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...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
2018-11-01
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| Series: | Brazilian Journal of Medical and Biological Research |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2019000100603&lng=en&tlng=en |
| _version_ | 1819102106090471424 |
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| author | Guan Shi Pu Jia Hao Chen Li Bao Fei Feng Hai Tang |
| author_facet | Guan Shi Pu Jia Hao Chen Li Bao Fei Feng Hai Tang |
| author_sort | Guan Shi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T01:29:17Z |
| format | Article |
| id | doaj.art-81c4b4d0091b41c799d0a6792451ba57 |
| institution | Directory Open Access Journal |
| issn | 1414-431X |
| language | English |
| last_indexed | 2024-12-22T01:29:17Z |
| publishDate | 2018-11-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-81c4b4d0091b41c799d0a6792451ba572022-12-21T18:43:32ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research1414-431X2018-11-0152110.1590/1414-431x20187844S0100-879X2019000100603Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibitionGuan ShiPu JiaHao ChenLi BaoFei FengHai TangNecroptosis 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2019000100603&lng=en&tlng=enNecroptosisMouse osteoblastZ-IETD-FMKNecrostatin-1TNF-α |
| spellingShingle | Guan Shi Pu Jia Hao Chen Li Bao Fei Feng Hai Tang Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition Brazilian Journal of Medical and Biological Research Necroptosis Mouse osteoblast Z-IETD-FMK Necrostatin-1 TNF-α |
| title | Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition |
| title_full | Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition |
| title_fullStr | Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition |
| title_full_unstemmed | Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition |
| title_short | Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition |
| title_sort | necroptosis occurs in osteoblasts during tumor necrosis factor α stimulation and caspase 8 inhibition |
| topic | Necroptosis Mouse osteoblast Z-IETD-FMK Necrostatin-1 TNF-α |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2019000100603&lng=en&tlng=en |
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