62

Biochimica et Biophysica Acta, 1034 (1990) 62-66 Elsevier

BBAGEN 23286

Inhibition of glucose-regulated and heat shock protein induction by low temperature Karl W. Lanks Department of Pathology, S U N Y Health Science Center at Brooklyn, Brooklyn, N Y (U.S.A,) (Received 8 November 1989)

Key words: Heat shock protein; Stress; Protein induction; Low temperature

The present study evaluating induction of the major stress proteins in the subphysiological temperature range (25-33 ° C) shows that none of the agents used could effectively induce the heat shock proteins (hsp) or the glucose related protein grp95 at low temperature. However, grp82 was still induced by some amino acide analogs and by glucose deprivation while certain oxygen-regulated proteins were still induced by hypoxia at 25 o C. Analogs were incorporated and protein turnover was increased at low temperature even though most stress proteins were not induced. Synthesis of hsps, but not that of grps, was induced if cultures containing analog-substituted proteins were shifted to 37 ° C. Temperature dependence of hsp induction by arsenite showed a sharp threshold between 30°C and 33 ° C. Low temperature inhibition of induction points to the existence of a temperature-dependent mechanism operating within the normal physiological temperature range and may be a useful parameter in evaluating proposed mechanisms of stress protein regulation.

Introduction A variety of stressful conditions is capable of inducing the synthesis of specific proteins. Thus, heat shock proteins (hsps) are induced by moderate temperature elevation, amino acid analogs and sulfhydryl-reactive compounds, including heavy metals, while glucose-regulated proteins (grps) are induced by glucose deprivation, certain amino acid analogs and hypoxia (for reviews see Refs. 1-5). Although induction of these proteins has been viewed as a response to physiological stress, several of the 'stress proteins', including grp95, grp82, hsp85 and one of the hsp70 isoforms, are expressed at high levels in cultured mammalian cells that are not exposed to any apparent stress. If constitutive synthesis is taken into account, then stress protein synthesis is regulated between 37 °C (a temperature at which synthesis is induced by chemical

Abbreviations: DME, Dulbecco's high glucose modification of Eagle's medium; AC, L-azetidine-2-carboxylic acid; CAN, L-canavanine; TACB, L-threo-anfinochlorobutyric acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; grp, glucose-regulated protein; hsp, heat shock protein. Correspondence: K.W. Lanks, Department of Pathology, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A.

agents) and 43-45 °C (a temperature range in which hsp synthesis is induced in the absence of exogenous chemical agents). This point of view leads to the prediction that there might exist a subphysiological temperature range within which stress protein synthesis is reduced or unresponsive to chemical inducers. The present study tests this hypothesis and examines its implications for future proposed models of stress protein regulation.

Materials and Methods L929 cells were grown in polystyrene petri dishes containing DME plus 10% newborn calf serum as previously described [6]. All cultures were maintained in fresh serum-free DME for 24 h before exposure to stress protein inducers. Media lacking specific amino acids or glucose were made up from the individual constituents of DME. Cultures were exposed to hypoxia by incubation in an atmosphere of 90% nitrogen 10% carbon dioxide in chambers that had been exhaustively purged with this gas mixture [7]. Unless stated otherwise, cultures were exposed to 1 mM amino acid analogs (AC and CAN from Sigma Chem. Co., St. Louis, MO; TACB from Calbiochem, San Diego, CA) for 18 h prior to labeling. Temperature shift experiments to assess stress protein induction were

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

63 performed by incubation with analog (1 m M in medium lacking the corresponding normal amino acid) for 48 h at 25 ° C followed by incubation in analog-free D M E at 37 o C for various periods of time. Turnover experiments were performed similarly except that cultures were incubated in medium with or without 1 mM TACB and 10/xCi/ml [35S]methionine for 18 h, washed and chased in DME. Duplicate cultures were assayed for trichloroacetic acid precipitable radioactivity and analyzed by SDS-PAGE. The fraction of initial radioactivity remaining was calculated and data are expressed as the ratio of fraction remaining in TACB-treated cultures relative to controls incubated and labeled at the same temperature. Cultures for amino acid analysis were scraped into cold 0.6 M perchloric acid and centrifuged. Acid insoluble material was hydrolyzed by heating at 100 ° C for 3 h in 6 M HC1, lyophilized and derivatized by heating with N,O-bis(trimethylsilyl)trifluoroacetamide (Pierce Chemical Co., Rockford, IL) at 60 ° C for 30 min. The resulting trimethylsilyl derivatives were analyzed by GC-MS as previously described [8]. Derivatives of natural amino acids and analogs were identified on the basis of retention time and spectra of authentic compounds and the M-115 ions were used for quantitation. Temperature dependence of arsenite induction was assessed using cultures exposed to 50 /zM sodium arsenite in D M E containing 20 m M Hepes incubated in air for 3 h at the indicated temperature followed by labeling with [35S]methionine at the same temperature. Labeling and one-dimensional SDS-PAGE were performed as previously described [6,9]. Gels were stained with Coomassie blue to visualize protein bands or dried and autoradioagraphed using Kodak XAR-5 film.

C

I

AC 25 =

37 =

1

Results

L929 cells maintained in D M E M at 25 ° C remained alive for at least 1 week and were morphologically indistinguishable from parallel cultures maintained at 37 o C. Preliminary SDS-PAGE analysis of control cultures labeled with [35S]methionine at the two temperatures showed essentially identical protein synthesis patterns. Temperature-dependence of stress protein induction was examined in this system using analogs of three different amino acids: i.e., L-canavanine (CAN), an analog of L-arginine that induces hsp synthesis [10]; L-azetidine-2-carboxylic acid (AC), an analog of L-proline that induces both grp and hsp synthesis [11]; and L-threo-aminochlorobutyric acid (TACB), an analog of L-valine that had not been previously reported to induce stress protein synthesis. AC at 37 ° C induced all of the major stress proteins including h s p l l 0 (Fig. 1). At 25 o C it had little effect except for moderate induction of grp82. CAN strongly induced hsp synthesis at 37 ° C, but had no effect at 25 ° C. Taking into account the slight induction seen in valine-free medium, the effects of TACB were similar to those of AC at the two temperatures. Several trivial explanations for these observations could be eliminated out of hand: (1) the cells were in good condition, since they remained viable for many days at the low temperature; (2) protein synthesis was sufficient for stress protein synthesis because [35S] methionine incorporation was reduced only 50%; and (3) the cells were transcriptionally active at 25 ° C since grp82 and 40-60 kDa oxygen-regulated proteins (see

TACB

CAN C

'25 =

37 ='

C

'25 °

37 ~

hspllO grp 95 hsp85 grp 82 hsp 70

acfin

Fig. 1. SDS-PAGEanalysis of stress proteins induced by amino acid analogs at 25°C and 37o C. L929 cultures were incubated for 18 h with 1 mM analog in DME lacking the correspondingnormal amino acid. Control cultures were incubated at 25 o C in the same medium: AC, normal DME; CAN, DME lacking arginine; and TACB, DME lacking valine. Labeling and autoradiography were performed as described in Materials and Methods.

64

Oh

Sh

6h 1.0"

0.9"

F

- - 0.8-

I10

O

O

85

"-- 0.7-

70 0.6. . . . . . . . . .

0.5-

0.4

I

18

i

24

Hours Fig. 2. SDS-PAGE analysis of stress protein induction by TACB following a shift from 25 o C to 37 o C. Cultures were incubated for 36 h at 25 °C in valine-free DME containing 1 mM TACB and labeled either at the low temperature or at intervals following incubation at the high temperature in analog-free DME.

below) were still induced under some of the experimental conditions. The possibility that steady state levels of analog substitution were not sufficient for induction at 25 ° C was examined using AC and TACB, the two compounds yielding trimethylsilyl derivatives suitable for GC-MS analysis. Analysis of proteins synthesized during 18 h incubations at 25 ° C and 37 ° C in the presence of the analogs showed that the extent of proline replacement by AC was 1.4% and 3.3%, while that of valine by TACB was 1.5% and 2.2% at 2 5 ° C and 37°C, respectively. Not only were the extents of analog substitution similar at the two temperatures, but hsp synthesis was strongly induced within 3 h when cultures were incubated with TACB at 25 ° C for 36 h and then stepped up to 37 ° C in analog-free medium containing valine (Fig. 2). This sort of step up experiment was performed using TACB, CAN, and AC with similar results. Incubation at 25 ° C in D M E alone followed by a shift up to 37 °C did not result in detectable grp or hsp induction. These findings support the assumption that the observed differences in the extent of analog substitution were not sufficiently large to account for inhibition of stress protein synthesis at 25 o C. Chase experiments in which TCAB was incorporated simultaneously with [3SS]methionine at 2 5 ° C or 3 7 ° C (Fig. 3) showed that proteins labeled in the presence of

Fig. 3. Turnover of L929 cell proteins containing TACB relative to that of analog-free controls. Cultures were labeled with [35S]methionine for 24 h in medium with or without 1 mM TACB, washed and chased in DME: (© . . . . . . ©), labeling and chase both at 25°C; (A . . . . . *), both at 37°C; or (o O), labeling at 25°C and chase at 37 o C. Radioactivity remaining in TACB-substituted and control proteins was determined at each time point and calculated as a fraction of that initially present at the end of the labeling period. Data points represent the mean ratio of TACB-substituted/control proteins.

TACB turned over significantly faster than proteins labeled in the absence of analog, i.e., the ratio T A C B / control was significantly less than 1.0. Cultures first TABLE I

Temperature-dependent induction of stress protein synthesis Inducer

Amino acid analogs AC (1 mM) CAN (1 mM) TACB (1 mM)

Stress proteins induced 25°C

37°C

grp82

grp95, hsp85, grp82, hsp70 hsp85, hsp70 grp95, hsp85, grp82, hsp70

grp82

Hypoxia

grp95, grp82

Calcium agents EGTA (0.1 mM in Ca~÷-free DME) A23187 (7.10 -6 M) Glucose deprivation

grp95, grp82 grp95, grp82 grp95, grp82

grp82

grp95, grp82

Tunicamycin (5/~gfml) SH reagents Iodoacetamide (1-10-5 M) Cu 2+ (8-10-'* M) Arsenite (50 jaM)

-

hsp70 hsp85, hsp70 hsp85, hsp70

65 labeled at 2 5 ° C and then chased at 37 ° C, showed an initial brief increase in turnover rate followed by a return to control levels within 24 h. A temperature shift experiment in which [a~S]methionine was incorporated for 1.5 h during analog exposure at 2 5 ° C showed increased turnover at 37°C, but no significant difference between analog treated ceils and controls. SDSpolyacrylamide gel analysis showed no difference in rate of grp or hsp turnover relative to total protein in any of these chase experiments (data not shown). Table I shows that low temperature inhibited hsp induction by both of the major classes of inducers, i.e., amino acid analogs and sulfhydryl reagents. Arsenite acts relatively rapidly [12] and, as a classical thiol reagent, its rate of reaction should be independent of metabolic energy generation and remain nearly constant between 25 ° C and 37 o C. Therefore, a more detailed analysis of temperature-dependent hsp induction was undertaken using this agent. Fig. 4 shows that whereas arsenite strongly induced synthesis of both hsp85 and hsp70 at 3 3 ° C or 37 o C, it was completely ineffective at 2 5 ° C or 30°C. Similar results were obtained after exposure to 50/~M arsenite for 6 h (data not shown). Temperature dependence of grp induction alone was also examined. The effects of hypoxia and glucose deprivation are shown in Fig. 5. At 37 ° C, both conditions strongly induced grp95 and grp82. Hypoxia also induced synthesis of a group of low molecular weight

25*

30*

33*

37*

15 0

Fig. 4. SDS-PAGE analysis of hsp induction by arsenite. Cultures were incubated for 3 h at the indicated temperature in DME containing 20 mM Hepes and 50 /~M sodium arsenite. [35S]Methionine labeling was at the same temperature in methionine-free DME containing 20 mM Hepes.

-Glucose

-Oz c

I

25*

37*

I

I 250

3701

Na:)~

p95 p82

tin

Fig. 5. SDS-PAGE analysis of grp induction by hypoxia and glucose deprivation. Cultures were incubated for 24 h at the indicated temperature either in the absence of oxygen or in glucose-freeDME. Labeling and autoradiography were as described in Materials and Methods.

(40-55 kDa) proteins corresponding to the oxygen-regulated proteins of Heacock and Sutherland [7]. In contrast, glucose deprivation at 2 5 ° C led to moderate induction of grp82 and no induction of grp95. Neither grp was induced by hypoxia at 25 o C, but induction of the lower molecular weight oxygen-regulated proteins was undiminished. Table I shows that similar results were obtained using a variety of inducing stimuli. Temperature shift experiments in which cultures were incubated for 48 h at 25 ° C in glucose-free medium or D M E containing 5 /~g/ml tunicamycin followed by D M E alone at 37 ° C showed no detectable grp induction (data not shown).

Discussion Consistent with the initial hypothesis, synthesis of the major heat shock proteins was not induced at or below 30 o C. This finding expands the range of temperatures over which stress protein synthesis is regulated and suggests that induction by heat shock represents one extreme of this regulatory range rather than an unphysiological stress response. Induction by chemical agents can also be viewed as a special case in which hsp synthesis is increased to a rate that would otherwise occur only at a higher temperature. Surprisingly, grp synthesis induction was also found to be temperature dependent. This was most apparent

66

for grp95 suggesting that this protein bears either a functional or a regulatory similarity to the hsps. In fact, sequences of grp95 and hsp85 genes are similar [13] and it has been suggested that grp95 is doing in membranes whatever hsp85 is doing in cytosol [14]. Despite the similarities, temperature shift experiments indicated that the hsp inducing stimulus persisted at low temperature, while the grp inducing stimulus did not. Which step of stress protein induction is suppressed at low temperature? This question addresses induction exclusively, since temperature-dependent changes in constitutive synthesis could be evaluated only by directly measuring mRNA synthetic rates. Preliminary experiments suggest that stress protein mRNA synthesis is inhibited relatively selectively at 25 °C (Buchhagen, D. and Lanks, K.W., unpublished observations). Additional work will allow the mechanism by which low temperature inhibits stress protein synthesis to be identified with certainty. One explanation of hsp regulation implicates accelerated protein degradation resulting from denaturation by heat, amino acid analog incorporation or reaction with sufhydryl reagents. This explanation is not sufficient to explain low temperature inhibition because: (1) turnover of TACB substituted proteins was significantly increased at 25°C but hsp synthesis was not induced; (2) grp synthesis was also suppressed at low temperature even though it was not induced by heat shock or sulfhydryl reagents; (3) arsenite reaction products and analog substituted proteins could be generated at 25 °C without causing hsp induction; and (4) hsp induction by arsenite showed a sharp threshold between 30 °C and 33 ° C, that would not be predicted simply from thermodynamic considerations of denaturation or turnover. To what extent can known stress protein functions account for low temperature inhibition of induction? Proteins such as immunoglobulin heavy chain bind to grp82 in the endoplasmic reticulum before glycosylation and assembly are complete [15]. Since induction by glucose deprivation and other agents is thought to be related to accumulation of complexes between underglycosylated proteins and this grp, failure of complexes to accumulate at low temperature could explain lack of induction. In fact, secretion of invertase that is incompletely glycosylated in the presence of tunicamycin has been found to occur at 25 °C [16]. Various hsp70 iso-

forms have been shown to facilitate polypeptide translocation [17,18] and uncoating of clathrin-coated vesicles [19]. However, these proposed functions cannot yet account for the present findings because the underlying processes have not been examined over the entire relevant temperature range. Thus, inhibition by low temperature can now serve as a useful parameter in future studies seeking to identify processes that are correlated with stress protein induction.

Acknowledgements T h e technical assistance of V. Shah a n d J.-P. G a o is greatly appreciated. I t h a n k N e n a C h i n for critically r e a d i n g the m a n u s c r i p t . This work was supported in part b y grants from N I H (GM32725), the A m e r i c a n C a n c e r Society (PDT-269) a n d the Research F o u n d a tion of S U N Y .

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Craig, E.A. (1985) CRC Crit. Rev. Biochem. 18, 239-280. Lanks, K.W. (1986) Exp. Cell Res. 165, 1-10. Lindquist, S. (1986) Ann. Rev. Biochem. 55, 1151-1191. Neidhardt, F.C., VanBogelen, R.A. and Vaughn, V. (1984) Ann. Rev. Genet. 18, 295-329. Nover, L., Hellmund, D., Neumann, D., Scharf, K.D. and Serfling, E. (1984) Biol. Zbl. 103, 357-435. Lanks, K.W., Shah, V. and Chin, N.W. (1986) Cancer Res. 46, 1382-1387. Heacock, C.S. and Sutherland, R.M. (1986) Int. J. Radiat. Oncol. Biol. Phys. 12, 1287-1290. Lanks, K.W. (1987) J. Biol. Chem. 262, 10093-10097. Chin, N.W. and Lanks, K.W. (1980) J. Cell Biol. 85, 402-413. Hightower, L.E. and White, F.P. (1981) J. Cell. Physiol. 108, 261-275. Welch, W.J. and Feramisco, J.R. (1982) J. Biol. Chem. 257, 14949-1495. Johnston, D., Opperman, H., Jackson, H. and Levinson, W. (1980) J. Biol. Chem. 255, 6975-6980. Sorger, P.K. and Pelham, H.R.B. (1987) J. Mol. Biol. 194, 341-344. Pelham, H.R.B. (1986) Cell 46, 959-961. Munro, S. and Pelham, H.R.B. (1986) Cell 46, 291-300. Bergh, M.L.E., Cepko, C.L., Wolf, D. and Robbins, P.W. (1987) Proc. Natl. Acad. Sci. USA 84, 3570-3574. Chirico, W.J., Waters, M.G. and Blobel, G. (1988) Nature 332, 805-810. Deshaies, R.J., Koch, B.D., Werner-Washburne, M., Craig, E. and Sheckman, R. (1988) Nature 332, 800-805. Chappell, T.G., Welch, W.J., Schlossman, D.M., Palter, K.B., Schlesinger, M.J. and Rothman, J.U. (1986) Cell 45, 3-13.

Inhibition of glucose-regulated and heat shock protein induction by low temperature.

The present study evaluating induction of the major stress proteins in the subphysiological temperature range (25-33 degrees C) shows that none of the...
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