Induction of cyclooxygenase-2 by heat shock protein 60 in
macrophages and endothelial cells
Blase
Billack1,
Diane E.
Heck1,
Thomas M.
Mariano2,
Carol R.
Gardner1,
Runa
Sur1,
Debra L.
Laskin1, and
Jeffrey D.
Laskin2
1 Department of Pharmacology and Toxicology, Rutgers
University and 2 Department of Environmental and
Community Medicine, University of Medicine and Dentistry of New
Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey
08854
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ABSTRACT |
The 60-kDa heat shock protein (HSP60), an
endogenous ligand for the toll-like 4 receptor, is generated in
response to inflammation, tissue injury, and/or stress and stimulates
macrophages to produce cytotoxic and proinflammatory mediators
including nitric oxide, tumor necrosis factor (TNF)-
, interleukin
(IL)-6, and IL-12. In the present studies we report that HSP60 is an
effective inducer of cyclooxygenase-2 (COX-2) in macrophages, as well
as endothelial cells. In both cell types, the synthesis of COX-2 was
coordinate with induction of nitric oxide synthase (NOS)-2 and
with nitric oxide production. With the use of promoter constructs in
transient transfection assays, optimal expression of COX-2 in
macrophages was found to require nuclear factor (NF)-
B, the
cAMP-response element (CRE), and NF-IL-6, but not the E-box. Mobility
shift assays revealed that HSP60 induced NF-
B and CRE binding
activity, while CCAAT/enhancer binding protein (C/EBP), which
binds to NF-IL-6, was constitutively active in the cells. Both c-Jun
and CRE binding protein (CREB) bound to the CRE, while
C/EBP-
bound to NF-IL-6. These data indicate that NF-
B,
C/EBP-
, c-Jun, and CREB are important in HSP60-induced expression of
COX-2. The c-Jun-NH2-terminal kinase (JNK), p44/42
mitogen-activated protein (MAP) kinase [extracellular signal-regulated
kinase 1/2 (ERK1/2)], and p38 MAP kinase were rapidly activated
by HSP60 in the macrophages. PD-98059, an inhibitor of
phosphorylation of ERK1/2, caused a marked inhibition of
HSP60-induced COX-2 and NOS-2 expression. Unexpectedly,
SB-203580, a p38 kinase antagonist, was found to block HSP60-induced
expression of COX-2, but not NOS-2. These data indicate that both
ERK1/2 kinase and p38 kinase play a role in regulating HSP60-induced
expression of COX-2.
nitric oxide; inflammation; cytokines; monocytes/macrophages
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INTRODUCTION |
THE 60-KDA HEAT SHOCK
PROTEIN (HSP60) has been identified as a danger signal to the
innate immune system (6). It appears to be a key
endogenous inflammatory mediator generated in response to tissue injury
and/or stress and is presumably released by damaged cells. HSP60
stimulates macrophages to produce cytotoxic and proinflammatory mediators, including nitric oxide, tumor necrosis factor (TNF)-
, interleukin (IL)-6, and IL-12 (6, 29). HSP60 functions by activating the toll-like 4 receptor, a transmembrane protein that is
also important in intracellular signaling initiated by
lipopolysaccharide (LPS) from gram-negative bacteria (1, 4, 26,
39, 41, 47, 52-54). Similarities in the intracellular
domain structure of the toll-like 4 receptor and the IL-1 receptor form
the basis of current models to explain the mechanism of action of HSP60 and LPS (39). Engagement of the toll-like 4 receptor
causes the adapter protein MyD88 to associate with the receptor
(27, 42, 63). This results in activation of an IL-1
receptor-associated kinase (IRAK), followed by phosphorylation and
activation of TNF receptor (TNFR)-associated factor (TRAF)-6 (20,
34, 48, 62). This pathway is known to be critical for the
activation of nuclear factor (NF)-
B, as well as the
mitogen-activated protein (MAP) kinases, signaling molecules that are
important in the transcriptional regulation of many cytotoxic and
proinflammatory mediators in macrophages, including the inducible form
of nitric oxide synthase (NOS-2) (7, 15, 52, 54).
It is well recognized that macrophages release lipid mediators,
including prostaglandins (PGs) and leukotrienes that promote inflammation (32, 37, 45). The synthesis of PGs is
dependent on the activity of cyclooxygenase (COX), an oxidoreductase
that converts arachidonic acid into the common PG precursor,
PGH2 (61). Two isoforms of the enzyme have
been identified, a constitutive form referred to as COX-1 that is
expressed in most cell types, and COX-2, an inducible form that is
thought to be important in inflammation (22, 55).
Macrophages express COX-2 in response to a variety of cytokines as well
as LPS (22, 37, 68). In the present studies, we determined
if COX-2 was also induced by HSP60. We found that HSP60 was an
effective inducer of COX-2 in macrophages, as well as endothelial
cells. Moreover, activation of MAP kinases, c-Jun/cAMP-response element
(CRE) binding protein (CREB), and NF-
B signaling pathways are
important in this activity. These data provide additional support for
the idea that HSP60 is important in nonspecific host defense and can
act as an endogenous mediator of inflammation.
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MATERIALS AND METHODS |
Chemicals.
Recombinant mouse and human interferon (IFN)-
were kindly provided
by Dr. S. Pestka, University of Medicine and Dentistry of New Jersey
(UMDNJ)-Robert Wood Johnson Medical School (Piscataway, NJ). LPS
derived from Escherichia coli (serotype O55: B5) was purchased from Sigma Chemical (St. Louis, MO). Recombinant human HSP60
(lot no. 008404) and antibodies against p65 Rel A were from StressGen
Biotechnologies (Vancouver, BC).
L-N5-(iminoethyl)ornithine (NIO) was
obtained from Alexis Biochemicals (San Diego, CA) and
4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236) from Monsanto/Searle (St. Louis, MO). Antibodies against NOS-2, p50, COX-2, CREB, c-Jun, and CCAAT/enhancer binding
protein (C/EBP)-
and all secondary antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2,
c-Jun-NH2-terminal kinase (JNK)/stress-activated protein
kinase (SAPK), phospho-JNK/SAPK, p38 MAP kinase, and phospho-p38
MAP kinase were from Cell Signaling Technology (Beverly, MA).
4-(4-Fluorophenyl)-2- (4-methylsulfinylphenyl)-5-(4-pyridil)1H-imidazole (SB-203580) and
2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one (PD-98059)
were from Calbiochem (La Jolla, CA). Unless otherwise indicated, all
other chemicals were from Sigma Chemical.
Cells, treatments, and measurements of nitric oxide production.
Primary cultures of rat endothelial cells and alveolar macrophages were
prepared as described previously (16, 43). RAW 264.7 mouse
macrophages were from Dr. D. Wolff, UMDNJ-Robert Wood Johnson Medical
School (Piscataway, NJ). Cells were cultured in DMEM supplemented with
10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Nitric oxide production by the cells was
quantified by the accumulation of nitrite in the cell culture medium
using the Greiss reagent with sodium nitrite as standard
(21). For these studies, cells (5 × 105)
were plated into 24-well plastic culture dishes. Each well contained 0.3 ml of culture medium. After 24 h, the medium was changed to serum- and phenol red-free growth medium without and with appropriate concentrations of HSP60 (1-10 µg/ml). Several experiments were performed to exclude the possibility that LPS contamination was responsible for the biological effects of HSP60 (6, 30). Using a Limulus amebocyte lysate assay (E-toxate kit,
Sigma), we found that treatment medium with HSP60 contained
<0.08-0.09 ng/ml of LPS, concentrations well below those required
to induce COX-2 and NOS-2 in the cells (data not shown). Moreover, LPS- but not HSP60-induced expression of COX-2, as well as NOS-2 and nitric
oxide production, was inhibited by the addition of polymyxin B (up to
30 µg/ml) to the cultures. The activity of HSP60, but not LPS, was
also found to be heat labile. Table
1 summarizes the effects of
polymyxin B and heat treatment on nitric oxide production induced by
HSP60 in RAW264.7 macrophages. In these studies, polymyxin B (0.3 and 3 µg/ml) reduced LPS- but not HSP60-induced nitrite accumulation in the
culture medium of the macrophages. Conversely, heat treatment (85°C,
15 min) inhibited the activity of HSP60, but not LPS. These data
indicate that the biological effects of HSP60 were not due to LPS
contamination.
Western blotting.
Methods for the preparation of cell lysates and Western blotting have
been described previously (21). Briefly, cells were solubilized in buffer containing 1% Triton X-100, 10% glycerol, 50 mM
Tris · HCl, pH 7.4, 50 mM NaCl, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. Equal amounts of protein (10-30 µg/ml) were
electrophoresed on 7.5 or 10% polyacrylamide gels, transferred to
nitrocellulose membranes, and then probed with a specific primary
antibody followed by a horseradish peroxidase-conjugated secondary
antibody. A chemiluminescence-based detection kit was used to visualize
protein expression (Renaissance Plus, Perkin-Elmer Life Sciences).
RNA isolation and RT-PCR of COX-2.
RNA was isolated from the macrophages using Trizol reagent (GIBCO BRL,
Rockville, MD) according to the manufacturer's instructions. First-strand cDNA synthesis was performed with SuperScript II for
RT-PCR (GIBCO BRL) using 0.4 µg RNA in each reaction. PCR was run
using 20 ng cDNA, COX-2 mouse upstream primer (5'-
CATTCTTTGCCCAGCACTTCAC-3') and downstream primer
(5'-GACCAGGCACCAGACCAAAGAC-3'; Ambion Gene-Specific Relative RT-PCR
kit), and Taq DNA polymerase (GIBCO BRL). PCR amplification
was performed using a modification of the manufacturer's protocol to
allow for COX-2 quantification using 18S as an internal standard.
Varying cycles were used to optimize quantitation. The PCR products
were amplified using a 1-min hot start at 94°C and 22 cycles of
30 s at 94°C, 30 s at 56°C, and 60 s at 72°C and a
final extension at 72°C for 10 min. Products were separated by
electrophoresis using 8 M urea polyacrylamide gels followed by autoradiography.
Transfection of macrophages.
Reporter plasmids driving the expression of firefly luciferase fused to
either wild-type or mutated promoter constructs of the murine COX-2
gene were the generous gift of Dr. H. Herschman, Univ. of California
(Los Angeles, CA). Mutated promoter constructs for the CRE (mCRE),
E-box (mE-box), NF-
B (mNF-
B), and two NF-IL-6 (mNF-IL-6-1,
mNF-IL-6-2) sites were used (68). The reporter construct in which the mutated NF-IL-6 site was located further upstream from the start of the luciferase gene was designated mNF-IL-6-1, while the mutated site further downstream was
designated mNF-IL-6-2 (68). To assess transfection
efficiency, cells were cotransfected with a
Renilla-luciferase cDNA. Plasmids were introduced into cells
by electroporation using a Bio-Rad Gene Pulser II system (200 V, 1,000 µF) and then seeded into 24-well culture plates (106
cells/well). After incubation at 37°C for 24 h, the cells were treated with HSP60 (10 µg/ml). Luciferase activity was then assessed 6 h later with a Turner model 20/20 luminometer by measuring the conversion of a firefly-luciferase- or
Renilla-luciferase-sensitive substrate to a luminescent
species (Dual Luciferase Assay, Promega). Results are reported as the
ratio of firefly luciferase activity to Renilla luciferase
activity in the extract.
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as described by Schreiber et al.
(59). Oligonucleotide consensus sequences for either
NF-IL-6 (5'-TGCAGATTGCGCAATCTGCA-3'), CRE (5'-TCCACATGAGATCATGGTTT-3'), NF-
B (5'-AGTTGAGGGGACTTTCCCAGGC-3') or IFN-
activating sequence (GAS) (5'-GATCGATTTCCCCGAAAT-3') (10, 13, 40, 70) were labeled with 32P and incubated at room temperature for 30 min with 15 µg of nuclear protein. Binding reactions were performed
in a total volume of 25 µl in a buffer containing 5 mM HEPES, pH 8.0, 5% glycerol, 1 mM MgCl2, 40 mM NaCl, 0.05 mM
dithiothreitol, 0.4% BSA, and 0.1 µg/ml salmon sperm DNA. In
competitor experiments, nonradioactive oligonucleotide was added to the
binding reaction mixture in a 100:1 ratio relative to labeled
oligonucleotide. The protein/DNA complexes were then fractionated on
4.5 or 7% polyacrylamide gels. Gels were transferred to Whatman filter
paper, covered with plastic wrap, dried for 1 h, and then exposed
to Kodak MS film (
70°C, 18 h). For p65 Rel A, c-Jun, CREB, or
c/EBP-
supershifts, nuclear extracts were incubated with a 1:50
dilution of anti-p65 Rel A, anti-c-Jun, or anti-CREB, or a 1: 5 dilution of anti-c/EBP-
antibodies, respectively, for 30 min on ice
before addition of the labeled oligonucleotide.
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RESULTS |
Effects of HSP60 on COX-2 expression.
The present studies report for the first time that HSP60 is an
effective inducer of COX-2 in macrophages and endothelial cells. In
both cell types, HSP60 readily induced COX-2 protein expression in a
concentration- and time-dependent manner (Fig.
1, A and B, top; Fig. 2, A
and B; and not shown). Whereas in the macrophages, expression of COX-2 was observed within 4 h, in endothelial cells, it was delayed for 16 h (Fig. 1, A and B,
top). In macrophages, induction of COX-2 protein was found
to be due to increased steady-state expression of COX-2 mRNA (Fig.
2D).

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Fig. 1.
Heat shock protein 60 (HSP60) induces cyclooxygenase (COX)-2 and
nitric oxide synthase (NOS)-2 in macrophages and endothelial cells.
Cells were treated without or with HSP60 (10 µg/ml) and/or
lipopolysaccharide (LPS; 10 ng/ml) as described in MATERIALS AND
METHODS. Cell lysates were probed for COX-2 and NOS-2 expression
by Western blotting (A and B, top) and
culture supernatants were analyzed for nitrite content (A
and B, bottom). Data represent means ± SE
of triplicate samples. , Control; ,
LPS; , HSP60; , LPS + HSP60.
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Fig. 2.
Effects of HSP60 on COX-2 and
NOS-2 expression in macrophages. Macrophages were treated without or
with HSP60 (10 µg/ml) in the presence or absence of inhibitors as
described in MATERIALS AND METHODS. A: Western
blots showing concentration-dependent induction of COX-2 and NOS-2 in
the cells. B: concentration-dependent production of nitrite
24 h after treatment with HSP60. Data are means ± SE of
triplicate samples. C: effect of inhibitors on HSP60-induced
expression of COX-2 and NOS-2. Macrophages were treated for 18 h
without or with HSP60 in the presence or absence of the NOS-2 inhibitor
L-N5-(iminoethyl)ornithine (NIO; 1 mM) or the COX-2 inhibitor SC-236 (20 µM). D: induction of
COX-2 mRNA by HSP60. Macrophages were pretreated for 60 min without or
with PD-98059 (40 µM), a specific inhibitor of mitogen-activated
protein kinase kinase (MEK) 1/2, and then stimulated without or with
HSP60 (3 or 6 h) or LPS (1 µg/ml, 6 h), as a control. RNA
was then isolated and analyzed by RT-PCR for COX-2 mRNA. 18S RNA served
as an internal standard.
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Previous studies have demonstrated that many inducers of COX-2 also
upregulate the expression and activity of NOS-2 (32, 37, 45,
61). We found that HSP60 readily induced nitric oxide production
in both macrophages and endothelial cells and was as effective as LPS
(Fig. 1, A and B, bottom). The
combination of LPS and HSP60 was, in general, more effective in
inducing nitric oxide production than LPS or HSP60 alone. The time
course of HSP60-induced NOS-2 expression was similar to induction of
COX-2 in both cell types (Fig. 1, A and B,
top). Induction of COX-2 and NOS-2 by HSP60 was also
observed in normal primary cultures of alveolar macrophages, indicating
that the effects of HSP60 were not limited to a macrophage tumor cell
line (data not shown).
In macrophages, maximal induction of NOS-2 occurs when cytokines such
as IFN-
are combined with LPS (50). Similarly, we found
that treatment of the macrophages with the combination of IFN-
and
HSP60 caused a significantly greater induction of nitric oxide
production (Fig. 3, top) and
NOS-2 compared with either agent alone (Fig. 3). In contrast,
IFN-
had no effect on HSP60-induced COX-2 expression in the cells
(Fig. 3, bottom). These data suggest that there are distinct
mechanisms regulating expression of COX-2 and NOS-2 in macrophages.

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Fig. 3.
Interferon (IFN)- enhances HSP60-induced expression of
NOS-2. Macrophages were treated for 24 h without or with LPS (10 µg/ml) and/or HSP60 (10 µg/ml) in the absence or presence of
IFN- (100 U/ml). Top: nitric oxide production by control
and treated cells. Bars represent means ± SE of triplicate
samples. Middle: Western blot showing expression of NOS-2.
Bottom: Western blot showing expression of COX-2.
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Previous studies have suggested that the activity of NOS-2 may be
important in regulating the expression of COX-2 (23, 38, 56). We found that the well-characterized NOS-2 inhibitor NIO did not alter HSP60-induced COX-2 expression (Fig. 2C).
Similarly, SC-236, a specific inhibitor of COX-2, had no effect on
either the expression of NOS-2 or production of nitric oxide by the
cells (Fig. 2C and not shown). These data indicate that in
mouse macrophages, neither COX-2 nor NOS-2 activity is required for
HSP60-induced expression of these proteins and further support the idea
that their expression is regulated independently.
Regulation of HSP60-induced COX-2 expression.
To examine regulatory elements required for HSP60-induced
transcriptional activation of COX-2, we transfected macrophages with
wild-type or mutated COX-2 promoters fused to a luciferase reporter
gene. The wild-type mouse promoter contains two NF-IL-6 regulatory
regions (mNF-IL-6-1, mNF-IL-6-2) and one site for the E-box,
CRE, and NF-
B elements (68). HSP60 was found to
readily induce luciferase activity in cells transfected with the
wild-type COX-2 reporter. Activity was significantly decreased in
HSP60-treated cells transfected with mutated constructs for
NF-
B, CRE, or NF-IL-6 (Fig. 4).
Interestingly, the two NF-IL-6 response elements were not equally
active. Compared with the mutated site near the 5'-end of the promoter
(mNF-IL-6-1), mutation of the site near the 3'-end (mNF-IL-6-2) resulted in a sharper decline in HSP60-induced
luciferase activity. The luciferase activity in extracts from cells
transfected with an NF-IL-6 construct mutated at both sites
(mNF-IL-6-1 and mNF-IL-6-2) was similar to the activity
observed in extracts from cells transfected with mNF-IL-6-2 alone.
These data demonstrate a role for the NF-
B, CRE, and NF-IL-6
regulatory elements in transcriptional activation of COX-2 and
also emphasize the importance of the location of these elements within
the promoter in controlling COX-2 expression.

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Fig. 4.
Luciferase activity of wild-type and mutant COX-2
reporter plasmids. A: schematic of COX-2 promoter cloned
into reporter plasmid as described by Wadleigh et al.
(68). The promoter spanned nucleotides 724 to +7. The
positions of the mutated sites for nuclear factor (NF)- B,
NF-interleukin (IL)-6-1, NF-IL-6-2, cAMP-response element
(CRE), and the E-box are shown. Note that the consensus sequences for
CRE and E-box overlap. B: transfection of promoter
constructs into RAW 264.7 cells. Macrophages were transiently
transfected with wild-type or mutant COX-2 promoter constructs along
with a Renilla luciferase control. The cells were then
treated without (closed bars) or with 10 µg/ml HSP60 (open bars).
After 6 h, cell lysates were prepared and assayed for firefly and
Renilla luciferase activities. Bars represent means ± SE of triplicate samples from 1 of 3 experiments. * Significantly
different (P 0.05) from HSP60-treated wild-type reporter
activity. m, Mutated promoter construct.
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On the basis of these results, we next examined the activity of
transcription factors that bind to the regulatory elements that appear
to be involved in induction of COX-2 by HSP60. Using mobility shift
assays, we found that HSP60, like LPS, readily activated NF-
B in the
cells (Fig. 5B). The
combination of HSP60 and LPS did not further increase NF-
B activity
(data not shown). Western blot analysis confirmed nuclear translocation
of both the p50 and p65 subunits of NF-
B after treatment of cells
with HSP60 (Fig. 5A). C/EBP is known to bind to NF-IL-6
regulatory regions in gene promoters (44-47). This
transcription factor was found to be constitutively active in the
macrophages (Fig. 5, C and D). Supershift assays
indicated that the
-isoform of C/EBP was predominately expressed in
the cells (Fig. 5D). Western blotting with antibodies to
C/EBP-
confirmed nuclear localization of the protein both before and
after HSP60 treatment (Fig. 5C). CREB and c-Jun bind CRE
regulatory regions in responsive gene promoters (13, 60).
HSP60, like LPS, readily activated CRE in macrophages. Supershift
assays confirmed the presence of both CREB and c-Jun in the CRE binding
complex (Fig. 6A). These data
are in accord with the transfection studies described above and further
demonstrate the importance of NF-
B, C/EBP, and c-Jun/CREB in
regulating HSP60-induced expression of COX-2.

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Fig. 5.
Activation of NF- B and
CCAAT/enhancer binding protein (C/EBP) by HSP60 in macrophages.
Macrophages were treated without or with HSP60 (10 µg/ml) for
increasing periods of time (15-45 min). LPS (1 µg/ml) was used
as a positive control. Nuclear extracts were prepared and analyzed by
Western blotting or in mobility shift assays as described in
MATERIALS AND METHODS. In mobility shift assays, the first
lane of each gel shows the probe (PROBE) run in the absence of nuclear
extract and the second lane (CTL), nuclear extract from buffer-treated
control cells. In these gels, lanes marked CC are nuclear extracts
analyzed in the presence of a ×100 excess of appropriate unlabeled
probe. A: Western blot of nuclear extracts showing
expression of the p50 and p65 subunits of NF- B in control (CTL) and
HSP60-treated cells. B: mobility shift assay for NF- B.
The p50 and p65 lanes show supershifts using antibodies to these
proteins. B, inset: another sample analyzed with the p65
antibody that more clearly demonstrates a supershift. The p50
supershifted band was not readily visible due to its spreading over the
higher molecular weight ranges in the gel. C: Western blot
of nuclear extracts showing expression of C/EBP- in CTL and
HSP60-treated cells. D: mobility shift assay for C/EBP. SS,
supershift with an antibody to C/EBP- .
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Fig. 6.
Effects of HSP60 on CRE binding protein (CREB), c-Jun, and signal
transducer and activator of transcription (STAT)-1 . Macrophages were
treated for 15 min without or with HSP60 (10 µg/ml), IFN- (100 U/ml), or LPS (1 µg/ml) as a control. Nuclear extracts were then
assayed for transcription factor activity. The first lane in each panel
represents labeled probe in the absence of extract, whereas the lanes
labeled CC were nuclear extracts analyzed in the presence of a ×100
excess of the appropriate unlabeled probe. Control (CTL) lanes were
from cells treated with buffer alone. A:
c-Jun/CREB. For supershift assays, extracts were treated with specific
antibodies to CREB or c-Jun. B: STAT-1 .
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As indicated above, HSP60-induced expression of NOS-2, but not COX-2,
was enhanced by IFN-
. To determine if HSP60 modulated IFN-
-induced signaling for NOS-2, we examined DNA binding of the
transcription factor signal transducer and activator of transcription (STAT)-1
to the GAS. This is a key element regulating NOS-2 gene expression (44). STAT-1
binding to GAS was detected in
nuclear extracts from macrophages stimulated with IFN-
, but not with HSP60 (Fig. 6B). The STAT-1
:GAS complex was supershifted
with antibodies to STAT-1
(data not shown). HSP60 had no
additional effect on IFN
-induced STAT-1
binding activity (Fig.
6B). These data indicate that HSP60 does not
function by activating STAT-1
and that other transcription factors
are responsible for enhancing NOS-2 expression in cells treated with
the combination of HSP60 and IFN-
.
Role of MAP kinases in HSP60-induced COX-2 expression.
Binding of LPS to the toll-like 4 receptor leads to activation of the
MAP kinase pathways, including the p44/p42 MAP kinase (ERK1/2), JNK,
and p38 kinase, each of which has been implicated in the regulation of
COX-2 (2, 11, 17, 31, 46, 68). These enzymes can directly
phosphorylate and activate transcription factors and/or other
intracellular substrates such as protein kinases that result in
transcription factor activation (31, 49). For example, the
JNK kinase phosphorylates c-Jun on its NH2-terminal
activating domain, resulting in the formation of c-Jun-c-Fos
heterodimers and c-Jun homodimers, which can upregulate genes via AP-1
and CRE regulatory elements (31, 49). The p38 kinase has
also been reported to activate a number of transcription factors,
including CREB and c-Jun (31, 49), while the activity of
ERK1/2 has been reported to be important in activation of NF-
B in
the COX-2 promoter (5). We found that the macrophages
constitutively expressed ERK1/2 kinase, JNK kinase, and p38 kinase
(Fig. 7, B and C,
and Fig. 8, B and
E). HSP60 caused a rapid time-dependent phosphorylation of
each of these proteins (Fig. 7A and Fig. 8, A and
E). ERK1/2 was phosphorylated, presumably, through the
action of MAP kinase kinase (MEK) 1 and MEK2
(31). PD-98059, a selective inhibitor of these kinases
(2), completely blocked HSP60-induced phosphorylation of
ERK1/2 (Fig. 7C). PD-98059 also caused a marked decrease in
HSP60-induced COX-2 mRNA and protein expression as well as NOS-2
protein expression (Fig. 2D and Fig. 7C).
SB-203580, a selective inhibitor of the p38 kinase (11),
blocked p38 phosphorylation in the macrophages (Fig. 8A).
However, in contrast to the ERK1/2 inhibitor, the p38 kinase inhibitor
was found to block HSP60-induced expression of COX-2, but not NOS-2
(Fig. 8, C and D).

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Fig. 7.
Activation of ERK1/2 kinases by HSP60. Macrophages were
treated without or with HSP60 (10 µg/ml) or LPS (1 µg/ml) as a
control for increasing periods of time. Cell lysates were analyzed by
Western blotting. A: time-dependent induction of
phosphorylated ERK1/2 (phospho-ERK1/2). B: effects of
increasing concentrations of PD-98059 on HSP60-induced phospho-ERK1/2
and total ERK1/2 expression. The cells were pretreated with a specific
inhibitor of ERK1/2 for 60 min before HSP60 was added to the cultures.
C: abrogation of HSP60-inducible COX-2 and NOS-2 by a
specific inhibitor of ERK1/2. Cells were pretreated without or with
PD-98059 for 60 min and then with HSP60 for 0-18 h. Cell lysates
were then assayed for phospho-ERK1/2, total ERK1/2, COX-2, and NOS-2 by
Western blotting.
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Fig. 8.
Activation of p38 and JNK kinases by HSP60. Macrophages were
treated without or with HSP60 (10 µg/ml) or LPS (1 µg/ml) as a
control and analyzed by Western blotting. A: time-dependent
phosphorylation of p38 kinase by HSP60. Note that cells were pretreated
without or with the p38 inhibitor SB-203580 (10 µM) for 60 min before
the addition of HSP60. B: total p38 expression in
macrophages treated without or with HSP60 for 30 min.
C: effect of the p38 kinase inhibitor on NOS-2 expression.
Cells were treated with HSP60 for 18 h. D: effects of
increasing concentrations of p38 kinase inhibitor on HSP60-induced
COX-2 expression after 6 h. E: time-dependent
phosphorylation of the p48 and p54 isoforms of JNK (phospho-JNK) and
total JNK expression in cells treated with HSP60 or LPS.
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DISCUSSION |
The present studies demonstrate that HSP60 is an effective inducer
of both COX-2 and NOS-2 in macrophages and endothelial cells. These
enzymes are responsible for the production of cytotoxic and
inflammatory mediators, and their ability to be induced by HSP60
provides support for the idea that this stress protein is an important
endogenous activator of innate immunity (1, 6, 47).
Increased expression of COX-2 in response to HSP60 was associated with
an increase in steady-state expression of COX-2 mRNA. In many cells,
increases in COX-2 mRNA are due to upregulation of COX-2 gene
transcription (22). However, it should be noted that the
COX-2 message can also be regulated posttranscriptionally. For example,
the 3'-untranslated region of the murine COX-2 message contains
multiple regulatory elements controlling mRNA stability and
translational efficiency (9). Our studies also showed that macrophages and endothelial cells were distinct with respect to the
timing of their responses to HSP60. Thus macrophages responded much
more quickly to induction of COX-2 and NOS-2 than did endothelial cells, which may reflect the different origins and functions of these cells.
Within each cell type, the kinetics of expression of COX-2 and NOS-2
were similar, suggesting that there are common aspects in the signaling
pathways mediating induction of these proteins. HSP60 binds the
toll-like 4 receptor, a transmembrane protein with an IL-1
receptor-like intracellular domain (47). Activation of
this receptor causes it to associate with MyD88 and activate several
receptor-associated kinases, leading to the induction of the MAP kinase
signaling cascades that participate in regulating COX-2 and NOS-2
(25, 27, 42, 63). Binding of HSP60 to the toll-like 4 receptor also activates transcription factors known to be important in
regulating expression of COX-2 and NOS-2, including NF-
B (1,
4, 47). In our studies, transfection of the macrophages with
reporter constructs for cis-acting elements of the 5'-flanking sequence
of COX-2 revealed that NF-
B was required for optimal HSP60-induced
expression of COX-2. Similar results were observed with CRE and two
NF-IL-6 sites. In contrast, mutation of an E-box site in the COX-2
promoter had no effect on HSP60-induced reporter activity. Consistent
with these findings, mobility shift assays showed that HSP60 readily
induced NF-
B and CRE binding activity in the macrophages, while
C/EBP, which binds to NF-IL-6, was constitutively active in the cells.
The activated NF-
B complex was comprised of both the p50 and p65
subunits of this transcription factor while both c-Jun and CREB bound
to the CRE, and C/EBP-
bound to NF-IL-6. These data indicate that
NF-
B, C/EBP-
, c-Jun, and CREB play a role in HSP60-induced
expression of COX-2. C/EBP-
has also been reported to be involved in
the transcriptional activation of COX-2 in activated murine osteoblasts
(67) and mouse skin carcinoma cells (28). In
macrophages, modulation of the activity of C/EBP-
also alters COX-2
reporter activity, further demonstrating the importance of this
transcription factor in regulating COX-2 expression (68).
Constitutive activity of C/EBP-
has been observed in many cell types
(13, 33, 51, 64). Transcriptional activity apparently
requires phosphorylation (33, 51), and it is possible that
HSP60 induces signaling kinases that activate this transcription factor
in macrophages.
At least six enhancer elements have been identified that regulate
maximal induction of NOS-2, including two IFN-stimulated response
elements, a GAS element, two NF-
B elements, and an octamer DNA
sequence motif (OCT) site, the latter binding the basal
transcriptional element octomer (44). Thus, at the present
time, it appears that NF-
B is critical in mediating induction of
both COX-2 and NOS-2 by HSP60, although it is likely that additional
common regulatory elements for these two genes will be identified as
the fine structure of the promoter elements are further characterized
(44, 61). Previous studies using the same reporter
constructs indicated that the CRE and NF-IL-6 sites, but not NF-
B,
are required for optimal activity of the COX-2 promoter after LPS
treatment of RAW 264.7 macrophages (68). In these cells,
inhibition of LPS-induced activation of NF-
B by expression of an
inhibitor
B-
(I
B-
) also had no effect on
LPS-dependent COX-2 reporter activity (64). On the basis
of these data, it appears that HSP60 and LPS induce COX-2 by distinct
mechanisms. That NF-
B is not required for endotoxin-induced activation of COX-2 in RAW 264.7 cells was surprising since both HSP60
and LPS utilize the toll-like 4 receptor (30, 47, 54). Moreover, as indicated above, we found that HSP60 and LPS were equally
effective in inducing NF-
B in RAW 264.7 macrophages, and LPS was
able to induce COX-2. Vabulas et al. (65) also reported that HSP60 activates I
B kinase, causing degradation of I
B-
and
inducing an NF-
B luciferase reporter in RAW 264.7 macrophages. Induction of NF-
B by HSP60 has also been described in human
endothelial cells (29). Moreover, in many cell types,
NF-
B has been reported to mediate COX-2 induction, for example, by
endotoxin in J774 macrophages (12, 66) and differentiated
U937 cells (24) and by TNF-
in MC3T3-E1 cells
(71) and in gastric AGS cells (35). Thus it
appears that there may also be distinct mechanisms for transcriptional
regulation of COX-2 in different cell lines.
Our data demonstrate that HSP60 readily activates the stress-activated
protein kinases JNK and p38 and the MAP kinases ERK1/2 in RAW264.7
macrophages. Specific inhibitors of the p38 kinase or ERK1/2 kinase
inhibited HSP60-induced COX-2 expression, indicating that these enzymes
are required for the process. Kol et al. (30) also
reported activation of the p38 kinase by HSP60 in human peripheral blood mononuclear cells. SB-202190, a selective p38 MAP kinase antagonist, inhibited IL-6 production in response to HSP60
(30). The ERK1/2 kinase inhibitor used in the present
studies also inhibited expression of HSP60-induced NOS-2, suggesting
that HSP60 signaling via these kinases also regulates expression of
this enzyme. In contrast, an inhibitor of the p38 kinase had no effect
on HSP60-induced NOS-2, indicating that there are distinct mechanisms
regulating HSP60-induced expression of COX-2 and NOS-2. Taken together,
these data suggest that p38 kinase mediates multiple functions of
HSP60. Recently, Vabulas et al. (65) demonstrated that
HSP60-induced activation of JNK proceeds via the toll-like 4 receptor
and involves MyD88 and TRAF-6. In addition, the signaling pathway
involved required receptor-mediated endocytosis of HSP60 in a process
inhibited by serum components (65). Endocytosis of the
receptor-ligand complex may be a mechanism for downregulating toll-like
4 receptors and suppressing the innate immune response in the presence
of excess HSP60.
Several laboratories have suggested that NOS-2 activity is essential
for cytokine and LPS induction of COX-2 or vice versa, although many of
these studies are conflicting (23, 38, 50, 56). For
example, NOS inhibitors have been found to suppress PG production in
LPS-treated rats (56). In human fetal fibroblasts, nitric
oxide treatment enhances IL-1-induced PG production (57), while peritoneal macrophages from NOS-2 knockout mice produce significantly less PGE2 when challenged with LPS and
IFN-
(38). In contrast, nitric oxide has been reported
to inhibit COX-2 expression and PGE2 release in
LPS-stimulated mouse macrophages (8), whereas inhibition
of NOS has been reported to enhance COX-2 protein expression and PG
production in rat peritoneal macrophages (18). These data
suggest that COX-2 activity can be regulated by nitric oxide, but the
specific biological response may depend on the animal and/or tissue
model used. In RAW264.7 macrophages, we found that HSP60-induced
expression of COX-2 was unaltered by an inhibitor of NOS-2. Similarly,
an inhibitor of COX-2 did not affect HSP60-induced expression of NOS-2.
These data indicate that, after HSP60 treatment, COX-2-generated
prostanoids are not required for expression of NOS-2. Similarly, nitric
oxide induced by HSP60 does not appear to regulate expression of COX-2.
It should be noted that COX-2 and NOS-2 are not always coordinately
expressed. For example, we found that IFN-
can selectively induce
NOS-2, but not COX-2, in the macrophages. IFN-
, but not HSP60, also
induces NOS-2 in PAM 212 keratinocytes (unpublished observations).
These findings are presumably due to the activation of transcription
factors by IFN-
, but not HSP60, that are important in regulating
expression of this enzyme in these two cell types. This idea is
supported by our findings that IFN-
was effective in inducing
phosphorylation, nuclear translocation, and DNA binding of STAT-1 in
macrophages while HSP60 was inactive. IFN-
was also found to enhance
HSP60-induced expression of NOS-2 and nitric oxide production without
altering COX-2. Presumably, transcription factors induced by IFN-
can synergize with those induced by HSP60 for maximal expression of
NOS-2 (29, 30, 47). This synergism may be due to
simultaneous activation of the NF-
B and Janus kinase (JAK)-STAT pathways, proteins which may physically interact to augment NOS-2 transcription (44, 58).
In summary, we have demonstrated that HSP60 is an effective inducer of
COX-2 and NOS-2 in macrophages and endothelial cells, providing
additional evidence that this protein has the capacity to function as a
proinflammatory mediator. On the basis of our data, we propose a model
for the action of HSP60 (Fig. 9).
According to this model, HSP60 released during injury and inflammation
binds to receptors on cells in surrounding tissues (19, 30, 36, 47, 54, 65). These receptors include those associated with the
actions of endotoxin as well as
3
1 integrin (3).
This in turn causes activation of the transcription factor NF-
B.
HSP60 also activates p38, ERK1/2, and JNK MAP kinases. This results in
activation of additional transcription factors important in the
regulation of COX-2 and NOS-2, including CREB, C/EBP, and c-Jun. It is
likely that additional transcription factors regulating expression of
COX-2 and NOS-2 are also activated by HSP60. Expression of COX-2
requires the ERK1/2 kinase and p38 kinase. Two lines of evidence
suggest that HSP60-induced signaling leading to expression of COX-2 and
NOS-2 occurs via distinct mechanisms. First, as indicated above,
IFN-
synergizes with HSP60 to induce NOS-2, but not COX-2; second,
p38 kinase activity appears to differentially regulate COX-2 and NOS-2.
Presumably, these differences are due to the different regulatory
elements that need to be activated to control COX-2 and NOS-2 gene
expression. Synthesis of COX-2 and NOS-2 leads to production of
prostanoids and nitric oxide, respectively, which contribute to and
amplify the inflammatory process. Further studies that more precisely
define the signaling pathways induced by HSP60 in macrophages are
needed to better elucidate mechanisms regulating expression of COX-2
and NOS-2 in tissues.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institutes of Health
Grants ES-06897, ES-05022, ES-03647, GM-34310, and ES-04738.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J. D. Laskin, Dept. of Environmental & Community Medicine,
UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ
08854 (E-mail:
jlaskin{at}eohsi.rutgers.edu).
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.
22 May 2002;10.1152/ajpcell.00609.2001
Received 20 December 2001; accepted in final form 15 May 2002.
 |
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