Gu Shi-Hong, Hsieh Yun-Chih, Lin Pei-Ling
Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung 404, Taiwan, ROC.
Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung 404, Taiwan, ROC.
J Insect Physiol. 2016 Jul;90:8-16. doi: 10.1016/j.jinsphys.2016.04.003. Epub 2016 Apr 16.
A complex signaling network appears to be involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). Less is known about the genomic action of PTTH signaling. In the present study, we investigated the effect of PTTH 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 PTTH in vitro resulted in a rapid increase in HR38 expression. Injection of PTTH 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 PTTH's 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 PTTH-stimulated 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 PTTH-stimulated HR38 gene expression. A Western blot analysis showed that PTTH 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 PTTH 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 PTTH-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 PTTH and ecdysone signaling to regulate insect development and lays a foundation for a better understanding of the mechanisms of PTTH's actions.
一个复杂的信号网络似乎参与了昆虫前胸腺（PG）中促前胸腺激素（PTTH）刺激的蜕皮激素合成。关于PTTH信号的基因组作用了解较少。在本研究中，我们调查了PTTH对家蚕HR38表达的影响，HR38是在昆虫系统中鉴定出的一个立即早期基因（IEG）。我们的结果表明，体外使用PTTH处理家蚕PG会导致HR38表达迅速增加。将PTTH注射到5龄末幼虫体内也大大增加了HR38表达，证实了体外实验结果。放线菌酮不影响HR38表达的诱导，这表明PTTH的作用不需要蛋白质合成。一种丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）激酶（MEK）抑制剂（U0126）和一种磷脂酰肌醇3激酶（PI3K）抑制剂（LY294002）部分抑制了PTTH刺激的HR38表达，这意味着ERK和PI3K信号通路都参与其中。当用直接提高细胞内Ca(2+)浓度的试剂（A23187或毒胡萝卜素）处理PG时，也检测到HR38表达增加，表明Ca(2+)参与了PTTH刺激的HR38基因表达。蛋白质印迹分析表明，PTTH处理增加了HR38蛋白水平，并且在末龄幼虫后期蛋白水平显著增加。HR38转录对PTTH的反应似乎经历了发育特异性变化。体外使用蜕皮激素处理不影响HR38表达。然而，20-羟基蜕皮激素处理降低了HR38表达。综上所述，这些结果表明HR38是一个PTTH刺激的IEG，至少部分是通过Ca(2+)/ERK和PI3K信号诱导的。本研究提出了PTTH和蜕皮激素信号之间潜在的相互作用机制以调节昆虫发育，并为更好地理解PTTH作用机制奠定了基础。
