(Received for publication, April 22, 1996, and in revised form, August 28, 1996)
From the Departments of Surgery,
§ Pharmacology, and
Cell Biology and Physiology,
School of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
Nitric oxide (NO) and tumor necrosis factor-
(TNF
) play important roles in the pathogenesis of liver disease
during acute inflammation. The present study was designed to elucidate
the effect of NO pre-exposure on TNF
-induced hepatotoxicity.
Pretreatment of primary cultures of rat hepatocytes with the NO donor
S-nitroso-N-acetylpenicillamine (SNAP) induced
the expression of heat shock protein 70 (HSP70) mRNA and protein,
which was associated with thermotolerance and cytoprotection from
TNF
+actinomycin D-induced hepatotoxicity and apoptosis. SNAP
transiently changed the intracellular redox state by inducing
glutathione (GSH) oxidation associated with the formation of
S-nitrosoglutathione (GSNO). HSP70 mRNA was also induced by the GSH-oxidizing agent diamide and the GSH-conjugating agent N-ethylmaleimide, suggesting that NO induces HSP70
expression through GSH oxidation.
The protective effect of SNAP pretreatment on TNF-induced apoptosis
correlated with the level of HSP70 expression. SNAP pretreatment inhibited reactive oxygen intermediate generation and lipid
peroxidation effects that were reversed by blocking HSP70 expression
using an antisense oligonucleotide to HSP70. Finally, endogenous NO formation, induced in hepatocytes stimulated with interferon-
and
interleukin-1
, led to the formation of GSNO and GSSG, induced HSP70,
and attenuated TNF
-mediated cytotoxicity. These findings demonstrated that NO can induce resistance to TNF
-induced
hepatotoxicity, possibly through the stimulation of HSP70
expression.
Many cell types have the capacity to generate NO from L-arginine. However, the level of NO production and the functional role of NO can vary between cell types. Two constitutive calcium-dependent NO synthases (cNOS)1: neuronal cNOS (nNOS or NOS1) and the endothelial cNOS (eNOS or NOS3), generate small quantities of NO sufficient only for cellular signaling under most circumstances. However, some cells that are exquisitely sensitive to NO, such as neurons, have been shown to exhibit toxicity in response to NOS1 activation (1). A third isoform, NOS2, typically expressed after exposure of cells to inflammatory stimuli (e.g. cytokines and microbial products), originally was referred to as the inducible NOS (iNOS). More recently, it has been recognized that NOS2 is expressed in some resting epithelial cells (2). NO production by NOS2 occurs independent of elevations in basal intracellular calcium concentration (3), and the quantities produced are sufficient to damage or kill susceptible cells (4) or microorganisms (5).
The precise factors that determine cellular sensitivity to NO-mediated
toxicity are not clear; however, a number of molecular targets for NO
and its reaction products have been identified. It is through the
interaction with these targets, typically sulfhydryl-containing molecules (6, 7) or redox metal-containing proteins (8), that NO
affects its biological action. The quantities of NO generated by cNOS
isoforms are adequate to activate soluble guanylate cyclase by
dislocation of the heme iron within the enzyme (9). Other signaling
actions of NO mediated via redox-sensitive sites include inhibition of
protein kinase C, activation of tyrosine kinase, inactivation of
NF-B, activation of SoxRS, and activation of G proteins (reviewed in
Ref. 10). NO also causes several metabolic alterations by inhibiting
the actions of certain thiol- and iron-containing enzymes and thus
inhibits mitochondrial respiration, the tricarboxylic acid cycle, DNA
synthesis, and antioxidant and DNA repair enzymes. NO and superoxide
react together at a diffusion-controlled rate to yield peroxynitrite
(ONOO
), which inflicts cellular injury through oxidation
of many biological molecules. Furthermore, ONOO
has also
been implicated in the inactivation of Mn and Fe superoxide dismutase
(11) and aconitase (12, 13). In contrast, NO may protect cells from
reactive oxygen intermediate (ROI)-mediated cytotoxicity by scavenging
superoxide anions which are implicated in toxicity through the
formation of hydrogen peroxide or hydroxyl radical via the Fenton
reaction (14). NO also reduces toxic ferryl species to ferrous ion,
thereby blocking the hemoprotein-mediated Fenton-like reaction (15).
Furthermore, NO has been shown to terminate the propagation of
radical-mediated lipid peroxidation (16).
Hepatocytes express NOS2 and produce large amounts of NO in response to
the synergistic combination of cytokines such as TNF, IL-1
, and
IFN
(17) or IL-1
alone in sufficient concentrations (18). This NO
synthesis has been associated with DNA condensation, inhibition of
protein synthesis, and decreased levels of cytochrome P450 and catalase
activity in vitro (19). Despite these changes, hepatocytes
are rather resistant to NO toxicity. Exposure to NO also protects
primary cultured hepatocytes from the cytotoxic effects of higher doses
of NO and H2O2 (20). This protective action
appears to be mediated by the induction of cytoprotective stress
proteins such as heme oxygenase, which, in turn, results from
NO-induced alteration in iron homeostasis.
In vivo, large quantities of NO can be generated in the
liver in acute inflammation (21, 22). We have suggested that this NO
can have protective actions. TNF is also produced in the liver in
inflammation (23), most likely by the resident macrophages. Studies
into the mechanism of hepatic TNF
toxicity have shown that TNF
in
the presence of RNA or protein synthesis inhibitors induces DNA
fragmentation characteristic of apoptosis (24). Furthermore, TNF
is
thought to mediate liver injury in acute inflammation (25) and
contribute to fulminant hepatic failure (26, 27). In other cell types,
NO has been shown to either promote (28, 29) or inhibit (30, 31)
apoptosis. Both NO and TNF
would be expected to be present in the
liver under many inflammatory liver conditions; however, it is unknown
if NO exposure potentiates or prevents TNF
toxicity in hepatocytes.
In this study, we examined the consequences of NO exposure on
TNF
-induced apoptosis in cultured rat hepatocytes. We report that NO
exposure prevents subsequent TNF
-induced cell death through the
induction of heat shock protein 70 (HSP70).
Williams medium E, penicillin, streptomycin,
L-glutamine, and HEPES were purchased from Life
Technologies, Inc. Insulin was purchased from Lilly, and calf serum was
obtained from HyClone Laboratories (Logan, UT). Murine macrophage NOS2
monoclonal antibody was obtained from Transduction Laboratories
(Lexington, KY). TNF was purchased from Genzyme (Cambridge, MA);
LipofectAMINE was purchased from R&D Systems. Anti-HSP70 monoclonal
antibodies were obtained from Sigma or Stress Gen
(Victoria, BC, Canada). Dichlorofluorescein diacetate (DCF-DA) was
purchased from Molecular Probes (Eugene, OR), and protein assay reagent
was purchased from Pierce. 125I-Labeled human TNF
was
purchased from Amersham; NF-
B-specific oligonucleotides and T4
polynucleotide kinase were obtained from Stratagene (La Jolla, CA) and
Boehringer Mannheim, respectively. S-Nitroso-N-acetylpenicillamine (SNAP) was
synthesized every 2 months as described previously (32), stored frozen
as a solid aliquot in the dark, and checked for stoichiometric
S-nitrosothiol content by the method of Saville (33).
NG-Monomethyl-L-arginine
(NMA) was purchased from Cyclopss (Salt Lake City, UT). Luciferase
assay kits and lysis buffer were purchased from Promega (Madison, WI).
HSP70 antisense oligomer (TGTTTTCTTGGCCAT), HSP70 sense oligomer
(ATGGCCAAGAAAACA), HSC70 antisense oligomer (AGGTCCCTTAGACAT), and
HSC70 sense oligomer (ATGTCTAAGGGACCT) were synthesized from sequences
complementary to the initiation codon and four downstream codons of rat
HSP70 mRNA (34) and rat HSC70 mRNA (35). All other chemicals
and proteins were purchased from Sigma, unless
indicated otherwise.
Purified hepatocytes were
isolated from male Sprague-Dawley rats (200-300 g, Harlan
Sprague-Dawley) by collagenase perfusion using the method of Seglen
(36). Hepatocytes were purified to >98% purity by repeated
centrifugation at 50 g, followed by further purification over 30%
Percoll. Viability at time of plating was consistently 90-95% by
trypan blue exclusion. Hepatocytes were cultured in Williams medium E
supplemented with 1 µM insulin, 2 mM
L-glutamine, 15 mM HEPES, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 10% low endotoxin calf serum
in 100-mm Petri dishes (5 ml/dish) at a concentration of 1 × 106 cells/ml for 16 h, which represents near-confluent
culture conditions. Some cells were pretreated with SNAP (as described
in the figure legends) or with a cytokine mixture (CM: 200 units/ml
IFN and 200 units/ml IL-1
) with or without 1.5 mM NMA
in supplemented Williams medium E containing 5% calf serum. The cells
were then incubated with 28 ng/ml TNF
and 325 nM
actinomycin D (ActD) for 9 h at various intervals after the SNAP
or CM addition, as indicated under "Results." To examine the effect
of antisense oligonucleotides against HSP70, hepatocytes were
preincubated with HSP70 sense or antisense oligomers (10 µM) for 4 h and then treated with 750 µM SNAP for 16 h. The cells were washed twice with
fresh medium and then exposed to TNF
and ActD in medium containing
oligonucleotides. Cell viability was determined by crystal violet
staining, as described (20).
Cultured
hepatocytes were washed with PBS, harvested using a plastic scraper,
and pelleted by centrifugation at maximum speed in a microcentrifuge
for 10 s at 4 °C. Cytosolic DNA was prepared by method of Leist
et al. (27). Briefly, the pellets were resuspended in 750 µl of lysis buffer (20 mM Tris-HCl, 10 mM
EDTA, 0.5% Triton X-100, pH 8.0) and occasionally shaken while on ice
for 45 min. The cytosolic fraction was collected by centrifugation at
13,000 × g for 20 min at 4 °C and protein
concentrations determined. Cytosol aliquots containing equal amounts of
protein were extracted with mixture of phenol and chloroform. One-tenth
volume of 3 M sodium acetate was added to the solution, and
DNA was precipitated by adding an equal volume of isopropanol. After
storing at 20 °C overnight, a DNA pellet was obtained by
centrifugation at 13,000 × g for 15 min at 4 °C and
washed twice with 75% ethanol. The pellet was dried and resuspended in
100 µl of 20 mM Tris-HCl, pH 8.0. After digesting RNA
with RNase (0.1 mg/ml) at 37 °C for 1 h, samples (15 µl) were
electrophoresed through a 1.2% agarose gel in 450 mM Tris
borate + EDTA (TBE), pH 8.0 buffer. DNA was photographed under
visualization with UV light.
Total RNA was isolated from the cultured hepatocytes as described previously (17). The RNA (20 µg) was electrophoresed on 1% agarose gel containing 1% formaldehyde, transferred to GeneScreen, hybridized with human HSP70 [32P]cDNA and mouse macrophage NOS2 [32P] cDNA, and exposed to autoradiography film. Relative mRNA levels were quantitated by PhosphorImager.
Western Blot AnalysisWestern blot analysis was performed using a method modified in our laboratories as described previously (37). Briefly, harvested hepatocytes (5 × 106 cells) were lysed in 100 µl of 20 mM Tris-HCl buffer, pH 7.4, containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 1 µg/ml cymostatin) by three freeze-thaw cycles, and the cytosolic fraction was obtained by centrifugation at 13,000 × g for 20 min at 4 °C. Samples (40 µg of protein) were separated on 8% SDS-polyacrylamide gels, and gels were transferred to nitrocellulose membrane. The membranes were blotted by 5% milk in PBS, pH 7.5, Tween (0.01%) for 1 h at room temperature and then hybridized with one of two different monoclonal anti-HSP70 antibodies (1:1000 dilution). After three washes with PBS-Tween, the blots were hybridized with goat anti-mouse IgG (1:1000 dilution) linked to horseradish peroxidase. Membranes were developed with chemiluminescence reagents (DuPont NEN) and exposed to Kodak X-Omat film for 2-10 min.
Enzyme Activity AssaysHepatocytes were collected from
Petri dishes, resuspended in PBS containing protease inhibitors, and
sonicated (Sonic & Materials, Danbury, CT) with three 15-s bursts while
on ice. The solution was centrifuged at maximum speed for 15 min at
4 °C in the microcentrifuge and the supernatant used for enzyme
activity assays. Antioxidant enzyme activities were measured as
described by Wheeler et al (38). Catalase activity was
spectrophotometrically determined by measuring decreased absorbance at
240 nm using hydrogen peroxide as a substrate. The activity was
calculated from molecular extinction coefficient of 43.6 M1 cm
1 for
H2O2. GSH peroxidase activity was determined in
the presence of GSH reductase by the decrease in NADPH concentration.
Superoxide dismutase activity was measured spectrophotometrically by
monitoring the inhibition of the reduction of ferricytochrome
c at 550 nm in the xanthine oxidase and hypoxanthine
system.
Intracellular ROI
were measured following formation of a fluorescent derivative of DCF-DA
(39). Briefly, hepatocytes were incubated with 100 µM
DCF-DA (in ethanol) with or without TNF+ActD or the equivalent
amount of ethanol for 80 min in a 5% CO2 incubator at
37 °C. After incubation, the cells were washed with PBS, harvested, and immediately used to determine the level of fluorescence in a
spectrophotofluorimeter (excitation, 488 nm; emission, 520 nm). Cell
numbers were determined in parallel, and fluorescent values were
normalized to the number of cells in each sample. Lipid peroxidation was assayed by measuring thiobarbituric acid reactive substances (TBARS) at 535 nm (40). Butylated hydroxytoluene (0.04%) was added to
the thiobarbituric acid solution to prevent lipid autooxidation during
the assay procedure.
Hepatocytes (2 × 107 cells) were resuspended in 400 µl of 20 mM Tris-HCl buffer, pH 7.4, containing 50 µg/ml lysophosphatidylcholine and protease inhibitors. The cytosolic fraction was obtained by microcentrifugation at maximum speed at 4 C for 10 min after three cycles of freeze-thaw. Cytosol (150 µl) was mixed with 7.5 µl of 100% trichloroacetic acid and incubated in ice for 5 min. Immediately after the supernatant was obtained by centrifugation, cellular glutathione (GSH) and GSSG were assayed by the GSH reductase recycling method (41). S-Nitrosoglutathione (GSNO) was measured by the method of Clancy et al. (42). Briefly, cytosol was incubated with 0.1 M NaBH4 at 37 °C for 5 min to break the S-nitroso bond. The solution was acidified to remove excess NaBH4, and acid-soluble fraction was obtained by centrifugation. Total glutathione was assayed as described above. The concentration of GSNO was calculated as the difference in GSH levels between untreated and NaBH4-treated cytosolic GSH.
Electromobility Shift AssayNuclear extracts were prepared
by the method of Staal et al. (43) from hepatocytes
stimulated with 500 units/ml TNF for 1 h. The cells were washed
with ice-cold PBS, scraped into PBS, and centrifuged at 3,000 rpm in a
microcentrifuge at 4 °C for 5 min. After discarding the supernatant,
the pelleted cells were resuspended in 5 volumes of Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40).
The cells were disrupted with 10 hand strokes in a Dounce homogenizer,
and nuclei were recovered by centrifugation at 5,000 rpm for 15 min and
resuspended in the same volume of Buffer B (Buffer A without Nonidet
P-40). After another 15-min centrifugation at 5,000 rpm, nuclear
proteins were extracted at 4 °C by gently mixing the nuclei in 150 µl of Buffer C (20 mM HEPES, pH 7.9, 10 mM
KCl, 1.5 mM MgCl2, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA,
and 0.5 mM dithiothreitol) and adding 50 µl of Buffer D
(Buffer C with 400 mM KCl) in a dropwise fashion.
Supernatants were collected after 1 h by centrifugation at 13,000 rpm for 30 min. For the electromobility shift assay, NF-
B-specific
oligonucleotide were end-labeled with [
-32P]ATP using
T4 polynucleotide kinase and purified on a G-50 Sephadex column.
Nuclear extracts (10 µg of protein) were incubated with ~40,000 cpm
(~0.5 ng) of 32P-labeled oligonucleotide for 20 min at
room temperature in a buffer containing 2 µg of poly(dI-dC), 4.2 mM HEPES, pH 7.4, 2.5% glycerol, 4.2 mM KCl, 1 mM MgCl2, 0.02 mM EDTA, 2% Ficoll,
and 21 mM dithiothreitol (final volume of 30 µl).
DNA-protein complexes were resolved on a 5% nondenaturing
polyacrylamide gel in TBE running buffer. Following electrophoresis,
gels were dried and subjected to autoradiography.
This
reporter (NF-B-pT109) was constructed by inserting three copies of
the NF-
B response element
(5
-GGGGACTTTCCCGGGGACTTTCCCGGGGACTTTCCC-3
, Life Technologies,
Inc.) into pTK-Luc (44), a plasmid carrying a portion of the herpes
thymidine kinase promoter (
109 to +52) ligated upstream of
luciferase. DNA transfections into hepatocytes were carried out in
six-well plates using LipofectAMINE, as described (44). The transfected
cells were allowed to recover overnight, exposed to 750 µM SNAP for 16 h, and stimulated with TNF
(500 units/ml) for 6 h. After washing twice with PBS, cells were lysed with Reporter lysis buffer. Luciferase activity was assayed using 20 µl of lysate in an AutoLumat LB953 luminometer (Berthold, Nashua, NH)
using a luciferase assay kit. Luciferase activity was normalized to
protein concentration.
TNF binding to cell
surface receptors was assayed as described previously (45). Briefly,
hepatocytes (2.5 × 105 cells/well in 12-well plates)
were pretreated with 750 µM SNAP for 16 h, washed
with serum-free medium twice, and incubated with 125I-labeled human TNF
(0.2-1.6 nM, 780 Ci/mmol) in the presence or absence of excess unlabeled TNF
(200-fold). After a 3-h incubation at 4 °C, the cells were washed
three times with prewarmed medium containing 5% calf serum, and
cell-bound radioactivity was counted in a
counter after
solubilizing in 5% Triton X-100. Specific binding of
125I-labeled TNF
was calculated as the difference in
binding in the absence and presence of excess unlabeled TNF
.
To analyze both the number and morphology of the apparent apoptosis within the cell population, a combination of light and electron microscopic methods were used. Monolayers of cells were treated as described earlier, fixed lightly in 2% paraformaldehyde, and counterstained with Hoescht 33258 (2 mg/ml) for 3 min. The cells were then mounted without washing in Gelvatol (Monsanto, St. Louis, MO) and observed using a Nikon FXL photomicroscope. Random images were collected using a 3-Chip Sony color camera, and stored with a coded identification number. The apoptotic nuclei in each field were counted in a blinded fashion. For electron microscopy, the treated cells were fixed in 2.5% glutaraldehyde in 0.1 M PBS for 1 h, washed in PBS, postfixed for 1 h in 1% aqueous osmium tetroxide, dehydrated through graded alcohols, and embedded in Epon (Energy Beam Science, Ahawam, MA). Following embedment, thin (60 nm) sections were cut using a Raichert Ultracut S (Leica, Chicago IL) microtome, mounted on copper grids, counterstained with 2% uranyl acetate (7 min) and 1% lead citrate (2 min), dried, and observed using a JEOL 100CX microscope.
Statistical AnalysisData are presented as mean ± S.D. of at least three separate experiments. Comparisons between two values were performed using paired Student's t test. Differences were considered significant when the p value was equal to or less than 0.05.
Our laboratory has shown previously that pretreatment of
hepatocytes with an NO-donor protects hepatocytes from subsequent H2O2 toxicity (20). To determine if NO
pretreatment also protects against TNF-induced toxicity, freshly
isolated rat hepatocytes were exposed to 750 µM SNAP as
an NO donor, and 18 h later the cells were washed, TNF
was
added, and viability was determined by crystal violet staining (Fig.
1). Pretreatment with 750 µM SNAP alone
did not reduce hepatocyte viability, while TNF
(28 ng/ml) induced a
12% and 5% cytotoxicity in untreated and SNAP-pretreated hepatocytes,
respectively. It has been shown previously that the transcriptional
inhibitor ActD markedly increases TNF
toxicity (24). As shown in
Fig. 1, viability measured at 14 h after TNF
addition was
decreased by 38% when ActD was combined with TNF
. Pretreatment with
SNAP for 16 h protected cultured hepatocytes from TNF
+ActD
toxicity. If cells were exposed to TNF
+ActD at intervals of 1-6 h
after SNAP exposure, no protection was seen (data not shown).
Since TNF-mediated cytotoxicity has been shown to be associated with
increased production of superoxide (46) and blocked by superoxide
dismutase (47), we measured intracellular levels of antioxidative
enzymes to determine if SNAP pretreatment increased levels of specific
intracellular antioxidant(s). As shown in Table I, the
cellular levels of the major antioxidants (including catalase, superoxide dismutase, and GSH peroxidase) were not elevated in SNAP-treated hepatocytes, indicating that the SNAP-induced
cytoprotective effect was not associated with increases in these
cellular antioxidative activities.
|
Since TNF+ActD toxicity in hepatocytes is associated
with the induction of apoptosis, we examined the effect of SNAP
pretreatment on TNF
+ActD-induced DNA fragmentation. Fig.
2A shows that TNF
+ActD, but not TNF
or
ActD alone, induced DNA fragmentation characteristic of apoptosis in
cultured hepatocytes. Pretreatment with 750 µM SNAP at 4 or 8 h prior to TNF
+ActD addition had no effect; however, if
TNF
+ActD were added 12 h after SNAP treatment, the degree of
DNA fragmentation was markedly reduced, and by 16 h apoptosis was
almost completely inhibited. When concentrations of SNAP ranging from
100 to 750 µM were added to hepatocytes 18 h prior
to TNF
+ActD, only SNAP concentrations of 250 µM or
greater were found to protect hepatocytes from apoptosis (Fig.
2B).
SNAP Induces HSP70 Expression
The major heat shock protein,
HSP70, has been shown to protect various types of cells from
TNF-induced injury (48, 49, 50). To determine if SNAP treatment induced
hepatocyte HSP70 expression, we exposed hepatocytes to increasing
concentrations of SNAP for different time periods and examined HSP70
expression by Northern and Western blot. Untreated hepatocytes
expressed a 2.8-kilobase mRNA consistent with the constitutive heat
shock protein 70, or HSC70 (Fig. 3A). SNAP
treatment resulted in a concentration-dependent increase in
the expression of HSC70 as well as the appearance of a 3.1-kilobase
mRNA band consistent with inducible HSP70 (51). HSP70 mRNA
expression was detected at concentrations of SNAP as low as 100 µM and was maximal at 750 µM. Western blot
analysis, using a monoclonal antibody (Sigma) that
detects both HSC70 and HSP70, demonstrated a dose-dependent
increase in expression of HSC70 and appearance of HSP70 protein in
hepatocytes treated with SNAP 16 h previously (Fig.
3B). Fig. 3C shows a time course for HSP70
mRNA levels following exposure to 750 µM SNAP and
demonstrates that mRNA levels progressively increased until 12 h and then declined. Similarly, the level of HSP70 protein measured
using an HSP70-specific monocloncal antibody (Stress Gen) following
SNAP pretreatment increased progressively up to 16 h and then
decreased (Fig. 3D).
Analysis of SNAP-mediated HSP70 induction by Northern and Western blot. Hepatocytes were treated with increasing concentrations of SNAP for 12 h (A) or 16 h (B), or for different time periods with 750 µM SNAP (C and D). Cells were washed twice with ice-cold PBS and harvested. For Northern blot analysis (A and C), RNA was isolated, electrophoresed, hybridized with a probe to HSP70, and relative levels of RNA were quantitated by PhosphorImager. For Western blots (B and D), cells were lysed with 20 mM Tris buffer, pH 7.4, containing protease inhibitors. The cytosolic fraction was obtained after centrifugation at maximum speed in microcentrifuge at 4 °C. Protein (40 µg) was separated on 8% SDS-polyacrylamide gels, transferred to nitrocellulose membrane, hybridized with monoclonal anti-HSP70 antibody (Sigma for B, which detects both HSC70 and HSP70; Stress Gen for D, which detects only HSP70), and anti-mouse IgG-linked horseradish peroxidase, and exposed to x-ray film after developing with chemiluminescence reagent.
SNAP Pretreatment Induces GSH Oxidation and GSNO Formation Responsible for HSP70 Induction
Because NO reacts with
intracellular thiols such as GSH (10), we examined intracellular GSH
and its derivatives. As shown in Fig. 4A,
intracellular GSH levels in hepatocytes exposed to 750 µM
SNAP decreased by about 50% at 4-6 h following exposure to 750 µM SNAP and then slowly recovered to control level (15 h). Intracellular GSNO levels increased to a maximum of 28% of total
GSH at 4 h, while GSSG levels became maximal at 34% of total GSH
at 6 h. Since it is known that conjugation, depletion, or oxidation of GSH increases the levels of cytoprotective heat shock proteins, including HSP70 (52, 53), in some cell types we studied the
level of HSP70 mRNA in hepatocytes with three thiol-modulating agents, each with a different mode of action. HSP70 mRNA was
induced by exposure to diamide (a GSH-oxidizing agent) and
N-ethylmaleimide (a GSH-conjugating agent), but not by
buthionine sulfoximine, which blocks GSH synthesis (Fig.
4B).
SNAP Pretreatment Induces Resistance to Heat Shock
Since our
data showed that SNAP exposure induced HSP70 expression, we next
examined whether pretreatment of hepatocytes with SNAP could result in
the expected resistance to subsequent heat challenge. Hepatocytes were
pretreated with concentrations of SNAP ranging from 0 to 1 mM for 16 h and then exposed to a heat challenge of
43.5 °C for 4 h. Viability was assayed after culturing the
cells with fresh medium for another 12 h at 37 °C. Fig.
5 reveals that hepatocytes developed resistance to
heat-induced killing following exposure to SNAP. Increases in viability
were seen following exposure to SNAP concentration of 250 µM, and maximum resistance was observed at 750 µM SNAP pretreatment.
Heat Exposure Mimics SNAP Treatment
The capacity for SNAP
pretreatment to protect against TNF+ActD-mediated apoptosis
correlated well with the stimulation of inducible HSP70 expression,
suggesting that HSP70 prevented the apoptosis. Therefore, we
hypothesized that induction of HSP70 by heat should also prevent
apoptosis by TNF
+ActD. Hepatocytes were exposed to heat (43.5 °C)
for 0-1.5 h and HSP70 protein expression determined 16 h later by
Western blot analysis (Fig. 6A). A
time-dependent increase in inducible HSP70 protein
expression was noted, and the degree of HSP70 expression correlated
well with protection from TNF
+ActD-induced DNA fragmentation (Fig.
6B). No significant injury (>90% viability as judged by
crystal violet staining) was observed during heat treatment.
Antisense Oligomer to HSP70 Blocks HSP70 Expression and Protection against TNF
To determine if HSP70 was
directly responsible for the inhibition of apoptosis induced by SNAP
exposure, hepatocytes were incubated with HSP70 antisense
oligonucleotide (10 µM) during a 750 µM
SNAP pretreatment, followed by TNF+ActD exposure. Antisense oligomers blocked induction of HSP70 expression (Fig.
7A) and inhibited SNAP-induced protection
from TNF
+ActD-mediated apoptosis (Fig. 7B). Sense
oligomers had no effect on induction of HSP70 protein expression and
did not inhibit the SNAP-induced cytoprotection. In contrast, HSC70
antisense oligomers (10 µM), which blocked HSC70
expression, had no effect on SNAP-stimulated HSP70 protein expression
or protection from TNF
cytotoxicity following SNAP exposure (data
not shown).
Microscopic Examination of Apoptosis
We further confirmed the
protective effect of SNAP pretreatment on TNF+ActD-induced apoptosis
by light and electron microscopy. Chromatin condensation and peripheral
and nuclear blebbing, typical characteristics of apoptotic morphology,
were apparent on electron microscopic examination of
TNF
+ActD-treated hepatocytes (Fig. 8, A
and B), but not control cells (Fig. 8C).
Similarly, the percentage of apoptotic hepatocytes following
TNF
+ActD treatment was significantly increased from 7% to 53%
(Fig. 8D). Pretreatment with SNAP or heat shock reduced
TNF
+ActD-induced apoptosis to 17% and 15%, respectively. Antisense
oligomers to HSP70 inhibited the antiapoptotic effect of SNAP
pretreatment.
TNF
Since TNF cytotoxicity can be reduced by
inhibiting TNF
binding to its receptors (54) and blocking its signal
transduction (55), we examined whether NO modified TNF
surface
binding or TNF
signal transduction. SNAP pretreatment did not change
the specific binding of TNF
to cell surface receptors (Fig.
9A). Similarly, TNF
-mediated NF-
B
activation as determined by electromobility shift assay was unchanged
by SNAP pretreatment (Fig. 9B). The lack of an effect of
SNAP treatment on TNF
-induced NF-
B activation was further
confirmed in cultured hepatocytes transfected with an NF-
B-reporter
(luciferase) construct. Induction of luciferase activity in response to
TNF
was not significantly different between SNAP-treated and control
hepatocytes (Fig. 9C), indicating no change in the NF-
B
activation in this assay which tests NF-
B function.
SNAP Pretreatment or Heat Inhibits ROI Formation and Lipid Peroxidation
TNF cytotoxicity is associated with
overproduction of activated oxygens (O
2 and
H2O2) from the mitochondrial respiratory chain
(46). Therefore, we next examined whether SNAP pretreatment inhibited
TNF
-induced ROI formation as measured by the oxidation of the
cell-permeable fluoregenic marker DCF-DA. TNF
exposure enhanced
ROI-induced oxidation of DCF-DA in untreated hepatocytes, whereas
pretreatment with SNAP or heat shock significantly reduced the
TNF
-induced oxidation of DCF-DA (Fig.
10A). Antisense oligomers of HSP70 prevented
the effect of SNAP on DCF oxidation.
Lipid peroxidation is another indicator of cellular damage by TNF
and is detected earlier than DNA fragmentation (56). We next measured
the formation of TBARS as an estimation of lipid peroxidation. TNF
increased TBARS in hepatocytes 5 h after treatment (Fig.
10B). However, pretreatment with SNAP or heat shock
significantly attenuated lipid peroxidation, whereas the addition of
HSP70 antisense oligonucleotide reversed the effect of SNAP on lipid
peroxidation.
To determine
whether endogenous NO could protect hepatocytes from TNF toxicity,
we examined TNF
cytotoxicity in hepatocytes induced to produce NO by
cytokine exposure. When incubated with IFN
and IL-1
, rat
hepatocytes expressed NOS2 protein as confirmed by Western blot
analysis (Fig. 11A) and produced NO as
judged by the accumulation of stable end products of NO, nitrite and
nitrate (Fig. 11B). NO synthesis was nearly completely
inhibited by the NOS inhibitor NMA. Although total GSH levels
(GSH+GSSG+GSNO) were unchanged in IFN
+IL-1
-treated cells,
intracellular GSNO and GSSG levels were elevated and GSH significantly
reduced. This change was also reversed by NMA (Fig. 11C).
Under these conditions, HSP70 protein levels were increased by
IFN
+IL-1
exposure and this increase was attenuated by NMA (Fig.
12A). When hepatocytes were subsequently
exposed to TNF
+ActD, IFN
- and IL-1
-stimulated hepatocytes were
protected from both TNF
-induced DNA fragmentation (Fig.
12B) and cytotoxicity (Fig. 12C). The protective
effects were inhibited by NMA.
In the present study, we have shown that pretreatment with the
NO-generating compound SNAP protects cultured rat hepatocytes from
TNF+ActD-induced cytotoxicity and apoptosis. Both time-course and
dose-response studies revealed that induction of HSP70 mRNA and
protein occurred in parallel to protection from TNF
+ActD-induced apoptosis. Antisense oligomers to HSP70, but not HSP70 sense nor HSC70
antisense oligomers, inhibited NO-induced HSP70 expression and rendered
hepatocytes again susceptible to TNF
+ActD-induced apoptosis.
Expression of HSP70 induced by hyperthermia also protected hepatocytes
from TNF
+ActD cytotoxicity. SNAP pretreatment did not increase
antioxidant enzyme activities, strongly suggesting that the protective
effect of SNAP pretreatment was not due to enhanced cellular
antioxidant capacity through these enzyme systems. Furthermore, the
decomposition products of SNAP (1 mM),
N-acetylpenicillamine (1 mM, the parent compound
of SNAP), and 8-bromo-cGMP (500 µM) had no effect on HSP
induction or the cytoprotection (data not shown), suggesting that NO
liberated from SNAP was the mediator through a cGMP-independent
mechanism. TNF
receptor binding and TNF
-mediated signal
transduction in hepatocytes were unchanged, as analyzed by the specific
binding of iodinated TNF
and NF-
B activation. Finally, endogenous
NO formation following IFN
+IL-1
exposure caused increased HSP70
expression and protection from TNF
+ActD-induced cytotoxicity. Thus,
we conclude that one mechanism by which NO protects hepatocytes from
TNF
-induced cytotoxicity is through the induction of HSP70.
The cytotoxic action of TNF has been associated with the activation
of phospholipase A2, the cytosolic release of ceramide, and
the formation of ROI. Overproduction of ROI has been identified as a
key component of apoptotic pathways involving activation of endogenous
endonucleases (57) and direct DNA fragmentation (58). The potential
importance of ROI in TNF
-induced cytotoxicity and apoptosis has been
demonstrated in studies where overexpression of Mn-superoxide
dismutase, radical scavengers, and inhibitors of mitochondrial electron
transfer block TNF
toxicity (59). In our study, the levels of
antioxidant enzymes, including superoxide dismutase, catalase, and GSH
peroxidase, were not changed in hepatocytes following SNAP
pretreatment. Furthermore, even though intracellular GSH levels were
transiently suppressed by SNAP exposure, the levels had returned to
base line by the time of TNF
exposure. Instead, SNAP pretreatment
induced the expression of HSP70, which has been shown to have
anti-apoptotic or cytoprotective effects against TNF
toxicity. Thus,
it is likely that the preinduction of HSP70 by SNAP protected the cells
from TNF
+ActD-mediated apoptosis.
NO can act as an antioxidant by scavenging O2 directly.
Therefore, another possible explanation of our findings is that NO neutralized O
2. This seems unlikely because the NO donor was added 16 h before the TNF
+ActD. Furthermore, no protective
effect from DNA fragmentation was seen when hepatocytes were treated simultaneously with 750 µM SNAP and TNF
(data not
shown). In fact, the protection from TNF
+ActD-induced apoptosis was
not detected until 12 h following SNAP exposure. Because SNAP has a half-life of only about 4.5 h under our experimental conditions (data not shown), no SNAP-derived NO should remain at the time of
TNF
treatment. We also could not detect the presence of possible delayed NO-generating sources in hepatocytes, such as GSNO (Fig. 4) or
iron-nitrosyl complexes (data not shown) 16 h following SNAP
pretreatment. In contrast to our findings, others have presented evidence that simultaneous NO production protects from apoptosis (30,
31). Thus, it is likely that the mechanism of NO-mediated protection
varies depending on the cell type and the quantity, as well as the
timing and duration, of NO exposure.
NO can have adverse effects on host survival, ranging from direct
cellular cytotoxicity (28, 29) to the damage of cellular components
leading to the mutagenesis (60). Therefore, it is not surprising that a
high level of NO exposure induces protective stress responses. Although
our observation that NO stimulates HSP70 expression in hepatocytes has
not been shown before, Kim et al. (19) have reported
previously that NO induces heme oxygenase, or HSP32, in rat
hepatocytes. The increased expression of HSP32 protects the cells from
subsequent toxic concentrations of NO (20). However, the inhibitor of
HSP32, Sn-protoporphyrin, did not reverse the induction of HSP70 or the
SNAP-induced protection from TNF toxicity (data not shown). Thus, it
is likely that HSP70 and HSP32 are both induced by NO exposure, but
while HSP32 protects from oxidative injury, HSP70 protects against
TNF
-induced apoptosis. Although the protection in both instances may
involve antioxidant properties, it is likely that HSP32 and HSP70
protect through distinct mechanisms.
HSP70 is induced by several environmental stimuli such as free radicals, heat, heavy metals (61), serum-free culture media (62), and agents that modulate intracellular ratio of GSSG to GSH in hepatocytes (52, 53, 63). Van Remmen et al. (62) recently reported that the type of serum-free culture medium profoundly influenced the spontaneous induction of HSP70 and HSC70 in cultured rat hepatocytes. Incubation in L15 medium resulted in marked spontaneous induction associated with a decrease in the GSH/GSSH ratio, whereas incubation in Williams medium E had little effect on HSP expression. Here we used Williams medium E and serum and, in agreement with this previous study, found no spontaneous induction of HSP70. The mechanism by which NO stimulates the expression of HSP70 may involve the interaction of NO with thiol-containing molecules. Ample evidence exists to support the view that NO readily oxidizes low molecular weight thiols, forming S-nitrosothiols and disulfide. Of cellular low molecular weight thiols, glutathione is the most abundant as well as being one of the intracellular targets of NO. Here, pretreatment of hepatocytes with NO was shown to alter the redox state accompanied by oxidation of GSH and formation of GSNO (Fig. 4A). A GSH oxidizing agent (diamide) and a GSH alkylating agent (N-ethylmaleimide) both induced HSP70 mRNA, but a GSH synthesis inhibitor (buthionine sulfoximine) did not (Fig. 4B). In addition, we show here that hepatocytes stimulated to produce NO by cytokine exposure expressed HSP70 associated with an intracellular GSH redox change. This cellular effect was attenuated by the NOS inhibitor NMA, strongly indicating that, like SNAP, cytokine-induced endogenous NO is also capable of inducing HSP70 expression in vitro. It is worth noting that even in the presence of NMA there was still a significant increase in HSP70 induction, which may indicate cytokine-dependent, but NO-independent, up-regulation (64). Taken together, these results suggest that it is possible that induction of HSP70 could be regulated by GSH-dependent cellular redox changes in response to NO.
Although heat exposure can cause apoptosis, it has been shown that heat
shock also induces resistance to a subsequent challenge of other
apoptotic agents, including ROI, NO, and glucocorticoid in mouse
thymocytes (65, 66), as well as serum withdrawal in neuroblastoma ND7
cells (67). HSPs may protect cells by acting as molecular chaperons,
guiding the folding and trafficking of damaged proteins (68). Induction
of HSP protects cells not only from damage due to heat but also from
damage due to oxidative injury and cytokine-mediated cytotoxicity. We
show here that both ROI production and lipid peroxidation are inhibited
by SNAP-induced HSP70 expression. Jäättelä et
al. (48) demonstrated that pretreatment of mouse fibrosarcoma
cells (WEHI) with heat-protected cells from TNF-induced cytolysis,
and that the protective effect roughly correlated with the kinetics of
HSP induction. Furthermore, only cells overexpressing HSP70 were found
to be protected from both ROI- and TNF
-induced cytotoxicity, whereas
overexpression of HSP27 protected only from exogenous ROI exposure but
not TNF
cytotoxicity (49, 50). Recent data have shown that HSP may protect from TNF
toxicity by inhibiting the action of ROI on mitochondrial membrane potential (69). Inhibition of TNF
toxicity by
SNAP pretreatment could occur through the inhibition of ROI production
in mitochondria, preventing ROI-mediated alterations in mitochondrial
membrane potential. This could prevent cytochrome c release,
which is involved in apoptosis through activation of cysteine protease
such as CPP32/Yama (70); however, since HSP70 is not a mitochondrial
protein, it is unlikely that HSP70 acts directly as a mitochondrial
antioxidant. HSP70 may instead block signal transduction to the
mitochondria, resulting in the inhibition of mitochondrial ROI
production by inhibiting either second lipid messenger(s) to
mitochondria (61) or by preventing the interaction between the death
domain of TNF
receptor and signal molecule(s) (71). Alternatively,
it is also possible that HSP70 may enhance the chaperon-mediated import
of precursor proteins into mitochondria which control mitochondrial
function (72, 73, 74) leading to decreased ROI formation. Further
experiments will be required to establish whether any of these
mechanisms account for the protection from TNF
-mediated
apoptosis.
We thank Debra Williams and Qi Wang for excellent technical assistance.