Ligand-independent requirements of steroid receptors EcR and USP for cell survival

Abstract

The active form of the Drosophila steroid hormone ecdysone, 20-hydroxyecdysone (20E), binds the heterodimer EcR/USP nuclear receptor to regulate target genes that elicit proliferation, cell death and differentiation during insect development. Although the 20E effects are relatively well known, the physiological relevance of its receptors remains poorly understood. We show here that the prothoracic gland (PG), the major steroid-producing organ of insect larvae, requires EcR and USP to survive in a critical period previous to metamorphosis, and that this requirement is 20E-independent. The cell death induced by the downregulation of these receptors involves the activation of the JNK-encoding basket gene and it can be rescued by upregulating EcR isoforms which are unable to respond to 20E. Also, while PG cell death prevents ecdysone production, blocking hormone synthesis or secretion in normal PG does not lead to cell death, demonstrating further the ecdysone-independent nature of the receptor-deprivation cell death. In contrast to PG cells, wing disc or salivary glands cells do not require these receptors for survival, revealing their cell and developmental time specificity. Exploring the potential use of this feature of steroid receptors in cancer, we assayed tumor overgrowth induced by altered yorkie signaling. This overgrowth is suppressed by EcR downregulation in PG, but not in wing disc, cells. The mechanism of all these cell death features is based on the transcriptional regulation of reaper. These novel and context-dependent functional properties for EcR and USP receptors may help to understand the heterogeneous responses to steroid-based therapies in human pathologies. Steroid hormones control multiple biological processes and, consequently, their faulty regulation underlies many pathologies. 1,2 The main Drosophila steroid hormone, ecdysone, is synthesized in the larval prothoracic gland (PG) using dietary sterols and cytochrome P450 enzymes. 3 Following its pulsated secretion into the hemolymph, peripheral tissues convert ecdysone into biologically active 20-hydroxyecdysone (20E) to control larval molting and metamorphosis. 4 The 20E signal is transduced by heterodimers of two nuclear receptors, ecdysone receptor (EcR) and Ultraspiracle (USP). 5 USP exhibits a single form throughout development. By contrast, EcR encodes three protein isoforms (EcRA, EcRB1 and EcRB2). These show common DNA-and hormone-binding domains but different N-termini although all of them can heterodimerize USP. 6,7 Each EcR isoform is hypothesized to have specific functions based on their distinct spatial and temporal patterns and N-termini. [8][9][10] EcR and USP DNA-binding domains recognize ecdysone response sequences which are short palindromes. 11 Like its vertebrate counterparts, 12 the ligand-EcR/USP complex activates transcription, whereas the unliganded receptor act as a repressor. [13][14][15] The in vivo validation of repressor activities for unliganded complexes, however, is mostly indirect. For example, EcR or USP loss...

Drosophilaenhancer-Gal4 lines show ectopic expression during development

“…This gene is thought to be specifically expressed in neurosecretory cells of the prothoracic gland (PG). However, we recently showed that the phm-Gal4 driver is expressed in margin cells of the wing disc [33]. The G-TRACE experiments confirm that cells in third instar larvae activate the phm enhancer in the PG and in the wing disc (figure 5 a – d ).…”

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

“…In D. melanogaster , a dominant‐negative mutation of EcR in the PG of larvae leads to reduced transcript levels of phm , dib and sad and lowers ecdysteroid biosynthesis (Moeller et al , ). Moreover, EcR is required for the survival of PG cells during metamorphosis via an ecdysone‐independent pathway (Mansilla et al , ). Furthermore, knockdown of EcR lowers circulating ecdysteroid levels in a hemimetabolan B. germanica (Cruz et al , ).…”

“…First, RNAi of USP in non‐drosophilid insects (Martin et al , ; Tan and Palli, ; Cao et al , ; Lenaerts et al , ; Xu et al , ) causes arrest in development, similar to D. melanogaster (Oro et al , ). Since reduction of USP in the PG cells also causes cell death, prevents ecdysone production and brings about the developmental arrest at late ultimate larval instar in D. melanogaster (Mansilla et al , ), an interesting question then rises: Do the developmental arrests in USP RNAi non‐drosophilid insects result from, at least partially, deficiency of 20E?…”

Conserved and Exapted Functions of Nuclear Receptors in Animal Development

“…EcR and usp also function in the development of male-specific neuronal architecture and male-mating behaviors [65]. More recently, usp and EcR have been shown to be required for the survival of prothoracic gland cells during metamorphosis via an ecdysone-independent pathway [66]. …”