUSION: The down-regulation of Eph A5 mRNA and protein in entorhinal cortex and dentate gyrus, and ephrin A3 mRNA in dentate gyrus after status epilepticus may be part of the endogenous molecular mechanism of mossy fiber sprouting to the inner molecular layer of dentate gyrus.
Asunto(s)
Axones/fisiología , Giro Dentado/fisiopatología , Efrina-A3/biosíntesis , Epilepsia del Lóbulo Temporal/metabolismo , Receptor EphA5/biosíntesis , Animales , Emparejamiento Cromosómico/fisiología , Regulación hacia Abajo , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/fisiopatología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/fisiopatologíaRESUMEN
MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.
Asunto(s)
Células Endoteliales/metabolismo , Efrina-A3/biosíntesis , Regulación de la Expresión Génica , MicroARNs/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/genética , Células Endoteliales/citología , Efrina-A3/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , ARN sin Sentido/genética , Transfección , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Androgenic alopecia (AGA) is the most common type of baldness in men. Although etiological studies have proved that androgen is one of the causes of this symptom, the defined molecular mechanism underlying androgen-related actions remains largely unknown. OBJECTIVES: To clarify the difference in the gene expression profile of dermal papilla cells (DPCs) in skin affected by baldness. METHODS: DNA macroarray study was carried out on cultured DPCs from AGA skin comparing with DPCs from skin that is not affected by baldness. RESULTS: From DNA macroarray analysis, we observed that 107 of the 1185 analyzed genes had differing expression levels. A marked difference was observed in the decreased gene expression of BMP2 and ephrin A3 and up-regulated in NT-4 gene. In order to clarify the roles of BMP2 and ephrin A3 in the hair follicles, we examined the proliferation of hair follicle keratinocyte and expression of a hair acidic keratin gene. Both BMP2 and ephrin A3 raised the proliferation rate of the outer root sheath cells (ORSCs) and induced gene expression in acidic hair keratin 3-II. CONCLUSION: These results lead us to the hypothesis that both BMP2 and ephrin A3 function as hair growth promoting factors in the hair cycle.