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 the bone 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 heat stress on HSP60 release by human osteoblasts. Normal human osteoblasts (NHOst) were exposed to heat stress at 40°C to 46°C for 5-15 min and then cultured for 24 h. Cell viability was analyzed using the MTT assay. HSP60 protein expression and release were analyzed by Western blotting of cell lysates and conditioned medium. HSP60 subcellular localization was analyzed using immunocytochemistry. Annexin-V-FITC/propidium iodide staining and the lactate dehydrogenase (LDH) assay were used to investigate the mechanisms of cell death. We found that heat-stress significantly reduced NHOst cell viability in a dose- and time-dependent manner (p<0.05). Heat stress did not induce HSP60 protein expression or release by human osteoblasts; however, freeze-thawed necrotic human osteoblasts released HSP60 into the medium. Immunocytochemistry revealed modest changes in the subcellular localization of HSP60 in human osteoblasts after heat stress. Both apoptosis and necrosis were induced in human osteoblasts after heat stress. In conclusion, hyperthermia at temperatures as low as 43°C induced both apoptotic and necrotic cell death in human osteoblasts; however, heat treatment did not induce HSP60 protein expression or release into the extracellular milieu.