RESUMEN
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)
Ameloblastos/citología , Células Madre Pluripotentes Inducidas/citología , Transducción de Señal/fisiología , Proteína Smad1/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Western Blotting , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero , Técnica del Anticuerpo Fluorescente , Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad1/análisis , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
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
OBJECTIVES: To evaluate the associations between breastfeeding duration, age at solids introduction, and their interaction in relation to infant (age 9-15 months) above normal body mass index (BMI). STUDY DESIGN: Cross-sectional, population-based study with 3153 infants from Melbourne (2007-2011). Above normal BMI (z score > 2, equivalent to >97.7th percentile) defined using the World Health Organization standard. RESULTS: Both longer duration of full and any (full or partial) breastfeeding were associated with lower odds of above normal BMI (eg, aOR, 0.37 [95% CI, 0.22-0.60] for full breastfeeding 4-5 months versus 0-1 months). Compared with introduction of solids at 5-6 months, both early and delayed introduction were associated with increased odds of above normal BMI (aOR for 4 months, 1.75 [95% CI, 1.10-2.80] and for ≥7 months, 2.64 [95% CI, 1.26-5.54] versus 6 months). Such associations differ by breastfeeding status at 4 months (interaction P = .08). Early introduction of solids was associated with increased odds of above normal BMI in both infants fully or partially breastfed for ≥4 months (aOR, 3.66; 95% CI, 1.41-9.51) and those breastfed for <4 months (aOR, 3.11; 95% CI, 1.39-6.97). Introduction of solids at ≥7 months was associated with increased odds of above normal BMI (aOR, 5.79; 95% CI, 1.91-17.49) among infants breastfed for <4 months only. CONCLUSION: Introduction of solids at 5-6 months, compared with either early or delayed introduction, is associated with decreased odds of above normal BMI at 1 year of age, regardless of infants' breastfeeding status at 4 months. These results may have implications for public health guidelines with regard to recommendations about the optimal timing of the introduction of solid foods in infancy.