The Mechanisms Of Cathepsin D And Phosphatidylinositol 3-kinase In Regulating Insect Growth And Metamorphosis

Background and scientific questionsDuring the metamorphosis,the steroid hormone ecdysone(20-hydroxyecdysone,20E)and insulin-like peptides(ILPs)antagonistically regulate the larval growth and initiation of metamorphosis.ILPs,via insulin/insulin-like growth factor(IGF)signaling(IIS),mainly promote cell proliferation,tissue growth,and development.20E titer is higher during molting and metamorphosis.20E promotes the complex formation of ecdysone nuclear receptor(Ec R)-ultraspiracle isoform 1(USP1),which can activate the expression of autophagy-and apoptosis-related genes,resulting in degradation of larval tissues and the regeneration of adult tissues.Moreover,different tissues have different fates,such as apoptosis of larval midgut and fat body,adult midgut,and fat body occur proliferation,and epidermis remodeling through transcriptome reprogramming,but the regulatory mechanism is still unclear.Cathepsin D(CTSD)is a lysosomal aspartic protease.CTSD firstly synthesizes inactive proenzyme precursor containing a signal peptide and precursor peptide in the rough endoplasmic reticulum(ER)and then released signal peptide,which was glycosylated to form pro-CTSD.Then pro-CTSD is transported to the Golgi body and then transported to the lysosome,where mature CTSD(m-CTSD)is formed by cleavage of unknown protease or self-activation.Selective permeabilization of lysosome makes m-CTSD enter the cytoplasm from the lysosome,resulting in Caspases activation and inducing apoptosis;pro-CTSD secrets out the cells and promotes cell proliferation.Therefore,the expression,maturation,and secretion of CTSD play key roles in promoting apoptosis or proliferation,but its regulatory mechanism is unclear.Recent studies have shown that estrogen promotes the expression of CTSD in breast cancer.20E promotes CTSD expression and induces apoptosis of larval fat body cells in Bombyx mori,but the specific mechanism of CTSD regulating tissue remolding is unclear.Phosphatidylinositol 3 kinases(PI3Ks)are key kinases in IIS.Class IA PI3Ks are heterodimers composed of different catalytic subunits P110?/?/? and different regulatory subunits P85?/? and P55?,which phosphorylate phosphatidylinositol 4,5diphosphate(PIP2)converted into phosphatidylinositol 3,4,5-trisphosphate(PIP3),which activates protein kinase B(AKT)to promote cell proliferation.Recent studies suggest 20E and ILPs antagonistically regulate insect growth and development.However,how 20E regulates PI3K to counteract the IIS remains unclear.Here,we use agricultural pest Helicoverpa armigera,as a model,to study the functions of CTSD and PI3K in the metamorphosis and to investigate the mechanisms of CTSD expression,maturation,and secretion,and the mechanism of 20E that promotes cell apoptosis,then complement the mechanism of 20E and ILPs antagonizing the insect growth and metamorphosis,and systemically clarify the molecular mechanisms of insect growth and metamorphosis.ResultsThe expression of CTSD was tissue-and time-specific during development.CTSD was expressed as glycosylated-mature-CTSD(G-m-CTSD)in the midgut in metamorphosis,as pro-CTSD in the epidermis at pupal stages.Pro-CTSD from epidermis secreted into hemolymph as glycosylated-pro-CTSD(G-pro-CTSD).The glycosylation of asparagine at position 233 determined the secretion of pro-CTSD.20E,via EcR,promoted CTSD expression and induced the maturation and localization inside cells of CTSD through autophagy,which promoted the activation of CASP3 and induced apoptosis.G-pro-CTSD in the hemolymph promoted DNA replication,cell proliferation,and reassociation of the adult fat body through the PI3K-AKT pathway.The tissue differences of 20E titer and autophagy-related genes(Atgs)may be related to the tissue differential expression of CTSD.ILPs induced P60 and P110 phosphorylation and cell proliferation in the growth stage;however,in the metamorphosis,20E promoted P60 expression and induced P60 and P110 dephosphorylation,but 20E inhibited P110 expression.20E upregulated the expression of protein tyrosine phosphatase non-receptor type 6(PTPN6)to induce PTPN6 to interact with P60 and P110 to dephosphorylate P60 and P110,inhibit the membrane localization of P60 and P110 under ILPs induction,and reduce the interaction between P60 and P110.Phosphorylated P60 promoted the phosphorylation of AKT and foxhead box protein O(FOXO)and induced the cytoplasmic localization of FOXO,thus promoting cell proliferation and tissue growth.P60 or P110 knockdown in larval growth stages inhibited the phosphorylation of AKT and FOXO and production of 20E,leading to delayed pupation.P60 knockdown in metamorphosis inhibited apoptosis and delayed pupation.However,20E promoted P60 expression via ErGPCRs-EcR-FOXO signal axis,an excessive expression of P60 interacted with phosphatase and tensin homolog(PTEN)to induce apoptosis.These suggested that phosphorylated P60 is essential for larval growth and metamorphosis.Conclusion and SignificanceThis paper reveals that CTSD tissue-and time-specific expression,glycosylation at N233 determines the secretion of CTSD,autophagy induces CTSD maturation and induces larval midgut apoptosis,and G-pro-CTSD promotes adult fat body proliferation and reassociation.In the IIS and 20E pathways,P60 has the dual role of promoting cell proliferation and apoptosis by changing its phosphorylation state.This paper clarifies the mechanism of CTSD expression,maturation and secretion,and its function in growth and metamorphosis,and reveals the molecular mechanism of different tissues with different fates during the metamorphosis of lepidopteran insects.Lepidopteran adult fat body cell proliferation is reported for the first time;the molecular mechanism of 20E antagonizing IIS to promote metamorphosis is supplemented,which provides a theoretical basis and genes target for pest control and has important theoretical and practical significance.