The effect of redoxmodulation on osteoclastogenesis

Abstract: During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS. The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation. Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.