RESUMO
OBJECTIVES: Osteoblasts are derived from Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), which play an indispensable role in bone formation. In this study, the authors aim to investigate the role of IRF4 in the osteogenic differentiation of BM-MSCs and its potential molecular mechanism. METHODS: The authors used lentivirus infection to overexpress IRF4 in BM-MSCs. The expression of IRF4 and osteogenesis-related genes were detected by qRT-PCR and western blot analysis. The osteogenic differentiation of BM-MSCs was evaluated by Alkaline Phosphatase (ALP) activity, Alizarin red staining, and Alkaline Phosphatase (ALP) staining. Chromatin Immunoprecipitation (ChIP), Dual-Luciferase reporter assay and RNA Immunoprecipitation Assay were applied to confirm the regulatory mechanism between IRF4, miR-636 and DOCK9. RESULTS: The authors found IRF4 was down-regulated during the osteogenic differentiation of BM-MSCs, and IRF4 overexpression could decrease the osteogenic differentiation of BM-MSCs by specifically promoting the reduction of Alkaline Phosphatase (ALP) activity and down-regulating osteogenic indicators, including OCN, OPN, Runx2 and CollA1. Mechanistically, IRF4 activated microRNA-636 (miR-636) expression via binding to its promoter region, and Dedicator of Cytokinesis 9 (DOCK9) was identified as the target of miR-636 in BM-MSCs. Moreover, the damage in the capacity of osteogenic differentiation of BM-MSCs induced by IRF4 overexpression could be rescued by miR-636 inhibition. CONCLUSIONS: In summary, this paper proposed that IRF4/miR-636/DOCK9 may be considered as targets for the treatment of osteoporosis (OP).
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Fatores Reguladores de Interferon , Células-Tronco Mesenquimais , MicroRNAs , Fosfatase Alcalina , Diferenciação Celular/genética , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores Reguladores de Interferon/metabolismo , MicroRNAs/genética , Osteogênese/genéticaRESUMO
Abstract Objectives Osteoblasts are derived from Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), which play an indispensable role in bone formation. In this study, the authors aim to investigate the role of IRF4 in the osteogenic differentiation of BM-MSCs and its potential molecular mechanism. Methods The authors used lentivirus infection to overexpress IRF4 in BM-MSCs. The expression of IRF4 and osteogenesis-related genes were detected by qRT-PCR and western blot analysis. The osteogenic differentiation of BM-MSCs was evaluated by Alkaline Phosphatase (ALP) activity, Alizarin red staining, and Alkaline Phosphatase (ALP) staining. Chromatin Immunoprecipitation (ChIP), Dual-Luciferase reporter assay and RNA Immunoprecipitation Assay were applied to confirm the regulatory mechanism between IRF4, miR-636 and DOCK9. Results The authors found IRF4 was down-regulated during the osteogenic differentiation of BM-MSCs, and IRF4 overexpression could decrease the osteogenic differentiation of BM-MSCs by specifically promoting the reduction of Alkaline Phosphatase (ALP) activity and down-regulating osteogenic indicators, including OCN, OPN, Runx2 and CollA1. Mechanistically, IRF4 activated microRNA-636 (miR-636) expression via binding to its promoter region, and Dedicator of Cytokinesis 9 (DOCK9) was identified as the target of miR-636 in BM-MSCs. Moreover, the damage in the capacity of osteogenic differentiation of BM-MSCs induced by IRF4 overexpression could be rescued by miR-636 inhibition. Conclusions In summary, this paper proposed that IRF4/miR-636/DOCK9 may be considered as targets for the treatment of osteoporosis (OP).
RESUMO
Icariin, a prenylated flavonol glycoside isolated from Epimedium, has been considered as a potential alternative therapy for osteoporosis. The present study aimed to clarify the detailed molecular mechanisms of action of icariin on osteoblast function, using bone marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were first stimulated by icariin. Then, gene and protein expression of cAMP/PKA/CREB signaling molecules were analyzed by RT-PCR and western blotting (WB), and alkaline phosphatase (ALP) was analyzed in cell lysates by ELISA. MTT assays indicated that icariin did not have significant effects on cell viability up to 1 µM. Icariin showed a dose-dependent effect on the alkaline phosphatase activity of BM-MSCs. WB analysis showed that icariin treatment of BM-MSCs significantly enhanced the protein expression of protein kinase A (PKA) and cAMP-responsive element binding protein (CREB), while RT-PCR results showed that icariin dose-dependently increased the mRNA levels of PKA and CREB. Icariin induced BM-MSC differentiation by BMP2, Smad1, and Runx2. RT-PCR and WB results indicated that icariin significantly increased the expression of BMP2, Smad1, and Runx2 in BM-MSCs. These results suggest that icariin is an agonist of the cAMP/PKA/CREB pathway in BM-MSC differentiation, raising the possibility that it could be used in the treatment of osteoporosis.