(Received for publication, July 8, 1996, and in revised form, November 22, 1996)
From the Induction of heat shock gene expression is
mediated by specific heat shock transcription factors (HSFs), but the
signaling pathways leading to activation of HSFs are poorly understood. To elucidate whether protein kinase C-responsive signaling pathways could be involved in the regulation of heat shock gene expression, we
have examined the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) on the
heat-induced stress response in K562 cells. We demonstrate that TPA
treatment markedly enhances heat shock gene expression during heat
stress, although TPA alone does not induce the heat shock response.
This TPA-mediated enhancement can initially be detected as an
accelerated acquisition of DNA binding and transcriptional activity of
HSF1 resulting in elevated Hsp70 protein concentrations. In the
presence of TPA, the attenuation of HSF1 DNA binding activity during
continuous exposure to heat shock occurs more rapidly and in concert
with the appearance of newly synthesized Hsp70, which supports earlier studies on the autoregulatory role of Hsp70 in deactivation of HSF1.
During heat stress, a correlation between the hyperphosphorylation of
HSF1 and its transcriptional activity was observed, in both the
presence and the absence of TPA. Our results show that the heat-induced
stress response can be significantly modulated by activation of protein
kinase C-responsive signaling pathways.
In eukaryotic cells, the common cellular response to heat shock
and other types of stress is a rapid transcriptional activation of heat
shock genes resulting in increased synthesis of the heat shock proteins
(Hsps).1 The initial signaling events in
this well characterized physiological process, also called the stress
response, have not been identified. Hence, it is not known how cells
sense stress and how the signal is conveyed to the transcriptional
apparatus. However, it is well established that the transcriptional
activity of heat shock genes in eukaryotes is regulated by specific
pre-existing transcription factors (HSFs). Upon exposure to various
forms of stress, HSFs bind to the heat shock element (HSE) located in
the promoter regions of heat shock genes (for review see Ref. 1). The
mechanisms regulating the DNA binding and transcriptional activity of
HSFs are currently the focus of intense investigation.
In mammalian cells, two HSFs, HSF1 and HSF2, have been identified as
regulators of heat shock gene expression (2-4). HSF1 is activated in
cells exposed to elevated temperatures and other environmental stress
conditions (5-7), whereas HSF2 appears to function in cells involved
in processes of differentiation and development (8-10). In response to
stress, HSF1 undergoes oligomerization from a non-DNA-binding monomer
to a DNA-binding trimer and translocates into the nucleus to interact
with HSEs of the heat shock gene promoters (5, 7, 11-14). In addition,
mammalian HSF1 undergoes inducible phosphorylation upon activation (7,
15-17). The inducible phosphorylation has also been shown for the
yeast Saccharomyces cerevisiae HSF, which unlike other
eukaryotic HSFs is constitutively bound to the HSE but remains
transcriptionally inactive prior to heat shock (18). Recent evidence
suggests that the hyperphosphorylated state of HSF1 is not required for
DNA binding activity but might be required for transcriptional
activation. For example, treatment with the anti-inflammatory drugs
sodium salicylate and indomethacin induces DNA binding activity of HSF1
but fails to activate transcription of heat shock genes (15, 19-21).
In neither case is HSF1 hyperphosphorylated. In contrast, treatment
with arachidonate leads to HSF1 DNA binding activity,
hyperphosphorylation of HSF1, and activation of hsp70 gene
transcription (22). Both activating and inactivating phosphorylation sites may be present on HSF1, because a study in the yeast
Kluyveromyces lactis proposes that phosphorylation of HSF
may also serve as a regulatory mechanism to inactivate HSF (23). Hence,
there is substantial evidence indicating that phosphorylation is
involved in the activation of HSF1, although the exact role of
phosphorylation in the regulation of this transcription factor has not
been established.
Significant progress has been made to define the signaling pathways
that lead to transcriptional activation of eukaryotic gene expression.
Although protein phosphorylation has been identified as a major
post-translational mechanism responsible for regulating the activity of
many known transcription factors, such as NF- To elucidate the signaling pathways involved in the regulation of
stress response, we have used the phorbol ester
12-O-tetradecanoylphorbol 13-acetate (TPA) to examine the
effects of PKC activation on heat-induced HSF1 activation and
expression of the heat shock genes hsp70 and hsp90. Our results show that although TPA treatment alone
does not induce the stress response in K562 cells, TPA treatment in combination with heat shock markedly enhances the stress response. This
enhancement is detected initially on the transcriptional level,
subsequently leading to increased levels of the hsp70
mRNA and protein. The TPA-mediated effect on the heat-induced
stress response also shows gene specificity, because the effects on
hsp70 and hsp90 gene expression are distinct.
Human K562 erythroleukemia cells and
HeLa cervix carcinoma cells were grown in RPMI 1640 (HyClone) and
Dulbecco's modified Eagle's medium (HyClone), respectively,
supplemented with 10% heat-inactivated fetal calf serum (BioClear) in
a humidified 5% CO2 atmosphere at 37 °C. Cells were
seeded at 5 × 106 cells/10-cm-diameter plate prior to
exposure to TPA (Sigma), 4 Whole cell extracts containing 15 µg of protein were incubated with For in
vitro run-on transcription reactions, isolated nuclei
(approximately 5 × 106 nuclei/reaction) were
incubated with 100 µCi of [ Total RNA was isolated by acid
guanidinium thiocyanate and phenol-chloroform extraction as described
previously (47). 10 µg of total RNA was separated on a 1% agarose
gel, fixed to a nylon membrane (Hybond-N, Amersham Corp.), and
hybridized with a 32P-labeled DNA probe containing either
the human hsp70 gene (pH2.3), human hsp90 gene
(pUCHS801), or rat GAPDH gene (pGAPDH). The hybridization and washing
conditions were as specified by the manufacturer (Amersham Corp.). The
plasmid DNA was radiolabeled using a nick translation kit
(Promega).
1 × 106 cells/ml were
washed with methionine- and cysteine-free medium, resuspended in 0.5 ml
of methionine-free medium containing 10% dialyzed fetal calf serum,
and exposed to treatment. During the last 30 min of treatment, 50 µCi
of [35S]methionine (1190 Ci/mmol,
TRAN35S-LABELTM, ICN) was added. Whole cell extracts
containing 15 µg of protein were analyzed by 8% SDS-PAGE and
fluorography. For Western blot analysis, whole cell extracts (10 µg)
were subjected to an 8% SDS-PAGE and transferred to nitrocellulose
membrane by using a semi-dry transfer apparatus as specified by the
manufacturer (Bio-Rad). After blocking for 90 min in 3% nonfat dry
milk in phosphate-buffered saline, the filters were incubated with
rabbit polyclonal antibodies to HSF1 or HSF2 (1:10,000 dilution; Ref. 7), mouse monoclonal antibodies to Hsp70 (1:20,000 dilution) (4g4, a
generous gift from Dr. R. I. Morimoto, Northwestern University, IL),
rat monoclonal antibodies to Hsp90 (1:5,000 dilution; SPA-835, StressGen), or rat monoclonal antibodies to Hsc70 (1:5,000 dilution; SPA-815, StressGen). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:20,000 dilution; Promega), goat anti-mouse immunoglobulin G (1:20,000 dilution; Amersham Corp.), and goat anti-rat
immunoglobulin G (1:20,000 dilution; Amersham Corp.) were used as
secondary antibodies. The detection was performed by using the ECL
system (Amersham Corp.).
To
examine the effect of TPA on the activation of HSF1 DNA binding
activity, K562 cells were exposed to heat shock at 42 °C, 100 nM TPA, or to both TPA and heat shock for various time
periods extending to 6 h. Whole cell extracts were prepared and
analyzed by gel mobility shift assay. As shown earlier (48), the HSE binding activity of HSF1 was transiently activated in cells exposed to
a continuous heat shock at 42 °C (Fig. 1). The
maximum level (about 6-fold induction above the control level) of
heat-induced HSF1 HSE binding was obtained within 1 h, after which
the binding activity decreased to the basal level within 4 h. TPA
treatment alone did not induce any HSE binding activity of HSF1 (Fig.
1), although TPA was obviously effective in these cells as reflected by
a 2-3-fold stimulation of AP-1 DNA binding activity upon TPA treatment
over a time period of 15 min to 4 h.2
To our surprise, TPA treatment during continuous heat shock markedly affected the kinetics of HSF1 HSE binding activity. The maximum HSF1
HSE binding activity (approximately 7-fold induction) was detected
within 15 min followed by a rapid attenuation of HSF1 HSE binding
activity that returned to the pre-heat shock level by 3 h.
Furthermore, a moderate increase (approximately 1.5-fold induction) in
the level of HSF1 HSE binding was detected within the first 60 min of
incubation in TPA-treated heat-shocked cells. To ensure that the
TPA-mediated effect was not due to down-regulation of PKC, a lower
concentration (10 nM) of TPA was used and identical results, including kinetics, were obtained.2 Furthermore,
the TPA-mediated enhancement of the heat-induced HSF1 HSE binding
activity occurs too fast to be accounted for by down-regulation of PKC.
The faster attenuation of HSF1 HSE binding activity in TPA-treated
heat-shocked cells appears not to be a cell-specific phenomenon,
because similar effects were observed using HeLa cells.2
None of the treatments had any effect on cell viability as determined by trypan blue exclusion.
We next studied the phosphorylation states of HSF1, based on its
migration on SDS-PAGE. By using specific anti-HSF1 antibodies in
Western blot analysis, a slower migrating band on an 8% SDS-PAGE could
be observed in cell extracts from heat-shocked K562 cells compared with
the HSF1 band from untreated cells (Fig. 2). The slower
migration is presumably due to increased phosphorylation of HSF1 as has
been previously suggested (7, 15).2 Upon exposure to heat
shock, a minor retardation of HSF1 migration could be observed at 15 min, whereas the major slower migrating HSF1 band was detected at 1-2
h, after which it returned back to the control form by 6 h (Fig.
2, upper panel). In comparison, simultaneous treatment with
TPA and heat shock induced hyperphosphorylation of HSF1 more rapidly,
because the slower migrating HSF1 form was predominant already at 15 min. By 3 h, the slower migrating HSF1 form in TPA-treated
heat-shocked cells shifted back to a faster migrating form (called the
putative intermediate form, see below; Fig. 2, upper panel),
which seems to be distinct from the control form. Consequently, the
hyperphosphorylated form of HSF1 appears and disappears in parallel
with the acquisition and attenuation of HSF1 DNA binding activity,
respectively (Fig. 1). Consistent with the studies on DNA binding
activity, TPA treatment by itself did not induce the
hyperphosphorylated form of HSF1. However, in TPA-treated samples, a
slightly slower migrating form of HSF1 as compared with HSF1 in control
sample was detected from 15 min to 6 h of incubation, suggesting
that a putative intermediate phosphorylation state of HSF1 can be
induced by TPA (Fig. 2, upper panel).
For Western blot analysis, HSF2 was used as an internal control,
because no change in HSF2 migration on SDS-PAGE upon treatment with
heat or hemin has been detected (7, 49). Indeed, heat shock, TPA, or
combined treatment with TPA and heat shock did not affect the migration
of HSF2, but TPA treatment seemed to decrease the amount of HSF2 after
4-6 h (Fig. 2, lower panel).
To ensure that the TPA-mediated
effect on the DNA binding activity of HSF1 was not due to unknown side
effects of TPA instead of PKC activation, the inactive TPA analogue
4
Because simultaneous treatment of K562 cells with
TPA and heat shock affected the DNA binding activity of HSF1, we wanted to analyze the consequences of this effect on the expression of HSF1-regulated heat shock genes. First, we examined transcription by
performing nuclear run-on analysis on nuclei isolated from K562 cells
that were exposed to heat shock, TPA, or a combination of TPA and heat
shock. As shown in Fig. 4, the maximal, 20-fold induction of hsp70 transcription was obtained after 60 min
of heat shock. At this time point, a gradual attenuation of
hsp70 transcription occurred, with transcription returning
to its basal level after 6 h of continuous exposure to 42 °C.
Upon exposure to TPA and heat shock, an additional 3-fold increase in
the transcription rate of hsp70 was detected after 15 min
(Fig. 4). This difference between TPA-treated heat-shocked cells and
cells exposed only to heat shock was maintained until the 2-h time
point. Hence, the induction of hsp70 transcription was both
accelerated and enhanced by the TPA treatment during a continuous heat
shock as compared with the induction of hsp70 transcription
by heat shock alone (Fig. 4B).
hsp90, another HSF1-regulated heat shock gene, has earlier
been shown to be transcriptionally induced upon heat shock (10, 43,
51), but the extent of heat-induced transcription of hsp90 is usually less dramatic relative to that of hsp70. In this
study, the maximal increase of about 3-fold in hsp90
transcription was detected during the first hour at 42 °C (Fig. 4).
In analogy with the enhanced hsp70 transcription upon the
combined treatment with TPA and heat shock, the transcriptional
activity of hsp90 was further increased by approximately
2-fold at 15 min of treatment as compared with cells exposed to heat
shock alone. In addition, a moderate increase in the transcriptional
activity of hsp60 and hsc70 genes was detected
(Fig. 4). TPA treatment by itself did not induce heat shock gene
transcription, which is consistent with the results of DNA binding and
phosphorylation state of HSF1 in TPA-treated cells. However,
transcription of Following the analysis of transcriptional activities, we examined the
steady-state levels of hsp70 and hsp90 mRNA
by Northern blot analysis (Fig. 5). Upon heat shock, the
amount of hsp70 mRNA was induced by approximately
80-fold reaching the maximum level at 2-3 h of treatment. In cells
exposed to both TPA and heat shock, an additional 4-fold increase in
hsp70 mRNA amount above the levels in heat-shocked cells
was detected within the first hour of treatment (Fig. 5). Likewise,
following 1-6 h of incubation, the steady-state levels of
hsp90 mRNA were approximately 2-fold higher in
TPA-treated heat-shocked cells than in cells exposed to heat shock
alone (Fig. 5). At the attenuation phase, a clear difference between
the decline of hsp70 and hsp90 mRNA was
observed. After the peak at 2-3 h exposure to heat shock in both the
presence and the absence of TPA, the hsp70 mRNA levels
steadily decreased up to 6 h of incubation, whereas the maximally
induced levels of hsp90 mRNA were maintained throughout
this time period. Taken together, our results indicate that the
TPA-mediated increase in the steady-state levels of hsp70 mRNA in heat-shocked cells is mainly due to transcriptional
induction of hsp70.
Because it had been earlier implied
that heat shock gene expression can be regulated at multiple levels and
that increased transcription does not necessarily result in increased
protein synthesis (22), we wanted to examine the effect of TPA on
heat-induced expression of heat shock proteins. The rate of Hsp70 and
Hsp90 synthesis was examined by labeling K562 cells with
[35S]methionine during the last 30 min of treatment with
heat shock, TPA, or TPA and heat shock together. A 4-fold induction in
Hsp70 synthesis was detected in cells exposed to the combined treatment with TPA and heat shock for 1 h, as compared with the increase in
Hsp70 synthesis during a 1-h heat shock (Fig. 6,
A and B). The difference in the induction of
Hsp70 between TPA-treated heat-shocked cells and cells subjected to
heat shock decreased at later time points. In contrast to Hsp70, no
similar enhancement in Hsp90 synthesis was detected in cells exposed to
both TPA and heat shock (Fig. 6, A and B). TPA
treatment alone did not induce synthesis of Hsp70 or Hsp90.
To examine the effects of simultaneous treatment with TPA and heat
shock on the kinetics of Hsp70 accumulation, whole cell extracts
prepared from K562 cells treated for various time periods up to 6 h were analyzed by Western immunoblotting. As shown in Fig.
6C (top panel), simultaneous treatment with TPA
and heat shock resulted in markedly elevated levels of Hsp70 already
after 2-3 h of incubation. These levels were comparable with the
amount of Hsp70 detected in cells that were subjected to a 6-h heat
shock in the absence of TPA (Fig. 6C, top panel).
The kinetics of Hsp70 accumulation correlate closely with HSF1
dephosphorylation and attenuation of HSF1 DNA binding activity, as seen
in Figs. 1 and 2. Consistent with the results on Hsp90 synthesis, as
presented above, no difference in the kinetics of Hsp90 accumulation
was observed between TPA-treated heat-shocked cells and cells exposed to heat shock alone (Fig. 6C, middle panel).
Hsc70 (Fig. 6C, bottom panel) was used an an
internal control for equal loading because no significant induction in
Hsc70 accumulation occurs upon exposure to heat shock. Taken together,
our results on the synthesis and accumulation of Hsp70 and Hsp90
suggest that the expression of the corresponding genes is, at the
translational level, differentially regulated upon TPA treatment during
continuous heat shock.
The results from the present study show that a remarkable
enhancement of the heat shock response can be achieved by stimulating the TPA-responsive signaling pathways during continuous heat shock. The
observed amplification in heat shock gene expression is mainly a
consequence of effects on the transcriptional level, because the
induction of both DNA binding activity of HSF1 and transcription of the
HSF1-regulated target genes are accelerated and enhanced. Subsequently,
treatment with TPA during continuous heat shock leads to elevated
synthesis and accumulation of Hsp70. Furthermore, the deactivation or
attenuation of HSF1 DNA binding activity during continuous heat shock
is accelerated in the presence of TPA. Because this faster attenuation
is tightly correlated with the accelerated effects on transcription and
protein synthesis, it is likely that some of the downstream effects
turn off the initial response. The TPA-induced signal is therefore not
only sufficient to amplify the heat shock response but will also
accelerate both induction and suppression of the response.
TPA per se is not sufficient for induction of the heat shock
response. Hence, an initial triggering signal by heat shock is required
to obtain the TPA-induced amplification. Because considerable evidence
exists that phorbol esters function principally by persistent stimulation of PKC (for review see Ref. 55), the results from our study
imply that a PKC-regulated pathway appears to be operative in
regulation of the heat shock response. This is in agreement with an
earlier report showing that treatment with the PKC inhibitors H-7 and
calphostine C inhibits hsp70 mRNA induction in cells
exposed to heat shock (56). These observations are important in
revealing a new mechanism involved in regulation of the heat shock
response. It is also significant from the point of view that
PKC-mediated signaling pathways are involved in regulation of a
multitude of different cellular processes (for review see Ref. 57). The
TPA-induced effects on the heat shock response could be mediated
directly by PKC or by some of the downstream signaling cascades
activated by PKC. In concordance with previous studies (58), our data show that TPA causes a significant increase in AP-1 DNA binding activity, suggesting possible activation of a MAPK cascade (for review
see Ref. 26). The different MAPKs embrace a central position in the
interactive signaling pathways regulating transcription. For example,
extracellular signal regulated kinase 1/2, the stress-activated protein
kinase, also called Jun N-terminal kinase, and p38 kinase, all members
of the MAPK family, have been shown to participate in the regulation of
c-fos and c-jun transcription, although their activities are clearly initiated by different signals (for review see
Refs. 26 and 59). Considering the preceding documentation demonstrating
MAPKs as key elements in transcriptional regulation, it will be of
major interest to elucidate the role of MAPKs in the regulation of the
heat shock response.
Previous studies have indicated that the transcriptionally active HSF1
is hyperphosphorylated, as reflected by the appearance of slower
migrating forms of HSF1 on SDS-PAGE (7, 15). In this study, a close
correlation between the activation-specific, slower migrating form of
HSF1 and the DNA binding activity of HSF1 was observed, coupled with
the transcriptional induction of hsp70 and hsp90
genes. These data support the previously raised hypothesis that
phosphorylation may play an important role in the regulation of heat
shock gene expression (7, 15, 16, 17). The accelerated activation and
attenuation of the HSF1 DNA binding in TPA-treated heat-shocked cells
correlate well with HSF1 hyperphosphorylation and dephosphorylation.
Earlier studies have shown that the process of HSF1 activation
potentially involves multiple steps, including various
post-translational modifications of HSF1, such as trimerization,
nuclear localization, and hyperphosphorylation (for review see Ref. 1).
These steps can be uncoupled because certain stimuli, such as the
nonsteroidal anti-inflammatory compounds sodium salicylate and
indomethacin, activate the heat shock response only partially so that
HSF1 acquires DNA binding activity without induction of heat shock gene
expression (15, 20, 21). In contrast to the fully activated
heat-inducible form of HSF1, the drug-activated HSF1 does not undergo
hyperphosphorylation. Recently, two studies have indicated that HSF1 is
phosphorylated on serine residues upon heat shock, whereas the
stress-induced serine-directed phosphorylation cannot be induced by the
anti-inflammatory agents (15, 19). Furthermore, Cotto and co-workers
(15) have shown that acquisition of the trimeric DNA binding state of
HSF1 occurs independently of hyperphosphorylation and that HSF1
trimerization precedes the inducible phosphorylation of HSF1. The
inducible phosphorylation of HSF1 is likely not required for the
acquisition of DNA binding activity but may be required for the
transcriptional activation. Although evidence is accumulating in
support for the hypothesis of a multistep activation of HSF1, the exact
phosphorylation sites of the inactive and active forms of HSF1, as well
as the kinases and/or phosphatases regulating these sites remain to be identified.
Several reports have indicated that heat shock proteins themselves may
be involved in the activation and deactivation of HSF1 by an
autoregulatory loop (60-65). Hsp70 has been implicated in the negative
regulation of HSF1 in mammalian cells, and this autoregulatory mechanism has been proposed to affect the DNA binding and
oligomerization of HSF1. According to Mosser and co-workers (64),
constitutive overexpression of hsp70, as obtained by
transfection techniques, results in a reduction in the level of HSF1
activation following heat shock. In their study, the DNA binding
activity of HSF1 was regulated in a dose-dependent manner
so that the extent of inhibition was greater in cells expressing higher
levels of Hsp70. Furthermore, Kim and co-workers have shown that
overexpression of Hsp70 accelerates the recovery of heat-shocked
mammalian cells through its modulation of HSF1 (65). The autoregulatory
role of Hsp70 is further supported by the observations that Hsp70 binds
to the active form of HSF1 and that excess exogenous Hsp70 prevents the
activation of HSF1 in vitro (60, 61). However, the
autoregulatory model is contradicted by a finding that Hsp70 and Hsc70
associate to similar extents with both the latent non-DNA-binding form
and the active DNA-binding form of HSF1 and that the induction of HSF1
DNA binding is not affected by overexpression of Hsp70 (66). In our
study, extensive genetic manipulation to obtain overexpression of Hsp70
was avoided, and the results show that the gradual HSF1
dephosphorylation and attenuation of HSF1 DNA binding activity follow
the kinetics of elevated Hsp70 accumulation. Although we have not
determined whether the accumulated Hsp70 exists free or is bound to a
substrate, our results give further support to the hypothesis that
Hsp70 plays a role in the attenuation of HSF1 DNA binding activity. Moreover, the close correlation of increased Hsp70 synthesis with decreased phosphorylation of HSF1 suggests that Hsp70 may have a role
not only in the regulation of HSF1 DNA binding activity but also in the
regulation of HSF1 phosphorylation.
We demonstrate that the expression of two classic heat shock genes,
hsp70 and hsp90, is differentially regulated upon
simultaneous treatment with TPA and heat shock. Although
hsp70 is known to be the most highly stress-inducible heat
shock gene, our experiments show that the transcription of both
hsp70 and hsp90 genes is markedly stimulated in
response to heat stress and that this transcriptional activation is
further enhanced by the combined treatment of heat and TPA. In the case
of Hsp70, the transcriptional enhancement results in accelerated and
increased protein synthesis and accumulation, whereas no such additive
effects can be detected on Hsp90. The distinct effects of TPA and heat
shock on the expression of hsp70 and hsp90 genes
raise the question of whether Hsp90 has a role in the autoregulatory
model of Hsp70. Assuming that both hsp70 and
hsp90 are transcriptionally activated by a common factor, HSF1, as earlier suggested (51), it would be important to know whether
Hsp90, in analogy to Hsp70, can form a complex with the DNA-bound form
of HSF1 and thereby inhibit the DNA binding and transcriptional
activities of HSF1. According to Rabindran and co-workers (66), the
association of Hsp90 with HSF1 cannot be detected in a
co-immunoprecipitation assay. The distinct features of Hsp70 and Hsp90
interactions with HSF1 lead to another question, i.e. what
is the mechanism behind attenuation of the inducible hsp90
transcription during continuous exposure to heat stress? Although the
synthesis and accumulation of Hsp90 are not further enhanced by
combined treatment with TPA and heat shock as compared with heat shock
alone, the induction and attenuation of hsp90 transcription
are equally accelerated as in the case of hsp70, suggesting
that Hsp70 could also be involved in the regulation of other HSF1
target genes, in addition to hsp70.
The complex regulation of eukaryotic heat shock gene transcription is
reflected by the presence of binding sites for transcription factors
other than HSFs in the heat shock gene promoters. For example, the
hsp70 promoter contains several consensus elements such as
TATA, GC, CCAAT, AP-2, and activating transcription factor- or
AP-1-like elements (67, 68, 69). These elements are involved in basal
expression of the hsp70 gene and mediate the induction in
response to nonclassical stress stimuli such as serum (70) and
adenovirus E1a (69). In addition, the basal promoter elements have been
shown to be required for maximal stress-induced transcription of the
hsp70 gene (71). The involvement of basal transcription factor activities in the TPA-enhanced heat shock response cannot be
excluded, although TPA alone is not capable of inducing transcription of heat shock genes in K562 cells. The question of a synergism between
the basal transcription factors and HSF1 in TPA-treated heat-shocked
cells needs to be addressed in the further studies.
Although activation of PKC in the present study has been achieved by
artificial means, the results obtained are highly relevant in terms of
the wide spectrum of cellular processes regulated by PKC. PKC-mediated
signaling pathways are used by a number of different cell membrane
receptors that can trigger either cell growth or differentiation in
various cellular systems. It is plausible that some of these processes
could involve a modified stress response as a consequence of activated
PKC-responsive signaling pathways. It is well established that PKC is a
family of several different isoenzymes with specific regulatory
functions, and the specificity is likely to be achieved by the
differential activation of PKC isoenzymes. It remains to be determined
which PKC isoenzyme(s) is involved in the regulation of the heat shock
response.
We thank Rick Morimoto for valuable comments
on the initial results of this study and Eleanor Coffey, Michael
Courtney, Jari Heikkilä, and Ann-Sofi
Härmälä-Braskén for their critical comments on
the manuscript. We are also grateful to Iina Elo and Laura Seppä
for expert technical assistance.
Turku Centre for Biotechnology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
B, AP-1, and CREB (for
review see Refs. 24-26), no specific protein kinases or protein
phosphatases have yet been identified to be directly involved in the
regulation of HSF1 activation or in the induction of the heat shock
response. However, involvement of phosphorylation in the regulation of
heat shock gene expression is supported by several studies. Various
modifiers of kinase/phosphatase activities can modulate different
regulatory steps of the heat shock response (27-32. Heat stress
per se has also been reported to activate major signaling
processes, such as the mitogen-activated protein kinase (MAPK; Ref.
33), protein kinase C (PKC; Refs. 34 and 35), and Jun N-terminal kinase
(36). Furthermore, oxidative stress and heat shock have been reported
to induce expression of the CL100 gene encoding a
tyrosine/threonine-specific protein phosphatase (37, 38).
Cell Culture, TPA Treatment, Heat Shock Conditions, and
Preparation of Cell Extracts
-12-O-tetradecanoylphorbol 13-acetate (4
-TPA; LC
Laboratories), or heat shock. For heat shock treatment, plates were
sealed with Parafilm and immersed in a 42 °C water bath. Whole cell
extracts were prepared as described previously (39), and the protein concentration was measured by a protein assay (Bio-Rad).
-32P-labeled HSE
oligonucleotide corresponding to a sequence in the human
hsp70 promoter and analyzed on a native 4% PAGE gel as
described previously (39). For analysis of AP-1 DNA binding activity, a
double-stranded TPA-responsive element-containing oligonucleotide was
used (40). The synthetic oligonucleotides were 32P-labeled
with T4 polynucleotide kinase (Promega). Quantitative analyses of the
HSE protein and TPA-responsive element-protein complexes were performed
using a Fujix Bas 1000 PhosphorImager.
-32P]UTP (3000 Ci/mmol,
Amersham Corp.) as described previously (41). The radiolabeled mRNA
was hybridized with nitrocellulose-fixed plasmids for the following
genes: human hsp70 (pH2.3; Ref. 42), human
hsp90
(pUCHS801; Ref. 43), human hsc70
(pHA7.6; Ref. 44), human grp78 (pHG23.1.2; Ref. 44), human
hsp60 (SPD-920; StressGenes), human hsp27
(SPD-910; StressGenes), human
-actin (pHF
A-1; Ref. 45), rat GAPDH
(pGAPDH; Ref. 46), and Bluescript vector (Stratagene). The
hybridization and washing conditions were as described previously (10).
The intensities of the radioactive signals were quantified with a Fujix
Bas 1000 PhosphorImager.
TPA Treatment during Continuous Heat Shock Results in Accelerated
Acquisition and Attenuation of HSF1 DNA Binding Activity
Fig. 1.
Analysis of the HSF1 DNA binding activity by
gel mobility shift assay. K562 cells were exposed to heat shock
(HS, 42 °C), TPA treatment (TPA 100 nM), or combined treatment with TPA and heat shock
(TPA + HS) for the indicated time periods. A, 15 µg of whole cell extracts were analyzed by gel mobility shift assay using an HSF-specific 32P-labeled oligonucleotide
(HSE). HSF indicates the inducible HSF-HSE complex, and CHBA denotes the constitutive HSF DNA binding
activity reported previously (39). NS indicates nonspecific
DNA binding activity, free indicates unbound HSE
oligonucleotide, and C denotes extracts from untreated
cells. B, quantitation of the levels of HSF HSE binding
activity was performed using a Fujix Bas 1000 PhosphorImager. The
HSF-HSE values were normalized against the respective nonspecific
values, which were presumed to be unaffected by the various treatments.
The values for the fold induction of HSF levels are shown relative to
the control level, which was arbitrarily assigned a fold induction
value of 1.
[View Larger Version of this Image (43K GIF file)]
Fig. 2.
Examination of the phosphorylation states of
HSF1 by Western blot analysis. 10 µg of whole cell extracts from
untreated K562 cells (C), cells exposed to heat shock
(HS, 42 °C), TPA treatment (TPA, 100 nM), and combined treatment with TPA and heat shock (TPA + HS) for the indicated time periods were run on an 8%
SDS-PAGE. Antibodies against HSF1 and HSF2 were used for Western blot
analysis. The distinct phosphorylation states of HSF1 were detected as
slower migrating complexes in the gel (see text for further
details).
[View Larger Version of this Image (37K GIF file)]
-TPA Treatment during Continuous Heat Shock Does Not Affect the
DNA Binding Activity of HSF1
-TPA was used (50). Combined treatment with 100 nM of
4
-TPA and heat shock neither altered the heat-induced HSE binding
activity nor the heat-induced hyperphosphorylation of HSF1 (Fig.
3, A and B, respectively). Furthermore, 4
-TPA treatment by itself did not induce the putative intermediate phosphorylation state of HSF1.2
Fig. 3.
Examination of the effects of 4-TPA on the
HSF1 DNA binding activity and on the phosphorylation states of HSF1.
A, K562 cells were exposed to heat shock (HS,
42 °C), 4
-TPA treatment (4
-TPA, 100 nM), or combined treatment with 4
-TPA and heat shock (4
-TPA + HS) for the indicated time periods. Whole cell
extracts were subjected to gel mobility shift assay as described in the legend to Fig. 1. CHBA, constitutive HSF DNA binding
activity; free, unbound HSE oligonucleotide; C,
extracts from untreated cells. B, Western blot analysis with
antibodies against HSF1.
[View Larger Version of this Image (64K GIF file)]
Fig. 4.
Analysis of the transcription rate of
hsp70 and hsp90 genes by nuclear run-on assay.
A, Nuclei were isolated from K562 cells exposed to heat
shock (HS, 42 °C), TPA treatment (TPA, 100 nM), or combined treatment with TPA and heat shock
(TPA + HS) for the indicated time periods.
C1 and C2 denote
separately prepared samples from untreated cells. GAPDH and -actin
were used as examples of nonheat shock genes, and BS
indicates the Bluescript plasmid used as a vector control.
B, quantitation of the transcription rates of
hsp70 and hsp90 genes as analyzed using a Fujix
Bas 1000 PhosphorImager.
[View Larger Version of this Image (55K GIF file)]
-actin was transiently induced by TPA (Fig. 4) as
has been previously shown (52, 53). The lack of TPA-induced
-actin
gene transcription in TPA-treated heat-shocked cells is presumably due
to down-regulation by heat shock, as has been previously shown
(54).
Fig. 5.
Analysis of the steady-state level of
hsp70 and hsp90 mRNA by Northern blot
hybridization. A, total RNA was extracted from K562 cells
exposed to heat shock (HS, 42 °C), TPA treatment (TPA, 100 nM), or combined treatment with TPA
and heat shock (TPA + HS) for the indicated time periods.
The samples containing 10 µg of total RNA were run on 1% agarose
gel, transferred to a nylon membrane, and hybridized with
32P-labeled DNA probes for hsp70,
hsp90, and GAPDH. C indicates control sample from
untreated cells. Note that the left and right panels showing hsp90 mRNA are not equally exposed.
kb, kilobases. B, quantitative analysis of the
levels of hsp70 and hsp90 mRNA as measured
using a Fujix Bas 1000 PhosphorImager.
[View Larger Version of this Image (43K GIF file)]
Fig. 6.
Analysis of Hsp70 and Hsp90 synthesis and
accumulation kinetics by metabolic labeling and Western immunoblotting,
respectively. A, K562 cells exposed to heat shock
(HS, 42 °C), TPA treatment (TPA, 100 nM), or combined treatment with TPA and heat shock
(TPA + HS) for the indicated time periods were labeled with
50 µCi of [35S]methionine during the last 30 min of
each treatment. Whole cell extracts were run on an 8% SDS-PAGE
followed by fluorography. C indicates sample from untreated
cells, and the indicated molecular mass markers are in kilodaltons.
B, quantitative analysis of Hsp70 and Hsp90 synthesis in
heat-shocked and TPA-treated heat-shocked cells. Note the difference in
scales between Hsp70 and Hsp90. C, Western blot analysis
with antibodies against Hsp70, Hsp90, and Hsc70.
[View Larger Version of this Image (36K GIF file)]
*
This work was supported by funds from the Academy of Finland
(to J. E. E., S. L., and L. S.), the Emil Aaltonen Foundation (to S. L.), and the Sigrid Jusélius Foundation (to L. S.).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.
¶
Financed by the Graduate School of Biomedical Sciences at the
Turku University.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.
To whom correspondence should be addressed: Turku Centre for
Biotechnology, P.O. Box 123, FIN-20521 Turku, Finland. Tel.: 358-2-333-8028; Fax: 358-2-333-8000; E-mail:
lea.sistonen{at}btk.utu.fi.
1
The abbreviations used are: Hsp, heat shock
protein; HSF, heat shock transcription factor; HSE, heat shock element;
PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol
13-acetate; 4-TPA, 4
-12-O-tetradecanoylphorbol 13-acetate; PAGE, polyacrylamide gel electrophoresis; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated
protein kinase.
2
C. I. Holmberg, I. Elo, J. E. Eriksson, and L. Sistonen, unpublished observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.