The Mechanisms Of The G Protein-coupled Receptor-mediated Non-genomic Pathway Of Ecdysone And The Juvenile Hormone Inhibiting Metamorphosis

Background and unsolved questionsThe development of insect, including larval growth and metamorphosis, is regulated by two hormones, the20-hydroxyecdysone (20E) and juvenile hormone (JH). During larval growth stage,20E activates the larval molting in the presence of high JH titers. When the JH titers are decreased or disappeared,20E activates the larval-pupal metamorphosis.20E promotes the molting and metamorphosis, however JH maintains the larval status. The balance of two hormones regulates the larval development.20E is lipid-soluble conventionally known to initiate signaling via a genomic pathway.20E enters the nucleus by freely diffusing through cell membranes to combine with intracellular nuclear receptors. With the aid of heat shock proteins,20E-EcR-USP bind to the DNA promoter, initiate the transactivation of downstream genes, and induce the molting and metamorphosis. Some reports show that20E induces various fast cellular responses via G protein-coupled receptors (GPCRs) on plasma membrane, such as the rapid intracellular calcium increase, the increase in cyclic adenosine monophosphate, suggesting the existence of20E-mediated non-genomic pathway. JH is also known to regulate genes transcript through the intracellular receptor methoprene-tolerant (Met) via freely diffusing into the cell.Various proteins regulated by20E and JH are involved in insect development. Some studies report that the insulator body protein modifier of mdg4[mod(mdg4)] modulates genes expression and is related to apoptosis by interaction with the transcription factor Suppressor of Hairy-wing [Su(Hw)]. During metamorphosis,20E induces the midgut programmed cell death (PCD). However, whether mod(mdg4) is involved in regulating PCD-related genes expression is little reported. G protein-coupled receptors are the largest membrane family, and they sense the moleculars outside the cell and activate inside signal transduction pathways and cellular responses. In Drosophila, DmDopEcR binds to20E. In Bombyx, some GPCR is involved in20E-mediated PCD of silk gland. However, the signaling pathway mediated by GPCR in20E signaling is little reported. Broad (Br) is a key transcription factor.20E initates the metamorphosis through upregulating Br expression. During larval growth, JH prevents20E-induced metamorphosis by suppressing the transcript of Br. However, the application of JH induces the expression of Br to inhibit metamorphosis. The mechanism of JH action on Br is not well studied. In additional, the stabilization of Br remains an enigma. The mod(mdg4), GPCRs and Br are chosen to study the the molecular basis of the20E-induced non-genomic pathway and the JH inhibiting metamorphosis. These studies enrich the theory of hormones regulating the insect development and provide new targets for pest control.Results and conclusionsl.Mod(mdg4) participates in hormonally regulated midgut programmed cell death during metamorphosisMod(mdg4)lA was localized in the larval midgut and was highly expressed during metamorphosis. Knockdown of mod(mdg4)1A by feeding dsRNA to the larvae suppressed midgut PCD and delayed metamorphosis. The silence of mod(mdg4)1A decreased the transcript levels of genes involved in PCD and metamorphosis.20E upregulated the transcript of mod(mdg4)la not through the classic EcRBl-USPl pathway. Methoprene upregulated mod(mdg4)1a via the intracellular receptor Met. The transcript level of mod(mdg4)la was independently upregulated by20E or JH analog methoprene. Overlapped20E and methoprene counteractively regulated the transcript level of mod(mdg4)la. Conclusion:mod(mdg4)1A participates in hormonally regulated midgut programmed cell death during metamorphosis by modulating the genes expression under the20E and JH direction.2. G protein-coupled receptor participates in20-hydroxyecdysone signaling on the plasma membraneThe transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by20E regulation. Knockdown of ErGPCR in vivo blocked larval-pupal transition and suppressed20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the20E-induced gene expression. The silence of ErGPCR suppressed20E-modulated Calponin nuclear translocation and phosphorylation, and20E-induced rapid increase in cytosolic Ca2+levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the20E-induced Ca2+increase and regulated gene expression and protein phosphorylation. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and20E-induced gene expression. ErGPCR was not detected to bind to the steroid hormone analog [3H]Pon A.20E regulated the EcRB1-USP1genomic pathway through ErGPCR. However20E upregulated mod(mdg4)1A expression through ErGPCR not through EcRB1-USP1. Conclusion:ErGPCR participates in20E-initiated gene expression ane larval-pupal transition, rapid Calponin nuclear translocation and phosphorylation, fast intracellular Ca2+increase.3. Juvenile hormone prevents20-hydroxyecdysone-induced metamorphosis by regulating a novel Broad phosphorylationA novel Broad protein Broad Ⅱ was identified in H. armigera. Knockdown of BrZ-Ⅱ by RNA interference in larvae inhibited metamorphosis and repressed the expression of20E-response genes. BrZ-Ⅱ was low expressed and phosphorylated during larval growth, and highly expressed in nonphosphorylated status during metamorphosis. The inhibitors of G protein-coupled receptor, phospholipase C, and protein kinase C suppressed JH-induced BrZ-Ⅱ phosphorylation, respectively. The phosphorylated BrZ-Ⅱ induced by JH bound to the5’ regulatory region of Calponin. Exogenous JH application to larvae induced BrZ-Ⅱ phosphorylation, suppressed20E-related gene transcript and blocked the metamorphosis. JH-induced Calponin interacted with USP1and inhibited the formation of20E transcription complex. Conclusion:During the larvae growth stage, natural JH in vivo regulates BrZ-Ⅱ lower expression and phosphorylation for JH-response gene to direct the larval growth. At the metamorphic stage,20E regulates BrZ-Ⅱ higher expression and maintains BrZ-Ⅱ non-phosphorylation to direct metamorphosis. Exogenous JH Ⅲ induces BrZ-Ⅱ high expression and phosphorylation to regulate Calponin expression. JH-induced Cal interacts with USP1to activate JH pathway and block the20E signaling pathway and metamorphosis.4. Heat shock protein90keeps the stabilization and function of transcription factor Broad Ⅱ protein by interacting with its BTB domainHeat shock protein90(Hsp90) interacted with transcription factor BrZ-Ⅱ in vivo and in vitro. The middle domain of Hsp90directly interacted with the BTB domain of BrZ-Ⅱ. Hsp90inhibitor,17-allylamino-17-desmethoxygeldanamycin(17-AAG) repressed the interaction between Hsp90and BrZ-Ⅱ, decreased the protein level of BrZ-Ⅱ, but did not affect the mRNA level of BrZ-Ⅱ. The addition of the proteasome inhibitor MG-132suppressed the17-AAG-induced degradation of BrZ-Ⅱ. The deletion of BTB domain of BrZ-Ⅱ and17-AAG treatment suppressed the interaction of Hsp90and BrZ-Ⅱ, and inhibited the function of BrZ-Ⅱ in gene regulation in the20E and JH pathways. Conclusion:The middle domain of Hsp90interacts with the BTB domain of BrZ-Ⅱ. The association between BrZ-Ⅱ and Hsp90is required for BrZ-Ⅱ stabilization and the function in gene regulation in the20E and JH pathways.Innovations and significationsThe findings about mod(mdg4)1A reveal the molecular mechanism of midgut PCD mediated by20E and JH, and indicate the existence of other signaling pathways except for EcR-USP pathway. The studies on ErGPCR provide a new insight that20E regulates the genomic pathway through GPCR-mediated non-genomic pathway, and reveal the existence of various pathways in20E signaling. JH blocks20E-induced genes expression and metamorphosis by regulating BrZ-Ⅱ phosphorylation. These results reveal a new mechanism by which JH suppresses20E-regulated metamorphosis, and enrich the function of Br during insect development. What’s more, Hsp90interacts with BrZ-Ⅱ to maintain BrZ-Ⅱ stabilization and the function of gene regulation in20E and JH pathways. It is the first report about BrZ-Ⅱ stability.