Báo cáo khoa học: Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells pptx

Identification of sodium salicylate as an
hsp
inducer using a simple
screening system for stress response modulators in mammalian cells
Keiichi Ishihara, Kenji Horiguchi, Nobuyuki Yamagishi and Takumi Hatayama
Department of Biochemistry, Kyoto Pharmaceutical University, Japan
As heat shock proteins (Hsps) are involved in protecting cells
and also in the pathophysiology of diseases such as inflam-
mation, cancer and neurodegenerative disorders, modula-
tors of Hsp expression in mammalian cells would seem to be
useful for the treatment of various diseases. In this study, we
isolated mammalian cell lines for screening of Hsp modu-
lators; mouse C3H10T1/2 cells stably transfected with a
plasmid containing the mouse Hsp105 or human Hsp70B
promoter upstream of a luciferase or b-galactosidase
reporter gene, respectively. Using these cells, we examined
the effect of sodium salicylate (SA), which may induce the
transcription of hsp genes, on stress response in mammalian
cells. When these cells were treated with SA for 1 h at 37 °C,
both promoter activities were up-regulated by SA at con-
centrations of more than 45 m
M
. The activation of heat
shock factor and the subsequent accumulation of Hsp105a
and Hsp70 were detected in cells treated with SA at con-
centrations of more than 20 and 45 m
M
, respectively. Fur-
thermore, SA induced resistance against a subsequent lethal
stress. These findings suggested that SA is a potent hsp
inducer, and may be used to protect cells against deleterious

stressors.
Keywords: cytoprotection; heat shock factor; heat shock
promoter; heat shock proteins; sodium salicylate.
Heat shock proteins (Hsp) are a set of highly conserved
proteins that are produced in response to physiological and
environmental stress [1]. Hsps are also expressed under
physiological conditions and play important roles in normal
cellular events such as the synthesis, translocation and
degradation of proteins [2]. Hsps protect cells from the
cytotoxic effects of aggregated proteins produced by various
types of stress, and play a vital role in cell survival under
both physiological and stressed conditions. Because cellular
resistance against stress appears to be regulated by the
expression levels of Hsps, selective modulators of Hsp
expression could have medicinal applications. For instance,
the induction of Hsps seems to improve the prognosis of
patients after a massive operation. Geranylgeranylacetone,
a nontoxic Hsp70 inducer, is suggested to prevent acute liver
failure after massive hepatectomy, at least in part, by
enhancing cellular levels of Hsp70 [3]. Moreover, as Hsp70
and Hsp40 protect against the aggregation of mutated
proteins and cell death in neurodegenerative disorders such
as Parkinson’s and Huntington’s disease [4,5], Hsp inducers
are expected to be useful for the treatment of these diseases.
On the other hand, a major problem with hyperthermia,
which is one of the therapies applied for advanced cancers,
is the development of a transient thermoresistance in cancer
cells with recurrent heat treatments [6,7]. The acquisition of
thermotolerance is expected to be suppressed by reducing
the expression levels of Hsps in the cells.

In mammalian cells, the transcription of hsp genes is
mediated by the conversion of a pre-existing heat shock
factor (HSF) from an inactive to an active form [8]. HSF
presents as an inactive monomeric form in the cytoplasm
under physiological conditions, and is converted to an
active trimeric form that has sequence-specific DNA-
binding activity under stressed conditions. Activated HSF
relocalizes to the nucleus and binds to heat shock element
(HSE) in the 5¢-flanking region of hsp genes, resulting in the
trans-activation of hsp genes [8]. In the present study, we
isolated mouse C3H10T1/2 cells stably transfected with a
plasmid containing the mouse Hsp105 or human Hsp70B
promoter upstream of a luciferase or b-galactosidase repor-
ter gene, respectively, as a simple system for screening Hsp
modulators.
Furthermore, although sodium salicylate (SA) is widely
used as a nonsteroidal anti-inflammatory drug, the mech-
anism of action of SA is still a subject of debate. Several
suggestions such as inhibition of cyclooxygenase, which is
the rate-limiting enzyme in the conversion of arachidonic
acid to prostaglandins [9] and inhibition of the activation of
transcription factor nuclear factor-kappa B [10], have been
made to describe how SA exerts its anti-inflammatory effects
and also its side effects. In addition, SA has been found to
activate HSF in mammalian cells, although the induction of
transcription of hsp genes may not be induced by SA [11].
Correspondence to T. Hatayama, Department of Biochemistry,
Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi,
Yamashina-ku, Kyoto 607-8414, Japan.
Fax: + 81 75 595 4758, Tel.: + 81 75 595 4653,

E-mail:
Abbreviations: SA, sodium salicylate; hsp(s), heat shock protein(s);
Hsp70, 70 kDa heat shock protein; Hsp40, 40 kDa heat shock pro-
tein; Hsp105, 105 kDa heat shock protein; Hsp105a, a isoform
of Hsp105; HSF, heat shock factor; HSE, heat shock element;
Luc, luciferase; b-gal, b-galactosidase; DMEM, Dulbecco’s
modified Eagle’s medium.
(Received 14 April 2003, revised 30 June 2003,
accepted3July2003)
Eur. J. Biochem. 270, 3461–3468 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03740.x
Here we examined the effects of SA on stress response
in mammalian cells using a simple screening system, and
revealed that SA is a potent Hsp inducer in mammalian
cells, thereby protecting cells against deleterious stress.
Experimental procedures
Cell culture
Mouse fibroblast C3H10T1/2 and mouse embryonic F9 cell
lines were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supple-
mented with 10% foetal bovine serum in a humidified
atmosphere of 5% (v/v) CO
2
in air at 37 °C. Human HeLa
cells were grown in Eagle’s Minimum Essential medium
(Nissui Pharmaceutical) containing 10% bovine serum in a
CO
2
incubator at 37 °C.
Screening for Hsp modulators
A reporter plasmid containing the Hsp105 promoter

upstream of a luciferase (luc) reporter gene was constructed
by subcloning a 1.2-kb fragment of the 5¢-flanking region of
the hsp105 gene [12] to pGL2-basic vector (Promega). The
p173OR plasmid, which contains the hsp70B promoter
upstream of the b-galactosidase (b-gal) reporter gene, was
obtained from StressGen Biotechnologies (San Diego, CA,
USA). pGL105 or p173OR plasmid (7 lg each) and pBK/
neo plasmid (Stratagene) containing a geneticin resistant
gene (3 lg) were cotransfected into C3H10T1/2 cells
(1 · 10
7
cells per 100 mm dish) with 30 lL lipofectAMINE
reagent (Invitrogen) according to the manufacturer’s
instructions, and incubated for 48 h. Cells were then
maintained in DMEM containing 0.4 mgÆmL
)1
G418
antibiotic reagent (Wako Pure Chemical, Osaka, Japan)
for 3 weeks, and C3H10T1/2 cell lines stably transfected
with pGL105 or p173OR plasmid, designated as pGL105/
C3H and p173OR/C3H, respectively, were obtained and
maintained in DMEM containing 0.2 mgÆmL
)1
G418.
Measurement of Luc activity
pGL105C3H cells (2 · 10
5
cells per 35 mm dish) were
washed with NaCl/P
i

three times, lysed in 50 lLCellLysis
Regent (Promega), and centrifuged at 20 000 g for 10 min.
Aliquots (5 lL) of cell extracts were added to 50 lL
Luciferase Assay Reagent (Promega), and the Luc activity
was measured using a Turner Designs model TD-20/20
Luminometer.
Measurement of b-gal activity
p173OR/C3H cells (2 · 10
5
cells per 35 mm dish) washed
with NaCl/P
i
were suspended in 50 lL0.25
M
Tris/HCl
pH 8.0, and lysed by freeze-thawing (frozen at )80 °Cfor
30 min and thawed at 37 °Cfor3min,fivetimes).After
centrifugation at 20 000 g for 10 min, aliquots of cell
extracts (5 lg protein) were added to a final volume of
125 lL Z buffer (0.2
M
sodium phosphate buffer pH 7.5,
10 m
M
KCl, 1 m
M
magnesium sulfate, 0.05 m
M
2-merca-
ptoethanol). Then, 25 lL15m

M
chlorophenolred-b-
D
-galactosidase were added, and the mixture was incubated
at 37 °C for 30 min. The reaction was stopped by adding
60 lL1
M
Na
2
CO
3
, and absorbance at 574 nm was
measured.
Gel mobility shift assay
C3H10T1/2 cells (5 · 10
5
cells per 60 mm dish) were
washed with NaCl/P
i
, and quickly frozen at )80 °C. Frozen
cells were suspended in 100 lL extraction buffer (20 m
M
Hepes/KOH pH 7.9, 1.5 m
M
MgCl
2
,0.2m
M
EDTA,
0.5 m

M
phenylmethanesulfonyl fluoride, 0.5 m
M
dithiothre-
itol, 0.42
M
NaCl and 25% glycerol, v/v), kept at 4 °Cfor
15 min, and vortexed for 15 min at 4 °C. After centrifuga-
tion at 50 000 g for 5 min, aliquots of the supernatant
(15 lg protein) were incubated in 25 lL buffer containing
10 m
M
Tris/HCl pH 7.8, 1 m
M
EDTA, 50 m
M
NaCl,
0.5 m
M
dithiothreitol, 5% (v/v) glycerol, 0.2 mgÆmL
)1
BSA, 40 lgÆmL
)1
poly[dI-dC] and 0.4 ngÆmL
)132
P-labelled
HSE corresponding to nucleotides )115 to )81 of the
human hsp70 gene [13] at 25 °C for 20 min. The mixtures
were then electrophoresed on a native 4% polyacrylamide
gel, and the gel was dried and subjected to autoradiography.

To define the specific HSF–HSE complex, unlabelled HSE
was added to the reaction mixture in a 100-fold molar excess
of the labelled HSE.
Western blot analysis
C3H10T1/2, F9 or HeLa cells were lysed in 100 lL0.1%
SDS. Cellular proteins (15 lg) were separated by SDS/
PAGE, and blotted onto nitrocellulose membrane. The
membrane was washed with Tris-buffered saline (0.1
M
Tris/HCl pH 7.5, 0.9% NaCl) containing 0.1% Tween 20
(TTBS), and reacted with rabbit anti-Hsp105 [14,15] or
mouse anti-Hsp70 Ig (Sigma) at room temperature for 1 h.
After a wash with TTBS, the membrane was further
incubated with horseradish peroxidase-conjugated anti-
rabbit or anti-mouse IgG (Santa Cruz Biotechnology) at
room temperature for 1 h. Hsp105a and Hsp70 were
detected using enhanced chemiluminescence reagent (Santa
Cruz Biotechnology). For quantification, films were digit-
ized by scanning into Adobe
PHOTOSHOP
5(AdobeSystems),
and the intensities of the bands (Hsp105 and Hsp70) were
quantified using the software program
NIH IMAGE
(http://
rsb.info.nih.gov/nih-image/).
Examination of cell morphology
C3H10T1/2 cells (7 · 10
4
cells per well) grown in 24-well

plates containing collagen-coated coverslips were washed
with NaCl/P
i
three times, fixed with 4% paraformaldehyde
at room temperature for 20 min, and then observed using
a phase-contrast microscope.
Neutral red uptake assay
C3H10T1/2 cells (7 · 10
4
cells/well) in 24-well plates were
incubated for 3 h in the presence of 50 lgÆmL
)1
neutral red,
and fixed with 1% formaldehyde containing 1% CaCl
2
for
1 min. The dye incorporated into viable cells was extracted
with 50% ethanol containing 1% acetic acid, and absorb-
ance at 540 nm was measured.
3462 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Results
Isolation of mammalian cell lines for screening of Hsp
modulators
To facilitate the measurement of heat shock promoter
activity, we isolated mouse C3H10T1/2 cells that were
stably transfected with pGL105 reporter plasmid containing
the luc gene linked to a 1.2 kb fragment of the 5¢-flanking
region of the hsp105 gene [12] or p173OR reporter plasmid
containing the b-gal gene linked to the Hsp70B promoter,
and designated them pGL105/C3H and p173OR/C3H cells,

respectively (Fig. 1A,C).
Under nonstressed conditions, the Luc activity in
pGL105/C3H cells was detected at low levels (Fig. 1B).
When pGL105/C3H cells were incubated at 37 °Cfor6h
after heat shock at 39, 41 or 43 °C for 1 h, Luc activity was
enhanced approximately 4 and 10 times in cells heat-
shocked at 41 and 43 °C, respectively, compared to control
levels. During continuous heat shock at 39, 41 or 43 °Cfor
6 h, Luc activity was only enhanced in cells treated at 41 °C.
Because firefly luciferase is thermosensitive and may be
rapidly inactivated at high temperature, we analyzed the
amount of Luc protein in the soluble and insoluble fractions
of these cells treated at 39, 41 and 43 °Cfor6h(Fig.1B,
part b, upper panel). Luc protein was detected in the soluble
fractions but not in the insoluble fractions under these
conditions, and the amounts of the protein were directly
proportional to the Luc activity in cells, suggesting that
levels of Luc activity at high temperatures also reflect the
levels of transcription and translation of Hsp105.
Furthermore, when these cells were treated with chem-
ical stressors such as sodium arsenite, cupric chloride and
zinc chloride, Luc activity was also enhanced in a dose-
dependent manner. As Hsp105a, a major product of
hsp105 gene, is constitutively expressed and also induced by
various forms of stress in mammalian cells, the expression
of Luc activity in pGL105/C3H cells seemed to reflect the
Fig. 1. Stress-inducible Hsp105 and Hsp70B promoters in pGL105/C3H and p173OR/C3H cells. (A) The structure of pGL105 plasmid containing
the Hsp105 promoter upstream of the luciferase reporter gene is shown schematically. (B) pGL105/C3H cells were incubated at 37 °Cfor6hafter
heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium arsenite (c), cupric chloride (d)
or zinc chloride (e) at 37 °C for 6 h. Then, Luc activity was assayed, and relative activities are shown as ratios to that of untreated control cells. For

detection of Luc protein, cells incubated at 37–43 °C for 6 h were lysed and the lysates were centrifuged at 20 000 g for 15 min, Luc protein in the
supernatant (s) and pellet (p) fractions were detected by Western blotting using anti-Luc Ig [upper panels in (b)]. (C) The structure of p173OR
plasmid containing the Hsp70B promoter up-stream of the b-galactosidase reporter gene is shown schematically. (D) p173OR/C3H cells were
incubated at 37 °C for 6 h after heat shock at various temperatures for 1 h (a), incubated at various temperatures for 6 h (b), or treated with sodium
arsenite (c), cupric chloride (d) or zinc chloride (e) at 37 °C for 6 h. Then, b-gal activity was assayed, and relative activities are shown (arbitrary unit,
AU).
Ó FEBS 2003 Sodium salicylate is a potent hsp inducer (Eur. J. Biochem. 270) 3463
expression of endogenous Hsp105a in mammalian cells
[15,16].
In p173OR/C3H cells, b-gal activity was not detected
under nonstressed conditions (Fig. 1D). The b-gal activity
in the cells was induced by heat shock but not by chemical
stressors. The induction of the enzyme activity was consis-
tent with that of the hsp70B gene in mammalian cells [17].
Thus, the promoter activities of Hsp105 and Hsp70B can be
measured easily using the pGL105/C3H and p173OR/C3H
cells, and pGL105/C3H cells seemed to be more useful for
screening modulators of stress response in mammalian cells.
Induction of stress response by SA
Enhancement of heat shock promoter activity by SA. SA
induces the activation of HSF but does not enhance the
transcription of hsp genes in human HeLa cells and
Drosophila [11,18], whereas the drug is also shown to
induce Hsp70 synthesis in mouse L929 cells [19]. Because
transcription of hsp genes may be induced by SA in
mammalian cells, we first examined the effect of SA on the
heat shock promoter using pGL105/C3H or p173OR/C3H
cells (Fig. 2). When pGL105/C3H cells were treated with
15–60 m
M

SA at 37 °C for 1 h and incubated further at
37 °C for 6 h without SA, the Luc activity increased
approximately 10- and 30-fold in cells treated with 45 and
60 m
M
SA, respectively, compared with that of untreated
cells (Fig. 2A). When the amounts of Luc protein in cells
treatedwithSAat37°C were examined by Western
blotting, the amounts of the protein were directly propor-
tional to the Luc activity in cells, suggesting that the
increase of Luc activity by SA reflect the levels of
transcription and translation of Hsp105, not due to an
indirect effect of SA on the basal activity of Luc. Enhance-
ment of Luc activity was also detected at 45 and 60 m
M
SA,
when pGL105/C3H cells were incubated at 39 °Cfor6h
after the SA treatment. However, the enzyme activity was
not enhanced in cells incubated at 41 °Cfor6hafter
treatment with 60 m
M
SA, due to the markedly reduced
viability of the cells.
Furthermore, when p173OR/C3H cells were treated with
15–60 m
M
SA at 37 °C for 1 h and incubated further at
37 °C for 6 h without SA, the activity significantly increased
in cells treated with 45 and 60 m
M

SA (Fig. 2B). Enhance-
ment of b-gal activity was also observed in cells incubated at
39 or 41 °C for 6 h after SA treatment similarly to the Luc
activity in pGL105/C3H cells. The enzyme activity was also
not enhanced in cells incubated at 41 °Cfor6hafter
treatment with 60 mm SA, due to the markedly reduced
viability of the cells. These results suggested that Hsp105
and Hsp70B promoters were activated even at 37 °Cincells
treatedwith45and60m
M
SA, followed by transcription
and translation of the gene products.
When the effects of other known Hsp-inducing com-
pounds such as geldanamycin, curcumin and geranylgera-
nylacetone on the heat shock promoter were examined
using pGL105/C3H cells [20–22], the Luc activity increased
approximately two- to fivefold in cells treated with these
compounds at 37 °C for 6 h compared with that of
untreated cells. SA seemed to activate the heat shock
promoter markedly than these Hsp inducers in mammalian
cells (Table 1).
Fig. 2. Effect of SA on Hsp promoters in pGL105/C3H and p173OR
cells. pGL105/C3H (A) or p173OR (B) cells were incubated with or
without 15, 30, 45 and 60 m
M
SA at 37 °C for 1 h, and further incu-
bated at 37, 39 or 41 °C for 6 h without SA. Then, luciferase or
b-galactosidase activity was assayed. Each value represents the
mean ± SD of three independent experiments. Statistical significance
was determined with Student’s t-test; *, P < 0.01 vs. control cells

incubated at 37, 39 or 41 °C for 6 h. Upper panel in (A) is Western blot
of Luc protein in the supernatant of cells treated with or without 30,
45 and 60 m
M
SA at 37 °C.
Table 1. Effect of geldanamycin, curcumin, or geranylgeranylacetone on
Hsp105 promoter in pGL105/C3H cells. Cells were treated with com-
pounds at 37 °C for 1 h, and further incubated at 37 °Cfor6h.Each
value is average of results from two independent experiments.
Treatment Concentration
Relative
luciferase
activity
Control 1.0
Sodium salicylate 45 m
M
9.2
60 m
M
27.6
Geldanamycin 10 l
M
3.4
15 l
M
4.5
Curcumin 50 l
M
1.7
75 l

M
4.2
Geranylgeranylacetone 0.5 l
M
2.0
1 l
M
2.3
3464 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Activation of HSF by SA and accumulation of Hsp105a
and Hsp70
To examine whether SA enhances heat shock promoter
activity by activating HSF, a gel mobility shift assay using
32
P-labelled HSE was performed. When C3H10T1/2 cells
were treated with 10–60 m
M
SA at 37 °C for 1 h, HSF was
activated in cells treated with SA at concentrations of more
than 20 m
M
(Fig. 3A). The kinetics of activation of HSF by
SA revealed that the HSF–HSE complex was detected
immediately after 30 m
M
SA treatment, and quickly
diminished in 1–2 h at 37 °C (Fig. 3B). Furthermore, SA
affected the activation of HSF by heat shock (Fig. 3C).
When activated by heat shock at 41 °C for 1 h, the HSF
decreased to the basal level within 2 h. However, when cells

treated with 30 m
M
SA at 37 °C were continuously heat-
shocked at 41 °C, the SA-activated HSF remained at a high
level for 2 h, then diminished to the basal level within 3 h of
heat shock. Thus, SA seems not only to activate HSF at
37 °C, but also to enhance the activation of HSF by heat
shock.
Next, we examined whether Hsp105a and Hsp70 proteins
accumulatedincellstreatedwithSA(Fig.4).Mouse
C3H10T1/2, mouse F9 and human HeLa cells were treated
with 15–60 m
M
SA at 37 °C for 1 h and further incubated
for 6 h at 37 °C. The levels of Hsp105a and Hsp70
significantly increased in cells treated with 45 and 60 m
M
SA. However, the levels remained unchanged or decreased,
when these cells were incubated at 41 °C for 6 h after the SA
treatment. These results suggested that SA treatment at
37 °C induced the expression of endogenous heat shock
proteins such as Hsp105a and Hsp70 in various mammalian
cells.
Enhancement of thermoresistance of cells by SA
Upon exposure to a sublethal heat treatment, mammalian
cells acquire transient resistance to a subsequent heat shock
that would normally be lethal [6,7]. The phenomenon is
known as thermotolerance, and much evidence supports the
idea that Hsps, especially Hsp70, play important roles in its
development. Since SA seemed to induce the expression of

Hsp105a and Hsp70 in mammalian cells, we examined
whether SA induced resistance against subsequent lethal
heat shock. As shown in Fig. 5A, the treatment of cells with
30 or 45 m
M
SA for 1 h did not induce marked changes of
cell morphology and numbers of cells attached to culture
dishes, although the attached cells were slightly reduced in
number by 60 m
M
SA treatment. When these cells were
incubated at 37 °C for 6 h and exposed to a lethal heat
shock at 45 °C for 45 min, cells attached to dishes markedly
decreased in number regardless of SA treatment. However,
when these cells were further incubated at 37 °C for 24 h,
numbers attached to culture dishes were increased in cells
treatedwith45or60m
M
SA, but not 30 m
M
SA.
Furthermore, the viability of cells was assessed based on
the ability of living cells to incorporate neutral red into
lysozomes (Fig. 5B). The uptake of neutral red into cells
decreased gradually after heat shock at 45 °C for 45 min,
although uptake of the dye was only slightly affected by
30–60 m
M
SA at 37 °C. However, the uptake again
increased in cells pretreated with 45 and 60 m

M
SA for 24
and 120 h, respectively, after the heat shock. Thus, the
resistance of cells against a subsequent heat shock seemed to
be enhanced by 45 or 60 m
M
SA treatment.
Discussion
As Hsps play important roles in the folding, regulation and
degradation of cellular proteins and also cellular resistance
against stress as molecular chaperones, drugs that can
regulate the expression levels of Hsps in cells seem to have
various medicinal applications. In the present study, we
isolated cell lines for screening of stress response modula-
tors: mouse pGL105/C3H and p173OR/C3H cells.
pGL105/C3H cells have a plasmid containing the Hsp105
promoter upstream of a luc reporter gene, while p173OR/
C3H cells have a plasmid containing the Hsp70B promoter
upstream of a b-gal reporter gene. The Luc or b-gal activity
in these cells was expressed and induced similarly to
endogenous Hsp105a or Hsp70B, respectively, in mamma-
lian cells. Using these cells, the activities of Hsp105 and
Hsp70B promoters could be easily measured.
Fig. 3. Effect of SA on activation of HSF in C3H10T1/2 cells. (A)
C3H10T1/2 cells were treated with or without 10, 20, 30 and 60 m
M
SA at 37 °C for 1 h, or heat-shocked at 42 °C for 3 h as a positive
control. (B and C) C3H10T1/2 cells were incubated with or without SA
at 37 °C for 1 h, then further incubated at 37 °C (B) or at 41 °C(C)
without SA for up to 6 h. Cell extracts from these cells were subjected

to gel mobility shift assay using a radioactive HSE probe. HSF–HSE
complexes were determined by adding a 100-fold excess of unlabelled
HSE. Arrows indicate specific HSF–HSE complexes.
Ó FEBS 2003 Sodium salicylate is a potent hsp inducer (Eur. J. Biochem. 270) 3465
Using these cells, we examined the effects of SA, a
nonsteroidal anti-inflammatory drug, on the stress response
of mammalian cells. Jurivich et al. have shown that 20 m
M
SA induces activation of HSF but not transcription of hsp
genes in human cells [11]. In contrast, Liu et al. have shown
that 60 m
M
SA induces not only activation of HSF but also
accumulation of Hsp70 in mouse cells [19]. Here we showed
that relatively high doses of SA (45–60 m
M
) induced the
activation of promoter activities of Hsp105 and Hsp70 as
well as the accumulation of Hsp105a and Hsp70 in mouse
and human cells, although relatively low doses of SA (20–
30 m
M
) induced the activation of HSF but not transcription
of hsp genes in cells. Thus, relatively high doses of SA
seemed to be required for the induction of the transcription
of hsp genes and the accumulation of Hsp in mammalian
cells.
HSF is a transcription factor which is converted to a
trimeric and hyper-phosphorylated active form from inac-
tive monomers in response to various forms of stress and

induces the transcription of hsp genes [8]. SA is shown to
trigger HSF differently than heat shock [20]. The SA-
induced form of HSF is not hyperphosphorylated like the
heat-induced form, and SA-induced threonine phosphory-
lation of HSF, whereas heat shock led to a predominance
of HSF serine phosphorylation [23]. However, the HSF
activated by relatively high doses of SA did induce
transcription of hsp genes in cells. The HSF activated by
SA may be somehow different at levels of modification such
as phosphorylation depending on the doses of SA, although
further study is needed to elucidate the different effects of
the SA-activated HSF on the induction of transcription of
hsp genes.
Hsps are suggested to play important roles in the
acquisition of resistance of cells against various forms of
stress [1]. Here we showed that SA induced the activation
of HSF, the transcription of hsp genes and the accumu-
lation of Hsps in various mammalian cells and a concomi-
tant increase of thermoresistance of cells. Thus, SA may
be used for the protection of cells against deleterious
stressors. Furthermore, several neurodegenerative disorders
including Alzheimer’s, polyglutamine and Parkinson’s
disease are though to be caused by an accumulation of
protein aggregates in the brain [24], and Hsps such as
Hsp70 and Hsp40 are shown to suppress the toxicity of
these diseases [25,26]. Recently, long-term use of non-
steroidal anti-inflammatory drugs was shown to prevent
the occurrence of Alzheimer’s disease [27,28]. Our finding
that SA induces the expression of Hsps in mammalian cells
may explain the protective effect of SA on Alzheimer’s

disease.
SA activated heat shock promoter and induced the
expression of Hsps in mammalian cells at concentrations
higher than those used for its anti-inflammatory effects.
However, long-term use of therapeutic doses of the drug
may induce the expression of Hsps in mammalian cells. SA
has a potent anti-inflammatory effect mediated by suppres-
sion of the production of inflammatory mediators by
inhibition of cyclooxygenase and nuclear factor-kappa B
activation [9,10]. In addition to the anti-inflammatory
effects, it is noteworthy that SA which can activate HSF
Fig. 4. Effect of SA on accumulation of
Hsp105 and Hsp70 in mammalian cells.
C3H10T1/2 (A), F9 (B), and HeLa cells (C)
were treated with or without SA at 37 °Cfor
1 h, then further incubated at 37 or 41 °Cfor
6 h without SA. Cellular proteins (15 lg) were
separated by SDS/PAGE (10% polyacryl-
amide), blotted onto nitrocellulose
membranes, and immunostained using anti-
Hsp105 or anti-Hsp70 Ig (upper panels).
Bands were quantified by densitometry, and
relative levels of Hsp105a or Hsp70 are shown
as ratios to that of untreated cells at 37 °Cor
cells heat-shocked at 41 °C for 6 h, respect-
ively (lower graphs). Each value in (A) and
(B) represents the mean ± SD of three
independent experiments. Statistical
significance was determined with Student’s
t-test; *, P < 0.01 vs. control cells incubated

at 37 or 41 °Cfor6h.
3466 K. Ishihara et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 5. Induction of thermoresistance of cells by SA. (A) C3H10T1/2 cells were treated with or without 30, 45 and 60 m
M
SA at 37 °C for 1 h (a).
These cells were further incubated without SA at 37 °C for 24 h (b) or heat shocked at 45 °C for 45 min after incubation at 37 °C for 6 h (c). The
heat-shocked cells were further incubated at 37 °C for 24 h (d). Then, these cells were fixed with 4% paraformaldhyde and observed using a phase
contrast microscope (· 100). (B) C3H10T1/2 cells treated with or without 30, 45 and 60 m
M
SA at 37 °C for 1 h were incubated at 37 °Cfor6h
without SA. Then, these cells were heat-shocked at 45 °C for 45 min, and further incubated at 37 °C for the indicated periods. Viability of cells was
assessed by neutral red uptake assay.
Ó FEBS 2003 Sodium salicylate is a potent hsp inducer (Eur. J. Biochem. 270) 3467
and induce the transcription of hsp genes may be used as
an Hsp inducer for treatment of diseases.
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