Signaling of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

• PTTH-stimulated ROS production in Bombyx PGs appears to be Ca2+- and PLC-dependent.
• Pretreatment with either NAC or mitochondrial inhibitors blocked PTTH-regulated signaling pathways.
• Treatment with 1 mM of H2O2 activated the phosphorylation of ERK and 4E-BP.
• ROS appear to lie upstream of PTTH-regulated phosphorylation of key signaling proteins.

Our previous study demonstrated that mitochondria-derived reactive oxygen species (ROS) generation is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the mechanism of ROS production and the signaling pathway mediated by ROS. PTTH-stimulated ROS production was markedly attenuated in a Ca2+-free medium. The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated ROS production, indicating the involvement of Ca2+ and PLC. When the PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)), a great increase in ROS production was observed. We further investigated the action mechanism of PTTH-stimulated ROS signaling. Results showed that in the presence of either an antioxidant (N-acetylcysteine, NAC), or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)), PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK was blocked. Treatment with 1 mM of H2O2 alone activated the phosphorylation of ERK and 4E-BP, and inhibited AMPK phosphorylation. From these results, we conclude that PTTH-stimulated ROS signaling is Ca2+- and PLC-dependent and that ROS signaling appears to lie upstream of the phosphorylation of ERK, 4E-BP, and AMPK.

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