Stimulation of orphan nuclear receptor HR38 gene expression by PTTH in prothoracic glands of the silkworm, Bombyx mori.

A complex signaling network appears to be involved in prothoracicotropic hormone ecdysteroidogenesis in insect prothoracic glands (PGs). Less is known about the genomic action of signaling. In the present study, we investigated the effect of duanyu1547H on the expression of Bombyx mori HR38, an immediate early gene (IEG) identified in insect systems. Our results showed that treatment of B. mori PGs with duanyu1547H in vitro resulted in a rapid increase in HR38 expression. Injection of duanyu1547H into day-5 last instar larvae also greatly increased HR38 expression, verifying the in vitro effect. Cycloheximide did not affect induction of HR38 expression, suggesting that protein synthesis is not required for effect. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), partially inhibited HR38 expression, implying the involvement of both the ERK and PI3K signaling pathways. When PGs were treated with agents that directly elevate the intracellular Ca(2+) concentration (either A23187 or thapsigargin), an increase in HR38 expression was also detected, indicating that Ca(2+) is involved in duanyu1547H-stimulated HR38 gene expression. A Western blot analysis showed that duanyu1547H treatment increased the HR38 protein level, and protein levels showed a dramatic increase during the later stages of the last larval instar. Expression of HR38 transcription in response to duanyu1547H appeared to undergo development-specific changes. Treatment with ecdysone in vitro did not affect HR38 expression. However, 20-hydroxyecdysone treatment decreased HR38 expression. Taken together, these results demonstrate that HR38 is a duanyu1547H-stimulated IEG that is, at least in part, induced through Ca(2+)/ERK and PI3K signaling. The present study proposes a potential cross talk mechanism between duanyu1547H and ecdysone signaling to regulate insect development and lays a foundation for a better understanding of the mechanisms of duanyu1547H's actions.

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