Fluorescence Imaging of Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Calcium Oscillations in Osteoclasts

Abstract

Background: Numerous bone diseases are caused by abnormal activity of osteoclasts, cells responsible for physiological bone degradation. Understanding the mechanisms of osteoclast formation and activation is important for developing diagnostic tools and treatments for various bone diseases. Receptor activator of nuclear factor κB ligand (RANKL), a key osteoclastogenic cytokine, induces changes in intracellular Ca2+ con- centration ([Ca2+]i) that can be visualized and measured with a fluorescent Ca2+ binding dye. The objective of the study was to characterize the changes in [Ca2+]i induced by acute application of RANKL in osteoclast precursors.

Methods: We performed calcium imaging in osteoclast precursors generated from RAW 264.7 cells loaded with Fura-2 fluorescent dye using an inverted microscope, Nikon TE2000-U. Data was collected with Volocity software and analysed in Excel and MATLAB.

Results: In osteoclast precursors, RANKL induced oscillations in [Ca2+]i within 2 minutes of exposure. The main frequency of oscillations was approximately 37.7 mHz. However, no significant change in the mean level of intracellular Ca2+ was observed. Interestingly, when ATP was applied to RANKL-treated osteoclast precursors, it induced a long-lasting increase in [Ca2+]i compared to control cells.

Limitations: The limitations of our study included the small number of replicates and the short duration of fluorescence recording under each condition.

Conclusions: Short exposure of osteoclast precursors to RANKL not only induced oscillations in calcium con- centration, but also modulated cellular response to the subsequent application of ATP.