Stimulation of orphan nuclear receptor HR38 gene expression by PTTH in prothoracic glands of the silkworm, Bombyx mori

Journal of Insect Physiology 90 (2016) 8–16

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Stimulation of orphan nuclear receptor HR38 gene expression by PTTH in prothoracic glands of the silkworm, Bombyx mori Shi-Hong Gu ⇑, Yun-Chih Hsieh, Pei-Ling Lin Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung 404, Taiwan, ROC

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Article history: Received 21 January 2016 Received in revised form 12 April 2016 Accepted 13 April 2016 Available online 16 April 2016 Keywords: Ecdysone PTTH Signaling Nuclear receptor HR38 Immediate early gene

a b s t r a c t A complex signaling network appears to be involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). Less is known about the genomic action of PTTH signaling. In the present study, we investigated the effect of PTTH on the expression of Bombyx mori HR38, an immediate early gene (IEG) identified in insect systems. Our results showed that treatment of B. mori PGs with PTTH in vitro resulted in a rapid increase in HR38 expression. Injection of PTTH into day-5 last instar larvae also greatly increased HR38 expression, verifying the in vitro effect. Cycloheximide did not affect induction of HR38 expression, suggesting that protein synthesis is not required for PTTH’s effect. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), partially inhibited PTTH-stimulated HR38 expression, implying the involvement of both the ERK and PI3K signaling pathways. When PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187 or thapsigargin), an increase in HR38 expression was also detected, indicating that Ca2+ is involved in PTTH-stimulated HR38 gene expression. A Western blot analysis showed that PTTH treatment increased the HR38 protein level, and protein levels showed a dramatic increase during the later stages of the last larval instar. Expression of HR38 transcription in response to PTTH appeared to undergo development-specific changes. Treatment with ecdysone in vitro did not affect HR38 expression. However, 20-hydroxyecdysone treatment decreased HR38 expression. Taken together, these results demonstrate that HR38 is a PTTH-stimulated IEG that is, at least in part, induced through Ca2+/ERK and PI3K signaling. The present study proposes a potential cross talk mechanism between PTTH and ecdysone signaling to regulate insect development and lays a foundation for a better understanding of the mechanisms of PTTH’s actions. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Nuclear receptors (NRs) comprise a superfamily of structurally related transcriptional factors that play important roles in regulating eukaryotic cell growth, development, and homeostasis (Evans and Mangelsdorf, 2014). The family includes ligand-inducible NRs and a number of so-called orphan receptors that lack identified physiological ligands (Benoit et al., 2006; Evans and Mangelsdorf, 2014). In mammals, it was reported that the NR4A receptor family belongs to the orphan NR superfamily and consists of three highly homologous mammalian members known as Nur77 (also referred to as NR4A1 and NGFI-B), Nurr1 (NR4A2), and NOR-1 (NR4A3) (Close et al., 2013; Kurakula et al., 2014). All of the NR4A subfamilies are encoded by immediate early genes (IEGs) the ⇑ Corresponding author. E-mail address: [email protected] (S.-H. Gu). http://dx.doi.org/10.1016/j.jinsphys.2016.04.003 0022-1910/Ó 2016 Elsevier Ltd. All rights reserved.

expressions of which are rapidly induced in response to a variety of signals including mitogens and cellular stress (Maxwell and Muscat, 2006). Moreover, NR4A receptors are widely expressed in several tissues, including the testis, ovary, muscle, thymus, adrenal gland, and brain (Maxwell and Muscat, 2006; Pei et al., 2006). In line with pleiotropic physiological stimuli that induce NR4A expression, the NR4A family appears to play important roles in regulating cell metabolism and homeostasis. The role of NR4A in regulating mammalian steroidogenesis was also documented (Maxwell and Muscat, 2006). Nur77 and Nurr1 were shown to play key roles in regulating basal and hormone-induced gene expressions in steroidogenic cells, including testicular Leydig cells (Maxwell and Muscat, 2006). In insects, both Drosophila HR38 (DHR38) and Bombyx HR38 belong to the NR4A subfamily of NRs and appear to disrupt ecdysone receptor (EcR)-Ultraspiracle (USP) heterodimers in vitro and in cell culture (Sutherland et al., 1995). Dimerization of HR38 with

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USP blocks ecdysone signaling in vivo during the arrest that precedes a blood meal in Aedes aegypti (Zhu et al., 2000). Evidence suggests that DHR38 participates with USP in an unusual ecdysteroid signaling pathway in Drosophila (Baker et al., 2003). DHR38 also plays pivotal roles in adult cuticle formation, carbohydrate metabolism, and glycogen storage (Kozlova et al., 1998, 2009; Ruaud et al., 2011). More recently, HR38 was identified as a novel IEG that is transiently expressed in the male by sex pheromone in both Drosophila and Bombyx and thus HR38 can be used as a neural activity marker in insect brains (Fujita et al., 2013). Insects molting and metamorphosis are initiated by ecdysteroids synthesized and secreted by prothoracic glands (PGs) (Thummel, 2001; De Loof, 2011; Smith and Rybczynski, 2012; De Loof et al., 2013, 2015). A neuropeptide, known as the prothoracicotropic hormone (PTTH), produced by brain neurosecretory cells, activates ecdysteroidogenesis in PGs (Ishizaki and Suzuki, 1994; Marchal et al., 2010; Smith and Rybczynski, 2012; De Loof et al., 2013, 2015). PTTH appears to bind to receptor tyrosine kinase to initiate a signaling transduction network (Rewitz et al., 2009, 2013; Marchal et al., 2010; Smith and Rybczynski, 2012; De Loof et al., 2013, 2015). Previous studies indicated that PTTHstimulated cAMP and Ca2+ are intracellular second messengers in both Manduca sexta (Smith et al., 1984, 1985; Fellner et al., 2005) and Bombyx mori (Gu et al., 1996, 1998). In M. sexta, p70S6 kinase (S6K) and ribosomal protein S6 are related to PTTH-stimulated ecdysteroidogenesis (Song and Gilbert, 1997). In addition, extracellular signal-regulated kinase (ERK) phosphorylation, phosphatidylinositol 3-kinase (PI3K)/adenosine 50 -monophosphate-acti vated protein kinase (AMPK)/target of rapamycin (TOR) signaling, and reactive oxygen species were found to be involved in PTTH’s stimulation of ecdysteroidogenesis (Rybczynski et al., 2001; Lin and Gu, 2007; Gu et al., 2010, 2011, 2012, 2013; Hsieh et al., 2013, 2014). Our recent study further characterized the nuclear localization of phosphorylated ERK stimulated by PTTH (Gu and Hsieh, 2015). We demonstrated that PTTH stimulates phosphorylation of histone H3 at ser10 in B. mori PGs both in vitro and in vivo (Gu and Hsieh, 2015). In mammalian cells, it was well documented that phosphorylation of histone H3 at ser10 in response to a mitogenic stimulus closely corresponds to transient expressions of activated IEGs, suggesting that this histone modification is linked to transcription activation (Prigent and Dimitrov, 2003; Sawicka and Seiser, 2014). Although PTTH appears to regulate expression of some ecdysteroidogenic genes (Niwa et al., 2005; Yamanaka et al., 2007; McBrayer et al., 2007), no IEG has been identified in PTTHstimulated PGs. In addition, transcriptional changes stimulated by the activation of EcR have been well characterized in Drosophila (Thummel, 2001; King-Jones and Thummel, 2005; Niwa and Niwa, 2014, 2016). It is hypothesized that some components of the EcR may be involved in the biosynthesis of ecdysone. EcRA, but not the two other EcR isoforms, is expressed in Drosophila PGs (Talbot et al., 1993). In Manduca, USP appears to modulate PTTH-stimulated ecdysteroidogenesis (Song and Gilbert, 1998). A null mutation in E75A results in a dramatic decrease in ecdysone levels, implying that E75A plays a dual role, acting both downstream of ecdysone signaling and upstream of ecdysone biosynthesis signaling (Bialecki et al., 2002; Li et al., 2015). Other NRs, such as bFTZ-F1 and Broad, appear to play similar roles in regulating both ecdysone’s action and its biosynthesis (Parvy et al., 2005; Xiang et al., 2010). It was also reported that PTTH signaling mediates nucleocytoplasmic trafficking of the NR DHR4, which acts as a repressor of ecdysteroidogenesis (Ou et al., 2011). More recently, it was shown that EcR-dependent positive feedback operating downstream of PTTH signaling plays a critical role in generating the high-level pulse that triggers pupariation in response to PTTH (Moeller

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et al., 2013). Complex interaction among the NRs, EcR, E75, DHR3, and bFTZ-F1, which mediate ecdysone signaling (King-Jones and Thummel, 2005), appears to be involved in regulating ecdysteroidogenesis (Parvy et al., 2014). Although those studies clearly indicated the involvement of the NRs in regulating ecdysteroidogenesis, the link from PTTH signaling to the NRs is not very clear. Considering that NR4A expression is induced by pleiotropic physiological stimuli and that NR4A plays an important role in regulating steroidogenesis in mammals, in the present study, we performed a detailed analysis of HR38 expression at both mRNA and protein levels in response to PTTH signaling in B. mori PGs. We demonstrate that HR38 is an IEG downstream of PTTH signaling, and thus appears to be involved in PTTH-stimulated ecdysteroidogenesis. 2. Materials and methods 2.1. Experimental animals Larvae of an F1 racial hybrid, Guofu  Nongfong, were reared on fresh mulberry leaves at 25 °C under a 12-h light: 12-h dark photoperiod. Newly-ecdysed 4th and last instar larvae were collected and used for each experiment. 2.2. Reagents and antibodies Ecdysone, 20-hydroxyecdysone, A23187, and thapsigargin were supplied by Sigma-Aldrich (St. Louis, MO, USA). Grace’s insect cell culture medium was obtained from Invitrogen (Carlsbad, CA, USA). A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor (U0126), a PI3K inhibitor (LY294002), and a protein synthesis inhibitor (cycloheximide, CHX) were purchased from Calbiochem (San Diego, CA, USA). All other reagents used were of analytical grade. Recombinant B. mori PTTH (PTTH) was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as previously described (O’Reilly et al., 1995). The same PTTH as that previously reported (O’Reilly et al., 1995; Gu et al., 2010; Gu and Hsieh, 2015) was used in the present study. In the present study, extracellular fluid from cells infected with vWTPTTHM rather than pure PTTH was used as the PTTH source, and it was diluted 500 times with medium. Each incubation (50 ll) contained about 0.15 ng PTTH. An antibody directed against Bombyx HR38 was kindly provided by Dr. Taketoshi Kiya (Fujita et al., 2013). An anti-a-tubulin antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). A horseradish peroxidase (HRP)-linked goat anti-rabbit second antibody was purchased from PerkinElmer Life Sciences (Boston, MA, USA). 2.3. In vitro incubation of PGs and in vivo injection of PTTH PGs from day-5 last instar larvae or other stages were dissected in lepidopteran saline (Gu et al., 2010). Following dissection, the medium was replaced with fresh medium (with or without inhibitors), and a 30-min preincubation period was initiated. After preincubation, glands were rapidly transferred to fresh medium (with or without experimental materials, such as an inhibitor or PTTH) and then incubated with gentle shaking. After incubation, glands were flash-frozen at 70 °C for a subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. To study the in vivo effect of PTTH on HR38 expression, day-5 last instar larvae were injected with 10 ll saline containing 0.3 ll of the original PTTH solution. Larvae injected with only 10 ll saline were used as the controls.

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2.4. Western blot analysis, RNA extraction, and quantitative real-time polymerase chain reaction (PCR) The Western blot analysis followed protocols as described in previous studies (Lin and Gu, 2007; Gu et al., 2010, 2011). Total RNA from PGs was extracted using the TRI Reagent (Molecular Research Center, OH, USA) according to the manufacturer’s protocol. The quantity of extracted RNA was assessed with a UV1101 photometer (Biotech, Cambridge, UK) and/or by electrophoresis on 1% (w/v) agarose gels. First-strand complementary DNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, CA, USA). For the quantitative real-time PCR analysis, total RNA was extracted from PGs (Young et al., 2012). The PCR was carried out in a 20-ll reaction volume containing 10 ll of SYBR1 Green Realtime PCR Master Mix (Bio-Rad), 2 ll of a first-strand cDNA template, and 8 ll of the primers. The iQ5 Real-Time PCR Detection System (Bio-Rad) was used according to the manufacturer’s instructions. The PCR primers were designed according to parameters (no primer dimers and a product length of no more than 200 bp) outlined in the manual of the SYBR1 Green Real-time PCR Master Mix. The annealing temperature for all reactions was 59.5 °C. Transcript levels were normalized to the Bombyx ribosomal protein 49 (rp49) mRNA levels. CT values were set against a calibration curve. The DDCT method was used to calculate the relative abundances. The quantitative real-time PCR was performed using the following primers: HR38 forward, 50 -GCCGTGGGTATGGT GAAAGA-30 , HR38 reverse, 50 -ATCAGAGAGATGGGTGGGCT-30 ; bFTZ-F1 forward, 50 -GATTAGTCCTTGGTTTTG-30 , bFTZ-F1 reverse, 50 -CGTCAATGATGATACTTG-30 ; E75A forward, 50 -GCTCCTCTTAA TAGTATCA-30 , E75A reverse, 50 -AAGTAGAATCAACGAGAA-30 ; and RP49 forward, 50 -CAGGCGGTTCAAGGGTCAATAC-30 , RP49 reverse, 50 -TGCTGGGCTCTTTCCACGA-30 .

Fig. 1. Changes in HR38 mRNA levels upon treatment with PTTH. PGs were preincubated in medium for 30 min, then transferred to medium containing PTTH (P) or control medium (C). Incubation was maintained for 30 min, 1, 2, 8, and 24 h, respectively. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, **p < 0.01).

2.5. Data analysis Results are expressed as the mean + standard error of the mean (S.E.M.). Data for each mRNA level were compared using Student’s t-test. p < 0.05 was considered significant. 3. Results 3.1. Expression of HR38 in B. mori PGs is stimulated by PTTH in vitro In the first experiment, we studied the in vitro stimulation of HR38 in PGs by PTTH. Fig. 1 shows that in vitro treatment of PGs from day-5 last instar larvae with PTTH resulted in a timedependent increase in HR38 gene expression. A slight increase in HR38 expression was detectable 30 min after treatment, and the expression was dramatically increased upon treatment with PTTH for 1 h. The highest increased expression of HR38 was detected at 2 h after treatment, the level then declined at 8 h, and returned to the basal level at 24 h. To identify whether HR38 is a PTTHinduced IEG, PGs were pretreated with 50 lM of CHX for 30 min and then treated with PTTH for 2 h. CHX pretreatment did not inhibit PTTH-induced HR38 expression (Fig. 2). In addition, no stimulation of HR38 expression was observed when PGs were treated with extracellular fluid from cells infected with wt AcMNPV (without PTTH) (O’Reilly et al., 1995), indicating the specificity of the recombinant PTTH (data not shown).

were injected with PTTH. One hour later, PGs were quickly dissected out and immediately flash-frozen after dissection, and HR38 expression was examined and compared to that of control larvae. Results (Fig. 3) show that a PTTH injection stimulated HR38 expression of PGs compared to that of a saline injection, verifying the in vitro stimulation of HR38 expression.

3.2. In vivo effect of a PTTH injection on HR38 expression

3.3. Involvement of Ca2+ in PTTH-stimulated HR38 expression

In subsequent experiments, we examined the in vivo stimulation of HR38 expression of PGs by PTTH. Day-5 last instar larvae

The involvement of Ca2+ in regulating PTTH-stimulated HR38 expression was next investigated. Fig. 4A shows that

Fig. 2. Effect of CHX on PTTH-stimulated HR38 expression. PGs from day 5-last instar larvae were pretreated with either 50 lM CHX or vehicle alone for 30 min, then transferred to media containing the same dose of the inhibitor, with or without PTTH. Incubation was maintained for 2 h. C, PGs incubated in control medium; CHX, PGs incubated in medium containing CHX only; P, PGs incubated in medium containing PTTH only; P + CHX, PGs incubated in medium containing both PTTH and CHX. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s ttest, **p < 0.01). n.s., no significant difference.

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Fig. 3. In vivo stimulation of HR38 expression by PTTH. P, larvae injected with saline containing PTTH; C, larvae injected with saline only. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, *p < 0.05).

PTTH-stimulated HR38 expression was completely blocked in Ca2+-free saline (with 5 mM EGTA). The existence of PTTHstimulated Ca2+-dependent HR38 expression prompted us to study the effects of agents that directly increase the intracellular Ca2+ concentration. The Ca2+ ionophore, A23187, was used, because it was previously reported that A23187 increases ERK phosphorylation and ecdysteroidogenesis in Bombyx PGs (Gu et al., 2010). Fig. 4B shows that treatment with A23187 greatly stimulated HR38 expression. Similar to A23187, thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, also caused an increase in HR38 expression. 3.4. Effects of the inhibitors of MEK and PI3K on PTTH-stimulated HR38 expression Our previous study showed that PTTH stimulates ERK phosphorylation of silkworm PGs (Lin and Gu, 2007). To examine whether or not PTTH-stimulated HR38 expression is linked to ERK phosphorylation, a specific MEK inhibitor, U0126, was used. PGs from day-5 last instar larvae were pretreated with 10 lM of U0126 (a concentration that completely inhibited PTTH-stimulated ERK phosphorylation (Lin and Gu, 2007)), and then they were stimulated with PTTH. As shown in Fig. 5A, U0126 partially inhibited PTTHstimulated HR38 expression. This result clearly showed that PTTH-stimulated HR38 expression is, at least in part, ERKdependent. The effect of the PI3K inhibitor, LY294002, on PTTH-stimulated HR38 expression was further examined. PGs from day-5 last instar larvae were pretreated with 50 lM of LY294002 (a concentration that completely inhibited PTTH-stimulated PI3K/TOR signaling (Gu et al., 2012)), and then they were challenged with PTTH. As shown in Fig. 5B, LY294002 partially inhibited PTTH-stimulated HR38 expression, indicating that PI3K signaling is, at least in part, involved in PTTH-stimulated HR38 expression. 3.5. Effect of PTTH on HR38 protein levels and changes in HR38 protein levels in the last larval instar To examine the effect of PTTH on HR38 protein levels, PGs were treated with PTTH for 2 and 3 h, and protein levels were analyzed by a Western blot analysis. As shown in Fig. 6A, HR38 protein levels increased 3 h after PTTH treatment. Fig. 6B shows developmental changes in HR38 protein levels between days 2 and 10 of

Fig. 4. Effect of external Ca2+ (A) on PTTH-stimulated HR38 expression and the effects of A23187 and thapsigargin (B). (A) PGs were preincubated in Ca2+-free saline (with 5 mM EGTA) for 30 min and then transferred to either control saline (+Ca2+), saline with the PTTH (+Ca2++PTTH), Ca2+-free saline (Ca2+), or Ca2+-free saline with PTTH (Ca2++PTTH). (B) Effects of A23187 and thapsigargin. PGs were preincubated with control medium for 30 min, and then transferred to control medium (C), or medium containing 50 lM A23187 (A23) or 10 lM thapsigargin (Thap). Incubations were maintained for 60 min. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, *p < 0.05; **p < 0.01).

the last larval instar. Low levels were detected between days 2 and 6. The protein level increased on day 7 when larvae began spinning, and the highest level was observed on day 10, 1 day before pupation. This temporal pattern shows that the increase in HR38 protein level is related to the increased ecdysteroid biosynthetic activity during the later stage of the last larval instar. 3.6. Developmental changes in PTTH-stimulated HR38 gene expression To examine whether PTTH-stimulated expression of HR38 undergoes changes in different developmental stages, PGs from day-2 4th instar larvae, and day-1, -6, and -10 last instar larvae were isolated and then challenged with PTTH. As shown in Fig. 7, PTTH significantly stimulated HR38 expression in PGs from day-2 4th instar larvae. PTTH did not stimulate HR38 expression PGs from day-1 last instar larvae. However, PTTH dramatically increased HR38 expression in PGs from day-6 last instar larvae. For day-10 last instar larvae (1 day before pupation), a decreased response in HR38 expression upon PTTH stimulation was detected.

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Fig. 6. Western blot analysis of HR38 protein in PGs of B. mori. (A) Effect of PTTH on the HR38 protein level. PGs were preincubated in control medium for 30 min and then transferred to control medium (C) or medium containing PTTH (P) for different times. (B) Changes in the HR38 protein level during the last larval instar. PGs of last larval instar larvae were isolated from day-2 (D2) to 10 (D10), and then immediately flash-frozen after dissection. Gland lysates were prepared and subjected to an immunoblot analysis with anti-HR38 (HR38) and anti-a-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments. Molecular weight markers are shown on the right side of the gel.

Fig. 5. Effects of U0126 (A) and LY294002 (B) on PTTH-stimulated HR38 expression. PGs were pretreated with either 10 lM U0126, or 50 lM LY294002, or incubated in control medium for 30 min, and then transferred to medium containing the same dose of each inhibitor, with or without PTTH. C, PGs incubated in control medium; P, PGs incubated in medium containing PTTH only; U, PGs incubated in medium containing U0126 only; P + U, PGs incubated in medium containing both PTTH and U0126; LY, PGs incubated in medium containing LY294002 only; P + LY, PGs incubated in medium containing both PTTH and LY294002; Incubations were maintained for 2 h. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, **p < 0.01; #p < 0.05).

3.7. Effects of ecdysone and 20-hydroxyecdysone on HR38 gene expression In Bombyx larvae, PGs predominantly secrete ecdysone (Kiriishi et al., 1990). Upon secretion into the hemolymph, ecdysone is converted to 20-hydroxyecdysone in peripheral tissues (Smith and Rybczynski, 2012). To rule out the possibility that HR38 expression is indirectly induced by ecdysone or 20-hydroxyecdysone as a result of PTTH-stimulated biosynthetic activity, PGs were treated in vitro with either ecdysone or 20-hydroxyecdysone at different concentrations (0, 10, 100, and 1000 ng/ml) for 2 h, and HR38 mRNA expression was examined. As shown in Fig. 8A, HR38 expression did not change due to treatment with ecdysone. However, 20-hydroxyecdysone treatment decreased HR38 expression (Fig. 8C). In addition, as a positive control, our results showed that ecdysone treatment mildly increased bFTZ-F1 expression (Fig. 8B), a target gene downstream of ecdysone signaling (King-Jones and

Fig. 7. Changes in PTTH-stimulated HR38 expressions during the 4th and last larval instars. PGs were isolated from day-2 4th instar, day-1, day-6, and day-10 last larval instar larvae, respectively, preincubated with control medium for 30 min, and then transferred to control medium (C) or medium containing PTTH (P). Incubation time was 60 min. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, *p < 0.05; **p < 0.01).

Thummel, 2005). Treatment with 20-hydroxyecdysone increased E75A expression (Fig. 8D). This result suggests that 20hydroxyecdysone negatively affects HR38 expression and further confirmed that the action of PTTH on HR38 expression is direct.

4. Discussion The present study clearly showed that PTTH stimulated HR38 expression in silkworm PGs in a time-dependent manner. A significantly increased HR38 mRNA level was detected 1 h after treatment with PTTH in vitro, the highest level was reached at 2 h, after which, the expression of HR38 decreased at 8 h, and returned to a basal level by 24 h. Moreover, in vitro stimulation of HR38 expression by PTTH was also verified by in vivo experiments: injection of PTTH into day-5 last instar larvae stimulated HR38 expression 1 h after PTTH injection. In addition, we further demonstrated the Ca2+-dependency of PTTH-stimulated HR38 expression: when

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Fig. 8. Effects of ecdysone (A, B) and 20-hydroxyecdysone (C, D) on gene expressions. (A) and (B), Effects of ecdysone on HR38 (A) and bFTZ-F1 (B) expressions. (C) and (D), Effect of 20-hydroxyecdysone on HR38 (C) and E75A (D) expressions. PGs were pretreated with control medium for 30 min, and then transferred to control medium (C), or medium containing 10, 100, or 1000 ng/ml of ecdysone or 20-hydroxyecdysone. Incubations were maintained for 2 h. Each bar represents the mean + SEM of four separate assays. An asterisk indicates a significant difference compared to the control (by Student’s t-test, *p < 0.05; ** p < 0.01).

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extracellular Ca2+ was removed, PTTH-stimulated HR38 expression was greatly reduced compared to that incubated in saline containing Ca2+ (Fig. 4). PTTH treatment also stimulated the HR38 protein level. HR38 expression did not change after treatment with ecdysone. However, 20-hydroxyecdysone treatment decreased HR38 expression. These results suggest direct stimulation of HR38 expression by PTTH. Although the roles of Drosophila HR38 in participating with USP in the ecdysteroid signaling pathway, in adult cuticle formation, in carbohydrate metabolism, and in glycogen storage were demonstrated in previous studies (Kozlova et al., 1998, 2009; Ruaud et al., 2011), to our knowledge, this is the first report to demonstrate that PTTH directly stimulates HR38 expression in silkworm PGs. We hypothesize that PTTH-stimulated HR38 expression lies upstream of ecdysone biosynthesis signaling. It was also interesting to note that HR38 is an IEG in pheromone-elicited response in insect brains (Fujita et al., 2013). To clarify the signaling pathway involved in PTTH-stimulated HR38 expression, we used U0126, which is known to block ERK activation, and LY294002, a PI3K inhibitor. Our results showed that PTTH-stimulated HR38 expression was partially inhibited by pretreatment with U0126, indicating the involvement of ERK signaling. In addition, A23187, a Ca2+ ionophore, and thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, which can cause an increase in the phosphorylation of both ERK and histone H3 phosphorylation at serine 10 (Gu et al., 2010; Gu and Hsieh, 2015) appeared to stimulate the HR38 expression. Pretreatment with LY294002 also partially inhibited activation of HR38 expression, indicating that PI3K signaling is partially involved. Only partial inhibition of PTTH-stimulated HR38 expression by either U0126 or LY294002 implies the possibility that the combination of multiple signaling pathways, not a single signaling pathway is responsible for HR38 expression. This result is consistent with previous reports on the induction of NR4A receptors in mammalian systems. It is well documented that NR4A gene expression is differentially regulated in a stimulus- and cell type-specific manner. Luteinizing hormone induces NGF-I B expression via protein kinase A (PKA), calmodulin kinase II, and MAPK pathways (Song et al., 2001). Parathyroid hormone induces NR4A receptors through PKA and protein kinase C (PKC) pathways (Pirih et al., 2003). We hypothesized that PTTH-stimulated HR38 expression is a point of convergence for multiple signaling pathways including Ca2+, ERK, and PI3K signaling. In the present study, we showed that PTTH rapidly stimulates HR38 expression and that pretreatment with CHX does not block subsequent induction by PTTH. These results showed that HR38 can be classified as a PTTH-inducible IEG. As an IEG that functions as a transcription factor, it is assumed that HR38 is a prime candidate for mediation of PTTH-stimulated ecdysteroidogenesis because of its potential to affect expression of secondary response genes. In vertebrate steroidogenesis, previous studies have demonstrated that numerous nuclear transcription factors, such as SF-1, members of the AP-1 family (c-FOS and c-JUN), and NR4A1, participate in the transcriptional regulation of steroidogenic enzyme genes in endocrine tissues (Stocco et al., 2001; Sirianni et al., 2002; Manna et al., 2003; Manna and Stocco, 2005; Martin and Tremblay, 2008; Nishida et al., 2008). Among them, NR4A1 has received increased consideration as an essential regulator of the expression of a number of steroidogenic enzyme genes in both the hypothalamo-pituitary–adrenal and hypothalamo-pituitary–g onadal axes. In the adrenal gland, a role for NR4A1 in regulating CYP21, CYP11B2, and HSD3B2 gene transcription was documented (Bassett et al., 2004a,b; Kelly et al., 2004), suggesting that it may regulate cortisol, aldosterone, and androgen production. It was reported that NR4A1 activates the promoter of several genes involved in testosterone biosynthesis in Leydig cells, including the human HSD3B2, rat CYP17, and mouse StAR promoter (Zhang

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Fig. 9. Signaling network in PTTH-stimulated PGs and the cross talk mechanism between PTTH and ecdysone signaling.

and Mellon, 1997; Martin and Tremblay, 2005; Martin et al., 2008). In ovarian theca cells, evidence shows that NR4A1 is a key regulator of StAR, CYP11A1, CYP17, and HSD3B2 transcription and thus plays an important role in determining the capacity of theca cells to produce androgen (Li et al., 2010). Similar to vertebrate steroidogenesis, PTTH rapidly stimulates ecdysteroidogenesis within minutes, and increased translation and/or phosphorylation of some key molecules appear to be involved in this acute effect of PTTH (Smith and Rybczynski, 2012; Rewitz et al., 2013). In addition, PTTH upregulates longterm transcriptional levels of certain ecdysteroidogenic enzymes in PGs (Keightley et al., 1990; Niwa et al., 2005; Yamanaka et al., 2007; Niwa and Niwa, 2014, 2016). It was demonstrated that in vitro PTTH treatment stimulates expressions of ecdysteroidogenic genes in B. mori (Niwa et al., 2005; Yamanaka et al., 2007). Ablating PTTH-producing neurons of Drosophila results in reduced expression of Halloween genes (McBrayer et al., 2007). These

results suggest a link between PTTH signaling and transcriptional regulation of ecdysteroidogenic genes. The target genes for HR38 in silkworm PGs are not known at the present time, and functional mapping of regulatory elements in the promoters of ecdysteroidogenic genes should determine whether HR38-binding sites are contained therein. In addition, the present study shows that in vitro PTTH treatment also stimulates the amount of the HR38 protein found in PGs. High levels of the HR38 protein found late in the last larval instar coincide with the major peak of ecdysteroid biosynthetic activity. This result further confirms the possible involvement of HR38 in PTTH-stimulated ecdysteroid biosynthesis. In the present study, we also examined changes in the responses of PGs in different stages to PTTH treatment. Our results showed that PTTH did not stimulate HR38 expression in PGs from day-1 last instar larvae. This result is consistent with previous results. Previous studies demonstrated that in the presence of juvenile hormone, PGs from early stages of the last larval instar do not

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respond to PTTH, due to a deficiency in PTTH signaling (Okuda et al., 1985; Gu et al., 1996, 1997). In addition, PTTH did not stimulate HR38 expression in PGs from day-10 last instar larvae, relative to day-6 last instar larvae, possibly due to the refractory state of PGs to PTTH stimulation by day-10 (Gu and Chow, 2005). PTTH-stimulated HR38 expression was also detected in PGs from day-2 fourth instar larvae, suggesting the possible involvement of HR38 in PTTH-stimulated ecdysteroid biosynthesis in fourth instar larvae. Ecdysteroids not only play important roles in regulating insect growth and development, but also have growth-promoting effects on many insect cells in vitro (Smith and Rybczynski, 2012; De Loof et al., 2013, 2015). Thus the possibility exists that ecdysteroids secreted by PGs themselves may rapidly modify HR38 expression. However, in the present study, we did not find that ecdysone affected HR38 expression in vitro. We did observe a decrease in HR38 expression in response to 20-hydroxyecdysone, suggesting a negative feedback by ecdysteroids once they are converted to an active form by peripheral tissues. These results support our hypothesis that PTTH directly stimulates HR38 expression. In addition, it was reported that DHR38 can compete with the EcR for dimerization with USP and consequently interferes with ecdysone-dependent transcription (Sutherland et al., 1995; Fahrbach et al., 2012). Induction of HR38 gene expression and protein level in B. mori PGs by PTTH suggests a potential cross talk mechanism between PTTH and ecdysone signaling in regulating ecdysteroidogenesis (Fig. 9). Recently, it was reported that EcRdependent positive feedback operating downstream of PTTH in Drosophila appears to play a role in generating a sustained major ecdysone peak before larval-pupal metamorphosis (Moeller et al., 2013). In conclusion, we demonstrated that PTTH rapidly and transiently induces mRNA expression of HR38 in B. mori PGs. The induction appears to be mediated, at least in part, through activation of Ca2+/ERK and PI3K signaling and to be independent of both ecdysone and 20-hydroxyecdysone. In light of these and recently published data of NR4A regulation of steroidogenesis in mammalian systems, we hypothesize that the HR38 gene may be a critical mediator of PTTH’s downstream effects on PGs. Acknowledgements The authors thank Dr. Taketoshi Kiya (Kanazawa Univ., Japan) for the antibody directed against Bombyx HR38, Taiwan Ministry of Science and Technology for grants (MOST 104-2311-B-178002-MY3), and the National Museum of Natural Science Taiwan for their financial support. References Baker, K.D., Shewchuk, L.M., Kozlova, T., Makishima, M., Hassell, A., Wisely, B., Caravella, J.A., Lambert, M.H., Reinking, J.L., Krause, H., Thummel, C.S., Willson, T.M., Mangelsdorf, D.J., 2003. The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway. Cell 113, 731–742. Bassett, M.H., Suzuki, T., Sasano, H., De Vries, C.J., Jimenez, P.T., Carr, B.R., Rainey, W. E., 2004a. The orphan nuclear receptor NGFIB regulates transcription of 3betahydroxysteroid dehydrogenase. Implications for the control of adrenal functional zonation. J. Biol. Chem. 279, 37622–37630. Bassett, M.H., Suzuki, T., Sasano, H., White, P.C., Rainey, W.E., 2004b. The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production. Mol. Endocrinol. 18, 279–290. Benoit, G., Cooney, A., Giguere, V., Ingraham, H., Lazar, M., Muscat, G., Perlmann, T., Renaud, J.P., Schwabe, J., Sladek, F., Tsai, M.J., Laudet, V., 2006. International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58, 798–836. Bialecki, M., Shilton, A., Fichtenberg, C., Segraves, W.A., Thummel, C.S., 2002. Loss of the ecdysteroid-inducible E75A orphan nuclear receptor uncouples molting from metamorphosis in Drosophila. Dev. Cell 3, 209–220. Close, A.F., Rouillard, C., Buteau, J., 2013. NR4A orphan nuclear receptors in glucose homeostasis: a minireview. Diabetes Metab. 39, 478–484.

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