RESUMEN
In this study, the effect of fibronectin on the neurite outgrowth from embryoid bodies (EBs) in neurodifferentiated embryonal carcinoma P19 cells was examined. The neurite outgrowth on fibronectin was maintained for a longer time in comparison with those on collagen or laminin. Quantitative RT-PCR revealed that mRNA level corresponding to sonic hedgehog (Shh) in neurodifferentiated P19 cells was upregulated on fibronectin, whereas collagen or laminin did not affect. Further knockdown of integrin αv subunit in P19 cells demonstrated that expression of Shh was mediated through interaction between fibronectin and integrin. Additionally, exogenous Shh agonist accelerated neurite outgrowth from embryonic stem cell-derived EBs without large change of neuronal phenotype expression. Taken together, fibronectin could maintain neurite outgrowth via increased Shh expression.
Asunto(s)
Fibronectinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Neuronas/fisiología , Tretinoina/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Proteínas Hedgehog/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa ß-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at -29/-17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.