Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 65:143–151 (2007) Tissue-Specific Regulation of Juvenile Hormone Esterase Gene Expression by 20-Hydroxyecdysone and Juvenile Hormone in Bombyx mori Manabu Kamimura,* Michiyoshi Takahashi, Kyoko Kikuchi, A.M.S. Reza, and Makoto Kiuchi Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH. Arch. Insect Biochem. Physiol. 65:143–151, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: juvenile hormone; esterase; ecdysone; Bombyx mori; silkworm; silk gland; fat body INTRODUCTION In insect, molt and metamorphosis are orchestrated by ecdysteroids and juvenile hormone (JH). Ecdysteroids, primarily 20-hydroxyecdysone (20E), initiate molting and JH determines the molt characteristics: pulses of 20E in the presence of JH trigger larval-larval molts, and in its absence cause a larval-pupal molt followed by pupal-adult transformation (Riddiford, 1985). Hemolymph JH titer is controlled by synthesis in the corpora allata and degradation in hemolymph and other periph- eral tissues (Hammock, 1985). The primary route of JH degradation is hydrolysis of its methyl ester by the highly specific JH esterase (JHE). Another route of JH degradation is hydration of the 10,11epoxide by JH epoxide hydrolase. The importance of JHE in the metabolism of JH, control of hemolymph JH titer, and induction of metamorphosis has been demonstrated by enzymatic inhibition using transition state analogue inhibitors (AbdelAal and Hammock, 1990; Browder et al., 2001) and molecular genetic manipulation of JHE gene expression using baculovirus expression systems or National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan Contract grant sponsor: Ministry of Agriculture, Forestry and Fisheries of Japan; Contract grant sponsor: Bio-oriented Technology Research Advancement Institution (PROBRAIN) of Japan. A.M.S. Reza’s present address is Department of Zoology, Rajshahi University, Rajshahi-6205, Bangladesh. *Correspondence to: M. Kamimura, Invertebrate Gene Function Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. E-mail: firstname.lastname@example.org © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20186 Published online in Wiley InterScience (www.interscience.wiley.com) 144 Kamimura et al. transgenic insects (Bonning et al., 1997; Hajos et al., 1999; Tan et al., 2005). In the silkworm Bombyx mori, JHE is mainly synthesized in the fat body and secreted into the hemolymph in the last larval instar, where it degrades remaining JH and potentiates the larval-pupal transition (Hirai et al. 2002). JHE expression is induced by JH in several insects (Feng et al., 1999; Kethidi et al., 2005; Venkataraman et al., 1994; Vermunt et al., 1999; Wroblewski et al., 1990). In the spruce budworm Choristoneura fumiferana (Feng et al. 1999) and in D. melanogaster (Kethidi et al., 2005), 20E suppresses JH-induced JHE expression. In a previous study, we reported that feeding 20E in the early5th instar suppressed the increase of JHE activity in hemolymph and application of an imidazole compound KK-42, which blocks ecdysone synthesis in the prothoracic gland and causes a precocious metamorphosis, in the 4th instar induced JHE mRNA expression in the fat body and the appearance of JHE activity in hemolymph (Hirai et al., 2002). Thus, it appears that JH expression is also hormonally controlled in the silkworm; however, the regulatory mechanisms involved are still mostly unknown. In the course of a screen to identify ecdysteroidregulated genes from the Bombyx anterior silk gland by mRNA differential display (Kamimura et al., 1999), we have identified JHE as an ecdysteroidinducible gene independently of the JHE expression study mentioned above. A 1.1-kbp RT-PCR fragment encoding a part of the cloned JHE cDNA (942-2035; Hirai et al., 2002) was strongly amplified from the anterior silk gland treated with JH plus 20E and weakly amplified from those treated only with 20E but not amplified from the control. These results suggested that JHE mRNA expression was positively regulated both by 20E and JH in the Bombyx anterior silk gland. So far, there has been no report showing that 20E can induce JHE expression in any insect species. To clarify this point, we investigated temporal expression profiles and hormonal regulation of JHE and mRNA in multiple tissues. The results suggest that the different responses to the hemolymph ecdysteroid titer yield tissue-specific JHE expression profiles. MATERIALS AND METHODS Insects and Hormonal Treatment F1 hybrid silkworms, strain C145 × N140, were reared on an artificial diet (Silkmate, Nihon Nosan Kogyo, Yokohama, Japan) under a 12-h light:dark photoperiod at 25 ± 1°C. The penultimate (4th) instar was staged at the onset of the scotophase and last (5th) instar at the photophase. Only female-determined larvae were used for experiments. Fifty micrograms of 20-hydroxyecdysone (20E; Sigma, St. Louis, MO) dissolved in 20 µl of distilled water were injected into the silkworm through the first abdominal leg with a microsyringe. One or 100 ng of a JH analog, fenoxycarb, supplied by Sankyo Corporation (Tokyo, Japan), were dissolved in 5 µl acetone and applied topically to the silkworm with a micropipet along the dorsal midline. Physiological responses of silkworms to those hormonal treatments were described in Kamimura and Kiuchi (1998) and Takahashi et al. (2003) in detail. Northern Blot Hybridization and Quantitative RT-PCR Total RNA was extracted by the acid guanidinium-phenol-chloroform method using TRIzol (Gibco BRL, Rockville, MD). Twenty micrograms of each RNA sample was separated on a guanidine thiocyanate 1% agarose gel (Goda and Minton, 1995) and transferred to a Hybond NX nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were hybridized with DNA probes labeled with alpha [α-32P]-dATP using a Strip-EZ™ DNA kit (Ambion, Austin, TX). A 1.1kbp PCR fragment encoding a part of the JHE cDNA (942-2035; GenBank No. AF287267) was used as a probe for JHE. Hybridization, washing, and stripping of blots were based on the manufacturer’s instructions. Signals were detected using an image analyzer (Molecular Imager GS-250, BioRad, Hercules, CA). Five micrograms of total RNA were reverse-transcribed with an oligo (dT) primer using Ready-ToGo You-Prime First-Strand Beads (Amersham PharArchives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Hormonal Regulation of Silkworm JHE macia Biotech, Uppsala, Sweden) and used for quantitative PCR on a real-time thermal cycler (model 7700, Applied Biosystems). Serial dilutions of a pBluescript II plasmid containing the full coding region of JHE cDNA were used as standards. A fluorogenic TaqMan probe JHE-pro1 5′-TGCCGA GAGTGAAACGATTGAGCAGG-3′. forward primer JHE-51 5′-CACATGACGATCACGACCACT-3′, and reverse primer JHE-31 5′-CCGTGATGTTGTCCA CTCTCTTT-3′ were designed in the 3′ UTR. A ribosomal protein gene rp49 (GenBank: AB048205) was chosen as a reference gene. A TaqMan probe rp49-pro1 5′-TGGTTACGGTTCCAACAAGAAGAC CCG-3′, forward primer rp49-51 5′-GGTCAATAC TTGATGCCCAACA-3′, and reverse primer rp49-31 5′-GGAATCCATTTGGGAGCATATG-3′ were used as in a previous report (Reza et al., 2004). PCR condition was based on the standard procedure on the manufacturer’s instructions. The molar amounts of JHE and rp49 cDNAs were calculated on the basis of a crossing point analysis, with standard curves generated from the standard plasmids. JHE cDNA amounts were normalized with rp49 cDNA amounts in the same samples. Independent sets of RNA were used for northern blot analysis and quantitative RT-PCR. 145 gradually decreased through the instar (Fig. 1). In the 5th instar, JHE mRNA expression had doublepeaked at wandering and just before pupation. This expression profile is consistent with a previous report (Hirai et al., 2002). JHE mRNA expression in the wing disc in the 5th larval instar was similar to that in the fat body, except that overall expression levels were much lower (Fig. 1). Developmental Regulation of JHE mRNA in the Anterior Silk Gland Detailed Northern blot analysis showed that JHE mRNA was expressed strongly on day 3.0 in the 4th larval instar and from day 10.0 to 11.0 in the 5th larval instar in the anterior silk gland (Fig. 2). The JHE expression periods coincided with the hemolymph ecdysteroid peaks. Trace amounts of JHE mRNA could be detected in the early 4th instar but not in the 5th instar, indicating that the basal JHE expression level is higher in the 4th instar than in the 5th larval instar. Quantitative RTPCR analysis showed a very similar JHE mRNA expression profile (Fig. 2). Hormonal Regulation of JHE mRNA in the Anterior Silk Gland RESULTS Developmental Regulation of JHE mRNA in Six Tissues In the anterior silk gland, a trace level of JHE mRNA was detected by Northern blot during the first 3 days of the 4th larval instar (Fig. 1). Its expression increased on day 3 and then decreased to the basal level on the next day. In the 5th larval instar, JHE mRNA was detected only on day 10, just before pupation. Very similar profiles of JHE mRNA expression were observed in the middle and posterior silk glands (Fig. 1). In the mid gut, strong JHE expression was also observed before each molting, although it peaked not on day 3 but on day 4 in the 4th instar (Fig. 1). In the fat body, JHE mRNA was expressed strongly at the beginning of the 4th instar and Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. When 20E was injected into the last instar larvae of day 4, JHE mRNA appeared 12 h post injection (p.i.) and was expressed most strongly 24 h p.i. (Fig. 3A). Topical application of 100 ng fenoxycarb just after the 20E injection strengthened and accelerated JHE mRNA: JHE mRNA expression was induced 2 h p.i. and peaked 12 h p.i. (Fig. 3A). Application of fenoxycarb only also induced JHE expression within 2 h (Fig. 3B). JHE mRNA gradually increased until 36 h and remained at least until 48 h after the application. The maximum level of JHE expression after fenoxycarb treatment was lower than after 20E alone or 20E plus fenoxycarb treatments (data not shown). In the control larvae, JHE mRNA was undetectable throughout the analyzed period (from day 4.0 to 6.0 of the 5th instar: Fig. 2). We topically applied 1 ng of fenoxycarb to the 146 Kamimura et al. Fig. 1. Tissue-specific expression profiles of JHE mRNA in the 4th and 5th larval instar of the silkworm as detected by Northern blot analysis. Ethidium bromide staining of rRNA is shown as a control for equal loading of RNA samples. last instar larvae on day 4 and analyzed JHE mRNA expression in one- or two-day intervals (Fig. 3C). This treatment induced a supernumerary molt into an abnormal 6th instar larvae with evaginated pupal antennae and often with evaginated wings (Kamimura and Kiuchi, 1998). JHE mRNA expression was induced immediately but transiently. JHE mRNA would then reappear on day 11. The tim- ing of the second JHE expression coincided with the hemolymph ecdysteroid peak. Topical application of 1 ng of fenoxycarb on day 0 of the last instar larvae delayed the larvalpupal metamorphosis by 6 days (Kamimura and Kiuchi, 1998). This JH treatment also induced JHE mRNA expression at two different times, 2 days after the application and just before pupation (Fig. Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Hormonal Regulation of Silkworm JHE 147 Fig. 2. Temporal expression profiles of JHE mRNA in the anterior silk gland. In quantitative RT-PCR analysis (middle panel), quantities of JHE cDNA in cDNA pools were normalized to those of internal standard gene rp49. Bars represent SE (N = 2). In Northern blotting analysis (bottom panel), ethidium bromide staining of rRNA is shown as a control for equal loading of RNA samples. Hemolymph ecdysteroid and JH titers (top panel) are based on Kiuchi (1992) and Niimi and Sakurai (1997), respectively. 3D). The timing of the second JHE expression also coincided with the hemolymph ecdysteroid peak. larvae on day 4 suppressed JHE mRNA expression (Fig. 4B). JHE mRNA disappeared 12 h p.i., while JHE expression was approximately constant in water-injected control larvae. Hormonal Regulation of JHE mRNA in the Fat Body We also investigated the effects of hormonal treatments on JHE mRNA expression in the fat body, which showed a distinct JHE expression profile compared to that in the anterior silk gland (Fig. 1). When only acetone was applied to the last instar larvae on day 0, JHE mRNA started increasing gradually from day 4 in the fat body (Fig. 4A) in the same fashion as when no hormone was applied (Fig. 1). Topical application of 100 ng fenoxycarb enhanced this JHE mRNA expression: JHE mRNA was expressed earlier and more strongly than in the control (Fig. 4A). In contrast, injection of 20E into the last instar Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. DISCUSSION Northern blot and quantitative RT-PCR analyses showed that the timing of the maximum JHE expression in the anterior silk gland coincides with the peak of hemolymph ecdysteroid titer in both the 4th and 5th larval instar (Fig. 2), as in the case of some ecdysteroid-inducible genes, such as chitinase (Takahashi et al., 2002), chitinase-related gene 1 (Takahashi et al., 2002), and a nuclear receptor BHR3 (unpublished data). 20E injection into the mid-5th instar larvae induced JHE expression (Fig. 3A). In addition, JHE mRNA was ex- 148 Kamimura et al. Fig. 3. Effects of 20E and fenoxycarb treatments on JHE mRNA expression in the anterior silk gland. A: Northern blot analysis of JHE expression in last instar larvae of day 4 that were injected with 50 µg of 20E (+20E), injected with 20E and topically applied with 100 ng of a JH analog fenoxycarb (+20E+JH). Ethidium bromide staining of rRNA is shown as a control for equal loading of RNA samples. B: Quantitative RT-PCR analysis of JHE expression in last instar larvae of day 4 that were topically applied with 100 ng of fenoxycarb. Quantities of JHE cDNA in cDNA pools were normalized to those of internal standard gene rp49. Values followed by the same lowercase letters do not differ significantly in statistical analysis (P < 0.05, Tukey-Kramer test after logarithmic transformation). Bars represent SE (N = 2–3). C: Northern blot analysis (bottom) of JHE expression in last instar larvae of day 4 that were topically applied with 1 ng of fenoxycarb. Hemolymph ecdysteroid titers (top) are based on Kamimura and Kiuchi (1998). D: Northern blot analysis (bottom) of JHE expression in last instar larvae of day 0 that were topically applied with 1 ng of fenoxycarb. Hemolymph ecdysteroid titers (top) are based on Kamimura and Kiuchi (1998). pressed at the hemolymph ecdysteroid peak in two types of fenoxycarb-applied larvae in which a supernumerary larval molt was induced (Fig. 3C) or the last larval period was prolonged (Fig. 3D). These results strongly indicate that high JHE expression at each molt is induced by the hemolymph ecdysteroid surge. Because JHE mRNA was expressed a few days later than those of the ecdysone receptors A and B1 isoforms (Kamimura et al., 1997) and early ecdysteroid-inducible genes, such as Broad-complex (Reza et al. 2004) and E75A (unpublished data) in the 5th larval instar, induction of JHE mRNA by 20E is indirect, probably depending on early ecdysteroid-inducible gene products. Fenoxycarb application also induced JHE mRNA expression in the anterior silk gland (Fig. 3B–D). The Bombyx JHE is, thus, a JH-inducible gene like the JHE gene of several other insects (Feng et al., 1999; Kethidi et al., 2005; Venkataraman et al., 1994; Vermunt et al., 1999; Wroblewski et al., 1990). This induction was rapid (within 2 h), suggesting that the transcription is directly induced by JH via a putative JH response element in the promoter region as is the ChoristoArchives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Hormonal Regulation of Silkworm JHE Fig. 4. Effects of 20E and fenoxycarb treatments on JHE mRNA expression in the fat body as detected by Northern blot analysis. Last instar larvae of day 4 were injected with 50 µg of 20E (A) or last instar larvae of day 0 were topically applied with 100 ng of fenoxycarb (B). Ethidium bromide staining of rRNA is shown as a control for equal loading of RNA samples. neura fumiferana JHE gene (Feng et al., 1999; Kethidi et al., 2004). We surmise that induction of JHE mRNA by fenoxycarb reflects the positive regulation of basal JHE expression by the hemolymph JH titer. Basal JHE mRNA expression levels in the 4th instar was higher than in the 5th instar (Fig. 2), supporting this hypothesis. Taken all together, we gather that hemolymph JH titer controls the basal expression level of JHE and ecdysteroid surges induce its high expression at molting in the anterior silk gland. JHE expression is probably regulated in similar manners in the remaining parts of silk glands and the mid gut, because the JHE mRNA expression profiles in these tissues were similar to that in the anterior silk gland. 20E and JH positively interact in regulating JHE expression in the anterior silk gland, because JHE expression was accelerated and strengthened after Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. 149 a combined 20E plus fenoxycarb treatment compared to 20E or fenoxycarb single treatment (Fig. 3A,B). In C. fumiferana and Drosophila melanogaster, 20E reversely suppressed JH-induced JHE expression (Feng et al., 1999; Kethidi et al., 2005). In C. fumiferana, 20E suppressed JH-induced binding of nuclear proteins to JHRE (Kethidi et al., 2004). 20E might act on putative JHRE also in the silkworm JHE. JHE expression was not induced by the ecdysteroid surge of the last larval instar in the C. fumiferana mid gut (Feng et al. 1999), in contrast to what we have observed in the B. mori mid gut (Fig. 1). These results suggest that the response of JHE to 20E is species-specific. Developmental JHE mRNA expression profile in the fat body was much different from that in the anterior silk gland, suggesting that its hormonal regulation is different between these two tissues. Indeed, 20E injection suppressed JHE expression in the fat body on day 4 of the 5th instar (Fig. 4B), although the same treatment induced it in the anterior silk gland (Fig. 3A). This result suggests that the rising titer of hemolymph ecdysteroid suppresses JHE expression in the fat body from day 7 to 8 of the 5th larval instar (Fig.1). However, JHE mRNA increases again in the 5th instar just before pupation in the fat body. This second JHE peak of expression might be the result of upregulation by the high ecdysteroid titer as in the anterior silk gland at the same time. We surmise that the JHE expression was directly downregulated by a small rise of ecdysteroids (20–100 ng/ml) on day 7–8 and then indirectly upregulated by the same hormone at its peak (ca. 1,500 ng/ml) via ecdysteroidinduced transcriptional factors. What is the biological significance of the high JHE mRNA expression at each molt in the silk gland and mid gut? We do not have enough evidence at this time to give a clear answer to this question. What we can say is that the translated JHE proteins in these tissues at the end of the 4th instar are probably not secreted into the hemocoel because neither JHE proteins nor JHE activities were detected in hemolymph throughout the 4th instar (Hirai et al., 2002). One possibility is that JHE proteins expressed at that time are retained in 150 Kamimura et al. the cytoplasm, the plasma membrane, or in the extracellular matrix and degrade JH in or around cells. At the larval stage, the primary function of JH is to modify ecdysteroid activity and to determine the molt characteristics (Riddiford, 1985). JHE expression during molting may enable the tissues to degrade the remaining JH in or around tissues and prepare to respond to hemolymph JH in the next instars. This hypothesis will be verified by examining JHE activity and JHE protein expression in these tissues. ACKNOWLEDGEMTS We thank Dr. Yasushi Kanamori with the assistance of quantitative RT-PCR. 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