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-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, 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-α-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-α 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-α alone increased the cell population of AV+PI-, while Z-IETD-FMK caused a shift in the cell population from AV+PI- to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved Caspase 3. TNF-α 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-α induced preferentially Apoptosis in osteoblast cell line and Necroptosis played a decisive role when TNF-α-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-α.