Molecular Mechanisms Underlying Osteocyte Apoptosis and the Associated Osteoclastogenesis in Cx43-Deficiency and Aging

Abstract

Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43^ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43^def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43^def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21^ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or toll-like receptor (TLR4) are involved in osteoclast recruitment in Cx43^ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms underlying osteocyte apoptosis in Cx43-deficiency and aging and identify the signaling molecules that link osteocyte apoptosis and osteoclast recruitment. We hypothesize that enhanced extracellular release of the signaling factors HMGB1 and miR21 recruits osteoclasts to areas containing apoptotic osteocytes and promotes osteoclast activity, leading to targeted bone resorption. Overall, these studies will provide insights into the mechanisms governing osteocyte apoptosis and the associated targeted osteoclast resorption, allowing for the development of novel therapeutics for preserving the structure and strength of the aging skeleton.