Thermotolerant Cells Show an Attenuated Expression of Hsp70 after Heat Shock*

Nicholas G. TheodorakisDagger §, Doreen DrujanDagger , and Antonio De MaioDagger parallel

From the Dagger  Division of Pediatric Surgery and the  Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of heat shock proteins (hsps) results in the protection of cells from subsequent stresses. However, hsps are also toxic when present within cells for a prolonged time period. Thus, the expression of hsps should be tightly regulated. In the present study, the expression of Hsp70 after heat shock was compared between thermotolerant cells, which contain a large concentration of Hsp70, and nonthermotolerant cells (naive). Accumulation of Hsp70, assessed by Western blotting, was negligible when thermotolerant cells were heat-shocked a second time. Hsp70 transcription was similar between thermotolerant and naive cells during heat shock. However, Hsp70 transcription was attenuated more rapidly in thermotolerant than naive cells immediately upon return to non-heat shock conditions. In addition, Hsp70 mRNA stability was reduced in thermotolerant cells as compared with naive cells following the stress. New synthesis of Hsp70 and the efficiency of Hsp70 mRNA translation were similar between thermotolerant and naive cells during the post-stress period. These results suggest that thermotolerant cells limit Hsp70 expression by transcriptional and pretranslational mechanisms, perhaps to avoid the potential cytotoxic effect of these proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of heat shock or stress proteins (hsps)1 is a common response to a variety of adverse conditions, including the increase in normal environmental temperatures, incubation with amino acid analogues, exposure to oxygen radicals, UV radiation, heavy metals, and ethanol (1, 2). Cells containing hsps induced after a primary stress show resistance to a secondary insult, a phenomenon termed stress tolerance. Several observations suggest that hsps are directly involved in stress tolerance. An increase in the number of Hsp70 gene copies in Drosophila melanogaster larvae results in higher protection from stress (3). Inhibition of the heat shock expression renders cells highly susceptible to damage (4). Microinjection of antibodies against hsps also increases the sensitivity of cells to stress (5). Cells transfected with the Hsp70 gene are more resistant to thermal stress (6). Incubation of artificially denatured enzymes with Hsp70, Hsp90, and Hsp40 results in the recovery of their enzymatic activities (7). Protein aggregates that accumulate within the cell after a stress are resolubilized by Hsp104 (8, 9). In addition, the presence of hsps has been correlated with the preservation or repair of cellular structures such as microfilaments (10) and centrosomes (11). Moreover, cellular processes such as splicing (12, 13) and translation (14-16) are stabilized by hsps during stress conditions. The latter seems to be mediated by the direct interaction of Hsp70 with ribosome subunits (17).

The expression of hsps is mainly regulated at the level of transcription in mammalian cells. Heat shock transcription factor 1 (HSF1) is present in a monomeric form in non-stressed cells. After stress, HSF1 trimerizes gaining DNA binding activity (18, 19). The interaction of this factor with the promoter region of the hsp genes activates polymerase II to continue transcription of these genes, which is in a paused state during normal conditions (19). Hsp70 has been found associated with HSF1, suggesting that Hsp70 may autoregulate its own expression (20-22). Several studies support this hypothesis. In Escherichia coli, the interaction of DnaK (Hsp70) and DnaJ (Hsp40) regulates the transactivator factor sigma 32, which is involved in the transcription activation of heat shock genes in bacteria (23-26). Functionally defective mutants of Hsp70 result in cells that overexpress this gene (27). Overexpression of Hsp70 in the absence of stress results in a reduced expression of this protein after heat shock (28). Since thermotolerant cells contain a large concentration of hsps, the expression of hsp genes may be limited when these cells are subjected to a secondary heat shock. In the present study, the expression of Hsp70 was found to be attenuated in thermotolerant HepG2 cells. The reduced expression of Hsp70 in these cells was apparently due to a rapid decrease in Hsp70 transcription and an increase in Hsp70 mRNA degradation upon return to non-heat shock conditions, but not during heat shock.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Metabolic Labeling-- HepG2 cells, a human hepatoblastoma cell line, were maintained in Eagle's minimal essential medium supplemented with nonessential amino acids (1×), sodium pyruvate (2 mM), L-glutamine (2 mM), penicillin (10 IU/ml), streptomycin (10 µg/ml), and heat-inactivated fetal calf serum (10%), at 37 °C with 5% CO2 in a humidified incubator. Pulse-labeling of cells was performed by incubation with [35S]methionine (50 µCi, 800 Ci/mmol, Tran35S-label, ICN Biomedicals) in methionine-free, serum-free minimal essential medium for 15 min. Immunoprecipitations were carried out as described previously (15).

RNA Isolation and Northern Blot/Hybridization-- Total RNA was isolated by the acid-guanidinium thiocyanate-phenol-chloroform method (29). RNA (10 µg) was separated in formaldehyde-agarose gels and transferred onto nylon modified membranes (GeneScreen Plus). Human Hsp70 DNA (30) was labeled by the random primer method (31) using [alpha -32P]dATP and [alpha -32P]dCTP (17). Membranes were hybridized for 16 h at 42 °C, washed with 50 mM Tris, pH 8.6, 1 M NaCl, 2 mM EDTA, 1% SDS at 42 °C, 30 mM sodium citrate, pH 7.0, 300 mM NaCl, 0.1% SDS at 42 °C and 65 °C, and exposed to x-ray films (Kodak X-Omat AR) at -70 °C in the presence of intensifying screens. To demonstrate equal loading of the RNA samples into the gel, blots were stained with methylene blue (0.03%) in 3 M NaOAc, pH 5.2, prior to hybridization. The signal corresponding to Hsp70 mRNA was quantitated using a laser scanner densitometer and normalized to the signal of the 18 S rRNA detected by methylene blue staining prior to hybridization.

Hsp70 mRNA Half-life Determination-- Hsp70 mRNA levels were quantified by scanning densitometry. The mRNA level from each time point at which transcription was negligible was plotted as a function of time. A line was fitted to graph of the log[RNA level] as a function of time using Microsoft Excel to calculate the slope and intercept and the time at which half the RNA would have decayed.

Transcript Run-off Analysis-- Nuclei were isolated according to the following procedure. Monolayers (90% confluent) of HepG2 cells (3 × 100-mm dishes) were rinsed with phosphate-buffered saline, scraped in phosphate-buffered saline (1 ml), and centrifuged (9,000 × g for 1 min). The pellet was resuspended in 10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40 (1 ml), and nuclei were isolated by centrifugation (9,000 × g for 3 s). Nuclei were resuspended in one volume of storage buffer (50 mM Tris, pH 8, 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol).

Transcript Run-off Reaction-- Nuclei in storage buffer (50 µl) were mixed with an equal volume of 50 mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM dithiothreitol, 150 mM KCl, 10% glycerol, 0.7 mM ATP, GTP and CTP, 0.8 µM UTP, and 0.1 mCi of [alpha -32P]UTP and incubated at 25 °C for 30 min. The reaction was stopped by incubation with DNase I (10 units) for 10 min at 37 °C, followed by the addition of four volumes of 10 mM Tris, pH 8, 0.35 M LiCl, 1 mM EDTA, 7 M urea, and 2% SDS. The reaction was further incubated with proteinase K (500 µg/ml) at 45 °C for 60 min. At the end of the incubation period, tRNA (50 µg) was added and the total mixture was precipitated with trichloroacetic acid (10%). The nucleic acid precipitate was resuspended in 10 mM Tris, pH 8, 1 mM EDTA, 0.5% SDS and hybridized to DNA targets. The DNA targets include pBluescript SK- (vector control), pHFbeta A-1, pHHsp70 (human Hsp70), and prRNA (human ribosomal 28 S). Hybridization was carried out in 50% formamide, 6× SSC, 10× Denhardt's, and 0.2% SDS at 42 °C for 72 h. After hybridization, filters were washed in 50 mM Tris, pH 8.6, 1 M NaCl, 2 mM EDTA, and 1% SDS at 42 °C for 30 min and 65 °C for 15 min. Washes were continued with 2× SSC, 0.1% SDS at 65 °C for 15 min twice, and 0.1× SSC, 0.1% SDS at 65 °C for 5 min. Filters were exposed to x-ray films (Kodak X-Omat AR) at -70 °C in the presence of intensifying screens.

Polysome Localization of Hsp70 mRNA-- Cells were lysed in 10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 µg/ml cycloheximide, 1% Nonidet P-40. The lysate was centrifuged at 13,000 × g for 5 min, and the supernatant (postmitochondrial fraction) was sedimented through a 15-45% linear sucrose gradient at 178,000 × g (average) for 90 min. The gradients were fractionated using an automatic system (Brandel Biomedical), with continuous recording of the absorbance at 254 nm. Total RNA was purified from each fraction (16 fractions), slot-blotted, probed with radiolabeled probes for Hsp70 and beta -actin, and visualized by autoradiography. The signal intensity in the film (linear range of exposure) was quantitated using a scanning densitometer and the tracing was plotted versus fraction number.

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INTRODUCTION
MATERIALS AND METHODS
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Accumulation of Hsp70 in Thermotolerant Cells-- HepG2 cells were made thermotolerant by a nonlethal pre-heat shock treatment (43 °C, 1.5 h), followed by incubation at 37 °C for 24 h (recovery period) (15). Thermotolerant cells showed over 90% incorporation of radioactive amino acids as compared with control cells maintained at 37 °C, in contrast to naïve cells, which had a 30% incorporation of radioactive amino acids with respect to controls as described previously (15). In addition, these cells showed viability 2-fold higher than naïve cells after incubation at 45 °C for 2 h. At the end of the recovery period, thermotolerant cells were thermally stressed (43 °C, 1.5 h) for a second time. Cells were lysed 3 h after the first stress (T1), immediately before the second stress (T2), and 6 h after the second stress (T3). For comparison, HepG2 cells were heat-shocked at 43 °C for 1.5 h and lysed after 6 h of recovery at 37 °C (naive cells, N). Non-stressed HepG2 cells (C) were also included in the analysis. The accumulation of Hsp70 in thermotolerant cells was analyzed by Western blotting. The levels of Hsp70 were similar between thermotolerant cells before (T2) and after (T3) the second heat shock and naive cells (Fig. 1). This observation suggests that there is no additional detectable accumulation of Hsp70 in thermotolerant cells after a second heat shock.


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Fig. 1.   Detection of Hsp70 in thermotolerant cells by Western blotting. HepG2 cells were incubated at 43 °C for 1.5 h (heat shock pretreatment), transferred to 37 °C, and allowed to recover for 24 h. Then cells were stressed again at 43 °C for 1.5 h and recovered at 37 °C. Cells were lysed after 3 h of recovery following the first stress (T1), and immediately before (T2) or 6 h after (T3) the second heat shock. Another set of cells were incubated at 43 °C for 1.5 h, recovered at 37 °C for 6 h, and lysed (N). Cells were also kept at 37 °C and lysed (C). Equal amounts of proteins (50 µg) were analyzed by Western blotting using a monoclonal antibody specific for Hsp70 and 125I-labeled goat anti-mouse antibody as secondary antibody. The blot was washed and exposed to x-ray films.

Transcription of Hsp70 in Thermotolerant Cells Is Attenuated Rapidly-- Naive and thermotolerant HepG2 cells were thermally stressed at 43 °C for 1.5 h. Nuclei were isolated from cells harvested immediately at the end of the heat shock period, or after different times during incubation at 37 °C following the stress. Transcription rates were measured by the transcript run-off technique using nuclei isolated from the same number of cells for each reaction. In naive cells, transcription of Hsp70 was maximal between the end of the heat shock (0) and 1 h of incubation at 37 °C after the stress, followed by a steady decrease between 2 and 8 h of incubation at 37 °C. The rate of Hsp70 transcription at the end of the heat shock was not different between thermotolerant and naive cells. However, Hsp70 transcription in thermotolerant cells showed a sharp decrease in activity during the period immediately after the heat shock (Fig. 2, A and B). Thus, transcription at 0.5 h of incubation at 37 °C was less than 10% of the rate of transcription at the end of the heat shock in thermotolerant cells (Fig. 2B). The transcription rates were also evaluated during the heat shock period (60 and 90 min at 43 °C). No differences in Hsp70 transcription were found between naïve and thermotolerant cells (Fig. 2C). Transcription of the 28 S rRNA decreased immediately after heat shock in naive cells but not in thermotolerant cells. There was no hybridization to pSK used as a negative control (not shown).


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Fig. 2.   Transcription of Hsp70 after heat shock of naive and thermotolerant cells. Naive or thermotolerant HepG2 cells were incubated at 37 °C (CON) or at 43 °C for 1.5 h (HS), followed by incubation at 37 °C for the indicated times. Nuclei were isolated and used for in vitro transcription elongation reaction (run-off). Radiolabeled run-off transcripts were hybridized to filter-bound DNA targets corresponding to pSK, Hsp70, or 28 S rRNA, washed, and exposed to x-ray films (A). The autoradiographic signals in A were quantitated by laser scanning densitometry and plotted as the relative transcription rate of the Hsp70 gene in naive (solid line) and thermotolerant (dotted line) cells as a function of time after heat shock (B). Naive or thermotolerant HepG2 cells were incubated at at 43 °C for 60 or 90 min, or maintained at 37 °C. Nuclei were isolated and used for transcription reactions as described above (C).

The Half-life of Hsp70 mRNA Is Reduced in Thermotolerant Cells-- Analysis of the Hsp70 mRNA levels by Northern blotting showed a pattern similar to the transcription of the Hsp70 gene. In naive cells, Hsp70 mRNA levels increased from the end of the heat shock to 5 h of return to non-heat shock conditions, which were then followed by a steady decrease (Fig. 3A). In thermotolerant cells, Hsp70 mRNA levels were identical to naive cells at the end of the heat shock. A mild increase in Hsp70 mRNA levels was observed within 1 h of the stress. There was also a sharp decrease in the message level after 1 h of post-stress recovery (Fig. 3A). The half-life of Hsp70 mRNA was determined in conditions of negligible Hsp70 transcription to avoid the use of transcriptional inhibitors. In naive cells, the Hsp70 mRNA half-life was 1 h (data not shown) at 37 °C after heat shock, which is consistent with previous observations (32). On the other hand, the half-life of Hsp70 mRNA in thermotolerant cells during incubation at 37 °C after the stress was 0.5-06 h from three separate experiments (a representative experiment is presented in Fig. 4).


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Fig. 3.   Steady-state levels of Hsp70 mRNA after heat shock of naive and thermotolerant cells. Naive or thermotolerant HepG2 cells were incubated at 37 °C (CON) or at 43 °C for 1.5 h (HS), followed of by recovery period at 37 °C for the indicated times. Total RNA was isolated and analyzed by Northern blot/hybridization using a radiolabeled DNA probe for human Hsp70. After hybridization, blots were washed and exposed to x-ray films (A). The autoradiographic signals were quantitated by laser scanning densitometry and plotted as the relative transcription rate of the Hsp70 gene in naive (solid line) and thermotolerant (dotted line) cells as function of time after heat shock (B).


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Fig. 4.   Determination of Hsp70 mRNA half-life in thermotolerant cells. Thermotolerant HepG2 cells were heat-shocked at 43 °C for 1.5 h and allowed to recover at 37 °C for different time periods. Hsp70 mRNA half-life was determined during conditions of negligible Hsp70 transcription as described under "Materials and Methods."

Translation of Hsp70 mRNA Is Not Affected in Thermotolerant Cells-- The preceding observations suggest that the reduced expression of Hsp70 in thermotolerant cells is due to a rapid decrease in transcription and an increase in Hsp70 mRNA degradation. To investigate whether translation of Hsp70 could also be affected in thermotolerant cells, synthesis of Hsp70 was analyzed by pulse-labeling experiments. Naive or thermotolerant cells were pulse-labeled with [35S]methionine for 15 min at 37 °C immediately after heat shock (0), or after 1 or 2 h of post stress recovery. After the pulse labeling, cells were lysed. Lysates were immunoprecipitated with a monoclonal antibody specific for Hsp70, and the immunoprecipitate was visualized by SDS-polyacrylamide gel electrophoresis and fluorography (Fig. 5, top panel). For comparison, Hsp70 mRNA levels in the pulse-labeled cells were also analyzed (Fig. 5, bottom panel). There were no significant differences in the synthesis of Hsp70 between naive and thermotolerant HepG2 cells when newly synthesized Hsp70 (immunoprecipitation) and Hsp70 mRNA levels were compared.


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Fig. 5.   Synthesis of Hsp70 after heat shock of naive and thermotolerant cells. Naïve (1-4) or thermotolerant (5-8) HepG2 cells were incubated at 37 °C or at 43 °C for 1.5 h (HS), and recovered at 37 °C for 0 (2, 6), 1 (3, 7), and 2 (4, 8) h. As control, nonstressed naive cells were used (1). In addition, thermotolerant cells without a second stress were used (5). Cells were lysed after pulse-labeling with [35S]methionine for 15 min. Part of the cell lysate (50 µg of protein) was immunoprecipitated with a monoclonal antibody specific for Hsp70 (C92, StressGen) and visualized by SDS-polyacrylamide gel electrophoresis and fluorography (A). Total RNA was isolated from the rest of the cell lysate and analyzed by Northern blot/hybridization using radiolabeled probes for Hsp70 and beta -actin. Blots were washed and exposed to x-ray films (B).

To further investigate whether there were differences in translation of Hsp70 mRNA between naive and thermotolerant cells, the translational efficiency of Hsp70 mRNA was analyzed by determining the message localization in polysomes isolated from these cells after heat shock. Polysomes were obtained by density centrifugation through sucrose gradients. Fractions of these gradients were collected, total RNA was isolated from each fraction, and analyzed by slot blot/hybridization using radiolabeled probes for Hsp70 and beta -actin. The presence of the particular mRNA in the heavier polysome fractions is an indicator of a message that is well translated, whereas the presence of the mRNA in the monosome or disome fractions suggests that the message is translated less efficiently. beta -Actin mRNA was efficiently translated in non-stressed cells and less efficiently translated after heat shock at 43 °C for 1.5 h (Fig. 6). This observation is consistent with reports indicating that non-heat shock proteins are synthesized poorly after heat shock (15, 33). In conjunction with the decrease in the translation of beta -actin mRNA, Hsp70 mRNA was translated with good efficiency (Fig. 6). After 1 or 2 h of incubation at 37 °C following heat shock, Hsp70 mRNA is translated with high efficiency, whereas a slow increase in the translation efficiency of beta -actin mRNA was observed (Fig. 6). A similar analysis of thermotolerant cells showed that Hsp70 mRNA is always translated with high efficiency (Fig. 6), probably with higher efficiency as compared with naive cells immediately after heat shock. Interestingly, the translational efficiency of beta -actin mRNA was much better preserved in thermotolerant cells (Fig. 6). This observation is consistent with the protection of protein synthesis observed in thermotolerant cells (14, 15, 17).


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Fig. 6.   Polysome localization of Hsp70 and beta -actin mRNAs in naive and thermotolerant cells. Naive (A) or thermotolerant (B) HepG2 cells were incubated at 37 °C or at 43 °C for 1.5 h (HS), and recovered at 37 °C for 0, 1, and 2 h. Cells were lysed, and cytoplasmic extracts were sedimented on 15-45% sucrose gradients at 180,000 × g for 1.5 h. The gradient was fractionated, and the absorbance at 254 nm was monitored continuously (position of the 80 S ribosomal subunit and polysomes is indicated). Total RNA was isolated from each fraction (0.75 ml) and analyzed by slot blot/hybridization using radiolabeled probes for Hsp70 and beta -actin. Blots were washed and exposed to x-ray films. The autoradiographic signals were quantitated by laser scanning densitometry.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In the present study, a limited expression of Hsp70 after heat shock was observed in thermotolerant cells as compared with naive cells. This reduced expression of Hsp70 in thermotolerant cells was due to the combination of a decline in Hsp70 transcription and an increase in Hsp70 mRNA degradation during the return to non-heat shock conditions after the stress. In contrast, there were no significant differences in the transcription rate of Hsp70 during heat shock between thermotolerant and naive cells. This transcriptional and posttranscriptional regulation of Hsp70 expression may be part of a mechanism to limit the cellular concentration of this protein. A similar mechanism has been previously proposed to regulate the expression of Hsp70 in naive Drosophila cells after heat shock (34).

Previous studies have shown that the expression of hsp is attenuated proportionally to the initial stress and occurs during the recovery period in naive cells (20, 33). Attenuation of Hsp70 expression was not observed in conditions of continuous stress, such as in the case of incubation with amino acid analogues (34, 35). The expression of Hsp70 and Hsp90 during stress has been reported to be reduced in cells that contain conditionally induced Hsp70 (28). Functional mutation of Hsp70 results in overexpression of this gene in yeast (27).

A mechanism that has been proposed for autoregulation or self-limiting expression of Hsp70 is based on the observation that Hsp70 binds to HSF1 (20-22). Although the association of Hsp70 with HSF1 does not affect its DNA binding activity, Hsp70 transcription is negatively regulated (28). A similar model has been proposed in E. coli, in which hsps also directly interact with the transactivator factor sigma 32 (23-26). Our observations cannot be completely explained on the basis of this model. The level of Hsp70 transcription was similar between thermotolerant and naive cells during heat shock. Thus, Hsp70 does not block its own transcription, at least in thermotolerant cells during heat shock. As mentioned above, attenuation of Hsp70 was only observed during incubation at 37 °C after the stress. A possible explanation for this observation is that a temperature labile protein is necessary to attenuate Hsp70 transcription. This protein may be synthesized during the recovery period following the stress, resulting in the reduction of Hsp70 transcription. In thermotolerant cells, the attenuation of Hsp70 transcription is achieved more rapidly, probably because translation is protected in these cells (15, 17). An alternative explanation of our results could be formulated on the assumption that the inducer of the heat shock expression is the presence of denatured polypeptides within the cell. Indeed, previous studies have shown that hsps are expressed after microinjection of unfolded polypeptides into unstressed cells (36). Thus, it is possible that polypeptides are rapidly denatured in cells during heat shock regardless of whether the cells do or do not contain Hsp70 (thermotolerant). The indirect activation of HSF1 by the presence of unfolded polypeptides is faster than the interaction and potential solubilization of the unfolded polypeptides by hsps. Thus, the initial stimuli, the presence of denatured polypeptides, is the same for thermotolerant and naive cells. Upon return to non-heat shock conditions, hsps limit the amount of denatured polypeptides, resulting in a decrease of Hsp70 expression.

In addition to a decrease of Hsp70 transcription, Hsp70 mRNA is less stable in thermotolerant cells with respect to naive cells. The half-life of Hsp70 mRNA in naive cells at 37 °C is on the order of 1 h, whereas it is 0.5-0.6 h in thermotolerant cells. This accelerated degradation of Hsp70 mRNA is consistent with a mechanism to limit the expression of Hsp70. For example, the attenuation of transcription decreases further accumulation of Hsp70 mRNA. However, the remaining cytosolic Hsp70 mRNA can still be translated. Thus, degradation of Hsp70 mRNA is necessary to limit the cellular level of Hsp70. Although this hypothesis may explain the reduction in Hsp70 mRNA half-life in thermotolerant cells, the mechanism by which Hsp70 mRNA is destabilized is not clear. Many cellular mRNAs contain elements located within the message that are involved in the stability of the message (37). However, Hsp70 mRNA is identical between naive and thermotolerant cells. Thus, it is possible that a destabilizing protein that interacts with Hsp70 mRNA is denatured during heat shock in naive cells while protected in thermotolerant cells. In support of this hypothesis, the half-life of Hsp70 mRNA has previously been reported to be longer during heat shock in comparison to the recovery after the stress period (32). An alternative explanation of our observations is that Hsp70 mRNA is unstable under normal conditions (37 °C in mammalian cells). During heat shock, Hsp70 mRNA is apparently more stable because mRNA degradation is affected by the stress. In thermotolerant cells, degradation seems to happen immediately after returning to 37 °C. It is possible that mRNA degradation is accelerated in thermotolerant cells, resulting in a reduced half-life of Hsp70 mRNA.

Hsp70 mRNA is translated with high efficiency in both thermotolerant and naive cells suggesting that translational control does not play a role in the attenuation of Hsp70 expression. On the contrary, the translation efficiency of beta -actin mRNA is dramatically reduced in naive cells during heat shock, while preserved in thermotolerant cells during identical conditions. This observation is consistent with the protection of protein synthesis in thermotolerant cells during a second heat shock that has previously been reported (14-16). Recent evidence has shown that Hsp70 is associated with polysomes by the direct binding to ribosome subunits rather than nascent polypeptides (17). Thus, the improved translation of beta -actin in thermotolerant cells during heat shock may be related to the interaction of Hsp70 with ribosomes.

In summary, our study shows that the expression of Hsp70 in thermotolerant cells is attenuated by a decrease in Hsp70 transcription during incubation at 37 °C (recovery period) after heat shock. An increase in Hsp70 mRNA degradation also contributes to limit the expression of this gene. The presence of Hsp70 and other hsps has been associated with cellular protection to different stresses: stress tolerance (2). However, the accumulation of Hsp70 in the absence of stress has been shown to affect cell division (38). Thus, the presence of Hsp70 seems to be detrimental in the long term. A mechanism to limit the expression of Hsp70 may have evolved to preserve the cell from the toxic effects of this protein. The direct role of Hsp70 in this process remains to be demonstrated.

    ACKNOWLEDGEMENTS

We thank Michael P. McCluskey for technical assistance and the members of the De Maio laboratory for critically reading the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health NIGMS Grant GM-50878 and the Garrett Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Surgery, Georgetown University Medical School, Washington, DC 20007.

parallel To whom correspondence should be addressed: Div. of Pediatric Surgery, Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Fax: 410-502-5093; E-mail: ademaio{at}welchlink.welch.jhu.edu.

    ABBREVIATIONS

The abbreviation used is: hsp, heat shock protein.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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