Transfection and overexpression of 11-ß-hydroxysteroid kinase type 1 in human osteosarcoma cells (HOS 58) and primary human osteoblasts

Objectives: The enzyme 11-beta-hydroxysteroiddehydrogenase (11ßHSD1) converts inactive cortisone in peripheral cells in active cortisol. Our aim was to establish a transfection method in a human osteosarcoma cell line (HOS 58 cells) and primary human osteoblasts (pHOB) for overexpression of 11ßHSD1 to investigate the role of high levels of this enzyme on osteoblast differentiation.

Methods: HOS 58 osteosarcoma cells and primary human osteoblasts (pHOB) were grown and subcultured as described before. Transfection of ßHSD1 plasmid DNA was performed via electroporation, lipofectamien (Qiagen) and effectene (Invitrogen). pUC19 plasmid was used as negative control vector. RNA was extracted from transfected cells 48h past transfection and applied to semiquantitative RT-PCR to evaluate transfection efficiency and overexpression of ßHSD1.

Results: The basal expression of ßHSD1 was very low in pHOB and not detectable in HOS 58 cells. Transfection experiments were performed to compare the transfection efficiency of the three transfection methods in use. In HOS 58 cells lipofectamine and effectene were both very effective and we observed equally high expression of ßHSD1 while after electroporation 90% of the cells died. In pHOB cells ßHSD1 expression was increased up to 200fold when lipofectamine-tranfected but only threefold by effectene transfection. Electroporation enhanced ßHSD1 expression only twofold and decreased the viability of the cells by 60%. In HOS 58 cells expression of ßHSD1 was detectable up to seven days after transient transfection.

Conclusion: The cell line HOS 58 as well as pHOB cells are suitable for transfection experiments with ßHSD1. For further experiments HOS 58 cells could be transfected using either lipofectamine or effectene, pHOB cells should be transfected by lipofectamine. Electroporation reduces viability in both systems highly and, therefore, seems to be inappropriate. Since ßHSD1 overexpression lasts for up to seven days there is a high potential for successfull differentiation experiments.

Supported by the Elsbeth-Bonhoff-Foundation