20-hydroxyecdysone upregulatesAtggenes to induce autophagy in the Bombyx fat body

Abstract

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. in the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. injection of 20E into the feeding larvae upregulated Atg genes and reduced torC1 activity resulting in autophagy induction in the fat body. Conversely, rnAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcR DN ) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, rnAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. taken together, we conclude that in addition to blocking torC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

“…Silkworms and Cells-Bombyx larvae (p50 strain) were provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (Zhenjiang, China), and fed fresh mulberry leaves at 25°C under 14-h light/10-h dark cycles (25,29). Bm-N cells were maintained in TC-100 medium (PAN-BIOTECH, Germany) supplemented with 10% heat-inactivated fetal bovine serum (Gibco).…”

“…Fluorescence Microscopy and Transmission Electron Microscopy-The prothoracic glands and fat body were dissected and processed for fluorescence microscopy and transmission electron microscopy analyses as described previously (25,29,31,40). TUNEL (Beyotime, China) labeling and LysoTracker Red (Invitrogen) staining were used to estimate caspase activity and autophagy, respectively.…”

“…The relative luciferase activity was calculated by normalizing the reporter firefly luciferase level to the reference Renilla luciferase level. Dual-luciferase assays were conducted using the dual-luciferase assay system (Promega) and a Modulus luminometer (Turner BioSystems) (29,31,42,43). For some experiments, two constructs of EGFP, E75A, E75B, E75C, or coE75A/B/C were co-transfected into HEK 293 cells equally or in a dose-dependent manner.…”

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis

“…The SP2-GAL4 and UAS-EcR DN transgenic lines were produced via germline transformation using the p50 strain as donors (24). The transgenic silkworm SP2-GAL4ϾUAS-EcR DN was obtained by crossing SP2-GAL4 with UAS-EcR DN as described previously; SP2-GAL4 was used as a control here (24).…”

“…During larval-pupal metamorphosis in Bombyx, the fat body undergoes significant remodeling (involving progressive autophagy, caspase activity, and cell dissociation) that is mainly controlled by the 20E-triggered transcriptional cascade (21)(22)(23)(24)(25). The wing discs grow and differentiate during this period, and the pupal-specific wing disc cuticle protein genes (WCPs) apparently contribute to wing disc differentiation.…”