RESUMEN
Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Asunto(s)
Transducción de Señal/fisiología , Proteína Smad1/fisiología , Células Madre Pluripotentes Inducidas/citología , Ameloblastos/citología , Fosforilación , Factores de Tiempo , Expresión Génica , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Células Cultivadas , Western Blotting , Técnica del Anticuerpo Fluorescente , Medio de Cultivo Libre de Suero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Interferencia de ARN , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Proteína Smad1/análisisRESUMEN
Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Asunto(s)
Transducción de Señal/fisiología , Proteína Smad1/fisiología , Células Madre Pluripotentes Inducidas/citología , Ameloblastos/citología , Fosforilación , Factores de Tiempo , Expresión Génica , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Células Cultivadas , Western Blotting , Técnica del Anticuerpo Fluorescente , Medio de Cultivo Libre de Suero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Interferencia de ARN , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Proteína Smad1/análisisRESUMEN
Estudaram-se os efeitos da remoçäo das glândulas salivares maiores sobre a incorporaçäo de 3H-prolina pelos ameloblastos secretores e odontoblastos de incisivos de camundongos. Animais sialoadenectomizados e controles foram injetados com 3H-prolina, sendo sacrificados e perfundidos 30 min, 2h, 12h e 24h após. Cortes de 1*m obtidos de incisivos inferiores incluídos em Polybed foram radioautografados. A distribuiçäo percentual de gräos de prata reduzida/100*m* sobre as referidas células, assim como em suas respectivas matrizes, foi sempre maior nos animais sialoadenectomizadas, nos diferentes intervalos de tempo. Duas hipóteses poderiam explicar esses resultados: 1. um aumento na biossíntese protéica provavelmente devida à falta do fator submandibular inibidor da insulina (SII) associada a um relativo aumento do pool de 3H-prolina devido à remoçäo das glândulas salivares; 2. um decrécimo na degradaçäo protéica devido ao hipotireoidismo que eventualmente ocorre nos animais sialoadenectomizados