(Received for publication, April 26, 1995; and in revised form, July 20, 1995)
From the
Sodium salicylate has the unusual property of partially
inducing the human heat shock response (Jurivich, D. A., Sistonen, L.,
Kroes, R., and Morimoto, R. I.(1992) Science 255,
1243-1245). Salicylate induces the DNA binding state of the human
heat shock transcription factor (HSF), but this is insufficient to
elevate heat shock gene expression. Because it is not known how HSF
enhances heat shock gene expression, further analysis of the
transcriptionally inert, salicylate-induced HSF was undertaken to
potentially identify components of the heat shock response that are
necessary for full transcriptional induction. Like thermal stress,
exposure of HeLa cells to salicylate led to the induction of HSF1 into
a DNA-bound state. Despite continued exposure of cells to salicylate,
HSF1DNA binding attenuated much more rapidly than a continuous
heat shock. Western blot analysis revealed that the salicylate-induced
form of HSF1 was not hyperphosphorylated like the heat-induced form.
Furthermore, supershifts of the HSF1 bound to an heat shock element
(HSE) oligonucleotide by monoclonal antibodies to phosphoamino acids
revealed that salicylate induced threonine phosphorylation of HSF1,
whereas heat led to a predominance of HSF1 serine phosphorylation.
These data suggest that salicylate-independent signals are necessary to
convert HSF1 into a transactivator of heat shock gene expression and
that brief acquisition of DNA binding by this factor is insufficient to
maximally enhance transcription.
Pharmacological agents can alter gene expression. This property
can be useful for understanding normal controls of transcription as
well as providing insight into how drugs exert their therapeutic effect
at the molecular level. Recently, anti-inflammatory drugs such as
sodium salicylate have been found to alter specific transcriptional
controls in human cells, as well as in plants and
bacteria(1, 2, 3, 4) . How
salicylate exerts its effect on the transcriptional machinery is not
fully understood. Some studies indicate that drug-induced perturbations
of DNA-binding proteins that act as enhancers or silencers of gene
expression may be one anti-inflammatory drug
target(1, 2) . Because the primary action of
salicylate and other non-steroidal anti-inflammatory compounds (NSAIDs) ()has been attributed to disruption of eicosanoic acid
metabolism(5) , these agents could modify gene expression
indirectly by modulating the levels of prostaglandins, leukotrienes,
and other associated lipids. Alternatively, salicylate and NSAIDs may
regulate gene expression by altering certain components of cellular
signal transduction such as G-regulatory proteins(6) . Finally,
as these drugs have the capacity to bind proteins, an additional
consideration is that DNA-binding proteins are directly affected by one
or more NSAIDs.
Compelling evidence for salicylate in regulating gene expression has been identified in plant cells responding to viral injury(7, 8, 9) . Induction of pathogen resistance genes is mediated by accumulation of salicylate both locally and systemically in disease-ridden plants. Whether salicylate directly induces transactivators of gene expression is not certain. A recent study has implicated salicylate's effect on gene expression through an indirect mechanism. By binding and inhibiting catalase, salicylate causes hydrogen peroxide to increase, which in turn induces expression of defense-related genes (10) . Salicylate has also been found to stimulate gene expression in bacteria associated with antibiotic resistance(4) .
In contrast to stimulating
transactivation of some genes, salicylate can also inhibit gene
expression. In plants, certain genes expressed during leaf wounding are
inhibited by salicylate(11) . -Interferon-induced HLA-DR
expression is altered by salicylate in human cells(12) . Both
these observations suggest that salicylate has the capacity to
selectively block gene expression in addition to activating molecular
switches for enhanced gene expression.
Given the relationship of
salicylate to induction of genes in response to cellular injury in
plants, recent investigations have been directed toward the possibility
that this agent mimics signals in human cells associated with
physiological stress. Thus far, salicylate has been found to affect two
stress-associated transcription factors, NFB and the heat shock
transcription factor, HSF(1, 2) . NF
B is induced
by inflammation, viral, and UV-induced
stress(13, 14) . Salicylate apparently blocks NF
B
by sustaining I
B levels and thus preventing translocation of the
transcription factor to the nucleus(2) . In contrast to its
effects on NF
B, salicylate was found to induce HSF into a DNA
binding state without enhancement of heat shock gene
expression(1) . The partial activation of the heat shock
response by salicylate suggests that signals in addition to HSF
multimerization are necessary to fully transactivate heat shock gene
expression. This fact is underscored by the ability of heat shock to
convert the salicylate-induced HSF into a transcriptionally competent
enhancer of heat shock gene expression.
Several possibilities may
account for salicylate's inability to fully induce the heat shock
response. Because there are several known members of the heat shock
factor gene family, it is possible that salicylate does not activate
the heat-inducible factor, HSF1. Alternatively, salicylate-induced HSF1
may not be post-translationally modified in the same manner as the
heat-inducible factor. Furthermore, salicylate may induce the DNA
binding domain of HSF, but it may not affect the transactivation
domain. In examining these possibilites, we found both salicylate and
heat induced HSF1 and not HSF2
DNA binding. Despite
induction of the same transcription factor as heat, the
salicylate-induced form of HSF1 exhibited different properties. The
duration of binding activity in vivo is different and the
salicylate-induced form of HSF1 does not appear to undergo extensive
phosphorylation as does the heat-inducible form. Phosphothreonine
modification of HSF1 by salicylate is noted, whereas heat shock
primarily causes phosphoserine modification of HSF1. Furthermore, the
salicylate-induced HSF1 appears to be extensively associated with heat
shock protein 70 (hsp70), a factor that may explain the incomplete
activation of the human heat shock response by this anti-inflammatory
compound. These data suggest that both the duration of HSF1
DNA
binding and additional modifications of HSF1 are linked to enhancement
of heat shock gene expression.
Column chromatography was performed on
whole cell extracts (1 10
cells) after dialysis
against salt-free, whole cell extract buffer. DEAE-Sepharose columns (1
ml) were loaded with dialyzed cell extracts from HeLa S3 cells treated
for 30 min at 37 °C with 20 mM salicylate or at 42 °C
in the absence of salicylate. The columns were washed with 2 column
volumes of elution buffer (10 mM Tris, 1 mM EDTA) and
then developed with a NaCl gradient (0-500 mM).
Fractions (0.5 ml) were collected, and 10 µl from each aliquot were
used for EMSA. Fractions containing the multimerized HSF1 were
identified by binding to the radiolabeled HSE oligonucleotide.
HSF
DNA binding activity in each fraction was evaluated by
scanning densitometry and expressed numerically as a percentage of
HSF1
DNA binding contained in 10 µg of protein from whole cell
extracts of heat-shocked cells.
Immunoprecipitation of P-labeled HSF1 from HeLa S3 cells was performed after 1
10
cells were exposed to 750 µCi of inorganic,
radiolabeled phosphorus in phosphate-free Dulbecco's modified
Eagle's medium for 6 h. Cells were washed three times in
PBS- before treatment with 20 mM salicylate or a 42
°C heat shock for 30 min. After treatment, cells were washed once
in PBS- and the pellet was solubilized in 100 µl of boiling 1
SDS solubilization buffer (10% glycerol, 2% SDS, 5%
2-mercaptoethanol, 50 mM Tris, pH 6.8). Extracts were
sonicated for 10 s and diluted 1:10 with NTE buffer (10 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA). After a 30-min
incubation of the lysate with 50 µl of a 50% solution of
Sepharose-protein A in NTE buffer, the clarified extract was incubated
2 h with 2 µl of polyclonal rabbit serum specific for HSF1. A total
of 2 µl of goat anti-rabbit IgG (1 mg/ml, Southern Biotechnology,
4010-01) was added to each extract for 30 min, followed by 70 µl of
Sepharose-protein A solution for 60 min. The Sepharose-protein A was
pelleted by centrifugation and washed six times with NTE buffer. The
immunoglobulin-HSF1 complexes were solubilized in boiling 2
SDS
buffer (35 µl) and then boiled in distilled H
O (35
µl) and the fractions combined for a total volume of 70 µL.
HeLa S3 cells were exposed to different concentrations of
salicylate, and whole cell extracts were made to analyze HSFDNA
binding activity by EMSA. In Fig. 1A, HSF
DNA
binding activity in the salicylate-treated cells was measured by
scanning densitometry and expressed as a percentage of HSF
DNA
binding induced by a 42 °C heat shock. 20 mM salicylate
induced HSF
DNA binding comparable to a 42 °C heat shock,
while lower concentrations produced less HSF
DNA binding in a
dose-dependent fashion.
Figure 1:
A, graphic display of HSFDNA
binding in HeLa S3 cells induced by various concentrations of sodium
salicylate at 37 °C. HSF
DNA binding was determined by
scanning densitometry of EMSA, and arbitrary units of measurement were
converted into a percentage of HSF
DNA binding measured from 42
°C heat-shocked cells (40 min). B, nuclear run-on analysis
of hsp70 gene expression in HeLa S3 cells. Scanning densitometry of
radiolabeled transcripts hybridized to plasmid pH2.3, which contains
the human heat shock protein 70 gene was done on samples taken at
various time intervals over a 1-h period. Maximal heat shock protein 70
gene expression occurred in this experiment between 20 and 40 min. None
of the salicylate-treated cells revealed increased hsp70 gene
expression as measured by this assay.
To test whether any HSFDNA binding
activity induced by salicylate led to increased heat shock gene
expression, nuclei were harvested at numerous time points for run-on
analysis. Fig. 1B shows that a 42 °C heat shock
induces hsp70 gene expression within 15-30 min, whereas none of
the salicylate treated cells exhibited increased transcription of this
gene. Because salicylate-induced HSF
DNA binding did not enhance
RNA polymerase II activity for heat shock genes, additional studies
were undertaken to identify how heat-induced HSF
DNA binding was
different from that in salicylate-treated cells.
To examine whether
salicylate was inducing the heat-regulated isoform of HSF, antibodies
specific for HSF1 and HSF2 were preincubated with whole cell extracts
prior to gel shift binding assay. Fig. 2A shows that
HSF1 antibodies supershifted both the heat- and salicylate-induced
HSFHSE binding, whereas HSF2 antibodies had no effect. Thus,
salicylate induces the same isoform of HSF as heat shock.
Figure 2:
A, electromobility supershift assay of
HSFHSE complexes from HeLa S3 cells treated for 30 min with
either a 42 °C heat shock or 20 mM salicylate at 37
°C. Preincubation of whole cell extracts with rabbit polyclonal
antibody to either HSF1 or HSF2 causes a supershift of the HSF
DNA
complex when HSF1 antibodies are used but not HSF2 antibodies. B, EMSA of HSF
DNA binding in control, heat-shocked (42
°C), and salicylate-treated cells (20 mM) in the presence
or absence of cycloheximide (10 µg/ml).
Because
previous studies showed cycloheximide-dependent and -independent
pathways of HSF activation, HeLa S3 cells were incubated with
cycloheximide (10 µg/ml) prior to stimulation with salicylate. Fig. 2B shows that cycloheximide inhibits HSFDNA
binding activity when cells are exposed to 20 mM salicylate.
Previous studies demonstrated the initial induction of HSF
DNA
binding by heat was not affected by cycloheximide(17) , thus
indicating that the salicylate-inducible form of HSF1 responds
differently than the heat-inducible form when protein synthesis is
inhibited.
Additional differences were noted in the salicylate- and
heat-inducible forms of HSF1 when HSFDNA binding activity was
examined over time. As can be seen in Fig. 3, both temporal
activation and maintenance of HSF by salicylate at 37 °C differed
from a 42 °C response. HSF
DNA binding activity reached a
maximum within 30-40 min during heat shock as compared with
20-30 min when cells were treated with salicylate. Despite
continuous exposure to heat, HSF
DNA binding activity attenuated
over a 3-4-h period, as has been previously
reported(19) . In contrast to the heat shock response and
despite the continued presence of 20 mM salicylate,
HSF
DNA binding activity attenuated rapidly. Within 90 min there
was little to no HSF
DNA binding activity in the
salicylate-treated cells. Even though salicylate-induced HSF
DNA
binding activity attenuated rapidly, the in vitro dissociation
of HSF
DNA binding of salicylate-treated cells was not different
from that in heat-shocked cells (data not shown).
Figure 3:
Graphic representation of HSF1DNA
binding activity induced by salicylate and heat shock over a 4-h
period. EMSA of HSF1 was conducted on whole cell extracts made of cells
during consecutive time points, and HSF1
DNA binding levels were
determined by scanning densitometry and expressed as a percentage of
maximal HSF
DNA binding activity at 42 °C for 40
min.
Additional in vitro analysis of salicylate-induced HSF1 was undertaken to identify potential differences relative to the heat-inducible form. When analyzed by ion-exchange chromatography, salicylate-induced HSF multimeric complexes or HSF-protein complexes elute differently than the heat-induced factor. Fig. 4A shows that salicylate-induced HSF begins to elute from a DEAE column at a lower salt concentration than the heat-induced form. Salicylate also appears to cause HSF to elute over a wider range of salt concentrations than the heat-induced form.
Figure 4:
A, DEAE
column chromatography of HSF1 in 42 °C heat-shocked cells compared
to 20 mM salicylate-treated cells at 37 °C. HSFDNA
binding was expressed as a percent of maximal HSF
DNA binding in
the EMSA. 10 µl of each column fraction was used for EMSA. B, Western blot analysis of HSF1 in control,
salicylate-treated, and heat-shocked HeLa S3 cells. Both the 42 °C
heat shock and the 20 mM salicylate treatments were 30 min. 20
µg of protein from whole cell extracts was subjected to SDS-PAGE,
and the separated proteins were transfered to nitrocellulose for
Western blotting with rabbit polyclonal antibody to HSF1. C,
semi-quantitative Western blot analysis of HSF1 from control and 20
mM salicylate-treated HeLa S3 cells (30 min). Samples from
salicylate-treated cells exhibit a slightly higher chemiluminescence
signal for HSF1 relative to samples from control
cells.
To determine if differences noted on DEAE chromatrography were due to post-translational changes of HSF1, Western blot analysis was performed. Previous studies revealed that the hyperphosphorylated form of HSF1 migrates more slowly in a 5% SDS-polyacrylamide gel than the less phosphorylated control form(16) . When this assay was performed on extracts from control, heat-shocked, and salicylate-treated cells, the Western blot revealed migration of HSF1 to be retarded as expected for heat-shocked samples, whereas control and salicylate forms migrated in the gel to a similar degree. However, when a 4% SDS-PAGE was employed, the Western blot demonstrated a slightly slower migrating form of the salicylate-induced HSF1 than the control form (Fig. 4B). Because the salicylate-induced form of HSF1 appeared to have a slightly broader band on Western blot analysis than the heat-induced form, semi-quantitative Western blot analysis was performed on whole cell extracts to determine whether salicylate stimulated the accumulation of HSF1. Fig. 4C represents a Western blot of HSF1 in control and salicylate-treated cells at 10, 20, and 30 µg of protein equivalents from whole cell extracts. Neither this or other blots revealed marked increases in HSF1 levels from salicylate-treated cells relative to extracts from control cells.
The difference in migration between control, salicylate, and
heat-shocked forms of HSF1 suggested that post-translational
modification of HSF1 through phosphorylation was possibily less robust
in salicylate- than in heat-treated cells. To examine this possibility,
immunoprecipition of HSF1 was undertaken after cells were metabolically
labeled with radioactive phosphorus. Table 1shows that after 6 h
of exposure to PO
, the control form of HSF1
had low levels of radiolabel incorporation. A 42 °C heat shock for
30 min increased
PO
incorporation by nearly
20-fold, whereas salicylate treatment increased radiolabel
incorporation 2.6-fold.
To further examine possible differences
between heat-shocked and salicylate-treated cells, supershifted
HSF1DNA complexes were sought in whole cell extracts treated with
monoclonal antibodies to phosphoamino acids serine, threonine, and
tyrosine. As seen in Fig. 5, antibodies to phosphoserine
interfere with heat-inducible HSF1
DNA binding and cause a diffuse
supershift of the HSF
HSE oligonucleotide complex within a 10-min
heat shock at 42 °C. In extracts from salicylate-treated cells the
phosphoserine antibodies had no effect on HSF1
DNA binding
activity or mobility. However, phosphothreonine antibodies caused a
marked supershift of the salicylate-induced HSF1. These antibodies
appeared to slightly diffuse the otherwise distinct HSF
HSE
banding observed in extracts from heat-shocked cells, but there was no
frank retardation of the HSF
HSE complex in the gel as was
observed with extracts from salicylate-treated cells. Phosphotyrosine
antibodies had little to no effect on migration of either the
salicylate- or heat-induced HSF1.
Figure 5:
Electromobility supershift assay of
HSFHSE binding activity in the presence of monoclonal antibodies
to phosphoserine, phosphothreonine, and phosphotyrosine. Whole cell
extracts were made from HeLa S3 cells that were heat-shocked (HS) 10 min or salicylate-treated (SA) 30 min, and
the extracts were incubated with the monoclonal antibodies prior to
initiation of HSF1 binding with a radiolabeled HSE oligonucleotide. HSF represents the HSF
HSE complex, and NS is
the nonspecific HSE-protein band. Supershifted HSF
HSE complexes
are noted closer to the top of the autoradiograph than HSF
HSE
complexes formed in the absence of antibodies. Incubation of the heat
shock whole cell extract with anti-phosphoserine antibody led to both a
``knock-out'' or loss of HSF
HSE binding and a
supershifted complex.
In addition to altered patterns of
post-translational modifications of HSF1, some observable differences
between the heat- and salicylate-inducible forms of HSF1 might be
attributable to protein interactions with HSF. Indirect assessment of
this possibility was approached by subjecting HSFHSE
oligonucleotide complexes to electrophoresis in lower percentage
polyacrylamide gels for longer periods of time than usually employed
for EMSA. Utilizing this approach, salicylate-induced HSF
HSE
complexes migrated more slowly than heat-induced complexes (Fig. 6), suggesting that despite acquisition of DNA binding,
the salicylate-induced HSF was associated with another protein that
impeded its migration in the gel. One known candidate interacting with
HSF1 is hsp70(20) . To examine the relationship of hsp70
binding to HSF1, an antibody specific for the inducible form of hsp70
was incubated in whole cell extracts prior to EMSA. Fig. 6shows
that antibody to hsp70 ``supershifts'' nearly all of the
salicylate-induced HSF1
DNA binding complexes, whereas it only
partially supershifts some of the heat shock-induced complexes.
Antibodies to hsp90 did not appear to alter the HSF
HSE migration
when mixed with either the salicylate- or heat shock-induced HSF.
Antibody to
-immunoglobulin, which was used as a control for
nonspecific antibody-HSF interaction, had no effect in supershifting
the HSF1
DNA complex either.
Figure 6:
EMSSA of HSFHSE binding activity
employing monoclonal antibodies to hsp70 and hsp90. Whole cell extracts
of HeLa S3 cells were obtained after a 120 min heat shock at 42 °C
or a 40 min treatment at 37 °C with 20 mM sodium
salicylate. Extracts from these cells were preincubated with antibody
to hsp70 (C92, StressGen) and hsp90 (16F1, StressGen). These extracts
were compared to reaction mixtures that either had no antibody, but
were diluted by an equivalent volume with a 10 mM, pH 7 Tris
buffer or were exposed to an antibody with no known reactivity to heat
shock proteins or HSF1. In the latter case, either serum or polyclonal
antibodies to
-immunoglobulin (IgG) were tested and none
of these affected the HSF
HSE complex. The lack of HSF
HSE
reaction with antibody to immunoglobulin G (Southern Biotechnology) is
shown in this representative experiment. The HSF
HSE complex is
designated by HSF, the nonspecific HSE-protein complex is
indicated by NS, and HSF
HSE complexes supershifted by
hsp70 antibodies are noted by an arrow.
Salicylate is one of the few known inducers of HSFDNA
binding activity that does not enhance transcription of heat shock
genes at 37 °C. None of the salicylate concentrations that
increased HSF
DNA binding activity induced hsp70 gene expression.
This dissociation of HSF
DNA binding from gene expression is quite
different from heat shock, where hsp 70 gene expression can be induced
to various levels depending upon the degree and duration of thermal
stress(19) . In HeLa S3 cells, a 42 °C heat shock leads to
maximal heat shock gene expression by 30-40 min. HSF
DNA
binding activity reaches an apex at this time also. As HSF
DNA
binding attenuates, the rate of heat shock gene expression also
declines in a proportionate fashion. By comparison, salicylate can
induce HSF
DNA binding up to 100% of that induced by a 42 °C
heat shock and yet not result in the enhanced expression of heat shock
genes. This observation suggests that salicylate can trigger
HSF
DNA binding without inducing some other event that renders HSF
transcriptionally competent.
Perhaps a simple explanation of salicylate's inability to induce a transcriptionally competent factor is that it does not activate the same factor as heat shock. Several isoforms of heat shock factor exist (21, 22) . Heat shock induces HSF1, whereas hemin induces HSF2(23) . Furthermore, transcription rates of the heat shock genes are substantially less when HSF2 is compared to the heat-inducible isoform, HSF1. Because salicylate-induced HSF reacts with antibodies directed against HSF1 and not HSF2, this observation indicates that salicylate affects the same transcription factor as heat. Salicylate either partially induces HSF1 modifications necessary for transactivation of the heat shock genes or it modifies HSF1 differently than heat shock.
Even though salicylate and heat induce the DNA binding state of
HSF1, it is not clear that these stimuli share the same pathway of
induction. Previous work has suggested that all inducers of heat shock
genes share a common triggering mechanism, namely abnormal
proteins(24) . More recent work has indicated that HSF1 can
assume a DNA-bound state simply in response to elevated
temperatures(25) , thus suggesting that temperature itself can
regulate HSF1DNA binding in addition to unspecified signals
generated by abnormal proteins. Unlike heat, salicylate does not
directly induce HSF1
DNA binding activity when examined by in
vitro assays, thus implying an indirect action. Recently,
salicylate has been found to inhibit catalase activity in plants, and
it is thought that increased H
O
levels
stimulate trans-acting factors that enhance expression of the so-called
defense related genes(10) . A similar mechanism might be evoked
in HeLa cells exposed to salicylate; however, we were unable to
prevent salicylateinduced HSF1
DNA binding by preincubating cells
with various antioxidants. (
)
Another difference between
heat and salicylate induction of HSF1 is revealed by the pretreatment
of cells with cycloheximide. Blockage of protein synthesis prevents
HSF1DNA binding induced by 20 mM salicylate, whereas
cycloheximide delays the heat induction of HSF1, but not overall
binding levels(17) . If one were to evoke the protein-damage
model for triggering HSF1, then salicylate would trigger HSF1 through
production of abnormal, newly synthesized polypeptides whereas heat
would work through accumulation of damaged, preexisting proteins. The
paradox here is that if the model of protein damage as a common
mechanism of induction for heat shock gene expression is correct, then
salicylate ought to fully induce the heat shock response. One
interpretation is that damaged proteins can contribute toward the
acquistion of HSF1
DNA binding, but additional signals are needed
to trigger gene expression.
Additional signal(s) necessary for HSF1 to fully enhance heat shock gene expression are not fully understood. Even though HSF1 can bind to DNA, it may have to undergo additional modifications in order to be a transactivator of heat shock gene expression. These modifications may include i) release of a regulatory protein, ii) interaction with another DNA-binding protein, and iii) post-translational modifications.
In considering the role of a
negative regulator of HSF1 function, some investigators have suggested
hsp70 as a modulator of HSF1 function(26, 27) . Hsp70
does not prevent HSF1 from binding DNA(28) , but it could
possibly block the transactivation domain of HSF1. Our observations
with salicylate are consistent with the hypothesis that hsp70 bound to
HSF1 suppresses transactivation of the heat shock genes. If one
examines HSF1HSE complexes on low percentage polyacrylamide gel
shift assays, the salicylate-induced HSF1 migrates differently than the
heat-induced form and the former is almost completely supershifted by
antibodies to hsp70. The ability of hsp70 antibodies to supershift most
of the salicylate-induced HSF1 complexes (
80% by scanning
densitometry) suggests that salicylate is incapable of inducing a pool
of HSF1 free of hsp70 such as seen during heat shock. An inference can
be made from this finding that maximal expression of heat shock genes
requires a nominal pool of HSF1, which is not associated with hsp70.
According to this hypothesis, salicylate would have the capacity to
trigger HSF1
DNA binding activity but would be unable to cause
dissociation of hsp70 from the multimerized transcription factor.
Unresolved is whether salicylate somehow increases the association of
hsp70 with HSF1 or whether it does not promote the dissociation of
preexisting hsp70 and HSF1 complexes. Thus far, we have not detected
hsp70 in immunoblots of HSF1 from non-stimulated cells, but this
observation does not preclude the possibility of transient interactions
of hsp70 and HSF1 whereby salicylate shifts the equilibrium toward more
stable interactions.
On the other hand, transcriptional incompetency
of salicylate-induced HSF1 may be due to the lack of post-translational
modifications or posssibly inappropriate modifications of HSF1. Heat
shock induces HSF1 phosphorylation(16, 29) . This
modification results in the altered mobility of HSF1 on SDS-PAGE
putatively by phosphorylated residues displacing SDS binding which
retard the mobility of this protein. When compared to the heat shock
form of HSF1, the salicylate-induced form shows little to no
retardation on SDS-PAGE. HSF1 incorporation of P
subsequent to salicylate treatment is less than heat shock treatment.
These data suggest that salicylate does not induce maximal HSF1
phosphorylation usually observed in heat shock samples. Whether
increased phosphorylation of HSF1 is associated with maximal expression
of heat shock genes is not certain. In yeast, a constitutively bound
HSF becomes phosphorylated as heat shock gene expression increases.
However, HSF phosphorylation has recently been associated with
attenuation of HSF
DNA binding activity(30) . Given that
as little as one phosphorylated amino acid can confer transactivation
of some transcription factors(31) , it is possible that certain
phosphorylated residues on HSF1 are associated with transactivation
whereas others are linked to attenuation.
Interestingly, salicylate
may actually induce an unique form of HSF1 phosphorylation as suggested
by supershifted complexes of HSF1HSE oligonucleotides in the
presence of monoclonal antibodies to phosphoamino acids. HSF1 induced
by heat appears to interact primarily with phosphoserine and, to a
lesser degree, phosphotyrosine antibodies. In contrast,
salicylate-induced HSF1 leads to an HSF1
HSE complex that
supershifts primarily with phosphothreonine and to a lesser degree with
phosphotyrosine. The supershifting of the HSF
HSE complexes by
monoclonal antibodies to phosphoamino acids may be the result of HSF1
phosphorylation or some other phosphoprotein complexed with HSF1. In
either case, it is curious that salicylate causes a dramatically
different pattern of phosphorylation than heat shock. Our data suggest
that salicylate may block a threonine-specific phosphatase, activate a
threonine protein kinase, or a combination of both. On the other hand,
salicylate may activate a serine/threonine kinase and the conformation
of HSF1 may be such that only threonine residues are affected. Because
salicylate-induced NSF1
DNA binding is so brief relative to heat
shock, it is possible that threonine phosphorylation of HSF1 may
regulate the duration of DNA binding activity.
One final consideration about the inability of salicylate to induce heat shock gene expression entails conformational changes in HSF1. As some investigators have reported HSF1 unfolding as a step toward multimerization(32) , it is possible that further conformational changes in HSF1 are necessary to transactivate the heat shock genes. For example, the leucine zipper repeats located in the carboxyl terminus of HSF1 have been cited as a domain of HSF1 possibly contributing to transactivation of heat shock genes(32) . A sequential unfolding of different domains of HSF1 would first allow multimerization and then transactivation. The ``hinge model'' of HSF1 whereby the carboxyl terminus swings outward and unmasks the transactivation domain has been proposed in yeast where HSF1 is constitutively bound(30) . In human cells, the unmasking of this regulatory domain could be insensitive to the action of salicylate. In this model, salicylate would induce HSF1 conformational changes but would be unable to create the proper signals for unmasking the transactivation domain. The inability of salicylate to induce this final step could account for a transcriptionally inert HSF1 that binds to the heat shock element.
While salicylate is a useful tool in
dissecting the properties of HSF1 that are associated with maximal
expression of the heat shock genes, we have also been interested in the
effect of salicylate on the heat-inducible expression of the heat shock
genes. We have found that concentrations of salicylate that by
themselves do not induce HSF1DNA binding activity will act
synergistically with heat in regulating heat shock gene expression. (
)These synergistic actions include enhancement of
HSF1
DNA binding levels at physiologically relevant temperatures
and concomitant increases in the rate of heat shock gene expression
relative to thermal induction alone. These observations suggest that
the action of salicylate is different under heat stress than at 37
°C, and this may have important implications for the efficacy of
salicylate and perhaps other NSAIDs in modulating inflammation and
possibly increasing cellular protection through the heat shock
response.