From the
Human peripheral blood monocytes (PBM) produce superoxide anions
(O) by a process involving electron transfer from NADPH to
O
Exposure of cells to elevated temperatures or to a wide variety
of physical and chemical injuries activates the expression of stress
response genes, coding for the heat shock
(HS(
Intracellular accumulation of abnormal
or degraded proteins has been proposed as a common signal for the
induction of stress protein synthesis
(8) , while we have
suggested that reactive oxygen species would represent a ubiquitous
second messenger for HSP induction. One model to study the effects of
endogenous reactive oxygen species production on HSP induction is
phagocytosis, during which the activation of the respiratory burst
enzyme NADPH oxidase leads to the massive generation of superoxide
anions (O)
(9) . We found that phagocytosis of inactivated
Staphylococcus aureus by human monocytes-macrophages induces
the selective synthesis of hsp70, which is enhanced in the presence of
iron, while infection with Leishmania major, which does not
activate the respiratory burst, fails to induce a host cell stress
response
(10, 11) . These and other results suggest that
reactive oxygen species, among which hydroxyl radicals are generated
via the iron-dependent O-driven Fenton reaction, play a specific role
in hsp70 induction
(12) .
Since, however, many activation
events, distinct from reactive oxygen species production, such as
calcium mobilization and activation of protein kinases
(13) ,
occur during phagocytosis, we investigated the role of O in HSP
regulation using phorbol 12-myristate 13-acetate (PMA), a
non-particulate, non-receptor-mediated activator of NADPH
oxidase
(14) . PMA is a potent activator of protein kinase C
(PKC) and may mimic in vitro a number of activation events
occurring during inflammation. We thus compared the expression of HSP
in human peripheral blood monocytes (PBM) after HS or exposure to PMA.
We also compared the inducing effects of HS and PMA on the stress
response in cells from normal donors and in cells from patients with
chronic granulomatous disease (CGD). CGD is characterized by a genetic
defect in NADPH oxidase leading to a selective lack of O production
upon phagocytosis or activation with PMA, while PKC activation is
unaltered. To further differentiate between the respective roles of O
and PKC in the regulation of HSP expression, we analyzed the effects of
PKC inhibitors on HSP induction by HS or PMA.
Our results indicate
that there is a differential expression of hsp70 and hsp90 in human PBM
following exposure to the two stresses considered here, HS and PMA.
This differential expression is the consequence of the involvement of
both distinct second messengers and distinct mechanisms for molecular
regulation of HS gene expression.
In this study, we report that HS and PMA lead to differential
expression of HSP in human PBM. Our data support the distinct
regulation of various members of the HSP families and provide new
insight into the respective roles of O and PKC as second messengers in
the stress response.
While heat-shocked PBM synthesized all
classical HSP (hsp110, hsp90, hsp70, as well as hsc70, hsp65, etc.),
PMA-treated cells mostly increased the expression of hsp90 and, to a
lesser extent, hsc70 and hsp70. Mitogen-induced HSP synthesis had been
described before in human lymphocytes and in the human premonocytic
line U937
(24, 25) . While hsc70 was predominantly
induced in lymphocytes, both hsc70 and hsp70 were only slightly and
transiently induced by mitogens in the U937 cells, which is in
agreement with our results in PBM. Hsp90, which also is inducible by
mitogens, actually consists of at least two proteins,
We propose that the
PMA-induced hsc70, hsp70, and hsp90 synthesis did not result from the
activation of NADPH oxidase and the resulting production of O, but was
mediated by the activation of PKC inasmuch as it was prevented by PKC
inhibitors. This was not the case for HS-induced HSP synthesis,
although protein kinases, mitogen-activated protein kinase, and PKC
have been described to be activated by HS
(27) . The finding that
O alone is neither necessary nor sufficient to induce HSP synthesis was
further supported by our observations in cells from patients with CGD.
Indeed, these cells, which lack a functional NADPH oxidase, were able
to mount a HS response following both elevated temperature and exposure
to PMA. The addition of iron during PMA treatment (but not during HS)
significantly increased the synthesis of hsp70 in normal PBM (data not
shown). These results suggest that O production is not directly
responsible for HSP induction during phagocytosis, while its resulting
transformation into hydroxyl radicals in the presence of iron may be
essential as inducers of HSP synthesis because of the oxidative
alteration of non-self (or self) proteins. Only hydroxyl radicals, and
not O, may have the ability to oxidatively alter proteins in a way
similar to HS, with the presence of altered proteins being the ultimate
signal for hsp70 induction. In contrast, the expression of hsc70 and
hsp90 appears to be tightly regulated by PKC activity, at least during
mitogen stimulation.
On the other hand, our data also indicate that
while HS-induced HSP synthesis is principally controlled at the
transcriptional level (activation of HSF), the PMA-induced HSP
synthesis resulted from a stabilization of HSP mRNA (Fig. 7). The
temporal delay in the accumulation of hsp70 and hsp90
In eukaryotes, cellular growth, differentiation, and
response to environmental stimuli are associated with differential mRNA
stability (29). Many transiently expressed genes including cytokines,
oncogenes (c-myc and c-fos), and transcriptional
activators accumulate in activated cells secondary to enhanced
stability of specific mRNA
(30, 31, 32) . A
number of these mRNAs contains AU-rich sequences as regulatory motifs
in their 3`-untranslated region. The presence of such AU-rich sequences
modulates the turnover of mRNA by targeting it to the cytoplasm for
rapid degradation
(33, 34) .
A similar mechanism of
degradation could be employed to reduce the constitutive level of HSP
mRNA expression under normal conditions. Directly after their
synthesis, HSP mRNAs could be targeted to and degraded in the cytosol
before accumulation. Indeed, Moseley et al.(35) have
demonstrated that in addition to transcriptional regulation, the human
gene coding for hsp70 can also be regulated post-transcriptionally
through the 3`-untranslated region by HS. The 3`-untranslated region of
the hsp70 message is AU-rich and contains an AUUUA-like
motif
(6, 36) . Furthermore, several groups have recently
shown that cell activation by phorbol esters or calcium ionophores
stabilizes AU-rich mRNA sequences by controlling, via PKC-mediated
phosphorylation, the activation of a cytoplasmic protein called
AU-binding factor
(37) . AU-binding factor forms in vitro complexes with a variety of labile RNA containing multiple
reiterations of the pentamer AUUUA
(38) . From data concerning
the inhibition by staurosporine and H-7 of HSP synthesis and HSP mRNA
accumulation in PMA-treated PBM, we propose that a similar
translational mechanism involving cycles of phosphorylation
consecutively to the PMA-mediated activation of PKC could lead to
stabilization and increased levels of HSP mRNA. However, we cannot
exclude that during physiological events such as cell proliferation and
differentiation, HSP synthesis results from a growth factor-mediated
pathway that would activate serum- and/or growth factor-responsive
elements in the promoters of hsp70 and hsp90
genes
(39, 40) .
According to the nature of the
stimuli (HS, phagocytosis, mitogen, etc.), a given cell could use
either genomic 5`-transcriptional regulatory elements or 3`-mRNA
post-transcriptional elements, or both, to finely control HSP
synthesis. Under conditions associated with a sustained HSP expression
such as cell proliferation and differentiation, inflammation, ischemia,
or carcinogenesis, stabilization of mRNA could be the essential
mechanism for HSP regulation, as we observed in cells stimulated with
PMA. In contrast, stresses such as HS that lead to a rapid alteration
of cellular proteins induce a more rapid and transient transcriptional
regulation of HS genes and subsequent HSP synthesis.
Taken together,
our results indicate that regulation of HSP induction after HS
principally involves transcriptional events, whereas PMA-mediated HSP
synthesis, as a paradigm for HSP induction during inflammation and cell
activation and differentiation, results from a post-transcriptional
regulatory mechanism.
Extracellular O production was determined by reduction of cytochrome
c monitored at 550 nm. Intracellular O was determined by NBT
assay performed as described. Values are means ± S.E. from three
distinct experiments.
We thank I. Maridonneau-Parini for stimulating our
interest in PMA regulation of HSP, R. Morimoto for helpful comments and
for the hsp90 cDNA probe, P. D. Lew for providing the cells from the
CGD patients, and L. Mudespacher for skillful technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, catalyzed by the respiratory burst enzyme NADPH oxidase.
We have previously shown that phagocytosis, while activating NADPH
oxidase, induced in PBM the synthesis of heat shock (HS) proteins
(HSP). The present study was undertaken to establish whether this
increase in HSP expression was related to O and/or to classical second
messengers such as protein kinase C (PKC). Thus, the effects of the PKC
activator phorbol 12-myristate 13-acetate (PMA) were compared with
those of heat shock on the expression, in PBM, of the major HSP, hsp70
and hsp90, using biometabolic labeling, Western and Northern blotting,
and gel mobility shift assays. PMA induced the accumulation of mRNA and
an increased expression of hsp90 and, to a lesser extent, hsp/hsc70
(hsc is the cognate, constitutive form). This induction was also
observed in PBM from patients with chronic granulomatous disease, a
genetic defect in NADPH oxidase, and was abolished by the PKC
inhibitors staurosporine and H-7. PMA did not cause activation of the
HS factor, and the PMA-induced overexpression of HSP was not blocked by
the transcriptional inhibitor actinomycin D. HSP-specific mRNA
stability was increased after PMA exposure as compared with heat shock.
These results suggest that O is not involved in the PMA-mediated
induction of hsp70 and hsp90 and that, in contrast to HS, PMA increases
the expression of HSP as a result of PKC-induced mRNA stabilization
rather than of transcriptional activation of HS genes.
))
(
)
/stress proteins (HSP) (1).
These proteins are generally classified into families according to
their apparent molecular weight and respective inducers and play
essential roles in protein chaperoning and cellular protection. HSP
expression is not limited to cells undergoing acute stress, and several
members of HSP families are constitutively expressed (HS cognates).
Furthermore, the expression of several members of the HSP families
(hsp/hsc70, hsp90) has been described to be modulated during
nonstressful conditions such as cell cycle
(2) and
differentiation
(3) , in response to serum
stimulation
(4) , and during the early stages of
embryogenesis
(5) . The expression of HSP has been shown to be
regulated by both transcriptional and translational
events
(6, 7) .
Cells and Media
PBM were isolated from normal
volunteers and from two patients with CGD by gradient centrifugation
and were purified by adherence as described previously
(15) . The
freshly isolated cells were cultured at 37 °C in antibiotic-free
RPMI 1640 medium (Gibco, Paisley, Scotland) supplemented with 10% fetal
calf serum (Gibco) and 1% glutamine (Gibco) in a humidified atmosphere
(95% air, 5% CO).
Reagents and Exposure to HS
PBM were incubated for
3.5 h at 37 °C with 50 ng/ml PMA (Sigma). For HS, PBM were
incubated in a water bath at 44 °C for 20 min, followed by a
recovery period of 2 h at 37 °C. The transcriptional inhibitor
actinomycin D (Sigma; 5 µg/ml) was added to PBM 10 min prior to HS
or exposure to PMA and was present throughout the experiments. In other
experiments, PKC inhibitors were added 30 min prior to HS or exposure
to PMA. Staurosporine was used at 200 nM and H-7
(1-(5-isoquinolinyl)-2-methylpiperazine) at 100 µM. Both
PKC inhibitors were from Calbiochem.
Superoxide Measurement
PBM were washed and
resuspended in buffer containing 138 mM NaCl, 6 mM
KCl, 1 mM MgSO7H
O, 1.1
mM CaCl
2H
O, 0.2 mM
EGTA, 5.5 mM glucose, and 20 mM HEPES (pH 7.4) at 20
10
cells/ml. 10
PBM were stimulated
with PMA (1.6
10
M) for 30 min at
37 °C or exposed to HS for 20 min at 44 °C. The extracellular
production of O was measured by the superoxide dismutase-inhibitable
reduction of cytochrome c as described previously
(14) .
In parallel experiments, the intracellular production of O was
qualitatively determined by nitro blue tetrazolium (NBT) reduction as
described previously
(16) . Briefly, 4
10
PBM
were incubated in phosphate-buffered saline containing 0.04% NBT and
10% fetal calf serum. After PMA (100 ng/ml) or HS stress, PBM were
cytocentrifuged on a glass slide, fixed with methanol, and stained with
safranin for 1 min. 200 cells/slide were counted under a light
microscope.
Analysis of Protein Synthesis
In these
experiments, RPMI 1640 medium was replaced by RPMI 1640 medium without
methionine (Gibco). PMA-treated PBM were metabolically labeled with
[S]methionine (6 µCi/ml; specific activity
of >1000 Ci/mmol; Amersham International, Buckinghamshire, United
Kingdom) added during the last 90 min of treatment with mitogen. For
heat-shocked PBM, labeling was performed for 90 min at 37 °C after
the 2 h of recovery. After labeling, aliquots corresponding to equal
cell numbers were resolved by SDS-PAGE (10% polyacrylamide) according
to Laemmli (17) and revealed by autoradiography. Hsc70, hsp70, and
hsp90 were characterized by Western blotting. Proteins were
electrophoresed; transferred to nitrocellulose membranes; and probed
with mouse monoclonal antibodies against human constitutive hsc70
(SPA820), inducible hsp70 (SPA810), or hsp90 (SPA840; all from
Stressgen Biotech Corp., Victoria, Canada). Bound antibodies were
revealed with anti-mouse IgG-peroxidase conjugated (Sigma) in the
presence of H
O
and 4-chloro-1-naphthol (Sigma).
RNA Extraction and Northern Blotting
Cells were
harvested immediately after HS or PMA treatment and lysed in buffer
containing 5 M guanidium isothiocyanate, 5 mM sodium
citrate (pH 7.0), 0.5% sodium N-laurylsarcosine, and 0.1
M 2-mercaptoethanol. Total RNA was purified and stored in
Tris/EDTA. For Northern analysis, total RNA (5 µg) was denatured
and fractionated by electrophoresis on 1% agarose gels in 5%
formaldehyde as described by Maniatis et al.(18) .
After electrophoresis, the RNA was transferred onto a Biodyne membrane
in 20 SSC (1
SSC = 0.15 M NaCl, 0.015
M trisodium citrate (pH 7)). The membranes were
UV-cross-linked; backed for 2 h at 80 °C; prehybridized for at
least 5 h at 55 °C in 5
SSC, 10 mM
NaPO
, 50% formamide, 5
Denhardt's solution (1
Denhardt's solution = 0.002% polyvinylpyrrolidone,
0.02% Ficoll, and 0.02% bovine serum albumin), 0.1% SDS, and 250
µg/ml salmon sperm; and hybridized overnight in the same buffer at
65 °C with a [
P]UTP-labeled antisense RNA
transcript (2
10
cpm/ml). This RNA probe was
prepared as described
(19) . For analysis of hsp90 mRNA, the
membranes were hybridized with a DNA probe labeled using the multiprime
labeling kit from Amersham International in the same buffer as
described above at 42 °C for 12-16 h. This probe was an
isolated HindIII-restricted fragment derived from an entire
human hsp90
gene (kindly provided by R. Morimoto). After
hybridization, the membranes were washed sequentially at 65-70
°C in 2
SSC and in 0.2
SSC, 0.1% SDS. The membranes
were then subjected to autoradiography on Hyperfilm (Amersham
International) at -70 °C using intensifying screens. As an
internal control for the loading of RNA, we analyzed the band
corresponding to 28 S mRNA.
HSP mRNA Stability after HS and PMA
Treatment
After HS (20 min at 44 °C, followed by a recovery
period of 30 min at 37 °C) or PMA exposure (50 ng/ml, 6 h),
actinomycin D was added to the culture medium at a final concentration
of 5 µg/ml to block RNA transcription as described
previously
(19) . The cells were lysed 2, 4, 6, and 8 h after
administration of actinomycin D, and total RNA was extracted and
analyzed by Northern blotting as described above. Results were
expressed as the percentage of the mRNA values obtained before the
addition of actinomycin D (time 0).
Preparation of Nuclear Extracts and Gel Mobility Shift
Assays
Preparation of nuclear protein extracts was adapted from
the method of Dignam et al. (20). Briefly, cells were
collected and kept on ice for 10 min in hypotonic lysis buffer
containing 10 mM HEPES (pH 7.9), 1.5 mM
MgCl, 10 mM KCl, 0.5 mM dithiothreitol,
0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each
antipain, leupeptin, and pepstatin. The cells were homogenized in a
Dounce homogenizer, and the nuclei were pelleted at 3000 rpm and
resuspended in extraction buffer (10 mM HEPES (pH 7.9), 400
mM NaCl, 1.5 mM MgCl
, 0.1 mM
EDTA, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, and 5% glycerol) with constant mixing at
4 °C for 30 min. The samples were centrifuged at 14,000 rpm for 15
min at 4 °C, and the supernatant was dialyzed against buffer
containing 20 mM HEPES (pH 7.9), 75 mM NaCl, 0.1
mM EDTA, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, and 20% glycerol. Samples were
quick-frozen in liquid nitrogen and stored at -80 °C. Protein
concentration was determined with a bicinchoninic acid protein assay
kit (Pierce). Nuclear extracts were then analyzed for HS element (HSE)
binding activity by gel mobility shift assays. Binding reactions were
performed for 30 min at 25 °C by adding 5 µg of nuclear
proteins to a mixture containing 10
cpm of
[
-
P]ATP end-labeled, double-stranded HSE
oligonucleotide (5`-GCCTCGAATGTTCGCGAAGTT-3`) in 15 µl of dialysis
buffer containing 2 µg of poly(dI-dC) and 10 µg of bovine serum
albumin. For the competition experiments, a 100-fold molar excess of
nonradioactive HSE or a 100-fold molar excess of a tumor-promoting
agent-responsive element oligonucleotide (5`-CTTGTGAGTCATTCC-3`) was
added. Samples were electrophoresed on a nondenaturing 4%
polyacrylamide gel, dried, and autoradiographed.
Differential Induction of Stress Proteins following
Exposure to HS or PMA
The induction of HSP expression in PBM
following HS or PMA exposure is shown in Fig. 1A.
Exposure of PBM to 44 °C for 20 min induced both hsp70 and hsp90
(Fig. 1A, lane 2) as well as hsp65 and hsp110
(Fig. 3A, lanes 4-6), whereas exposure to
PMA essentially induced the expression of hsp90
(Fig. 1A, lane 3). Time course experiments
indicated that PMA-induced HSP synthesis was maximal at 3.5 h of
incubation before reaching a plateau (data not shown). The differential
induction of HSP observed in Fig. 1A was confirmed by
Western blot analysis performed with specific monoclonal antibodies
against hsc70, hsp70, and hsp90 (Fig. 1B). Both HS and
PMA slightly increased the expression of constitutive hsc70
(Fig. 1B, panela); the antibody used
to detect hsc70 cross-reacts with inducible hsp70. Using an antibody
specific for inducible hsp70, we confirmed that HS strongly increased
the expression of this protein, while only a weak induction was
observed with PMA (Fig. 1B, panelb).
In contrast, both stresses stimulated the expression of hsp90 with a
similar intensity (Fig. 1B, panelc).
Figure 1:
Differential induction
of HSP in human PBM following exposure to HS or PMA. A,
SDS-PAGE analysis of protein synthesis by PBM after HS (20 min at 44
°C) (lane 2) or treatment with PMA (50 ng/ml, 3.5 h, 37
°C) (lane 3). Lane 1 is the control. B,
Western blot analysis of hsc70 (panela), hsp70
(panelb), and hsp90 (panelc)
after exposure to HS (lanes 2) or PMA (lanes 1).
Lanes 3 are the controls. While HS induced major classical HSP
synthesis, PMA essentially increased the expression of hsp90.
a, actin.
Figure 3:
PMA-mediated HSP synthesis in human PBM is
inhibited by PKC inhibitors. A, SDS-PAGE analysis of protein
synthesis by PBM after HS (lanes 4-6) or treatment with
PMA (lanes 7-9) in the absence (lanes 4 and
7) or presence of staurosporine (200 nM) (lanes 5 and 8) or H-7 (100 µM) (lanes 6 and
9). PKC inhibitors were added to PBM 30 min before exposure to
HS or PMA. B, Western blot analysis of hsp70 (panela) and hsp90 (panelb) protein
induction in PBM exposed to HS (lanes 2-4) or incubated
with PMA (lanes 5-7) in the absence (lanes 2 and 5) or presence of H-7 (100 µM)
(lanes 3 and 6) or staurosporine (200 nM)
(lanes 4 and 7). Lanes 1 are the controls
(C).
Superoxide Anion Production during Exposure to HS or
PMA
As an approach to understand this differential induction of
HSP following HS and PMA exposure, we first compared the production of
O by PBM exposed to HS or treated with PMA. In are
summarized the results of O production as determined by cytochrome
c (extracellular O production) and NBT (intracellular O
production) reduction. While PMA induced in PBM both extracellular and
intracellular O production (38.4 ± 1.9 nmol of O/10 cells, respectively; 152 ± 9.5 NBT-positive cells), HS did
not induce O production at least as detected by the methods used. On
the other hand, as described previously
(21) , pre-exposure of
PBM to HS abolished the PMA-induced O production ().
Involvement of PKC in PMA-induced hsp90
Expression
To determine whether the PMA-induced HSP synthesis
resulted from the activation of PKC (rather than from the subsequent
activation of the respiratory burst), we compared PBM from normal
donors and PBM from patients with CGD with respect to the induction of
HSP by PMA. In the latter cells, PMA increased hsp90 expression as in
control cells (Fig. 2, compare lanes 5 and 2).
To provide further arguments in favor of the hypothesis that
PMA-mediated hsp90 synthesis relates to PKC activation rather than to O
production, we analyzed the effects of the PKC inhibitors staurosporine
and H-7 on HSP expression. Control experiments indicated that at the
concentrations used, these inhibitors were not toxic for the cells
(Fig. 3A, lanes 1-3). The PKC inhibitors
had no effect on HS-induced HSP (Fig. 3A, lanes
4-6), while in contrast, they abolished the PMA-induced HSP
synthesis (lanes 7-9). Western blot analysis confirmed
both the lack of inhibition in HS-mediated HSP expression in the
presence of staurosporine and H-7 (Fig. 3B, lanes
2-4) and the inhibition of PMA-mediated hsp90 expression
(lanes 5-7). The effect of staurosporine on the
inhibition of HSP synthesis was greater than that of H-7, an
observation that might relate to the fact that staurosporine inhibits
PKC more specifically than H-7.
Figure 2:
Comparison of HSP induction in PBM from
normal donors and from patients with CGD. Shown is SDS-PAGE analysis of
protein synthesis by PBM from normal donors (lanes 1-3)
and from patients with CGD (lanes 4-6) exposed to HS
(lanes 3 and 6) or to PMA (lanes 2 and
5). Unstressed cells (control (C)) are shown in
lanes 1 and 4. PMA induced the expression of hsp90 in
both normal and CGD cells.
Differential Kinetics of hsp70 and hsp90 mRNA Levels
following Exposure to HS or PMA
We then addressed the
possibility of a differential level of regulation in HSP synthesis
following HS or PMA exposure. We first investigated the effects of PMA
on hsp70 and hsp90 mRNA levels. Cytoplasmic RNA was isolated at
different times following PMA treatment, and HSP mRNA levels were
determined by Northern blot analysis with specific probes for hsp70 or
hsp90 (Fig. 4). In unstressed cells, neither hsp70 nor hsp90
mRNA was detected (Fig. 4, lane1 in A and B). HS induced a rapid increase in hsp70 mRNA levels
at 30 min, whereas PMA led to a delayed accumulation of hsp70 mRNA,
detectable after 2 h and increasing over time (up to 6 h)
(Fig. 4A). Even at 6 h, the levels of hsp70 mRNA were
still lower with PMA than with HS. In contrast, both stresses induced
hsp90 mRNA accumulation, and in this case, the levels of mRNA at 6 h
were higher with PMA than with HS (Fig. 4B). The
kinetics of hsp90 mRNA induction by PMA appeared similar to those
obtained with the same stressor for hsp70 mRNA. Both hsp70- and
hsp90-specific mRNA induced at 6 h by PMA were suppressed by
pretreatment of the cells with the PKC inhibitors staurosporine and H-7
(Fig. 4, A and B, compare lane 7 with
lanes 8 and 9).
Figure 4:
Kinetics of HSP mRNA induction in PBM
following exposure to HS or PMA. Total RNA (5 µg/lane) from control
PBM (C; lanes 1), heat-shocked PBM (lanes
2), or PBM treated with PMA for the indicated time periods
(lanes 3-7) was analyzed using the
P-radiolabeled hsp70 (A) and hsp90
(B) cDNA probes as described under ``Materials and
Methods.'' Both hsp70- and hsp90-specific mRNA induced by PMA (6
h) were suppressed by pre-exposure of the cells to the PKC inhibitors
staurosporine (compare lanes 7 and 8) and H-7
(compare lanes 7 and 9).
Differential Activation of HSF following Exposure to HS
or PMA
To determine the effects of PMA on the activation of HSF
and its binding to HSE, we performed gel mobility shift assays using a
synthetic oligonucleotide containing the consensus HSE-binding site
from the human HSP promoter. Gel mobility shift assays were done with
nuclear protein extracts prepared from cells treated for different
periods of time with PMA. HS induced the appearance of typical
HSFHSE complexes (Fig. 5, A, lane 2;
B, lanes 2 and 4), while following exposure
to PMA, no specific HSF
HSE complexes were detected (A,
lanes 3-8)
(22) . Specificity of HSF binding was
tested by competition experiments with a molar excess of 100-fold
nonradioactive HSE or tumor-promoting agent-responsive element
(Fig. 5B). Under the conditions of our experiments, both
the constitutive HSE binding activity (CHBA) (when expressed,
which was not the case in all experiments; see Fig. 5A)
and the inducible forms of DNA binding activity were suppressed by an
excess of cold HSE.
Figure 5:
PMA fails to induce HSFHSE binding
activity in human PBM. A, PBM were exposed to HS (lane
2) or to PMA for the indicated time periods (lanes
3-8). Five µg of nuclear proteins were subjected to gel
mobility shift assay using the HSE sequence from the human hsp70
promoter. As expected, HS induced HSF
HSE binding activity. In
contrast, after exposure to PMA, no HSF
HSE binding activity was
detected. Unstressed cells are shown in lane 1 (control
(C)). B, the specificity of HSF
HSE binding
activity was controlled by competition experiments with a 100-fold
molar excess of cold HSE (lanes 2 and 6) or
tumor-promoting agent-responsive element (TRE; lanes 4 and 7). In this experiment, we also observed constitutive
HSF
HSE binding activity
(CHBA).
Effects of Actinomycin D on HSP Synthesis following
Exposure to HS or PMA
Because of the observed lack of HSF
activation in PMA-mediated HSP synthesis, we also tested the effects of
the transcriptional inhibitor actinomycin D. Western blotting performed
with specific antibodies against hsp70 and hsp90 indicated that hsp70
synthesis was inhibited by pretreatment with actinomycin D in
heat-shocked cells only, with no effect in PMA-treated PBM
(Fig. 6, compare lanes 3 and 4). In contrast to
hsp70, both HS- and PMA-induced hsp90 synthesis were unaffected by
actinomycin D (Fig. 6, compare lanes 5 and 6),
which suggests that during HS-mediated induction of HSP synthesis,
different regulatory mechanisms could be involved for different HSP.
Figure 6:
Effects of actinomycin D on the expression
of HSP induced by HS or PMA. Shown are the Western blots for hsp70
(A) and hsp90 (B) in PBM exposed to HS (lanes 3 and 4) or incubated with PMA (lanes 5 and
6). Actinomycin D (5 µg/ml) prevented the HS-mediated
hsp70 expression, but had no effects on hsp90 and PMA-induced hsp70
expression. Lanes 1, control cells; lanes 2,
control cells with actinomycin D
(AD).
PMA Increases HSP mRNA Stability
To further
establish that the increased HSP expression following PMA was caused by
a post-transcriptional rather than a transcriptional mechanism, we
compared the hsp70 and hsp90 mRNA half-lives in HS- and PMA-treated
cells. As shown in Fig. 7, PMA increased HSP mRNA stability
compared with HS, an effect more important for hsp70 mRNA than for
hsp90 mRNA. In heat-shocked cells, we observed a 95% decrease in hsp70
mRNA levels 8 h after the addition of actinomycin D, compared with a
48% decrease in PMA-treated cells (Fig. 7A). With
respect to hsp90 mRNA, which has been described to be more stable than
hsp70 mRNA
(23) , the difference in the two conditions was less:
8 h after treatment with actinomycin D, there was a 35% decrease
following HS, compared with a 20% decrease in PMA-treated cells
(Fig. 7B).
Figure 7:
Effects of HS and PMA on HSP mRNA
stability. After exposure of PBM to HS or PMA, actinomycin D was added
(5 µg/ml final concentration), and the culture was continued for 2,
4, 6, or 8 h at 37 °C. At each time point, the cells were lysed,
and both hsp70- and hsp90-specific mRNA levels were determined by
Northern blot analysis. Results (means ± S.E.) are expressed as
the percentage of the mRNA values obtained before the addition of the
inhibitor (time 0 (T0)).
and
,
each encoded by its own gene
(26) . Both forms of hsp90 are
regulated by HS and mitogenic stimuli, with hsp90
being more
inducible by HS than by mitogens, while the opposite is true for
hsp90
. These HSP genes share similar promoter regions; thus,
regulatory mechanisms involving a distinct unique secondary messenger
or a cascade of messengers must exist to explain the differential HSP
expression that we and others observed.
mRNA during
PMA treatment stands in marked contrast to the rapid increase in hsp70
and hsp90
mRNA levels upon HS. In the latter case, increased
steady-state levels of mRNA were detectable within the first 30 min.
Furthermore, in contrast to HS, PMA failed to induce activation,
translocation, and DNA binding activity of HSF
(Fig. 5)
(7, 28) . The lack of effect of the
transcriptional inhibitor actinomycin D on PMA-mediated HSP synthesis
further supports a predominantly post-transcriptional regulation of HSP
expression by PMA (Fig. 6). We also found that in contrast to
hsp70, hsp90 induction upon HS was not inhibited by actinomycin D,
which suggests that the expression of this specific HSP family is
regulated post-transcriptionally even during HS, which is in agreement
with the increased hsp90 mRNA stability as compared with hsp70 mRNA
(Fig. 7).
Table:
Superoxide production by human monocytes
following HS or PMA treatment: colorimetric measurement and NBT assay
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.