Nitric oxide (NO) has biphasic effects on regulating osteoblast survival and death. This study was aimed to evaluate the effects of NO pretreatment on hydrogen peroxide (HP)-induced insults of rat osteoblasts and the possible mechanisms. Exposure of osteoblasts prepared from rat calvarias to HP significantly increased intracellular reactive oxygen species levels, decreased alkaline phosphatase activity and cell survival, and ultimately induced cell apoptosis. However, NO pretreatment lowered HP-induced oxidative stress and apoptotic insults. In parallel, HP increased Bax levels and its translocation from the cytoplasm to mitochondria. NO pretreatment caused significant attenuations in HP-induced modulations in Bax synthesis and translocation. In contrast, pretreatment with NO enhanced levels and translocation of antiapoptotic Bcl-XL protein in rat osteoblasts. RNA analyses further revealed that HP inhibited Bcl-XL mRNA expression without affecting Bax mRNA levels. In comparison, NO induced Bcl-XL mRNA production and alleviated HP-caused inhibition of this mRNA expression. As to the mechanism, HP suppressed RNA and protein levels of transcription factor GATA-5 in rat osteoblasts. Pretreatment with NO induced GATA-5 mRNA and protein expressions and simultaneously attenuated HP-induced inhibition of this gene’s expression. Consequently, GATA-5 knockdown using RNA interference inhibited Bcl-XL mRNA expression and concurrently lowered NO’s protection against HP-induced apoptotic insults. Therefore, this study showed that NO can protect rat osteoblasts from HP-induced apoptotic insults. The protective mechanisms are mediated by GATA-5-mediated transcriptional induction of Bcl-XL gene, and translocational modulation of Bcl-XL and Bax proteins.
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