Am J Physiol Renal Physiol 290: F1169 –F1176, 2006.
First published November 22, 2005; doi:10.1152/ajprenal.00378.2005.
Mitochondrial reactive oxygen species contribute to high NaCl-induced
activation of the transcription factor TonEBP/OREBP
Xiaoming Zhou,1 Joan D. Ferraris,2 and Maurice B. Burg2
1
Division of Nephrology, Department of Medicine, Uniformed Services University of
the Health Sciences, and 2Laboratory of Kidney and Electrolyte Metabolism, National
Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Submitted 20 September 2005; accepted in final form 14 November 2005
superoxides; rotenone; myxothiazol; transactivation; nuclear translocation; ATP release
THE TRANSCRIPTION FACTOR tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP),
when activated by high NaCl and other hypertonic stresses,
increases transcription of genes involved in osmoprotective
accumulation of organic osmolytes, including glycine betaine
and heat shock proteins (26, 34, 48). In the kidney, TonEBP/
OREBP also controls expression of a urea transporter (UT-A)
(36) and of aquaporin-2 (22, 26), thus regulating the urinary
concentrating mechanism. Hypertonicity, induced by high
NaCl, increases reactive oxygen species (ROS) in mIMCD3
Address for reprint requests and other correspondence: X. Zhou, Div. of
Nephrology, Uniformed Services Univ. of the Health Sciences, Bethesda, MD
20814 (e-mail: [email protected]).
http://www.ajprenal.org
(50), mIMCD-K2 (49), HEK293 cells (51), and, presumably,
also in the renal medulla, where interstitial NaCl normally is
high and there is oxidative stress that results in protein carbonylation (50). We previously found that high NaCl-induced
ROS contribute to activation of TonEBP/OREBP (51). The
purpose of the present study was to identify the source within
cells of the ROS that are produced in response to high NaCl
and whether it is necessary for full activation of TonEBP/
OREBP.
ROS are a by-product of mitochondrial generation of ATP
through oxidative phosphorylation. This is true even in the
renal papilla, where the oxygen tension is low (9). Under
physiological conditions, ⬃0.2–2% of the oxygen taken up by
cells is converted by mitochondria to ROS, initially through
production of superoxide that is subsequently dismutated to
hydrogen peroxide (17). There are two main sites of superoxide generation in the inner mitochondrial membrane: NADH
dehydrogenase at complex I, and the interface between ubiquinone and complex III (30).
The number of mitochondria in renal medullary cells apparently is affected by osmolality. Brattleboro rats have hereditary
diabetes insipidus, which reduces their medullary interstitial
osmolality because of lack of vasopressin. Their renal papillas
have a reduced number of mitochondria (29). Infusion of
vasopressin into the rats, which increases osmolality in the
renal medullary interstitium, increases the number of mitochondria (4). Similarly, low renal medullary osmolality in
aquaporin-1 null mice (27) is associated with reduced expression of several mitochondrial genes (31). The increased mitochondrial capacity associated with hyperosmolality presumably reflects increased energy demand. Furthermore, mitochondria are major sources of ROS in renal medullas where the
urine is concentrated (52).
In the present study, we examined whether high NaClinduced ROS were produced by mitochondria and whether the
ROS originating from mitochondria contributed to activation
of TonEBP/OREBP.
MATERIALS AND METHODS
Cells, cell culture, and chemicals. Human embryonic kidney 293
(HEK293) cells, purchased from American Type Culture Collection
(Manassas, VA) and grown as a monolayer, were incubated in Eagle’s
minimal essential medium plus 10% fetal bovine serum in 5% CO295% air at 37°C. Cells were used below passage 50. The osmolality
of the control, “isotonic,” medium was 290 mosmol/kgH2O. DihyThe 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.
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Zhou, Xiaoming, Joan D. Ferraris, and Maurice B. Burg.
Mitochondrial reactive oxygen species contribute to high NaCl-induced activation of the transcription factor TonEBP/OREBP. Am J
Physiol Renal Physiol 290: F1169 –F1176, 2006. First published
November 22, 2005; doi:10.1152/ajprenal.00378.2005.—Hypertonicity activates the transcription factor tonicity-responsive enhancer/
osmotic response element binding protein (TonEBP/OREBP), resulting in increased expression of genes involved in osmoprotective
accumulation of organic osmolytes, including glycine betaine, and in
increased expression of osmoprotective heat shock proteins. Our
previous studies showed that high NaCl increases reactive oxygen
species (ROS), which contribute to activation of TonEBP/OREBP.
Mitochondria are a major source of ROS. The purpose of the present
study was to examine whether mitochondria produce the ROS that
contribute to activation of TonEBP/OREBP. We inhibited mitochondrial ROS production in HEK293 cells with rotenone and myxothiazol, which inhibit mitochondrial complexes I and III, respectively.
Rotenone (250 nM) and myxothiazol (12 nM) reduce high NaClinduced ROS over 40%, whereas apocynin (100 ␮M), an inhibitor of
NADPH oxidase, and allopurinol (100 ␮M), an inhibitor of xanthine
oxidase, have no significant effect. Rotenone and myxothiazol reduce
high NaCl-induced increases in TonEBP/OREBP transcriptional activity (ORE/TonE reporter assay) and BGT1 (betaine transporter)
mRNA abundance ranging from 53 to 69%. They inhibit high NaClinduced TonEBP/OREBP transactivating activity, but not its nuclear
translocation. Release of ATP into the medium on hypertonic stress
has been proposed to be a signal that triggers cellular osmotic
responses. However, we do not detect release of ATP into the medium
or inhibition of high NaCl-induced ORE/TonE reporter activity by an
ATPase, apyrase (20 U/ml), indicating that high NaCl-induced activation of TonEBP/OREBP is not mediated by release of ATP. We
conclude that high NaCl increases mitochondrial ROS production,
which contributes to the activation of TonEBP/OREBP by increasing
its transactivating activity.
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fold-difference in RNA abundance between conditions (F) was calculated, as F ⫽ E(Ct1⫺Ct2). E is the efficiency of the reaction determined from the results of reactions containing 8 or 80 ng of cDNA
template. The efficiency was (means ⫾ SE) 1.98 ⫾ 0.056 in 18
experiments. Ct1 and Ct2 are the numbers of cycles required to reach
the threshold of amplicon abundance in respective experimental
conditions (13).
Measurement of TonEBP/OREBP transactivating activity. A yeast
binary GAL4 reporter assay system was used, as previously described
(13). Briefly, the assay system comprises an expression vector containing the TonEBP/OREBP transactivation domain (TAD),
GAL4dbd-TAD, and a reporter plasmid (GAL4UAS-GL3) that are
cotransfected. GAL4dbd-TAD contains in-frame insertion of cDNA
coding for amino acids 548 –1531 of TonEBP/OREBP in a vector
containing the neomycin resistance gene (pFA-CMV, Stratagene). An
otherwise identical construct, in which the GAL4dbd construct does
not contain a TAD, was used as a control for nonspecific effects.
GAL4UAS-GL3 contains five tandem repeats of the yeast GAL4
binding site (upstream activating sequence) and a minimal promoter
(TATATA) derived from pFR-Luc (Stratagene) and inserted into the
NheI/HindIII sites of pGL3 (Promega) upstream of the Photinus
pyralis luciferase gene GAL4UAS-GL3. It was further modified for
blasticidin resistance as described before (13). Stable reporter cell
lines were established by blasticidin and neomycin selection and used
as described above for assays of TonEBP/OREBP transcriptional
activity.
Measurement of TonEBP/OREBP nuclear translocation. TonEBP/
OREBP nuclear translocation was measured by Western blot analysis.
Briefly, after 6 ⫻ 106 cells/10-cm dish were seeded and incubated for
24 h, the medium was changed for 30 min to one still at 290
mosmol/kgH2O or osmolality was elevated to 500 mosmol/kgH2O by
the addition of NaCl. The cytoplasmic and nuclear proteins were
separated with a NE-PER kit (Pierce) with a protease inhibitor
cocktail tablet (Roche, Indianapolis, IN), 2.5 mM NaF and 2.5 mM
Na3VO4 added. The protein concentrations were determined with the
BCA reagent (Pierce). Thirty micrograms cytoplasmic proteins/lane
or 20 ␮g nuclear proteins/lane were loaded into a 15-well precast
4 –12% gradient polyacrylamide gel (Invitrogen, Carlsbad, CA). The
nitrocellulose membrane was probed with TonEBP/OREBP antibody
(Affinity BioReagents, Golden, CO) overnight at 4°C, followed by
incubation with Alexa fluophor-conjugated secondary antibody (Molecular Probes) for 60 min at 37°C. Display and quantification were by
infrared imaging (Odyssey, Li-Cor, Lincoln, NE).
Statistical analysis. Data are expressed as means ⫾ SE. Statistical
analyses were performed by paired t-test or repeated ANOVA, as
appropriate. Post hoc comparison was made by Dunnett’s test. P ⬍
0.05 is considered significant.
RESULTS
High NaCl-induced ROS come from mitochondria. We previously showed that high NaCl increases ROS in HEK293 cells
(51), renal epithelial cells of human origin (40). Rotenone
inhibits mitochondria complex I (42). Myxothiazol inhibits
mitochondria complex III (10). To examine whether hypertonicity-induced ROS are produced by mitochondria, we examined the effects of rotenone and myxothiazol on ROS activity
by confocal microscopy, using dihydroethidium as a probe.
Apocynin inhibits NADPH oxidase (41). Allopurinol inhibits
xanthine oxidase (45). To examine whether NADPH oxidase
and xanthine oxidase contribute to high NaCl-induced ROS,
we also examined the effect of apocynin and allopurinol on
high NaCl-induced ROS. Rotenone and myxothiazol reduce
high NaCl-induced ROS by 44 and 47%, respectively, whereas
apocynin and allopurinol have no significant effect (Fig. 1A).
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droethidium was purchased from Molecular Probes (Eugene, OR),
and all other chemicals were purchased from Sigma (St. Louis, MO).
All inhibitors and probes were freshly prepared. All controls, except
in Fig. 6, contained 0.1% DMSO in the medium.
Measurement of ROS. ROS were measured as previously described
(51). Briefly, 0.8 to 1 ⫻ 106 cells were grown overnight on the optical
glass bottom of a dish (MatTek, Ashland, MA) and preincubated with
30 ␮M dihydroethidium in phenol red-free culture medium for 30
min. Then, the medium was replaced with an otherwise identical one
at 290 or 450 mosmol/kgH2O (NaCl added) for 45 min. The fluorophore was excited at 488 nm. Fluorescent emission at 525 nm was
recorded by confocal microscopy (LSM510 Meta, Zeiss, Thornwood,
NY) and analyzed with MetaMorph software.
Measurement of ATP abundance. An ATP assay kit from Calbiochem (San Diego, CA) was used according to the manufacturer’s
instructions. This assay is based on oxidation of luciferin, which is
proportional to the amount of ATP present in the reaction buffer. For
measuring cellular ATP, 2–3 ⫻ 104 cells/well were seeded in 96-well
white view plates (Packard, Wellesley, MA) and incubated for 24 h at
290 mosmol/kgH2O. Then, the medium was changed for 16 h to one
still at 290 mosmol/kgH2O or the osmolality was elevated to 500
mosmol/kgH2O by the addition of NaCl. The cells were lysed, the
protein concentrations were estimated by the BCA reagent (Pierce,
Rockford, IL), and ATP/mg cell protein was measured. For measuring
ATP release to medium, the cells were seeded at 2–3 ⫻ 104 cells/well
in a 96-well plastic plate in phenol red-free cell culture medium.
Osmolality was increased to 500 mosmol/kgH2O (NaCl added) in the
presence of 200 ␮M suramin, which prevents the degradation of any
ATP that might be released (8, 25). In experiments involving depletion of medium ATP by apyrase, apyrase activity was measured at the
end of the experiment by incubating 10 ␮l medium with a standard
ATP solution to be sure that it had remained active.
Measurement of TonEBP/OREBP transcriptional activity. The osmotic responsive element (ORE) reporter, ⫺1233/⫺1105 IL2minGL3, was constructed by inserting nucleotides ⫺1233 to ⫺1105 of the
5⬘-flanking region of the human aldose reductase gene into MluI/NheI
sites upstream of the human IL-2 minimal promoter, as previously
described (51). An otherwise identical reporter to which binding of
TonEBP/OREBP is prevented by mutation of the potentiating and
ORE elements and the adjacent AP-1 site was used to control for
specificity for TonEBP/OREBP. Mutations were performed using a
QuikChange site mutagenesis kit (Stratagene, La Jolla, CA) according
to the manufacturer’s protocol. Accuracy of the mutations was confirmed by sequencing (51).
Stable reporter cell lines were established by transfection with
Effectene (Qiagen, Valencia, CA), blasticidin selection, and screening
for luciferase activity to select clones with the highest expression.
Next, 2–3 ⫻ 104 cells/well were seeded in 96-well white view plates
(Packard) and incubated for 24 h at 290 mosmol/kgH2O; then, the
medium was changed for 16 h to one still at 290 mosmol/kgH2O or
the osmolality was elevated to 500 mosmol/kgH2O by the addition of
NaCl. Luciferase activity was measured with Bright-Glo substrate
(Promega, Madison, WI) in a Victor luminometer (PerkinElmer,
Wellesley, MA). Cell proteins were determined with the BCA reagent
(Pierce).
Measurement of BGT1 mRNA expression. As previously described
(13), 2.0 ⫻ 105 cells/well were plated in a six-well dish, then
incubated at the indicated osmolality for 16 h. Total RNA was isolated
using an RNeasy Mini Kit (Qiagen) and reverse transcribed with
Taqman RT reagents (Applied Biosystems, Foster City, CA). Amplicons were detected with the ABI Prism 7900HT detection system
(Applied Biosystems). Primers for the human BGT1 mRNA are
5⬘-TGTTCAGCTCCTTCACCTCTGA-3⬘ and 5⬘-GCAATGCTCTGTGTTCCAAAAG-3⬘. The 6-carboxyfluorescein (FAM)-labeled
probe is 5⬘-CTGCCCTGGACGACCTGCAACAA-3⬘. As a control
for RT efficiency and loading, 18S RNA was measured at the same
time, using primers and a probe from Applied Biosystems. The
MITOCHONDRIAL REACTIVE OXYGEN SPECIES ACTIVATION OF TONEBP/OREBP
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These results point to a mitochondrial source of the high
NaCl-induced increase in ROS.
Mitochondrial ROS contribute to high NaCl-induced activation of TonEBP/OREBP. We next examined the influence of
mitochondrial ROS on high NaCl-induced activation of
TonEBP/OREBP by measuring the effect of rotenone and
myxothiazol on ORE/TonE luciferase reporter activity. High
NaCl increases ORE/TonE reporter activity by 58-fold (Fig.
2A). Rotenone (250 nM) and myxothiazol (12 nM) attenuate
the effect of high NaCl by 62 and 53%, respectively. As a
control for specificity to TonEBP/OREBP, we used an otherwise identical reporter in which the ORE/TonE sites are mutated. This prevents the binding of TonEBP/OREBP and thus
inhibits high NaCl-induced increase in reporter activity. Rotenone and myxothiazol have no significant effect on activity of
the mutated ORE reporter (Fig. 2B), excluding nonspecific
effects.
As an additional test, we measured the mRNA abundance of
BGT1, a transcriptional target of TonEBP/OREBP (16). Rotenone and myxothiazol inhibit the high NaCl-induced increase
of BGT1 mRNA expression by 69% (Fig. 2C), further evidence
of a mitochondrial origin of the ROS that contribute to activation of TonEBP/OREBP.
Rotenone and myxothiazol reduce cellular ATP. However,
rotenone and myxothiazol can reduce cellular ATP, which
complicates the interpretation. In fact, each greatly reduces
ATP in HEK293 cells (Fig. 1B). Therefore, we performed
additional experiments in an attempt to reduce mitochondriaderived ROS without decreasing ATP. Oligomycin at high
concentrations inhibits mitochondrial respiration and reduces
ATP synthesis (7). However, at low concentrations, oligomycin inhibits ROS without a reduction in ATP abundance (19).
Oligomycin (5 ng/ml) inhibits high NaCl-induced increase in
ROS (Fig. 3A) without significantly affecting cellular ATP
abundance (Fig. 3B). It also inhibits high NaCl-induced ORE
reporter activity by 54% (Fig. 3C), similar to the effects of
rotenone and myxothiazol. As a control for specificity, oligomycin does not significantly affect activity measured with the
mutated ORE reporter (Fig. 3D). Combined with our previous
investigation showing that antioxidants N-acetylcysteine
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(NAC) and manganese (III) tetrakis (4-benzoic acid)porphyrin
chloride (MnTBAP) inhibit high NaCl-induced activation of
TonEBP (51), we conclude that a reduction in mitochondrialderived ROS can decrease high NaCl-induced activation of
TonEBP/OREBP independently of any decrease in cellular ATP.
Mitochondrial ROS contribute to high NaCl-induced activation of TonEBP/OREBP by increasing its transactivation.
TonEBP/OREBP contains a TAD, whose activity is increased
by high NaCl (12, 13, 23). To test whether mitochondrial ROS
contribute to high NaCl-induced transactivating activity of
TonEBP/OREBP, we examined the effect of rotenone and
myxothiazol in HEK293 cells stably transfected with a binary
GAL4 transactivation reporter system. Raising osmolality from
290 to 500 mosmol/kgH2O by adding NaCl increases transactivating activity by 11.8-fold (Fig. 4A). Rotenone and myxothiazol, but not allopurinol, reduce high NaCl-induced transactivating activity by 66 and 46%, respectively (Fig. 4A). We
used an otherwise identical reporter that does not contain the
TAD of TonEBP/OREBP to exclude a general effect on transactivation. Rotenone and myxothiazol have no significant effect on the control reporter activity (Fig. 4B). We conclude that
ROS contribute to activation of TonEBP/OREBP by transactivating it.
Contribution of mitochondrial ROS to high NaCl-induced
activation of TonEBP/OREBP does not involve nuclear translocation. At ⬃300 mosmol/kgH2O, TonEBP/OREBP distributes between the cytosol and nucleus. On hypertonic stress,
TonEBP/OREBP translocates into the nucleus within 30 min
(5, 32). We tested whether mitochondrial ROS contribute to
TonEBP/OREBP nuclear translocation by examining the effect
of rotenone and myxothiazol on TonEBP/OREBP protein
abundance in cytoplasm and nucleus. Rotenone and myxothiazol do not significantly affect high NaCl-induced nuclear
translocation of TonEBP/OREBP, nor does the antioxidant
NAC (15 mM) (Fig. 5). We conclude that high NaCl-induced
TonEBP/OREBP nuclear translocation is independent of ROS
in general and of mitochondrial ROS in particular.
High NaCl does not induce release of cellular ATP. In Jurkat
T and neutrophil cells, hypertonicity induces release of cellular
ATP, which triggers cellular osmotic responses (8, 25). To
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Fig. 1. A: rotenone (250 nM) and myxothiazol (12 nM),
but not apocynin (100 ␮M) or allopurinol (100 ␮M),
significantly inhibit high NaCl-induced reactive oxygen
species (ROS) activity in HEK293 cells. ROS activity was
estimated by confocal microscopy, using dihydroethidium
(DHE) as a probe. Osmolality of medium bathing the cells
was increased from 290 to 450 mosmol/kgH2O (NaCl
added) for 45 min. The dye and inhibitors were added, as
indicated, 30 min before the increase in NaCl. Results are
expressed relative to control at 290 mosmol/kgH2O (*P ⬍
0.05, paired t-test, n ⫽ 4). B: rotenone (250 nM) and
myxothiazol (12 nM) significantly inhibit cellular ATP
abundance. HEK293 cells were incubated with inhibitors
for 30 min before osmolality was increased to 500
mosmol/kgH2O by addition of NaCl for 16 h. Then, the
cells were lysed, and cellular ATP was measured with a
bioluminescence assay (**P ⬍ 0.005, paired t-test, n ⫽ 3).
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MITOCHONDRIAL REACTIVE OXYGEN SPECIES ACTIVATION OF TONEBP/OREBP
determine whether this is also true of HEK293 cells, we
measured ATP in the medium. High NaCl does not induce
release of HEK293 cellular ATP (Fig. 6A). To exclude the
possibility that some ATP might be released, but maintain
measurable levels only at the surface of the cells, where it
could still act as a signal, we added the ATPase apyrase (20
U/ml) to eliminate any traces of ATP. Apyrase does not reduce
high NaCl-induced ORE reporter activity (Fig. 6B), excluding
a role for extracellular ATP. We confirmed that apyrase remains active throughout the 16-h incubation at 500 mosmol/
kgH2O by directly measuring the activity at the end of the
experiment (Fig. 6C). We conclude that high NaCl-induced
activation of TonEBP/OREBP does not depend on release of
ATP from the cells.
DISCUSSION
Rotenone and myxothiazol have been widely used to inhibit
mitochondrial production of ROS, despite the fact that they
have other effects as well. Rotenone and myxothiazol inhibit
high NaCl-induced increase in ROS (Fig. 1A) and increase in
TonEBP/OREBP transcriptional activity (Fig. 2). They also
attenuate the high NaCl-induced increase in TonEBP/OREBP
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transactivating activity (Fig. 4) but do not affect high NaClinduced TonEBP/OREBP nuclear translocation (Fig. 5). Thus
the ROS that are induced by high NaCl and contribute to the
activation of TonEBP/OREBP come from mitochondria, and
they mediate high NaCl-induced activation of TonEBP/
OREBP by increasing its transactivating activity, not its translocation to the nucleus.
In the renal cortex, ROS are primarily generated by NADH
oxidase (52). However, this differs in the renal medulla,
associated with higher interstitial osmolality. In the outer
portions of the renal medulla, NADH oxidase and mitochondria are both major sources of ROS, but in the renal papilla
mitochondria are the only major source (52), which we attribute to the higher interstitial NaCl concentration. An increased contribution of mitochondria to overall cellular production of ROS when NaCl is high can explain why rotenone
and myxothiazol reduce ROS at 500 but not at 290 mosmol/
kgH2O (Fig. 1A). Previously, hypoxia and mechanical stretch
were also found to increase mitochondrial production of ROS
(1, 6), and increased ROS were found to trigger cellular
responses (1, 6), analogous to the responses found in the
present studies.
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Fig. 2. A: rotenone (250 nM) and myxothiazol (12
nM), but not allopurinol (100 ␮M), inhibit high
NaCl-induced osmotic responsive element (ORE)
luciferase reporter activity in HEK293 cells. B:
rotenone (250 nM) and myxothiazol (12 nM) have
no significant effect with mutated ORE luciferase
reporter, used as a control for specificity. C: rotenone (250 nM) and myxothiazol (12 nM), but not
allopurinol (100 ␮M), inhibit high NaCl-induced
increase in betaine transporter (BGT1) mRNA expression. HEK293 cells were incubated with inhibitors for 30 min before osmolality was increased to
500 mosmol/kgH2O by addition of NaCl for 16 h.
Results are expressed relative to control at 290
mosmol/kgH2O (*P ⬍ 0.05 compared with the
corresponding control, paired t-test, n ⫽ 3).
MITOCHONDRIAL REACTIVE OXYGEN SPECIES ACTIVATION OF TONEBP/OREBP
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Fig. 4. A: rotenone (250 nM) and myxothiazol (12
nM), but not allopurinol (100 ␮M), inhibit high
NaCl-induced increase in tonicity-responsive enhancer/osmotic response element binding protein
(TonEBP/OREBP) transactivating activity. B: rotenone (250 nM), myxothiazol (12 nM), and allopurinol (100 ␮M) do not significantly affect transactivation when the reporter contains no TonEBP/
OREBP transactivation domain (TAD). HEK293
cells were incubated with chemicals for 30 min
before osmolality was increased to 500 mosmol/
kgH2O by addition of NaCl for 16 h. Results are
expressed relative to control at 290 mosmol/kgH2O
(*P ⬍ 0.05, paired t-test, n ⫽ 3).
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Fig. 3. A: oligomycin (5 ng/ml) inhibits high NaClinduced ROS in HEK293 cells. ROS were measured by confocal microscopy, using DHE as a
probe. Osmolality of medium bathing the cells was
increased from 290 to 450 mosmol/kgH2O by adding NaCl for 45 min. The dye and inhibitor were
added 30 min before exposure of cells to high NaCl.
Results are expressed relative to control at 290
mosmol/kgH2O (*P ⬍ 0.05, paired t-test, n ⫽ 3). B:
oligomycin (5 ng/ml) has no significant effect on
cellular ATP. HEK293 cells were incubated with
oligomycin for 30 min before osmolality was increased to 500 mosmol/kgH2O by addition of NaCl
for 16 h. Then, the cells were lysed, and cellular
ATP was measured. C: oligomycin (5 ng/ml) inhibits high NaCl-induced ORE luciferase reporter activity (*P ⬍ 0.05, paired t-test, n ⫽ 3). D: oligomycin (5 ng/ml) has no significant effect on activity
of the mutated ORE luciferase reporter. HEK293
cells were incubated with the inhibitor for 30 min
before osmolality was increased to 500 mosmol/
kgH2O by addition of NaCl for 16 h.
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MITOCHONDRIAL REACTIVE OXYGEN SPECIES ACTIVATION OF TONEBP/OREBP
Fig. 6. A: high NaCl does not cause detectable release
of ATP from HEK293 cells. Osmolality was increased
to 500 mosmol/kgH2O by addition of NaCl in the
presence of suramin (200 ␮M) to prevent loss of ATP.
B: apyrase (20 U/ml) has no significant effect on high
NaCl-induced ORE reporter activity. HEK293 cells
were incubated with apyrase for 30 min, and then
osmolality was increased to 500 mosmol/kgH2O by
addition of NaCl for 16 h. C: apyrase remains active
after 16 h at 500 mosmol/kgH2O. An aliquot of medium, taken at the end of experiments in B, was incubated with an ATP standard solution at 37°C for 30
min, and then ATP was measured (n ⫽ 3).
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Fig. 5. Rotenone (Rot; 250 nM; A and B), myxothiazol (Myx; 12 nM; A and B), and N-acetylcysteine (NAC; C and D) do not significantly affect high
NaCl-induced TonEBP/OREBP nuclear translocation in HEK293 cells. The cells were incubated with each chemical for 30 min, and then osmolality was
increased to 500 mosmol/kgH2O by addition of NaCl for an additional 30 min. TonEBP/OREBP was measured in cytoplasmic and nuclear extracts by Western
blot analysis (n ⫽ 3).
MITOCHONDRIAL REACTIVE OXYGEN SPECIES ACTIVATION OF TONEBP/OREBP
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prevented by the antioxidant NAC (37). Thus the high NaClinduced increase in mitochondrial ROS production might contribute to activation of p38 by inactivating a PTP that restricts
p38 phosphorylation and activity. Increased p38 activity could
then transactivate TonEBP/OREBP. The process could be
expedited by the presence of both PTPs (38) and p38 (21) in
mitochondria. A previous example of ROS affecting transactivation via phosphorylation by a MAPK is provided by the
effect of PDGF. Activation of PDGF increases ROS, which
contribute to activity of the transcription factor AP-1 (33),
mediated by ERK, which increases phosphorylation of a TAD
located at the COOH terminal of c-Fos, a partner in AP-1
heterodimers (33).
ACKNOWLEDGMENTS
The authors thank Drs. Chris Combs and Daniela A. Malide for expert help
with confocal microscopy.
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In Jurkat T and neutrophil cells, hypertonicity activates
NADPH oxidase to produce ROS. This follows release of
cellular ATP and phosphorylation of p38 kinase through a
putative osmoreceptor system (8, 25). This mechanism must be
cell type specific, however, because we do not observe any
high NaCl-induced release of ATP (Fig. 6), nor do we find
effects of apocynin in HEK293 cells, which express NADPH
oxidase (39) (Fig. 1A). Along the same line, the high NaClinduced increase in ROS is not reduced when either gp91phox
or p47phox, which are critical NADPH oxidase components, is
inactivated in primary cultures of inner medullary collecting
duct cells (49). In contrast, rotenone and other inhibitors of
mitochondrial ROS production block high NaCl-induced ROS
in these cells, consistent with a mitochondrial source of the
ROS (49).
Rotenone and myxothiazol inhibit ROS activity by only
44 – 47%, and higher concentrations of the inhibitors do not
increase the effect (data not shown), implying that there may
be other sources of high NaCl-induced ROS besides mitochondria. Other possible sources besides mitochondria and NADPH
oxidase include xanthine oxidase (46) and nitric oxide synthase
(44). However, NG-monomethyl-L-arginine (a general inhibitor of nitric oxide synthases) (2, 51), apocynin, and allopurinol (Fig. 1A) do not inhibit high NaCl-induced ROS
production, leaving the question as to what the other sources
of ROS might be.
How does hypertonicity increase mitochondrial ROS production? One possibility follows from the fact that mitochondria are anchored to the cytoskeleton via actin binding complexes in the outer membrane (3, 18, 24). This transmits forces
associated with cytoskeleton restructuring to the outer membrane, leading to altered respiratory chain activity and superoxide generation. Examples include increased mitochondrial
ROS production in response to actin cytoskeleton remodeling
triggered by cyclic strain (1) or antibody binding (47). Hypertonicity also induces actin cytoskeleton reorganization (11, 43),
which could produce the same effect. A direct effect of cellular
hyperosmolality is another possibility. Acute hypertonic stress
results in inhibition of substrate oxidation and decrease in ATP
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per se. Instead, hypertonicity compresses voids and decreases
the availability of voids in the membrane phase for quinone
diffusion (28). This could lead to an increase in the life of
electron transport intermediates such as ubisemiquinone,
which generates a major component of ROS, superoxide, by
transferring one electron to one oxygen molecule.
At this point, we can only speculate about how ROS contribute to high NaCl-induced activation of TonEBP/OREBP.
High NaCl is known to induce a MAPK cascade that phosphorylates p38, increasing its kinase activity (43). This activation of p38 contributes to high NaCl-induced increase in
TonEBP/OREBP transcriptional activity (20, 35) by transactivating TonEBP/OREBP (20). Phosphorylation of p38 is a
dynamic and reversible process that is dictated by the balance
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phosphatases, including protein tyrosine phosphatase (PTP)s.
ROS inhibit PTPs through either direct oxidation of the thiol
group in the active cysteine residue or glutathionylation of it
due to an increased oxidized glutathione level (14). Hypertonicity inhibits protein phosphatase activity, which can be
F1175
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