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INTRODUCTION |
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
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.
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MATERIALS AND METHODS |
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
[
-32P]dATP and [
-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 [
-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), pHF
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
-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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RESULTS |
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.
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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).
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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."
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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
-actin. Blots were washed and exposed to x-ray films
(B).
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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
-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.
-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
-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
-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
-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
-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 -actin. Blots were washed and exposed to x-ray films. The
autoradiographic signals were quantitated by laser scanning
densitometry.
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DISCUSSION |
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
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
-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
-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.