A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila

Abstract

Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.

Keywords: OATP; SLCO; ecdysone; membrane transporter; nuclear receptor; steroid hormone.

Figure 1.. Identification of a Putative Ecdysone Importer through Two Independent Genetic Screens.

(A) Scheme of the in vivo RNAi screening to identify ecdysone importers. Candidate genes are listed in Table S1. (B) Knockdown of EcR or Oatp74D causes the loss of ecdysone-dependent glue-GFP expression in salivary glands in wandering third instar larvae. fkh-Gal4 > UAS-dicer2 was used to induce RNAi in salivary glands. Scale bars, 1 mm (upper panels), 200 µ m (lower panels). (C) Knockdown of EcR or Oatp74D in the fat body blocks ecdysone-dependent fat body cell migration into the pupal head (arrowheads). Cg-Gal4 > UAS-dicer2, UAS-2xEGFP was used to induce RNAi and label cells in the fat body. hAPF, hours after puparium formation. Scale bar, 1 mm. (D) Schematic of the in vitro CRISPR screening in S2R+ cells to identify sgRNAs that render resistance to 20E. Cells with mutations on genes essential for 20E-dependent cell cycle arrest (shown in red and blue) are expected to be over-represented in the 20E-treated population. (E) Gene-level CRISPR score distribution for the in vitro CRISPR screening. Computed CRISPR score (mean Log2[fold-change] of all sgRNAs targeting the same gene) for each gene from 20E-treated versus untreated populations is plotted. Size of bubbles represents number of active sgRNAs for each gene, where active sgRNAs are defined as those conferring 20E context-specific resistance in the top 2% of all sgRNAs in the screen.

Figure 2.. EcI Is a Conserved Membrane Transporter That Is Expressed Ubiquitously during Development.

(A, B) EcI/Oatp74D has orthologs in a broad range of insects and other arthropods that utilize ecdysteroids as the molting hormone. (A) Neighbor-joining unrooted phylogenetic tree constructed using entire amino acid sequences of 8 Drosophila melanogaster Oatps (asterisks) and 82 other OATP proteins from vertebrates, a tunicate, arthropods, and a nematode. Protein names and GenBank accession numbers are listed in Table S2. Shaded area indicates the clade containing EcI/Oatp74D. (B) Neighbor-joining unrooted phylogenetic tree constructed using entire amino acid sequences of EcI/Oatp74D clade proteins that are shaded in (A). Scale bars indicate an evolutionary distance of 0.2 amino acid substitutions per position. (C, D) EcI is expressed ubiquitously during development. (C) Relative expression levels of EcI/Oatp74D in various tissues and S2 cells, as assessed by qRT-PCR. Tissues were dissected from wandering third instar larvae (w¹¹¹⁸). CNS, central nervous system; ID, imaginal disc; SG, salivary gland; FB, fat body; MT, Malpighian tubule. (D) Developmental changes in the relative expression level of EcI in the whole body, as assessed by qRT-PCR. Samples were collected from w¹¹¹⁸ animals. hAEL, hours after egg laying; hAH, hours after hatching; hAPF, hours after puparium formation. Adult cDNA samples were prepared from flies at 24 hours after eclosion. Values are shown as percentages relative to the maximum level. All values are the means ± SD (n = 3). (E) HA-tagged EcI is localized at the plasma membrane. fkh-Gal4 or Cg-Gal4 > UAS-EcI-HA wandering third instar larvae were immunostained for HA (red) and nuclei (green). Scale bars, 100 µm.

Figure 3.. EcI Is Required for the Larval Developmental Transition.

(A) Developmental changes in body size of control (w¹¹¹⁸), EcI mutant (EcI¹/EcI²) and EcI mutant rescued by weak ubiquitous expression of EcI (arm-Gal4 > UAS-EcI; EcI¹/EcI²). Representative images of animals at different stages were combined into a single panel. hAH, hours after hatching. Scale bar, 1 mm. (B) Developmental progression and survival rate (%) of control (w¹¹¹⁸), EcI mutants (EcI¹/EcI¹, EcI²/EcI², and EcI¹/EcI²) and EcI transheterozygous mutant rescued by weak ubiquitous expression of EcI (arm-Gal4 > UAS-EcI; EcI¹/EcI²). Color bars indicate percentages of first instar larvae (white), second instar larvae (blue), third instar larvae (green), and prepupae/pupae (yellow). Percentages of arrested first instar larvae with double mouth hooks (DM) are shown in light blue. Larval stages were determined by stage specific morphology of larval mouth hooks and posterior spiracles (Figure S4). (C) Larval mouth hook morphology of control (w¹¹¹⁸), EcI transheterozygous mutant (EcI¹/EcI²) and EcI transheterozygous mutant rescued by weak ubiquitous expression of EcI (arm-Gal4 > UAS-EcI; EcI¹/EcI²) at 96 hAH. Arrows indicate second instar larval mouth hooks observed in the double mouth hooks larva (right lower panel). Scale bars, 25 µm.

Figure 4.. Developmental Arrest Phenotype of EcI Mutant Can Be Rescued by a Non-Steroidal Ecdysone Agonist.

(A) Structures of the endogenous EcR ligand 20-hydroxiecdysone (20E) and a non-steroidal ecdysone agonist chromafenozide (CF). (B) Transient compound feeding scheme. Larvae were fed on yeast paste with 2% ethanol (EtOH) with or without 1 mM (final concentration) 20E or CF from 6 to 18 hAH. Larvae were then cultured on normal food for an additional 12 hours and larval morphology was observed at 30 hAH. About 80–85% of control larvae complete first-to-second instar molting at 24 hAH. (C) Representative images of mouth hooks and posterior (P) spiracles of control (w¹¹¹⁸) and EcI mutant (EcI¹/EcI²) larvae at 30 hAH after 20E or CF feeding. Enlarged mouth hook images correspond to the boxed areas. IM, incomplete molting, where the first instar cuticle remains attached (white arrow). DMS, double mouth hooks and spiracles. Black arrows indicate first instar mouth hooks; arrowhead indicates first instar posterior spiracles. Scale bars, 500 µm (white) and 50 µm (black). (D) Developmental stages of control (w¹¹¹⁸), EcI mutants (EcI¹/EcI¹, EcI²/EcI² and EcI¹/EcI²), ecdysone-deficient larvae (phm22-Gal4 > UAS-phm RNAi, UAS-dicer2) and ecdysone-deficient larvae in EcI mutant background (phm22-Gal4>UAS-phm RNAi, UAS-dicer2; EcI¹/EcI²) after transient feeding of 20E or CF. phm22-Gal4 > UAS-dicer2 was used as a prothoracic gland-specific Gal4 driver. Color bars indicate percentages of first instar larvae (white), first instar larvae with the DMS phenotype (light blue), second instar larvae with IM phenotype (blue), and second instar larvae (dark blue). Numbers of animals observed are shown on top of each bar.

Figure 5.. EcI Cell-Autonomously Regulates Ecdysone Signaling and Facilitates Cellular Uptake of Ecdysteroids in Vivo.

(A) Relative expression levels of ecdysone-inducible genes (E74A, E75A and E75B) in the fat body at 72 and 96 hAH, as assessed by qRT-PCR. Two independent UAS-RNAi lines for EcI and EcR were used. Cg-Gal4 > UAS-dicer2 was used as a fat body-specific Gal4 driver. Values were calculated relative to the expression level of each gene at 72 hAH in control. (B) Cell-autonomous requirement of EcI and EcR for ecdysone signaling in the fat body. Clones of fat body cells expressing EcI-RNAi #1 or EcR-RNAi #1 with dicer2 were labeled with GFP. EcRE-LacZ reporter gene was used to monitor the activity of ecdysone signaling. hs-flp;; Act>CD2>GAL4, UAS-nlsGFP was used to generate GFP-marked flip-out clones. Flippase activity was induced by 10 min heat shock. The fat body from white prepupae at 96 hAH was immunostained for LacZ (red), GFP (green) and nuclei (blue). Scale bars, 50µm. (C) Quantification of ecdysteroids in the hemolymph and fat body at 72 and 96 hAH, as assessed by ELISA. UAS-RNAi and Gal4 lines used were the same as in (A). All values are the means ± SD (n = 3). *p < 0.05, **p < 0.01 from Student’s t test compared to control.

Figure 6.. EcI Is Both Necessary and Sufficient for Ecdysone Action in EcR-Expressing Cells.

(A, B) Luciferase (Luc) reporter activity was monitored in Drosophila S2 cells treated with EcI double-stranded RNA (A) or overexpressing EcI (B) after 24-hour treatment with 20E, ecdysone (E) or CF. S2 cells were co-transfected with EcREx3-Luc and EcR. usp is endogenously highly-expressed in S2 cells (Baker et al., 2000). O/E, overexpression. (C) Luc reporter activity in S2 cells treated for 0, 4, 8 or 16 hours with 10⁻⁷ M or 10⁻⁶ M of 20E. (D) Luc activity in HEK293 cells treated for 24 hours with 20E, E or CF. EcREx5-Luc was used as a reporter construct. HEK293 cells overexpressing modified EcR (VgEcR) and co-receptor RXR were used for the assay. Values are relative to the control level (0 M or 0 hour). All values are the means ± SD (n = 3). *p < 0.05, **p < 0.01 from Student’s t test compared to control.

Figure 7.. Facilitated Diffusion Model of Ecdysteroid Uptake by Target Cells.

EcI/Oatp74D is required for facilitating cellular uptake of ecdysteroids (blue circles) from the hemolymph into cytoplasm down the concentration gradient. The nuclear receptor complex composed of EcR and Usp regulates the transcription of genes for molting and metamorphosis in the nucleus.