Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster

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Abstract

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called “Black Box”, has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14α-ol (Δ4-diketol) and 5β [H]cholesta-7-ene-3,6-dione-14α-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Δ4-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Δ4-diketol or diketol. These results indicate that Δ4-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok.

Highlights

► We characterize candidate intermediates of the ecdysteroid biosynthetic pathway. ► The Δ4-diketol and diketol trigger molting of ecdysteroid-defective larvae. ► Conversion of the Δ4-diketol or diketol into ecdysteroids is observed in vivo. ► 20E-inducible genes are induced by application of the Δ4-diketol or diketol.

Introduction

In insects, developmental transitions including molting and metamorphosis are triggered by pulses of steroid hormones. The most common hormone is the ecdysteroid, 20-hydroxyecdysone (20E), which is derived from ecdysone (E). E is biosynthesized from dietary cholesterol in the prothoracic gland (PG), released into hemolymph and converted into 20E in the peripheral tissues. The biosynthetic pathway has been extensively studied, but the critical steps including a rate limiting step, the so-called “Black Box”, have not been elucidated [1].

The first step in the biosynthetic pathway in the PG is the conversion of cholesterol to 7-dehydrocholesterol (7dC) [2], [3], [4]. The steps from 7dC to the diketol help to build the ecdysteroid skeleton, the structure of which is characterized by a cis junction of rings A and B, a 7-ene-6-one chromophore, and a trans junction of rings C and D (Fig. 1A). However, no intermediates in these Black Box reactions have been isolated or detected from arthropods, including insects, because of their instability and/or small concentration in the PG. From tracer experiments using radiolabeled compounds, the 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14α-ol (Δ4-diketol) and its reduced derivative, 5β [H]cholesta-7-ene-3,6-dione-14α-ol (diketol), have been considered as the first ecdysteroid-like precursors after the subsequent oxidative modifications of 7dC [5], [6], [7], [8]. The Δ4-diketol is converted into ecdysteroids, including 3-dehydroecdysone, in the crustacean Y-organ which corresponds to the PG of insects [6]. The diketol is efficiently converted into 3-dehydroecdysone and E in the PG of Locusta migratoria [8]. However, neither of these 3-oxo steroids have been detected in insects and no biological activity of metabolite(s) derived from them has been reported. In Drosophila and several other higher flies, the diketol undergoes reduction at C-3 to form the ketodiol [1]. The three terminal hydroxylation reactions at C-25, C-22 and C-2 of the ketodiol in the PG lead to E [9]. The synthesized E is released into hemolymph and then hydroxylated at C-20, which leads to the final product 20E in the peripheral tissues [10].

In the last decade, ecdysteroid biosynthetic enzymes have been characterized in Drosophila melanogaster. The first step from cholesterol to 7dC is catalyzed by a Rieske oxygenase, Neverland [11], [12]. The sequential terminal oxygenations from the ketodiol to 20E are catalyzed by several cytochrome P450 enzymes, Phantom, Disembodied, Shadow and Shade [10], [13], [14], [15]. All of these genes encoding P450 enzymes have been identified from embryonic lethal mutants and named the Halloween genes. Of the Halloween genes, spook and spookier (spok) have been hypothesized to code for P450 enzymes which catalyze one of the Black Box reactions [16]. Recently, Cyp6t3 has been reported as an ecdysteroid biosynthetic enzyme that also acts on a step in the Black Box [17]. RNAi-mediated knockdown of expression of a gene which codes for an ecdysteroid biosynthetic enzyme results in arrest of molting, the phenotype of which can be rescued by feeding E or appropriate intermediate(s) to the larvae [11], [16], [17], [18]. Therefore, we assume that feeding-rescue experiments for larvae in which spok is knocked down in the PG (hereafter called spok-RNAi larvae) could be applicable to clarify the intermediates in the Black Box. We focus on intermediates downstream of a step catalyzed by Spok in the Black Box and show here that application of candidate 3-oxo intermediates, the Δ4-diketol and diketol, triggers molting of spok-RNAi larvae. We also detect an enhancement of ecdysteroids, E and 20E, in the spok-RNAi larvae fed with these 3-oxo steroids, indicating that the dietary Δ4-diketol or diketol is converted into ecdysteroids in vivo. Finally, we show that induction of transcripts of 20E-inducible transcription factors occurs in Δ4-diketol- or diketol-fed spok-RNAi larvae. These results reinforce that the Δ4-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok.

Section snippets

Drosophila strains

UAS-spok-IR; UAS-spok-IR and phm-Gal4/TM3, sb, GFP (gifts from M.B. O’Connor) were crossed to generate spok-RNAi animals [16]. Flies were cultured on a standard cornmeal/yeast extract/dextrose medium.

Chemicals

The Δ4-diketol, ketol and ketodiol were synthesized from 7dC as described previously [6], [19], [20], [21]. The diketol was synthesized from the ketodiol by chromic acid oxidation in acetone. E was purchased from Sigma. Each compound was purified by reverse-phase HPLC before experiments.

Ecdysteroid feeding experiments

Eggs were

Candidate ecdysteroid precursors triggered molting of spok-RNAi first instar larvae

Spok is thought to be the rate-limiting enzyme in the Black Box of the ecdysteroid biosynthetic pathway during larval-pupal transitions in D. melanogaster, as recent study has shown that only Spo, paralog of Spok, of the Halloween ecdysteroid biosynthetic enzymes is phosphorylated in the PG cells of the moth, Manduca sexta, following stimulation by the neuropeptide known as prothoracicotropic hormone (PTTH) [22]. Because intermediates downstream of the conversion step catalyzed by Spok are

Discussion

Extensive studies over several decades have so far failed to completely elucidate the chemistry involved in ecdysteroid biosynthesis, but it has long been thought that the oxidation of the steroidal 3β-OH function to the 3-ketone occurs early in this process, i.e. 3-oxo-7dC may be the first step in these unknown Black Box oxidations [28]. Immediately subsequent intermediates eventually leading to the secretion of 3-dehydroecdysone likely remain oxidized at C-3. We addressed the involvement of

Acknowledgments

We are grateful to James T. Warren for critical reading. We also thank Michael B. O’Connor for stocks. This work was partly supported by a Grant-in-Aid for Young Scientists (B) [No. 21780047 to HO] from the Japan Society for the Promotion of Science (JSPS) and the Asahi Glass Foundation (to HO).

References (28)

Cited by (21)

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    However, recent observations have provided important insights. Key intermediates such as 3-oxo-steroids and Δ4-diketol have been confirmed in the Black Box [16–18]. The results unambiguously show that 7dC is first oxidized at carbon 3 to form 3-oxo-7dC, and the unstable 3-oxo-7dC is then isomerized into the more stable 3-oxo-Δ4,7C. Feeding Δ4-diketol rescues neverland mutants, suggesting 3-oxo-Δ4,7C is converted to Δ4-diketol by hydroxylation of carbon 14 and oxidation of carbon 6, though the intermediates remain a mystery [17,18].

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    The first enzymatic reaction of the pathway, the conversion of cholesterol to 7-dehydrocholesterol (7 dC) is catalyzed by Neverland (Nvd) (Yoshiyama et al., 2006; Yoshiyama-Yanagawa et al., 2011). 7 dC is then converted to 5β-ketodiol (KD) through the ‘Black Box’, a biosynthetic step not yet characterized, in which Shroud (Sro), Spook (Spo) and Spookier (Spok) are involved (Namiki et al., 2005; Ono et al., 2006, 2012; Niwa et al., 2010). Phantom (Phm) transforms KD in ketotriol (KT), Disembodied (Dib) converts KT in 2-deoxyecdysone (2 dE) and Shadow (Sad) converts 2 dE to ecdysone (E) (Chavez et al., 2000; Warren et al., 2002, 2004; Petryk et al., 2003; Niwa et al., 2004).

  • Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae

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    Previous studies have shown that the terminal hydroxylations, C-25, C-22 and C-2, do not have strict substrate specificities, i.e. the 5α-ketodiol and 5β-cholest-7-ene-3β,6α,14α-triol were hydroxylated at C-25, C-22 and C-2 in the PG as shown in 5β-ketodiol, but neither of them were converted into E by isomerization at C-5 or oxidation at C-6, respectively (Bollenbacher et al., 1977; Schwab and Hetru, 1991). Hence, 3-oxo-Δ4,7C might be converted to a ketodiol-like compound by enzymes in the Black Box, and then hydroxylated to an uncharacterized compound possessing a molting activity as shown in 14-deoxyecdysone derived from 3β-hydroxy-5β-cholest-7-en-6-one (ketol) (Bollenbacher et al., 1977; Ono et al., 2012). Regardless of whether 3-oxo-Δ4,7C is the intermediate or not, identification of any metabolite derived from 3-oxo-Δ4,7C could provide a critical clue to understand reactions in the Black Box.

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These authors contributed equally to this work.

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