Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

( )Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-alpha (INF-alpha)-treated osteoblasts. Therefore, we conducted the study with TNF-alpha, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-alpha-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-alpha inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-alpha alone increased the cell population of AV+ Pl-, while Z-IETD-FMK caused a shift in the cell population from AV+ Pl- to AV+ PI + . Furthermore, TNF-alpha significantly increased protein cleaved caspase 3. TNF-alpha plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-alpha induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-alpha-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-alpha.