Subunit P60 of phosphatidylinositol 3-kinase promotes cell proliferation or apoptosis depending on its phosphorylation status.

The regulatory subunits (P60 in insects, P85 in mammals) determine the activation of the catalytic subunits P110 in phosphatidylinositol 3-kinases (PI3Ks) in the insulin pathway for cell proliferation and body growth. However, the regulatory subunits also promote apoptosis via an unclear regulatory mechanism. Using Helicoverpa armigera, an agricultural pest, we showed that H. armigera P60 (HaP60) was phosphorylated under insulin-like peptides (ILPs) regulation at larval growth stages and played roles in the insulin/ insulin-like growth factor (IGF) signaling (IIS) to determine HaP110 phosphorylation and cell membrane translocation; whereas, HaP60 was dephosphorylated and its expression increased under steroid hormone 20-hydroxyecdysone (20E) regulation during metamorphosis. Protein tyrosine phosphatase non-receptor type 6 (HaPTPN6, also named tyrosine-protein phosphatase corkscrew-like isoform X1 in the genome) was upregulated by 20E to dephosphorylate HaP60 and HaP110. 20E blocked HaP60 and HaP110 translocation to the cell membrane and reduced their interaction. The phosphorylated HaP60 mediated a cascade of protein phosphorylation and forkhead box protein O (HaFOXO) cytosol localization in the IIS to promote cell proliferation. However, 20E, via G protein-coupled-receptor-, ecdysone receptor-, and HaFOXO signaling axis, upregulated HaP60 expression, and the non-phosphorylated HaP60 interacted with phosphatase and tensin homolog (HaPTEN) to induce apoptosis. RNA interference-mediated knockdown of HaP60 and HaP110 in larvae repressed larval growth and apoptosis. Thus, HaP60 plays dual functions to promote cell proliferation and apoptosis by changing its phosphorylation status under ILPs and 20E regulation, respectively.

The steroid hormone 20-hydroxyecdysone counteracts insulin signaling via insulin receptor dephosphorylation.

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.

The steroid hormone 20-hydroxyecdysone induces phosphorylation and aggregation of stromal interacting molecule 1 for store-operated calcium entry.

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.

Protein kinase C delta phosphorylates ecdysone receptor B1 to promote gene expression and apoptosis under 20-hydroxyecdysone regulation.

The nuclear receptor EcRB1, which is activated by the insect steroid hormone 20-hydroxyecdysone (20E), is reportedly phosphorylated by a protein kinase after 20E induction. However, the protein kinase has not been identified, and the significance of EcRB1 phosphorylation is unclear. In this study, we identified a protein kinase C δ (PKCδ) isoform (the E isoform) that phosphorylates EcRB1 in the lepidopteran Helicoverpa armigera, a serious agricultural pest worldwide, to promote apoptotic gene expression and apoptosis during metamorphosis. Through activation of the EcRB1/USP1 transcription complex by 20E, PKCδ expression was up-regulated in several tissues during the metamorphic stage. Knockdown of PKCδ caused failure to transition from larvae to pupae, prevented tissues from undergoing programmed cell death (PCD), and down-regulated the expression of the transcription factor Brz-7 and the apoptosis executors caspase-3 and caspase-6 The threonine residue at position 1343 of PKCδ was phosphorylated and was critical for its proapoptotic function. Overexpression of the PKCδ catalytic domain was localized to the nuclei in HaEpi cells, which increased caspase-3 activity and apoptosis. PKCδ directly phosphorylated a threonine residue at position 468 in the amino acid sequence of EcRB1. The phosphorylation of EcRB1 was critical for its heterodimeric interaction with the USP1 protein and for binding to the ecdysone response element. The data suggested that 20E up-regulates PKCδ expression to regulate EcRB1 phosphorylation for EcRB1/USP1 transcription complex formation, apoptotic gene transcription, and apoptosis.

Insulin and 20-hydroxyecdysone oppose each other in the regulation of phosphoinositide-dependent kinase-1 expression during insect pupation.

Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.

Autophagy triggers CTSD (cathepsin D) maturation and localization inside cells to promote apoptosis.

CTSD/CathD/CATD (cathepsin D) is a lysosomal aspartic protease. A distinguishing characteristic of CTSD is its dual functions of promoting cell proliferation via secreting a pro-enzyme outside the cells as a ligand, and promoting apoptosis via the mature form of this enzyme inside cells; however, the regulation of its secretion, expression, and maturation is undetermined. Using the lepidopteran insect Helicoverpa armigera, a serious agricultural pest, as a model, we revealed the dual functions and regulatory mechanisms of CTSD secretion, expression, and maturation. Glycosylation of asparagine 233 (N233) determined pro-CTSD secretion. The steroid hormone 20-hydroxyecdysone (20E) promoted CTSD expression. Macroautophagy/autophagy triggered CTSD maturation and localization inside midgut cells to activate CASP3 (caspase 3) and promote apoptosis. Pro-CTSD was expressed in the pupal epidermis and was secreted into the hemolymph to promote adult fat body endoreplication/endoreduplication, cell proliferation, and association. Our study revealed that the differential expression and autophagy-mediated maturation of CTSD in tissues determine its roles in apoptosis and cell proliferation, thereby determining the cell fates of tissues during lepidopteran metamorphosis.Abbreviations: 20E: 20-hydroxyecdysone; 3-MA: 3-methyladenine; ACTB/ß-actin: actin beta; AKT: protein kinase B; ATG1: autophagy-related 1; ATG4: autophagy-related 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG14: autophagy-related 14; BSA: bovine serum albumin; CASP3: caspase 3; CQ: choroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DPBS: dulbecco's phosphate-buffered saline; DsRNA: double-stranded RNA; EcR: ecdysone receptor; EcRE: ecdysone response element; EdU: 5-ethynyl-2´-deoxyuridine; G-m-CTSD: glycosylated-mautre-CTSD; G-pro-CTSD: glycosylated-pro-CTSD; HaEpi: Helicoverpa armigera epidermal cell line; HE staining: hematoxylin and eosin staining; IgG: immunoglobin G; IM: imaginal midgut; JH: juvenile hormone; Kr-h1: krueppel homologous protein 1; LM: larval midgut; M6P: mannose-6-phosphate; PBS: phosphate-buffered saline; PCD: programmed cell death; PNGase: peptide-N-glycosidase F; RFP: red fluorescent protein; RNAi: RNA interference; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SYX17: syntaxin 17; USP1: ultraspiracle isoform 1.