1 Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring 20910-7500; and Departments of 2 Medicine and 3 Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799
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ABSTRACT |
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The nitric oxide (NO) synthase inhibitor
N-nitro-L-arginine
(L-NNA) inhibits heat stress (HS)-induced NO production and
the inducible 70-kDa heat shock protein (HSP-70i) in many rodent
organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i
expression. Intracellular Ca2+ concentration
([Ca2+]i) was measured using fura-2, and
protein kinase C (PKC), and PKA activities were determined. HS
increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for
10 min and allowed to recover for 6 h. L-NNA treatment
for 1 h before HS inhibited the induction of HSP-70i mRNA and
protein, with an IC50 of 0.0471 ± 0.0007 µM.
Because the HS-induced increase in HSP-70i mRNA and protein is
Ca2+ dependent, we measured
[Ca2+]i after treating cells with
L-NNA. L-NNA at 100 µM significantly decreased resting [Ca2+]i. Likewise,
treatment with 1 µM GF-109203X or H-89 (inhibitors of PKC and PKA,
respectively) for 30 min also significantly decreased [Ca2+]i and inhibited HS-induced increase in
HSP-70i. GF-109203X- or H-89-treated cells failed to respond to
L-NNA by further decreasing [Ca2+]i and HSP-70i. L-NNA
effectively blocked heat shock factor-1 (HSF1) translocation from the
cytosol to the nucleus, a process requiring PKC phosphorylation. These
results suggest that L-NNA inhibits HSP-70i by reducing
[Ca2+]i and decreasing PKC and PKA activity,
thereby blocking HSF1 translocation from the cytosol to the nucleus.
heat; nitric oxide; heat shock protein; protein kinase C; protein kinase A
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INTRODUCTION |
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NITRIC OXIDE (NO) is produced by constitutive and inducible NO synthase (cNOS and iNOS, respectively) in many cell types, including endothelial cells, neurons, and macrophages (34). NO is involved in a wide range of physiological processes, including blood vessel relaxation (14), neurotransmitter transduction (26), memory functions (31), and host defense against microbial pathogens (28). Increased levels of NO (16, 41, 43) and NOS activity (1, 16, 30, 43, 47) have been measured during hemorrhagic shock. Inhibition of NO production reduces the damage caused by hemorrhage (2, 16, 54). It appears that activation of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through cNOS activation to alleviate the damaged brain area (for review, see Ref. 3). A number of in vitro studies (11, 13, 48, 49) have demonstrated that heat stress inhibits iNOS gene expression.
Interactions between NO and heat shock protein (HSP) production have been studied previously. Reports have demonstrated that increases in NO production result in elevation of HSP-70 expression in smooth muscular cells (50), Hep G2 cells (32), and hepatocytes (25). In smooth muscle cells, NO-induced HSP-70 expression is mediated by heat shock factor-1 (HSF1) activation (50). HSPs are overexpressed under stress and display cytoprotection against various insults in many different types of organs and cells (23, 24). Our laboratory has found that inhibition of NO production by a NOS inhibitor can protect rat small intestine from injury caused by ischemia-reperfusion and that treatment with NOS inhibitors attenuates HSP-70 expression induced by heat stress. However, the underlying protective mechanism has not been studied.
The mechanism underlying the cytoresistance provided by HSPs is not fully clear. Evidence indicates that overexpression of inducible HSP 70 kDa (HSP-70i; a member of the HSP 70-kDa family) diminishes the intracellular Ca2+ response to stress or toxins (23, 24), downregulates the basal enzymatic activities of protein kinase A (PKA), PKC (7), c-Jun amino-terminal kinase, and p38 kinase (9), upregulates the basal enzymatic activities of protein phosphatase-1 and -2A (7, 29), and enhances the immunogenicity of tumor cells (33). These observations suggest that diminished signal transduction may mediate HSP-70-induced cytoprotection. NOS inhibitors may interrupt the signal pathway so that the cytoprotection is diminished.
Studies of human epidermoid A-431 cells (5, 18), Madin-Darby canine kidney cells (51), rat luteal cells (17), human breast cancer T-47D cells (20), MCF7 cells (21), and MDA-MB-231 cells (22) have indicated that HSP-70 is regulated by intracellular Ca2+ concentration ([Ca2+]i). Only cNOS is Ca2+ dependent (8). However, the interaction between NO generation and [Ca2+]i has not been investigated.
In this study, we found that heat stress increased HSP-70i expression in human colon carcinoma T84 cells. Our experiments provide a link between NOS, PKC, and PKA activities, placing NOS activity upstream of PKC and PKA activity. Blockade of NOS eventually causes decreased HSP-70i expression by inhibiting HSF1 translocation from the cytosol to the nucleus.
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METHODS |
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Cell culture. Human colon carcinoma T84 cells (American Type Culture Collection, Rockville, MD) were grown in 75-cm2 tissue culture flasks (Costar, Cambridge, MA) containing DMEM supplemented with 0.03% glutamine, 4.5 g/l glucose, 25 mM HEPES, 10% fetal bovine serum, penicillin (50 µg/ml), and streptomycin (50 U/ml; GIBCO-BRL, Gaithersburg, MD) and incubated in a 5% CO2 atmosphere at 37°C. Cells were fed every 3-4 days.
RNA extraction. T84 cells (5 × 106/sample) were combined with 0.8 ml of TRIzol reagent (GIBCO-BRL), sonicated, and then combined with 0.2 ml chloroform. The mixture was vortexed and centrifuged at 12,000 g for 15 min at 4°C. The clear aqueous liquid containing RNA (450 µl) was transferred to a new Eppendorf tube, and isopropanol (225 µl) was added, vortexed, and centrifuged at 12,000 g for 15 min at 4°C. The RNA pellets were collected and washed with 75% ethanol. The quantity of extracted RNA dissolved in water was determined spectrophotomically.
RT-PCR.
The reaction mixture was 1× reverse transcription buffer containing 1 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 U RNasin, 0.5 µg
oligo(dT)15 primer, 1 µg total RNA, and 15 U avian
myeloblastosis virus RT (Promega, Madison, WI) in a final volume of 20 µl. The mixture was incubated at 37°C for 10 min and then at 42°C
for 20 min. The transcription reaction was terminated by heating the mixture at 95°C in a water bath for 10 min before chilling on ice.
The primers specific for HSF1, HSP-70, and -actin were as follows:
HSF1, 5'-CTG GCC ATG AAG CAT GAG AA-3' and 5'-GAG AAC TGC CGG CTA TAC
TA-3'; HSP-70, 5'-AAG GTG GAG ATC ATC GCC AA-3' and 5'-GCG ATC TCC TTC
ATC TTG GT-3'; and
-actin, 5'-ATG GAT GAT GAT ATC GCC GC-3' and
5'-AGG AAT CCT TCT GAC CCA TG-3'. Each length of the single-stranded
primer was 20 bp and had a ratio of 1:1 for guanine-to-cytosine and
adenine-to-thymine to maximize specificity.
Western blots. To investigate the synthesis of HSPs, we incubated cells in medium at different temperatures for different periods of time, returning them to 37°C for different periods of time. Cells were then removed from the culture plate with trypsin-EDTA (GIBCO-BRL) and pelleted by centrifugation at 750 g for 10 min. The pellet was lysed in Tris buffer (pH 6.8) containing 1% SDS and 1% 2-mercaptoethanol. Aliquots containing 20 µg protein were resolved on SDS-polyacrylamide slab gels (Novex precasted 10% gel, San Diego, CA). Protein was blotted onto a nitrocellulose membrane (type NC, 0.45 µm, Schleicher & Schuell), using a Novex blotting apparatus according to the manufacturer's suggested protocol. After the nitrocellulose was blocked by incubation for 90 min at room temperature in PBS containing 5% nonfat dried milk, the blot was incubated for 60 min at room temperature with mouse polyclonal antibody against cNOS, HSP-72, and HSF1 (Stressgen, Calgary, AB, Canada) at a 500× dilution in PBS-5% BSA containing 0.1% thimerosal. The blot was then washed three times (for 10 min each) in PBS-0.1% Tween 20 before being incubated for 60 min at room temperature with a 1,000× dilution of rabbit anti-mouse IgG peroxidase conjugate (Amersham Life Science, Arlington Heights, IL) in PBS-1% gelatin. The blot was washed six times (for 5 min each) in PBS-0.1% Tween 20 before detection of the peroxidase activity using enhanced chemiluminenscence (Amersham).
NO measurements. NO production indicated as nitrite was measured using the Griess reagent system with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride under acidic conditions (Promega).
[Ca2+]i measurements. Suspended cells were loaded with 5 µM fura 2-AM plus 0.2% Pluronic F-127 at 37°C for 60 min. Cells were then washed with Na+ Hanks' solution before fluorescence measurements. The method used to determine [Ca2+]i was as described previously (18).
Activity assays of PKC and PKA.
The samples were extracted from both cytosol and membrane-bound
fractions according to the manufacturer's suggested protocol (Life
Technologies, Gaithersburg, MD). The cells were homogenized in
extraction buffer without detergent and centrifuged at 100,000 g for 30 min. The supernatant contained the cytosolic
enzyme, and the pellet contained the membrane-bound enzyme. The amount of protein in the samples was measured with Bio-Rad protein assay dye
(Bio-Rad, Richmond, CA). The activities of PKC and PKA were measured using commercial assay kits (Life Technologies). The PKA
activity was measured by phosphorylation of a specific substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly, and the enzymatic activity of PKC was
measured by phosphorylation of
Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, as described by Yasuda
et al. (53). [-32P]ATP (6,000 Ci/mmol stock solution) was used in this study. The substrate for PKA
and PKC was incubated with protein samples at 30°C for 5 min and then
transferred to the phosphocellulose disc. The disc was washed twice for
3-5 min with phosphoric acid solution and dried. The radioactivity
was measured with an automatic liquid scintillation counter. The
activities of enzymes were calculated according to the manufacturer's
suggested protocol and expressed as the percentage of untreated cells.
Statistical analysis. All data are expressed as means ± SE. One-way ANOVA, two-way ANOVA, Studentized-range test, Bonferroni's inequality test, and Student's t-test were used for comparison of groups with P < 0.05 considered significant.
Chemicals.
Chemicals used in this study were albumin,
N-nitro-L-arginine
(L-NNA), aminoguanidine, L-arginine,
N
-nitro-L-arginine methyl ester
(L-NAME),
L-N6-(1-iminoethyl)lysine
(L-NIL),
S-nitroso-N-acetylpenicillamine (SNAP; Sigma),
H-89, GF-109203X (Calbiochem, La Jolla, CA),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM
(BAPTA-AM), fura 2-AM, and Pluronic F-127 (Molecular Probes, Eugene, OR).
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RESULTS |
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L-NNA inhibits heat stress-induced increases in HSP-70i. Previously, we (18, 20-22) have established that human carcinoma T84 cells behave similarly to many other types of cells in producing HSP-70i in response to heat stress (38). Therefore, the following experiments were conducted with T84 cells exposed to 45°C for 10 min followed by recovery at 37°C for 6 h to induce optimal levels of HSP-70i.
In preliminary experiments, we found that intravenous administration of L-NNA but not aminoguanidine (an inhibitor of iNOS) to rats 1 h before exposure to heat stress effectively inhibited HSP-70i expression in rat small intestine (unpublished data). Hence, we treated T84 cells with L-NNA at different concentrations prior to heat stress. Figure 1 shows that L-NNA inhibited the heat stress-induced increase in HSP-70i production in a concentration-dependent manner with an IC50 of 0.0471 ± 0.0007 µM. Similar to the results observed with the in vivo studies, aminoguanidine did not alter the heat stress-induced increase in HSP-70i production in T84 cells (data not shown). These results suggest that L-NNA regulates HSP-70i expression.
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L-NNA inhibits increase in HSP-70i at mRNA level. It has been shown (23, 24) that the heat stress-induced HSP-70i increase in other cell types occurs at the mRNA level. To determine whether the increase in HSP-70i in T84 cells exposed to heat stress occurred at the mRNA or translational level, RNA was collected at different time points after cells were exposed to heat stress at 45°C for 10 min. Using RT-PCR, we observed that the HSP-70i mRNA levels started increasing within 5 min after exposure to heat stress, reached the maximum at 2 h, and remained above the baseline 8 h later (data not shown).
Treatment with actinomycin D (an inhibitor for transcription) or cycloheximide (an inhibitor for translation) but not genistein (an inhibitor for protein tyrosine kinase) 10 min before heat stress effectively blocked heat stress-induced HSP-70i production (Fig. 4A). Using RT-PCR, we found that actinomycin D but not cycloheximide inhibited increases in HSP-70 mRNA caused by heat stress (Fig. 4B), supporting the concept that the heat stress-induced HSP-70i increase occurs at the mRNA level. Treatment with 100 µM L-NNA 1 h before heat stress significantly inhibited HSP-70 mRNA, indicating that L-NNA inhibits HSP-70i at the mRNA level (Fig. 4C).
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L-NNA decreases resting
[Ca2+]i.
Our laboratory (5, 6, 18) reported previously that the
heat stress-induced increase in HSP-72 in human A-431 cells was
regulated by [Ca2+]i. We aimed to determine
whether the heat stress-induced increase in HSP-70i production in T84
cells was also Ca2+ dependent. Cells treated with 10 mM
EGTA 30 min before exposure to heat stress exhibited greatly diminished
levels of HSP-70i production, whereas cells treated with 100 µM BAPTA
displayed slightly attenuated HSP-70i expression. Treatment of cells
with both EGTA and BAPTA caused an additive reduction of HSP-70i
expression (Fig. 5).
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L-NNA inhibits heat stress-induced translocation of
HSF1 from cytosol to nucleus.
HSP-70i expression is mediated by the phosphorylation, trimerization,
and translocation of HSF1, which normally resides in the cytosol
(24), from the cytosol to the nucleus. To determine whether L-NNA inhibition of heat stress-induced HSP-70i
overexpression is preceded by inhibition of HSF1, we separated
cytosolic extracts from nuclear extracts from cells treated with 100 µM L-NNA 1 h before heat stress. We found that heat
stress increased total HSF1 protein to 220 ± 18% and mRNA to
195 ± 5% (n = 3). L-NNA treatment
blocked this increase (Fig. 6,
A and B). Heat stress also elevated cytosolic
HSF1 translocation to the nucleus, whereas treatment with
L-NNA blocked this translocation (Fig. 6, C and D). These results suggest that L-NNA inhibits
heat stress-induced increase in HSP-72 by inhibiting HSF1 synthesis and
translocation.
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L-NNA inhibits PKC and PKA activities.
Our laboratory (20-22) has reported previously that
PKC and PKA regulate HSP production in human breast cancer cells.
Because PKC and PKA are known to be involved in HSP synthesis, we
studied the interactions between HSP-70i, L-NNA, PKC, and
PKA. We found that cells treated with GF-109203X (a PKC inhibitor) or
H-89 (a PKA inhibitor) failed to increase HSP-70i levels in response to heat stress. L-NNA also failed to further inhibit the
expression of HSP-70i in cells pretreated with GF-109203X or H-89 (Fig.
7).
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DISCUSSION |
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In this study, we observed that exposure of human intestinal epithelial T84 cells to heat stress increases HSP-70i levels in a time- and temperature-dependent manner. Like other cells, the increase occurs at the mRNA level and is regulated by [Ca2+]i (5, 6, 18). Furthermore, we also found that the NOS inhibitor L-NNA blocked HSP-70i production at the mRNA level. The IC50 of L-NNA was 0.0471 ± 0.0007 µM, which is similar to that reported (27) in the nervous system, suggesting that L-NNA exerts its action on cNOS. Data from the experiments in which L-NAME and L-NIL were used further reinforce the view that cNOS regulates HSP-70i expression. In addition, the results from the experiments involving treatment with NO donors strongly suggest that NO production is involved in HSP-70i expression.
L-NNA downregulates HSP-70i overexpression, which has been associated with protection against ischemia-reperfusion injury (3, 40). It is known (18, 35, 36) that the increase in [Ca2+]i promotes the phosphorylation of cytosolic HSF1 and its translocation to the nucleus followed by its binding to heat shock elements located on the promoter region of the HSP-70 gene. These results are in agreement with findings (5) in human epidermoid A-431 cells. Therefore, the ability of L-NNA to block HSP-70i expression induced by heat stress is probably mainly due to its ability to lower [Ca2+]i. Data obtained from experiments showing that L-NNA failed to additionally reduce [Ca2+]i and HSP-70i expression in cells pretreated with inhibitors of PKC or PKA further reinforce this view. Moreover, the inhibitory effects of L-NNA on the enzymatic activities of PKC and PKA are highly likely to be the result of its ability to decrease [Ca2+]i since these two enzymes are known to be Ca2+ dependent.
On the basis of data from the current study and findings reported
previously (18), we propose a model to explain the
mechanisms involved in L-NNA-induced inhibition of HSP-70i
expression (Fig. 10). At
first, heat stress promotes Ca2+ entry via the reversed
mode of the Na+/Ca2+ exchanger located at the
cell membrane (18). The increases in
[Ca2+]i stimulate the
Ca2+-dependent cNOS to generate more NO as well as other
Ca2+-dependent enzymes such as PKC and PKA. The generation
of NO is also promoted by activation of nuclear factor-B (NF-
B),
which is initiated by increases in free radicals due to heat stress. Activation of PKC and PKA phosphorylates HSF1 in the cytosol, which
afterward trimerizes, enters the nucleus, and initiates the process of
transcription and translation. L-NNA alone decreases [Ca2+]i. This decrease is thought to
attenuate the enzymatic activities of cNOS, PKC, and PKA, which leads
to diminished HSF1 phosphorylation, trimerization, and translocation,
resulting in a blockade of HSP-70i production.
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This is the first published study showing that treatment of cells with
L-NNA attenuates the heat stress-induced increase in HSF1
expression. This attenuation also represents a reduction of NO
production because L-NNA but not L-NIL inhibits
cNOS. Currently, it is not clear how NO interacts with HSF1 synthesis.
It is documented that heat stress (5) and treatment of
cells with a PKC stimulator (5) cause increased HSF1
expression, which is found here in T84 cells as well. Because NO is
required to regulate the SP1 binding site of the tumor necrosis
factor- (TNF-
) promoter in U-937 cells treated with TNF-
(45), it is likely that NO can also regulate the SP1
binding site on the promoter region of the HSF1 gene. The inhibitory
effect of L-NNA on cNOS results in decreased NO production,
therefore leading to downregulation of the HSF1 gene.
It has been reported (12) that enhanced NO production
contributes to the production of proinflammatory molecules such as the
cytokines interleukin-6, granulocyte colony-stimulating factor, NF-B, and signal transducer and activator of transcription 3 in rat
liver and lungs. In the ischemia-reperfusion-induced tissue injury model, NO displays a dual function. In local tissues, NO appears
to combine with superoxide to form peroxynitrite (ONOO
),
a powerful oxidant that can be cleaved into free radicals such as
OH
and NO
It has been reported (4, 10, 37, 38, 44) that tumor cells exposed to sublethal heat shock before chemotherapy are less responsive to treatment. Under such conditions, the presence of HSPs is not desired in cancer patients, and remedies that enable the reduction of HSP production are targeted. Additionally, some HSPs are known to act as molecular chaperones in the cell. The understanding of this link between NOS, PKA, PKC, and HSPs may offer insight into the ability of NO to regulate the activities of molecular chaperones in the cell. HSPs are expressed in many infectious diseases and autoimmune diseases and act as the immunodominant antigen (15, 24). Therefore, the ability of L-NNA, GF-109203X, and H-89 to downregulate HSP-70i may be of therapeutic use for treating patients with cancer, infectious diseases, or autoimmune diseases.
In conclusion, we have shown that cNOS is involved in the induced expression of HSP-70i by affecting the required transcription factor HSF1. cNOS is involved in the regulation of [Ca2+]i and therefore in regulating the activities of PKC and PKA, which are also involved in regulating the activities of HSF1 and HSP-70i.
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ACKNOWLEDGEMENTS |
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We thank R. L. Collins for making the graphs.
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FOOTNOTES |
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This work was supported by Department of Defense Research Area Management II Science Technology Objective C and R. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense.
Address for reprint requests and other correspondence: J. G. Kiang, Dept. of Cellular Injury, Walter Reed Army Institute of Research, 503 Robert Glen Ave., Silver Spring, MD 20910-7500 (E-mail: Juliann.Kiang{at}na.amedd.army.mil).
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.
10.1152/ajpgi.00138.2001
Received 29 March 2001; accepted in final form 8 November 2001.
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