| BackgroundsInsects,as the most diverse species in the world,experience the two major development physiological processes:molting and metamorphosis.The development and metamorphosis are mainly controlled by two hormones:20-hydroxyecdysone(20E)and juvenile hormone(JH).In the larvae stage,JH has a high titer to maintain insect larvae state;in the molting and metamorphosis stage,JH titer gradually decrease and 20E titer become high to start insect molting and metamorphosis.The two major hormones coordinately control insect molting and metamorphosis.So,studying the mechanism of molecular regulation of this process not only provides scientific basis for pest control,but also presents the possible references for some human diseases.Previous studies showed that 20E exerts its action via the genomic pathway,that is 20Efuses into cells and binds to intracellular nuclear ecdysone receptor(EcR),then bind to DNA to initiate gene transcription.Recent years,as the increasing molecular biology techniques,studies suggest that animal steroid hormones existing non-genomic pathway.20E can activate receptors in the cell membrane to initiate rapid non-genomic interactions,such as rapid cellular calcium increase,proteins rapid phosphorylation and subcellular translocation.At the same time,20E-inducedrapid non-genomic pathway and the membrane receptors are also studied as hot spots.G-protein-coupled receptors(GPCR)are proposed involved in animal steroidhormone signaling,but their mechanism is unclear.Calcium ions are essential secondary messengers that regulate diverse cellularprocesses including gene transcription,cell proliferation,and apoptosis.The steroid hormone 20E promotes programmed cell death during insect metamorphosis,whereas juvenile hormone(JH)counteracts 20E activity to prevent metamorphosis.Both 20E and JH can induce cellular calcium increase;however,the mechanisms and physiological consequences resulting from calcium increase caused by the two counter-acting hormones are unclear.In this research,we report ErGPCR-2 as a target gene to study the function of GPCR in the 20E non-genomic pathway,as well as the molecular mechanism of calcium ions as a second messenger at 20E-regulated apoptosis.The results may enrich and improve the theoretical knowledge of 20E signal pathways and insect growth and development,provide the target gene and scientific basis for the pest control and human disease treatment.Research results1.GPCR controls steroid hormone signaling in cell membrane.In this research,we report that a GPCR called ErGPCR-2 controls steroid hormone 20E signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera.ErGPCR-2 was highly expressed during molting and metamorphosis.20E,via ErGPCR-2,regulated rapid intracellular calcium increase,protein phosphorylation,gene transcription,and insect metamorphosis.ErGPCR-2 was located in the cell membrane and was internalized by 20E induction.GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization.The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling.ErGPCR-2 knockdown suppressed the entrance of 20E analog[3H]ponasterone A([3H]Pon A)into the cells.ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of[3H]Pon A into the cells.However,ErGPCR-2 did not bind to[3H]Pon A.Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane.2.20E promotes higher calcium mobilization to induce apoptosis20E via GPCR induced a major calcium level rise in the cells,whereas JH via receptor tyrosine kinases induced a minor calcium increase.The calcium release-activated calcium modulator 1(Orail)and transient receptor potential(TRP)channels were necessary for 20E-induced rapid calcium influx.A higher calcium level was maintained in a long time and more genes including Orail and TRP channels showed elevated expression after the treatment of 20E than did after JH treatment.Caspase3/7 activation,cell death and pro-apoptotic gene expression were elicited by 20E induction,but not by JH.JH could repress 20E-induced calcium influx,caspase3/7 activation and gene expression.Higher calcium levels induced apoptosis.These results suggest that 20E and JH via different pathways regulate calcium mobilization and homeostasis at different levels,thus inform different gene expression and cellular responses.Conclusions and Significances1.ErGPCR-2 controls 20E into cells on the cell membraneErGPCR-2 was localized on the cell membrane to control[3H]Pon A into cells.ErGPCR-2 regulated the 20E signaling pathway on the cell membrane.This study complements the theoretical basis of the 20E non-genomic pathway,which establishing a link between the genomic pathway and the non-genomic pathway.This study also provides a new theoretical basis for the study of 20E signal pathway in the future.2.20E promoting higher calcium mobilization to induce apoptosis.20E via GPCR induced a major calcium level rise in the cells,whereas JH via receptor tyrosine kinases induced a minor calcium increase.A higher calcium level was maintained in a long time and more calcium channel-related genes showed elevated expression after the treatment of 20E.20E induced Caspase3/7 activation levels,promoted cell death and upregulated the expression of pro-apoptotic genes.Higher calcium levels induced apoptosis.Calcium ions are essential secondary messengers in cells that play important roles in cell proliferation and apoptosis.20E via GPCR pathway regulates higher level of calcium homeostasis to induce cell apoptosis.This study confirms the function of calcium ions in cell apoptosis,which not only contributes to improve the molecular mechanism of insect apoptosis,but also to promote scientific studies for cancer treatment. |
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