Signaling pathway underlying autocrine activation of ecdysteroidogenesis in prothoracic glands of Bombyx mori

Autocrine activation of ecdysteroidogenesis in the prothoracic glands (PGs) of the silkworm, Bombyx mori, was previously demonstrated. In the present study, the signaling cascade involved in this autocrine activation was further investigated. I found that phosphorylation of the translational repressor 4E-binding protein (4E-BP) increased in a time-dependent manner when PGs were incubated in a small volume (10 μl) of medium, compared to those incubated in a larger volume (100 μl). Higher levels of 4E-BP phosphorylation were also observed in PGs incubated either in PG-conditioned medium or under group incubation conditions. Treatment with LY294002 and rapamycin reduced 4E-BP phosphorylation, indicating the involvement of the PI3K/TOR pathway. I further investigated the downstream target genes involved in autocrine activation. Results showed that the expression levels of several sugar transporter (St) and trehalase (Treh) genes were upregulated in PGs incubated in a small volume (10 μl) of medium. Treatment with LY294002 and rapamycin suppressed the autocrine activation of St1 and Treh1 expressions, suggesting that their regulation is mediated via the PI3K/TOR pathway. Time-dependent autocrine activation of Treh enzyme activity was observed, and this effect was blocked by pretreatment with validamycin A, a specific Treh inhibitor, suggesting that the autocrine factor directly enhances Treh activity. Treatment with either validamycin A or the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) suppressed autocrine activation of ecdysteroidogenesis, clearly indicating that both Treh activity and glycolysis are involved. To investigate potential upstream ligands, I examined the gene expressions of bombyxins and epidermal growth factor (Egf) signaling components. Results showed that only bombyxin-Z1 expression was upregulated in a time-dependent manner under an autocrine condition, while other genes remained unchanged. Using a specifically generated anti-bombyxin-Z1 antibody, the presence of bombyxin-Z1 protein was confirmed. To my knowledge, this is the first study to elucidate the signaling cascade involved in autocrine activation of ecdysteroidogenesis in an insect system.