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

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.

SUBMITTER: Gu SH

PROVIDER: S-EPMC5807416 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Gu Shi-Hong SH Li Gen G Hsieh Hsiao-Yen HY Lin Pei-Ling PL Li Sheng S

Frontiers in physiology 20180205

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 ins ...[more]

PMID: 29459829

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Project description:In this study, we investigated inhibition of the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPK? phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-?-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPK?, ?, and ? by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis.

Dataset Information

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

Publications

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, <i>Bombyx mori</i>.

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