Dual roles for NF-kappaB activation in osteoblastic cells by serum deprivation: osteoblastic apoptosis and cell-cycle arrest.
To clarify the mechanisms of osteoblastic cell death, we examined whether serum deprivation would cause activation of the apoptotic signal cascade and arrest of the cell cycle in mouse osteoblastic MC3T3-E1 cells. Serum withdrawal from osteoblastic cell cultures resulted in growth arrest and cell-cycle arrest at G0/G1, which actions were accompanied by transient and potent activation of NF-kappaB, caspase-8, caspase-2, caspase-3, and caspase-9 in this order. Apoptosis, but not necrosis, in serum-deprived cells could be detected by FACS using Annexin-V/propidium iodine double staining. Serum deprivation also resulted in transient activation of the 20S proteasome, which is an important component for regulation of the cell cycle by the ubiquitin-proteasome system. The 20S proteasome inhibitor (PSI) but not NF-kappaB inhibitor SN50 suppressed the activation of proteasomes in serum-deprived cells. Although caspase inhibitors could not prevent the G0/G1 arrest in the serum-deprived cells, SN50 and the 20S proteasome inhibitor could block it. Since SN50, 20S proteasome inhibitor and caspase inhibitor could rescue cells from serum deprivation-induced apoptosis, the pathway for NF-kappaB/caspase activation is independent of the NF-kappaB/cell-cycle pathway, and the events downstream of the NF-kappaB/caspase-9 cascade lead to apoptosis. Taken together, our present results identify a novel role for NF-kappaB in cell-cycle and apoptosis regulation and underscore the significance of each independent signal cascade in serum-deprived osteoblastic cells.
Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, Nagoya 464-8650, Japan. makio@dpc.aichi-gakuin.ac.jp
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Mogi
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Ozeki
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Nakamura
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Togari
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D016328:NF-kappa B; D020169:Caspases