Investigation of molecular regulators in osteoblast differentiation

Bones provide mechanical support for movement and normal daily functions, which can only be provided when healthy. Bone diseases such as osteoporosis and cancer are a worldwide problem affecting millions of people and causing significant financial impact. This study should indicate possible solutions that might contribute to combatting bone diseases by understanding the expression of bone markers that are known to be expressed differentially at different stages of osteoblastic differentiation. Thus a reliable, easily available and easy to maintain bone cell model was selected using osteosarcoma cell lines (MG-63, TE-85 and SaOS-2). The effect of ATP and PTH treatments on the expression of these markers was also tested to determine differences with primary osteoblasts (HOBs). We also determined the differential expression of miRNAs between the cell lines and HOBs and identified miRNAs involved in regulating expression of sclerostin, examining also their effect on the properties of trabecular bone in a mouse model. We hypothesized that these models were appropriate for studying the biology of osteoblastic cells and that miRNAs could control bone cell phenotype. In the first objective of this study, the expression of seven bone markers actively involved in bone development (OPG, ALP, COL1A2, COL6A3, SOST, OSX and RANKL) were investigated. The expression of OPG, COL1A2 and COL6A3 were significantly higher in the least differentiated cell line, MG-63, while expression of ALP, SOST, OSX and RANKL were highest in SaOS-2, the most differentiated. ATP and PTH exerted the expected inhibitory or inductive effects on the cells at the osteoblastic maturation stage confirming that the cell lines were a suitable model for the study of osteoblasts. MicroRNA profiling of cell lines and HOBs revealed hundreds of differentially expressed miRNAs. Six that were predicted to target the regulation of SOST were chosen for further validation. Mimics and antagomirs of these were transfected into TE-85 and SaOS-2 cells to confirm their involvement in SOST expression regulation. SOST gene expression and protein production were quantified by qPCR and ELISA, revealing that miR-1231, miR-1254 and miR-1914 decreased SOST protein expression in TE-85 and SaOS-2 cells and miR-378a-3p in just TE-85 cells, but these effects were not reversed by the corresponding antagomir. Adult and elderly mice were treated with miR-378a-3p mimic and antagomir by injection into the tail vein. Few differences could be observed in trabecular bone three weeks following treatment, except for significantly increased trabecular pattern factor and decreased trabecular separation in elderly mice treated with miR-378a-3p mimic. In conclusion, this thesis demonstrates that ATP and PTH regulate osteosarcoma cell lines similarly to HOBs and that miRNAs can be used to control SOST protein expression. Potential future studies should investigate miRNAs as novel therapies for bone diseases