Effects of the RANKL Inhibitor OPG-Fc on Osteoclast Dynamics

Objectives: Although much progress has been made in defining molecular pathways critical for osteoclast function and identifying molecular targets for anti-resorptive therapies, interrogating osteoclast biology by live cell imaging can reveal novel insights into their function. The RANKL inhibitor, Denosumab, is used in osteoporosis patients to inhibit bone resorption. However, it is unknown how manipulation of RANKL signaling affects real-time osteoclast cell and resorption dynamics. Using live cell imaging and computational analysis, the goal of this study was to determine the real-time responses of osteoclasts to RANKL inhibition.
Methods: Time lapse confocal live cell imaging was performed using calvarial bone explants from transgenic mice expressing a LysM-Cre-dependent TdTomato fluorescent reporter in osteoclasts. Explants were treated with the RANKL inhibitor OPG-Fc (500ng/ml) as a surrogate for Denosumab, or vehicle for 44h, then subsequently treated for 16h with RANKL (200ng/ml). Computational analysis was performed on thresholded and skeletonized time lapse images to measure morphological parameters.
Results: Long term live cell imaging (~3 days) revealed complex osteoclast dynamics including fission, fusion, recycling, amoeboid-like streaming and extension/retraction of cell processes. OPG-Fc caused a rapid change within 1-2h to an elongated, intricately branched morphology accompanied by loss of sealed zones and arrest of bone resorption. Computational analysis showed that OPG-Fc induced significant increases in osteoclast perimeter and cell branching and significant decreases in circularity and solidity, measures that reflect increased branching/complexity. OPG-Fc also induced significant increases in geodesic diameter and longest-shortest path, reflecting elongation. These effects were most prominent within the first 24 hours of OPG-Fc treatment and were partially reversed by subsequent RANKL treatment.
Conclusions: Live cell imaging showed rapid effects of OPG-Fc on osteoclasts, inducing a change to a stellate, elongated, and intricately branched morphology and suggests that altering RANKL signaling causes osteoclasts to transition between active and inactive states that are morphologically recognizable. Supported by UMKC SOD Summer Scholars program.