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Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone
Molecular and Cellular Endocrinology 184 (2001) 1 – 11 www.elsevier.com/locate/mce
Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone Robert Rybczynski *, Stephanie C. Bell, Lawrence I. Gilbert Department of Biology, Coker Hall CB c 3280, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599 -3280, USA Received 30 May 2001; accepted 5 September 2001
Abstract Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70S6k). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Steroid hormone (insect); Extracellular signal-regulated kinase (ERK); Prothoracicotropic hormone (PTTH); Phosphorylation; Signal transduction; Ecdysteroidogenesis
1. Introduction Episodic increases in circulating levels of the steroid hormone 20-hydroxyecdysone (20E) result in significant changes in gene activity that are necessary for insect post-embryonic development (see Henrich et al., 1999). In many insects, surges in the titer of 20E result from the conversion of the prohormone 3-dehydroecdysone, synthesized in the prothoracic gland, to ecdysone via a hemolymph reductase (Warren et al., 1988; Kiriishi et al., 1990), and subsequent metabolism of ecdysone to 20E by intracellular monooxygenases found in a variety of tissues (see Grieneisen, 1994). The production of 3-dehydroecdysone is positively and acutely regulated by the homodimeric neuropeptide hormone prothoracicotropic hormone (PTTH) (see Gilbert et al., 1996). Current evidence suggests that PTTH interacts with a G-protein-coupled receptor, resulting in Ca2 + influx, * Corresponding author. Tel.: + 1-919-966-5535; fax: +1-919-9621344. E-mail address: [email protected] (R. Rybczynski).
activation of a Ca2 + -calmodulin-sensitive adenylyl cyclase, cAMP generation and subsequent protein kinase A activity (see Gilbert et al., 1996). PTTH further stimulates activation of the p70S6 kinase and phosphorylation of ribosomal protein S6, perhaps via protein kinase A. PTTH-stimulated ecdysteroid synthesis requires protein synthesis (Keightley et al., 1990; Rybczynski and Gilbert, 1995) that itself is dependent upon S6 phosphorylation (Song and Gilbert, 1995). The PTTH-prothoracic gland axis has close parallels with the vertebrate adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) steroidogenic pathways. In all these systems, the protein hormone that elicits increased steroid hormone synthesis does so via a transductory cascade that generates cAMP, and rapidly increases protein synthesis (see Gilbert et al., 1996; Cooke, 2000). However, a number of observations suggest that both the vertebrate and invertebrate transductory cascades are not linear, but instead are complex and involve additional messengers and proteins not typically considered to be involved in G-protein-coupled receptor signaling that is linked to cAMP generation.
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The mitogen-activated protein kinases (MAPK) comprise a family of signaling molecules that play important roles in regulating many cellular events. In vertebrates, four parallel MAPK pathways have been discovered, all of which involve a three-level kinase module (see Garrington and Johnson, 1999; Murga et al., 1999). The archetypal MAPK path flows from an external signaling molecule such as a growth factor or hormone, to receptor tyrosine kinases to Ras (a small GTP-binding protein) to Raf-1 (MAP kinase kinase kinase) to MEK (MAP kinase kinase) to one or two extracellular signal-regulated kinases (ERK1/2). Recent work has shown that G-protein coupled receptors can also activate the ERK pathway through G-protein- or cAMP-dependent processes (see Lewis et al., 1998). In a number of adrenal and ovarian model systems, steroidogenic peptide hormones activate the ERK pathway resulting in ERK-dependent effects on cellular proliferation and differentiation (Chabre et al., 1995; Balla et al., 1998; Lotfi et al., 1997; Das et al., 1996). However, the role of ERKs has not been analyzed thoroughly in regard to a possible role in modulating insect steroid hormone synthesis. The present study examines the possibility that MAPKs, specifically those of the ERK family, are activated in the prothoracic gland by PTTH, and, if so, whether or not they are involved in the regulation of ecdysteroid synthesis. This problem is of intrinsic value, given the importance of ecdysteroid hormones in insect development. Furthermore, since prothoracic gland cells do not undergo mitosis following embryonic development, they may provide a model for understanding the role of ERKs in rarely dividing or post-mitotic endocrine cells, i.e. those in the vertebrate central nervous system.
Jolla, CA). Grace’s medium was from GibcoBRL (Grand Island, NY). Pure recombinant Manduca sexta PTTH, expressed in E. coli, was a gift of Dr H. Kataoka (University of Tokyo, Japan) (Gilbert et al., 2000). Partially purified PTTH was prepared from brains of day 1 pupae as described previously (Rybczynski and Gilbert, 1995).
2.2. Organ incubations Manduca sexta larvae were group-reared and developmentally staged as described previously (Rybczynski and Gilbert, 1994). Prothoracic glands from day 3 fifth instar larvae were extirpated under insect saline and transferred to individual wells (one gland/well) of a spot test plate containing 50 ml Grace’s medium (Rybczynski and Gilbert, 1994). Two prothoracic glands are found in each animal and one was used as a matched control for each gland receiving an experimental treatment. After completion of a dissection series (5–10 pairs of prothoracic glands), the medium was replaced rapidly with fresh medium9 inhibitors, and the preincubation period was initiated. At the end of the preincubation period, the medium was replaced rapidly with 25 ml of fresh Grace’s medium 9experimental materials (PTTH, inhibitors). Pure recombinant PTTH was used at a standard dose of 0.25 ng per gland and partially purified PTTH was used at 0.25 brain equivalents per gland; these two preparations have identical effects on prothoracic gland signal transduction and ecdysteroidogenesis (Gilbert et al., 2000). At the end of the incubation period, 20 ml medium from each well were removed, diluted into phosphatebuffered saline and frozen at − 20 °C until analyzed for ecdysteroids by RIA. Prothoracic glands were flash frozen and stored at − 80 °C until analysis by SDSPAGE and immunoblotting.
2. Materials and methods
2.3. Immunoblots 2.1. Materials Monoclonal antibodies against dually phosphorylated ERK mammalian and Drosophila melanogaster sequences were obtained from Cell Signaling Technology (Beverly, MA), and Sigma (St. Louis, MO), respectively. Phosphorylation state-independent ERK polyclonal antibodies were obtained from Stressgen (Victoria, BC, Canada) and a blocking peptide containing a dually phosphorylated ERK1/2 sequence was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-linked goat anti-rabbit and goat anti-mouse antibodies were acquired from Jackson ImmunoResearch Laboratories (West Grove, PA). The ERK inhibitor apigenin was purchased from Sigma while the MAP kinase kinase (MEK) inhibitors PD 98059 and UO 126 were obtained from Calbiochem (La
Prothoracic glands were boiled in SDS sample buffer for 10 min followed by centrifugation at 15 800×g for 3 min. Aliquots of the supernatants were loaded onto 10% SDS gels. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, blocked for 1 h in phosphate-buffered saline with 2% bovine serum albumen, 5% non-fat powdered dry milk and 0.1% Tween 20. Blots were incubated overnight at 4 °C with a primary antibody (1:3000) in phosphatebuffered saline with 2% bovine serum albumen, 2% non-fat powdered dry milk and 0.1% Tween 20. Blots were then washed three times in phosphate-buffered saline with 0.1% Tween 20 for 10 min each and then incubated with an appropriate horseradish peroxidaselinked second antibody in phosphate-buffered saline with 2% bovine serum albumen, 2% non-fat powdered
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dry milk and 0.1% Tween 20. Following three additional washes, immunoreactivity was visualized using chemiluminescence. The resultant films were scanned and quantified using a Molecular Dynamics Computing Densitometer (Sunnyvale, CA) and the IMAGEQUANT program.
2.4. ERK acti6ity assay ERK activity was determined in single gland lysates using reagents from Cell Signaling Technology, with slight modifications of the manufacturer’s protocol. Briefly, following incubation of prothoracic glands in vitro, as described above, single gland lysates were made by sonicating glands (4×5 s) in 200 ml cell lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 1 mM PMSF]. Lysates were centrifuged at 15 800×g at 4 °C for 5 min and 10 ml of a suspension of agarose bead-linked anti-phosphorylated-ERK 1/2 monoclonal antibodies was added to the supernatant in a fresh tube. This suspension was incubated overnight at 4 °C with gentle shaking, followed by centrifugation for 30 s. The pellet was washed twice with 500 ml cell lysis buffer (4 °C) and twice with 500 ml kinase buffer [25 mM Tris (pH 7.5), 5 mM b-glycerophosphate, 0.1 mM Na3VO4, and 10 mM MgCl2] at 4 °C. The pellet was then resuspended in 50 ml kinase buffer containing 200 mM ATP and 1.5 mg of an ELK-1 transcription factor fusion protein to serve as an ERK substrate. After incubation for 30 min at 30 °C, the reaction was terminated by the addition of 25 ml SDS sample buffer, followed by centrifugation for 3 min. Reactions were analyzed by SDS-PAGE (25 ml/sample) and immunoblotting, using chemiluminescent detection of an anti-phospho ELK antibody. ELK phosphorylation was quantified using scanning densitometry and the IMAGEQUANT program.
2.5. Protein dephosphorylation Prothoracic gland lysates were treated with lambda protein phosphatase (New England Biolabs: Beverly, MA), which removes phosphates from serine, threonine or tyrosine residues (Zhuo et al., 1993). Prothoracic glands were sonicated directly in phosphatase buffer [50 mM Tris –HCl (pH 7.5), 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, 2 mM MnCl2] with added protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 0.75 mg/ml pepstatin, 0.5 mg/ml leupeptin). Following sonication, samples were centrifuged for 3 min at 15 800× g (4 °C) and the supernatant was incubated for 90 min at 25 °C with 400 units of lambda protein phosphatase per 50 ml of supernatant (1 prothoracic gland equivalent per 10 ml).
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Samples were then flash frozen and stored at −80 °C until PAGE analysis.
2.6. Radioimmuoassay of ecdysteroids Thawed samples were vortexed, centrifuged at 15 000× g for 5 min and 15–45 ml aliquots of the supernatant assayed in duplicate for ecdysteroid content as described previously (Warren and Gilbert, 1986, 1988). The antibody (S-3) used for RIA (courtesy of Prof. Sho Sakurai, Kanazawa University, Japan) has similarly high affinity for ecdysone, 20-hydroxyecdysone and 3-dehydroecdysone (Kiriishi et al., 1990). The results of the RIA experiments are expressed as percent of control, i.e. the ratios of the synthetic activity of treated glands to their control contralateral partners multiplied by 100%. This ratio corrects for inter-animal variation by relating the response of each treated gland to ecdysteroid production by the control contralateral gland. RIA results are presented as the mean9 S.E.M.
3. Results
3.1. The prothoracic gland expresses an ERK MAP kinase Polyclonal antibodies against a conserved, non-phosphorylated region of the mammalian ERK 1 and ERK 2 map kinases were used to probe immunoblots of prothoracic gland lysates. The results revealed a strongly immunoreactive protein with a molecular weight of about 42 000 (Fig. 1A), readily detectable in a lysate of a single prothoracic gland from a day 3 fifth instar larva (mean total extractable protein= 12.6 mg) (Rybczynski and Gilbert, 1994). A second less abundant, or less immunoreactive, protein was detected at about 40 000 MW. The molecular weights of the detected ERKs are similar to, but slightly smaller than, the molecular weights expected from studies on vertebrate tissues (44 000 and 42 000) (see Lewis et al., 1998). In Drosophila melanogaster, immunoblot analysis and molecular studies suggest that there is a single ERK1/2 homologue, of : 44 kDa (Biggs and Zipursky, 1992; Gabay et al., 1997; Morrison et al., 2000). Immunoblots of prothoracic gland lysates probed with monoclonal antibodies raised against either dually phosphorylated mammalian (data not shown) or Drosophila ERK amino acid sequences yielded identical results, i.e. a strongly reactive protein at about 42 kDa and a weaker signal from a protein of about 40 kDa (Fig. 1A: all phosphorylated ERK immunoblot data presented in this report derive from transfers probed with the monoclonal antibody raised against the Drosophila sequence).
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When prothoracic gland lysates were incubated with lambda protein phosphatase prior to electrophoresis, the immunoreactivity detected by the two anti-phospho-ERK monoclonal antibodies was virtually eliminated, indicating that these antibodies, raised against mammalian and Drosophila sequences, were indeed recognizing a phosphorylated epitope in the Manduca samples (Fig. 1B). Further evidence that these phosphorylated proteins are ERKs, and that the antibodies are specifically recognizing ERKs, comes from the use of specific MAP kinase kinase (MEK) inhibitors that prevent the phosphorylation of ERKs by MEKs (Alessi et al., 1995; Favata et al., 1998). Treatment of prothoracic
Fig. 1. Immunoblot of ERK in the Manduca prothoracic gland. (A) Following SDS-PAGE and immunoblotting, prothoracic gland extracts were probed with pan-ERK antibodies (panERK Ab; not selective as to the phosphorylation state of the ERK) or with a monoclonal phosphorylated ERK antibody (pERK Ab; selective for dually phosphorylated ERKs and raised against the Drosophila sequence). The arrow indicates the second possible ERK protein (see text). (B) Treatment of prothoracic gland lysates with lambda protein phosphatase prior to electrophoresis eliminates the signal recognized by the phosphorylated ERK antibody in subsequent immunoblotting. (C) Incubation of prothoracic glands with MEK inhibitors in vitro greatly diminishes the signal recognized by the phosphorylated ERK antibody in subsequent immunoblotting of extracts from these glands. Glands were incubated for 2 h with 10 mM PD 098059 or for 150 min with 1 mM UO 126. (D) ERK phosphorylation is stimulated by both partially purified PTTH (brain) and recombinant PTTH (rPTTH), using equivalent doses (0.25 brain equivalents and 0.5 ng, respectively) as determined by prothoracic gland in vitro ecdysteroid synthesis (Gilbert et al., 2000). Glands were treated 9 either PTTH for 1 h, followed by SDS-PAGE and immunoblotting with the pERK Ab.
glands in vitro with the MEK inhibitors PD 98059 and UO 126 resulted in a substantial decrease in antigen recognition by the anti-phospho-ERK monoclonal antibodies (Fig. 1C). Further, preincubation of the monoclonal antibodies with a peptide containing the mammalian phosphorylated ERK sequence, prior to their use in immunoblotting analyses, resulted in a diminished ability to detect the insect ERK immunoreactivities (data not shown). Recombinant Manduca PTTH has recently become available and found to have the same effects on second messenger systems, ribosomal protein S6 phosphorylation, and protein and ecdysteroid synthesis as does PTTH partially purified from Manduca brains (Gilbert et al., 2000). Similarly, no difference was found between recombinant PTTH and partially purified PTTH in the stimulation of ERK phosphorylation (Fig. 1D), using doses previously shown to have equal effects on steroidogenesis (Gilbert et al., 2000). These data do not determine conclusively whether Manduca prothoracic glands express two ERK proteins, encoded by two genes as in vertebrates (ERK1 and ERK2), or a single ERK encoded by a single gene, as apparently is the case for Drosophila melanogaster (Biggs and Zipursky, 1992; Morrison et al., 2000). Therefore, this report will deal exclusively with the 42 kDa protein, which will be referred to as Manduca ERK. Another reason for this decision is that the level of the 40 kDa protein single gland lysates was usually too low to quantify with the available antibodies. Immunoblot analysis revealed that the behaviors of the 42 and 40 kDa proteins, and other smaller polypeptides detected after long immunoblot-film exposures, were highly positively correlated, especially in regard to PTTH stimulation, lambda phosphatase exposure and inhibitor sensitivity. The possibility that the 40 kDa and smaller immunoreactive proteins are proteolytic fragments of the 42 kDa protein cannot be ruled out and may be likely, given the evidence from Drosophila suggesting that insects might only possess one ERK gene.
3.2. PTTH stimulates ERK phosphorylation and acti6ity The prothoracic glands respond to PTTH with a consistent set of intracellular changes, culminating in increased ecdysteroid synthesis, e.g. Ca2 + influx, cAMP generation, phosphorylation of ribosomal protein S6, and protein synthesis (see Gilbert et al., 2000). Immunoblot analysis of cell lysates of PTTH-treated glands revealed that PTTH also evoked an increase in ERK phosphorylation after only 15 min of prothoracic gland exposure to PTTH. Glands treated with PTTH for 30 or 60 min also exhibit a large increase in ERK phosphorylation, and an apparent peak in ERK phos-
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3.3. ERK acti6ity and steroidogenesis
Fig. 2. PTTH stimulates an increase in the phosphorylation of the Manduca ERK in the prothoracic gland in vitro. (A) Representative immunoblot showing the increase in ERK phosphorylation after 15 min of PTTH treatment in vitro. The non-PTTH treated controls were excised from the same animals as were the PTTH-treated glands. (B) Scanning densitometric analysis of immunoblots showing PTTHdependent prothoracic gland ERK phosphorylation as a function of time. The fold-increase was determined relative to the phosphorylation level detected in each of the paired control glands removed from the same animals; N = 4 –8 gland pairs per time point. All blots were probed with a pERK antibody raised against a Drosophila ERK sequence.
phorylation occurs at 30 min (Fig. 2). It should be noted that phosphorylated ERK was also readily detectable in the control or ‘basal’ glands in this and other experiments. Dual phosphorylation of ERKs on threonine and tyrosine residues, as detected by the anti-phosphorylated ERK antibodies used, is required for maximal kinase activity. To assess the actual increases in ERKdependent phosphorylation activity that accompanies PTTH stimulation, ERK phosphorylation activity was measured in lysates from control and PTTH-treated prothoracic glands. Immune-precipitated, dually phosphorylated ERKs were incubated with an ELK-1 fusion protein in a kinase-permissive buffer; ELK-1 is a wellcharacterized (mammalian) ERK target (Marais et al., 1993; Hill and Treisman, 1995). SDS-PAGE followed by immunoblotting with a phospho-ELK-1-specific antibody and scanning densitometry revealed that 1 h of PTTH treatment increased ELK-1 phosphorylation 41.8-fold (9 14.7: n =6).
Under standard in vitro conditions, prothoracic glands produce ecdysteroids at a rate that is correlated roughly with gland size, which changes during development (Smith and Pasquarello, 1989), and is increased 4–6 fold by physiological doses of PTTH (Gilbert et al., 2000). The possibility that basal or PTTH-stimulated ecdysteroid synthesis is dependent on ERK activity was tested by using the specific MEK inhibitors UO 126 and PD 98059, and the ERK inhibitor, apigenin. UO 126 inhibits MEK activation as well as the activity of already active, i.e. phosphorylated, MEK (Favata et al., 1998) and by doing so, prevents the phosphorylation-dependent activity of ERKs. The rates of ecdysteroid synthesis were determined by RIA for day 3 fifth instar prothoracic glands incubated either in the presence or absence of partially purified PTTH, and with concentrations of UO 126 ranging from 0.1 to 20 mM. The results of these experiments demonstrated that both basal and PTTH-stimulated ecdysteroidogenesis were inhibited substantially by concentrations of UO 126 in the low mM range (Fig. 3). Under these experimental conditions, the IC50s for UO 126 in the presence or absence of PTTH were similar, being 4.2 mM (Fig. 3A) and 3.3 mM (Fig. 3B), respectively. The data suggest also that low doses (B1 mM) of inhibitor may stimulate basal ecdysteroidogenesis slightly but there was considerable variation in these data and the possible stimulatory effect was not explored further. The effect of UO 126 on ecdysteroid synthesis stimulated by recombinant PTTH (rPTTH) was not characterized in detail, due to time-dependent activity loss by the recombinant protein, under all tested storage conditions (data not shown), following reconstitution from the lyophilized state. However, when prothoracic glands were incubated with freshly prepared rPTTH (0.5 ng: see Gilbert et al., 2000) 94.2 mM UO 126, the IC50 concentration determined with the partially purified PTTH, the inhibitor had the same effect on the action of the rPTTH, i.e. 4.2 mM UO 126 inhibited rPTTHstimulated ecdysteroidogenesis by 509 3% (n=5). The effect of UO 126 on ERK phosphorylation was assessed by immunoblot analysis of the glands used in the ecdysteroid synthesis experiment described above. For glands stimulated with partially purified PTTH, the inclusion of UO 126 resulted in a substantial inhibition of PTTH-dependent ERK phosphorylation, even at the lowest concentration of inhibitor tested, i.e. 100 nM (Fig. 4). The percent inhibition was similar from 100 to 400 nM (85–90%) and a second plateau of inhibition was evident with concentrations of 800 nM to 3.2 mM, with the percent inhibition of ERK phosphorylation ranging from 95 to 99%. Note that PTTH still elicits an increase in ERK phosphorylation in the presence of 100–400 nM UO 126 relative to PTTH-free controls,
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i.e. after 1 h of PTTH stimulation, ERK phosphorylation is increased :16-fold (see Figs. 2B and 3B), and thus basal ERK phosphorylation is approximately 6% of that seen in PTTH-stimulated glands. At 5 and 20 mM, phosphorylated ERKs were not detectable in single gland lysates. The effect of UO 126 on ERK phosphorylation in glands not treated with PTTH appeared to follow a similar trend. For instance, at 800 nM, phosphorylated ERK levels were decreased 87% ( 9 2%) relative to inhibitor-free, matched controls, and by 5 mM, phosphorylated ERKs were again undetectable in single gland lysates. The effect of 4.2 mM UO 126 on rPTTH-stimulated ERK phosphorylation was also determined as described above. ERK phosphorylation in glands treated with both rPTTH and UO 126 was 1.39 0.3% of that seen in glands treated only with rPTTH, again indicating that rPTTH and Fig. 4. The MEK inhibitor UO 126 blocks PTTH-dependent ERK phosphorylation. Prothoracic glands were challenged in vitro with PTTH for 1 h, subsequent to a 90 min preincubation 9the MEK inhibitor UO 126. ERK phosphorylation was determined by scanning densitometry following SDS-PAGE and immunoblotting of gland lysates with a pERK antibody raised against a Drosophila sequence. ERK phosphorylation in glands treated with both UO 126 and PTTH was compared to that detected in PTTH-treated but inhibitorfree, contralateral glands. The stars indicate that the amount of dually phosphorylated ERK in single gland lysates was below the resolution of our immunoblotting technique. Inhibition less than 94% (dashed line) indicates that PTTH was still able to stimulate ERK phosphorylation above that seen in basal glands (no inhibitor or PTTH: see text and Fig. 3B). N= 5 – 10 gland pairs per inhibitor concentration.
Fig. 3. Prothoracic gland ecdysteroid synthesis is inhibited by low micromolar levels of the MEK inhibitor UO 126. Prothoracic glands were preincubated for 90 min 9 UO 126, prior to medium change and subsequent 1 h incubation (A) with PTTH9 UO 126 (IC50 : 4.2 mM) or (B) 9 UO 126 in the absence of PTTH (IC50 : 3.3 mM). Ecdysteroid synthesis by inhibitor-treated glands is expressed as a percent of that produced by the untreated, contralateral glands. N =5 – 10 gland pairs per inhibitor concentration.
partially purified PTTH have indistinguishable effects on prothoracic glands. Two other inhibitors of the ERK pathway were used to explore the relationship between ERK activity and ecdysteroid synthesis although they were not tested with as wide a range of concentrations as was UO 126. One, PD 98059, inhibits MEK activation in vertebrate systems but, unlike UO 126, does not inhibit the activity of already activated MEK and is effective only at higher concentrations than is UO 126 (Favata et al., 1998). The second inhibitor, apigenin, inhibits the activity of ERKs in vertebrate tissues; however, apigenin can also inhibit tyrosine kinases (Kuo and Yang, 1995; Huang et al., 1996), complicating the interpretation of its effect on ecdysteroid synthesis. At 40 mM, PD 98059 inhibited PTTH-stimulated ecdysteroid synthesis by 299 13% (n= 5), while at 100 mM PD 98059, PTTHstimulated ecdysteroid synthesis was inhibited by 589 7% (n =10). No consistent effect of PD 98059 was seen on basal ecdysteroid synthesis at these concentrations. Apigenin had no effect on basal ecdysteroid synthesis at 30 mM (92922% of control; n=6) but at 50 mM, apigenin resulted in a 449 13% inhibition (n=7).
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PTTH-stimulated ecdysteroid synthesis was inhibited by 62 910% by 30 mM apigenin (n = 5) and 50 mM yielded an inhibition of 919 3% (n = 6). It is possible that, even in the presence of inhibitor, PTTH may evoke some increase in ERK phosphorylation. This was tested explicitly using 4.2 mM UO 126, the IC50 concentration for UO 126 (see Fig. 4A). In glands treated with 4.2 mM UO 126, ERK phosphorylation was 1.090.1% (n = 6) of that measured in inhibitor-free glands. However, a comparison of ERK phosphorylation in UO 126-treated glands with that found in glands treated with both UO 126 and PTTH showed that PTTH evoked an 11.393.1-fold (n= 7) increase in ERK phosphorylation, indicating that even at this relatively high inhibitor level, MEK activation is not completely suppressed. The possibility that UO 126 and PD 98059 exerted their effects via cell toxicity rather than through inhibition of the ERK pathway was addressed by performing pulse –chase experiments with these compounds. Following standard pre-incubation and incubation periods 9 100 mM PD 98059 or 920 mM UO 126, glands were rinsed with Grace’s medium twice, incubated for 4 or 24 h in Grace’s medium and then challenged for 1 h with PTTH in fresh Grace’s medium. PTTH-stimulated ecdysteroid synthesis recovered rapidly from these inhibitor treatments. Following 4 h of chase, ecdysteroid synthesis by glands that had been treated with UO 126 was 3.19 0.4 times that of matched controls (n= 5), while synthesis following a 24 h chase was 2.19 0.3 times that of control glands (n =5). The notably higher rate of ecdysteroid synthesis by glands that had been treated earlier with UO 126 presumably reflects a larger available precursor (cholesterol) pool remaining in these glands than in the inhibitor-free glands. The control glands had synthesized considerable amounts of ecdysteroid during the pulse phase. Ecdysteroid synthesis declines with time in culture and evidence indicates that this reflects depletion of a finite precursor pool (Song and Gilbert, 1998). Immunoblot analysis of glands held in vitro for 24 h showed no difference between control glands and those that had been treated with UO 126 in the PTTH-stimulated ERK phosphorylation response; the level of ERK phosphorylation in glands treated earlier with UO 126 was 93912% of that measured in control glands (n = 5). Prothoracic glands showed a similar recovery from treatment with 100 mM PD 98059 after either a 4 or 24 h chase (data not shown), but the overshoot in ecdysteroid synthesis by glands treated with PD 98059 was not as dramatic ( :30% more at 4 h and no difference after 24 h). Visual inspection of the prothoracic glands with a dissecting microscope did not reveal any obvious differences between control glands and those treated with either MEK inhibitor, even after 24 h in vitro.
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4. Discussion MAPKs, especially those belonging to the ERK subfamily, play a pivotal role in transducing intercellular signals like hormones, growth factors and neurotransmitters into intracellular events (see Lewis et al., 1998; Grewal et al., 1999). ERK activity regulates events as disparate as cytoskeletal stability, steroid hormone receptor function, rate of translation and selective transcription of genes. The data reported here showed that PTTH elicited a rapid and large increase in ERK phosphorylation in the principal steroidogenic organ of insects, the prothoracic gland, that was readily detectable after 15 min of PTTH exposure and persisted for at least 60 min under conditions of continuous PTTH presence. The MEK inhibitors UO 126 and PD 098059, at mM concentrations, blocked both PTTHstimulated ERK phosphorylation and PTTH-stimulated ecdysteroid synthesis. Consistent with the finding that control (basal) glands contain measurable amounts of a phosphorylated ERK, the MEK inhibitors UO 126 and the ERK inhibitor apigenin also blocked basal ecdysteroid synthesis. Immunoblot studies revealed that the phosphorylation of the Manduca ERK homologue expressed in the prothoracic gland was detectable in glands incubated in vitro for more than 24 h and which had not been challenged with the neuropeptide hormone PTTH. This indicates that some ERK phosphorylation is maintained in the absence of extra-cellular signaling molecules and, further, that such ERK activity may be required for the functioning of one or more intracellular pathways. It is not currently known if this is unique to prothoracic gland cells. It is broadly accepted that lepidopteran prothoracic gland cells do not divide after the embryonic stage (see Beaulaton, 1990). Some cell count and DNA synthesis measures suggest that post-embryonic mitosis might occur in Manduca but mitotic figures have not been observed (Hanton et al., 1993; Lee et al., 1995). If larval Manduca prothoracic gland cells do divide, it seems likely that they do so rarely and perhaps at only very specific developmental times. Thus, the current data suggest that PTTHstimulated ERK activity is not obligately involved in cell cycling events, since PTTH-stimulated ERK phosphorylation has been observed at all fifth larval instar and early pupal-adult stages tested (Rybczynski, Bell and Gilbert, unpublished observations). An unexpected finding of this study was that the concentrations of MEK inhibitors sufficient to substantially block the PTTH-stimulated increase in ERK phosphorylation were not sufficient to inhibit PTTHstimulated ecdysteroid synthesis. That is, at concentrations of 100– 400 nM UO 126, 1 h of PTTH stimulation was able to increase ERK phosphorylation only 1–2fold, rather than the 15-fold increase seen in inhibitor free-glands (Figs. 3B and 5), but PTTH-stimulated
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ecdysteroid synthesis was unaffected (Fig. 4A). Rather, ecdysteroid synthesis was noticeably affected only at inhibitor levels of :1 mM or more, at which concentrations ERK phosphorylation was decreased below levels characteristic of resting or ‘basal’ glands. These data suggest that the large increase in ERK phosphorylation stimulated by PTTH is not necessary for PTTH-stimulated ecdysteroid synthesis, but these data also do not rule out a role for ERKs in ecdysteroidogenesis. There are at least three possible explanations for the discordance between ERK phosphorylation and ecdysteroid synthesis revealed by the MEK inhibitor studies. One possibility is that PTTH-stimulated ERK phosphorylation regulates a non-steroidogenic effect of PTTH and the inhibition of ecdysteroid synthesis is due to unanticipated effects of the MEK inhibitors on processes other than MEK activity, e.g. UO 126 inhibits another kinase involved in PTTH signal transduction or one of the P450 enzymes that mediates the synthesis of ecdysteroids from cholesterol. A second possibility is that PTTH-independent ERK activity regulates one or more processes fundamental to both basal and PTTH-stimulated ecdysteroid synthesis, in addition to regulating non-steroidogenic effects of PTTH. The nature of such regulation is conjectural at present but could involve phosphorylation-dependent, rate-limiting transport of an ecdysteroid precursor between different intracellular locations. Evidence suggests that movement of a precursor between intracellular compartments may be rate-limiting in Drosophila ecdysteroid synthesis (Warren and Gilbert, 1996; Warren et al., 1996). In vertebrate steroidogenic systems, functional analogs of PTTH, e.g. ACTH, elicit increased steroidogenesis primarily by stimulating translation of one or two small proteins, namely the steroidogenic acute regulatory protein (StAR) and the diazepam-binding inhibitor (DBI), which are believed to facilitate translocation of cholesterol across the mitochondrial membrane and delivery to the P450 side-chain cleavage enzyme (see Stocco and Clark, 1997; Papadopoulos et al., 1997). StAR is a phosphoprotein and phosphorylation may be necessary for StAR to function fully (see Stocco and Clark, 1997). A search of the Drosophila genome database suggests that insects do not possess a StAR homologue but data indicate that Manduca does express a DBI which may be involved in the regulation of basal ecdysteroid synthesis (Snyder and Van Antwerpen, 1998). Since consensus ERK phosphorylation sites are not found in the Drosophila DBI sequence [Prosite and Phosphobase analyses (Hofmann et al., 1999; Kreegipuu et al., 1999)], it is unlikely that DBI is a direct substrate of ERK activity. A third explanation is that prothoracic glands contain several MEK-ERK populations which vary in their vulnerability to inhibition, and that a population less vulnerable to inhibition regulates ecdysteroid synthesis. The observation that
PTTH can elicit an 11-fold increase in ERK phosphorylation at levels of MEK inhibitor sufficient to lower ERK phosphorylation to 1% of basal is consistent with this hypothesis. This population could be defined by intracellular location, for which there is precedence in vertebrate cells. For instance, injection of rats with epidermal growth factor (EGF) causes the association of ERKs with rat liver endosomes and plasma membrane, apparently due, at least in part, to ERK and EGF receptor complexing (Faure et al., 1999). Experiments are currently underway to explicitly test which, if any, of the above postulated relationships between ecdysteroid synthesis and ERK phosphorylation are present in the prothoracic gland. A role for ERKs in the regulation of vertebrate steroidogenesis remains to be demonstrated. In aldosterone-producing cells, angiotensin II-stimulated ERK activation appears to modulate the mitogenic response to angiotensin II. However, MEK inhibitors do not have an effect on aldosterone production (Chabre et al., 1995; Coˆ te´ et al., 1998). An ACTH-dependent activation of ERKs has been found in Y1 mouse adrenocortical tumor cells but activation appears again to be involved in proliferation rather than steroidogenesis (Lotfi et al., 1997). In granulosa cells, FSH, LH and insulin-like growth factor-1 stimulate the phosphorylation and activity of one or more ERKs (Cameron et al., 1996; Makarevich et al., 2000) but the possibility that ERKs regulate granulosa cell progesterone or estradiol synthesis appears to be untested. These observations, indicating that ERK activation is not involved in the regulation of steroidogenesis, are suggestive but not conclusive since these studies with MEK inhibitors employed only a single inhibitor concentration, as well as single pre-incubation and incubation periods. Discovering if and how ERK activation is connected to ecdysteroid synthesis, and to already described PTTH-induced events like Ca2 + influx, cAMP generation, protein kinase A activation, translation and transcription will form the focus of our future work on this system. A simple model for the involvement of ERKs in the regulation of PTTH-stimulated ecdysteroid synthesis is presented in Fig. 5. MAP kinases are important regulators of nuclear transcription factor function (see Lewis et al., 1998). It seems likely that PTTH-stimulated ERK phosphorylation plays a significant role in regulating the transcription of the genes coding for steroidogenic enzymes, as seen in homologous vertebrate systems like the ACTH-adrenal axis (see OrmeJohnson, 1990). The regulation of transcription factors by phosphorylated ERKs takes place within the nucleus (Chen et al., 1992) and it is probable that phosphorylated ERKs move into the nuclei of prothoracic glands, as they do in other cell types. However, this topic remains to be rigorously tested in the prothoracic glands, where the extra-cellular matrix (basal lamina)
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Fig. 5. A model of major intracellular events triggered by PTTH in the Manduca prothoracic gland. PTTH is believed to interact with a plasma membrane-bound G-protein coupled receptor (R), resulting in Ca2 + influx and Ca2 + -dependent cAMP generation, presumably via a Ca2 + -calmodulin-dependent adenylyl cyclase. Protein kinase A (PKA) and p70S6 kinase are activated, resulting in phosphorylation of ribosomal protein S6 and S6-dependent protein synthesis. PTTH-stimulated ecdysteroid synthesis is dependent on new protein synthesis. Preliminary data indicate that PTTH-stimulated ERK activity is regulated by PTTH-stimulated Ca2 + influx in a manner independent of PTTH-stimulated cAMP generation (R. Rybczynski, unpublished observations). The sensitivity of ERK phosphorylation to the ERK pathway inhibitors PD 98059 and UO 126 implies the presence of a MAPK kinase (MAPKK) and a MAPKK kinase immediately upstream of ERK but these are not shown on the diagram. Solid lines connect molecules and processes whose contributions to the regulation of ecdysteroid synthesis are well-documented while dashed lines indicate hypothesized interactions. Further details and speculations on the regulation of ecdysteroid synthesis are reviewed elsewhere (Henrich et al., 1999; Gilbert et al., 1996, 1997).
and very large nucleus have made phospho-ERK immunocytochemistry and subcellular fractionation problematic (R. Rybczynski and L.I. Gilbert, unpublished observations). Other aspects of prothoracic gland physiology not acutely tied to ecdysteroidogenesis may also be modulated by PTTH-stimulated ERK activity. For example, the complex controls of eukaryotic protein synthesis includes regulation by the ERK pathway (see Rhoads, 1999) and PTTH-stimulated general protein synthesis (Rybczynski and Gilbert, 1994) could be linked to PTTH-stimulated ERK activity. The PTTH-prothoracic gland axis offers a unique model for discovering potential signaling pathway intersections in a classic steroidogenic cell that occurs in a natural monoculture. Because lepidopteran prothoracic gland cells do not divide during post-embryonic life or may do so only rarely, this system might be useful in suggesting novel roles for MAP kinases in other steroidogenic cells by, in essence, removing the complexity of the proliferative response. Likewise, these insect cells may provide a useful model for elucidating the mechanisms that control steroid hormone synthesis by rarely dividing and post-mitotic glia and neurons, a topic of growing interest and importance (see Balthazart and Ball, 2000).
Acknowledgements This work was supported by NIH grant DK-30018 and NSF grant IBN 9603710 (to L.I.G.) and NIH grant GM63198-01 (to R.R.). We thank Susan Whitfield for graphics and Dr James T. Warren for helpful discussions.
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Hill, C.S., Treisman, R., 1995. Transcriptional regulation by extracellular signals: mechanism and specificity. Cell 80, 199 – 211. Hofmann, K., Bucher, P., Falquet, L., Bairoch, A., 1999. The PROSITE database, its status in 1999. Nucleic Acids Res. 27, 215 – 219. Huang, Y.T., Kuo, M.L., Liu, J.Y., Huang, S.Y., Lin, J.K., 1996. Inhibitions of protein kinase C and proto-oncogene expressions in NIH 3T3 cells by apigenin. Eur. J. Cancer 32A, 146 – 151. Keightley, D.A., Lou, K.J., Smith, W.A., 1990. Involvement of translation and transcription in insect steroidogenesis. Mol. Cell. Endocrinol. 74, 229 – 237. Kiriishi, S., Rountree, D.B., Sakurai, S., Gilbert, L.I., 1990. Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3b-reductase mediated conversion to ecdysone in representative insects. Experientia 46, 716 – 721. Kreegipuu, A., Blom, N., Brunak, S., 1999. PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res. 27, 237 – 239. Kuo, M.-L., Yang, N.-C., 1995. Reversion of v-H-ras-transformed NIH 3T3 cells by apigenin through inhibiting mitogen activated protein kinase and its downstream oncogenes. Biochem. Biophys. Res. Commun. 212, 767 – 775. Lee, C.Y., Lee, K.J., Chumley, P.H., Watson, C.J., Abdur-Rahman, A., Watson, R.D., 1995. Capacity of insect (Manduca sexta) prothoracic glands to secrete ecdysteroids: relation to glandular growth. Gen. Comp. Endocrinol. 100, 404 – 412. Lewis, T.S., Shapiro, P.S., Ahn, N.G., 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74, 49 –139. Lotfi, C.F.P., Todorovic, Z., Armelin, H.A., Schimmer, B.P., 1997. Unmasking a growth-promoting effect of the adrenocorticotropic hormone in Y1 adrenocortical tumor cells. J. Biol. Chem. 272, 29886 – 29891. Makarevich, A., Sirotkin, A., Chrenek, P., Bulla, J., Hetenyi, L., 2000. The role of IGF-1, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits. J. Steroid Biochem. Mol. Biol. 73, 123 – 133. Marais, R., Wynne, J., Treisman, R., 1993. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73, 381 – 393. Morrison, D.K., Murakami, M.S., Cleghorn, V., 2000. Protein kinases and phosphatases in the Drosophila genome. J. Cell Biol. 150, F57 – F62. Murga, C., Fukuhara, S., Gutkind, J.S., 1999. Novel molecular mediators in the pathway connecting G-protein-coupled receptors to MAP kinase cascades. Trends Endocrinol. Metab. 10, 122 – 127. Orme-Johnson, N.R., 1990. Distinctive properties of adrenal cortex mitochondria. Biochim. Biophys. Acta 1020, 213 – 231. Papadopoulos, V., Amri, H., Boujrad, N., Cascio, C., Culty, M., Garnier, M., Hardwick, M., Li, H., Vidic, B., Brown, A.S., Reversa, J.L., Bernassau, J.M., Drieu, K., 1997. Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21 – 28. Rhoads, R.E., 1999. Signal transduction pathways that regulate eukaryotic protein synthesis. J. Biol. Chem. 274, 30337 –30340. Rybczynski, R., Gilbert, L.I., 1994. Changes in general and specific protein synthesis that accompany ecdysteroid synthesis in stimulated prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 24, 175 – 189. Rybczynski, R., Gilbert, L.I., 1995. Prothoracicotropic hormone elicits a rapid, developmentally specific synthesis of b tubulin in an insect endocrine gland. Dev. Biol. 169, 15 – 28. Smith, W.A., Pasquarello, T.J., 1989. Developmental changes in phosphodiesterase activity and hormonal response in the prothoracic glands of Manduca sexta. Mol. Cell Endocrinol. 63, 239 – 246.
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Warren, J.T., Gilbert, L.I., 1988. Radioimmunoassay: ecdysteroids. In: Gilbert, L.I., Miller, T.A. (Eds.), Immunological Techniques in Insect Biology. Springer, New York, pp. 181 – 214. Warren, J.T., Gilbert, L.I., 1996. Metabolism in vitro of cholesterol and 25-hydroxycholesterol by the larval prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 26, 917 – 929. Warren, J.T., Bachman, J.S., Dai, J.-D., Gilbert, L.I., 1996. Differential incorporation of cholesterol and cholesterol derivatives by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd 1 mutation. Insect Biochem. Mol. Biol. 26, 931 – 943. Warren, J.T., Sakurai, S., Rountree, D.B., Gilbert, L.I., Lee, S.-S., Nakanishi, K., 1988. Regulation of the ecdysteroid titer of Manduca sexta: reappraisal of the role of the prothoracic glands. Proc. Natl. Acad. Sci. USA 85, 958 – 962. Zhuo, S., Clemens, J.C., Hakes, D.J., Barford, D., Dixon, J.E., 1993. Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase. J. Biol. Chem. 268, 17754 – 17761.
Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone Robert Rybczynski *, Stephanie C. Bell, Lawrence I. Gilbert Department of Biology, Coker Hall CB c 3280, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599 -3280, USA Received 30 May 2001; accepted 5 September 2001
Abstract Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70S6k). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Steroid hormone (insect); Extracellular signal-regulated kinase (ERK); Prothoracicotropic hormone (PTTH); Phosphorylation; Signal transduction; Ecdysteroidogenesis
1. Introduction Episodic increases in circulating levels of the steroid hormone 20-hydroxyecdysone (20E) result in significant changes in gene activity that are necessary for insect post-embryonic development (see Henrich et al., 1999). In many insects, surges in the titer of 20E result from the conversion of the prohormone 3-dehydroecdysone, synthesized in the prothoracic gland, to ecdysone via a hemolymph reductase (Warren et al., 1988; Kiriishi et al., 1990), and subsequent metabolism of ecdysone to 20E by intracellular monooxygenases found in a variety of tissues (see Grieneisen, 1994). The production of 3-dehydroecdysone is positively and acutely regulated by the homodimeric neuropeptide hormone prothoracicotropic hormone (PTTH) (see Gilbert et al., 1996). Current evidence suggests that PTTH interacts with a G-protein-coupled receptor, resulting in Ca2 + influx, * Corresponding author. Tel.: + 1-919-966-5535; fax: +1-919-9621344. E-mail address: [email protected] (R. Rybczynski).
activation of a Ca2 + -calmodulin-sensitive adenylyl cyclase, cAMP generation and subsequent protein kinase A activity (see Gilbert et al., 1996). PTTH further stimulates activation of the p70S6 kinase and phosphorylation of ribosomal protein S6, perhaps via protein kinase A. PTTH-stimulated ecdysteroid synthesis requires protein synthesis (Keightley et al., 1990; Rybczynski and Gilbert, 1995) that itself is dependent upon S6 phosphorylation (Song and Gilbert, 1995). The PTTH-prothoracic gland axis has close parallels with the vertebrate adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) steroidogenic pathways. In all these systems, the protein hormone that elicits increased steroid hormone synthesis does so via a transductory cascade that generates cAMP, and rapidly increases protein synthesis (see Gilbert et al., 1996; Cooke, 2000). However, a number of observations suggest that both the vertebrate and invertebrate transductory cascades are not linear, but instead are complex and involve additional messengers and proteins not typically considered to be involved in G-protein-coupled receptor signaling that is linked to cAMP generation.
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The mitogen-activated protein kinases (MAPK) comprise a family of signaling molecules that play important roles in regulating many cellular events. In vertebrates, four parallel MAPK pathways have been discovered, all of which involve a three-level kinase module (see Garrington and Johnson, 1999; Murga et al., 1999). The archetypal MAPK path flows from an external signaling molecule such as a growth factor or hormone, to receptor tyrosine kinases to Ras (a small GTP-binding protein) to Raf-1 (MAP kinase kinase kinase) to MEK (MAP kinase kinase) to one or two extracellular signal-regulated kinases (ERK1/2). Recent work has shown that G-protein coupled receptors can also activate the ERK pathway through G-protein- or cAMP-dependent processes (see Lewis et al., 1998). In a number of adrenal and ovarian model systems, steroidogenic peptide hormones activate the ERK pathway resulting in ERK-dependent effects on cellular proliferation and differentiation (Chabre et al., 1995; Balla et al., 1998; Lotfi et al., 1997; Das et al., 1996). However, the role of ERKs has not been analyzed thoroughly in regard to a possible role in modulating insect steroid hormone synthesis. The present study examines the possibility that MAPKs, specifically those of the ERK family, are activated in the prothoracic gland by PTTH, and, if so, whether or not they are involved in the regulation of ecdysteroid synthesis. This problem is of intrinsic value, given the importance of ecdysteroid hormones in insect development. Furthermore, since prothoracic gland cells do not undergo mitosis following embryonic development, they may provide a model for understanding the role of ERKs in rarely dividing or post-mitotic endocrine cells, i.e. those in the vertebrate central nervous system.
Jolla, CA). Grace’s medium was from GibcoBRL (Grand Island, NY). Pure recombinant Manduca sexta PTTH, expressed in E. coli, was a gift of Dr H. Kataoka (University of Tokyo, Japan) (Gilbert et al., 2000). Partially purified PTTH was prepared from brains of day 1 pupae as described previously (Rybczynski and Gilbert, 1995).
2.2. Organ incubations Manduca sexta larvae were group-reared and developmentally staged as described previously (Rybczynski and Gilbert, 1994). Prothoracic glands from day 3 fifth instar larvae were extirpated under insect saline and transferred to individual wells (one gland/well) of a spot test plate containing 50 ml Grace’s medium (Rybczynski and Gilbert, 1994). Two prothoracic glands are found in each animal and one was used as a matched control for each gland receiving an experimental treatment. After completion of a dissection series (5–10 pairs of prothoracic glands), the medium was replaced rapidly with fresh medium9 inhibitors, and the preincubation period was initiated. At the end of the preincubation period, the medium was replaced rapidly with 25 ml of fresh Grace’s medium 9experimental materials (PTTH, inhibitors). Pure recombinant PTTH was used at a standard dose of 0.25 ng per gland and partially purified PTTH was used at 0.25 brain equivalents per gland; these two preparations have identical effects on prothoracic gland signal transduction and ecdysteroidogenesis (Gilbert et al., 2000). At the end of the incubation period, 20 ml medium from each well were removed, diluted into phosphatebuffered saline and frozen at − 20 °C until analyzed for ecdysteroids by RIA. Prothoracic glands were flash frozen and stored at − 80 °C until analysis by SDSPAGE and immunoblotting.
2. Materials and methods
2.3. Immunoblots 2.1. Materials Monoclonal antibodies against dually phosphorylated ERK mammalian and Drosophila melanogaster sequences were obtained from Cell Signaling Technology (Beverly, MA), and Sigma (St. Louis, MO), respectively. Phosphorylation state-independent ERK polyclonal antibodies were obtained from Stressgen (Victoria, BC, Canada) and a blocking peptide containing a dually phosphorylated ERK1/2 sequence was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-linked goat anti-rabbit and goat anti-mouse antibodies were acquired from Jackson ImmunoResearch Laboratories (West Grove, PA). The ERK inhibitor apigenin was purchased from Sigma while the MAP kinase kinase (MEK) inhibitors PD 98059 and UO 126 were obtained from Calbiochem (La
Prothoracic glands were boiled in SDS sample buffer for 10 min followed by centrifugation at 15 800×g for 3 min. Aliquots of the supernatants were loaded onto 10% SDS gels. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, blocked for 1 h in phosphate-buffered saline with 2% bovine serum albumen, 5% non-fat powdered dry milk and 0.1% Tween 20. Blots were incubated overnight at 4 °C with a primary antibody (1:3000) in phosphatebuffered saline with 2% bovine serum albumen, 2% non-fat powdered dry milk and 0.1% Tween 20. Blots were then washed three times in phosphate-buffered saline with 0.1% Tween 20 for 10 min each and then incubated with an appropriate horseradish peroxidaselinked second antibody in phosphate-buffered saline with 2% bovine serum albumen, 2% non-fat powdered
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dry milk and 0.1% Tween 20. Following three additional washes, immunoreactivity was visualized using chemiluminescence. The resultant films were scanned and quantified using a Molecular Dynamics Computing Densitometer (Sunnyvale, CA) and the IMAGEQUANT program.
2.4. ERK acti6ity assay ERK activity was determined in single gland lysates using reagents from Cell Signaling Technology, with slight modifications of the manufacturer’s protocol. Briefly, following incubation of prothoracic glands in vitro, as described above, single gland lysates were made by sonicating glands (4×5 s) in 200 ml cell lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 1 mM PMSF]. Lysates were centrifuged at 15 800×g at 4 °C for 5 min and 10 ml of a suspension of agarose bead-linked anti-phosphorylated-ERK 1/2 monoclonal antibodies was added to the supernatant in a fresh tube. This suspension was incubated overnight at 4 °C with gentle shaking, followed by centrifugation for 30 s. The pellet was washed twice with 500 ml cell lysis buffer (4 °C) and twice with 500 ml kinase buffer [25 mM Tris (pH 7.5), 5 mM b-glycerophosphate, 0.1 mM Na3VO4, and 10 mM MgCl2] at 4 °C. The pellet was then resuspended in 50 ml kinase buffer containing 200 mM ATP and 1.5 mg of an ELK-1 transcription factor fusion protein to serve as an ERK substrate. After incubation for 30 min at 30 °C, the reaction was terminated by the addition of 25 ml SDS sample buffer, followed by centrifugation for 3 min. Reactions were analyzed by SDS-PAGE (25 ml/sample) and immunoblotting, using chemiluminescent detection of an anti-phospho ELK antibody. ELK phosphorylation was quantified using scanning densitometry and the IMAGEQUANT program.
2.5. Protein dephosphorylation Prothoracic gland lysates were treated with lambda protein phosphatase (New England Biolabs: Beverly, MA), which removes phosphates from serine, threonine or tyrosine residues (Zhuo et al., 1993). Prothoracic glands were sonicated directly in phosphatase buffer [50 mM Tris –HCl (pH 7.5), 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, 2 mM MnCl2] with added protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 0.75 mg/ml pepstatin, 0.5 mg/ml leupeptin). Following sonication, samples were centrifuged for 3 min at 15 800× g (4 °C) and the supernatant was incubated for 90 min at 25 °C with 400 units of lambda protein phosphatase per 50 ml of supernatant (1 prothoracic gland equivalent per 10 ml).
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Samples were then flash frozen and stored at −80 °C until PAGE analysis.
2.6. Radioimmuoassay of ecdysteroids Thawed samples were vortexed, centrifuged at 15 000× g for 5 min and 15–45 ml aliquots of the supernatant assayed in duplicate for ecdysteroid content as described previously (Warren and Gilbert, 1986, 1988). The antibody (S-3) used for RIA (courtesy of Prof. Sho Sakurai, Kanazawa University, Japan) has similarly high affinity for ecdysone, 20-hydroxyecdysone and 3-dehydroecdysone (Kiriishi et al., 1990). The results of the RIA experiments are expressed as percent of control, i.e. the ratios of the synthetic activity of treated glands to their control contralateral partners multiplied by 100%. This ratio corrects for inter-animal variation by relating the response of each treated gland to ecdysteroid production by the control contralateral gland. RIA results are presented as the mean9 S.E.M.
3. Results
3.1. The prothoracic gland expresses an ERK MAP kinase Polyclonal antibodies against a conserved, non-phosphorylated region of the mammalian ERK 1 and ERK 2 map kinases were used to probe immunoblots of prothoracic gland lysates. The results revealed a strongly immunoreactive protein with a molecular weight of about 42 000 (Fig. 1A), readily detectable in a lysate of a single prothoracic gland from a day 3 fifth instar larva (mean total extractable protein= 12.6 mg) (Rybczynski and Gilbert, 1994). A second less abundant, or less immunoreactive, protein was detected at about 40 000 MW. The molecular weights of the detected ERKs are similar to, but slightly smaller than, the molecular weights expected from studies on vertebrate tissues (44 000 and 42 000) (see Lewis et al., 1998). In Drosophila melanogaster, immunoblot analysis and molecular studies suggest that there is a single ERK1/2 homologue, of : 44 kDa (Biggs and Zipursky, 1992; Gabay et al., 1997; Morrison et al., 2000). Immunoblots of prothoracic gland lysates probed with monoclonal antibodies raised against either dually phosphorylated mammalian (data not shown) or Drosophila ERK amino acid sequences yielded identical results, i.e. a strongly reactive protein at about 42 kDa and a weaker signal from a protein of about 40 kDa (Fig. 1A: all phosphorylated ERK immunoblot data presented in this report derive from transfers probed with the monoclonal antibody raised against the Drosophila sequence).
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When prothoracic gland lysates were incubated with lambda protein phosphatase prior to electrophoresis, the immunoreactivity detected by the two anti-phospho-ERK monoclonal antibodies was virtually eliminated, indicating that these antibodies, raised against mammalian and Drosophila sequences, were indeed recognizing a phosphorylated epitope in the Manduca samples (Fig. 1B). Further evidence that these phosphorylated proteins are ERKs, and that the antibodies are specifically recognizing ERKs, comes from the use of specific MAP kinase kinase (MEK) inhibitors that prevent the phosphorylation of ERKs by MEKs (Alessi et al., 1995; Favata et al., 1998). Treatment of prothoracic
Fig. 1. Immunoblot of ERK in the Manduca prothoracic gland. (A) Following SDS-PAGE and immunoblotting, prothoracic gland extracts were probed with pan-ERK antibodies (panERK Ab; not selective as to the phosphorylation state of the ERK) or with a monoclonal phosphorylated ERK antibody (pERK Ab; selective for dually phosphorylated ERKs and raised against the Drosophila sequence). The arrow indicates the second possible ERK protein (see text). (B) Treatment of prothoracic gland lysates with lambda protein phosphatase prior to electrophoresis eliminates the signal recognized by the phosphorylated ERK antibody in subsequent immunoblotting. (C) Incubation of prothoracic glands with MEK inhibitors in vitro greatly diminishes the signal recognized by the phosphorylated ERK antibody in subsequent immunoblotting of extracts from these glands. Glands were incubated for 2 h with 10 mM PD 098059 or for 150 min with 1 mM UO 126. (D) ERK phosphorylation is stimulated by both partially purified PTTH (brain) and recombinant PTTH (rPTTH), using equivalent doses (0.25 brain equivalents and 0.5 ng, respectively) as determined by prothoracic gland in vitro ecdysteroid synthesis (Gilbert et al., 2000). Glands were treated 9 either PTTH for 1 h, followed by SDS-PAGE and immunoblotting with the pERK Ab.
glands in vitro with the MEK inhibitors PD 98059 and UO 126 resulted in a substantial decrease in antigen recognition by the anti-phospho-ERK monoclonal antibodies (Fig. 1C). Further, preincubation of the monoclonal antibodies with a peptide containing the mammalian phosphorylated ERK sequence, prior to their use in immunoblotting analyses, resulted in a diminished ability to detect the insect ERK immunoreactivities (data not shown). Recombinant Manduca PTTH has recently become available and found to have the same effects on second messenger systems, ribosomal protein S6 phosphorylation, and protein and ecdysteroid synthesis as does PTTH partially purified from Manduca brains (Gilbert et al., 2000). Similarly, no difference was found between recombinant PTTH and partially purified PTTH in the stimulation of ERK phosphorylation (Fig. 1D), using doses previously shown to have equal effects on steroidogenesis (Gilbert et al., 2000). These data do not determine conclusively whether Manduca prothoracic glands express two ERK proteins, encoded by two genes as in vertebrates (ERK1 and ERK2), or a single ERK encoded by a single gene, as apparently is the case for Drosophila melanogaster (Biggs and Zipursky, 1992; Morrison et al., 2000). Therefore, this report will deal exclusively with the 42 kDa protein, which will be referred to as Manduca ERK. Another reason for this decision is that the level of the 40 kDa protein single gland lysates was usually too low to quantify with the available antibodies. Immunoblot analysis revealed that the behaviors of the 42 and 40 kDa proteins, and other smaller polypeptides detected after long immunoblot-film exposures, were highly positively correlated, especially in regard to PTTH stimulation, lambda phosphatase exposure and inhibitor sensitivity. The possibility that the 40 kDa and smaller immunoreactive proteins are proteolytic fragments of the 42 kDa protein cannot be ruled out and may be likely, given the evidence from Drosophila suggesting that insects might only possess one ERK gene.
3.2. PTTH stimulates ERK phosphorylation and acti6ity The prothoracic glands respond to PTTH with a consistent set of intracellular changes, culminating in increased ecdysteroid synthesis, e.g. Ca2 + influx, cAMP generation, phosphorylation of ribosomal protein S6, and protein synthesis (see Gilbert et al., 2000). Immunoblot analysis of cell lysates of PTTH-treated glands revealed that PTTH also evoked an increase in ERK phosphorylation after only 15 min of prothoracic gland exposure to PTTH. Glands treated with PTTH for 30 or 60 min also exhibit a large increase in ERK phosphorylation, and an apparent peak in ERK phos-
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3.3. ERK acti6ity and steroidogenesis
Fig. 2. PTTH stimulates an increase in the phosphorylation of the Manduca ERK in the prothoracic gland in vitro. (A) Representative immunoblot showing the increase in ERK phosphorylation after 15 min of PTTH treatment in vitro. The non-PTTH treated controls were excised from the same animals as were the PTTH-treated glands. (B) Scanning densitometric analysis of immunoblots showing PTTHdependent prothoracic gland ERK phosphorylation as a function of time. The fold-increase was determined relative to the phosphorylation level detected in each of the paired control glands removed from the same animals; N = 4 –8 gland pairs per time point. All blots were probed with a pERK antibody raised against a Drosophila ERK sequence.
phorylation occurs at 30 min (Fig. 2). It should be noted that phosphorylated ERK was also readily detectable in the control or ‘basal’ glands in this and other experiments. Dual phosphorylation of ERKs on threonine and tyrosine residues, as detected by the anti-phosphorylated ERK antibodies used, is required for maximal kinase activity. To assess the actual increases in ERKdependent phosphorylation activity that accompanies PTTH stimulation, ERK phosphorylation activity was measured in lysates from control and PTTH-treated prothoracic glands. Immune-precipitated, dually phosphorylated ERKs were incubated with an ELK-1 fusion protein in a kinase-permissive buffer; ELK-1 is a wellcharacterized (mammalian) ERK target (Marais et al., 1993; Hill and Treisman, 1995). SDS-PAGE followed by immunoblotting with a phospho-ELK-1-specific antibody and scanning densitometry revealed that 1 h of PTTH treatment increased ELK-1 phosphorylation 41.8-fold (9 14.7: n =6).
Under standard in vitro conditions, prothoracic glands produce ecdysteroids at a rate that is correlated roughly with gland size, which changes during development (Smith and Pasquarello, 1989), and is increased 4–6 fold by physiological doses of PTTH (Gilbert et al., 2000). The possibility that basal or PTTH-stimulated ecdysteroid synthesis is dependent on ERK activity was tested by using the specific MEK inhibitors UO 126 and PD 98059, and the ERK inhibitor, apigenin. UO 126 inhibits MEK activation as well as the activity of already active, i.e. phosphorylated, MEK (Favata et al., 1998) and by doing so, prevents the phosphorylation-dependent activity of ERKs. The rates of ecdysteroid synthesis were determined by RIA for day 3 fifth instar prothoracic glands incubated either in the presence or absence of partially purified PTTH, and with concentrations of UO 126 ranging from 0.1 to 20 mM. The results of these experiments demonstrated that both basal and PTTH-stimulated ecdysteroidogenesis were inhibited substantially by concentrations of UO 126 in the low mM range (Fig. 3). Under these experimental conditions, the IC50s for UO 126 in the presence or absence of PTTH were similar, being 4.2 mM (Fig. 3A) and 3.3 mM (Fig. 3B), respectively. The data suggest also that low doses (B1 mM) of inhibitor may stimulate basal ecdysteroidogenesis slightly but there was considerable variation in these data and the possible stimulatory effect was not explored further. The effect of UO 126 on ecdysteroid synthesis stimulated by recombinant PTTH (rPTTH) was not characterized in detail, due to time-dependent activity loss by the recombinant protein, under all tested storage conditions (data not shown), following reconstitution from the lyophilized state. However, when prothoracic glands were incubated with freshly prepared rPTTH (0.5 ng: see Gilbert et al., 2000) 94.2 mM UO 126, the IC50 concentration determined with the partially purified PTTH, the inhibitor had the same effect on the action of the rPTTH, i.e. 4.2 mM UO 126 inhibited rPTTHstimulated ecdysteroidogenesis by 509 3% (n=5). The effect of UO 126 on ERK phosphorylation was assessed by immunoblot analysis of the glands used in the ecdysteroid synthesis experiment described above. For glands stimulated with partially purified PTTH, the inclusion of UO 126 resulted in a substantial inhibition of PTTH-dependent ERK phosphorylation, even at the lowest concentration of inhibitor tested, i.e. 100 nM (Fig. 4). The percent inhibition was similar from 100 to 400 nM (85–90%) and a second plateau of inhibition was evident with concentrations of 800 nM to 3.2 mM, with the percent inhibition of ERK phosphorylation ranging from 95 to 99%. Note that PTTH still elicits an increase in ERK phosphorylation in the presence of 100–400 nM UO 126 relative to PTTH-free controls,
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i.e. after 1 h of PTTH stimulation, ERK phosphorylation is increased :16-fold (see Figs. 2B and 3B), and thus basal ERK phosphorylation is approximately 6% of that seen in PTTH-stimulated glands. At 5 and 20 mM, phosphorylated ERKs were not detectable in single gland lysates. The effect of UO 126 on ERK phosphorylation in glands not treated with PTTH appeared to follow a similar trend. For instance, at 800 nM, phosphorylated ERK levels were decreased 87% ( 9 2%) relative to inhibitor-free, matched controls, and by 5 mM, phosphorylated ERKs were again undetectable in single gland lysates. The effect of 4.2 mM UO 126 on rPTTH-stimulated ERK phosphorylation was also determined as described above. ERK phosphorylation in glands treated with both rPTTH and UO 126 was 1.39 0.3% of that seen in glands treated only with rPTTH, again indicating that rPTTH and Fig. 4. The MEK inhibitor UO 126 blocks PTTH-dependent ERK phosphorylation. Prothoracic glands were challenged in vitro with PTTH for 1 h, subsequent to a 90 min preincubation 9the MEK inhibitor UO 126. ERK phosphorylation was determined by scanning densitometry following SDS-PAGE and immunoblotting of gland lysates with a pERK antibody raised against a Drosophila sequence. ERK phosphorylation in glands treated with both UO 126 and PTTH was compared to that detected in PTTH-treated but inhibitorfree, contralateral glands. The stars indicate that the amount of dually phosphorylated ERK in single gland lysates was below the resolution of our immunoblotting technique. Inhibition less than 94% (dashed line) indicates that PTTH was still able to stimulate ERK phosphorylation above that seen in basal glands (no inhibitor or PTTH: see text and Fig. 3B). N= 5 – 10 gland pairs per inhibitor concentration.
Fig. 3. Prothoracic gland ecdysteroid synthesis is inhibited by low micromolar levels of the MEK inhibitor UO 126. Prothoracic glands were preincubated for 90 min 9 UO 126, prior to medium change and subsequent 1 h incubation (A) with PTTH9 UO 126 (IC50 : 4.2 mM) or (B) 9 UO 126 in the absence of PTTH (IC50 : 3.3 mM). Ecdysteroid synthesis by inhibitor-treated glands is expressed as a percent of that produced by the untreated, contralateral glands. N =5 – 10 gland pairs per inhibitor concentration.
partially purified PTTH have indistinguishable effects on prothoracic glands. Two other inhibitors of the ERK pathway were used to explore the relationship between ERK activity and ecdysteroid synthesis although they were not tested with as wide a range of concentrations as was UO 126. One, PD 98059, inhibits MEK activation in vertebrate systems but, unlike UO 126, does not inhibit the activity of already activated MEK and is effective only at higher concentrations than is UO 126 (Favata et al., 1998). The second inhibitor, apigenin, inhibits the activity of ERKs in vertebrate tissues; however, apigenin can also inhibit tyrosine kinases (Kuo and Yang, 1995; Huang et al., 1996), complicating the interpretation of its effect on ecdysteroid synthesis. At 40 mM, PD 98059 inhibited PTTH-stimulated ecdysteroid synthesis by 299 13% (n= 5), while at 100 mM PD 98059, PTTHstimulated ecdysteroid synthesis was inhibited by 589 7% (n =10). No consistent effect of PD 98059 was seen on basal ecdysteroid synthesis at these concentrations. Apigenin had no effect on basal ecdysteroid synthesis at 30 mM (92922% of control; n=6) but at 50 mM, apigenin resulted in a 449 13% inhibition (n=7).
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PTTH-stimulated ecdysteroid synthesis was inhibited by 62 910% by 30 mM apigenin (n = 5) and 50 mM yielded an inhibition of 919 3% (n = 6). It is possible that, even in the presence of inhibitor, PTTH may evoke some increase in ERK phosphorylation. This was tested explicitly using 4.2 mM UO 126, the IC50 concentration for UO 126 (see Fig. 4A). In glands treated with 4.2 mM UO 126, ERK phosphorylation was 1.090.1% (n = 6) of that measured in inhibitor-free glands. However, a comparison of ERK phosphorylation in UO 126-treated glands with that found in glands treated with both UO 126 and PTTH showed that PTTH evoked an 11.393.1-fold (n= 7) increase in ERK phosphorylation, indicating that even at this relatively high inhibitor level, MEK activation is not completely suppressed. The possibility that UO 126 and PD 98059 exerted their effects via cell toxicity rather than through inhibition of the ERK pathway was addressed by performing pulse –chase experiments with these compounds. Following standard pre-incubation and incubation periods 9 100 mM PD 98059 or 920 mM UO 126, glands were rinsed with Grace’s medium twice, incubated for 4 or 24 h in Grace’s medium and then challenged for 1 h with PTTH in fresh Grace’s medium. PTTH-stimulated ecdysteroid synthesis recovered rapidly from these inhibitor treatments. Following 4 h of chase, ecdysteroid synthesis by glands that had been treated with UO 126 was 3.19 0.4 times that of matched controls (n= 5), while synthesis following a 24 h chase was 2.19 0.3 times that of control glands (n =5). The notably higher rate of ecdysteroid synthesis by glands that had been treated earlier with UO 126 presumably reflects a larger available precursor (cholesterol) pool remaining in these glands than in the inhibitor-free glands. The control glands had synthesized considerable amounts of ecdysteroid during the pulse phase. Ecdysteroid synthesis declines with time in culture and evidence indicates that this reflects depletion of a finite precursor pool (Song and Gilbert, 1998). Immunoblot analysis of glands held in vitro for 24 h showed no difference between control glands and those that had been treated with UO 126 in the PTTH-stimulated ERK phosphorylation response; the level of ERK phosphorylation in glands treated earlier with UO 126 was 93912% of that measured in control glands (n = 5). Prothoracic glands showed a similar recovery from treatment with 100 mM PD 98059 after either a 4 or 24 h chase (data not shown), but the overshoot in ecdysteroid synthesis by glands treated with PD 98059 was not as dramatic ( :30% more at 4 h and no difference after 24 h). Visual inspection of the prothoracic glands with a dissecting microscope did not reveal any obvious differences between control glands and those treated with either MEK inhibitor, even after 24 h in vitro.
7
4. Discussion MAPKs, especially those belonging to the ERK subfamily, play a pivotal role in transducing intercellular signals like hormones, growth factors and neurotransmitters into intracellular events (see Lewis et al., 1998; Grewal et al., 1999). ERK activity regulates events as disparate as cytoskeletal stability, steroid hormone receptor function, rate of translation and selective transcription of genes. The data reported here showed that PTTH elicited a rapid and large increase in ERK phosphorylation in the principal steroidogenic organ of insects, the prothoracic gland, that was readily detectable after 15 min of PTTH exposure and persisted for at least 60 min under conditions of continuous PTTH presence. The MEK inhibitors UO 126 and PD 098059, at mM concentrations, blocked both PTTHstimulated ERK phosphorylation and PTTH-stimulated ecdysteroid synthesis. Consistent with the finding that control (basal) glands contain measurable amounts of a phosphorylated ERK, the MEK inhibitors UO 126 and the ERK inhibitor apigenin also blocked basal ecdysteroid synthesis. Immunoblot studies revealed that the phosphorylation of the Manduca ERK homologue expressed in the prothoracic gland was detectable in glands incubated in vitro for more than 24 h and which had not been challenged with the neuropeptide hormone PTTH. This indicates that some ERK phosphorylation is maintained in the absence of extra-cellular signaling molecules and, further, that such ERK activity may be required for the functioning of one or more intracellular pathways. It is not currently known if this is unique to prothoracic gland cells. It is broadly accepted that lepidopteran prothoracic gland cells do not divide after the embryonic stage (see Beaulaton, 1990). Some cell count and DNA synthesis measures suggest that post-embryonic mitosis might occur in Manduca but mitotic figures have not been observed (Hanton et al., 1993; Lee et al., 1995). If larval Manduca prothoracic gland cells do divide, it seems likely that they do so rarely and perhaps at only very specific developmental times. Thus, the current data suggest that PTTHstimulated ERK activity is not obligately involved in cell cycling events, since PTTH-stimulated ERK phosphorylation has been observed at all fifth larval instar and early pupal-adult stages tested (Rybczynski, Bell and Gilbert, unpublished observations). An unexpected finding of this study was that the concentrations of MEK inhibitors sufficient to substantially block the PTTH-stimulated increase in ERK phosphorylation were not sufficient to inhibit PTTHstimulated ecdysteroid synthesis. That is, at concentrations of 100– 400 nM UO 126, 1 h of PTTH stimulation was able to increase ERK phosphorylation only 1–2fold, rather than the 15-fold increase seen in inhibitor free-glands (Figs. 3B and 5), but PTTH-stimulated
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ecdysteroid synthesis was unaffected (Fig. 4A). Rather, ecdysteroid synthesis was noticeably affected only at inhibitor levels of :1 mM or more, at which concentrations ERK phosphorylation was decreased below levels characteristic of resting or ‘basal’ glands. These data suggest that the large increase in ERK phosphorylation stimulated by PTTH is not necessary for PTTH-stimulated ecdysteroid synthesis, but these data also do not rule out a role for ERKs in ecdysteroidogenesis. There are at least three possible explanations for the discordance between ERK phosphorylation and ecdysteroid synthesis revealed by the MEK inhibitor studies. One possibility is that PTTH-stimulated ERK phosphorylation regulates a non-steroidogenic effect of PTTH and the inhibition of ecdysteroid synthesis is due to unanticipated effects of the MEK inhibitors on processes other than MEK activity, e.g. UO 126 inhibits another kinase involved in PTTH signal transduction or one of the P450 enzymes that mediates the synthesis of ecdysteroids from cholesterol. A second possibility is that PTTH-independent ERK activity regulates one or more processes fundamental to both basal and PTTH-stimulated ecdysteroid synthesis, in addition to regulating non-steroidogenic effects of PTTH. The nature of such regulation is conjectural at present but could involve phosphorylation-dependent, rate-limiting transport of an ecdysteroid precursor between different intracellular locations. Evidence suggests that movement of a precursor between intracellular compartments may be rate-limiting in Drosophila ecdysteroid synthesis (Warren and Gilbert, 1996; Warren et al., 1996). In vertebrate steroidogenic systems, functional analogs of PTTH, e.g. ACTH, elicit increased steroidogenesis primarily by stimulating translation of one or two small proteins, namely the steroidogenic acute regulatory protein (StAR) and the diazepam-binding inhibitor (DBI), which are believed to facilitate translocation of cholesterol across the mitochondrial membrane and delivery to the P450 side-chain cleavage enzyme (see Stocco and Clark, 1997; Papadopoulos et al., 1997). StAR is a phosphoprotein and phosphorylation may be necessary for StAR to function fully (see Stocco and Clark, 1997). A search of the Drosophila genome database suggests that insects do not possess a StAR homologue but data indicate that Manduca does express a DBI which may be involved in the regulation of basal ecdysteroid synthesis (Snyder and Van Antwerpen, 1998). Since consensus ERK phosphorylation sites are not found in the Drosophila DBI sequence [Prosite and Phosphobase analyses (Hofmann et al., 1999; Kreegipuu et al., 1999)], it is unlikely that DBI is a direct substrate of ERK activity. A third explanation is that prothoracic glands contain several MEK-ERK populations which vary in their vulnerability to inhibition, and that a population less vulnerable to inhibition regulates ecdysteroid synthesis. The observation that
PTTH can elicit an 11-fold increase in ERK phosphorylation at levels of MEK inhibitor sufficient to lower ERK phosphorylation to 1% of basal is consistent with this hypothesis. This population could be defined by intracellular location, for which there is precedence in vertebrate cells. For instance, injection of rats with epidermal growth factor (EGF) causes the association of ERKs with rat liver endosomes and plasma membrane, apparently due, at least in part, to ERK and EGF receptor complexing (Faure et al., 1999). Experiments are currently underway to explicitly test which, if any, of the above postulated relationships between ecdysteroid synthesis and ERK phosphorylation are present in the prothoracic gland. A role for ERKs in the regulation of vertebrate steroidogenesis remains to be demonstrated. In aldosterone-producing cells, angiotensin II-stimulated ERK activation appears to modulate the mitogenic response to angiotensin II. However, MEK inhibitors do not have an effect on aldosterone production (Chabre et al., 1995; Coˆ te´ et al., 1998). An ACTH-dependent activation of ERKs has been found in Y1 mouse adrenocortical tumor cells but activation appears again to be involved in proliferation rather than steroidogenesis (Lotfi et al., 1997). In granulosa cells, FSH, LH and insulin-like growth factor-1 stimulate the phosphorylation and activity of one or more ERKs (Cameron et al., 1996; Makarevich et al., 2000) but the possibility that ERKs regulate granulosa cell progesterone or estradiol synthesis appears to be untested. These observations, indicating that ERK activation is not involved in the regulation of steroidogenesis, are suggestive but not conclusive since these studies with MEK inhibitors employed only a single inhibitor concentration, as well as single pre-incubation and incubation periods. Discovering if and how ERK activation is connected to ecdysteroid synthesis, and to already described PTTH-induced events like Ca2 + influx, cAMP generation, protein kinase A activation, translation and transcription will form the focus of our future work on this system. A simple model for the involvement of ERKs in the regulation of PTTH-stimulated ecdysteroid synthesis is presented in Fig. 5. MAP kinases are important regulators of nuclear transcription factor function (see Lewis et al., 1998). It seems likely that PTTH-stimulated ERK phosphorylation plays a significant role in regulating the transcription of the genes coding for steroidogenic enzymes, as seen in homologous vertebrate systems like the ACTH-adrenal axis (see OrmeJohnson, 1990). The regulation of transcription factors by phosphorylated ERKs takes place within the nucleus (Chen et al., 1992) and it is probable that phosphorylated ERKs move into the nuclei of prothoracic glands, as they do in other cell types. However, this topic remains to be rigorously tested in the prothoracic glands, where the extra-cellular matrix (basal lamina)
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Fig. 5. A model of major intracellular events triggered by PTTH in the Manduca prothoracic gland. PTTH is believed to interact with a plasma membrane-bound G-protein coupled receptor (R), resulting in Ca2 + influx and Ca2 + -dependent cAMP generation, presumably via a Ca2 + -calmodulin-dependent adenylyl cyclase. Protein kinase A (PKA) and p70S6 kinase are activated, resulting in phosphorylation of ribosomal protein S6 and S6-dependent protein synthesis. PTTH-stimulated ecdysteroid synthesis is dependent on new protein synthesis. Preliminary data indicate that PTTH-stimulated ERK activity is regulated by PTTH-stimulated Ca2 + influx in a manner independent of PTTH-stimulated cAMP generation (R. Rybczynski, unpublished observations). The sensitivity of ERK phosphorylation to the ERK pathway inhibitors PD 98059 and UO 126 implies the presence of a MAPK kinase (MAPKK) and a MAPKK kinase immediately upstream of ERK but these are not shown on the diagram. Solid lines connect molecules and processes whose contributions to the regulation of ecdysteroid synthesis are well-documented while dashed lines indicate hypothesized interactions. Further details and speculations on the regulation of ecdysteroid synthesis are reviewed elsewhere (Henrich et al., 1999; Gilbert et al., 1996, 1997).
and very large nucleus have made phospho-ERK immunocytochemistry and subcellular fractionation problematic (R. Rybczynski and L.I. Gilbert, unpublished observations). Other aspects of prothoracic gland physiology not acutely tied to ecdysteroidogenesis may also be modulated by PTTH-stimulated ERK activity. For example, the complex controls of eukaryotic protein synthesis includes regulation by the ERK pathway (see Rhoads, 1999) and PTTH-stimulated general protein synthesis (Rybczynski and Gilbert, 1994) could be linked to PTTH-stimulated ERK activity. The PTTH-prothoracic gland axis offers a unique model for discovering potential signaling pathway intersections in a classic steroidogenic cell that occurs in a natural monoculture. Because lepidopteran prothoracic gland cells do not divide during post-embryonic life or may do so only rarely, this system might be useful in suggesting novel roles for MAP kinases in other steroidogenic cells by, in essence, removing the complexity of the proliferative response. Likewise, these insect cells may provide a useful model for elucidating the mechanisms that control steroid hormone synthesis by rarely dividing and post-mitotic glia and neurons, a topic of growing interest and importance (see Balthazart and Ball, 2000).
Acknowledgements This work was supported by NIH grant DK-30018 and NSF grant IBN 9603710 (to L.I.G.) and NIH grant GM63198-01 (to R.R.). We thank Susan Whitfield for graphics and Dr James T. Warren for helpful discussions.
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