The Functions Of β-arrestin1 And Protein Kinase B(PKB/Akt) In The Ecdysone Signaling Pathway From Helicoverpa Armigera

Background, scientific question and significance:The insect development is regulated by three kinds of hormones:ecdysone (20-hydroxyecdysone,20E),juvenile hormone(JH)and insulin.20E is an important insect hormone which plays essential roles in insect molting and metamorphosis. In a genomic pathway,20E combines with an ecdysone nuclear receptor (EcR) and forms a transcription complex with the transcription factor ultraspiracle potein (USP) to regulate transcription of several genes, including hormone receptor 3 (HR3) and transcription factor Broad.Recent studies have found 20E signaling pathway activites a non-genomic signaling pathway before the genomic pathway.20E induces intracellular calcium increase and protein phosphorylation, and 20E also mediates USP phosphorylation through Ecdysone response G protein coupled receptors (ErGPCR), Phospholipase C and protein kinase C (PKC) signaling. However, the mechanism of 20E signal termination (desensitization) is unclear. β-arrestinl is an evolutionarily conserved molecules involved in GPCR desensitization. In Drosophila, β-arrestinl (Kurtz) is involved in a variety of receptors desensitization, but the function of β-arrestin1 in 20E signaling is still unclear.The conserved insulin signaling pathway regulates animal growth. Insulin binds to insulin receptor (InR) and transductes the extracellular signal into intracellular molecules. At the downstream of InR, the adapter protein Chico phosphorylates PI3K110 via its adaptor protein PI3K60. The activated PI3K110 activates protein kinase B (PKB, also named Akt) by phosphorylation modification. The FoxO can be phosphorylated by Akt, the phosphorylated FoxO locates in the cytoplasm and inhibits cell apoptosis. In Drosophila,20E signal antagonizes insulin signals through mediating Akt phosphorylation and the translocation of FoxO, but the mechanism on remains unclear.The cotton bollworm (Helicoverpa armigera) belongs to lepidoptera. As a severe agricultural pest, H.armgeria has gradually become an important experimental animal to investigate insect development. In this paper, H. armigera was used as the research model to explore the function of β-arrestinl in ecdysone signal desensitization. The studies on the function of β-arrestinl in 20E signaling provide further insights into animal steroid hormone signaling. The study on the function of Akt in 20E signaling provides us further understanding of the molecular mechanism of two hormones’ crosstalk in insect development regulation.Results and Conclusion:1. The function of P-arrestinl in 20E signal termination.Western blot showed β-arrestinl was increased expression during metamorphosis. β-arrestinl knockdown in larvae caused advanced pupation and larval-pupal chimera. The mRNA levels of 20E-response genes were increased after β-arrestinl was knocked down.20E induced the migration of β-arrestin1 from the cytosol to the cytoplasa membrane to interact with ErGPCR1. The inhibitors suramin and chelerythrine chloride repressed 20E-induced β-arrestinl phosphorylation and membrane migration. The double mutation of the amino acids Serl70 and Ser234 to asparagine inhibited phosphorylation and membrane migration of β-arrestinl in 20E induction. Theses results suggest 20E via ErGPCR1-, PKC-signal induces β-arrestin1 phosphorylation; phosphorylated β-arrestinl migrates to the cytoplasmic membrane to interact with ErGPCR1 to block 20E signaling via a feedback mechanism. The studies on the non-genomic pathway of 20E provide deep insights into animal steroid hormone signaling, and also offer us a further understand of the mechanism on GPCR desensitization.2.20E blocks the phosphorylation of Akt to antagonize insulin signal.The study on the HaEpi showed that high dose of 20 or knockdown Akt increased apoptosis-related genes mRNA levels and induced cell apoptosis. Western blots and immunocytochemistry results showed,20E blocked the insulin induced Akt transloction and phosphorylation. qRT-PCR and Western blots showed 20E up-regulated the expression of PTEN, and also knockdown PTEN removed the 20E-induced repression of Akt phosphorylation. Knockdown EcRB1 and USP1 block 20E-induced PTEN up-regulation. These results indicate that 20E via the transcription factors EcRB1 and USP1 increases the expression of PTEN. High expression of PTEN impedes Akt phosphorylation, which raise the apoptosis related gene mRNA levels, leading to cell apoptosis. This study reavled the mechanism of 20E signal antagonist insulin signal in details, give us a well understand on the 20E interaction with insulin regulation the insect development.