Indexed on: 30 Aug '16Published on: 30 Aug '16Published in: Biochemical and Biophysical Research Communications
Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. L-thyroxine was selected as a novel drug candidate. L-thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of L-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, L-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, L-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, L-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that L-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines, which suggests that L-thyroxine could be a potential bone production agent.