Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein

FEBS Letters 583 (2009) 655–660

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Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding protein Ikuko Fujii-Taira a,b,c, Shinji Yamaguchi a,c, Ryoko Iijima b,c, Shunji Natori b, Koichi J. Homma a,c,* a

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa 229-0195, Japan Natori Special Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan c Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan b

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Article history: Received 16 September 2008 Revised 11 December 2008 Accepted 29 December 2008 Available online 20 January 2009 Edited by Lukas Huber Keywords: Ecdysteroid Progesterone receptor membrane component Growth arrest Metamorphosis Drosophila

a b s t r a c t Ecdysteroid is a crucial steroid hormone in insects, especially during metamorphosis. Here, we show that the Drosophila membrane steroid binding protein (Dm_MSBP) is a novel structural homolog of the vertebrate membrane-bound receptor component for progesterone. Dm_MSBP exhibited binding affinity to ecdysone when expressed on the cell surface of Drosophila S2 cells. In S2 cells, the stable overexpression of Dm_MSBP suppressed the growth arrest triggered by 20-hydroxyecdysone and prevented the temporal activation of extracellular signal-regulated kinase proteins. These results suggest that Dm_MSBP is a membranous suppressor to ecdysteroid and blocks the signaling by binding it in extracellular fluid. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Ecdysteroid is a steroid hormone that is involved in multiple cellular responses during development, especially in the metamorphic reorganization in insects [1]. The ecdysone receptor (EcR), one of the nuclear receptor superfamily, is a functional nuclear receptor for ecdysteroid. The EcR gene encodes three isoforms of the EcR protein in Drosophila. The differences in their expression patterns produce the variation of in vivo cellular responses [2]. EcR resides in the cell nucleus, and is thus unable to directly mediate rapid non-genomic effects which occur in the cytoplasmic membrane [3–7]. In vertebrates, it has been reported that some non-genomic cellular responses to steroid hormones are initiated through plasma membrane receptors, and these responses do not require new pro-

Abbreviations: Dm_MSBP, Drosophila membrane steroid binding protein; ERK, extracellular signal-regulated kinase; EcR, ecdysone receptor; PGRMC, progesterone receptor membrane component; TMR, transmembrane region; SBD, steroid binding domain; DmDopEcR, Drosophila melanogaster dopamine/ecdysteroid receptor; 20HE, 20-hydroxyecdysone; S2 cell, Drosophila Schneider’s cell line 2; VIG, vasa intronic gene * Corresponding author. Address: Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa 229-0195, Japan. Fax: +81 42 685 3738. E-mail addresses: [email protected], [email protected] (K.J. Homma).

tein synthesis [8]. For example, the progesterone receptor membrane component (PGRMC) has been purified as a membrane protein that binds to progesterone [9]. PGRMC family proteins have a single transmembrane region (TMR) and a steroid binding domain (SBD). The overexpression study of rat PGRMC has provided evidence of resistance to apoptosis by progesterone in the cells which do not express a classical nuclear receptor for progesterone [10,11], suggesting that rat PGRMC mediates a non-genomic response. Apart from in vertebrates, non-genomic response pathways mediate some rapid actions of steroid hormones in invertebrates [3,12], and the membranous receptor for ecdysteroid has been suggested to play important developmental roles. For example, the Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) has been identified as a G-protein coupled receptor, which showed the binding activity for ecdysteroid [12]. DmDopEcR transmits catechol signals, and ecdysteroid acts as its antagonist. Since DmDopEcR expression was not detected in pupal stages, there seemed to be another signaling cue for ecdysteroid mediated by membrane receptors during insect metamorphosis. Here, we identify the Drosophila membrane steroid binding protein (Dm_MSBP) gene as a PGRMC homolog of Drosophila. When Dm_MSBP cDNA was introduced in Drosophila cells, the protein was expressed on the cell membrane and exhibited binding affinity to ecdysone. The stable overexpression of Dm_MSBP suppressed

0014-5793/$34.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.056

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cellular responses triggered by 20-hydroxyecdysone (20-HE). Dm_MSBP may thus be a modulator for ecdysteroid signaling.

2. Materials and methods 2.1. cDNA cloning EST-based DNA probe fragments (Supplementary Fig. 1A) were amplified from a Drosophila embryo cDNA library (Stratagene) by PCR. Primers used were 50 -GGATTCCAAAGTGGGTGCAGATCC-30 (sense) and 50 -CATTTCCCATTCCCGCACAGAGTCC-30 (antisense). Using plaque hybridization, two clones were isolated from 2.2  105 clones of a Drosophila embryo cDNA library, and their nucleotide sequences were determined. 2.2. Sequence analysis The transmembrane region was predicted using the SOSUI [13] (http://bp.nuap.nagoya-u.ac.jp/sosui/) and the Transmembrane Hidden Markov Model (TMHMM) [14] (http://www.cbs.dtu.dk/services/TMHMM/) programs. The multiple sequence alignments were carried out with the ClustalW program at http://clustalw.ddbj.nig.ac.jp. The phylogenetic tree was constructed on the basis of amino acid difference (p-distance) by the neighbourjoining method. 2.3. Cells The Drosophila Schneider’s cell line 2 (S2 cell) was cultured at 25C in Schneider’s Drosophila medium (GIBCO) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 lg/ml streptomycin, and 100 U/ml penicillin. 20-HE (SIGMA) was dissolved in sterile milliQ water and added directly to the culture medium to produce the final concentration. The transient expression experiments were performed as described previously [15]. To establish the stable Dm_MSBP transfectants, we used pIB/V5 vector (Invitrogen). The cells were selected through cultivation with Blasticidin– HCl (Invitrogen) and cloned by limiting dilution [16] after transfection of pIB-Dm_MSBP construct. 2.4. Ecdysone binding assay To prepare membrane fractions, the cells were homogenized in phosphate-buffered saline containing protease inhibitors by glass homogenizer and centrifuged (1000g, 10 min). The resulting supernatants were centrifuged (100 000g, 30 min), and the pellets were used as membrane fractions. The binding assay was performed according to the method of Meyer et al. [9] with some modifications. In brief, the membrane fractions were solubilized by 20 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid with 10 mg/ml bovine serum albumin. They were incubated in the presence of 10 nM a-[23, 24-3H(N)]-ecdysone (NEN) at 25C for 1 h, then collected on a Whatman GF/C glass filter. After washing with cold Tris–buffered saline, the radioactivities remaining on the filters were measured using a liquid scintillation counter. 2.5. Measurement of cell growth The S2 cells were suspended at a density of 1  105 cells/ml in the culture medium, and cultured for 2 days in the presence or absence of 20-HE. Growth of the S2 cells was assessed by manual cell counting using the hemocytometer as well as the AlamarBlue assay. The AlamarBlue assay is the method to measure the level of

cellular metabolism using the AlamarBlue reagent. In brief, 10% volume of AlamarBlue (TREK Diagnostic Systems, Cleveland, OH) was added to the culture medium, and the change in the color of the culture medium was monitored by measuring the absorbance at 570 nm and 610 nm. In this assay, the reducing activity generated by the proliferating cells changed the color of AlamarBlue. To confirm the correlation between cell number and metabolic activity, calibration curves were constructed in each experiment. 2.6. Immunoblotting and immunofluorescence Immunoblotting and immunofluorescence staining were performed, as described previously [15]. Anti-Dm_MSBP rabbit antibodies were raised against the peptides corresponding to the amino acid residues 182–197 and 203–218 of Dm_MSBP, and used for immunoblotting and immunofluorescence staining, respectively. These two antibodies are both suitable for immunofluorescent staining and immunoblotting. However, the antibody against the peptide corresponding to the amino acid residues 203–218 of Dm_MSBP worked better for immunoblotting and the other (antibody against amino acid 182–197) worked better for immunocytochemistry. Each antibody was affinity-purified with the recombinant Dm_MSBP produced by Escherichia coli. The antibody against b-galactosidase protein was purchased from Promega. The extracellular signal-regulated kinase (ERK) proteins were detected according to the method of Gabay et al. [17]. To prepare the samples for the short 20-HE-exposure experiments, cells were briefly washed by ice-cold PBS and lysed immediately in a RIPA buffer (50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 30 mM NaF, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol bis(2-aminoethylether) tetraacetic acid, 5 mM sodium diphosphate, 1 mM orthovanadate, 1 mM dithiothreitol, 10 lg/ml leupeptin, 10 lg/ml pepstatin A, 10 lg/ml aprotinin, 10 lg/ml E64, 0.1 mM PMSF). They were then frozen by liquid N2. ERK and phospho-ERK antisera were purchased from SIGMA. 3. Results 3.1. Cloning and characterization of Dm_MSBP cDNA Database analyses have indicated that there is an EST clone which has a similarity to vertebrate PGRMC (Genbank AI297635). We designed a 502-bp probe based on this EST clone (Supplementary Fig. 1), and obtained two cDNA clones by plaque hybridization. One of them completely matched to one locus, symbolized as CG9066 in Drosophila genome (Genbank AB290904). The other clone had a 68-nt deletion in 50 -UTR region (Genbank AB290905). This deletion started and ended with a 2-bp consensus sequence (GT at the 50 -end and AG at the 30 -end, see Supplementary Fig. 1A), suggesting that the clone appeared to be splicing variant from the original single gene (Supplementary Fig. 1A). They coded an identical protein which showed 43% homology to that of the human PGRMC1 overall. The region between amino acid residues 81 and 179 showed similarities with the SBD of the human PGRMC1 and the human PGRMC2 of 61% and 66%, respectively (Fig. 1A). Based on the BLAST searches, Dm_MSBP is the only gene which shows a significant similarity to both Hs_PGRMC1 and Hs_PGRMC2 in the Drosophila genome. This is true for other insects as well. The BLAST searches showed that other insects, Culex quinquefasciatus (southern house mosquito), Aedes aegypti, Anopheles gambiae and Bombyx mori, had one ortholog gene of Dm_MSBP. From these characteristics, we concluded that Dm_MSBP is a member of the PGRMC family, hence its name, D. melanogaster membrane steroid binding protein (Dm_MSBP).

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Fig. 1. Comparison of amino acid sequences between Dm_MSBP and PGRMCs. (A) Multiple alignment of Dm_MSBP and PGRMCs sequences. The predicted TMR is indicated with a thick horizontal line above the sequence alignment. The SBD (ProDom ID PD006731) of PGRMC is boxed, and the amino acid residues matching to Dm_MSBP are shaded. The open arrowhead indicates the Asp residue corresponding to Asp120 in rat PGRMC1. (B) A phylogenetic tree of PGRMCs based on amino acid sequences is generated by the neighbour-joining method.

In rat PGRMC1, Asp120 is essential for binding to the progesterone [10]. This residue was also conserved in Dm_MSBP (Fig. 1A, open arrowhead). The sequence analysis using SOSUI and TMHMM programs revealed that this protein had a single TMR (Fig. 1A). In phylogenetic terms, Dm_MSBP is located intermediate to PGRMC1 and 2 (Fig. 1B). Dm_MSBP may play roles similar to those of the two PGRMCs in vertebrates. The Dm_MSBP mRNA was ubiquitously expressed in larval and adult tissues (Supplementary Fig. 1), but was found in increased amounts of the Dm_MSBP protein in both the prepupal and early pupal stages (Fig. 2). Focusing on the prepupal stage, an immunoreactive protein with a molecular mass of 65 kDa was detected, besides the monomer of Dm_MSBP (Fig. 2, open arrowhead). In general, dimers can be separated under reducing condition. But the vertebrate PGRMC1 has been reported to form the homodimer even in reducing condition, suggesting the homodimer was linked covalently [9,18]. Dm_MSBP also may thus form a homodimer, and

Fig. 2. Expression of Dm_MSBP during metamorphosis. Extracts of Drosophila whole bodies were subjected to immunoblotting using the anti-Dm_MSBP antibody. The positions of the Dm_MSBP monomer and presumed dimer are indicated by the closed arrowhead and the open arrowhead, respectively.

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this dimerization may be involved in the function of Dm_MSBP at the beginning of metamorphosis. 3.2. Cellular localization of Dm_MSBP and its binding affinity to ecdysone To determine whether in fact Dm_MSBP is a membrane protein, we used the Drosophila S2 cell, an embryonically-derived cell line. The immunoblotting showed that the Dm_MSBP was detectable in S2 cells at low levels, but increased greatly following the introduction of Dm_MSBP cDNA (Fig. 3A). We then examined the cellular localization of Dm_MSBP by immunostaining under non-fixative conditions using an antibody against the Dm_MSBP C-terminal re-

gion. As a result, those cells expressing Dm_MSBP were strongly stained with the anti-Dm_MSBP antibody. But cells expressing the cytosolic b-galactosidase protein were not stained with the anti-b-galactosidase antibody under the same experimental conditions (Fig. 3B). Both the anti-Dm_MSBP antibody and the anti-bgalactosidase antibody reacted to the transformant, respectively, when the cells were fixed and permeabilized (data not shown). These results indicate that Dm_MSBP localizes on the cell surface, and exposes its C-terminus extracellularly. The fact that Dm_MSBP shows high sequence homology to the SBD of PGRMC (Fig. 1), suggests that ecdysone may be a ligand of Dm_MSBP, because ecdysone is a typical insect steroid hormone. Therefore, we tested whether Dm_MSBP has an affinity to an

Fig. 3. Expression of Dm_MSBP on the cell surface and its binding affinity to ecdysone. (A) Immunoblotting of Dm_MSBP cDNA-transfected cells. Transfectants and untransfected cells were subjected to immunoblotting using an anti-Dm_MSBP antibody. (B) Immunofluorescence staining under non-fixative conditions. The Dm_MSBPexpressing cells (left) and the b-galactosidase-expressing cells (right) were stained with the anti-Dm_MSBP antibody and the anti-b-galactosidase antibody, respectively. Strongly stained cells are indicated by arrowheads. Bars indicate 10 lm. (C) Ecdysone binding assay on the membrane fraction. Membrane fractions were prepared from Dm_MSBP-overexpressing and empty vector-transfected cells. Their binding affinities to [3H]-ecdysone were measured. Data represents mean ± S.E.M. of three determinations. *P < 0.05 (Student’s t-test).

Fig. 4. Suppression of 20-HE-triggered cell responses by Dm_MSBP. (A) The inhibitory effect of 20-HE on cell proliferation. The cells were suspended and the numbers of cells were manually counted after 2 days of culture in the presence of various concentrations of 20-HE. (B) The clonal cells highly expressing Dm_MSBP. The amounts of Dm_MSBP in the clonal cells were examined using immunoblotting. (C) Suppression of the 20-HE-triggered growth arrest by Dm_MSBP. Growth of the clonal cell lines in the presence of 0.1 lM 20-HE were compared. Data represent mean ± S.E.M. of three determinations. *P < 0.05, **P < 0.01 (Student’s t-test). (D and E) Changes in the quantity of phosphorylated ERK proteins in untransfected S2 cells (D), and Dm_MSBP-expressing cells (E) by a short-period stimulation of 20-HE. Cells were exposed to 20-HE for 0, 2, 5, 10, and 60 min, respectively.

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[3H]-ecdysone by performing a binding assay using the membrane fraction. As a result, the overexpression of Dm_MSBP elevated the binding ability to ecdysone (Fig. 3C), suggesting that ecdysone is indeed a ligand of Dm_MSBP. The Kd value of the Dm_MSBP protein was calculated to be about 300 nM. This value is very similar to that of porcine 28-kDa PGRMC1 [9], indicating that the binding to ecdysteroids is substantial enough to attenuate 20-HE signaling, and is physiologically meaningful. 3.3. Suppression of 20-HE-triggered cell growth arrest of S2 cells We examined the involvement of Dm_MSBP in ecdysteroid signaling using Dm_MSBP-overexpressing cells. It has been reported that 20-HE can induce G2 arrest [19] and apoptosis [20] of S2 cells. Therefore, we examined the effect of 20-HE on S2 cell growth regulation. When S2 cells were cultured in the presence of 20-HE for 2 days, the growth was clearly inhibited by 20-HE in a dose-dependent manner (Fig. 4A, Supplementary Fig. 2A). In addition, the cells treated with 20-HE were stained by calcein-staining (Supplementary Fig. 2B), indicating that the cells remained viable. There is a report which suggests that growth arrest following the exposure of S2 cells to 20-HE was based on the genomic response. In the paper, it is shown that the depletion of the ecdysone nuclear receptor by RNAi suppressed the G2 arrest of S2 cells [19]. We also observed the G2 arrest of S2 cells needed day-scale cell culture longer than 24 h in the presence of 20-HE. These findings suggest that the G2 arrest of S2 cells is required for the ecdysone signaling through a genomic pathway. After washing away the 20-HE, they started to reproliferate (Supplementary Fig. 2C). These results show that 20-HE-triggered growth arrest is reversible. Next, we examined whether Dm_MSBP is involved in the growth arrest induced by 20-HE. For this, we established clonal cell lines of high-expression of Dm_MSBP (Fig. 4B), and compared their growth in the presence of 0.1 lM of 20-HE. As the expression levels of Dm_MSBP increased, the growth arrest by 20-HE was suppressed (Fig. 4C). This suggests that Dm_MSBP has a competitive effect against 20-HE on S2 cell growth in a long-term genomic pathway. We also focused on short-term non-genomic effects by analyzing the intracellular signaling. There is significant evidence that progesterone signaling non-genomically activates ERK pathway in mammals [21–23]. Based on this evidence, we expected that ecdysteroid might be effective on ERK activity in insects. For this, we examined ERK activity in 20-HE-exposed cells and found that phosphorylated ERK levels increased 2–5 min after the addition of 20-HE, but decreased again 10 min post-stimulation (Fig. 4D). Conversely, the clonal cell line with highest expression of Dm_MSBP (clone E1) did not activate ERK but rather diminished it, following 20-HE stimulation (Fig. 4E). These results strongly suggest that Dm_MSBP down-regulates ecdysteroid signaling in a rapid non-genomic manner.

4. Discussion We have identified Dm_MSBP in Drosophila which is a single gene similar to vertebrate PGRMC1 and 2, suggesting that Dm_MSBP is the ortholog of vertebrate PGRMC. To our knowledge, this is the first report of an insect membrane ecdysone-binding protein being expressed during pupal stages. Considering the orientation of Dm_MSBP on the cell surface, Dm_MSBP captures the ecdysone in the extracellular fluid and competes with its signaling. Our results suggest that Dm_MSBP negatively regulates the ecdysteroid signaling pathway on the cell surface. Since the cytosolic region of Dm_MSBP is very short and has no structural features of a signal transducer, it is reasonable to speculate that Dm_MSBP does not transmit the ecdysteroid signals directly. We

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hypothesize that the function of Dm_MSBP is to capture ecdysteroid on the cell surface and passively attenuate the intracellular signaling of ecdysteroid. But we do not exclude the possibility that other cytosolic protein(s) bind to the cytosolic region of Dm_MSBP and thus actively down-regulate the ecdysteroid signaling. Whether Dm_MSBP passively attenuates or actively down-regulates ecdysteroid signaling will be tested by expressing the extracellular portion of Dm_MSBP together with transmembrane and cytoplasmic domains from another unrelated protein. If the effects of Dm_MSBP do not involve active down-regulation of signaling through the MSBP cytoplasmic domain, then these hybrid proteins should inhibit ecdysone signaling in a manner similar to normal Dm_MSBP. In vertebrates, PGRMC interacts with plasminogen activator inhibitor RNA binding protein-1 [10], which is known as a homolog of Drosophila vasa intronic gene (VIG), a member of RNA-induced silencing complex [24]. It is known that the expression of microRNAs are regulated by ecdysteroid in Drosophila [25]. If Dm_MSBP interacts with VIG, the interaction may affect the processing and function of microRNA. The expression patterns of Dm_MSBP provide some cues to its physiological function. The quantity of Dm_MSBP increased in the early stages of metamorphosis, and the up-regulated Dm_MSBP expression may suppress excessive ecdysteroidal signaling and contribute to cell proliferation of some populations exposed to high concentrations of ecdysteroid. According to the genome-wide gene expression profile in the Gene Expression Database of Berkeley Drosophila Genome Project [26], the gene locus of Dm_MSBP, CG9066, is expressed in several tissues such as the ring gland and embryonic corpus allatum in embryos at stage 13–16. The concentration of ecdysteroid also peaks during the embryonic stage, in addition to the commencement of the pupal stage in Drosophila [1]. Gene expression of CG9066 (We named as Dm_MSBP), increases in mbn2 cells following LPS-treatment [27], and downregulates the in adult ovary after mating [28], implying that Dm_MSBP is involved in immune responses, reproduction as well as the process of the metamorphosis. Thus Dm_MSBP may be a regulator of ecdysteroid signaling in a wide range of physiological events. The establishment of the Dm_MSBP mutant fly and RNAiexpressing transgenic flies targeted to Dm_MSBP [29] will allow for further investigation of the various functions of Dm_MSBP. Acknowledgments This work was supported by a Grant-in-Aid from the Special Postdoctoral Researchers Program of RIKEN (I.F.-T.), Scientific Research on Priority Areas (Molecular Brain Science) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (K.J.H.), Scientific Research from the Japan Society for the Promotion of Science (K.J.H.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.Y.), the Naito Foundation (K.J.H.), the Japan Foundation of Applied Enzymology (K.J.H.), the Uehara Memorial Foundation (S.Y.) and, the Sagawa Foundation for Promotion of Cancer Research (S.Y.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.12.056. References [1] Thummel, C.S. (2001) Molecular mechanisms of developmental timing in C. Elegans and Drosophila. Dev. Cell 1, 453–465. [2] Talbot, W.S., Swyryd, E.A. and Hogness, D.S. (1993) Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73, 1323–1337.

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