Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori.

Shi-Hong Gu,Sheng Li,Pei-Ling Lin,Gen Li,Hsiao-Yen Hsieh

doi:10.3389/fphys.2018.00043

Abstract

In this study, phosphorylation of c-Jun N-terminal kinase (JNK) by the prothoracicotropic hormone (PTTH) was investigated in prothoracic glands (PGs) of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC) and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin) that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126) of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and an inhibitor (LY294002) of phosphoinositide 3-kinase (PI3K) failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC) or diphenylene iodonium (DPI), PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

Highlights

We further examined whether prothoracicotropic hormone (PTTH) stimulates Jun N-terminal kinase (JNK) phosphorylation
The phosphorylation level of another immunoreactive protein with an molecular weight (MW) of ∼38 kDa was strongly increased upon PTTH stimulation, it remained low in the lysate of the control prothoracic glands (PGs)
Several studies demonstrated the involvement of the protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and AMPK/target of rapamycin (TOR) signaling pathways (Smith and Rybczynski, 2012; Gu et al, 2017)

Summary

Introduction

Ecdysteroids regulate insect growth, molting, and metamorphosis; they are synthesized and secreted by the prothoracic glands (PGs) (Marchal et al, 2010; De Loof, 2011; Smith and Rybczynski, 2012; De Loof et al, 2013, 2015; Yamanaka et al, 2013). A complex signaling transduction network is activated downstream of PTTH receptor activation (Rewitz et al, 2009; Marchal et al, 2010; Smith and Rybczynski, 2012). This network includes a rapid increase in Ca2+ (Gu et al, 1998; Birkenbeil and Dedos, 2002; Fellner et al, 2005), cAMP generation (Smith et al, 1984, 1985; Gu et al, 1996), and activation of protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), p70S6 kinase (S6K), ribosomal protein S6, and tyrosine kinase (Song and Gilbert, 1995, 1997; Smith et al, 2003; Rybczynski and Gilbert, 2006; Lin and Gu, 2011). Our recent studies further indicated that reactive oxygen species (ROS) and phosphoinositide 3-kinase (PI3K)/adenosine 5′-monophosphate-activated protein kinase (AMPK)/target of rapamycin (TOR) signaling are involved in PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs (Gu et al, 2011, 2012, 2013; Hsieh et al, 2013, 2014)

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Results

Conclusion