Abstract

Composition and Regulatory Activities of Extracellular Vesicles Released by Osteoclasts

Shannon Holliday, Associate Professor, University of Florida

Bone is constantly remodeled and healthy bone is maintained by communication between osteoclasts, the cells that remove bone, and osteoblasts, the cells that form bone. Extracellular vesicles (EVs), have emerged as important intercellular regulators. We have shown that hematopoietic cells grown in culture shed EVs as they differentiate from pre-osteoclasts into osteoclasts. Treatment of isolated hematopoietic cells with these EVs did not affect their RANKL-stimulated differentiation into osteoclasts. However, EVs from osteoclast progenitors promoted 1, 25-dihydroxyvitamin D3-dependent osteoclast and osteoblast formation in whole mouse marrow cultures, which contain both osteoclasts and osteoblasts, and EVs from mature osteoclasts inhibited osteoclastogenesis and osteoblast formation in the same cultures. EVs from mature osteoclasts contained receptor activator of nuclear factor kappa B (RANK). Immunogold labeling showed RANK was enriched in 1 in every 32 EVs shed by osteoclasts and isolated RANK-rich EVs inhibited 1, 25-dihydroxyvitamin D3-stimulated osteoclast formation. EVs from resorbing osteoclasts grown on bone slices and inactive osteoclasts on plastic were characterized by two-dimensional liquid chromatography/mass spectroscopy and characterization of selected microRNAs was also performed. Data from these studies show that large changes in the composition of EVs released occurs as osteoclasts activate to resorb bone. Among the changes are an absence of RANK and large increases in vacuolar H+-ATPase subunits and associated binding proteins in the EVs from resorbing osteoclasts compared with EVs from inactive osteoclasts. Efforts are underway to better understand the mechanisms by which EVs from osteoclasts regulate osteoblasts and to test the therapeutic potential of osteoclast EVs for bone regeneration in vivo.