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INTRODUCTION |
Manganese superoxide dismutase
(MnSOD),1 a vital
anti-oxidant enzyme localized to the mitochondrial matrix, catalyzes
the dismutation of superoxide anions (O
2) to hydrogen peroxide
(H2O2). In aerobic cells, the mitochondrial
electron transport chain is probably the most abundant source of
O
2. At atmospheric oxygen concentrations, it is estimated that
between 1 and 3% of the O2 reduced in the mitochondrial
electron transport chain during ATP production may form O
2
(1-3). Although O
2 and other ROS are by-products of normal
respiration, imbalance or loss of cellular homeostasis results in
oxidative stress, causing damage to cellular components (lipid
membranes, proteins, and nucleic acids) (4, 5). MnSOD acts as the first
line of cellular defense to detoxify these O
2 (6). Various
inflammatory mediators (TNF-
, IL-1
, IL-6, and LPS) in multiple
tissues have been demonstrated to elicit dramatic elevations of both
the messenger RNA and protein levels of MnSOD (7-12). The increased
levels of MnSOD have been shown to be cytoprotective (13-16). However,
the signaling pathways responsible for MnSOD expression are
numerous and are still far from being fully elucidated.
Elaborate intercommunications take place between the nucleus and
mitochondria coordinating not only mitochondrial gene expression and
genome maintenance but also nuclear gene expression (17). The classic
view has been that mitochondria simply function as organelles
responding to changes in energy demand. However, recent data would
suggest a more complex picture where mitochondria also function as
active signaling organelles in a number of important intracellular
pathways (18-21).
TNF-
binding to membrane receptors triggers complex signal
transduction cascades (22-24), some of which result in excess ROS production in the mitochondria (25, 26). The cytocidal effect of these
ROS is either direct or necessary for downstream signaling events
leading to cell death. The crucial toxic role of ROS was demonstrated
by the inhibition of mitochondrial electron transport at specific
sites, which differentially interferes with TNF-
-mediated cytotoxicity (25) and by the correlation between sensitivity to TNF-
cytotoxicity and mitochondrial activity in the cell (26). Pharmacological experiments revealed that the mitochondrial respiratory chain is the major source of TNF-
-induced ROS (25-27). Antioxidants inhibit various actions of TNF-
(transcription factor activation, gene expression, and cytotoxicity), and exogenously added ROS mimic its
biological action (28-30). Our data would agree with the literature
regarding ROS and TNF-
.
In this paper, we show that inhibition of mitochondrial electron
transport results in the loss of TNF-
-stimulated MnSOD
expression, most likely due to the loss of mitochondria-to-nucleus
signaling with ROS acting as the second messenger in the signal
transduction pathway. In addition, we demonstrate that although
inflammatory mediators (TNF-
, IL-1
, and LPS) may elicit similar
inducible mRNA levels of MnSOD, the signaling pathways
leading to this expression are very different.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human TNF-
(a gift from
Genentech), IL-1
(a gift from NCI, National Institutes of Health),
antimycin A, N-acetylcysteine, AACOCF3, SB
203580, PD 98059 (Calbiochem), I
kinase inhibitor, Bay 11-7082 (BioMol, Plymouth Meeting, PA), bacterial lipopolysaccharide (LPS),
amobarbital (Amytal), 2-heptyl-4-hydroxyquinoline N-oxide, oligomycin, and myxothiazol (Sigma) were used.
Cell Culture--
L2 cells, a rat pulmonary epithelial-like line
(ATCC CCL 149), were grown in Ham's F12K media (Life Technologies,
Inc.) with 10% fetal bovine serum (Flow Laboratories, McLean, VA), 10 mM L-glutamine, and antibiotic/antimycotic
solution (ABAM, Sigma) at 37 °C in room air, 5% CO2. VA
cells, a rat pulmonary artery endothelial cell line, isolated from
segments of pulmonary artery by mechanical methods (11), were grown in
Medium 199 with Earle's salts (Sigma) with sodium bicarbonate to pH
7.4, 10% fetal bovine serum, 10 mM
L-glutamine, and antibiotic/antimycotic solution at
37 °C in room air, 5% CO2. LA9 cells, a mouse
fibroblast antimycin-resistant mutant cell line (generously provided by
Dr. Neil Howell, see Ref. 31), were grown in Ham's F12K media (Life
Technologies, Inc.) with 10% fetal bovine serum (Flow Laboratories,
McLean, VA), 10 mM L-glutamine, and
antibiotic/antimycotic solution (ABAM, Sigma) at 37 °C in room air,
5% CO2. When the cells were 70-90% confluent they were
exposed to inflammatory mediators (10 ng/ml TNF-
, 2 ng/ml IL-1, or
0.5 µg/ml LPS) and/or inhibitors. At 8 h after exposure, total
RNA was isolated and evaluated by Northern analysis as described below.
RNA Isolation and Northern Analysis--
Total RNA was isolated
by the acid guanidinium thiocyanate/phenol/chloroform extraction method
described by Chomczynski and Sacchi (32) with modifications (7). Twenty
micrograms of total RNA was size-fractionated on a 1%
agarose-formaldehyde gel (33) and electrotransferred to a charged
nylon membrane (Zetabind, Cuno Laboratory Products, Cuno Inc., Meriden,
CT) and UV covalently cross-linked. The membrane was hybridized with
32P-labeled rat manganese superoxide dismutase or rat
cathepsin B (as an RNA loading control) cDNAs and subjected to
autoradiography. All autoradiographs depicted in the figures throughout
this paper are representative of at least three independent experiments.
Measurement of Intracellular Generation of ROS--
Flow
cytometric analysis of intracellular generation of ROS was performed
using dihydrorhodamine 123 as a probe (34, 35). Cells were cultured in
6-well plates, and at confluence (1 × 106 cells/well)
they were treated with TNF-
(10 ng/ml), antimycin A (4 µM), or a combination of TNF-
/antimycin A. After
8 h of incubation, dihydrorhodamine 123 (5 mM) was
added, and the incubation was prolonged for an additional 30 min. The
cells were harvested, washed, centrifuged for 5 min at 800 rpm,
resuspended in phenol red-free M199 medium, and analyzed by flow
cytometry (excitation, 488 nm; emission 530 nm).
Statistical Analysis--
All results are expressed as
means ± S.D. unless stated otherwise. The unpaired Student's
t test was used to evaluate the significance of differences
between groups, accepting p < 0.05 as the level of significance.
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RESULTS |
Mitochondrial Electron Transport Inhibitors Modulate
TNF-
-induced Expression of MnSOD in Pulmonary Epithelial
Cells--
Previous analysis of the effects of mitochondrial electron
transport chain inhibitors showed that, depending on the site of action
of the inhibitor, the cytotoxicity of TNF-
was either increased or
decreased (25). A schematic of the mitochondrial respiratory chain is
shown in Fig. 1A. Based on
earlier experiments demonstrating stimulated expression of
MnSOD mRNA following treatment of a rat pulmonary
epithelial-like cell line (L2 cells) with inflammatory mediators (7,
9), we initiated studies to evaluate the effect of TNF-
on
MnSOD expression in cells also treated with mitochondrial respiratory chain inhibitors. Fig. 1, B
D, illustrates the
effects of treatment of L2 cells with antimycin A or amobarbital and/or TNF-
. The control samples exhibit a low constitutive level of expression of MnSOD mRNA. Addition of antimycin A or
amobarbital at increasing concentrations (25) did not affect this
constitutive MnSOD mRNA expression. Maximal induction of
MnSOD mRNA levels with TNF-
occurs after 8 h,
and thus this time point was selected for isolation of RNA for Northern
analysis. Cotreatment with TNF-
and antimycin A simultaneously
caused a marked decrease in MnSOD mRNA expression at
both concentrations of antimycin A (Fig. 1, B and
C) (25). Amobarbital, which blocks electron transfer through complex I (Fig. 1A), had minimal effect on TNF-
-induced
expression of MnSOD mRNA compared with antimycin A (Fig.
1, B and D). Microscopic examination at 8 h
showed no apparent difference in the cellular viability between control
and treated cells.

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Fig. 1.
A, simplified scheme of the respiratory
chain showing sites of substrate entry, inhibitor action, and potential
sites of superoxide anion formation. Cyt, cytochrome;
UQ, ubiquinone; Fe-S, iron-sulfur center.
B, Northern analysis of RNA from pulmonary epithelial cells
exposed to TNF- and/or mitochondrial inhibitors for 8 h.
Inhibition of TNF- -stimulated induction of MnSOD
by amobarbital (50 and 400 µM) and antimycin A (12.5 and
50 µM) in rat pulmonary epithelial cells (L2 cells)
either untreated or treated with mitochondrial inhibitors alone or in
combination with TNF- (10 ng/ml) or with TNF- alone is shown.
Control lanes 2 and 3 contain 0.1 and 0.5%
ethanol, which were used as the solvent for all inhibitors not soluble
in water. All figures are scanned images of autoradiographs and not
from a PhosphorImager. C, densitometric analysis of
autoradiographs (n = 3) demonstrating fold changes of
TNF-inducible MnSOD levels in L2 cells relative to control
and in response to inhibitors, antimycin A or amobarbital. All
quantitation of Northern analysis (relative to cathepsin B as a loading
control) within this paper was performed using ScionImage software and
a Microtek ScanMaker 9600XL scanner with transparency adapter.
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Antimycin A Strongly Decreases TNF-
-inducible MnSOD Expression
in Pulmonary Endothelial Cells--
With the results shown in Fig.
1B, we proceeded to evaluate the cellular specificity of the
mitochondrial inhibitor, antimycin A, studying its effects on another
cell type, the rat pulmonary artery endothelial cell line (VA cells,
see Ref. 11). By observing that the two concentrations of antimycin A
used in the pulmonary epithelial cell experiments (Fig. 1B)
gave similar results in the inhibition of TNF-
-stimulated
MnSOD mRNA levels and based on that fact that much lower
concentrations of antimycin A are effective at inhibiting mitochondrial
electron transport, we tested concentrations from 0.5 to 20 µM to find the optimum inhibitory concentration in the VA
cells. Fig. 2, A and
C, illustrates the effects of increasing concentrations of
antimycin A on MnSOD mRNA levels. The maximal inhibition
was achieved at a relatively low concentration of 4 µM.
By using this concentration of antimycin A, we tested the effect on
TNF-
-stimulated expression over 24 h. The maximal inhibition
appears to occur between 8 and 12 h when both TNF-
and
antimycin A were added simultaneously (data not shown). Furthermore,
the sequence of addition of TNF-
or antimycin A to the cells was
important for the TNF-
-stimulated expression of MnSOD
mRNA. If TNF-
was added as little as 15 min prior to antimycin
A, the diminution of the inducible expression was dramatically reduced
(data not shown). Other investigators (34) have found that pretreatment
of L929 cells with mitochondrial inhibitors resulted in a significant
decrease in binding of TNF to cell surface receptors. Thus, all the
cell treatments in our experiments were done simultaneously.

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Fig. 2.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to TNF- and/or antimycin
A. Inhibition of TNF- -stimulated induction of
MnSOD by antimycin A in rat pulmonary endothelial cells (VA
cells) either untreated or treated with increasing concentrations of
antimycin A alone or in combination with TNF- (10 ng/ml) or with
TNF- alone for 8 h is shown. B, Northern analysis of
RNA from pulmonary artery endothelial cells exposed to TNF- and
myxothiazol. Inhibition of TNF- -stimulated induction of
MnSOD in rat pulmonary endothelial cells (VA cells) either
untreated or treated with increasing concentrations of myxothiazol
alone or in combination with TNF- (10 ng/ml) or TNF- alone is
shown. C and D, densitometric analysis of
autoradiographs (n = 3) demonstrating fold changes of
TNF-inducible MnSOD levels in VA cells relative to control
and in response to inhibitors, antimycin A (C) or
myxothiazol (D).
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Effects of Other Mitochondrial Respiratory Chain Inhibitors on
TNF-
-stimulated Expression of MnSOD--
Treatment of both
pulmonary epithelial and endothelial cells with the complex III
inhibitor, antimycin A, showed dramatic inhibition of TNF-
induction
of MnSOD mRNA. With this in mind, we decided to test whether
another complex III inhibitor would give similar results. The
inhibitors, antimycin A and myxothiazol, both block electron transport
at the cytochrome b-c1 segment of the
mitochondrial respiratory chain but at different binding sites (37,
38). Fig. 2, B and D, shows the effects of
increasing concentrations of myxothiazol on TNF-
-stimulated
expression of MnSOD mRNA. The extent of inhibition by myxothiazol
is very similar to the pattern observed for antimycin A, which might be
expected considering the proximity of the binding of antimycin and
myxothiazol in the cytochrome b1-c
crystal structure (38). Interestingly, the inhibition of gene
expression by these electron transport inhibitors is exquisitely
specific in that 2-heptyl-4-hydroxyquinoline N-oxide, which
also inhibits complex III but at a different site, does not alter TNF
induction of MnSOD (data not shown). These results would
suggest that inhibition of mitochondrial electron transport at complex
III alters production of ROS that can act in retrograde communication
with the nucleus.
The Signaling Pathway for TNF-
Is Different from the Pathways
for LPS- or IL-1-stimulated Expression of MnSOD--
We have shown
previously that both lipopolysaccaride (LPS) and interleukin-1 (IL-1)
also induce expression of MnSOD in both pulmonary epithelial
and endothelial cells (7, 9). Maximal induction occurs at 8-12 h
similar to TNF-
. To evaluate whether signaling pathways for all
three inflammatory mediators were similar when mitochondrial
respiration is inhibited with antimycin A, we examined the effect that
increasing concentrations of antimycin A had on the LPS- and
IL-1-stimulated expression of MnSOD in VA cells
(Fig. 3, A, C and
D). Only at the highest concentration of antimycin A did the
level of stimulated expression of MnSOD vary even slightly
from LPS or IL-1 alone. This would suggest that the intracellular
signaling pathway of TNF-
is different from that of LPS or IL-1.

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Fig. 3.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to lipopolysaccaride or
interleukin-1 and/or antimycin A for 8 h. Inhibition of
LPS- and IL-1-stimulated induction of MnSOD in rat pulmonary
endothelial cells (VA cells) either untreated or treated with
increasing concentrations of antimycin A alone or in combination with
LPS (0.5 µg/ml) or IL-1 (2 ng/ml) or with LPS or IL-1 alone is
shown. B, Northern analysis of RNA from antimcyin-resistant
murine fibroblast cells exposed to TNF- and/or antimycin A. Inhibition of TNF- -stimulated induction of MnSOD
in a murine fibroblast antimycin-resistant cell line (LA9, see Ref. 31)
either untreated or treated with increasing concentrations of antimycin
A alone or in combination with TNF- (10 ng/ml) or TNF- alone is
shown. C-E, densitometric analysis of autoradiographs
(n = 3) demonstrating fold changes of LPS-inducible
(C) or IL-1-inducible (D) MnSOD levels
in VA cells or LA9 cells (E) relative to control and in
response to the inhibitor, antimycin A.
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TNF-
-stimulated Expression of MnSOD Is Unaffected by Antimycin A
in a Resistant Fibroblast Cell Line--
To demonstrate that the
effects of antimycin A are directly associated with mitochondrial
inhibition and not due to other possible side effects, we obtained an
antimycin-resistant mouse fibroblast mutant cell line, LA9 (31). In
this mutant, the rate of respiration is normal, but electron transport
through the succinate-cytochrome c oxidoreductase segment of
the mitochondrial respiratory chain, which includes cytochrome
b, shows resistance to inhibition by antimycin A. Fig. 3,
B and E, illustrates the effect of TNF-
on the
expression of MnSOD in the mutant LA9 cells. We should point
out that we previously demonstrated that the five separate MnSOD mRNA transcripts in the rat result from
alternative polyadenylation (39); however, there are only two murine
MnSOD transcripts at 1 and 4 kilobase pairs. At
concentrations of antimycin A varying from 0.5 to 20 µM,
TNF-
continued to induce MnSOD mRNA levels demonstrating the
specificity of the antimycin action in VA cells.
Inhibition of Mitochondrial ATPase with Oligomycin Also Represses
TNF-
-stimulated Expression of MnSOD--
Oligomycin, which inhibits
F1FO-ATPase, causes uncoupling of mitochondrial
respiratory electron transport and ATPase activity. Previous work by
other investigators (40) has demonstrated that cells treated with
TNF-
results in an increase in oligomycin-sensitive mitochondrial
respiration, with the resultant increase in ROS. However, cells treated
with both TNF-
and oligomycin resulted in decreased levels of
cellular ATP as well as blockade of the increase in ROS generation
(40). To evaluate whether oligomycin treatment of VA cells would
inhibit TNF-
induction of MnSOD, we treated VA cells with
increasing concentrations of oligomycin in the presence and absence of
TNF-
. The results shown in Fig. 4,
A and B, would indicate that oligomycin inhibits
TNF-
-inducible MnSOD expression and that this inhibition
may be due to the decreased mitochondrial ATP and/or ROS levels.

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Fig. 4.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to TNF- and oligomycin
for 8 h. Inhibition of TNF- -stimulated induction of
MnSOD in rat pulmonary endothelial cells (VA cells) either
untreated or treated with increasing concentrations of oligomycin alone
or in combination with TNF- (10 ng/ml) or TNF- alone is shown.
B, densitometric analysis of autoradiographs
(n = 3) demonstrating fold changes of TNF-inducible
MnSOD levels in VA cells relative to control and in response
to the inhibitor, oligomycin.
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Reactive Oxygen Species Are Important for TNF-
-stimulated
Expression of MnSOD--
Since levels of ROS have been shown
previously to be increased in cells undergoing oxidative stress (41,
42), we made use of the antioxidant, N-acetylcysteine (NAC),
to evaluate whether ROS scavenging can modulate TNF-
-inducible
expression of MnSOD. NAC caused a dose-dependent
decrease in the TNF-
-stimulated expression of MnSOD to
base-line levels with no detectable effect on cell viability (Fig.
5, A and C).
However, NAC did not cause any change in the IL-1-inducible expression
of MnSOD (Fig. 5, B and D), further evidence that the signaling pathways are different for TNF and IL-1. Of
note, the concentrations of NAC capable of decreasing MnSOD
expression are far below the millimolar levels used in much of the
literature, demonstrating the potential importance of ROS in
TNF-
-mediated signal transduction and the sensitivity of ROS in
retrograde mitochondria-to-nucleus communication.

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Fig. 5.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to TNF- and/or NAC for
8 h. Inhibition of TNF- -stimulated induction of
MnSOD in pulmonary artery endothelial cells (VA cells)
either untreated or treated with various increasing concentrations of
NAC alone or in combination with TNF- (10 ng/ml) or TNF- alone is
shown. B, Northern analysis of RNA from pulmonary artery
endothelial cells exposed to IL-1 and/or NAC. Inhibition of
IL-1 -stimulated induction of MnSOD in pulmonary artery
endothelial cells (VA cells) either untreated or treated with various
increasing concentrations of NAC alone or in combination with IL-1
(2 ng/ml) or IL-1 alone is shown. C and D,
densitometric analysis of autoradiographs (n = 3)
demonstrating fold changes of TNF-inducible (C) or
IL-1-inducible (D) MnSOD levels in VA cells
relative to control and in response to NAC.
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TNF-
-stimulated Expression of MnSOD in Endothelial Cells Is
Dependent on Cytoplasmic Phospholipase A2
(cPLA2)--
Several studies (43-46) have demonstrated
the connection between arachidonic acid and mitochondrial ROS
production. To explore whether inhibition of cPLA2 and thus
mitochondrial ROS affect MnSOD expression, we utilized the potent and
selective cPLA2 inhibitor, arachidonyltrifluoromethyl
ketone (AACOCF3) (44). This inhibitor caused a
dose-dependent repression of MnSOD expression in
TNF-
-stimulated endothelial cells (Fig.
6, A and C),
whereas AACOCF3 had no effect on IL-1
-stimulated cells
(Fig. 6, B and D).

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Fig. 6.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to TNF- and/or
cPLA2 inhibitor (AACOCF3) for 8 h.
Inhibition of TNF- -stimulated induction of MnSOD
in pulmonary artery endothelial cells (VA cells) either untreated or
treated with increasing concentrations of AACOCF3 alone or
in combination with TNF- (10 ng/ml) or TNF- alone is shown.
B, Northern analysis of RNA from pulmonary artery
endothelial cells exposed to IL-1 and cPLA2 inhibitor
(AACOCF3). Inhibition of IL-1-stimulated induction
of MnSOD in pulmonary artery endothelial cells either
untreated or treated with increasing concentrations of
AACOCF3 alone or in combination with IL-1 (2 ng/ml) or
IL-1 alone is shown. C and D, densitometric
analysis of autoradiographs (n = 3) demonstrating fold
changes of TNF-inducible (C) or IL-1-inducible
(D) MnSOD levels in VA cells relative to control
and in response to AACOCF3.
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TNF-
-inducible Expression of MnSOD Is Not Dependent on
NF-
B--
Other investigators (45) have shown that ROS activation
by diverse conditions is important for gene activation by NF-
B. To
investigate whether NF-
B is important in TNF-
signaling of MnSOD, we utilized the I
K inhibitor, BAY 11-7082 (47). At
increasing concentrations of the I
K inhibitor, MnSOD
expression in IL-1-stimulated endothelial cells could be reduced to
base line (Fig. 7, B and E), with no effect on TNF-
-stimulated cells (Fig. 7,
A and D). These data suggest that NF-
B is
activated in the IL-1
signaling pathway but not in the TNF-
signaling pathway of MnSOD. Other kinase signaling pathways
were also investigated by using specific inhibitors. The
mitogen-activated protein (MAP) kinases are a group of protein
serine/threonine kinases that are activated in response to a variety of
extracellular stimuli and mediate signal transduction from the cell
surface to the nucleus. Two of the MAP kinase pathways that have been
implicated in TNF-
and IL-1 signal transduction are JNK/SAPK
and p38. The c-Jun kinase/stress-activated protein kinase cascade is
activated following exposure to UV radiation, heat shock, or
inflammatory cytokines. The p38 kinase (reactivating kinase) is the
newest member of the MAP kinase family. It is activated in response to
inflammatory cytokines, endotoxins, and osmotic stress. Selective
inhibitors of the MKK1/2 (PD 98059) (48) and p38 (SB 203580) (49) were
utilized at increasing concentrations to treat endothelial cells with
or without TNF-
. No differences were seen between inducible
expression of MnSOD with either of the inhibitors
(representative example, Fig. 7, C and F)
suggesting that TNF-
signal transduction for MnSOD likely
does not occur through these MAP kinase pathways.

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Fig. 7.
A, Northern analysis of RNA from
pulmonary artery endothelial cells exposed to TNF- and/or I K
inhibitor (Bay 11-7082) for 8 h. Inhibition of
TNF- -stimulated induction of MnSOD in pulmonary artery
endothelial cells (VA cells) either untreated or treated with
increasing concentrations of I K inhibitor alone or in combination
with TNF- (10 ng/ml) or TNF- alone is shown. B,
Northern analysis of RNA from pulmonary artery endothelial cells
exposed to IL-1 and/or I K inhibitor (Bay 11-7082).
Inhibition of IL-1 -stimulated induction of MnSOD
in pulmonary artery endothelial cells (VA cells) either untreated or
treated with increasing concentrations of I K inhibitor alone or in
combination with IL-1 (2 ng/ml) or IL-1 alone is shown.
C, Northern analysis of RNA from pulmonary artery
endothelial cells exposed to TNF- and/or MAP kinase inhibitor (PD
98059). Levels of TNF- -stimulated induction of MnSOD in
pulmonary artery endothelial cells (VA cells) either untreated or
treated with increasing concentrations of the MAP kinase inhibitor (PD
98059) alone or in combination with TNF- or TNF- alone are shown.
Also, when the MAP kinase inhibitor, SB 203580, was used in similar
experiments, no alteration in TNF- -inducible pattern of
MnSOD was observed, almost identical to the above experiment
with PD 98059. D-F, densitometric analysis of
autoradiographs (n = 3) demonstrating fold changes of
TNF-inducible (D and E) or IL-1-inducible
(E) MnSOD levels in VA cells relative to control
and in response to I K inhibitor (D and E) or
PD 98059 (F).
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ROS Production from Mitochondria in Response to TNF and/or
Antimycin A--
Intracellular generation of ROS by cultured VA cells
in response to TNF-
treatment was measured by flow cytometry using
the fluorescent probe, dihydrorhodamine 123. As shown in Fig.
8, treatment of cells with 10 ng/ml
TNF-
for 8 h resulted in significantly higher rhodamine 123 fluorescence, indicating increased ROS generation. Of note, cells
treated with 4 µM antimycin A did demonstrate increased fluorescence compared with control cells. However, cells treated simultaneously with TNF and antimycin A showed no higher fluorescence than cells treated with antimycin A alone. Cells treated with IL-1
did not have any more fluorescence than control cells.

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Fig. 8.
Effect of TNF- on
intracellular generation of ROS. Cellular generation of ROS was
determined by flow cytometry using rhodamine 123 fluorescence. Cells
were incubated in the absence (control) or presence of 4 µM antimycin A or 10 ng/ml TNF- or 10 ng/ml TNF-
plus 4 µM antimycin A or 2 ng/ml IL-1 . Results are
means ± S.D. of four independent experiments. *,
p < 0.01 and; **, p < 0.001 between
control and experimental treatments.
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DISCUSSION |
Manganese superoxide dismutase plays an important role in the
cellular defense against superoxide produced by the mitochondrial electron transport chain during normal cellular metabolism (13-16). Reduction or deficiency of MnSOD has been shown to promote
cytotoxicity under conditions of oxidant stress (50, 51). A number of
laboratories, including our own (51-53), have begun to understand the
workings of the promoter and the intronic enhancer in causing the
dramatic inducible expression of MnSOD. However, the
molecular intracellular signaling pathways and the nature of the
induction of MnSOD by various inflammatory mediators are
still being unraveled.
Retrograde communication from the mitochondria to the nucleus likely
consists of metabolic signals and transduction pathways that function
across the inner mitochondrial membrane. Since ROS are very short lived
molecules closely regulated by a coordinated enzyme system, they could
be potential signal transducers of putative mitochondria-to-nucleus
signaling pathways. Production of ROS in the mitochondria is related to
changes in electron flux through the respiratory chain, brought about
by various physiological conditions such as heat shock (55), variations
in oxygen tension (56), and exposure to nitric oxide (57). ROS have
been found to act as second messengers in cellular functions such as
cell growth and differentiation (58, 59). Mitochondrial respiration has
been linked to the expression of the mammalian gene,
GLUT1 (60). Expression of the GLUT1 gene,
one of the isoforms of the glucose transporter, is enhanced by hypoxia
and by exposing cells to inhibitors of mitochondrial respiration (60).
Therefore, we postulated that inhibition of mitochondrial respiration
might also regulate MnSOD expression possibly through a
mechanism involving intracellular levels of ROS.
Our data show that mitochondrial respiratory chain inhibitors,
antimycin A (Fig. 1, B and C, and Fig. 2,
A and C) and myxothiazol (Fig. 2, B
and D), as well as the F1FO-ATPase
inhibitor, oligomycin (Fig. 4, A and B), can
repress TNF-
-inducible expression of nuclear-encoded MnSOD. We have also addressed the specificity of the
antimycin effects with studies in antimycin A-resistant LA9 mutant
cells stimulated with TNF-
(Fig. 3, B and D).
Northern analysis utilizing the antioxidant, NAC, would suggest that
TNF-
-mediated pathways required intracellular ROS to function (Fig.
5, A and C), whereas the IL-1
pathway did not
(Fig. 5, B and D). In addition, the rhodamine 123 fluorescence data (Fig. 8) further implicate ROS, ROS by-products,
and/or lipid peroxides as likely candidates for signal transduction
from the mitochondria to the nucleus in the TNF-
signal pathway. In
addition, other investigators (45, 46) have shown that submicromolar
concentrations of arachidonic acid cause a substantial increase in ROS
production in mitochondria. The data in Fig. 6, A and
C, would suggest that decreased levels of arachidonic acid,
occurring as a result of selective blockade of cPLA2
enzyme, are sufficient to inhibit the TNF-
-inducible expression of
MnSOD. Thus oxidative events generated in the mitochondrion, not simply inhibition of energy-coupled processes, are crucial in
TNF-
-induced MnSOD gene expression.
ROS or other mitochondrial intermediates may control both the cytotoxic
and gene-regulatory effects of TNF-
, thus providing a basis for a
mitochondria-to-nucleus signaling pathway, which requires bidirectional
communication between the nucleus and the mitochondria. Although the
cytotoxic activity of TNF-
seems to be rather restricted to tumor
cells, nearly every cell type responds to TNF-
by the activation of
a wide range of different genes (61, 62). Transcriptional regulation of
genes involves interaction of cis-acting elements of DNA
with their cognate DNA-binding proteins. TNF-
induces activation of
nuclear factors that act as "third" messenger molecules
specifically binding to cis-acting sequences. Perhaps the
best understood example of the second messenger function of ROS is the
activation of the mammalian transcription factor, NF-
B (63). When
activated, NF-
B induces the expression of various genes involved in
inflammatory responses, immune cell regulation, and differentiation
(64). A diverse set of conditions can cause NF-
B activation (64,
65). Intracellular ROS are involved in the TNF-
signaling of
MnSOD expression (Figs. 1-3); however, our experiments with
the I
kinase inhibitor (Fig. 7, A and B, and
D and E) would indicate that although NF-
B may
be involved in the IL-1
signaling pathway, it is not involved in TNF-
-inducible expression of MnSOD. In addition, our data
would also suggest that the MAP kinase pathways involving c-Jun
kinase/stress-activated protein kinase and p38 probably do not regulate
expression of MnSOD (Fig. 7, C and
F).
Thus, our data would suggest that multiple signaling pathways result in
stimulated expression of MnSOD. Clearly, inhibition of
mitochondrial electron transport alters inducible expression of
MnSOD by TNF-
but not IL-1 or LPS. Thus, it would appear
that the TNF-
signaling pathway requires retrograde communication from the mitochondria to the nucleus, probably involving intracellular ROS. However, due to the high reactivity of ROS and their production within the mitochondrial membrane, it is much more likely that the
cytoplasmic signaling molecule of the TNF-
signaling pathway may be
a protein acted upon by a lipid peroxide or other ROS or even the lipid
peroxide itself. Our data with the cPLA2 inhibitor would
seem to bolster this argument in the TNF-
pathway. Other investigators (46) have shown that TNF-
-induced ROS production requires cPLA2 and 5-lipoxygenase activity but not
cyclooxygenase activity in the Rac signal transduction cascade. This
would suggest that ROS generation is dependent on synthesis of
arachidonic acid and its subsequent metabolism to leukotrienes. In
fact, exogenously applied leukotriene B4 could increase
mitochondrial ROS (46). A model of our proposed MnSOD
signaling pathways is shown in Fig. 9,
detailing the differences between the TNF-
and IL-1
pathways. It
may be that a yet unidentified signal pathway is activated by
intracellular reactive oxygen species, ultimately leading to DNA-protein interactions within the intronic enhancer of
MnSOD to produce the dramatic inductions seen with TNF-
(Fig. 9).

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Fig. 9.
Model of TNF- and
IL-1 signal transduction pathways involved in
inducible MnSOD expression. Mitochondrial
inhibitors (antimycin A and myxothiazol) and
F1F0-ATPase inhibitor (oligomycin) are shown to
negatively affect mitochondrial production of ROS, which are involved
in the TNF- signaling pathway but not the IL-1 pathway. The
cPLA2 inhibitor (AACOCF3) diminishes production of
arachidonic acid (AA), which increases mitochondrial
production of ROS to produce potential lipid peroxide species. I K
inhibitor (Bay 11-7082) inhibits phosphorylation of the I subunit
thus preventing separation of the I subunit from nuclear factor B
(NF- B) and blocking it from translocating into the nucleus.
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