RESUMO
BACKGROUND: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-ß signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested. METHODS: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFß3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. RESULTS: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-ß3 in MEPM cells but significantly inhibited TGF-ß receptor II. CONCLUSIONS: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFß signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.
Assuntos
Apoptose/efeitos dos fármacos , Fissura Palatina/embriologia , Fissura Palatina/prevenção & controle , Ácido Fólico/uso terapêutico , Teratogênicos , Fator de Crescimento Transformador beta3/fisiologia , Tretinoína , Complexo Vitamínico B/uso terapêutico , Animais , Caspase 9/metabolismo , Células Cultivadas , Fissura Palatina/induzido quimicamente , Ácido Fólico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Complexo Vitamínico B/farmacologiaRESUMO
PURPOSE: Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of bone marrow stromal cells (BMSCs). Bone morphogenetic protein (BMP) signaling has important effects on the process of skeletogenesis. In the present study, we tested the role of bone morphogenetic protein receptor (BMPR) in the osteogenic differentiation of rat bone marrow stromal cells in osteogenic medium (OM) with or without BMP-2. MATERIALS AND METHODS: BMSCs were harvested from rats and cultured in OM containing dexamethasone, beta-glycerophosphate, and ascorbic acid, with or without BMP-2 in order to induce osteogenic differentiation. The alkaline phosphatase (ALP) activity assay and von kossa staining were used to assess the osteogenic differentiation of the BMSCs. BMPR mRNA expression was assessed using reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The BMSCs that underwent osteogenic differentiation in OM showed a higher level of ALP activity and matrix mineralization. BMP-2 alone induced a low level of ALP activity and matrix mineralization in BMSCs, but enhanced the osteogenic differentiation of BMSCs when combined with OM. The OM significantly induced the expression of type IA receptor of BMPR (BMPRIA) and type II receptor of BMPR (BMPRII) in BMSCs after three days of stimulation, while BMP-2 significantly induced BMPRIA and BMPRII in BMSCs after nine or six days of stimulation, respectively. CONCLUSION: BMSCs commit to osteoblastic differentiation in OM, which is enhanced by BMP-2. In addition, BMP signaling through BMPRIA and BMPRII regulates the osteogenic differentiation of rat BMSCs in OM with or without BMP-2.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Células Estromais/citologia , Células Estromais/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Masculino , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/efeitos dos fármacosRESUMO
It has been well established that all-trans-retinoic acid (ATRA) influences bone metabolism when given in the treatment or prevention of cancer. However, the molecular mechanisms underlying this are unknown. In the present study, we investigated the effect of ATRA on differentiation of rat bone marrow stromal cells (BMSCs). BMSCs were harvested from rats and induced to differentiate in the presence or absence of ATRA in either osteogenic (OM) or control medium (CM). BMSCs underwent osteogenic differentiation, showed alkaline phosphatase (ALP) activity, a high level of matrix mineralization, and expressed osteonectin when cultured in OM. Although ATRA induced ALP activity, it failed to induce matrix mineralization and osteonectin, decrease mineralization in OM, and induce lipid accumulation in BMSCs. Moreover, while ATRA induced the expression of BMP-RIA, both BMP-RII and Smad5 mRNA were induced by OM and ATRA. Thus, ATRA inhibited osteogenesis and promoted adipogenesis of BMSCs. BMP signaling cooperated with ATRA in the differentiation of BMSCs.
