Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland

e

bhcularsnd ccl lulrrr Endoclinoloey

Molecular and Cellular Endocrinology

I15 (1995) 73-85

Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland Robert Rybczynski, Lawrence I. Gilbert* Department of Biology, Coker Hall CB # 3280. University of‘ North Carolina, Chapel Hill, NC 27599, USA

Received 8 June 1995; accepted 28 August 1995

Abstract In Manduca sesta, ecdysteroids coordinate molting and metamorphosis of insects and are produced by the prothoracic glands under the acute control of the brain neuropeptide prothoracicotropic hormone (PTTH). PTTH stimulates rapid ecdysteroidogenesis accompanied by specific increases in the synthesis and accumulation of three proteins, including one with M, = 70 kDa. This 70-kDa protein is a constitutively expressed member of the heat shock protein 70 family (hsc 70). Levels of this hsc 70 vary in a prothoracic gland-specific manner during development as does its PTTH-stimulated synthesis when assayed in vitro. The accumulation of hsc 70 may be regulated by abrupt changes in its turnover rate. The PTTH-stimulated increase in hsc 70 synthesis is dependent upon both translational and transcriptional events. Hsc 70 expression in the prothoracic gland may be required for changes in gland growth, e.g., protein content, that underlie alterations in ecdysteroid production. Keywords:

Ecdysteroid;

Heat shock protein; Prothoracicotropic

hormone; Prothoracic

gland (Manduca

sexta);

Steroidogenesis;

Translation

1. Introduction Steroid hormones play important roles in the development and homeostasis of both vertebrates and invertebrates, and their synthetic control has many features in common. In both taxa steroid hormones are produced from cholesterol, and acute increases in steroid hormone synthesis are triggered by peptide hormones, produced in the case of insects by specific brain neurosecretory cells (see Gilbert et al., 1988). Upon interacting with target tissue receptors, these peptides initiate a cascade of events that results in increased steroid hormone synthesis and export; long-term effects documented in vertebrates include increased transcription and translation of some genes and mRNAs, such

Abbreviations: hsc 70, 70 kDa constitutively expressed (cognate) heat-shock protein; hsp 70, 70 kDa stress-induced heat-shock protein; PAGE, polyacrylamide gel electrophoresis; PTTH prothoracicotropic hormone; 1D PAGE, one-dimensional polyacrylamide gel electrophoresis; 2D PAGE, two-dimensional polyacrylamide electrophoresis. * Corresponding author. 030X-7207/95/$09.50

Q 1995 -

SSDl0303-7207(95)03672-T

as those encoding steroidogenic enzymes (see Simpson and Waterman, 1988). Among the common features of this cascade are: increased generation of CAMP and in some cases, increases in intracellular free Ca’+; activation of kinase(s) resulting in the phosphorylation of specific proteins, and changes in protein translation (see Gilbert et al., 1988; Orme-Johnson, 1990). Studies in vitro indicate that peptide-stimulated protein synthesis plays a crucial role in the phenomenon of peptide-stimulated steroid hormone synthesis in vertebrates and invertebrates. That is, translation blockers such as cycloheximide or puromycin inhibit peptide-elicited steroid synthesis although basal steroidogenesis is unaffected by such inhibitors (see Keightley et al., 1990; Orme-Johnson, 1990; Rybczynski and Gilbert, 1995). These and other observations led to the discovery of three small ( I 30 kDa) proteins in mammalian systems whose rapid syntheses are postulated to be necessary and rate-limiting for peptide-stimulated steroid hormone synthesis (see Orme-Johnson, 1990). The exact mechanism(s) by which any of these proteins facilitate steroidogenesis is not clear but these observations led us to examine the

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R. Ryhczynski, L.i. Gilbert / Molecular and Celluhr Endocrinology 115 (199.5) 73-85

possibility that analogous regulatory proteins exist in the insect system (Rybczynski and Gilbert, 1994). Ecdysteroids are polyhydroxylated steroids that in conjunction with juvenile hormones, control the molting process of insects and which, directly or indirectly. affect the transcription of many genes (e.g., Andres et al.. 1993; Hurban and Thummel, 1993). In moths, and presumably other insects prothoracicotropic hormone (PTTH), a brain neuropeptide stimulates acute increases in the synthesis of ecdysteroid hormones by the prothoracic glands, paired glands of about 220 cells each (see Gilbert et al., 1988). Our earlier studies of the tobacco hornworm, Munduca sexta. revealed that the increased syntheses of three medium-sized proteins are specifically associated with PTHH-stimulated ecdysteroid synthesis (Rybczynski and Gilbert, 1994). Their special roles in ecdysteroidogenesis are currently unknown but it seems unlikely that any of the three are functional homologs of the vertebrate proteins (Rybczynski and Gilbert, 1994). The smallest, 52 kDa has been recently identified as a /?-tubulin which may interact with the cytoskeleton of prothoracic gland cells so as to promote the inter-organelle movements of ecdysteroid precursors or export of the final products (Rybczynski and Gilbert, 1995). In this report, we identify a second protein, 70 kDa, as a constitutively expressed member of the heat shock protein 70 family (heat shock cognate or hsc 70), describe its developmental and PTTH-stimulated expression in the prothoracic gland of Munduccl under in vivo and in vitro conditions and suggest possible roles for hsc 70 in ecdysteroidogenesis. 2. Materials and methods 2.1. Animals Manduca sexta larvae were group reared under a 168 light-dark cycle at z 25°C; and at 2 60% humidity (Vince and Gilbert, 1977). Fifth instar larvae were staged by timing from head capsule slippage, weight class and morphological markers (Goodman et al., 1985). Pupae were staged by timing from the appearance of a complete pupal cuticle.

2.2. Reugen ts Grace’s medium (standard and methionine-free) was obtained from Gibco BRL (Grand Island, NY, USA) and brought to pH 6.8-7.0 with 3 M KOH or 2 M K+-Hepes (pH 8.0). Medium was filter-sterilized and stored at 4°C. [3H]Ecdysone (specific activity, 80 Ci/ mmol) was obtained from New England Nuclear (Boston, MA, USA). The monoclonal antibody to HSP 70 was obtained from Sigma (St. Louis, MO, USA). Tran3?S-Label ( > 1000 Ci/mmol; z 70% 3sS-methionine and z 15% 35S-cysteine: ICN, Irvine, CA, USA) was used to radiolabel proteins during in vitro tissue incubations. Bioactive, recombinant Bombyx mori PTH

was the generous gift of Dr. David R. O’Reilly (Imperial College of Science, Technology, and Medicine; London). 2.3. Prothoracicotropic hormone (PTTH) PTTH extracts were prepared from day 1 pupal (P,) Munduca brains. Brains were homogenized in methionine-free Grace’s medium at 4°C (10 or 20 pl/ brain) (Bollenbacher et al., 1979). The homogenate was heated to 95°C for 4-5 min, cooled to 4°C and centrifuged for 10 min at 15 000 x g at 4°C. This supernatant contains both big PTTH ( 2 25 000 kDa) and small PTTH ( < 10 000 kDa) (Bollenbacher et al., 1984). The two PTTHs were separated by ultrafiltration through a YMlO filter (Amicon) followed by two washes, after which the big PTTH concentrate was brought up to the starting volume (10 @l/brain) with methionine-free Grace’s medium. Experiments utilized big PTTH at a concentration of 0.25 to 1 brain equivalent per prothoracic gland. PTTH preparations were stored as aliquots at - 80°C and aliquots were discarded after two freezethaw cycles. 2.4. Dissection of prothoracic glands and 35S-labelting of proteins

Larvae were sacrificed as described previously (Rybczynski and Gilbert, 1994). Prothoracic glands were extirpated under insect Ringer’s (Weevers, 1966) at 25°C and transferred to individual wells (2 cm diameter) of a ‘spot test plate’ containing 100&200 ~1 Grace’s medium. The concentration of methionine in the medium was 5 Llg/rnl (10% of the concentration in complete Grace’s) to facilitate ?S-methionine labelling of newly synthesized proteins and support maximal ecdysteroid synthesis in stimulated glands (Rybczynski and Gilbert, 1994). Glands were equilibrated by preincubation at 25°C for 30-45 min after which the preincubation media was removed and replaced rapidly by 50 {tl of Grace’s medium (5 pg/ml methionine) containing any experimental substance(s). ?j-Methionine was included at lo-50 PCi per 50 ~1. The contralateral prothoracic gland from the same animal was used as the control. Preincubations and incubations were carried out under high humidity conditions in a covered container placed in a 25°C chamber. At the end of an experiment, glands were removed from the incubation medium frozen rapidly and stored at - 80°C. Pulse-chase experiments followed the above described protocol with additional steps to remove PTTH and/or ‘?S-methionine following the pulse period. At the end of the labelling period, the medium was rapidly removed and replaced with 100 ~1 of complete Grace’s medium (50 /lg methionineiml). After 2 min, this medium was removed and replaced with 200 ~1 of complete Grace’s medium (50 pg methionine/ml). At the end of this chase, the glands were collected and stored at - 80°C.

R. Ryhczynski, L.I. Gilbert

: Molecular and Cellular Endocrinolog! 115 (1995) 73.-85

Experiments to determine the transcription and translation requirements for PTTH-stimulated hsc 70 accumulation utilized the drugs cycloheximide and actinomycin D at 10 ,uM (Keightley et al., 1990; Rybczynski and Gilbert, 1995). Cycloheximide _t PTTH was added at the beginning of the 30-min experimental period along with “?S-methionine. Actinomycin D treatments were the same as for cycloheximide except for the additional inclusion of actinomycin D to a concentration of 10 ,L~M in the last 15 min of a 45-min preincubation period. 2.5. Heut shock Prothoracic glands or other tissues were removed from fifth instar larvae as described above. After a 30-min preincubation period at 25°C the medium was removed and replaced with medium containing ‘?S-methionine as usual. Control tissues were maintained at 25°C while the experimental samples were covered with a piece of H,O-saturated filter paper wrapped around a thin glass plate and partially submerged in a 42°C water bath. Under in vitro conditions 42°C evokes a full Man&cu heat shock response in isolated tissues (Fittinghoff and Riddiford, 1990); cells die rapidly at slightly higher temperatures (Fittinghoff and Riddiford, 1990; Rybczynski and Gilbert, unpublished data). Covering the heat shock samples with HzO-saturated filter paper minimized evaporative loss from the small volumes used for labeiling (50-100 ~1) since substantial evaporation was fatal to cells, presumably through hyperosmotic shock. 2.6. Sumple preparution

urzd analysis

oj~ protein

svnthesis

Prothoracic glands were homogenized in I 70 ~11 ice-cold prothoracic gland homogenization buffer (10 mM TrissHCl (pH 9.5), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 6 M urea 0.2% Triton X-100, 0.2% sodium deoxycholate) with a tube-fitting teflon pestle, or directly boiled in SDS sample buffer (see below). Homogenization was followed by sonication at 4°C with the tube in direct contact with the submerged microprobe of a Sonifier Cell Disrupter (Model W140; Branson Sonic Power Co. Danbury CT), for 20 s, at a setting of 5. The protein content of homogenized tissues was determined according to Bradford (1976) using bovine serum albumin as the standard. ?S-Methionine incorporation into newly translated protein was assayed by precipitation in trichloracetic acid (TCA) as described (Rybczynski and Gilbert, 1994). The remaining samples were stored at - 80°C. 35S-Methionine incorporation results are presented as the mean f SEM. 2.7. Gel electrophoresis The standard Laemmli (1970) SDS-polyacrylamide gel electrophoresis (PAGE) system was employed to

15

separate proteins by molecular weight and standard gels were 8.5% total acrylamide (acrylamide:bis-acrylamide = 36.5:1). Protein samples were boiled for 5 min in sample buffer (4.6% SDS). For most radiolabelled samples, the same amount of TCA precipitable counts/ min was loaded in each lane of the gel. After PAGE proteins were transfered to a membrane for immunoblotting (see below) or gels were fixed, stained with Coomassie Blue, dried onto filter paper and exposed to Kodak X-Omat film for 16 h to 5 days at room temperature as described (Rybczynski and Gilbert 1994). Autoradiographs were scanned with a Computing Densitometer (Model 300A; Molecular Dynamics; Sunnyvale, CA). Results are calculated as ratios: the densitometric band intensity in a sample from a stimulated bland divided by the band density in the sample from its unstimulated contralateral partner. Means are presented + SEM. Two-dimensional (2D) PAGE was performed essentially as described by O’Farrell (1975). A radiolabelled control or PTTH-stimulated prothordcic gland was homogenized directly in isoelectric focusing sample buffer at 20 /Al per gland. The homogenate was then centrifuged at 15 000 x g at room temperature for 5 min to remove insoluble debris. The supernatant was loaded onto IEF tube gels (9.0 M urea, 1.4% pH 4-6 ampholines and 0.3% pH 5-7 ampholines, 2% NP 40 and 0.9% CHAPS) that had been prefocused (15 min each at 200, 300 and 400 V); the tube gels were 6 cm long and 2 mm in diameter. Samples were focused for 6 h at 500 V using 50 mM NaOH and 50 mM H,PO, as the cathode and anode solutions. Extruded tube gels were incubated for 10 min in SDS sample buffer at room temperature with gentle shaking, and placed at - 80°C until needed. Stored tube gels were thawed to room temperature and shaken slowly for lo-15 min with a new aliquot of SDS sample buffer. SDS-equilibrated tube gels were sealed on top of the second dimension SDS gel (I .5 mm thick; 8 x 10 cm) with electrophoresis buffer containing 1%) agarose. Second dimension gels included a z 5-mm stacking gel (3.5% acrylamide). 2.8. Irnrnunoblotting Proteins separated by SDS-PAGE were electrotransferred to nitrocellulose or polyvinylidene difluoride membranes for 60-85 min at 250-200 mA constant current in 15% methanol, 25 mM Tris and 192 mM glycine. Filters were blocked 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. Filters were incubated with the primary monoclonal antibody dissolved in TBST (l/l 000) for 13- 15 h at 4°C followed by three 4-min washes in room temperature TBST and incubation with a horseradish peroxidase conjugated anti-mouse IgG (l/1000: Sigma) for l-l.5 h at room temperature. After two 4-min washes in TBST

76

R. Rybczynski. L.I. Gilbert 1 Molecular and Cellular Endocrinology 115 (1995) 73-85

B.

Hsp 70 lmmunostaining Prothoracic Gland

Fat Body

Dtub

Pm4

-

+

con CAMP HS

Fig. 1. Stimulation of p70 synthesis by PTTH and dibutyryl CAMP. The amount of protein loaded was normalized to equalize the amount (countsimin) of “S-methionine in each lane. (A) PTTH stimulates the specific synthesis of three proteins in V,,, prothoracic glands, including one of 70 kDa. Glands were incubated for 30 min with ?+methionine i_ PTTH and proteins were separated on an 8.5% SDS gel with 4 M urea. (B) An anti-hsp 70 antibody recognizes proteins of 70 and 72 kDa in Manduca tissues. Proteins were separated on an 8.5% SDS gel and transferred to a polyvinyhdene difluoride membrane. The membrane was probed with an anti-hsp 70 monoclonal antibody followed by a horseradish peroxidase-conjugated second antibody and diaminobenzidine staining. The 70-kDa hsp co-localizes with the PTTH-stimulated p70 of the prothoracic gland. A second immunoreactive protein at 72 kDa is readily detectable in tissues other than the prothoracic gland, such as the fat body. (C) Dibutyryl CAMP also stimulates p70 synthesis and p?O exhibits the same molecular weight as a TO-kDa heat shock protein. ~100 stimulation is also visible but these Vz glands are too young to exhibit the strong PTTH-stimulated /?-tub&n synthesis typical of older glands (Rybczynski and Gilbert, 1995). Control (con) and dibutyryl CAMP (CAMP)-treated glands were incubated for 1 h with j5S-methionine. Heat shocked glands (HS) were incubated for 1 h at 42°C with ‘sS-methionine.

and one 4-min wash in TBS, signals were developed in diaminobenzidine-HCl (0.8 mg/ml), NiCl, (0.4 mg/ml) and H,O, (0.3 ,~l of 30%/ml) in 10 mM Tris-HCl (pH 7.5). Immunoblots were scanned while wet. Autoradiographs of dry filters were obtained after immunostaining. Radioimmunoassay (RIA) The medium was removed at the end of a prothoracic gland incubation and stored at - 20°C. Thawed samples were centrifuged at 15 000 x g for 5 min and aliquots of the supernatant assayed in duplicate for ecdysteroid content as described previously (Warren and Gilbert, 1986 1988). When necessary, samples were diluted with Grace’s medium to place the sample values within the limits of the standard curve. The antibody (S-3) used for RIA (courtesy of Prof. Sho Sakurai, Kanazawa, Japan) has high affinity for ecdysone 20-hydroxyecdysone and 3-dehydroecdysone (Kiriishi et al. 1990).

3. Results 3.1. p 70 is an hsc 70 protein In the initial description of PTTH-stimulated synthesis of a 70-kDa protein in prothoracic glands (Rybczynski and Gilbert 1994), it was suggested that this protein (~70) is a heat shock 70 family member based on the following evidence: (1) the molecular weight was appropriate and was identical to an z 70-kDa polypeptide whose synthesis was strongly stimulated by conventional heat shock; (2) reports that a vertebrate steroidogenesis activating protein, while much smaller in size ( < 10 kDa), was highly homologous at the amino acid level with a portion of the heat shock family member GRP 78 (see Orme-Johnson, 1990); and (3) preliminary data suggesting that heat shock might stimulate ecdysteroid synthesis. Also suggestive was the observation that PTTH stimulated general protein synthesis in the prothoracic gland (Rybczynski and Gilbert, 1994; 1995) because heat shock cognate (hsc 70) proteins play a part in the folding of nascent proteins and their import

R. R.vhczynski, L.I. Gilherr I Moleculur and Cellular Endocrinology I15 (1995) 73-85

into

intracellular

organelles

(see Hendrick

and Hartl,

1993; Becker and Craig, 1994). Fig. 1A shows the effect of big PTTH on the synthesis of ~70 by larval prothoracic glands of day 3.5 of the fifth instar (V,,) as visualized by PAGE and autoradiogrdphy of 35S methionine-labelled proteins; in this example p70 synthesis has been stimulated by z 45”/0 over the control. Also visible are increases in the other two proteins (~100 and /I-tubulin) whose syntheses/accumulation are stimulated by PTTH (Rybczynski and Gilbert, 1994, 1995). Fig. 1B demonstrates that a monoclonal antibody that recognizes both stress-inducible and cognate hsp 70 family members in vertebrates and Drosophila recognizes two proteins in Manduca tissues. The 70-kDa protein whose synthesis is upregulated by PTTH (Fig. IA) co-localizes with the smaller ( z 70 kDa) of the two proteins. The larger protein ( z 72 kDa) does not co-localize with a PTTH- or heat shockinducible 35S-methionine-labelled protein and probably represents a constitutively expressed hsp 70 family member (heat shock cognate (hsc)), i.e., an hsc 70 protein such as hsc 3 or hsc 5 of Drosophila (Rubin et al., 1993). This 72-kDa hsp 70 immunoreactive band is difficult to detect in prothoracic gland homogenates but is apparently much more abundant in fat body (Fig. 1B). Fig. 1C reveals the effect of dibutyryl CAMP (CAMP) and 42°C heat shock (1 h) on V, prothoracic gland protein synthesis; note that the strong p-tubulin response to PTTH (Fig. 1A) (V,_, glands) is developmentally specific and is not evoked in V2 glands (Rybczynski and Gilbert, 1995). It is clear that the ~70, whose synthesis is stimulated by PTTH or dbcAMP at 25°C occurs at the same position in the gels as does an z 70-kDa heat shock protein. This protein is the largest of three proteins of z 62-70 kDa whose syntheses are strongly stimulated by heat shock of the prothoracic gland (Fig. 1B) as well as other larval tissues not shown (i.e., brain, salary gland, fat body and epidermis). The gel pattern of proteins whose syntheses are specifically stimulated by heat shock of the prothoracic gland in vitro is nearly the same as reported by Fittinghoff and Riddiford (1990) for several other Manduca tissues subjected to a similar regimen. However, we detected three heat shock proteins of z 62270 kDa M, rather than the two reported by Fittinghoff and Riddiford (1990); presumably this reflects differences in resolving power between our gels (8.5% acrylamide vs. 12% acrylamide) for this molecular weight range. A larger heat shock protein of z 84 kDa appears to be constitutively expressed by the prothoracic gland and other tissues but its synthesis did not change in a consistent manner in glands stimulated with PTTH or other ecdys-

teroidogenic agents. Several other prothoracic gland proteins also appear to change with PTTH or CAMP stimulation. e.g., = 50 kDa in Fig. 1A and z 50 and 80

71

kDa in Fig. lC, as well as two smaller proteins ( z 32 and 28 kDa) not shown. Whether or not changes in these four proteins are consistent and prothoracic gland-specific responses to PTTH has not yet been fully determined. To determine more rigorously if the p70 prothoracic gland protein whose synthesis is stimulated by PTTH is an hsp/hsc 70 family member, 2D PAGE followed by immunoblotting with the anti-hsp 70 antibody was employed to test for the possible identity of “S-methionine-labelled p70 from PTTH-stimulated prothoracic glands. This analysis (Fig. 2) indicates that the PTTH-stimulated p70 protein is indeed a heat shock protein of the hsp 70 family since the radiolabelled PTTH-stimulated p70 co-localizes exactly with the 70kDd hsp 70 immunoreactivity. When similar experiments were carried out using proteins from heat shocked prothoracic glands or other tissues, the strongly induced, radiolabelled p70 co-localized also with the 70-kDa hsp immunoreactivity. These data reveal that the prothoracic glands and other larval Munduca tissues constitutively express an hsp 70 gene whose translation is stimulated by heat shock but which is also uniquely stimulated by PTTH in the prothoracic glands (see below). Regardless of the observation that the hsp 70 family member whose translation in Manduca prothoracic glands is stimulated by PTTH shows a still greater response to heat shock it will be referred to as a heat shock cognate (hsc 70) based on its relatively high level of basal synthesis in the absence of overt stress, as judged by ?S-methionine incorporation (Rybczynski and Gilbert, 1994) and facile detectability in immunoblots. This 2D gel analysis also indicated that it would be valid to use ID SDS gels to assay hsc 70 since no other immunoreactive or ‘S-methionine incorporating protein of this molecular mass was detectable under this experimental regimen. The stimulation of both hsc 70 synthesis and ecdysteroid synthesis by the prothoracic glands can be also elicited by dibutyryl CAMP (Fig. 1C) or a calcium ionophore (A231 87) as well as by PTTH (Rybczynski and Gilbert, 1994; see also Gilbert et al., 1988). This response is characteristic of, and specific to the prothoracic glands. Other tissues such as salivary glands do not exhibit increased synthesis of hsc 70 in response to these agents (Fig. 3). Furthermore, the prothoracic gland itself does not up-regulate hsc 70 synthesis in response to peptide preparations from tissues other than brain. the only confirmed source of biologically active PTTH (Fig. 3). In fact recombinant Bombyx PTTH. which does not elicit steroidogenesis in Manduca prothoracic glands (unpublished data), is similarly ineffective at stimulating hsc 70 synthesis in that organ (Fig. 3). 20-Hydroxyecdysone also had no discernible elTect on prothoracic gland hsc 70 expression when glands were incubated with a series of concentrations of

R. Rvbczyski,

78

L.I. Gilbert 1 Molecular and Cellular Endocrinology I15 (1995) 73-85

Autoradiograph

lmmunoblot

Autoradiograph

lmmunoblot

c 0 N T R 0

L

P T T H

Fig. 2. Identification of p70 as a member of the hsp 70 protein family using 2D PAGE. Following electrophoresis proteins from control (upper panels) or PTTH-stimulated (lower panels) prothoracic glands were transferred to a polyvinylidene difluoride membrane and probed with an anti-hsp 70 monoclonal antibody. After autoradiography of the immunoblots, they were reprobed with an anti-P-tubulin (13T) monoclonal antibody (Amersham) to provide reference points. The PTTH-stimulated 35S-methionine-labe11ed protein p70 (left panels) exactly co-localized with the 70-kDa hsp/hsc 70 immunoreactivity (right panels). Note that, as described previously (Rybczynski and Gilbert, 1995), much of the tubulin failed to fully enter into and focus in the first dimension (isoelectric focusing) gel and is located in the vertical streak on the left side of the 2D blots.

20-hydroxyecdysone spanning those typically produced in vitro during incubations of 5 2 h. This conclusion is strengthened by developmental studies of hsc 70 and ecdysteroid synthesis. For example, the hsc 70 synthesis response in vitro to PTTH varies between stages of the fifth instar (e.g., V, vs. V,,,: presented below) that produce similar amounts of ecdysteroids. In addition,

Salivary Gland

Prothoracic Gland

3.2. Hsc 70 expression in the prothoracic gland during the last larval instar, pupal and pupal-adult stages

hsc 70 I -+

Treatment

stages with very different basal or stimulated ecdysteroid production can exhibit a similar hsc 70 response to PTTH (Rybczynski and Gilbert, 1994). The synthesis of an hsc 70 protein in prothoracic glands challenged by PTTH suggests that hsc 70 synthesis could be related to ecdysteroidogenesis. At present this problem can only be in situ or in vitro correlates with prothoracic gland ecdysteroid synthesis, with some other aspect of prothoracic gland biology, or with broad patterns of protein/hsc 70 expression that are not specific to the prothoracic gland.

Manduca PlTH

-+

Flight Muscle

-+

Bombyx PllH

Fig. 3. PTTH stimulation of hsc 70 expression is specific to prothoracic glands. Salivary glands (shown) and other tissues do not upregulate hsc 70 synthesis in response to Manduca PTTH. Challenging prothoracic glands with PTTH from the moth Bombp nrori or with peptide extracts from non-brain sources like adult Manduca flight muscle also fail to elicit increases in hsc 70 synthesis. Ninety-min incubation/treatment periods were used

If prothoracic gland hsc 70 levels influence ecdysteroid synthesis, then hsc 70 levels in the prothoracic gland should be correlated with hemolymph ecdysteroid levels in vivo. Fig. 4 shows the levels of hsc 70 in the prothoracic gland, brain and fat body, the typical ecdysteroid titers during the fifth larval instar, pupal and pupal-adult stages, as well as changes in both prothoracic gland and brain protein content. Based on the amount of tissue homogenate (pg protein) required to achieve a similar degree of immunostaining, the 70-kDa hsc is enriched in prothoracic glands ywo- to three-fold relative to brain, and five- to ten-fold relative to fat body. Peaks in hsc 70 levels either per ,~g protein

R. Rybcynski,

P

“1

“3

“5

“I

“9

L.I. Gilbert

79

I Molecular and Cellular Endocrinology 115 (1995) 73-85

These changes in hsc 70 levels in the prothoracic gland are specific to this tissue. During the same period (V,-P,) that prothoracic gland hsc 70 levels exhibit the complex changes illustrated in Fig. 4A and B hsc 70 levels in brain and fat body exhibit different, less dramatic variations (Fig. 4C). Brain hsc 70 levels per pg protein increase for the first 6 days of the fifth larval instar, as does brain protein content (Fig. 4C insert). However. brain protein content continues to increase during the last 4 days of the fifth instar while brain hsc 70 levels per pg protein decline. Changes in brain hsc 70 levels again correlate positively with brain protein content during pupal-adult development (PO-P,), when the brain undergoes very rapid growth (Fig. 4C insert). In the fat body, hsc 70 content per ,~lgprotein declines gradually during the first 5 days of the fifth instar to about 50% of that in V, larvae and no real changes are evident subsequent to V,. (It is not practical to analyze fat body on a per gland basis since this tissue does not form a compact organ but is distributed diffusely throughout the animal.) To complement these studies, the ability of PTTH to elicit hsc 70 synthesis under in vitro conditions was analyzed. A 40-min stimulation period was chosen to allow comparison with PTTH-stimulated j?-tubulin synthesis, which is transient and peaks I 1 h after initiating the PTTH challenge (Rybczynski and Gilbert, 1995). Fig. 5 shows the specific stimulation by PTTH of hsc 70 synthesis. It is important to note that in this and

PO

Developmental stage

Developmental stage

250 ,

0

1 “1

“3

“5

“7

“9

PO

l-

%

Developmental stage Fig. 4. Patterns of hsc 70 expression levels in the prothoracic gland, brain and fat body during the fifth farval instar, and pupal and early pupal-adult stages. Hsc 70 levels were determined from immunoblot analysis using a densitometer and are expressed as arbitrary absorbance units; each day is represented by tissue pooled from at least five individuals and all determinations were made at least twice. (A) Changes in prothoracic gland hsc 70 levels per pg protein. Error bars indicate k 1 SEM. Also shown are mean circulating ecdysteroid levels during the same period, based on radoimmunoassay data gathered by Bollenbacher et al. (1981) and Grieneisen et al. (1993). (B) Changes in hsc 70 levels per prothoracic gland. Also shown are mean levels of protein per prothoracic gland. (C) Changes in brain and fat body hsc 70 levels per pg protein. Insert shows the change in brain size during the same period as measured by mean brain protein content.

4A) or per gland (Fig. 4B), are evident on days 3-4 and 7 of the fifth instar, as well as on the first day of pupal life. Hemolymph ecdysteroids during this period exhibit a small peak on V,, the ‘commitment’ peak (see Riddiford, 1994), and a larger V, surge that elicits the pupal molt (Fig. 4A). (Fig.

v2

v3

v3.5

v7

v9

P

1

P

3

Developmental stage Fig. 5. Developmental changes in the ability of PTTH to stimulate hsc 70 synthesis in prothoracic glands. Glands were incubated with ‘%-methionine + indicate mean + - PTTH for 40 min. Columns SEM.

80

R. Rybczynski, L.I. Gilbert I Molecular and Celhlar Endocrinology Ii5 (1995) 73-85

the following analyses, the amount of protein loaded in each gel lane was adjusted to yield the same amount (countsjmin) of 35S-methionine incorporated into protein based on TCA precipitations (see Methods), Thus, these data do not indicate the total changes in hsc 70 synthesis but only the specific changes relative to other proteins. Depending on the stage and length of incubation, PTTH can stimulate increases in general protein synthesis of more than two-fold (Rybczynski and Gilbert 1994, 1995, and unpublished observations). PTTH-stimulated hsc 70 synthesis exhibits two peaks. The first on V, correlates with a period of rapid growth of the prothoracic gland (see Fig. 4B), and with a brief but biologically crucial period of ecdysteroid synthesis (the commitment peak: see Fig. 4A). The second broad peak of PTTH-stimulatable prothoracic gland hsc 70 synthesis that begins on V, and peaks on P, (Fig. 5) overlaps a period of slow but steady increase in gland protein content (Fig. 4B), as well as a period of moderate to very high ecdysteroid titer (Fig. 4A). This developmental pattern is similar to, but not identical with that seen in PTTH-stimulated /?-tubulin synthesis (Rybczynski and Gilbert, 1995).

240

Time after PlTH addition (min)

PredIcted accumulation

/’

/’

,

,’ 1’ I

,’ /’ /’

c’

-

3.3. Kinetics of PTTH-stimulated hsc 70 accumulation Under in vitro conditions, V,,, and older prothoracic glands respond very quickly ( I 10 min) and transiently ( z 2-3 h) to PTTH stimulation with increased /3-tubulin synthesis/accumulation that peaks 30-60 min after starting treatment (Rybczynski and Gilbert, 1995, unpublished observations). Similar studies of PTTH-stimulated hsc 70 synthesis/accumulation indicated a somewhat different response time course. Fig. 6A reveals that PTTH-stimulated hsc accumulation in V,,, prothoracic glands increases rapidly for 1- 1.5 h at which time a plateau is reached that persists for at least another 2.5-3 h. This plateau appears to be the result of a rapid increase in the degradation rate of hsc 70 with little change in the synthesis rate since 30-min pulse labelings with 35S-methionine indicate an almost constant level of synthesis for at least 2 h (Fig. 6B). This change in hsc 70 accumulation occurs between 60 and 90 min and can be seen when the predicted accumulation of hsc 70 (Fig. 6B), assuming no degradation at all, is compared with observed accumulation (Fig. 6A). These two values are in good agreement for the first 60 min of PTTH stimulation, both reaching z 150% of control values, but by 90 min the predicted value is z 200% of control while the actual accumulation averaged z 160% of the control value. Data from V, glands also reinforce the notion of the biphasic nature of the hsc 70 response to PTTH stimulation. Stimulating prothoracic glands from this stage for 40 min results in about twice the hsc 70 accumulation (50% more than control) seen in V,, glands (20% more than control) (cf. Figs. 4 and 5). However after 2 h of

B

250 I

0

30

/

60

/’

90

120

150

Time after PlTH addition (min)

Fig. 6. Synthesis and accumulation of PTTH-stimulated hsc 70 synthesis in V,, prothoracic glands. Values are mean f 1 SEM. (A) Accumulation of hsc 70 in prothoracic glands stimulated for I5 min to 4 h. The dashed lines are intended to emphasize the biphasic nature of hsc 70 accumulation and are not statistically derived fits to the data. (B) The PTTH-stimulated synthesis and accumulation of hsc 70 determined by 30-min pulse labellings. %-methionine was added at 0, 30, 60 or 90 min after PTTH addition. The dashed line indicates the accumulation of hsc 70 predicted by the addition of the accumulations measured in the four pulse labellings shown connected by the solid lines.

stimulation of V, prothoracic glands hsc 70 accumulated to z 190 + 13 % of the control, which is only slightly higher than that seen in stage V,,, (172 + 14% of control). Temporal aspects of PTTH-stimulated hsc 70 synthesis were further explored in several pulse-chase experiments. In one experiment, V,,, prothoracic glands were divided into two approximately equal portions. One prothoracic gland (two halves) from each animal was pulse labelled with 35S-methionine for 30 min without PTTH while the other gland (two halves) received PTTH. At the end of the pulse-labelling period, one half of each prothoracic gland was removed and frozen while the remaining halves were chased for 30 min without either 35S-methionine or PTTH. After 30 min of labelling, hsc 70 accumulation in PTTH-stimulated glands averaged 151 k 23% of the control value. Following the 30-min chase, hsc 70 accumulation in the PTTH-stimulated glands had not declined relative to levels at the beginning of the chase (147 i 14% of

R. Rybczynski, L.I. Gilbert / Moleculur and Cellular Endocrinology 115 (1995) 73-85

Chx

Chx+PTTH

ActD

81

ActD+PTTH

Treatment Fig. 7. The effect of cycloheximide (Chx) and actinomycin D (ActD), in the absence and presence of PTTH, on synthetic activities in the prothoracic gland. Overall protein synthesis (Protein), hsc 70 synthesis relative to other proteins (Hsc 70) net hsc 70 synthesis (NET: overall protein synthesis times specific hsc 70 synthesis); and ecdysteroid synthesis, relative to untreated controls (Ecdysteroids). Error bars = 1 SEM, and the dashed line indicates the control value (100%).

controls). During the same chase period accumulation of the p-tubulin synthesized in response to PTTH stimulation declined by z 50% (Rybczynski and Gilbert, 1995) indicating that the absence of change in hsc 70 levels following the chase was not due to failure to wash out PTTH and/or 35S-methionine. This experiment suggests a long half-life for PTTH-stimulated hsc 70, i.e., 1 l- 12 h, using a semilogarithmic plot of the densitometric data to estimate the half-life. In a second experiment, prothoracic glands were incubated with 35S-methionine _t PTTH for one hour, followed by a 35S-methionine- and PTTH-free 3-h chase. At the end of this chase, PTTH-stimulated hsc 70 accumulation was 24% of the level at the end of a l-h pulse-labelling and accumulation of the p-tubulin synthesized in response to PTTH stimulation had declined to control levels, indicating that the wash-out of PTTH and 35S-methionine was effective. This experiment suggests a short half-life for PTTH-stimulated hsc 70 ( z 1.5 h; half life estimated as above). Together these two pulse-chase experiments suggested quite different half-lives for hsc 70 in the prothoracic glands. The data reinforce the view that PTTH-stimulated hsc 70 accumulation is biphasic with accumulation equalling synthesis for a period of z 1 h, followed by a second phase in which hsc 70 turnover nearly balances synthesis (see Fig. 6A). PTTH-stimulated p-tubulin synthesis and accumulation by prothoracic glands in vitro are transient and drop very rapidly when PTTH is removed from glands (t 112 z 35 min; Rybczynski and Gilbert, 1995) The above-described pulse-chase experiments suggest that PTTH effects on hsc 70 persist longer. To determine if

hsc 70 synthesis remained altered in glands even after PTTH treatment had been withdrawn a modified pulsechase experiment was performed. V,,, prothoracic glands were stimulated with PTTH for 1 h, washed carefully and thoroughly, and incubated for 2 h in PTTH-free medium. 35S-Methionine was then added for a l-h labelling period. Glands that were challenged with PTTH accumulated 34 rfr 10% more hsc 70 than did contralateral control glands (no PTTH), suggesting that transient exposure of prothoracic glands in vivo to PTTH may have long-term effects on gene expression and/or protein turnover. In contrast, no difference in the accumulation of the ‘PTTH-stimulated’ B-tubulin was detectable between treated and control glands confirming earlier studies (Rybczynski and Gilbert, 1995; unpublished observations) 3.4. Efects of cycloheximide and actinomycin D on PTTH-stimulated

hsc accumulation

Keightley et al. (1990) showed that blocking translation or transcription in pupal prothoracic glands in vitro with cycloheximide or actinomycin D, respectively, had no effect on the basal rate of ecdysteroid synthesis, but that PTTH-stimulated increases in ecdysteroid synthesis were inhibited by these drugs. Rybczynski and Gilbert (1995) found that larval glands were similarly affected and that it was not necessary to block translation completely to inhibit PTTH-stimulated ecdysteroidogenesis. The same paradigm was used to study the synthesis of hsc 70 in prothoracic glands treated with either 10 ,uM cycloheximide or actinomycin D in the presence or absence of PTTH (30-min labelling period) (Fig. 7). At this concentration, there

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was no effect on basal ecdysteroid synthesis but the typical in vitro PTTH-stimulated increase in ecdysteroid synthesis was blocked completely (cycloheximide; 100% inhibition) or substantially (actinomycin D, > 75”/) inhibition), resulting in ecdysteroid production similar to that exhibited by control glands (no PTTH) (see Fig. 7). Overall protein synthesis in actinomycin- or cycloheximide-treated glands was partially inhibited at these doses (30-75% respectively: Fig. 7). The effect of cycloheximide and actinomycin on hsc 70 synthesis is shown in two ways in Fig. 7. The columns labelled ‘Hsc 70’ present the specific synthesis of hsc 70 during cycloheximide or actinomycin treatment relative to other proteins in the gels. The columns labelled ‘NET’ indicate the net changes in hsc 70 synthesis when the overall changes in protein translation per gland are integrated with specific changes in hsc 70. In the absence of PTTH, actinomycin D had no specific effect on hsc 70 synthesis relative to other proteins; glands treated with actinomycin accumulated hsc 70 to the same level as untreated glands (99 t_ 10% of control: Fig. 7). However, the normal PTTH-stimulated increase in hsc 70 synthesis relative to matched controls (experimentals = 129 f 6% of controls: Fig. 7) was inhibited completely by the inclusion of actinomycin (experimentals = 102 f 7% of controls: Fig. 7). In contrast to actinomycin, cycloheximide in the absence of PTTH had a specific, additional inhibitory effect on hsc 70 synthesis (PTTH-treated = 67 f 7% of controls: Fig. 7) relative to other proteins. This specific effect of cycloheximide on hsc 70 synthesis was also observed when glands were treated with PTTH. In this case, hsc 70 synthesis in glands receiving simultaneous treatment with PTTH and cycloheximide was 89 &- 6% of that in glands receiving cycloheximide alone or about 60 f 4% of the synthesis in control glands (no PTTH or drugs) (Fig. 7). When changes in overall protein synthesis are factored into the analysis of hsc 70 synthesis (columns labelled ‘NET’ in Fig. 7) a preferential translation of hsc 70 in prothoracic glands is suggested since its synthesis/ accumulation is depressed below that of other proteins with the application of cycloheximide (compare ‘Protein’ and ‘NET’ columns for cycloheximide f PTTH. in Fig. 7). In contrast, during the 30-min labelling period, actinomycin treatment did not result in net changes of hsc 70 synthesis that were different from other proteins suggesting that preferential hsc 70 transcription is not important in maintaining the early phase of the hsc 70 response to PTTH (compare ‘Protein’ and ‘NET’ columns for actinomycin + PTTH, in Fig. 7). The effect of PTTH on pre-existing hsc 70, in the absence of normal translation was also investigated. Prothoracic glands were incubated with “?S-methionine for 30 min, at which point cycloheximide was added to

a concentration of 10 PM and the incubation continued for 15 min. The medium was then changed to Grace’s without 35S-methionine, with 10 /IM cycloheximide k PTTH, and this incubation continued for 30 min. Under these conditions, PTTH had no effect on hsc 70 synthesized prior to the 30-min stimulation period (PTTH-treated = 102 + 13% of control). These results are consistent with the interpretation that hsc 70 changes elicited by PTTH are due primarily to changes in hsc 70 translation during PTTH stimulation. Note that actinomycin alone appeared to stimulate ecdysteroidogenesis slightly but this effect was not additive with PTTH. 4. Discussion Studies of the Manduca prothoracic gland in vitro have elucidated a portion of the transductory cascade that precedes and accompanies ecdysteroidogenesis. i.e., that PTTH stimulates increases in intracellular Ca2 + ; CAMP, and the phosphorylation of the ribosomal protein S6 (see Gilbert et al. 1988; Song and Gilbert, 1995), and that PTTH-stimulated increases in ecdysteroid synthesis require protein synthesis (Smith et al., 1986; Keightley et al., 1990; Rybczynski and Gilbert, 1995). The synthesis of at least three proteins in prothoracic glands is stimulated specifically by PTTH (100, 70 and 52 kDa: Rybczynski and Gilbert, 1994) and here the 70-kDa protein has been identified as a cognate (hsc) or constitutively expressed member of the heat shock protein 70 (hsp 70) family. The PTTH-stimulated 70-kDa protein was identified as a member of the hsp 70 family based on molecular weight, isoelectric point and immunoreactivity with an anti-hsp 70 monoclonal antibody. This protein is considered to be a heat shock cognate based on its high degree of expression in non-stressed prothoracic glands as assayed by immunodetection of in situ levels and by incorporation of 35S-methionine during in vitro incubations. Based on 1D and 2D PAGE, either the synthesis of this protein is also significantly inducible by heat shock or Manduca tissues express a second, true, hsp 70 with very similar biochemical characteristics, i.e., nearly identical molecular weight, isoelectric point and immunoreactivity. The degree of hsc 70 synthesis in response to heat shock is considerably greater than that due to PTTH stimulation under our standard in vitro experimental paradigm. It is clear that the expression of an hsc 70 protein in the prothoracic gland is unique in at least three ways relative to other tissues. First, increased synthesis of this hsc 70 occurs in response to stimulation of prothoracic glands by the same agents that stimulate ecdysteroid synthesis: PTTH, dibutyryl CAMP and calcium ionophore (see also Rybczynski and Gilbert 1994). Peptides isolated from tissues other than brain, or PTTH

R. Rybczyski,

L.I. Gilhert

/ Mole&w

from another species of moth, fail to elicit ecdysteroid synthesis or an increase in hsc 70 synthesis by the prothoracic glands. Second, challenges of tissues other than prothoracic glands with PTTH, dibutyryl CAMP and calcium ionophore fail to elicit hsc 70 synthesis in those tissues. Third, in situ levels of prothoracic gland hsc 70 are correlated uniquely with developmental changes in the size (protein content) and ecdysteroidogenic activity of the gland. During the same developmental period, hsc 70 levels in other organs like brain and fat body were nearly constant, i.e., no consistent changes with organ growth or the production or circulation of ecdysteroids. The prothoracic gland hsc 70 also exhibits a developmentally varying response to PTTH-stimulation, with a peak on day 3 of the fifth larval instar and a broader peak extending from the last day (V,) of the fifth larval instar through day 3 (P,) of pupal-adult development. V, glands are undergoing rapid growth and this is also the stage at which a small, biologically important pulse of ecdysteroid synthesis occurs that reprograms cells such that the next molt is larva-pupa rather than larvalarva (the commitment peak: see Riddiford, 1994). The second, multi-day peak of PTTH-stimulatable hsc 70 synthesis extending from V, to P, occurs during a period of less dramatic gland growth, but of moderate to very high ecdysteroid production. However, on day 7 of the fifth instar when the ecdysteroid titer is at its fifth larval instar maximum only a low level of PTTH-stimulated hsc 70 synthesis is noted. When in vivo hsc 70 levels are considered, either per pg protein or per gland peaks are evident on days 4 and 7 of the fifth instar, as well as on the first day of pupal life. The height of the V, peak is of the same order as the V, and P, peaks; in contrast, the V, hemolymph ecdysteroid peak is only a small fraction of the V, level. Taken together, these observations suggest that the level and synthetic rate of hsc 70 in the prothoracic gland are more likely to be involved in PTTH-stimulated growth and/or protein turnover in the gland rather than directly in ecdysteroid synthesis. Consistent with this view is the observation that PTTH-stimulated hsc 70 accumulation reaches a plateau at 1 h that remains for at least 3 h. Under the same in vitro conditions, increases in ecdysteroidogenesis and p-tubulin synthesis are detectable in lo- 15 min after the start of PTTH challenge, peak at 30-60 min and decline thereafter (Rybczynski and Gilbert, 1995; Song and Gilbert, 1995). Furthermore PTTH-stimulated hsc 70 synthesis can be detected at least 2 h after PTTH withdrawal, suggesting that PTTH can have long-term effects on prothoracic gland metabolism. However, it is important to note that the only demonstrated functions of the prothoracic gland are ecdysteroid synthesis and release. Hence any PTTH-elicited changes in hsc 70 levels and synthetic rates are likely to be ultimately of importance to ecdysteroidogenesis,

and Cellular Endocrinology 1 I5 (1995) 73-85

83

even if hsc 70 plays no direct role in the synthesis of ecdysteroids. Data from the cycloheximide and actinomycin D experiments suggest that early changes in hsc 70 synthesis in response to PTTH stimulation are mediated primarily by increases in transcription and translation. PTTH had no effect on hsc 70 synthesized prior to PTTH exposure, and other experiments with actinomycin and cycloheximide reinforce this interpretation. Actinomycin had no selective effect on hsc 70 accumulation relative to other proteins in control glands but did inhibit PTTH-stimulated hsc 70 synthesis, indicating that enhanced transcription characterizes the early response of prothoracic glands to PTTH. Actinomycin also appeared to stimulate ecdysteroid and /?-tubulin synthesis anomalously (Rybczynski and Gilbert, 1995). This effect may be mediated via actinomycin-influenced phosphorylation of the ribosomal protein S6 (unpublished data) which is hypothesized to be critical for PTTH-stimulated ecdysteroidogenesis (Gilbert et al., 1988) Cycloheximide had specific inhibitory effects on hsc 70 accumulation relative to other proteins in control glands as well as in stimulated glands. These data suggest that hsc 70 levels in the prothoracic gland are dynamic, selectively regulated and also support the concept that early PTTH effects on hsc 70 are mediated primarily by new synthesis rather than by decreased degradation of pre-existing molecules. These data also suggest that PTTH controls the synthesis or activation of one or more proteases that are relatively specific for hsc 70, and /I-tubulin as well. The observations indicate that activation or increased synthesis of a protease occurs about 1 h after the initiation of PTTH stimulation. or that such proteolytic activity is specifically inhibited during this period. In the first hour, hsc 70 accumulation approximates synthesis and hsc 70 has an apparent half-life of 1 l- 12 h. Subsequently, hsc 70 accumulation slows greatly although the rate of synthesis remains constant as judged by 0.5-h pulse-labellings, and the apparent half life is much shorter ( z 1.5 h). PTTH-stimulated /I-tubulin accumulation also shows a rapid increase followed by a decline (Rybczynski and Gilbert, 1995). suggesting that proteolysis of p-tubulin and hsc 70 may be regulated similarly in stimulated prothoracic glands. The PTTHstimulated hsc 70 and p-tubulin response in prothoracic cells share an additional trait. Our present data suggest that the transcription and translation of these two genes is not limited to the prothoracic gland (this report; Rybczynski and Gilbert, 1995; unpublished data). However. in both cases, it appears that prothoracic gland-specific control of transcription, translation, degradation and perhaps post translational modification, combine to yield a gland-specific response to PTTH and a gland-specific developmental program involving widely expressed genes.

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L.I. Gilbert / Molecular and Cellular Endocrinolog>, 115 (1995) 73-85

The selective stimulation of the expression of an hsp 70 protein by peptide hormones or neurotransmitters as opposed to the induction of the full suite of stress-response proteins, has been described for a number of other tissues and cell lines. Hsp 70 expression in the adrenal gland can be up-regulated by both ACTH and a dopaminergic agonist (Blake et al.. 1991, 1993) and insulin stimulates hsp 70 expression in a cultured hepatoma cell line (Ting et al. 1989). HeLa cells and embryonic kidney cell line respond similarly to serum stimulation (Wu and Morimoto 1985) as do human T lymphocytes stimulated with interleukin 2 (Ferris et al., 1988). In none of these cases is the precise role of increased hsp/hsc 70 expression in response to hormone stimulation known. Ting et al. (1989) suggest that hsp 70 expression may play an important but unknown role in the insulin transductory cascade. In HeLa cells, embryonic kidney cells and T lymphocytes, hsp 70 expression was tied to the cell cycle and a role in DNA replication has been suggested (e.g., Milarski and Morimoto, 1986). Since post-embryonic lepidopteran prothoracic glands are amitotic (see Beaulaton, 1990) this putative role is eliminated for the PTTH-stimulated protein. Blake and co-workers (Blake et al., 1991, 1993; Udelsman et al., 1993) propose that peptide hormoneor neurotransmitter-stimulated hsp 70 synthesis acts in a homeostatic capacity, ensuring that proteins synthesized in response to transmitter or hormone reach their appropriate intracellular location, i.e., hsp 70s support the cell-specific translational responses to intercellular messengers. In addition to these observations of hsp 70 synthesis changing in response to various stimuli, Pak et al. (1992) have reported that an ecdysteroidogenic extract prepared from Drosophila whole larvae or brain/ganglion complex elicits synthesis of heat shock protein 83 (83 kDa) in wing imaginal discs. At present, it is unclear whether or not this hsp 83 response is due to stimulation by Drosophila PTTH(s) or some other peptide (Pak et al., 1992). We found no evidence for a PTTH-related change in the expression of a putative Manduca homolog of this protein (hsp 83) in any of our studies of larval tissues. Members of the hsp 70 family are important components of several cellular pathways, including the folding of nascent proteins and their translocation into intracellular organelles (see Hendrick and Hartl, 1993; Becker and Craig, 1994). It is possible and perhaps likely that the PTTH-stimulated hsc 70 serves in such a manner since PTTH generally has a trophic effect on general protein synthesis in the prothoracic gland, in addition to its specific stimulation of p 100. hsc 70 and p-tubulin synthesis (Rybczynski and Gilbert, 1994, 1995: see Gilbert et al., 1995). Hsc 70 could thus be critical in supporting periods of gland growth (Fig. 2B) as well as high protein turnover that might be associated with high levels of ecdysteroid synthesis.

However, many other functions for hsp 70s have been demonstrated or hypothesized. Hsp 70s target some proteins to lysosomes and may facilitate proteolysis in lysosomes and within mitochondria (see Stuart et al., 1994; Terlecky, 1994). Other hsp 70 family members play roles in steroid receptor function (see Pratt, 1992, 1993) clathrin-coated vesicle cycling (Chappell et al.. 1986) and possibly phosphatase activation (Mivechi et al., 1993). We cannot rule out a role for hsc 70 in one of these other pathways. Mechanisms for feedback control or monitoring of ecdysteroid levels by the tissue of origin would be expected (Sakurai and Williams, 1989) and hsp 70s may be important in the correct assembly of steroid hormone receptor complexes (see Pratt, 1992, 1993). An hsp 70 is the uncoating ATPase of the clathrin-coated vesicle pathway that is critical for cholesterol uptake by vertebrate cells (Brown and Goldstein 1986). Since cholesterol is the precursor molecule for ecdysteroids, the clathrin pathway is also a reasonable candidate for the participation of a PTTHstimulated hsc 70. Mivechi et al. (1993) reported that purified hsp 70s could activate phosphoprotein phosphatases in vitro; such a role for hsp 70 in the prothogland is also reasonable, since the racic phosphorylation state of ribosomal protein S6, and perhaps other proteins, may regulate PTTH-stimulated ecdysteroidogenesis (Gilbert et al., 1988; Song and Gilbert, 1995). Finally, it is certainly possible that the PTTH-stimulated synthesis of hsc 70 by prothoracic glands might subserve some unexpected function in ecdysteroid synthesis. The resolution of which one or several of the candidate pathways are affected by PTTH-stimulated hsc 70 synthesis will require selective alteration of hsc 70 levels perhaps via anti-sense techniques, in conjunction with other approaches such as sub-cellular fractionation to determine the cellular compartments to which the PTTH-stimulated hsc 70 is targeted. Acknowledgements

We thank members of the laboratory for help in raising animals. This research was supported by grants from NSF (IBN-9300164) and NIH (DK-30118). References Andres, A.J., Fletcher. J.C., Karim, F.D. and Thummel, C.S. (1993) Dev. Biol. 160. 388--404. Beaulaton, J., (1990) In: Morphogenetic Hormones of Arthropods (Gupta. A.P.. ed.), Vol. I 1Pt. 2.. pp. 3433435, Rutgers Univ. Press, New Brunswick, NJ. Becker, J. and Craig, E.A. (1994) Eur. J. Biochem. 219, 1 I-23. Blake, M.J.. Udelsman, R., Feulner, G.J., Norton. D.D. and Holbrook, N.J. (1991) Proc. Natl. Acad. Sci. USA 88. 987339877. Blake, M.J.. Buckley, D.J. and Buckley, A.R. (1993) Endocrinology 132. 1063-1070.

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Boilenbacher. W.E.. Agui, N., &anger, N.A. and Gilbert, L.1. (1979) Proc. Nat]. Acad. Sci. USA 76, 5148-5152. Bollenbacher. W.E.. Smith, S.L.. Goodman, W. and Gilbert, L.!. (1981) Gen. Comp. Endocrine!. 44, 302-306. Brown, MS. and Goldstein. J.L. (1986) Science 232. 34447. Chappel!, T.G.. Welch, W.J., Schlossman. D.M.. Palter. K.B. Schlesinger, M.J. and Rothman. J.E. (1986) Cell 45, 3-13. Ferris, D.K., Hare!-Bellan. A., Morimoto. R.I., Welch, W.J. and Farrar, W.L. (1988) Proc. Nat]. Acad. Sci. USA 85, 3850-3854. Fittinghoff, C.H. and Riddiford, L.M. (1990) J. Comp. Physio!. B 160. 349-356. Gilbert, L.I.. Combest, W.L.. Smith, W.A.. Meller. V.H. and Rountree, D.B. ( 1988) BioEssays 8. 1533157. Gilbert, L.I., Rybczynski. R. and Tobe. S. (1995) In: Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Insect and Amphibian Cells. (3rd edn.) (Gilbert L.I. Atkinson, B. and Tata J., eds.). Academic Press, NY. in press. Goodman, W.G.. Carlson. R.O. and Nelson, K.L. (1985) Ann. Entomo!. Sot. Am. 78, 70-80. Grieneisen, M.L., Warren, J.T. and Gilbert. L.I. (1993) Insect Biochem. Mol. Bio!. 23, !3-~23. Hendrick, J.P. and Hart]. F.-U (1993) Ann. Rev. Biochem. 62 339-384. Hurban. P. and Thumme!, C.S. (1993) Mol. Cell. Bio!. I3 7101-71 I I. Keightley, D.A.. Lou. K.J. and Smith. W.A. (1990) MO!. Cell. Endocrine!. 74, 229-237. Kiriishi, S., Rountree. D.B., Sakurai. S. and Gilbert. L.I. (1990) Experientia 46. 716- 721. Laemmli. U.K. (1970) Nature 227, 680-6 85. Milarski, K.L. and Morimoto. R.I. (1986) Proc. Nat!. Acad. Sci. USA 83, 9517-9521. Mivechi. N.F.. Trainor, L.D. and Hahn, G.M. (1993) Biochem. Biophys. Res. Commun. 192, 9544963. O’Farrel!. P.H. (1975) J. Bio!. Chem. 250, 400774021.

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Orme-Johnson, N.R. (1990) Biochim. Biophys. Acta 1020, 213-231. Pak, J.-W., Chung, K.W., Lee, C.C.. Kim. K.. Namkoong, Y. and Koolman, J. (1992) J. Insect Physio!. 38. 167- 176. Pratt, W.B. (1992) BioEssays 14, 841-848. Pratt, W.B. (1993) J. Bio!. Chem. 268, 21455-21458. Riddiford, L.M. (1994) Adv. Insect Physio!. 24. 2133274. Rubin, D.M., Mehta, A.D.. Zhu, J., Shosam. S.. Chen. X., Wells Q.R. and Palter. K.B. (1993) Gene 128, 155.-163. Rybczynski, R. and Gilbert, L.I. (1994) Insect Biochem. MO!. Biol. 24, 175-189. Rybczynski, R. and Gilbert, L.I. (1995) Dev. Bio!., in press. Sakurai, S. and Williams. C.M. (1989) Gen. Comp. Endocrine!. 75 204-216. Simpson. E.R. and Waterman, M.R. (1988) Ann. Rev. Physio!. 50 427 - 440. Smith, W.A., Rountree, D.B., Bollenbacher, W.E. and Gilbert L.I. (1987) In: Progress in Insect Neurochemistry and Neurophysiology. (Borkovec. A. and Gelman, D. eds.), pp. 319-322, Humana Press. Clifton, NJ. Song, Q. and Gilbert. L.I. (1995) Insect Biochem. Mol. Biol., in press. Stuart, R.A., Cyr, D.M. and Neupert. W. (1994) Experientia 50 10022101 I. Terlecky, S.R. (1994) Experientia 50, 1021.. 1025. Ting, L.-P.. Tu, C.-L. and Chou. C.-K. (1989) J. Biol. Chem. 264, 340443408. Udelsman. R.. Blake, M.J., Stagg, C.A., Li. D.-G., Putney. J. and Holbrook, N.J. (1993) J. Clin. Invest. 91, 4655473. Vince, R.K. and Gilbert. L.I. (1977) Insect Biochem. 7 1155120. Warren, J.T. and Gilbert. L.I. (1986) Insect Biochem. 16 65582. Warren. J.T. and Gilbert, L.I. (1988) In: Immunological Techniques in Insect Biology (Gilbert. L.I. and Miller. T.A. eds.), pp. 181-214, Springer-Verlag. New York. Weevers. R.G. (1966) J. Exp. Bio!. 44, 1633184. Wu, B.J. and Morimoto, R.I. (1985) Proc. Nat!. Acad. Sci. USA 82, 6070 6074.