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cDNA cloning and expression of a hormone-regulated heat shock
protein (hsc 70) from the prothoracic gland of Manduca sexta
1Robert Rybczynski
*, Lawrence I. Gilbert
Department of Biology, Coker Hall CB #3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Received 20 September 1999; received in revised form 10 February 2000; accepted 17 February 2000
Abstract
The brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (hsc 70). To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm (Manduca sexta) prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc 70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands’ hormonal milieu showed that hsc 70 mRNA levels respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.2000 Elsevier Science Ltd. All rights reserved.
Keywords: Heat shock protein; Ecdysteroid; Transcription; Prothoracicotropic hormone; Juvenile hormone
1. Introduction
The finding that Drosophila chromosomal puffing occurred in response to thermal and chemical stress led to the discovery of the gene families coding for the pro-teins termed heat shock propro-teins (hsps) (see Petersen and Mitchell, 1985). Stress-induced puffing resulted in the transcription and translation of the hsps and the repression of normal, tissue-specific protein synthesis. Since the first studies in Drosophila, it has become clear that the heat shock response is conserved among organ-isms as diverse as bacteria and mammals, and also that homologues to the stress-induced hsps exist, which are constitutively-expressed and are abundant cellular components [heat shock cognates (hscs)]. Hscs partici-pate in a wide variety of intra-cellular processes, chiefly through their ability to bind other proteins and facilitate correct folding and/or mediate import into cellular
* Corresponding author. Tel.:+1-919-966-5535; fax: +1-919-962-1344.
E-mail address: [email protected] (R. Rybczynski). 1 The sequence has been deposited in the GenBank data base under
accession # AF194819.
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 3 1 - X
The function of hsc 70 in PTTH-stimulated ecdystero-idogenesis has proven difficult to determine. Hsc 70 pro-teins participate in a variety of cellular processes in addition to their roles in protein folding and import into organelles (see Mayer and Bukau, 1998; Fink, 1999). For instance, hsc 70s are also involved in clathrin uncoating (Chappell et al., 1986) and in vertebrate ster-oid hormone receptor assembly and function (see Pratt and Toft, 1997). Hsc 70s are also abundant proteins such that PTTH-stimulated changes in abundance must be measured against a very high background. For these reasons, a molecular approach was chosen, with the hope that determining the hsc 70 isoform distribution in the prothoracic gland and other tissues during development and under in vitro PTTH stimulation might afford further insight into the relationship between ecdysteroid syn-thesis and hsc 70 synsyn-thesis. The data indicate that the chief hsc 70 expressed in the prothoracic gland is a Man-duca homologue of the hsc 70-4 gene product of Droso-phila (Perkins et al., 1990). The translation of this hsc 70 undergoes short-term up-regulation by PTTH in the gland but longer exposure to PTTH or PTTH analogues and to 20-hydroxyecdysone (20E) result in down-regu-lation of both protein and mRNA levels, suggesting a role in the negative feedback control of ecdysteroid receptor complex assembly in the prothoracic gland (see Gilbert et al., 1997).
2. Methods and materials
2.1. Animal rearing, prothoracic gland dissections and in vitro manipulations
M. sexta were reared and staged as described pre-viously (Rybczynski and Gilbert, 1995a). The 24 h fol-lowing the molt to the fifth instar is designated as fifth instar day 1 (V1), while the 24 h following the pupal molt is designated as pupal day 0 (P0). Subsequent 24 h periods are numbered successively, e.g., V2, P1 etc.
Following dissection under insect Ringers (Weevers, 1966), glands to be treated with PTTH or other agents were transferred to spot test plate wells containing Gra-ce’s medium and pre-incubated at 25°C for 30–45 min. The medium was then removed and replaced rapidly by 25µl (incubations<2 h). For overnight incubations (14– 20 h), the glands were transferred to small Petri dishes containing 5 ml Grace’s medium. All incubations were performed under high humidity conditions at 25°C. For in vitro experiments, one of the two prothoracic glands present in each animal received the experimental treat-ment while the other gland served as a control. Control glands were incubated for the same time, in the same volume, and also experienced the same solvents or buf-fers, as the experimental glands. The juvenile hormone (JH) analogue hydroprene (gift of Zoecon/Novartis) was
dissolved in acetone (1.5 µg/15 µl) and applied to the dorsal surface of day 3 fifth instar (V3) larvae weighing 6.0 to 6.5 g. This dose of hydroprene consistently resulted in a 24 h delay in pupal commitment (Rountree and Bollenbacher, 1986; Rybczynski and Gilbert, unpub-lished observations).
2.2. Prothoracicotropic hormone (PTTH)
PTTH extracts were prepared from P1 brains of M. sexta homogenized in methionine-free Grace’s medium at 4°C (10 µl/brain) followed by boiling and centrifug-ation (Bollenbacher et al., 1979; Rybczynski and Gilbert, 1995a). The resultant supernatant contains big PTTH (>25,000 kDa) and small PTTH (,10,000 kDa) (Bollenbacher et al., 1984) which were separated by three cycles of ultrafiltration (YM10 filter: Amicon, Beverly, MA) after which the big PTTH concentrate was brought up to the starting volume with methionine-free Grace’s medium (estimated 1,000-fold dilution of small PTTH concentration in the big PTTH fraction). Methion-ine-free Grace’s medium was used to make experiments consistent with earlier studies in which 35S-methionine labeled protein synthesis was studied (e.g., Rybczynski and Gilbert, 1994; Rybczynski and Gilbert, 1995a) and PTTH aliquots were stored at280°C and discarded after two freeze-thaw cycles.
2.3. RNA isolation and construction and screening of a prothoracic gland cDNA library
Tissues were extirpated under insect Ringer at<25°C, frozen rapidly in pre-chilled (270°C) microfuge tubes and stored at280°C. Total RNA was isolated by either the hot phenol method or the SDS–urea method (Jowett, 1986).
1995) was used for additional analysis of the deduced amino acid sequence.
2.4. RNA and protein blot analysis
RNA was separated by formaldehyde gel electro-phoresis (Maniatis et al., 1982) and transferred by pass-ive diffusion to HyBond-N+ (Amersham) nylon mem-brane according to the manufacturer’s instructions. Random-primed, 32
P-labeled DNA probes were incu-bated overnight with the blots at 65°C in 50 mM PIPES (pH 6.5), 100 mM NaCl, 50 mM Na phosphate buffer (pH 7.0), 1 mM EDTA and 5% SDS, followed by 65°C and room temperature washes in 1×SSC with 5% SDS (Virca et al., 1990). Kodak X-OMAT film was exposed to hybridized filters and the resulting autoradiographs were scanned with a Molecular Dynamics Scanning Densitometer (ImageQuant program: Molecular Dynam-ics: Sunnyvale, CA).
Tissues were lysed in TEP buffer [10 mM Tris–HCl (pH 9.5), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 6 M urea, 0.2% Triton X-100, 0.2% sodium deoxycholate] by sonication at 4°C (Sonifier Cell Dis-rupter Model W140; Branson Sonic Power Co., Dan-bury, CT) for 20 s at a setting of 5 and protein content determined according to Bradford (1976) using bovine serum albumen as the standard. Proteins were then sep-arated by SDS–PAGE (Laemmli, 1970), electro-transferred to polyvinylidene difluoride membranes, fol-lowed by blocking for 1 h at room temperature in TBST [10 mM Tris–HCl (pH 7.5) with 0.9% NaCl and 0.1% Tween 20] with 1% non-fat milk. Blots were then incu-bated at 4°C for 14 to 16 h with an anti-hsp/hsc 70 monoclonal antibody (Sigma: clone BRM-22) dissolved in TBST (1/2,000). Blots were then washed 3×10 min in TBST, incubated with an alkaline phosphatase-conju-gated anti-mouse IgG (1/2,000: Sigma) for 1–1.5 h at room temperature, washed as above and signals developed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, as described previously (Rybczynski et al., 1996). Blots were scanned while wet using a Molecular Dynamic Computing Densitometer and the ImageQuant program.
2.5. Protein dephosphorylation
Prothoracic gland extracts were treated with lambda protein phosphatase (New England Biolabs), which removes phosphates from proteins phosphorylated at ser-ine, threonine or tyrosine residues (Zhuo et al., 1993), to determine if any apparent hsp/hsc 70 isoforms detected with immunoblotting were phosphorylated. Pro-thoracic glands were sonicated, as described above, 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 µg/ml aprotinin, 0.75 µg/ml pepstatin, 0.5 µg/ml leupeptin). Following sonication, samples were centrifuged for 3 min at 15,800
g at 4°C and the supernatant was incubated for 90 min
at 25°C with 400 units of lambda protein phosphatase per 50µl of supernatant (1 prothoracic gland equivalent per 10µl). Samples were then flash frozen and stored at
280°C until PAGE analysis.
3. Results
3.1. Isolation and sequence analysis of a hsc 70 cDNA clone from M. sexta
A Trichoplusia ni hsc 70 cDNA clone (Schelling and Jones, 1996) was used to screen at low stringency (i.e. 3×SSC at 50°C) <120,000 independent clones from a M. sexta prothoracic gland cDNA library. After two rounds of rescreening, 20 putative hsp/hsc 70 clones were chosen, phagemids containing the putative positive cDNAs excised and recovered, and the cDNA inserts subjected to reprobing, and restriction site mapping. Six apparent clone types resulted but partial sequencing of these clones showed that they were identical to one another. They were also highly homologous with the hsc 70-4 coding sequence of D. melanogaster (Perkins et al., 1990) and with the probe hsc sequence from T. ni (Schelling and Jones, 1996). One of these clones (phsc5) contained an insert of 2,124 base pairs, the largest among those characterized. Complete sequencing of phsc5 revealed an open reading frame of 652 amino acids (Fig. 1), coding for a protein with a deduced molecular weight of 71,431. The 85 bases preceding the putative initiation methionine contain no other initiation codon and the CAAA immediately upstream of this presumptive trans-lation initiation site matches the consensus sequence for an insect (D. melanogaster) initiation site (Caverner and Ray, 1991). A single conventional polyadenylation sig-nal (AATAA) occurs 54 bases after the stop codon (TAA), and 13 bases separate this AATAA from the polyA+ tail sequence of this clone (Fig. 1).
Comparisons of the translated sequence from clone phsc5 with D. melanogaster, T. ni and vertebrate hsp 70 and hsc 70 sequences are presented in Fig. 2 and Table 1. These analyses indicated that the sequence from M. sexta is nearly identical to the hsc gene product of T. ni, which was used at low stringency to screen our library. Like the T. ni sequence, the M. sexta hsc 70 deduced amino acid sequence shows the highest identity with the Drosophila hsc 70-4 gene product. Overall, the differ-ences between the moth sequdiffer-ences and the Drosophila sequence appear to be randomly spaced throughout their lengths, with a slight increase in frequency in the last 100 residues.
Fig. 2. Comparison of the deduced hsc 70 amino acid sequence from M. sexta (Msexta) with the hsc 70 from T. ni and hsc 70-4 from D.
melanogaster (Dmhsc4). Double dots indicate identity with the sequence from M. sexta. and dashes indicate gaps required to align the sequences.
70 contains multiple potential sites for phosphorylation by cAMP/cGMP-dependent kinases, protein kinase C and casein kinase II [Prosite analysis (Bairoch et al., 1995)] and phosphorylation of proteins of the hsp 70 family has been reported from vertebrates tissues (e.g.,
Table 1
A comparison of the deduced amino acid sequence of the M. sexta cognate heat shock protein with hsp 70 and hsc 70 sequences from
Trichoplusia ni, Drosophila melanogaster and humansa
Identity with M. (AAs)
sexta hsc
Trichoplusia hsc 98.2% 653
Drosophila hsc 4 90.5% 652
Drosophila hsc 1 82.1% 654
Drosophila hsp 1 74.5% 651
Drosophila hsp 2 74.3% 649
Drosophila hsc 3 63.7% 614
Drosophila hsc 5 50.5% 604
Human hsc 71 86.7% 652
Human hsp 70-1 82.5% 652
aThe percent identity with the M. sexta sequence (652 AAs) is
given in the middle column while the right column indicates the num-ber of amino acids in the compared sequence.
other changes using immunoblot detection. In the same samples, dephosphorylation-dependent band mobility shifts were readily detected for the ecdysone receptor components EcR and USP, as described previously by Song and Gilbert (1998). Prosite analysis also revealed the presence of a presumed ATP/GTP binding site (AEAYLGKT, at residues 131–138, that has been termed the “P-loop”: Saraste et al., 1990) and which is to be expected in a hsp/c 70 protein.
3.2. The expression of M. sexta hsc 70 in prothoracic glands and other tissues of M. sexta
Northern and immunoblot blot analyses were used to assess the abundance of hsc 70s in prothoracic glands relative to other tissues. Fig. 3 shows that the Manduca hsc 70-4 cDNA hybridizes strongly with an mRNA of about 2.4 kb in all tissues probed. In Drosophila, hsc 70-4 mRNA is <2.3 kb (Perkins et al., 1990), and the Trichoplusia hsc 70-4 homologue mRNA is about 2.0 kb (Schelling and Jones, 1996).
The expression of hsp/c 70 proteins in these same tissues was also assessed using an antibody that reacts with many cognate and stress-inducible hsp/c 70 pro-teins. These data show that several hsc 70 proteins appear to be expressed in most if not all non-stressed tissues (Fig. 3). All tissues surveyed express an hsc of 70 kDa and most also express a presumptive “hsc 70” of 72 kDa. Several tissues show additional immunoreac-tivity at<66 and 62 kDa (e.g., fat body and gut) while Malphigian tubules contain a 71 kDa putative hsp/c 70 family member. The identification of these immunoreac-tive proteins as member of the hsp/c 70 is tentaimmunoreac-tive and we can not rule out the possibility that the lower molecu-lar weight proteins are proteolytic fragments of one or more of the larger proteins. Heat shock of prothoracic glands in vitro (42°C for 1–2 h) followed by Northern
Fig. 3. The tissue distribution of hsc 70 mRNA as determined by Northern blot hybridization (5 µg total RNA/lane: lower panel) and hsc 70 protein as determined by immunoblot analysis (5 µg protein/lane: upper panel). The bottom panel shows the amount of ribosomal RNA loaded per lane (ethidium staining), as a control for loading variation. All tissues were from V4larvae. Abbreviations: PG,
prothoracic gland; M, Malphigian tubules; G, thoracic ganglia; Br, brain; SG, salivary gland; FB, fat body; Ep, epidermis; Gut, midgut; rRNA, ribosomal RNA.
analysis or immunoblotting revealed that the mRNA and hsc 70 protein changes after such treatment were insig-nificant to modest; per gland, heat shock increased hsc 70 mRNA only 13% (±20%) and hsc 70 protein increased 68% (±18%) relative to 25°C controls.
Previous work demonstrated that the levels of hsc 70 protein in the prothoracic gland change during late larval and early pupal development in a manner that suggests a role for hsc 70s in ecdysteroidogenesis and/or cell growth (Rybczynski and Gilbert, 1995a). Changes in the hsc 70 mRNA concentration in the prothoracic gland during the fifth larval instar and during early pupal-adult development were assessed using Northern blot analysis (Fig. 4A). Hsc levels were expressed per µg protein or RNA because the prothoracic gland cells undergo a large change in cell size during the developmental period stud-ied, e.g., <10-fold change in protein content (Rybczynski and Gilbert, 1995b), making per gland comparisons difficult to interpret. Hsc 70 mRNA and protein levels appear well correlated during the fifth lar-val instar but diverge somewhat in early pupal-adult development. The maximum hsc 70 mRNA level, per
Fig. 4. Hsc 70 mRNA and protein levels during the fifth larval instar and early pupal-adult development. Data are derived from densito-metric scans of immunoblots or autoradiographs of Northern blots probed with an anti-hsc 70 monoclonal antibody or with the radiolab-eled M. sexta hsc 70 cDNA, respectively. Levels are expressed as a percentage of the maximum concentration measured during this devel-opmental period. (A) hsc 70 mRNA and hsc 70 protein (protein data from Rybczynski and Gilbert, 1995a) in the prothoracic gland. (B) hsc 70 mRNA in the brain and fat body. Tissues from at least five animals were pooled for each time-point. Two separate series of prothoracic glands were collected for Northern blots and for the immunoblots. Note that the actual concentration of hsc 70 mRNA perµg RNA com-prising the peaks is tissue-specific (see also Fig. 3). The onset of larval wandering is denoted by the arrowhead.
levels were also determined for two non-steroidogenic organs, brain and fat body (Fig. 4B). Neither organ shows a pattern of hsc 70 mRNA expression matching that seen in the prothoracic gland. In brain, hsc 70 mRNA peaks occur just after the molt from fourth to fifth larval instar (V1) and just after the larval-pupal molt (P0and P1). In fat body, hsc 70 mRNA levels are highest just before, and shortly after, the larval-pupal molt. Earl-ier work indicated also that hsc 70 protein levels in the prothoracic gland were not correlated with levels
meas-ured in brain and fat body (Rybczynski and Gilbert, 1995a).
3.3. Hormonal regulation of hsc 70 mRNA in the prothoracic gland
The possibility that the developmentally-specific changes in hsc 70 mRNA described above were hor-monally controlled was explored in a series of in vitro and in vivo experiments using partially purified PTTH, 20E and hydroprene, a slowly metabolized JH analogue. Two developmental periods were investigated, the first being the three days centered on the commitment peak (V3in our colony). At this time a PTTH-stimulated small increase in circulating ecdysteroids occurs in the relative absence of juvenile hormone and results in the “commit-ment” of larvae to develop into pupae at the next molt (Riddiford, 1976). The second period chosen was the first day of pupal-adult development, a period charac-terized by extensive remodeling of the organism to pro-duce an adult moth.
Short-term (2 h) exposure in vitro of prothoracic glands to PTTH resulted in a decrease in hsc 70 mRNA levels on V3 but no changes were noted with V4 or P0 glands (Fig. 5A). However, a longer exposure (14 h) to dibutyryl cAMP, which mimics the effects of PTTH (Rybczynski and Gilbert, 1994), resulted in decreased prothoracic gland hsc 70 mRNA levels at all three stages studied (Fig. 5B). Dibutyryl cAMP was used to conserve PTTH because of the larger incubation volumes employed (5 ml vs 25 µl) during these longer incu-bations. Small incubation volumes may yield results reflecting the influence of ecdysteroids produced by the gland during the incubation rather than the direct effect of PTTH.
When V2 prothoracic glands were exposed for 20 h in vitro to 150 nM 20E, a level normally seen at the V3 commitment peak (Grieneisen et al., 1993), hsc 70 mRNA levels decreased by more than 60% (Fig. 5C). Exposure of P0 prothoracic glands to 1µM 20E, a level present normally at stages P3to P4 (Warren and Gilbert, 1986), also resulted in a large drop in prothoracic gland hsc 70 mRNA levels (<40%: Fig. 5C). The purpose of these 20E experiments was to determine the steroid’s possible effects on hsc 70 mRNA levels but the effect of 20E on hsc 70 protein was also addressed briefly. V5 prothoracic glands incubated for 10 to 24 h with 10 µM 20 E, a physiological concentration on V7, respond by changing expression of the ecdysone receptor proteins EcR and USP, an event that is associated with down-regulation of ecdysteroid synthesis (Song and Gilbert, 1998). Hsc 70 protein levels in glands so treated decline considerably (44±3% of control) in line with the hypo-thesized negative feedback of 20E on ecdysteroid syn-thesis (Song and Gilbert, 1998).
commit-Fig. 5. The effects of PTTH, dibutyryl cAMP and 20E on prothoracic gland hsc 70 mRNA levels in vitro. (A) hsc 70 mRNA levels after 2 h in vitro incubation with PTTH (0.25 brain equivalents) (B) hsc 70 mRNA levels after 14 h in vitro incubation with 5 mM dibutyryl cAMP. PTTH stimulates cAMP production and dibutyryl cAMP mim-ics the effect of PTTH on prothoracic gland protein synthesis and ecdy-steroid synthesis. (C) hsc 70 mRNA levels after 20 h incubation with 20E in vitro. V2and P0 prothoracic glands were incubated with 150
nM 20E and 1µM, respectively. In these experiments, one prothoracic gland from an animal received the experimental treatment while the other gland served as a control.
ment peak (early V3) retards development (Rountree and Bollenbacher, 1986) and a topical application of 1.5 µg hydroprene delayed larvae by about two days in reaching the aphagic, wandering phase (data not shown). No dif-ference was found between control and treated protho-racic gland hsc 70 mRNA levels when animals were sampled 24 h after the topical application (Fig. 6) but a large increase seen 48 h later in control animals was clearly inhibited by hydroprene application. Incubation of prothoracic glands in vitro (2–24 h) with a similar concentration of hydroprene did not result in detectable changes in hsc 70 mRNA levels (data not shown).
4. Discussion
Previous work demonstrated that translation and/or accumulation of a member of the 70 kDa heat shock protein family is specifically up-regulated, relative to nearly all other proteins, in prothoracic glands exposed to PTTH (Rybczynski and Gilbert, 1994, 1995a). Mem-bers of this protein group function in many cellular cesses including folding of nascent and misfolded pro-teins, protein translocation across intra-cellular membranes, protein degradation, vesicle trafficking, and, vertebrate steroid hormone receptor assembly and func-tion (see Pratt and Toft, 1997; Mayer and Bukau, 1998; Fink, 1999). To understand the possible function(s) of hsc 70 in the PTTH transductory cascade and ecdys-teroid synthesis, a molecular approach was used to deter-mine the hsc 70 forms present in the M. sexta prothoracic gland. A number of cDNA clones were isolated and par-tial sequences indicated that all clones were homologous to the Drosophila hsc 70-4 isoform (Perkins et al., 1990), with lesser but still high homology to other hsc/p 70
Fig. 6. The effects of the juvenile hormone agonist hydroprene on prothoracic gland hsc 70 mRNA levels in vivo. Hydroprene (HYD: 1.5µg/15µl acetone) was applied to the dorsal surface of V3larvae
family members. Northern blot analysis indicated that hsc 70 mRNA is expressed in M. sexta in all tissues surveyed. The data suggested also that this hsc 70 is the predominant constitutively expressed hsp 70 family member in Manduca tissues, as is the hsc 70-4 transcript in Drosophila (Palter et al., 1986). If other isoforms are expressed in the prothoracic gland or the other tissues probed, their mRNAs are either too rare to detect using a heterologous probe or are of the same size as hsc 70. The failure to find a very large increase in an hsc 70 cross-hybridizing mRNA following heat shock suggests that Manduca may lack the diversity of hsp 70 genes seen in Drosophila but this interpretation must be tem-pered by the observation that tissue-specific differences in true heat shock protein expression occur in Manduca (Fittinghoff and Riddiford, 1990). Regardless of hsp/c 70 gene diversity, it seems likely that Manduca exhibits a thermal stress response different from that of Droso-phila melanogaster, as suggested by observations that heat shock inhibits global protein translation in Manduca much less than in Drosophila (Fittinghoff and Riddiford, 1990; Rybczynski and Gilbert, 1995a).
Considerable variations in hsc 70 mRNA and hsc 70 protein levels were found among the organs examined, regardless of the apparent ubiquity of hsc 70 expression. At stage V4, one day past the ecdysteroid commitment peak, the hsc 70 mRNA level in the prothoracic gland was higher than that seen in the seven other organs sur-veyed and hsc 70 protein levels were also in the upper range. Developmental studies of hsc 70 mRNA levels in the M. sexta prothoracic gland revealed peaks at the onset of wandering (V5) and at V7, the period of fifth instar maximum ecdysteroid synthesis. A peak was also found during early pupal-adult development (P2), that was observed also in fat body. In contrast, hsc 70 protein in the prothoracic gland exhibited peaks at (1) V4, shortly after the “commitment peak” of ecdysteroids that determines that the result of the next molt will be a pupa rather than a larva (Riddiford, 1976); (2) V7, the period of maximum fifth instar ecdysteroid synthesis (Grieneisen et al., 1993); and (3) P1, just after the meta-morphic molt. Previous work revealed that hsc 70 pro-tein in the brain and fat body varied little during this developmental period, in contrast to the observed vari-ation in the prothoracic gland (Rybczynski and Gil-bert, 1995a).
The prothoracic gland is subjected to a variety of fac-tors that directly or indirectly affect its function as an endocrine gland. Demonstrated modulators of gland function are several hormones, i.e. PTTH, which directly stimulates ecdysteroidogenesis (see Henrich et al., 1999); JH, which may act directly or indirectly and usu-ally down-regulates basal and PTTH-stimulated ecdys-teroidogenesis (e.g., Rountree and Bollenbacher, 1986; and Lonard et al., 1996); and 20E, which directly down-regulates basal and PTTH-stimulated
ecdysteroidogen-esis under in vitro conditions (Song and Gilbert, 1998). These same hormones were found to have effects on hsc 70 mRNA levels after manipulations of the gland’s hor-monal environment in vitro or in vivo. Short-term exposure (2 h) of glands to PTTH resulted in a .40% decrease in hsc 70 mRNA levels in V3 glands but no effect was detectable with V4 or P0 glands. All three stages did respond with lower mRNA levels (30 to 70% of control) when dibutyryl cAMP was used as an ecdys-teroidogenic agent in longer (14 h) incubations. The JH analogue hydroprene had no in vitro effect on hsc 70 mRNA levels but in vivo application to fifth instar larvae did block a rise seen in control, wandering animals. These changes in hsc 70 mRNA levels do not reflect an overarching change in gland RNA levels sinceβ1 tubu-lin mRNA levels in these experiments did not change in the same way. For example, incubation of V2prothoracic glands with V3 levels of 20E evoked an increase in β1 tubulin mRNA contra to the effect on hsc 70 mRNA and in vivo hydroprene treatment had no effect onβ1 tubulin mRNA levels (Rybczynski and Gilbert, 1995b). These data indicate a potential for complex control of hsc 70 expression via hormones, all of which had a negative effect on hsc 70 RNA levels. However, since hsc 70 levels are not constantly decreasing in vivo during the developmental period studied it is obvious that additional factors or complex multi-hormone interactions are likely to participate in this control. It is also evident that hsc 70 mRNA and protein do not necessarily change in concordance during development (see Fig. 4) or dur-ing short-term in vitro manipulations, i.e. PTTH exposure had no effect or negative effects on hsc 70 mRNA levels in glands (this study) while hsc 70 protein synthesis is typically increased by about 60% under such conditions (Rybczynski and Gilbert, 1994, 1995a). These results suggest that there exists a threshold concentration of hsc 70 protein above which hsc RNA levels are down-regulated. Such a phenomenon has been reported for stress-induced hsp 70 mRNA in Drosophila cells (DiDomenico et al., 1982) but appears to be unknown for cognate members of the hsp 70 gene family.
receptor proteins EcR and USP, probably before the mature heteromeric, DNA-bound state is achieved (Q. Song, R. Rybczynski and L.I. Gilbert; unpublished observations). Thus, the hormonally-controlled changes in hsc 70 transcription and translation demonstrated here could be involved in controlling the dynamics of ecdy-sone receptor assembly and turnover. The abundance and isoform composition of the ecdysone receptor, in turn, may be crucial in modulating ecdysteroid synthesis since Song and Gilbert (1998) have shown that 20E can initiate a negative feedback loop resulting in decreased ecdysteroid synthesis in vitro. The resolution of the func-tion of hsc 70 in ecdysteroidogenesis or other cellular processes will require development of model systems in which hsc 70 levels can be readily manipulated under a variety of conditions.
Acknowledgements
We thank Virginia Mergner for her assistance with the molecular biology experiments and Susan Whitfield for graphics. This research was supported by NIH grant DK-30118.
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