【作者】 刘文；

【导师】 赵小凡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 研究背景由于昆虫个体发育阶段明显分为蜕皮和变态,所以科学家将其作为研究多细胞生物发育转变分子机理的理想模式系统。自从Stefan Kopec对舞毒蛾变态发育的研究开始(Kopec,1926),近一个世纪以来,科学家一直对昆虫内分泌的研究保持着浓厚的兴趣。由于在舞毒蛾中的开拓性研究,导致了包括神经内分泌细胞(Kopec,1922)等一系列新颖概念的提出,从而大大促进了现代内分泌学的发展。近年来,由于分子生物学等各项技术的快速发展,科学家对昆虫蜕皮和变态这两个重要发育转变的分子机理有了更加深刻的认识。研究发现,这些发育转变受到昆虫内分泌系统严格的调控,而控制蜕皮和变态的激素主要是蜕皮激素(20-hydroxyecdysone,20E)和保幼激素(juvenile hormone, JH)。本领域存在的科学问题和研究意义在JH存在下,20E诱导昆虫在幼虫期的多次蜕皮；而当JH滴度下降时,20E促进昆虫从幼虫到蛹的变态发育。因此,20E和JH信号通路通过复杂的相互作用共同调控昆虫的发育,但是其分子机理还有待于进一步的研究。在20E信号通路中,20E诱导核受体EcR/USP的形成及共激活子的募集,从而形成具有DNA结合活性的转录复合体,最终调节基因转录。越来越多的研究表明,除了经典的核受体途径,在20E信号转导通路中还存在一种细胞膜介导的非基因组途径。但是,其机制还没有深入研究。在JH通路中,由于其受体Met的鉴定,JH信号通路的研究蔚然成风,但是关于20E和JH信号通路之间相互作用的机理还并不清楚。人类周期蛋白依赖的激酶10(cyclin-dependent kinase10, CDK10)可以与Hsp90发生相互作用,但是机理并不清楚。己知Hsp90参与调控EcR的DNA活性,并调节20E信号通路。另外,胰岛素可以有效诱导CDK10的快速磷酸化。这些现象暗示,CDK10可能通过与Hsp90的相互作用参与20E通路。同时,我们有必要探索20E能否诱导CDK10的快速磷酸化参与20E激活的非基因组途径。在家蚕和果蝇中的研究表明,20E可以通过细胞膜上的G蛋白偶联受体(G-protein-coupled receptor, GPCR)诱导钙离子水平上调。果蝇中USP的磷酸化依赖于20E对PKC的激活。然而,20E诱导的钙信号与PKC激活的USP磷酸化之间的联系还没有报道。事实上,一种磷脂酶C(phospholipase C gamma1, PLCG1)可以承接GPCR信号并通过上调钙信号而激活PKC。所以,20E诱导的细胞膜信号与核受体信号之间的联系是否依赖于PLCG1则需要深入的探讨。在20E信号通路中,Hsp90和Hsc70(Hsp70同源蛋白)通过与果蝇EcR相互作用调节受体的DNA结合活性,但是当受体装配成转录复合体后,热休克蛋白则解离下来而并不出现在启动子区域的复合体中。然而也有文章报道,Hsp90可以调控盐皮质激素受体结合于启动子的不同位置,这暗示它可能参与到转录复合体中。另外Hsc70对于受体的循环和降解也起着至关重要的作用。因此,Hsp90和Hsc70是否参与棉铃虫20E信号转录复合体并调节转录,需要更进一步的研究。鉴于20E和JH能通过复杂的调控网络精密有效地控制昆虫发育,所以阐明20E和JH信号通路不仅能为害虫防治提供策略,也能够为研究人类内分泌系统的调节机理提供参考,最终服务于人类健康。研究方法与取得成果1.20E通过诱导蛋白激酶CDK10的快速磷酸化调节蜕皮激素核受体在启动子上的装配利用RNA干扰技术下调幼虫和表皮细胞系中的CDK10阻碍了幼虫发育并抑制20E可诱导基因的转录。20E诱导CDK10的快速磷酸化,但是G蛋白偶联受体、磷脂酶C、钙离子通道等蛋白的抑制剂都能抑制此磷酸化。磷酸化的CDK10促进其与热休克蛋白(heat shock proteins, Hsps)Hsc70和Hsp90的相互作用,随后有助于更多的Hsc70和Hsp90与蜕皮激素受体EcRB1的相互作用,并最终促进Hsps-EcRB1复合体结合到20E应答元件上调控基因转录。下调CDK10可以抑制Hsps-EcRB1复合体在HR3启动子上的形成。以上结果说明,20E通过非基因组途径诱导CDK10磷酸化,并最终调控细胞核中的基因转录。2. PLCG1将ErGPCR介导的20E膜信号与核受体信号有效连接通过RNA干扰沉默PLCG1后,20E诱导的昆虫化蛹被显著抑制,并导致幼虫死亡和化蛹缺陷,同时20E可诱导基因的表达被下调。20E诱导PLCG1发生酪氨酸磷酸化并向细胞膜迁移。Src激酶家族抑制剂PP2、沉默ErGPCR和Gaq都能抑制PLCG1的酪氨酸磷酸化。PLCG1参与调节20E诱导的Ca2+内流。抑制GPCR、PLC、IP3受体(1,4,5-trisphosphate receptor)或钙离子通道均有效抑制20E诱导的Ca2+内流和蜕皮激素应答元件EcRE激活的转录。通过PLCG1和钙离子信号途径,20E调控了PKC对USP1第21位丝氨酸(Ser21)磷酸化的催化,而这决定了USP1与EcRE的结合活性。这些结果说明,20E通过ErGPCR和Src激活PLCG1,从而诱导Ca2+内流和USP1的PKC磷酸化,最后调节基因转录。3.热休克蛋白Hsp90通过磷酸化状态的转换和蛋白质相互作用的改变调节20E和JH信号通路的相互作用激素诱导和免疫细胞化学实验显示,20E和JH Ⅲ以及JH类似物methoprene都能诱导Hsp90表达上调和向细胞核部分转移。qRT-PCR分析说明,干扰Hsp90后有效抑制了20E和JH信号通路中相关基因的表达。蛋白质免疫共沉淀结果显示,20E诱导Hsp90与USP1相互作用,而JH诱导Hsp90与Met1相互作用,20E和JH能互相拮抗各自诱导的相互作用。PKC磷酸化抗体分析说明,JH能诱导Hsp90发生PKC催化的磷酸化,而20E拮抗JH诱导的Hsp90磷酸化。因此,20E和JH通过调控Hsp90磷酸化状态的改变实现相互作用。所取得成果的创新点和意义本文的创新点主要表现在以下几个方面：(1)首次证明了Hsp90在JH信号通路中的功能,及其在20E和JH信号相互作用中的功能；(2)首次发现20E可以通过ErGPCR, PLCG1和钙信号诱导CDK10快速磷酸化,以及CDK10磷酸化对Hsp90和Hsc70介导的核受体装配的调节功能；(3)首次证明Hsp90和Hsc70可以定位于结合有蜕皮激素启动子的转录复合体中；(4)首次发现PLCG1通过激活PKC而磷酸化USP1,且这一过程依赖于ErGPCR和钙信号上调；(5)鉴定了棉铃虫USP1的PKC催化的磷酸化位点Ser21,及其对DNA结合的必要性。本研究将20E非基因组和基因组途径有效联系起来,并给出了理解20E和JH信号通路相互作用的新思路。更多还原

【Abstract】 BackgroundInsects are ideal model systems for studying the molecular mechanisms that regulate developmental transitions in multicellular organisms, largely because their individual developmental stages are clearly punctuated by molting and metamorphosis. These transition events are under strict endocrine control and have been of interest to entomologists for close to a century, since the classical study of Stefan Kopec on the metamorphosis of the gypsy moth, Lymantria dispar. Due to the rapid development of molecular biology technology in recent years, the molecular mechanism of insect molting and metamorphosis is uncovered partly. Data to date show that insect development transitions are strictly controlled by endocrine system. Ecdysone (20E) and juvenile hormone (JH) play the main function in the processes.Questions and significances20E regulates larval molting in the presence of JH, but promotes metamorphosis in the absence of JH at the stage of larval-pupal transition. Therefore,20E signaling interacts with JH signaling to control insect development, while the mechanism remains unclear. In the20E pathway,20E induces the formation of EcR/USP and the cofactors recruitment to assemble activated transcriptional complex for gene expression. More and more studies show that there is a cell membrane-mediated non-genomic pathway in addition to the classical nuclear receptor pathway in20E signaling. But, it is still far from enough mechanism research. In the JH pathway, the identification of JH intracellular receptor (Methoprene-tolerant, Met) results in the increased studies in JH signaling, while the mechanism of the crosstalk between20E and JH signalings remains unclear. Human cyclin-dependent kinase10(CDK10) can strongly interact with heat shock protein90(Hsp90), but the significance is uncovered. Meantime, by interacting with EcR, Hsp90regulates the DNA binding activating of EcR and the20E signaling. In Rat cells, insulin induces the rapid phosphorylation of CDK10. These results suggest that CDK10may take part in20E signaling by interacting with Hsp90. Meantime, we want to know whether20E could induce CDK10phosphorylation to regulate the non-genomic action in20E pathway. In Bombyx and Drosophila,20E induces calcium signaling through G-protein-coupled-receptor (GPCR). In Drosophila,20E regulates USP phosphorylation dependent on PKC activation. However, the link between the20E-induced calcium signaling and the PKC activation remains unknown.In fact, phospholipase C gamma1can regulate GPCR-triggered calcium to activate PKC. Therefore, whether the connection between the20E-induced membrane signal and the nuclear receptor signal relies on PLCG1needs an exhaustive study. In20E signaling, Hsp90and Hsc70interact with EcR and do not appear at the promoter-bound transcription complex in Drosophila. However, Hsp90does not depart from mineralcorticoid receptor and regulates the transcription sites, suggesting the existence in transcription complex. At the same time, Hsc70plays a key role in the regulation of receptor cycling and degradation. Therefore, whether Hsp90and Hsc70participate in the20E-induced transcriptional complex is still unclear and need further investigation.Given the20E-and JH-regulated precision network of insect development, the investigation of mechanisms of the20E and JH signalings not only provide pest control strategy, but also are beneficial to the study of human endocrine mechanisms.Results1.20E regulated the association of ecdysone nuclear receptor at promoter by inducing CDK10phosphorylation via a non-genomic pathwayThe downregulation of CDK10by RNA interference (RNAi) in larvae and the epidermal cell line delayed development and suppressed20E-induced gene transcription. The rapid phosphorylation of CDK10was induced by20E, whereas it was repressed by the inhibitors of G-protein-coupled-receptors, phospholipase C, and Ca2+channels. Phosphorylated CDK10exhibited increased interactions with heat shock proteins (Hsps) Hsc70and Hsp90and then promoted the interactions between Hsps and ecdysone receptor EcRB1and the binding of Hsps-EcRB1complex to the20E response element for the regulation of gene transcription. CDK10depletion suppressed the formation of the Hsps-EcRB1complex at HR3promoter. These results suggest that20E induces CDK10phosphorylation via a non-genomic pathway to regulate gene transcription in the nucleus.2. Phospholipase C gamma1connects the cell membrane pathway to the nuclear receptor pathway in20E signalingThe depletion of PLCG1by RNAi blocked20E-enhanced pupation; caused larvae death and pupation defects, and repressed20E-induced gene expression.20E induced the tyrosine phosphorylation of PLCG1and its migration toward the plasma membrane. The Src-familay kinase inhibitor PP2and the depletions of ErGPCR and Gaq inhibited PLCG1tyrosine phosphorylation. PLCG1participated in the20E-induced Ca2+influx. The inhibition of GPCR, PLC, inositol1,4,5-trisphosphate receptor, and calcium channels repressed the20E-induced Ca2+influx. Through calcium signaling, PLCG1mediated the transcriptional activation driven by the ecdysone response element (EcRE). Through PLCG1and calcium signaling,20E regulated PKC phosphorylation of USP1at Ser21to determine its EcRE binding activity. These results suggest that20E activates PLCG1via ErGPCR and Src-family kinases to regulate Ca2+influx and PKC phosphorylation of USP1in order to subsequently modulate gene transcription.3. Heat shock protein90regulated the crosstalk between20E and JH pathway by phosphorylation variation and different proteins interactions20E and JH induced Hsp90expression and its nuclear localization by using hormone injection in larvae and immunocytochemistry in HaEpi cells. Silencing of Hsp90by RNAi reduced the20E-or JH-induced gene expression.20E induced the interaction between Hsp90and the20E receptor USP1, and JH III or methoprene induced the interaction between Hsp90and the JH candidate receptor Metl. Both JH III and methoprene induced protein kinase C (PKC)-mediated phosphorylation of Hsp90; however,20E maintained non-phosphorylation of Hsp90. These results show that Hsp90participates in the crosstalk between the20E and the JH signaling pathways for gene expression by altering phosphorylation status and forming different transcriptional complexes.Conclusion and significanceThe innovation points of this research are described as following.(1) We firstly demonstrate that the function of Hsp90in JH pathway, and its key role in the crosstalk between20E and JH signaling.(2) We found20E induces the rapid phosphorylation of CDK10to regulate the Hsp90-and Hsc70-mediated association of nuclear receptor through an ErGPCR-PLCG1-Ca2+pathway.(3) This is the first report that Hsp90and Hsc70exist in the20E transcriptional complex.(4) We verify that PLCG1activates PKC to phosphorylate USP1through ErGPCR and calcium signaling.(5) We identify the USP1phosphorylation site Ser21in H. armigera, and demonstrate it’s essential for DNA binding. This work establishes a link between20E-induced non-genomic pathway and genomic pathway, and provides a new insight to understand the crosstalk between20E and JH signaling pathways.更多还原