Regulation of histone H3 phosphorylation at serine 10 in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori.

A complex signaling network appears to be involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we investigated the localization of phosphorylated extracellular signal-regulated kinase (ERK) in PTTH-stimulated PGs in Bombyx mori. The nuclear effect of PTTH was further studied by examining phosphorylation of histone H3 at serine 10. Results showed that in PTTH-stimulated PGs, higher phosphorylated ERK was detected in nuclear fraction compared to that in cytosolic fraction. PTTH treatment in vitro appears to rapidly enhance the transcriptional activation-associated histone H3 phosphorylation at serine 10. PTTH stimulated histone H3 phosphorylation in a time-dependent manner. Injection of PTTH into day-6 last instar larvae greatly increased histone H3 phosphorylation, verifying the in vitro effect. The stimulation of histone H3 phosphorylation by PTTH appears to be developmentally regulated. PTTH-stimulated histone H3 phosphorylation was greatly reduced in Ca(2+)-free saline or by pretreatment with a potent and specific inhibitor of phospholipase C (PLC), U73122. When PGs were treated with agents that directly elevate the intracellular Ca(2+) concentration (either A23187 or thapsigargin), a greatly increase in histone H3 phosphorylation at serine 10 was observed, indicating Ca(2+)-dependency of histone H3 phosphorylation stimulated by PTTH. In addition, PTTH-stimulated histone H3 phosphorylation was partially reduced by U0126, a specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, indicating the involvement of ERK. However, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, did not inhibit PTTH-stimulated histone H3 phosphorylation, implying that PI3K signaling is not related to PTTH-stimulated histone H3 phosphorylation. Taken together, these results suggest that PTTH-stimulated histone H3 phosphorylation at serine 10 is mediated by Ca(2+)/ERK signaling in B. mori PGs.