Inhibition of KCa3.1 decreases differentiated osteoclast function in RAW264 cells (893.21)

Abstract

Human intermediate conductance Ca2+‐activated K+ channels (KCa3.1) are found in many cell types including colonic and airway epithelia, vasculature smooth muscle and T‐lymphocytes. Recent work has shown Ca2+‐activated K+ channels may also be involved in the osteoclast spreading leading to the development of osteoporosis. The aim of this study was to further investigate the role of KCa3.1 in osteoclast differentiation and resorption. Cell culture studies were done using RAW264.7 cells differentiated into active osteoclasts using m‐CSF and RANKL. Initially, KCa3.1 protein was identified in undifferentiated and differentiated RAW264.7 cells using an antibody specific to the KCa3.1 channel. Analysis of the western blots showed similar levels of protein expression in undifferentiated and differentiate osteoclasts. To establish a relationship between KCa3.1 function and osteoclast differentiation, TRAP‐staining was done to visualize differentiated RAW264.7 cells. Cells were treated with m‐CSF and RANKL for five days to differentiate the macrophage cells. Results showed that treatment with DC‐EBIO or clotrimazole (CLT) did not significantly alter the number of differentiated osteoclast. Finally, to elucidate the role of KCa3.1 in osteoclast function a bone resorption assay was used. RAW264.7 cells were differentiated and passed onto an Osteolyse plate containing bone matrix. Cells were treated with CLT and DC‐EBIO and bone resorption was assessed using fluorescence imaging. Treatment of the differentiated cells with CLT at day 7 showed a statistically significant decrease in the resorption and osteoclast function when compared to control differentiated RAW264.7 cells. These results suggest that although KCa3.1 does not play a pivotal role in osteoclast differentiation, activation of the channel may lead to the development of osteoporosis. Moreover, inhibition of the channel showed a decline in osteoclast activity and maybe protective against developing osteoporosis.