Ca2+-PKC-caspase 3-like protease pathway mediates DNA and nuclear fragmentation in ecdysteroid-induced programmed cell death

20-Hydroxyecdysone (20E) induces programmed cell death in the anterior silk gland of the silkworm. Here, we report the direct interaction between Ca2+ and protein kinase C (PKC)-caspase 3-like protease pathway in the 20E-induced cell death. The calcium ionophore can mimic 20E effects in inducing DNA and nuclear fragmentation, but such mimicry is only possible in the glands precultured for 18 h with 20E. The simultaneous presence of translation inhibitor with 20E in the preculture showed that de novo protein synthesis was needed to mimic 20E effects by the calcium ionophore. Both a PKC inhibitor and a caspase 3 inhibitor inhibited the mimicking effects. After substitution of the calcium ionophore for 20E, caspase 3-like protease was fully activated 12 h later, and DNA and nuclear fragmentation occurred faster than continuous 20E stimuli. The results show the presence of a Ca2+-PKC-caspase 3-like protease pathway in 20E signaling, and possible involvement of the pathway up to the mobilization of Ca2+ in regulating the timing of cell death in vivo.