Davis, Hannah M.Valdez, SinaiGomez, LelandMalicky, PeterWhite, Fletcher A.Subler, Mark A.Windle, Jolene J.Bidwell, Joseph P.Bruzzaniti, AngelaPlotkin, Lilian I.2021-08-022021-08-022019-05-20Davis, H. M., Valdez, S., Gomez, L., Malicky, P., White, F. A., Subler, M. A., Windle, J. J., Bidwell, J. P., Bruzzaniti, A., & Plotkin, L. I. (2019). High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro. Journal of Cellular Biochemistry, 120(10), 16741–16749. https://doi.org/10.1002/jcb.289321097-4644https://hdl.handle.net/1805/26322Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the pro-inflammatory cytokine, high mobility group box1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/MCSF treated bone marrow cells (BMC). In bone marrow macrophages (BMMs), TLR4 inhibition with LPS-RS, but not RAGE inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly effects differentiation by activating TLR4 in BMMs and RAGE in pre-osteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.en-USosteoclastosteocyteHMGB1RAGETLR4apoptosiscytokineArticle