The steroid hormone 20-hydroxyecdysone counteracts insulin signaling via insulin receptor dephosphorylation
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100318
ISSN1083-351X
AutoresYan-Li Li, You-Xiang Yao, Yu‐Meng Zhao, Yuqin Di, Xiao‐Fan Zhao,
Tópico(s)Healthcare and Venom Research
ResumoThe insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph. The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph. Insulin, insulin-like growth factors (IGFs), and insulin-like peptides (ILPs) promote growth via insulin/IGF signaling (IIS) (1Nassel D.R. Vanden Broeck J. Insulin/IGF signaling in Drosophila and other insects: Factors that regulate production, release and post-release action of the insulin-like peptides.Cell. Mol. Life Sci. 2016; 73: 271-290Crossref PubMed Scopus (145) Google Scholar). The steroid hormones 20-hydroxecdysone (20E) and estrogen attenuate insulin signaling and the growth rate in Drosophila and humans, respectively (2Hyun S. Body size regulation and insulin-like growth factor signaling.Cell. Mol. Life Sci. 2013; 70: 2351-2365Crossref PubMed Scopus (46) Google Scholar). Insulin and 20E are the main regulators of insect growth (3Nijhout H.F. Riddiford L.M. Mirth C. Shingleton A.W. Suzuki Y. Callier V. The developmental control of size in insects.Wiley Interdiscip. Rev. Dev. Biol. 2014; 3: 113-134Crossref PubMed Scopus (154) Google Scholar). The insulin pathway determines the growth rate, and 20E determines the duration of growth (4Lin X. Smagghe G. Roles of the insulin signaling pathway in insect development and organ growth.Peptides. 2018; 122: 169923Crossref PubMed Scopus (34) Google Scholar). However, despite intensive research, how animals regulate growth and growth termination by the cross talk between insulin and steroid hormones remains unclear. In addition, alterations of the insulin pathway also result in diabetes via insulin insufficiency (type I diabetes) or insulin resistance and pancreatic β-cell dysfunction (type II diabetes) (5Elena C. Chiara M. Angelica B. Chiara M.A. Laura N. Chiara C. Claudio C. Antonella F. Nicola G. Hyperglycemia and diabetes induced by glucocorticoids in nondiabetic and diabetic patients: Revision of literature and personal considerations.Curr. Pharm. Biotechnol. 2018; 19: 1210-1220Crossref PubMed Scopus (13) Google Scholar, 6Kahn S.E. Cooper M.E. Del Prato S. Pathophysiology and treatment of type 2 diabetes: Perspectives on the past, present, and future.Lancet. 2014; 383: 1068-1083Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar). Insulin maintains normal blood glucose levels; however, the steroid hormones counteract insulin function and increase blood glucose levels, even cause diabetes (7Hwang J.L. Weiss R.E. Steroid-induced diabetes: A clinical and molecular approach to understanding and treatment.Diabetes Metab. Res. Rev. 2014; 30: 96-102Crossref PubMed Scopus (110) Google Scholar). For example, glucocorticoids, which are widely used anti-inflammatory and immunosuppressive drugs (5Elena C. Chiara M. Angelica B. Chiara M.A. Laura N. Chiara C. Claudio C. Antonella F. Nicola G. Hyperglycemia and diabetes induced by glucocorticoids in nondiabetic and diabetic patients: Revision of literature and personal considerations.Curr. Pharm. Biotechnol. 2018; 19: 1210-1220Crossref PubMed Scopus (13) Google Scholar), induce hyperglycemia and insulin-resistant diabetes (8Schultz H. Rasmussen B.K. Kristensen P.L. Jensen A.K. Pedersen-Bjergaard U. Early incidence of glucocorticoid-induced diabetes in patients with brain tumors: A retrospective study of the first 7 days of treatment.Neurooncol. Pract. 2018; 5: 170-175PubMed Google Scholar); however, the mechanisms are not fully understood. The regulation of hemolymph glucose levels by 20E and its mechanism are also unclear. The insulin receptor (INSR) is a receptor tyrosine kinase that plays important roles in the insulin pathway by binding its ligand (insulin) to regulate glucose, fatty acids, and protein metabolism to promote growth (9Gabbouj S. Ryhanen S. Marttinen M. Wittrahm R. Takalo M. Kemppainen S. Martiskainen H. Tanila H. Haapasalo A. Hiltunen M. Natunen T. Altered insulin signaling in Alzheimer's disease brain - special emphasis on PI3K-Akt pathway.Front. Neurosci. 2019; 13: 629Crossref PubMed Scopus (64) Google Scholar). INSR is a constitutive homodimeric transmembrane glycoprotein (10Mohammadiarani H. Vashisth H. All-atom structural models of the transmembrane domains of insulin and type 1 insulin-like growth factor receptors.Front. Endocrinol. 2016; 7: 68Crossref PubMed Google Scholar), comprising two α and two β subunits linked by disulfide bridges (11Tatulian S.A. Structural dynamics of insulin receptor and transmembrane signaling.Biochemistry. 2015; 54: 5523-5532Crossref PubMed Scopus (28) Google Scholar). INSR is encoded by the Insr gene as a single protein. A protease, furin, cleaves the protein into the α and β subunits, named INSRα and INSRβ, respectively (12Bass J. Turck C. Rouard M. Steiner D.F. Furin-mediated processing in the early secretory pathway: Sequential cleavage and degradation of misfolded insulin receptors.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11905-11909Crossref PubMed Scopus (46) Google Scholar). INSRα has insulin-binding sites and is located outside the cell membrane. INSRβ contains a transmembrane domain and the intracellular tyrosine kinase elements (13Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. The human insulin receptor cDNA: The structural basis for hormone-activated transmembrane signalling.Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (939) Google Scholar). Insulin binding causes a conformational change and autophosphorylation of INSRβ, resulting in phosphorylation of phosphoinositide-3-kinase (PI3K), which phosphorylates phosphatidylinositol 4, 5-diphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) (14Czech M.P. Yu K.T. Lewis R.E. Davis R.J. Mottola C. MacDonald R.G. Necessary P.C. Corvera S. Insulin receptor kinase and its mode of signaling membrane components.Diabetes Metab. Rev. 1985; 1: 33-58Crossref PubMed Scopus (5) Google Scholar). PIP3 attracts AKT/protein kinase B (PKB) to the cell membrane, where it is phosphorylated by the phosphoinositide-dependent protein kinase 1 (PDK1) (9Gabbouj S. Ryhanen S. Marttinen M. Wittrahm R. Takalo M. Kemppainen S. Martiskainen H. Tanila H. Haapasalo A. Hiltunen M. Natunen T. Altered insulin signaling in Alzheimer's disease brain - special emphasis on PI3K-Akt pathway.Front. Neurosci. 2019; 13: 629Crossref PubMed Scopus (64) Google Scholar). AKT phosphorylates AS160 protein, which promotes glucose transporter 4 (GLUT4) translocation to the cell membrane for glucose uptake into the cell from blood (9Gabbouj S. Ryhanen S. Marttinen M. Wittrahm R. Takalo M. Kemppainen S. Martiskainen H. Tanila H. Haapasalo A. Hiltunen M. Natunen T. Altered insulin signaling in Alzheimer's disease brain - special emphasis on PI3K-Akt pathway.Front. Neurosci. 2019; 13: 629Crossref PubMed Scopus (64) Google Scholar). AKT also phosphorylates Forkhead box O (FoxO), a negative regulator of the insulin pathway, to locate FoxO in the cytoplasm, thus blocking its transcriptional activity in the nucleus (15Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB.Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (301) Google Scholar). The above insulin-induced events can be reversed by the pathway's negative regulator, phosphatase, and tensin homolog deleted on chromosome 10 (PTEN), also named as MMAC1 (mutated in multiple advanced cancer 1), or TEP1 (TGF-regulated and epithelial cell-enriched phosphatase) (16Goberdhan D.C. Paricio N. Goodman E.C. Mlodzik M. Wilson C. Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway.Genes Dev. 1999; 13: 3244-3258Crossref PubMed Scopus (278) Google Scholar). The INSR-mediated insulin signaling pathway and its downstream signaling molecules are structurally and functionally conserved throughout evolution from worms to mammals (17Das D. Arur S. Conserved insulin signaling in the regulation of oocyte growth, development, and maturation.Mol. Reprod. Dev. 2017; 84: 444-459Crossref PubMed Scopus (59) Google Scholar). Insulin promotes growth and ecdysone production in the prothoracic gland (PG), and in turn, the increased 20E level counteracts insulin function and promotes insect metamorphosis (18Mirth C. Truman J.W. Riddiford L.M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster.Curr. Biol. 2005; 15: 1796-1807Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). 20E regulates the nuclear localization of transcription factor FoxO to counteract the role of insulin in promoting growth in Drosophila melanogaster (19Colombani J. Bianchini L. Layalle S. Pondeville E. Dauphin-Villemant C. Antoniewski C. Carre C. Noselli S. Leopold P. Antagonistic actions of ecdysone and insulins determine final size in Drosophila.Science. 2005; 310: 667-670Crossref PubMed Scopus (433) Google Scholar). In Helicoverpa armigera, 20E upregulates Pten expression to repress AKT phosphorylation; therefore, FoxO cannot be phosphorylated by AKT and enters the nucleus to induce downstream gene transcription in the 20E pathway (20Cai M.J. Zhao W.L. Jing Y.P. Song Q. Zhang X.Q. Wang J.X. Zhao X.F. 20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting.Development. 2016; 143: 1005-1015Crossref PubMed Scopus (30) Google Scholar). In addition, a high 20E titer inhibits Pdk1 expression and represses AKT and FoxO phosphorylation, resulting in FoxO nuclear localization to induce autophagy and repress cell proliferation in H. armigera (21Pan J. Di Y.Q. Li Y.B. Chen C.H. Wang J.X. Zhao X.F. Insulin and 20-hydroxyecdysone oppose each other in the regulation of phosphoinositide-dependent kinase-1 expression during insect pupation.J. Biol. Chem. 2018; 293: 18613-18623Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). All these studies indicate that 20E counteracts the insulin pathway; however, whether 20E exerts this effect from the beginning of the insulin pathway by repressing INSR phosphorylation and expression, and the consequence, is unknown. To understand the mechanisms by which steroid hormones counteract the insulin pathway, we used the lepidopteran insect H. armigera, an agricultural pest, as a model, and the well-known factors in insulin pathway as readouts for the study. Our study revealed that INSR plays roles in insect larval growth and therefore promotes 20E production to reach a critical titer during larval feeding stages. 20E induces the dephosphorylation of INSR to repress the insulin pathway. Thus, the steroid hormone 20E counteracts the insulin pathway to stop larval growth and accumulate hemolymph glucose. H. armigera completes six larval instars, pupae, and adult stages in about 1 month (22Zhao X.-F. Progress in understanding hormonal regulation during the postembryonic development of Helicoverpa armigera.J. Integr. Agric. 2020; 19: 1417-1428Crossref Scopus (4) Google Scholar). To study the function of INSR, its abundance and phosphorylation profiles during development were examined. Two commercially available antibodies against human non-phospho-INSRβ (INSRβ) and phospho-INSRβ (p-INSRβ) were used for western blotting to detect changes in the INSR protein levels and changes in protein phosphorylation. INSRβ was detected as a single band using the anti-INSRβ antibody in the epidermis, midgut, and fat body. INSRβ levels increased during feeding stages from the sixth instar 6 h to 48 h (sixth-6 h–sixth-48 h) and decreased during metamorphosis from the sixth instar larvae 72 h (sixth-72 h) to the adult (Fig. 1, A and B). The antibody against INSRβ recognized H. armigera INSRβ-His specifically, with a lower molecular band representing the endogenous INSRβ (Fig. S1A). The mRNA levels of Insr showed similar expression profiles to that of the protein (Fig. S1B). These data suggested that INSR expression was reduced during metamorphosis. To determine the profile of INSRβ phosphorylation, an antibody against p-INSRβ was used after enrichment of same amount of INSRβ to overcome the different expression levels of INSRβ during metamorphosis. Higher levels of p-INSRβ during the larval feeding stages (5F and sixth-6 h–sixth-48 h) compared with that during the metamorphic stages (sixth-72 h–sixth-120 h) were detected (Fig. 1C). INSRβ was detected as two bands using the anti-INSRβ antibody and a 7.5% low-concentration gel (Fig. 1D). Lambda protein phosphatase (λPPase) treatment decreased the intensity of the p-INSRβ band, suggesting that the upper band was the phosphorylated form of INSRβ (p-INSRβ) (Fig. S1C). These results suggested that phosphorylation of INSRβ occurs at a lower level during metamorphosis. The 20E titer in vivo appeared to increase from 0.5 μM to 10 μM from larval growth to metamorphosis in H. armigera (23Di Y.Q. Han X.L. Kang X.L. Wang D. Chen C.H. Wang J.X. Zhao X.F. Autophagy triggers CTSD (cathepsin D) maturation and localization inside cells to promote apoptosis.Autophagy. 2020; : 1-23Crossref PubMed Scopus (8) Google Scholar, 24Kang X.L. Zhang J.Y. Wang D. Zhao Y.M. Han X.L. Wang J.X. Zhao X.F. The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation.PLoS Genet. 2019; 15e1008331Crossref PubMed Scopus (21) Google Scholar). To address the mechanism of the lower level and phosphorylation of INSRβ during metamorphosis, we investigated the roles of insulin and 20E in these events by injecting different concentrations of insulin and 20E into the sixth instar 6 h larvae hemocoel. Increasing concentrations of insulin increased INSRβ levels and phosphorylation in the larval epidermis (Fig. 2A). The increased levels of phosphorylated INSRβ were confirmed using the anti-p-INSRβ antibody after normalization of the INSRβ levels by affinity enrichment of INSRβ (Fig. 2B). In contrast, a higher concentration of 20E (500 ng) decreased INSRβ levels (Fig. 2C) and phosphorylation, detected after affinity enrichment of INSRβ (Fig. 2D). In addition, a high concentration of 20E (5 μM) was confirmed to repress insulin-induced INSRβ levels and phosphorylation in HaEpi cells (Fig. 2, E and F), which represented a model to study INSR in cells. These results suggested that high concentration of 20E represses INSRβ levels and phosphorylation. INSRβ undergoes autophosphorylation by binding insulin (14Czech M.P. Yu K.T. Lewis R.E. Davis R.J. Mottola C. MacDonald R.G. Necessary P.C. Corvera S. Insulin receptor kinase and its mode of signaling membrane components.Diabetes Metab. Rev. 1985; 1: 33-58Crossref PubMed Scopus (5) Google Scholar). Therefore, a deficiency of insulin might be the reason for the decrease of INSRβ phosphorylation. However, eight ILPs were identified by BLAST searching of the H. armigera genome using the sequences of 38 Bombyx mori ILPs, 8 Drosophila ILPs (25Lin F. Hossain M.A. Post S. Karashchuk G. Tatar M. De Meyts P. Wade J.D. Total solid-phase synthesis of biologically active Drosophila insulin-like peptide 2 (DILP2).Aust. J. Chem. 2017; 70: 208-212Crossref PubMed Scopus (10) Google Scholar), and 10 Homo sapiens ILPs (Fig. S2), and which ILP binds to INSR was not known; therefore, the expression levels of all Ilp genes were detected to address the insulin levels. Surprisingly, the mRNA levels of eight Ilp genes increased in the epidermis, midgut, and fat body during metamorphosis from the wandering stage to the later pupal stage, as assessed using quantitative real-time reverse transcription PCR (qRT-PCR) (Fig. S3, A–H). The Ilp genes in the brain also exhibited increased expression during metamorphosis, in addition to B10-like, whose relationship with INSRβ requires further study (Fig. S3I). Compared with the dimethyl sulfoxide (DMSO) control, 20E-injected in larvae increased their expression of B2-like, B10-like, and C1-like in a dose and time-dependent manner (Fig. S3, J–O). These findings suggested that the expression levels of most Ilp genes are sufficient during metamorphosis and are thus not the reason for the decrease in INSRβ phosphorylation; therefore, INSRβ is likely dephosphorylated by some protein phosphatases under 20E regulation during metamorphosis. The protein synthesis inhibitor, cycloheximide, was used to test the above hypothesis in HaEpi cells. Insulin-induced INSRβ phosphorylation was compared with that in the PBS control. 20E inhibited the insulin-induced INSRβ phosphorylation, compared with that of the DMSO control; however, 20E could not inhibit INSRβ phosphorylation after the cells were preincubated with cycloheximide (Fig. 3, A and C), suggesting that 20E inhibited INSRβ phosphorylation by promoting the synthesis of certain proteins. Further experiments showed that phosphatase inhibitors (Phosphatase Inhibitor Cocktail, Cat. 20109ES05) repressed 20E-induced INSRβ dephosphorylation (Fig. 3, B and D), implying that 20E acts via a protein phosphatase to dephosphorylate INSRβ. To determine the phosphatase that phosphorylates INSRβ, we identified three highly expressed protein-tyrosine-phosphatase (PTPase) genes, Ptpn1, Mtmr6, and Ptprn2, during metamorphosis from the midgut transcriptomes at the sixth-24 h feeding stage and sixth-72 h metamorphic stage, using the Illumina sequencing platform, a database produced in our laboratory. The transcriptome had only been determined once; therefore, the phosphatases were further examined for their developmental expression profiles in tissues using qRT-PCR. The transcript levels of the three PTPases increased in the midgut during the metamorphic stages (sixth-72 h to adult) compared with that in the feeding stages (sixth-6 h–sixth-48 h) (Fig. 4A), and 20E increased their expression (Fig. 4B), suggesting that they might be involved in 20E-induced INSRβ dephosphorylation. To identify which of the three PTPases dephosphorylates INSRβ, we knocked down their expression in HaEpi cells using RNAi, separately. Insulin induced INSRβ phosphorylation, compared with PBS, and 20E decreased insulin-induced INSRβ phosphorylation compared with DMSO. However, knockdown of Ptpn1 maintained INSRβ phosphorylation, whereas after knockdown of Mtmr6 and Ptprn2, 20E still induced INSRβ dephosphorylation, compared with dsGFP (Fig. 4, C and D). The efficacy of PTPase knockdown was demonstrated using qRT-PCR analysis (Fig. S4). Coimmunoprecipitation (Co-IP) experiments using the antibody against INSRβ (anti-INSRβ) confirmed that the overexpressed PTP1B-GFP-His protein interacted with INSRβ under 20E induction (Fig. 4E). These data suggested that PTP1B dephosphorylates INSRβ. PTEN is a dual-function phosphatase playing roles in the cell membrane (26Jamaspishvili T. Berman D.M. Ross A.E. Scher H.I. De Marzo A.M. Squire J.A. Lotan T.L. Clinical implications of PTEN loss in prostate cancer.Nat. Rev. Urol. 2018; 15: 222-234Crossref PubMed Scopus (164) Google Scholar), and its expression is upregulated by 20E during metamorphosis (20Cai M.J. Zhao W.L. Jing Y.P. Song Q. Zhang X.Q. Wang J.X. Zhao X.F. 20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting.Development. 2016; 143: 1005-1015Crossref PubMed Scopus (30) Google Scholar); therefore, its involvement in INSRβ dephosphorylation was examined. The mRNA levels of Pten increased during metamorphosis from the wandering stage to the pupal stage in the epidermis, midgut, and fat body (Fig. 5A). A 20E receptor EcR-binding element (EcRE) 5′-AATGGCAATGACTAC-3′ (−1060 to −1074 bp, relative to ATG) was predicted (http://jaspardev.genereg.net/) in the promoter region of Pten. 20E increased the transcript level of Pten in a dose- and time-dependent manner, compared with that in the DMSO control (Fig. S5, A and B). In HaEpi cells, 20E reduced the insulin induced-p-INSRβ levels; however, knockdown of Pten blocked 20E-induced dephosphorylation of INSRβ, compared with dsGFP. In contrast, overexpression of Pten decreased INSRβ phosphorylation in the absence of 20E induction, compared with that in cells overexpressing green fluorescent protein (GFP) (Fig. 5B). The statistical analysis confirmed the data (Fig. 5C). The efficacy of Pten knockdown was demonstrated using qRT-PCR analysis (Fig. S5C), and the overexpression of Pten was indicated using western blotting (Fig. S5D). However, Co-IP experiments did not identify an interaction between INSRβ and PTEN (Fig. S5E). These results indicated that PTEN is involved in INSRβ dephosphorylation, but it cannot interact with INSRβ directly. A previous study showed that PTEN induces FoxO nuclear localization by repressing AKT and FoxO phosphorylation in H. armigera under 20E induction (20Cai M.J. Zhao W.L. Jing Y.P. Song Q. Zhang X.Q. Wang J.X. Zhao X.F. 20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting.Development. 2016; 143: 1005-1015Crossref PubMed Scopus (30) Google Scholar); therefore, PTEN was suspected to play roles in 20E-induced INSRβ dephosphorylation by regulating FoxO nuclear localization, thereby promoting Ptpn1 expression. Therefore, FoxO fused with a GFP tag was overexpressed in HaEpi cells to address the mechanism of PTEN's involvement in 20E-induced INSRβ dephosphorylation, with GFP showing the subcellular location of FoxO. FoxO-GFP and GFP were confirmed as overexpressed in HaEpi cells, separately (Fig. S6A). The overexpressed FoxO-GFP was localized in the nucleus in the PBS control and translocated into the cytoplasm after insulin induction. Compared with the insulin plus DMSO control, 20E reversed insulin's function and maintained FoxO-GFP's nuclear localization. However, compared with dsRFP plus insulin and 20E, after knockdown of Pten, 20E could not maintain FoxO-GFP's nuclear localization (Fig. 6A). The control group overexpressing the GFP tag did not show the subcellular variation in FoxO localization (Fig. S6B), confirming that PTEN determines FoxO-GFP nuclear localization. The effect of FoxO-GFP nuclear localization on the expression of PTP1B was examined to address its role in PTP1B expression. A FoxO-binding element (FoxOBE) 5′-TTGTTAAC-3′ (−980 to −973 bp, relative to ATG) was predicted in the promoter region of Ptpn1, which was similar to the FoxOBE sequence (5′-TTGTTTAC-3′) in the promoter region of H. armigera BrZ7 (20Cai M.J. Zhao W.L. Jing Y.P. Song Q. Zhang X.Q. Wang J.X. Zhao X.F. 20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting.Development. 2016; 143: 1005-1015Crossref PubMed Scopus (30) Google Scholar). In the FoxO-GFP overexpressing cells, the Ptpn1 level increased compared with that in the GFP overexpressing cells (Fig. 6B), suggesting that the overexpressed FoxO-GFP enhanced Ptpn1 expression. A chromatin immunoprecipitation (ChIP) assay showed that FoxO-GFP bound more FoxOBE under 20E treatment than it did in the DMSO treatment control using the primer FoxOBE of Ptpn1 (FoxOBE-Ptpn1-F/FoxOBE-Ptpn1-R primers targeting the Ptpn1 FoxOBE-containing sequence, as shown in the Table S1). The IgG negative control and Ptpn1 primers (Ptpn1-F/R located in the open reading frame (ORF) of Ptpn1) did not produce the same results (Fig. 6C). These results suggested that 20E via FoxO promotes Ptpn1 transcription. Next, the effect of FoxO-GFP nuclear localization on the level of INSRβ was examined because another kind of FoxOBE, 5′-TGTTTAC-3′ (−1835 to −1829 bp, relative to ATG), was predicted in the promoter region of Insr. Interestingly, in the FoxO-GFP overexpressing cells, the protein level of INSRβ decreased, in addition to decrease of INSRβ phosphorylation by PTP1B (Fig. 6D), suggesting that overexpression of FoxO-GFP repressed Insr expression. ChIP results further showed that FoxO-GFP bound more FoxOBE than GFP, using the primer FoxOBE of Insr (FoxOBE-Insr-F/FoxOBE-Insr-R primers targeting the Insr FoxOBE containing sequence, as shown in the Table S1). The IgG negative control and Insr primers (Insr-F/R, located in the open reading frame of Insr Table S1) did not produce the same results (Fig. 6E). These findings suggested that FoxO is the repressor of Insr transcription. To examine the function of PTP1B in vivo, we knocked down Ptpn1 by injecting dsPtpn1 into the sixth instar 6 h larval hemocoel. qRT-PCR showed that the expression of Ptpn1 was successfully knocked down in the epidermis (Fig. 7A). The level of phosphorylated INSRβ after injection of dsPtpn1 was significantly higher than that in the dsGFP control group (Fig. 7B), which confirmed the role of PTP1B in INSRβ dephosphorylation in vivo. As a consequence, the major effects of knockdown of Ptpn1 were the production of smaller pupae (Fig. 7, C–E) and earlier pupation, compared with that in the control group (Fig. 7F). In addition, the larval midgut appeared red, which normally occurs during larval midgut programmed cell death (PCD) during metamorphosis (27Hakim R.S. Baldwin K. Smagghe G. Regulation of midgut growth, development, and metamorphosis.Annu. Rev. Entomol. 2010; 55: 593-608Crossref PubMed Scopus (182) Google Scholar, 28Wang J.L. Jiang X.J. Wang Q. Hou L.J. Xu D.W. Wang J.X. Zhao X.F. Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera.BMC Dev. Biol. 2007; 7: 76Crossref PubMed Scopus (23) Google Scholar), in the dsPtpn1 group, but not in the dsGFP-treated larval midgut at the same time after injection of double-stranded RNA (dsRNA) (Fig. 7G). This suggested that the larvae of dsPtpn1 entered metamorphosis earlier than the control group. The 20E titer in the hemolymph was 5 μM, which was higher than that (2.5 μM) in the dsGFP control (Fig. 7H), indicating that maintaining phosphorylation of INSRβ increased 20E production, which accelerated pupation. To examine the function of INSR in insect growth and insulin function, we injected dsInsr into fifth instar 24 h larvae to knock down Insr. Western blotting showed that the level of the INSRβ protein was successfully reduced (Fig. 8A). This resulted in 30.9% death among the fifth instar and the sixth instar larvae and 59.5% small pupae (Fig. 8, B and C). Moreover, the pupal weight of the dsInsr-treated group decreased to an average of 0.26 g compared with 0.37 g of the dsGFP injection control (Fig. 8D). The pupation time was delayed significantly from 140 h to 190 h after knockdown of Insr (Fig. 8E), and the midgut appeared red later than that in the dsGFP control (Fig. 8F), suggesting that INSR plays roles in larval growth and pupation. The reason for the delayed pupation might because of insufficient 20E in the hemolymph (Fig. 8G) along with the delayed larval growth when Insr was knocked down. Meanwhile, the mRNA levels of insulin pathway genes- Insr, Pi3k, and Akt were downregulated after Insr knockdown (Fig. 8H), and western blottin
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