Regulating the Release of RANK-Containing Extracellular Vesicles From Osteoclasts

Objectives: Extracellular vesicles that contain receptor activator of nuclear factor kappa B (RANK-EVs) are crucial intercellular signals released by osteoclasts that couple bone resorption with bone formation. We examined the biogenesis of RANK-EVs, and how their release is regulated.
Methods: EVs were detected and characterized using an ExoView R100. EVs were isolated using ExoQuick, and by immunoprecipitation with an anti-CD9 antibody. EVs were characterized by quantitative Westerns. FAM21 was knocked down by RNAi; retromer activity was stimulated with the molecular chaperone R55. Osteoclasts were detected by tartrate-resistant acid phosphatase activity, osteoblasts by alkaline phosphatase activity, and in vitro mineralization was determined by the Orthoimage assay.
Results: By ExoView, we found that RANK-EVs are a small (2-4%) subset of the EVs that contain CD9. Immunoprecipitation studies were consistent with this analysis. In other cell types, CD9 EVs are known to be formed in association with plasma membrane receptor recycling. We tested for effects of the retromer and FAM21, regulators of plasma membrane receptor recycling, on RANK-EV release. Stimulation of retromer activity or knockdown of FAM21 triggered increased RANK-EV release from GST-RANKL stimulated osteoclasts in cell culture. In calcitriol-stimulated mouse marrow, which contains both osteoclasts and osteoblasts, R55 simultaneously inhibited osteoclast formation and promoted osteoblast differentiation.
Conclusions: RANK-EVs are a subset of CD9-containing EVs. Their release can be controlled by stimulation of the retromer or RNAi knock down of FAM21. R55 is a new type of bone active agent that simultaneously inhibits osteoclast formation and stimulates osteoblast maturation in mouse marrow cultures.