Regulation of Shedding of Coupling Extracellular Vesicles by Osteoclasts by the Retromer
Shivani S. Patel, Lorraine Perciliano de Faria, L. Shannon Holliday
Dr. Shannon Holliday
College of Dentistry
Bone is constantly removed by osteoclasts and replaced by osteoblasts. In healthy individuals, removal and formation are coupled to maintain constant bone mass. Diseases like osteoporosis occur when coupling is disrupted. Receptor activator of nuclear factor kappa B (RANK) is a membrane protein required for the formation of osteoclasts. Osteocyte-derived RANK-ligand (RANK-L), binds RANK on osteoclasts and stimulates them to resorb bone. RANK was also identified in extracellular vesicles (EVs) released by osteoclasts, where it binds RANK-L on osteoblasts and stimulates bone formation, thus suggesting RANK serves as a “coupling factor”. The retromer is a trimeric complex that compartmentalizes proteins internalized from the plasma membrane and releases EVs into the intercellular space. Here, its activity was manipulated using a membrane-permeable molecule, R55, which stimulates the retromer. We hypothesized that the retromer promotes packaging of RANK into EVs that are shed by osteoclasts. This could regulate the amount of RANK on the osteoclast surface that is able to receive pro-stimulatory signals from RANK-L, and the amount of RANK in EVs. Treatment of recombinant RANK-L-stimulated RAW 264.7 cells with 10μM R55 decreased the number of osteoclasts by 82%. In calcitriol-stimulated mouse bone marrow, which models the bone microenvironment, osteoclasts were reduced by 71%, but osteoblast numbers increased. Furthermore, we found that there was an increased amount of RANK in the EVs released from R55 treated RAW 264.7 cells. These results were significant (p-value<0.05) and identify R55 as a possible bone anabolic agent. In vivo studies are planned to test this idea further.
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