Nuclear FOXO1 localization and osteogenesis in MC3T3 cells and C3H10T12 cells
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FOXO1A, a human gene with genetic material that fits into the forkhead family of transcription factors, takes part in significant roles in regulating the expression of genes concerned in cell growth, propagation, differentiation, and prolonged existence. The exact function of FOXOIA protein (FKHR) has not so far been established. Alkaline phosphatase (ALP) is a basic marker of osteoblast maturation and osteogenesis. Its activity is commonly measured as an index of the presence of osteoblasts and osteogenic differentiation. Previous studies have suggested that FOXO1 is a regulator of gene expression for cell differentiation and maturation. Recent studies have shown that alkaline phosphatase levels and nuclear FOXO1 levels increase during osteoblast differentiation and maturation, specifically in the murine MC3T3 cell line. It has been suggested that alkaline phosphatase is a target gene regulated by FKHR and that FKHR plays an important role in the maturation and osteogenesis of osteoblasts. The first objective of this study is to confirm the previous results. To extend these results, we attempted to study the expression of FOXO1 in osteogenic cells and the effects of FOXO1 on transcription of the alkaline phosphatase gene. We believed that by silencing the FOXO1 gene, we could silence alkaline phosphatase activity. Murine MC3T3-E1 cells and C3H10T1/2 cells induced or enhanced by BMP-2 were used to test this hypothesis. Results suggest that alkaline phosphatase activity and nuclear FOXO1 levels increase in cultured MC3T3 cells and BMP-2 treated C3H10T1/2 cells. Maximum ALP activity was observed by day 21 of 4.9-fold compared to day 0 and maximum nuclear FOXO1 levels were observed by day 14 of 2.6-fold compared to day 0. Results suggest that alkaline phosphatase activity could decrease in cultured C3H10T1/2 cells transfected with FOXOI siRNA with significant silencing at days 14 and 17 (of 2.4-fold and 2.6-fold; respectively, compared to day 0). However, scrambled SiRNA also silenced alkaline phosphatase activity. In conclusion, the stimulated FOXO1 gene may indeed play a role in the regulation of ALP activity. Our experiments show a significant association between the nuclear localization of FOX01 and increased ALP activity. Future studies are necessary to confirm their relationship in more detail.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 42-48.
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