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The Juvenile Hormone Analogue, Methoprene, Inhibits Ecdysterone Induction of Small Heat Shock Protein Gene Expression DECATO
EDWARDM.BERGER,~KARYNGOUDIE,LOUISKLIEGER,MATTBERGER,ANDRODNEY Department
of Biology
and Molecular
Genetics Accepted
Center,
Dartmouth
February
College,
Hanover,
New
Hampshire
03755
24, 19%
The small heat shock protein (hsp) genes of Drosophila are expressed in cultured cells in response to the moulting hormone, ecdysterone. We show here that juvenile hormone (JHIII) and the juvenile hormone analogue, methoprene, inhibit that induction in a dose-dependent manner. Heat shock induction is not inhibited. In transient expression studies using S3 line cells transfected with EcRE-CAT constructs, methoprene inhibition was found to require a 2-hr pretreatment (before ecdysterone addition), and methoprene’s continued presence was essential. Farnesol, farnesyl acetate, and retinoic acid did not cause inhibition. Several models of methoprene inhibition are discussed. o 1992Academic press, I~C. INTRODUCTION
The development of holometabolous insects is under the control of two hormones; ecdysterone, a steroid, and juvenile hormone (JH), a sesquiterpene. While high ecdysterone titers are required to initiate each moult, the type of moult completed is largely determined by the JH level. During larval-to-larval moults relatively high JH titers are required. The commitment to pupal development in Lepidoptera appears to take place when the JH titer has fallen below detectable levels in final instar larvae as the ecdysterone levels rise (Riddiford, 1978). Later, during the prepupal period JH levels again rise (Riddiford, 1978) apparently to prevent adult differentiation of imaginal disc structures during pupal development. Over the past 10 years a great deal has been learned about the molecular biology of ecdysterone action (see Ashburner, 1990; Segraves and Richards, 1990; Riddihough and Pelham, 198’7; Martinez et ah, 1991). As in the case of many other steroid hormone-regulated systems, target gene expression is controlled by the presence or absence of a hormone-receptor complex bound to a palindromic DNA sequence situated near the transcription unit. Recent studies in Drosophila have identified a short, 15-bp imperfect palindrome located some 500 bp upstream of the heat shock protein 27 (hsp 27) gene that is sufficient to confer ecdysterone-inducible expression on a contiguous reporter gene (Cherbas et al., 1991). A version of this ecdysterone response element (EcRE) is found adjacent to other ecdysterone-inducible Drosophila genes including hsp 22, hsp 23, and EIP 28/29 (Do1 To whom correspondence should be addressed.
0012-1606/92$5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
bens et al., 1991; Cherbas et al, 1991). There is weak evidence that a different recognition sequence is involved in the case of ecdysterone-repressible genes (Wallrath et ah, 1990). Based on both biochemical and genetic evidence, the ecdysterone receptor appears to be localized primarily in the nucleus most likely associated with EcRE sequences (Cherbas et ab, 1991; Dobens et al, 1991; Koelle et al, 1991). Ecdysterone presumably diffuses into the cell and reaches the nucleus passively where it binds to a receptor and initiates transcription. This mechanism of action resembles that found with mammalian thyroid hormone (Damm et al., 1989). Here also unliganded receptor is constitutively bound to the target gene’s response element where it acts to repress basal expression. Binding of hormone converts the receptor into a transcriptional activator. With the recent isolation and characterization of the ecdysterone receptor, and its gene (Koelle et ab, 1991), a more detailed description of transcriptional activation and repression will develop. In contrast, the molecular biology of JH action is largely unknown. JH is believed to somehow antagonize or inhibit ecdysterone action by preventing the expression of adult specific genes, while still allowing or enabling the expression of those ecdysterone-responsive genes required for the execution of larval moults (Riddiford, 1985). In several insects, including Drosophila and the tobacco hornworm, Manduca, developmental variations in JH levels have been reported (Sliter et ab, 1987; Bownes and Rembold, 1987). JH-binding proteins have been described that display the specificity, saturability, and high hormone-binding affinity expected of a bona fide receptor (Klages et al., 1980; Chang, 1985; Chang et al, 1985; Engelmann et ah, 1987; Goodman and Chang, 410
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1985; Osir and Riddiford, 1988; Moore, 1990; Palli et ab, 1990; Shemshedini et al, 1990). These proteins, however, are generally smaller than steroid hormone receptors (Evans, 1988) and there is only circumstantial evidence that JH receptors bind to DNA in a sequence-specific way (Palli et al, 1990). In a recent report Shemshedini and Wilson (1990) have identified an 85-kDa juvenile hormone-binding protein from extracts of Drosophila fat body, In a Drosophila mutant strain, MET (methoprene tolerant), the level of juvenile hormone binding is greatly reduced. However, the mutant strain shows no discernable mutant phenotype in terms of development. We have begun to study the molecular biology of JH action using a well-defined ecdysterone-inducible system, the Drosophila hsp27 gene. This and the three other ecdysterone-inducible small hsp genes (Ireland and Berger, 1982) are a tightly linked multigene family located at cytogenetic band 67B on the polytene map. In this report we show, first, that the JH analogue, methoprene, inhibits the ecdysterone-induced expression of endogenous small hsp genes, but has no effect on small hsp gene regulation by heat shock. Second, we show that methoprene inhibition can occur in a transient expression assay in which EcRE sequences are regulating hormone-dependent reporter gene expression. EXPERIMENTAL PROCEDURES
Cell culture and transfection. Drosophila line S3 cells were cultured in Schneider’s medium (GIBCO) containing 10% fetal bovine serum at 25°C. For transfection, 20 pg of CsCl-purified plasmid DNA was added as a CaCl, coprecipitate (DiNocera and Dawid, 1983) to each 25cm2 flask of confluent cells in 4 ml of medium. Except where indicated ecdysterone was added to 1 palm final concentration 24 hr after DNA addition, and the flasks were cultured for an additional 24 hr at 25°C before cell harvesting. Hormones. Ecdysterone was purchased from Calbiothem and stored frozen in an aqueous solution at 1 mM. Methoprene was a gift from Dr. D. Cerf (Sandoz, Palo Alto). JHIII was purchased from Sigma Biochemical, as was retinoic acid. Farnesol and farnesyl acetate were purchased from Aldrich Chemical Co. Methoprene, JHIII, farnesol, and farnesyl acetate were stored at -20°C in glass vials in 95% ethanol and diluted into culture medium directly. Stock dilutions were made in 95% ethanol using plastic pipet tips. CAT assay. Following treatment the cells were harvested by centrifugation. After a wash in Drosophila Ringer’s, a cell pellet was recovered and resuspended in 250 ~1 of 0.25 M Tris (pH 8.0). Cell lysates, prepared by brief sonication, were clarified by a 5-min spin at 5°C in a microcentrifuge. Aliquots of the clear supernatant
were then assayed for CAT activity (Gorman et a& 1982), and the reaction products were visualized by ascending thin-layer chromatography and autoradiography. Quantitation of CAT activity was done by excising spots containing [14C]chloramphenicol and its acetylated products and conducting scintillation counting. The percentage of chloramphenicol acetylation was normalized for protein content in the lysate and is expressed as CAT-specific activity. Plasmids. For the purpose of constructing plasmids containing multiple copies of the EcRE, plasmid pUC18Ec, a gift from Guy Riddihough, was digested with SmaI, and a BgZII linker inserted. A 47-bp BamHIBgZII fragment containing the EcRE was purified from low-melting-point agarose and ligated in the presence of BamHI and BgZII enzyme at room temperature for 1 hr. Polymers with a head-to-tail arrangement of the BamHI-BgZII fragment were separated on and purified from low-melting-point agarose (FMC), and directionally cloned into the plasmid designated ptkAT0. This CAT-reporter plasmid contains the tk TATA box and start site of transcription on a 150-kp SalI-BgZII fragment from ptkS0 directionally cloned into the multicloning site of pBLCAT3. Full details of the construction of EcRE-2R-CAT, hsp 27-CAT, and hsp 70-CAT (DiNocera and Dawid, 1983) are provided in Dobens et al. (1991). Protein analysis. Cells incubated in the absence or presence of hormones were labeled for 4 hr with [35S]methionine at 100 &i/ml (New England Nuclear) in Schneider’s medium lacking methionine, yeast hydrolysate, and serum. Cells were maintained in suspension during labeling by shaking. Following the label period, the cells were pelleted, washed in cold saline, and prepared for one-dimensional gel electrophoresis in 15% acrylamide, 1% SDS gels, or for two-dimensional (isoelectric focusing/SDS) gel electrophoresis, and autoradiography according to published procedures (Ireland and Berger, 1982; Ireland et al, 1982). RESULTS 1. Endogenous
Small Heat Shock Protein
(s-hsp) Gene
Expression We first examined the level of small hsp gene expression in Drosophila S3 line cells following ecdysterone treatment, in either the presence or absence of the JH analogue, methoprene. Methoprene is a potent juvenoid in dipterans (see Cherbas et al., 1989) and is particularly useful in these studies because of its relatively high water solubility. We initially used Richards’ (1978) protocol, which involves a pretreatment with 10m5M methoprene 6 hr prior to ecdysterone addition. Twenty-four hours after ecdysterone addition cells were allowed to
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incorporate [35S]methionine for an hour and then protein was extracted. As seen in Fig. 1, and confirming several previous studies, the presence of ecdysterone alone led to the greatly enhanced synthesis of two low molecular weight polypeptides known to be hsp 23 and hsp 22 (Ireland and Berger, 1982). The slightly larger and more basic hsp 26 and hsp 27 are not resolved in this gel system but have been shown to be induced also (Ireland et al., 1982). In cells first pretreated with 10e5 M methoprene there was no evidence of enhanced hsp 22 or hsp 23 synthesis in five replicate experiments. Minor variations in the abundance of other polypeptides seen in Fig. 1 were observed between treatments in only some experiments but were not pursued further. To examine the specificity of the methoprene effect we looked at the expression of small hsp genes in response to heat shock using cells that were pretreated with methoprene. From the data presented in Fig. 2 we conclude that JH has no important effect on diminishing the level of small hsp synthesis in response to high temperature, so that methoprene inhibition of ecdysterone induced small hsp gene expression appears specific. 2. CAT-Reporter
Constructs
In an effort to extend the analysis of methoprene inhibition, several recombinant plasmid constructs made previously (Dobens et al., 1991) were analyzed functionally using a transient expression assay. The hsp 27-CAT construct contains the bacterial CAT gene ligated downstream of a 1.2-kb DNA fragment recovered from the hsp 27 promoter region. The fragment extends from position -1100 to +87, where +l refers to the transcription initiation site. The hsp 27 EcRE is located between position -546 to -532. When transfected into responsive S3 line cells, the hsp 27-CAT plasmid promoted a low level ecdysterone inducible CAT activity in the absence of methoprene. In the presence of methoprene, the ecdysterone-induced CAT activity was essentially abolished (Fig. 3). The conclusion, then, is that a plasmid containing the hsp 27 upstream region of DNA tested contains sequences required for both ecdysterone-inducible expression and for the methoprene-dependent inhibition of that induction. Methoprene inhibition could involve the association of a methoprene receptor complex to either the EcRE itself, perhaps in combination with the ecdysterone-receptor complex, or to a nonoverlapping binding site elseFIG. 1. Two-dimensional gel electrophoretic pattern of [%]methionine-labeled S3 cells protein showing methoprene inhibition of ecdysterone-dependent hsp 22 and hsp 23 synthesis. Cells were treated with (a) buffer alone, (b) 10e6 M ecdysterone for 24 hr, or (c) 6-hr pretreatment in 1O-5 M methoprene, and then with methoprene and
1O-6 M ecdysterone together for 24 hr. Approximately lo5 cpm of labeled protein extract was used for each gel, and autoradiography was for 48 hr. Arrows in (b) indicate hsp 23 and hsp 22.
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the ecdysterone-receptor complex to its target EcRE sequence, or that it prevents some step in the transcriptional activation process that follows ecdysterone-receptor binding. 3.
FIG. 2. Sodium dodecyl sulfate, 15% polyacrylamide gel electrophoretie analysis of [35S]methionine-labeled polypeptides extracted from S3 cells: untreated control (a), heat shock for 30 min at 37°C followed by a 45-min labeling period using cells pretreated (b), or not (c), with lo- M methoprene for 24 hr before heat shock. Top arrow points to the hsp 26 and hsp 2’7 pair, and lower arrow points to the hsp 22 and hsp 23 pair.
where in the upstream region. As a test we utilized a construct designated EcRE-2R-CAT that had been previously shown to display a high level of ecdysterone-dependent CAT activity in a transient expression assay (Dobens et al., 1991). The EcRE-2R-CAT construct contains a CAT gene just downstream of a herpes virus thymidine kinase minimal promoter. In the adjacent multicloning site we had inserted a tandem pair of 23 bp synthetic hsp 27 EcRE sequences in the orientation found in the endogenous gene (Dobens et al, 1991). Following transfection with the plasmid, S3 cells were treated with lO-‘j M ecdysterone in either the presence or absence of 10e5 M methoprene which had been added 6 hr earlier. The results, summarized in Fig. 3, clearly show that methoprene nearly abolishes the high level of CAT activity induced by ecdysterone in transfected cells. Therefore, we surmise that methoprene, perhaps in association with a receptor, somehow leads to the loss of ecdysterone receptor, interferes with the binding of
Specificity
In order to examine further the nature of methoprene inhibition, several experiments focusing on specificity were carried out. First, we looked at the effect of methoprene pretreatment on the induction of CAT activity in a transient expression assay using an hsp 70-CAT reporter construct and high temperature as the stimulus. The hsp 70-CAT plasmid used in this study has been described in detail elsewhere (DiNocera and Dawid, 1983). Following transfection cells were pretreated with or without low5 M methoprene for 24 hr, and then exposed to 37°C for 30 min. After a recovery period of 1.5 hr at 25°C cell lysates were assayed for CAT activity. As shown in Fig. 4, the high level of CAT activity measured in lysates of heat-shocked cells is little if at all affected by the methoprene pretreatment, which alone had no effect on inducing CAT activity. Comparable results were observed using shorter periods of heat shock, in which lower levels of CAT activity are induced (data not shown). This result is in agreement with previous experiments in which endogenous small hsp induction was examined (Fig. 2). In a second experiment we examined the effect of other reagents on the inhibition of CAT induction by ecdysterone, using the EcRE-2R-CAT construct. The compounds tested include JHIII, farnesol, farnesyl acetate, and retinoic acid, each added to a final concentration of 10m5M to the transfected cells 6 hr before a 24-hr ecdysterone treatment. The results, summarized in Fig. 5A, clearly show that the inhibition of CAT induction was extensive with JHIII but failed to occur in response to any of the other pretreatments. The addition of farnesol, farnesyl acetate, retinoic acid, JHIII, or
FIG. 3. CAT assays. Levels of CAT activity were assayed by using extracts of Droso@ila S3 cells that had been transfected with plasmids hsp 27CAT (a, c, d, g, h), or EcRE-2R-CAT (b, e, f, i, j) and then treated with buffer alone (a, b), with 10m6Mecdysterone alone (c-f), or with a 6-hr pretreatment with 1O-5 M methoprene and then ecdysterone (g-j).
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methoprene alone had no effect on inducing CAT activity (not shown). Finally, we examined the dose-response profile of methoprene inhibition using the standard protocol, one which involves a 6-hr methoprene pretreatment followed by the addition of ecdysterone. These results, also summarized in Figs. 5A and 5B, indicate that inhibition occurs over a range of methoprene concentrations higher than low8 M and with half maximum inhibition at about 10m7M.
A
a
b
c
d
efghi
i
k~lmn
o
~cf
B .07 r
4. Kinetics of Inhibition In order to evaluate the need for a methoprene pretreatment, in terms of inhibition, an experiment was done in which either the time of addition and or duration of methoprene exposure was varied. In all cases lop6 M ecdysterone was administered at experimental time zero. The results, summarized in Fig. 6, illustrate two important features of methoprene inhibition. First, there is a 2-hr period of pretreatment that is essential for the complete inhibition by methoprene to be established. Pretreatments started later than -2 hr lead to progressively lower levels of inhibition, until by +4 hr
.06 I
I
,. I\ Methoprene
Concentration
(M)
FIG. 5. (A) CAT assays to investigate the dose-response profile of methoprene inhibition and the specificity of inhibition. S3 cells were transfected with 15 pg of the plasmid EcRE-2R-CAT, then subjected to hormone treatment, and finally assayed for CAT activity. Treatments include (a) no treatment control, (b, c) 10m6Mecdysterone for 24 hr, (d) ecdysterone with 10e4 M methoprene, (e) ecdysterone with 10m5 M methoprene, (f) ecdysterone with 10m6M methoprene, (g) ecdysterone with 10e7 M methoprene, (h) ecdysterone with lo-’ M methoprene, (i) ecdysterone with 10m9Mmethoprene, (j, k) ecdysterone with 10m5M farnesyl acetate pretreatment, (1, m) ecdysterone with 10e5 M retinoic acid pretreatment, (n, o) ecdysterone with 10d5 M farnesol pretreatment, and (p, q) ecdysterone with 10d5 M JHIII pretreatment. Pretreatments began 6 hr before ecdysterone addition and continued until cell harvesting. (B) Results are plotted graphically. Values indicate the average and range of CAT specific activity values from three replicate experiments. The 10m9M methoprene point seen in A was only determined once and was not included in the summary figure.
FIG. 4. CAT assay. Levels of CAT activity were assayed by using extracts of Drosophila S3 cells that had been transfected with the plasmid hsp ‘IO-CAT and then maintained at 25°C (a, b), or heat shocked at 37°C for 30 min (c-f). In b, e, and f, cells were treated with 10e5 M methoprene for 24 hr before treatment at 25°C (b) or 37°C (e, 0
methoprene treatments become essentially ineffective. It is not clear what process or processes occurs during the pretreatment to establish subsequent inhibition. The second and rather surprising result was that a 4- or a 6-hr pretreatment with methoprene was ineffective if methoprene was removed from the cell culture medium before adding ecdysterone. This implies that while the mechanism of methoprene inhibition requires 2 hr to become established, inhibition also requires the continuous presence of methoprene during the period of ecdysterone treatment.
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B
Ok
11-:/J;, -6
-4
, ,
-2
0
Time of Addition
(hours)
+2
+4
+6
Methowene
FIG. 6. Effect of the time of methoprene pretreatment and pretreatment duration on the level of CAT enzyme activity induced by a 24-hr treatment with lObe’ M ecdysterone. Results in (A) are CAT assays, plotted in (B) as CAT specific activity. The time of ecdysterone addition is indicated as time 0. Lanes in A include CAT assay in which methoprene was added at the following times: (a) -6 hr, (b, c) -4 hr, (d) -2 hr, (e) -1 hr, (f, g) 0 hr, (h, i) +2 hr, (j) +4 hr, (k, 1) +6 hr, (m, n) between -6 and -2 hr only, (0, p) between -6 and 0 hr only. The horizontal bars in B correspond to CAT specific activities averaged from lanes m and n (0) and lanes o and p (0).
DISCUSSION
In three previous studies (Wyss, 1976; Cherbas et al., 1989; Yudin et aZ., 1982) both natural juvenile hormones (JH, JHII, JHIII) and the synthetic analogue, methoprene, were shown to partially inhibit several of the ecdysterone responses exhibited by Drosophila Kc line cells. Levels of inhibition ranging from 50 to 80% or higher were noted for ecdysterone-dependent cell elongation, cell aggregation, mitotic arrest, and acetylcholinesterase induction. The single exception was the failure of JHIII, or methoprene, to inhibit ecdysterone-dependent stimulation of EIP 28/29 synthesis. Cherbas et al. (1989) also reported that JHIII and methoprene incompletely inhibited ecdysterone-dependent cell elongation in an independently derived cell line, Schneider S3 cells, the line used in the work described here. We now report that methoprene and JHIII also inhibit ecdysterone-mediated induction of small heat shock protein gene expression. Inhibition was observed
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both for hsp 22 and hsp 23 synthesis, analyzed by [35S]methionine incorporation. Experiments designed to measure rates of hsp gene RNA synthesis and accumulation are underway. The ability to carry out DNA-mediated gene transfer with S3 cells allowed us to conduct transient expression studies using chimeric promoter-reporter gene constructs. We found that the ecdysterone-dependent induction of CAT gene expression was almost entirely inhibited by both methoprene and JHIII, when reporter gene expression was under the control of either a natural hsp 2’7 promoter region or a tandem pair of synthetic ecdysterone response elements (EcREs). In contrast, the heat-dependent induction of a CAT gene placed under the control of an hsp 70 heat shock promoter was not affected by a 24-hr methoprene pretreatment. Because transcription of CAT mRNA in the EcRE-2RCAT construct initiates from a herpes virus thymidine kinase promoter, there are no Drosophila sequences at all present in the final message. Thus, it is unlikely that methoprene is mediating its inhibition through a mechanism of selective mRNA turnover, unless, of course, the tk-CAT mRNA coincidentally contains the same unusual structure or sequence that confers methoprenedependent message instability on natural small hsp mRNAs. We suspect, rather, that methoprene inhibition is based on events occurring at the level of transcript initiation or elongation. For a number of reasons, juvenile hormone is thought to function through a specific receptor in much the way that thyroid hormone, retinoic acid, and the steroid hormones work (e.g., see Moore, 1990). As a consequence we anticipated that JH, in conjunction with its receptor, would bind to a specific recognition DNA sequence on or near its target gene and, as a consequence of binding, prevent ecdysterone from engaging its program of transcriptional activation. Therefore, we were somewhat surprised to find that methoprene inhibition of ecdysterone induction occurred using even the EcRE-2RCAT promoter-reporter construct. We have not yet tested other independently constructed EcRE-reporter gene constructs, so it is still possible that the synthetic 23-bp EcRE contains a JH-receptor binding site within, overlapping, or outside of the 15bp core EcRE sequence defined by Cherbas et al. (1991), or that cryptic vector or reporter gene sequences are functioning as fortuitous JH-receptor binding sites. We think that these possibilities are unlikely. The working model is that methoprene, probably bound to a receptor, somehow blocks or modifies the binding of the ecdysterone-receptor complex to an EcRE, or that the JH-receptor complex interferes with some subsequent step in the progress of transcriptional activation. Two important questions remain. First, what does the
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evidence allow us to deduce about the mechanism of methoprene inhibition? Second, is the phenomenon of methoprene inhibition in S3 cells interesting in the physiological sense? In terms of physiology, several points can be made in support of the significance of our results. First, methoprene (isopropyl[2E,4E] 11-methoxy-3,7,11trimethyl-2,4-dodecadienoate), also known as ZR 515, ZR 2008, and Altosid, has been used extensively for some time as a bona fide JH known to be particularly active in Diptera (Cherbas et al., 1989). Compared with the Cherbas’ results (1989) the methoprene concentrations required for inhibition in our study, 10-5-10-‘M, are high. However, juvenile hormone or JH analogue concentrations in the range of 10-5-10-7 M have been used previously in work with Drosophila cells in culture (Walker et al., 1991; Yudin et ah, 1982), with salivary glands (Richards, 1978), and with imaginal discs (Chihara et al., 1972). Because of methoprene’s low aqueous solubility, and its lipophilic tendency to stick to plastic and glass, it is not unlikely that the stated concentrations are overestimates of the actual hormone concentration in the medium (Giese et al., 1977; however, see Cherbas et al., 1989). Nevertheless, the inhibition of ecdysterone-mediated CAT induction has been shown to be a property of biologically active juvenoids, methoprene and JHIII, and did not occur using structurally related but biologically inactive compounds, such as farnesol, farnesyl acetate, and retinoic acid. The credibility of our interpretation also relies on the absence of toxic effects. As Cherbas et al. (1989) point out, 1O-5 M methoprene partially reverses the ecdysterone-induced onset of mitotic arrest, although they do note a slight inhibition of cell proliferation compared with untreated control cells. Methoprene-treated cells continue to show the normal pattern of protein synthesis and we noticed no structural abnormalities in the methoprene-treated cells. Based on these observations and on the results of trypan blue exclusion studies (data not shown), methoprene does not noticeably affect cell viability at the concentrations used. There is very little known about the mechanism of JH or methoprene action at the molecular level. Several reports have appeared demonstrating the presence of high affinity, saturable, and ligand-specific binding proteins. These proteins that we refer to generally as “receptors” have been found in a variety of JH target tissues and in a number of insect species. Tissues in which cytosolic or nuclear receptors have been identified include ovary (Van Mellaert et al, 1985; Koeppe et ah, 1981), epidermis (Klages et aZ., 1980; Wisniewski et al., 1988; Riddiford and Mitsui, 1987; Osir and Riddiford, 1988; Palli et al., 1990; Shemshedini et ah, 1990), fat body (Engelmann et ab, 1987; Roberts and Wyatt, 1983; Palli et ah, 1990; Shemshedini et aZ., 1990), silk gland (Wisniewski and
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Kochman, 1984), and Drosophila tissue culture cells (Chang et ab, 1980, 1985; Wang et ah, 1989; Klages et ab, 1980; Goodman and Chang, 1985). With the exception of one report (see below) the subunit molecular weight of the receptor is low, between 24 and 38 kDa, compared with the much larger receptor proteins belonging to the steroid receptor super family (Evans, 1988). Shemshedini et al. (1990) have identified a single major cytosolic JH-binding protein of molecular weight 85,000 from extracts of Drosophila larval fat body and integument (epidermis, cuticle, and muscle), both JH-responsive tissues. They show further that a strain of Drosophila selected for methoprene resistance (Wilson and Fabian, 1986) contains a JH-binding protein found normally in wild-type flies (Shemshedini et al., 1990), but that in the methoprene-resistant strain the binding protein had a lo-fold lower binding affinity for both methoprene and JHIII (Shemshedini and Wilson, 1990). The function of any JH-binding protein, however, is unknown. Palli et al. (1990) describe two 29-kDa nuclear proteins found in epidermis, one that specifically bound photoaffinity analogues of JH and one that bound to the DNA of two cuticle protein genes. Based on the developmental pattern of appearance of these proteins, they postulated them to be the same. A protein of the same approximate size is absent from nuclei at developmental stages when no high affinity JH-binding sites are detected (Riddiford et al., 1987) and a protein of comparable size is found in JH responsive epidermal cell nuclei (Osir and Riddiford, 1988). The significance of the protein and its binding properties, however, remains obscure. Despite these intriguing patterns of ligand and DNA binding, several mysteries remain. First, putative Manduca receptors that are high affinity JH binders are not competible with methoprene or related analogues (Riddiford et ah, 1987; Osir and Riddiford, 1988). Reciprocally, high affinity methoprene or iodovinylmethoprenol-binding proteins are not competible with natural JH’s (Chang, 1985; Palli et ah, 1990; Goodman and Chang, 1985; Osir and Riddiford, 1988). So it is not clear yet whether there are two or more different binding proteins in target cells or whether there are several isoforms derived from a single gene that differ in ligandbinding properties. It is possible, of course, that methoprene and its binding protein function through an entirely different mechanism. Our results, in contrast to those of Cherbas et al. (1989), show that in order to obtain inhibition methoprene must be present at least 2 hr before ecdysterone is added. This result does not immediately lend itself to the classical model unless 2 hr are needed to load the JH receptor in viva. The short half-life of the inhibition mechanism, in the absence of methoprene, also is enigmatic. We speculate that JH may act by establishing
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some catalytic activity, such as phosphorylation or dephosphorylation, either through the epigenetic activation of a kinase or phosphatase, or through the activation of a separate target gene that encodes an enzyme needed for inhibition of ecdysterone-mediated gene expression. These models are being explored experimentally, first, by examining the effect of protein kinase or phosphatase inhibitors on the activity of methoprene and ecdysterone and, second, by determining whether methoprene inhibition requires a primary transcription-translation event in order to develop. This work was supported by a grant from the National Science Foundation (DCB-8612483), and from the USDA (91-01222). We are grateful to Dr. David Cerf for his gift of methoprene.
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Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240,889-895. Geise, C., Spindler, K. D., and Emmerich, H. (1977). The solubility of insect juvenile hormone in aqueous solutions and its absorption by glassware and plastics. 2. Naturforsch. C 32,158-160. Goodman, W. G., and Chang, E. S. (1985). Juvenile hormone cellular and hemolymph binding proteins. Zn “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 7, pp. 491-510. Pergamon Press, Oxford. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2, 1044-1051. Ireland, R. D., and Berger, E. M. (1982). Synthesis of low molecular weight heat shock polypeptides stimulated by ecdysterone in a cultured Drosophila cell line. Proc. Natl. Acad. Sci. USA 79,855-859. Ireland, R., Berger, E., Sirotkin, K., Yund, M., Osterbur, D., and Fristrom, J. (1982). Ecdysone induces the transcription of four heatshock genes in Drosophila S3 cells and imaginal discs. Dev. Biol. 93, 498-507.
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Bownes, M., and Rembold, H. (1987). The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster. Eur. J. B&hem. 164,709-712. Chang, E. S. (1985). Cellular juvenile hormone binding proteins. Zn “Methods in Enzymology” (J. H. Law and H. C. Rilling, Eds.), Vol. 111, pp. 494-509. Academic Press, San Diego, CA. Chang, E. S., Bruce, M. J., and Prestwich, G. D. (1985). Further characterization of the juvenile hormone-binding protein from the cytosol of a Drosophila melanogaster cell line: Use of a photoaffinity label. Insect
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Chang, E. S., Courdron, T. A., Bruce, M. J., Sage, B. A., O’Connor, J. D., and Law, J. H. (1980). Juvenile hormone binding protein from the cytosol of Drosophila Kc cells. Proc. Natl. Acad. Sci. USA 77, 46574661. Cherbas, L., Koehler, M. M., and Cherbas, P. (1989). Effects of juvenile hormone on the ecdysone response of Drosophila Kc-cells. Dev. Genet.
10,177-188.
Cherbas, L., Lee, K., and Cherbas, P. (1991). Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene. Genes Dew. 5,120-131. Chihara, C. J., and Fristrom, J. W. (1973). Effects and interactions of juvenile hormone and P-ecdysone on Drosophila imaginal discs cultured in vitro. Dev. BioL 35,36-46. Chihara, C. J., Petri, W. H., Fristrom, J. W., and King, J. W. (1972). The assay of ecdysones and juvenile hormones on Drosophila imaginal discs in vitro. .I Insect PhysioL 18, 1115-1123. Damm, K., Thompson, C. C., and Evans, R. M. (1989). Protein encoded by v-erb A functions as a thyroid hormone receptor antagonist. Nature
(London)
339,593-597.
DiNocera, P. P., and Dawid, I. B. (1983). Transient expression of genes introduced into cultured cells of Drosophila. Proc NatL Acad. Sci. USA 80,7095-7098.
Dobens, L., Rudolph, K., and Berger, E. M. (1991). Ecdysterone regulatory elements function as both transcriptional activators and repressors. MoL Cell BioL 11,1846-1853. Engelmann, F., Mala, J., and Tobe, S. S. (1987). Cytosolic and nuclear receptors for juvenile hormone in fat bodies of Leucophaea maderae.
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Klages, G., Emmerich, H., and Peter, M. G. (1980). High affinity binding sites for juvenile hormone I in the larval integument of Drosophila hydei. Nature 286,282-285. Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P., and Hogness, D. (1991). The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59-77. Koeppe, J. K., Kovalick, G. E., and LaPointe, M. C. (1981). Juvenile hormone interactions with ovarian tissue in Leucophaea madera. Zn “Juvenile Hormone Biochemistry” (G. E. Pratt and G. T. Brooks, Eds.), pp. 215-231. Elsevier/North Holland, Amsterdam. Martinez, E., Givel, F., and Wahli, W. (1991). A common ancestor DNA motif for invertebrates and vertebrate hormone response elements. EMBO
J. 10,263-268.
Moore, D. D. (1990). Diversity and unity in the nuclear hormone receptors: A terpenoid receptor superfamily. New Biologist 2,100-105. Osir, E. O., and Riddiford, L. M. (1988). Nuclear binding sites for juvenile hormone and its analogs in the epidermis of the tobacco hornworm. J. BioL Chem. 263, 13,812-13,818. Palli, S., Osir, E., Eng, W., Boehm, M., Edwards, M., Kulcsar, P., Ujvary, I., Hiruma, K., Prestwich, G. D., and Riddiford, L. M. (1990). Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labelling. Proc. NatL Acad. Sci. USA 87, 796800. Palli, S. R., Riddiford, L. M., and Hiruma, K. (1991). Juvenile hormone and “retinoic acid” receptors in Manduca epidermis. Insect Biothem.
21,7-15.
Richards, G. (1978). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila me&waster. VI. Inhibition by juvenile hormones. Dev. BioL 66, 32-42. Riddiford, L. (1978). Ecdysone-induced charges in cellular commitment of the epidermis of the tobacco hornworm, Maduca se&a, at the initiation of metamorphosis. Gen. Comp. Endocrinol. 34, 438446. Riddiford, L. M. (1985). Hormone action at the cellular level. In “Comprehensive Insect Physiology, Biochemistry, and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 8,37-84. Pergamon Press, Oxford. Riddiford, L. M., and Mitsui, T. (1978). Loss of cellular receptors for juvenile hormone during the change in commitment of the epidermis of the tobacco hornworm, Manduca se&a. In “Comparative Endocrinology” (P. J. Gaillard and H. Boer, Eds.), p. 519. Elsevier, Amsterdam. Riddiford, L. M., Osir, E. O., Fittinghoff, C. M., and Green, J. M. (1987). Juvenile hormone analog binding in Manduca epidermis. Insect Birr them.
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Riddihough, G., and Pelham, H. R. B. (1987). An ecdysone response element in the Drosophila hsp 2’7 promoter. EMBO J. f&3729-3734. Roberts, P. E., and Wyatt, G. R. (1983). Juvenile hormone binding by components of fat body cytosol from vitellogenic locusts. Mol. Cell. Endocrinol. 31,53-69. Segraves, W. A., and Richards, G. (1990). Regulatory and developmental aspects of ecdysone-regulated gene expression. Invert. Reprod. Den
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Shemshedini, L., Lanoue, M., and Wilson, T. G. (1990). Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster. J. Biol Chem. 265, 1913-1918. Shemshedini, L., and Wilson, T. G. (1990). Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone binding protein. Proc. Natl. Acad. Sci. USA 87,2072-2076. Sliter, T. J., Sedlak, B. J., Baker, F. C., and Schooley, D. A. (1987). Juvenile hormone in Drosophila melanogaster. Identification and titer determination during development. Insect Biochem. 17, 161165. Van Mellaert, H., Theunis, S., and DeLoof, A. (1985). Juvenile hormone binding proteins in Sarcophaga bullata haemolymph and vitellogenie ovaries. Insect B&hem. 15,655-661. Vitek, M. P., and Berger, E. M. (1984). Transcriptional and post-transcriptional regulation of small heat shock protein synthesis. J. Mol. Biol.
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Walker, V. K., Schreiber, M. L., Purvis, C., George, J., Wyatt, G. R., and Bendena, W. G. (1991). Yolk polypeptide gene expression in cultured Drosophila cells. In Vitro Cell. Dev. Biol. A 27,121-127. Wallrath, L., Burnett, J., and Friedman, T. (1990). Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone repressible gene expressed only in Malphighian tubules. Mol.
Cell Biol.
10, 5114-5127.
Wang, X., Chang, E., and O’Conner, J. D. (1989). Purification of the Drosophila Kc cell juvenile hormone binding protein. Insect Biothem.
19,327-335.
Wilson, T., and Fabian, J. (1986). A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone. Dev. Biol 118, 190-201.
Wisniewski, R. J., and Kochman, M. (1984). Juvenile hormone-binding protein from the silk gland of Galleria mellonella. FEBS I&. 171, 127-130.
Wisniewski, R. J., Wawizenczyk, C., Prestwich, G. D., and Kochman, M. (1988). Juvenile hormone binding proteins from the epidermis of Galleria
mellonella.
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Biochem.
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Wyss, C. (1976). Juvenile hormone analogue counteracts growth stimulation and inhibition by ecdysone in clonal cell lines. Experientia 22, 1272-1274. Yudin, A. I., Clark, W. H., and Chang, E. S. (1982). Ecdysteroid induction of cell surface contacts in a Drosophila cell line. J. Exp. 2001. 219,399-403.
BIOLOGY
151,410-418 (19%)
The Juvenile Hormone Analogue, Methoprene, Inhibits Ecdysterone Induction of Small Heat Shock Protein Gene Expression DECATO
EDWARDM.BERGER,~KARYNGOUDIE,LOUISKLIEGER,MATTBERGER,ANDRODNEY Department
of Biology
and Molecular
Genetics Accepted
Center,
Dartmouth
February
College,
Hanover,
New
Hampshire
03755
24, 19%
The small heat shock protein (hsp) genes of Drosophila are expressed in cultured cells in response to the moulting hormone, ecdysterone. We show here that juvenile hormone (JHIII) and the juvenile hormone analogue, methoprene, inhibit that induction in a dose-dependent manner. Heat shock induction is not inhibited. In transient expression studies using S3 line cells transfected with EcRE-CAT constructs, methoprene inhibition was found to require a 2-hr pretreatment (before ecdysterone addition), and methoprene’s continued presence was essential. Farnesol, farnesyl acetate, and retinoic acid did not cause inhibition. Several models of methoprene inhibition are discussed. o 1992Academic press, I~C. INTRODUCTION
The development of holometabolous insects is under the control of two hormones; ecdysterone, a steroid, and juvenile hormone (JH), a sesquiterpene. While high ecdysterone titers are required to initiate each moult, the type of moult completed is largely determined by the JH level. During larval-to-larval moults relatively high JH titers are required. The commitment to pupal development in Lepidoptera appears to take place when the JH titer has fallen below detectable levels in final instar larvae as the ecdysterone levels rise (Riddiford, 1978). Later, during the prepupal period JH levels again rise (Riddiford, 1978) apparently to prevent adult differentiation of imaginal disc structures during pupal development. Over the past 10 years a great deal has been learned about the molecular biology of ecdysterone action (see Ashburner, 1990; Segraves and Richards, 1990; Riddihough and Pelham, 198’7; Martinez et ah, 1991). As in the case of many other steroid hormone-regulated systems, target gene expression is controlled by the presence or absence of a hormone-receptor complex bound to a palindromic DNA sequence situated near the transcription unit. Recent studies in Drosophila have identified a short, 15-bp imperfect palindrome located some 500 bp upstream of the heat shock protein 27 (hsp 27) gene that is sufficient to confer ecdysterone-inducible expression on a contiguous reporter gene (Cherbas et al., 1991). A version of this ecdysterone response element (EcRE) is found adjacent to other ecdysterone-inducible Drosophila genes including hsp 22, hsp 23, and EIP 28/29 (Do1 To whom correspondence should be addressed.
0012-1606/92$5.00 Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
bens et al., 1991; Cherbas et al, 1991). There is weak evidence that a different recognition sequence is involved in the case of ecdysterone-repressible genes (Wallrath et ah, 1990). Based on both biochemical and genetic evidence, the ecdysterone receptor appears to be localized primarily in the nucleus most likely associated with EcRE sequences (Cherbas et ab, 1991; Dobens et al, 1991; Koelle et al, 1991). Ecdysterone presumably diffuses into the cell and reaches the nucleus passively where it binds to a receptor and initiates transcription. This mechanism of action resembles that found with mammalian thyroid hormone (Damm et al., 1989). Here also unliganded receptor is constitutively bound to the target gene’s response element where it acts to repress basal expression. Binding of hormone converts the receptor into a transcriptional activator. With the recent isolation and characterization of the ecdysterone receptor, and its gene (Koelle et ab, 1991), a more detailed description of transcriptional activation and repression will develop. In contrast, the molecular biology of JH action is largely unknown. JH is believed to somehow antagonize or inhibit ecdysterone action by preventing the expression of adult specific genes, while still allowing or enabling the expression of those ecdysterone-responsive genes required for the execution of larval moults (Riddiford, 1985). In several insects, including Drosophila and the tobacco hornworm, Manduca, developmental variations in JH levels have been reported (Sliter et ab, 1987; Bownes and Rembold, 1987). JH-binding proteins have been described that display the specificity, saturability, and high hormone-binding affinity expected of a bona fide receptor (Klages et al., 1980; Chang, 1985; Chang et al, 1985; Engelmann et ah, 1987; Goodman and Chang, 410
BERGERETAL.
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Juvenile Hormone Action
1985; Osir and Riddiford, 1988; Moore, 1990; Palli et ab, 1990; Shemshedini et al, 1990). These proteins, however, are generally smaller than steroid hormone receptors (Evans, 1988) and there is only circumstantial evidence that JH receptors bind to DNA in a sequence-specific way (Palli et al, 1990). In a recent report Shemshedini and Wilson (1990) have identified an 85-kDa juvenile hormone-binding protein from extracts of Drosophila fat body, In a Drosophila mutant strain, MET (methoprene tolerant), the level of juvenile hormone binding is greatly reduced. However, the mutant strain shows no discernable mutant phenotype in terms of development. We have begun to study the molecular biology of JH action using a well-defined ecdysterone-inducible system, the Drosophila hsp27 gene. This and the three other ecdysterone-inducible small hsp genes (Ireland and Berger, 1982) are a tightly linked multigene family located at cytogenetic band 67B on the polytene map. In this report we show, first, that the JH analogue, methoprene, inhibits the ecdysterone-induced expression of endogenous small hsp genes, but has no effect on small hsp gene regulation by heat shock. Second, we show that methoprene inhibition can occur in a transient expression assay in which EcRE sequences are regulating hormone-dependent reporter gene expression. EXPERIMENTAL PROCEDURES
Cell culture and transfection. Drosophila line S3 cells were cultured in Schneider’s medium (GIBCO) containing 10% fetal bovine serum at 25°C. For transfection, 20 pg of CsCl-purified plasmid DNA was added as a CaCl, coprecipitate (DiNocera and Dawid, 1983) to each 25cm2 flask of confluent cells in 4 ml of medium. Except where indicated ecdysterone was added to 1 palm final concentration 24 hr after DNA addition, and the flasks were cultured for an additional 24 hr at 25°C before cell harvesting. Hormones. Ecdysterone was purchased from Calbiothem and stored frozen in an aqueous solution at 1 mM. Methoprene was a gift from Dr. D. Cerf (Sandoz, Palo Alto). JHIII was purchased from Sigma Biochemical, as was retinoic acid. Farnesol and farnesyl acetate were purchased from Aldrich Chemical Co. Methoprene, JHIII, farnesol, and farnesyl acetate were stored at -20°C in glass vials in 95% ethanol and diluted into culture medium directly. Stock dilutions were made in 95% ethanol using plastic pipet tips. CAT assay. Following treatment the cells were harvested by centrifugation. After a wash in Drosophila Ringer’s, a cell pellet was recovered and resuspended in 250 ~1 of 0.25 M Tris (pH 8.0). Cell lysates, prepared by brief sonication, were clarified by a 5-min spin at 5°C in a microcentrifuge. Aliquots of the clear supernatant
were then assayed for CAT activity (Gorman et a& 1982), and the reaction products were visualized by ascending thin-layer chromatography and autoradiography. Quantitation of CAT activity was done by excising spots containing [14C]chloramphenicol and its acetylated products and conducting scintillation counting. The percentage of chloramphenicol acetylation was normalized for protein content in the lysate and is expressed as CAT-specific activity. Plasmids. For the purpose of constructing plasmids containing multiple copies of the EcRE, plasmid pUC18Ec, a gift from Guy Riddihough, was digested with SmaI, and a BgZII linker inserted. A 47-bp BamHIBgZII fragment containing the EcRE was purified from low-melting-point agarose and ligated in the presence of BamHI and BgZII enzyme at room temperature for 1 hr. Polymers with a head-to-tail arrangement of the BamHI-BgZII fragment were separated on and purified from low-melting-point agarose (FMC), and directionally cloned into the plasmid designated ptkAT0. This CAT-reporter plasmid contains the tk TATA box and start site of transcription on a 150-kp SalI-BgZII fragment from ptkS0 directionally cloned into the multicloning site of pBLCAT3. Full details of the construction of EcRE-2R-CAT, hsp 27-CAT, and hsp 70-CAT (DiNocera and Dawid, 1983) are provided in Dobens et al. (1991). Protein analysis. Cells incubated in the absence or presence of hormones were labeled for 4 hr with [35S]methionine at 100 &i/ml (New England Nuclear) in Schneider’s medium lacking methionine, yeast hydrolysate, and serum. Cells were maintained in suspension during labeling by shaking. Following the label period, the cells were pelleted, washed in cold saline, and prepared for one-dimensional gel electrophoresis in 15% acrylamide, 1% SDS gels, or for two-dimensional (isoelectric focusing/SDS) gel electrophoresis, and autoradiography according to published procedures (Ireland and Berger, 1982; Ireland et al, 1982). RESULTS 1. Endogenous
Small Heat Shock Protein
(s-hsp) Gene
Expression We first examined the level of small hsp gene expression in Drosophila S3 line cells following ecdysterone treatment, in either the presence or absence of the JH analogue, methoprene. Methoprene is a potent juvenoid in dipterans (see Cherbas et al., 1989) and is particularly useful in these studies because of its relatively high water solubility. We initially used Richards’ (1978) protocol, which involves a pretreatment with 10m5M methoprene 6 hr prior to ecdysterone addition. Twenty-four hours after ecdysterone addition cells were allowed to
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incorporate [35S]methionine for an hour and then protein was extracted. As seen in Fig. 1, and confirming several previous studies, the presence of ecdysterone alone led to the greatly enhanced synthesis of two low molecular weight polypeptides known to be hsp 23 and hsp 22 (Ireland and Berger, 1982). The slightly larger and more basic hsp 26 and hsp 27 are not resolved in this gel system but have been shown to be induced also (Ireland et al., 1982). In cells first pretreated with 10e5 M methoprene there was no evidence of enhanced hsp 22 or hsp 23 synthesis in five replicate experiments. Minor variations in the abundance of other polypeptides seen in Fig. 1 were observed between treatments in only some experiments but were not pursued further. To examine the specificity of the methoprene effect we looked at the expression of small hsp genes in response to heat shock using cells that were pretreated with methoprene. From the data presented in Fig. 2 we conclude that JH has no important effect on diminishing the level of small hsp synthesis in response to high temperature, so that methoprene inhibition of ecdysterone induced small hsp gene expression appears specific. 2. CAT-Reporter
Constructs
In an effort to extend the analysis of methoprene inhibition, several recombinant plasmid constructs made previously (Dobens et al., 1991) were analyzed functionally using a transient expression assay. The hsp 27-CAT construct contains the bacterial CAT gene ligated downstream of a 1.2-kb DNA fragment recovered from the hsp 27 promoter region. The fragment extends from position -1100 to +87, where +l refers to the transcription initiation site. The hsp 27 EcRE is located between position -546 to -532. When transfected into responsive S3 line cells, the hsp 27-CAT plasmid promoted a low level ecdysterone inducible CAT activity in the absence of methoprene. In the presence of methoprene, the ecdysterone-induced CAT activity was essentially abolished (Fig. 3). The conclusion, then, is that a plasmid containing the hsp 27 upstream region of DNA tested contains sequences required for both ecdysterone-inducible expression and for the methoprene-dependent inhibition of that induction. Methoprene inhibition could involve the association of a methoprene receptor complex to either the EcRE itself, perhaps in combination with the ecdysterone-receptor complex, or to a nonoverlapping binding site elseFIG. 1. Two-dimensional gel electrophoretic pattern of [%]methionine-labeled S3 cells protein showing methoprene inhibition of ecdysterone-dependent hsp 22 and hsp 23 synthesis. Cells were treated with (a) buffer alone, (b) 10e6 M ecdysterone for 24 hr, or (c) 6-hr pretreatment in 1O-5 M methoprene, and then with methoprene and
1O-6 M ecdysterone together for 24 hr. Approximately lo5 cpm of labeled protein extract was used for each gel, and autoradiography was for 48 hr. Arrows in (b) indicate hsp 23 and hsp 22.
BERGER
ET AL.
Juvenile
Hormone
Actiml
413
the ecdysterone-receptor complex to its target EcRE sequence, or that it prevents some step in the transcriptional activation process that follows ecdysterone-receptor binding. 3.
FIG. 2. Sodium dodecyl sulfate, 15% polyacrylamide gel electrophoretie analysis of [35S]methionine-labeled polypeptides extracted from S3 cells: untreated control (a), heat shock for 30 min at 37°C followed by a 45-min labeling period using cells pretreated (b), or not (c), with lo- M methoprene for 24 hr before heat shock. Top arrow points to the hsp 26 and hsp 2’7 pair, and lower arrow points to the hsp 22 and hsp 23 pair.
where in the upstream region. As a test we utilized a construct designated EcRE-2R-CAT that had been previously shown to display a high level of ecdysterone-dependent CAT activity in a transient expression assay (Dobens et al., 1991). The EcRE-2R-CAT construct contains a CAT gene just downstream of a herpes virus thymidine kinase minimal promoter. In the adjacent multicloning site we had inserted a tandem pair of 23 bp synthetic hsp 27 EcRE sequences in the orientation found in the endogenous gene (Dobens et al, 1991). Following transfection with the plasmid, S3 cells were treated with lO-‘j M ecdysterone in either the presence or absence of 10e5 M methoprene which had been added 6 hr earlier. The results, summarized in Fig. 3, clearly show that methoprene nearly abolishes the high level of CAT activity induced by ecdysterone in transfected cells. Therefore, we surmise that methoprene, perhaps in association with a receptor, somehow leads to the loss of ecdysterone receptor, interferes with the binding of
Specificity
In order to examine further the nature of methoprene inhibition, several experiments focusing on specificity were carried out. First, we looked at the effect of methoprene pretreatment on the induction of CAT activity in a transient expression assay using an hsp 70-CAT reporter construct and high temperature as the stimulus. The hsp 70-CAT plasmid used in this study has been described in detail elsewhere (DiNocera and Dawid, 1983). Following transfection cells were pretreated with or without low5 M methoprene for 24 hr, and then exposed to 37°C for 30 min. After a recovery period of 1.5 hr at 25°C cell lysates were assayed for CAT activity. As shown in Fig. 4, the high level of CAT activity measured in lysates of heat-shocked cells is little if at all affected by the methoprene pretreatment, which alone had no effect on inducing CAT activity. Comparable results were observed using shorter periods of heat shock, in which lower levels of CAT activity are induced (data not shown). This result is in agreement with previous experiments in which endogenous small hsp induction was examined (Fig. 2). In a second experiment we examined the effect of other reagents on the inhibition of CAT induction by ecdysterone, using the EcRE-2R-CAT construct. The compounds tested include JHIII, farnesol, farnesyl acetate, and retinoic acid, each added to a final concentration of 10m5M to the transfected cells 6 hr before a 24-hr ecdysterone treatment. The results, summarized in Fig. 5A, clearly show that the inhibition of CAT induction was extensive with JHIII but failed to occur in response to any of the other pretreatments. The addition of farnesol, farnesyl acetate, retinoic acid, JHIII, or
FIG. 3. CAT assays. Levels of CAT activity were assayed by using extracts of Droso@ila S3 cells that had been transfected with plasmids hsp 27CAT (a, c, d, g, h), or EcRE-2R-CAT (b, e, f, i, j) and then treated with buffer alone (a, b), with 10m6Mecdysterone alone (c-f), or with a 6-hr pretreatment with 1O-5 M methoprene and then ecdysterone (g-j).
414
DEVELOPMENTALBIOLOGY V0~~~~151,1992
methoprene alone had no effect on inducing CAT activity (not shown). Finally, we examined the dose-response profile of methoprene inhibition using the standard protocol, one which involves a 6-hr methoprene pretreatment followed by the addition of ecdysterone. These results, also summarized in Figs. 5A and 5B, indicate that inhibition occurs over a range of methoprene concentrations higher than low8 M and with half maximum inhibition at about 10m7M.
A
a
b
c
d
efghi
i
k~lmn
o
~cf
B .07 r
4. Kinetics of Inhibition In order to evaluate the need for a methoprene pretreatment, in terms of inhibition, an experiment was done in which either the time of addition and or duration of methoprene exposure was varied. In all cases lop6 M ecdysterone was administered at experimental time zero. The results, summarized in Fig. 6, illustrate two important features of methoprene inhibition. First, there is a 2-hr period of pretreatment that is essential for the complete inhibition by methoprene to be established. Pretreatments started later than -2 hr lead to progressively lower levels of inhibition, until by +4 hr
.06 I
I
,. I\ Methoprene
Concentration
(M)
FIG. 5. (A) CAT assays to investigate the dose-response profile of methoprene inhibition and the specificity of inhibition. S3 cells were transfected with 15 pg of the plasmid EcRE-2R-CAT, then subjected to hormone treatment, and finally assayed for CAT activity. Treatments include (a) no treatment control, (b, c) 10m6Mecdysterone for 24 hr, (d) ecdysterone with 10e4 M methoprene, (e) ecdysterone with 10m5 M methoprene, (f) ecdysterone with 10m6M methoprene, (g) ecdysterone with 10e7 M methoprene, (h) ecdysterone with lo-’ M methoprene, (i) ecdysterone with 10m9Mmethoprene, (j, k) ecdysterone with 10m5M farnesyl acetate pretreatment, (1, m) ecdysterone with 10e5 M retinoic acid pretreatment, (n, o) ecdysterone with 10d5 M farnesol pretreatment, and (p, q) ecdysterone with 10d5 M JHIII pretreatment. Pretreatments began 6 hr before ecdysterone addition and continued until cell harvesting. (B) Results are plotted graphically. Values indicate the average and range of CAT specific activity values from three replicate experiments. The 10m9M methoprene point seen in A was only determined once and was not included in the summary figure.
FIG. 4. CAT assay. Levels of CAT activity were assayed by using extracts of Drosophila S3 cells that had been transfected with the plasmid hsp ‘IO-CAT and then maintained at 25°C (a, b), or heat shocked at 37°C for 30 min (c-f). In b, e, and f, cells were treated with 10e5 M methoprene for 24 hr before treatment at 25°C (b) or 37°C (e, 0
methoprene treatments become essentially ineffective. It is not clear what process or processes occurs during the pretreatment to establish subsequent inhibition. The second and rather surprising result was that a 4- or a 6-hr pretreatment with methoprene was ineffective if methoprene was removed from the cell culture medium before adding ecdysterone. This implies that while the mechanism of methoprene inhibition requires 2 hr to become established, inhibition also requires the continuous presence of methoprene during the period of ecdysterone treatment.
BERGERETAL.
Juvenile
B
Ok
11-:/J;, -6
-4
, ,
-2
0
Time of Addition
(hours)
+2
+4
+6
Methowene
FIG. 6. Effect of the time of methoprene pretreatment and pretreatment duration on the level of CAT enzyme activity induced by a 24-hr treatment with lObe’ M ecdysterone. Results in (A) are CAT assays, plotted in (B) as CAT specific activity. The time of ecdysterone addition is indicated as time 0. Lanes in A include CAT assay in which methoprene was added at the following times: (a) -6 hr, (b, c) -4 hr, (d) -2 hr, (e) -1 hr, (f, g) 0 hr, (h, i) +2 hr, (j) +4 hr, (k, 1) +6 hr, (m, n) between -6 and -2 hr only, (0, p) between -6 and 0 hr only. The horizontal bars in B correspond to CAT specific activities averaged from lanes m and n (0) and lanes o and p (0).
DISCUSSION
In three previous studies (Wyss, 1976; Cherbas et al., 1989; Yudin et aZ., 1982) both natural juvenile hormones (JH, JHII, JHIII) and the synthetic analogue, methoprene, were shown to partially inhibit several of the ecdysterone responses exhibited by Drosophila Kc line cells. Levels of inhibition ranging from 50 to 80% or higher were noted for ecdysterone-dependent cell elongation, cell aggregation, mitotic arrest, and acetylcholinesterase induction. The single exception was the failure of JHIII, or methoprene, to inhibit ecdysterone-dependent stimulation of EIP 28/29 synthesis. Cherbas et al. (1989) also reported that JHIII and methoprene incompletely inhibited ecdysterone-dependent cell elongation in an independently derived cell line, Schneider S3 cells, the line used in the work described here. We now report that methoprene and JHIII also inhibit ecdysterone-mediated induction of small heat shock protein gene expression. Inhibition was observed
Hormone
Action
415
both for hsp 22 and hsp 23 synthesis, analyzed by [35S]methionine incorporation. Experiments designed to measure rates of hsp gene RNA synthesis and accumulation are underway. The ability to carry out DNA-mediated gene transfer with S3 cells allowed us to conduct transient expression studies using chimeric promoter-reporter gene constructs. We found that the ecdysterone-dependent induction of CAT gene expression was almost entirely inhibited by both methoprene and JHIII, when reporter gene expression was under the control of either a natural hsp 2’7 promoter region or a tandem pair of synthetic ecdysterone response elements (EcREs). In contrast, the heat-dependent induction of a CAT gene placed under the control of an hsp 70 heat shock promoter was not affected by a 24-hr methoprene pretreatment. Because transcription of CAT mRNA in the EcRE-2RCAT construct initiates from a herpes virus thymidine kinase promoter, there are no Drosophila sequences at all present in the final message. Thus, it is unlikely that methoprene is mediating its inhibition through a mechanism of selective mRNA turnover, unless, of course, the tk-CAT mRNA coincidentally contains the same unusual structure or sequence that confers methoprenedependent message instability on natural small hsp mRNAs. We suspect, rather, that methoprene inhibition is based on events occurring at the level of transcript initiation or elongation. For a number of reasons, juvenile hormone is thought to function through a specific receptor in much the way that thyroid hormone, retinoic acid, and the steroid hormones work (e.g., see Moore, 1990). As a consequence we anticipated that JH, in conjunction with its receptor, would bind to a specific recognition DNA sequence on or near its target gene and, as a consequence of binding, prevent ecdysterone from engaging its program of transcriptional activation. Therefore, we were somewhat surprised to find that methoprene inhibition of ecdysterone induction occurred using even the EcRE-2RCAT promoter-reporter construct. We have not yet tested other independently constructed EcRE-reporter gene constructs, so it is still possible that the synthetic 23-bp EcRE contains a JH-receptor binding site within, overlapping, or outside of the 15bp core EcRE sequence defined by Cherbas et al. (1991), or that cryptic vector or reporter gene sequences are functioning as fortuitous JH-receptor binding sites. We think that these possibilities are unlikely. The working model is that methoprene, probably bound to a receptor, somehow blocks or modifies the binding of the ecdysterone-receptor complex to an EcRE, or that the JH-receptor complex interferes with some subsequent step in the progress of transcriptional activation. Two important questions remain. First, what does the
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evidence allow us to deduce about the mechanism of methoprene inhibition? Second, is the phenomenon of methoprene inhibition in S3 cells interesting in the physiological sense? In terms of physiology, several points can be made in support of the significance of our results. First, methoprene (isopropyl[2E,4E] 11-methoxy-3,7,11trimethyl-2,4-dodecadienoate), also known as ZR 515, ZR 2008, and Altosid, has been used extensively for some time as a bona fide JH known to be particularly active in Diptera (Cherbas et al., 1989). Compared with the Cherbas’ results (1989) the methoprene concentrations required for inhibition in our study, 10-5-10-‘M, are high. However, juvenile hormone or JH analogue concentrations in the range of 10-5-10-7 M have been used previously in work with Drosophila cells in culture (Walker et al., 1991; Yudin et ah, 1982), with salivary glands (Richards, 1978), and with imaginal discs (Chihara et al., 1972). Because of methoprene’s low aqueous solubility, and its lipophilic tendency to stick to plastic and glass, it is not unlikely that the stated concentrations are overestimates of the actual hormone concentration in the medium (Giese et al., 1977; however, see Cherbas et al., 1989). Nevertheless, the inhibition of ecdysterone-mediated CAT induction has been shown to be a property of biologically active juvenoids, methoprene and JHIII, and did not occur using structurally related but biologically inactive compounds, such as farnesol, farnesyl acetate, and retinoic acid. The credibility of our interpretation also relies on the absence of toxic effects. As Cherbas et al. (1989) point out, 1O-5 M methoprene partially reverses the ecdysterone-induced onset of mitotic arrest, although they do note a slight inhibition of cell proliferation compared with untreated control cells. Methoprene-treated cells continue to show the normal pattern of protein synthesis and we noticed no structural abnormalities in the methoprene-treated cells. Based on these observations and on the results of trypan blue exclusion studies (data not shown), methoprene does not noticeably affect cell viability at the concentrations used. There is very little known about the mechanism of JH or methoprene action at the molecular level. Several reports have appeared demonstrating the presence of high affinity, saturable, and ligand-specific binding proteins. These proteins that we refer to generally as “receptors” have been found in a variety of JH target tissues and in a number of insect species. Tissues in which cytosolic or nuclear receptors have been identified include ovary (Van Mellaert et al, 1985; Koeppe et ah, 1981), epidermis (Klages et aZ., 1980; Wisniewski et al., 1988; Riddiford and Mitsui, 1987; Osir and Riddiford, 1988; Palli et al., 1990; Shemshedini et ah, 1990), fat body (Engelmann et ab, 1987; Roberts and Wyatt, 1983; Palli et ah, 1990; Shemshedini et aZ., 1990), silk gland (Wisniewski and
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Kochman, 1984), and Drosophila tissue culture cells (Chang et ab, 1980, 1985; Wang et ah, 1989; Klages et ab, 1980; Goodman and Chang, 1985). With the exception of one report (see below) the subunit molecular weight of the receptor is low, between 24 and 38 kDa, compared with the much larger receptor proteins belonging to the steroid receptor super family (Evans, 1988). Shemshedini et al. (1990) have identified a single major cytosolic JH-binding protein of molecular weight 85,000 from extracts of Drosophila larval fat body and integument (epidermis, cuticle, and muscle), both JH-responsive tissues. They show further that a strain of Drosophila selected for methoprene resistance (Wilson and Fabian, 1986) contains a JH-binding protein found normally in wild-type flies (Shemshedini et al., 1990), but that in the methoprene-resistant strain the binding protein had a lo-fold lower binding affinity for both methoprene and JHIII (Shemshedini and Wilson, 1990). The function of any JH-binding protein, however, is unknown. Palli et al. (1990) describe two 29-kDa nuclear proteins found in epidermis, one that specifically bound photoaffinity analogues of JH and one that bound to the DNA of two cuticle protein genes. Based on the developmental pattern of appearance of these proteins, they postulated them to be the same. A protein of the same approximate size is absent from nuclei at developmental stages when no high affinity JH-binding sites are detected (Riddiford et al., 1987) and a protein of comparable size is found in JH responsive epidermal cell nuclei (Osir and Riddiford, 1988). The significance of the protein and its binding properties, however, remains obscure. Despite these intriguing patterns of ligand and DNA binding, several mysteries remain. First, putative Manduca receptors that are high affinity JH binders are not competible with methoprene or related analogues (Riddiford et ah, 1987; Osir and Riddiford, 1988). Reciprocally, high affinity methoprene or iodovinylmethoprenol-binding proteins are not competible with natural JH’s (Chang, 1985; Palli et ah, 1990; Goodman and Chang, 1985; Osir and Riddiford, 1988). So it is not clear yet whether there are two or more different binding proteins in target cells or whether there are several isoforms derived from a single gene that differ in ligandbinding properties. It is possible, of course, that methoprene and its binding protein function through an entirely different mechanism. Our results, in contrast to those of Cherbas et al. (1989), show that in order to obtain inhibition methoprene must be present at least 2 hr before ecdysterone is added. This result does not immediately lend itself to the classical model unless 2 hr are needed to load the JH receptor in viva. The short half-life of the inhibition mechanism, in the absence of methoprene, also is enigmatic. We speculate that JH may act by establishing
BERGER ET AL.
Juvenile
some catalytic activity, such as phosphorylation or dephosphorylation, either through the epigenetic activation of a kinase or phosphatase, or through the activation of a separate target gene that encodes an enzyme needed for inhibition of ecdysterone-mediated gene expression. These models are being explored experimentally, first, by examining the effect of protein kinase or phosphatase inhibitors on the activity of methoprene and ecdysterone and, second, by determining whether methoprene inhibition requires a primary transcription-translation event in order to develop. This work was supported by a grant from the National Science Foundation (DCB-8612483), and from the USDA (91-01222). We are grateful to Dr. David Cerf for his gift of methoprene.
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Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240,889-895. Geise, C., Spindler, K. D., and Emmerich, H. (1977). The solubility of insect juvenile hormone in aqueous solutions and its absorption by glassware and plastics. 2. Naturforsch. C 32,158-160. Goodman, W. G., and Chang, E. S. (1985). Juvenile hormone cellular and hemolymph binding proteins. Zn “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 7, pp. 491-510. Pergamon Press, Oxford. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2, 1044-1051. Ireland, R. D., and Berger, E. M. (1982). Synthesis of low molecular weight heat shock polypeptides stimulated by ecdysterone in a cultured Drosophila cell line. Proc. Natl. Acad. Sci. USA 79,855-859. Ireland, R., Berger, E., Sirotkin, K., Yund, M., Osterbur, D., and Fristrom, J. (1982). Ecdysone induces the transcription of four heatshock genes in Drosophila S3 cells and imaginal discs. Dev. Biol. 93, 498-507.
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