Heat stress induces both apoptosis and necrosis in normal human osteoblasts without heat shock protein-60 (HSP60) release

Thermal trauma can irreversibly damage bone cells; however, the mechanisms by which thermal trauma affects thebone microenvironment are poorly characterized. Heat shock protein-60 (HSP60) can be induced by stresses, including hyperthermia, and released from cells as an endogenous danger signal. The aim of this study was to determine the effects of heatstress on HSP60 release by human osteoblasts. Normal human osteoblasts (NHOst) were exposed to heat stress at 40°C to46°C for 5-15 min and then cultured for 24 h. Cell viability was analyzed using the MTT assay. HSP60 protein expressionand release were analyzed by Western blotting of cell lysates and conditioned medium. HSP60 subcellular localization wasanalyzed using immunocytochemistry. Annexin-V-FITC/propidium iodide staining and the lactate dehydrogenase (LDH) assaywere used to investigate the mechanisms of cell death. We found that heat-stress significantly reduced NHOst cell viabilityin a dose- and time-dependent manner (p<0.05). Heat stress did not induce HSP60 protein expression or release by humanosteoblasts; however, freeze-thawed necrotic human osteoblasts released HSP60 into the medium. Immunocytochemistryrevealed modest changes in the subcellular localization of HSP60 in human osteoblasts after heat stress. Both apoptosis andnecrosis were induced in human osteoblasts after heat stress. In conclusion, hyperthermia at temperatures as low as 43°Cinduced both apoptotic and necrotic cell death in human osteoblasts; however, heat treatment did not induce HSP60protein expression or release into the extracellular milieu.