Urea inhibits hypertonicity-inducible TonEBP expression and
action
Wei
Tian and
David M.
Cohen
Divisions of Nephrology and Molecular Medicine and Department of
Cell and Developmental Biology, Oregon Health Sciences University
and the Portland Veterans Affairs Medical Center, Portland, Oregon
97201
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ABSTRACT |
Tonicity-responsive genes are regulated by the TonE enhancer
element and the tonicity-responsive enhancer binding protein (TonEBP)
transcription factor with which it interacts. Urea, a permeant solute
coexistent with hypertonic NaCl in the mammalian renal medulla,
activates a characteristic set of signaling events that may serve to
counteract the effects of NaCl in some contexts. Urea inhibited the
ability of hypertonic stressors to increase expression of TonEBP mRNA
and also inhibited tonicity-inducible TonE-dependent reporter gene
activity. The permeant solute glycerol failed to reproduce these
effects, as did cell activators including peptide mitogens and phorbol
ester. The inhibitory effect of urea was evident as late as 2 h
after the application of hypertonicity. Pharmacological inhibitors of
known urea-inducible signaling pathways failed to abolish the
inhibitory effect of urea. TonEBP action is incompletely understood,
but evidence supports a role for proteasome function and p38 action in
regulation; urea failed to inhibit proteasome function or p38 signaling
in response to hypertonicity. Consistent with its effect on TonEBP
expression and action, urea pretreatment inhibited the effect of
hypertonicity on expression of the physiological effector gene, aldose
reductase. Taken together, these data 1) define a molecular
mechanism of urea-mediated inhibition of tonicity-dependent signaling,
and 2) underscore a role for TonEBP abundance in regulating
TonE-mediated gene transcription.
tonicity-responsive enhancer binding protein; stress; renal; kidney; cell culture; proteasome; mitogen-activated protein kinase
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INTRODUCTION |
ALL ORGANISMS ADAPT TO
HYPERTONICITY at the cellular level through intracellular
accumulation of osmotically active compounds. It is hypothesized that
these compounds function primarily to offset a detrimental decrement in
cell volume that accompanies profound hypertonic stress, however, other
protective roles have been proposed as well. Cells of the mammalian
kidney medulla are routinely subjected to a substantially elevated and
fluctuating ambient osmolarity by virtue of the renal concentrating
mechanism. Genes encoding proteins essential for osmolyte transport or
synthesis are regulated by hypertonicity at the transcriptional level
by a member of the nuclear factor of activated transcription (NFAT) family of transcription factors (12), tonicity-responsive
enhancer binding protein (TonEBP) (15). TonEBP binds to
its cognate DNA consensus sequence, which has been identified in the
promoters governing osmotically responsive expression of these
transporters and metabolic enzymes (reviewed in Ref. 4).
Comparatively little is known about the mechanism of TonEBP regulation
or action.
Urea coexists with hypertonic NaCl in the renal medulla. Some data have
suggested a mutually protective role for these two solutes. Santos et
al. (24) and Neuhofer et al. (20) have shown
that NaCl may protect renal epithelial cells from the potentially adverse effects of urea; we have shown the converse: urea protects medullary cells from the proapoptotic effect of NaCl
(33). Interestingly, earlier data suggested that urea may
abrogate the ability of (or necessity for) hypertonically stressed
cells to maximally accumulate organic osmolytes
(16-18).
For these reasons, we examined the effect of urea on tonicity-inducible
TonEBP expression and action. Urea (but not the permeant solute,
glycerol) suppressed hypertonicity-inducible TonEBP mRNA expression, TonE- mediated reporter gene transcription, and
tonicity enhancer element (TonE)-dependent transcription of aldose
reductase. These effects were not attributable to any of the previously
described actions of urea and were not a consequence of urea
influencing any of the known signaling events leading to enhanced
TonEBP expression.
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EXPERIMENTAL PROCEDURES |
General methods.
Cell culture and solute treatment were performed as previously
described (23, 26). The following inhibitors and stimuli (purchased from Calbiochem unless otherwise indicated) were used: 200 mM urea (Sigma); 100 mM NaCl (200 mosmol/kgH2O; Sigma); 200 mM mannitol, (Sigma); 200 mM glycerol (Sigma); 100 mM epidermal growth
factor (EGF; Sigma); 100 mM 12-O-tetradecanoyl
phorbol-13-acetate (TPA); 50 µM PD98059; 10 µM; LY294002; 30 mM
N-acetylcysteine (Sigma); 50 µM SB203580; 30-100 µg/ml
cycloheximide (Sigma); and 10 µM MG-132. Cells receiving pretreatment
with an inhibitor or solute (i.e., urea) were generally pretreated for
30 min unless otherwise indicated and remained exposed to the
pretreatment compound for the duration of the experiment until
determination of the experimental end point. Depicted data represent
means ± SE unless indicated; statistical significance is assigned
to P < 0.05 (Excel; Microsoft).
TonEBP RT-PCR and RNase protection assay.
Total cellular RNA was prepared using the Trizol reagent (Life
Technologies) in accordance with the manufacturer's directions. Murine
TonEBP partial cDNA was generated by RT-PCR from mouse kidney
poly-A+ RNA (Clontech) by using the Superscript
preamplification system (Life Technologies) for first-strand cDNA
synthesis in accordance with the manufacturer's directions. The RT
reaction was subjected to PCR amplification (30 cycles total at an
annealing temperature of 60°C and an extension time of 90 s)
using the OptiPrime system (buffer 1; Stratagene). 5' Primer
(Portland VA Molecular Biology Core Laboratory, Portland, OR) included
sequences from nucleotides 3570-3590 of the mRNA encoding human
TonEBP [GenBank accession AF089824 (15)]:
ctattgccgatgctcagaacc; 3' primer included nucleotides 4045-4065
from this sequence (read 5'
3'): tgcatggcaactattgagtgc. A ~500-bp
amplified fragment was identified, subcloned into pcDNA3.1/V5/His-TOPO vector [using Eukaryotic TOPO TA Cloning Kit (Invitrogen) according to
the manufacturer's directions], and its identity was confirmed by
sequencing (Core Facility, Vollum Institute for Advanced Biomedical Research, Portland, OR). Although not identical with human TonEBP, most
differences at the amino acid level were conservative (Fig. 1); it was identical at the amino acid
level with unpublished murine sequences corresponding to NFAT5/CAG-8
(GenBank accession nos. AF200687, MMAJ5159, and AF162853). Our sequence
differed from these murine sequences at two nucleotides, neither of
which affected the conceptual translation [identical with AF200687 from 3266 through 3770 (exclusive of primer sequences) except at
positions 3291 (c
g) and 3405 (t
a)]. Vector was linearized with
XhoI, gel-purified (QIAEX II kit; Qiagen), reconstituted in
diethylpyrocarbonate-treated H2O, and used for biotinylated antisense riboprobe preparation using biotin RNA labeling mix and T7
polymerase (Boehringer-Mannheim). Hybridization of probe (800 pg unless
otherwise indicated; see below) with total RNA (15 µg) was performed
at 45°C [RNase protection assay (RPA) II kit; Ambion]; subsequent
digestion with RNase A and T1 was performed in solution at 37°C.
Detection was achieved with the BrightStar BioDetect nonisotopic
detection kit (Ambion). Aldose reductase murine partial cDNA/expressed
sequence tag has been deposited in the public domain and was obtained
from Research Genetics (IMAGE clone 518609); plasmid was linearized
with EcoRI, and antisense riboprobe was transcribed from the
T7 promoter. In selected experiments, labeled riboprobe was increased
by 10- and 100-fold beyond that recommended by the manufacturer to
establish that probe was used in gross excess; results were
indistinguishable from those presented (data not shown).

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Fig. 1.
Conceptual translation of murine tonicity-responsive enhancer
binding protein (TonEBP) partial cDNA. Murine cDNA was RT-PCR amplified
from murine kidney poly-A+ RNA by using primers based on
the human TonEBP sequence (15); (see EXPERIMENTAL
PROCEDURES). , Identity between human
(15) and murine sequences using the single-letter amino
acid code; , conservative substitutions. The first
amino acid in this partial depiction represents amino acid 1008 in the
human sequence. Primer sequences (homologous to the human clone) were
omitted.
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Reporter gene assay.
Plasmid betaine-GABA transporter (BGT)-2X-luciferase (Luc), based on
the TonE element (14), has been previously described (26). Cells were transfected with the plasmid of
interest [10 µg, as well as a lacZ expression plasmid
under the control of the cytomegalovirus long-terminal repeat (CMV
LTR)] via electroporation; luciferase and
-galactosidase
activities were quantitated at 6 h of treatment as previously
described (7) except that for the latter, the Luminescent
-Galactosidase Detection Kit II (Clontech) was used in accordance
with the manufacturer's directions.
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RESULTS |
Approximately 500-bp partial murine TonEBP cDNA was PCR generated
for use in RPA with the murine inner medullary collecting duct
(mIMCD-3) cell line. The sequence was virtually identical with
subsequently deposited but unpublished GenBank sequences encoding
murine NFAT5/CAG-8. Comparison of the conceptual translation of the
partial murine clone to human TonEBP revealed an extremely high degree
of similarity (Fig. 1), apart from a three-amino acid insertion
contributing an additional negative charge.
By RPA, TonEBP expression in the murine renal medullary mIMCD-3 cell
line appeared constitutive; upregulation of expression was observed
with as little as 2 h of treatment with 100 mM (200 mosmol/kgH2O) NaCl (Fig. 2).
Expression was much more robust at 4 and 6 h of treatment.
Pretreatment with urea (200 mM × 30 min; see General
methods) blunted the ability of hypertonic NaCl to increase TonEBP
expression.

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Fig. 2.
Urea inhibits tonicity-inducible TonEBP expression. RNase
protection assay (RPA) of TonEBP (filled arrowhead) and actin (Control;
open arrowhead) mRNA expression from murine inner medullary collecting
duct (mIMCD-3) cells treated with 100 mM NaCl (200 mosmol/kgH2O) for the indicated interval, in the absence
(NaCl) or presence (Urea + NaCl) of 30-min pretreatment with 200 mM urea, is shown. P in this and subsequent figures indicates
undigested probe alone (~10% of the activity added to RNA samples in
other lanes). One of two replicate experiments is depicted.
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Previous data have indicated that some properties of the permeant
solute urea are mimicked by other permeant solutes whereas others are
not (e.g., Ref. 33). Therefore, the ability of urea to
prevent hypertonicity-inducible TonEBP expression was compared with
another permeant solute, glycerol. Urea pretreatment, but not
glycerol pretreatment, inhibited NaCl-inducible TonEBP expression (Fig.
3A). In all repeats of this
experiment, urea inhibited NaCl-inducible TonEBP expression by at least
25% (and generally much more) whereas glycerol, when applied at
equiosmolar amounts, failed to reproduce this effect. Prolonged
treatment with either 200 mM urea or glycerol failed to independently
increase TonEBP expression. The effect of urea (and lack of effect of
glycerol) was also apparent in the nonrenal 3T3 cell line (Fig.
3B). Results of densitometric reduction of autoradiograph
data from three independent experiments using mIMCD-3 cells are
depicted in Fig. 3C.

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Fig. 3.
Glycerol fails to block TonEBP expression. RPA of TonEBP
(filled arrowhead) and actin (Control; open arrowhead) mRNA from
mIMCD-3 cells (A) and 3T3 cells (B) treated with
100 mM NaCl × 6 h, in the absence ( ) or presence (+) of
30-min pretreatment with the indicated permeant solute (applied at 200 mM) is shown. C: densitometric reduction of autoradiograph
data from 3 separate experiments using mIMCD-3 cells.
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Because of the ability of urea to block TonEBP expression, the effect
of urea on TonEBP-dependent transcription, a downstream event, was
explored next. Cells were transfected with a luciferase reporter gene
(Fig. 4A) under the control of
two tandem repeats of the BGT tonicity-responsive enhancer element
(14), as well as a control plasmid harboring the
lacZ gene with expression directed by the constitutively
active CMV long-terminal repeat promoter. As anticipated, and
consistent with the data of others, NaCl treatment (100 mM × 6 h) increased normalized reporter gene activity ~10-fold in
mIMCD-3 cells (Fig. 4B). Urea pretreatment (200 mM × 30 min) substantially suppressed the effect of hypertonic NaCl, whereas glycerol pretreatment was again ineffective. By way of comparison, urea
pretreatment resulted in a modest increase (~50%) in transcription from the control CMV LTR promoter, arguing against a global inhibitory effect of urea in this context. Similarly, urea pretreatment followed by NaCl treatment resulted in essentially no net effect on
transcription from the CMV LTR (data not shown). As an additional
control, the effect of urea on a nonosmotically responsive but
specifically regulated promoter was examined. Cells (mIMCD-3) were
transfected with a plasmid harboring a luciferase gene under the
control of the glucocorticoid-responsive MMTV promoter (MMTV-Luc;
Clontech). Dexamethasone treatment (300 nM × 24 h) increased
luciferase activity from 170 ± 170 to 9,600 ± 2,400 relative light units; in the presence of urea pretreatment (200 mM × 30 min), dexamethasone increased luciferase activity from 590 ± 70 to 8,100 ± 300 relative light units (data not shown).
Neither control nor dexamethasone-inducible values in the presence of
urea pretreatment were statistically distinct from those obtained in
the absence of urea pretreatment. Because some but not all effects of
urea appear to be specific to cells of renal epithelial origin (e.g.,
MDCK and mIMCD-3 cells), the modulatory effect of urea was examined in
nonrenal fibroblastic 3T3 cell line. Similar to the mIMCD-3 model,
hypertonic NaCl (100 mM × 6 h) increased reporter gene
activity by >10-fold (Fig. 4C). Also consistent with
the mIMCD-3 model, urea pretreatment but not glycerol pretreatment
substantially blocked the effect of hypertonic NaCl.

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Fig. 4.
Urea inhibits TonEBP-mediated transcription. Luciferase
(Luc) activity (normalized to -galactosidase activity from a
lacZ-expressing constitutively active cotransfected plasmid)
representing transcription of a thymidine kinase promoter under the
control of 2 tandem repeats of the betaine-GABA transporter (BGT)
tonicity-responsive consensus element (A) in mIMCD-3
(B) and 3T3 (C) cells receiving no pretreatment
(Control) or 30-min pretreatment with either urea (+Urea; 200 mM) or
glycerol (+Glycerol; 200 mM) before treatment with NaCl (100 mM × 6 h) is shown. Data in each panel are normalized to control
pretreatment in the absence of NaCl treatment (first bar in graph) and
represent the means ± SE of at least 4 separate experiments, each
with determinations performed in duplicate or triplicate.
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Some data have suggested that there is a unique relationship between
urea and NaCl that does not extend to all hypertonic stressors. The
effect of urea in the context of mannitol-associated hypertonicity was
therefore examined in mIMCD-3 cells. The effect of mannitol (200 mM)
was indistinguishable from that of equiosmolar NaCl (100 mM) at
activating reporter gene activity (Fig.
5). Urea significantly blocked the effect
of hypertonic mannitol on BGT-2X-Luc activity; despite the tendency
toward a greater effect on NaCl-inducible transcription (84%
inhibition) than mannitol-induced transcription (60% inhibition),
there was no statistically significant difference between the two
solutes with respect to protection afforded by urea.

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Fig. 5.
Urea also blocks the effect of mannitol-associated
hypertonicity. Luc activity (normalized to -galactosidase activity)
in mIMCD-3 cells cotransfected with BGT-2X-Luc and cytomegalovirus
(CMV)-Gal, and treated with the indicated solute for 6 h in the
absence ( Urea) or presence (+Urea) of urea pretreatment (200 mM × 30 min) is shown. Data are normalized to control treatment in the
absence of urea treatment (first open bar in graph) and represent the
means ± SE of at least 3 separate experiments, each with
determinations performed in triplicate.
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The ability of urea to protect from hypertonicity-inducible
apoptosis resembled that of peptide growth factors such as EGF and insulin-like growth factors (33). Therefore, the
effect of other cell activators on hypertonicity-inducible
transcription was explored. Neither the mitogen, EGF, nor the classic
protein kinase C (cPKC)-activating TPA, affected tonicity inducibility of the luciferase reporter gene (Fig. 6).
Specifically, induction by NaCl with no pretreatment was 9.9-fold, with
EGF pretreatment, 9.2-fold, and with TPA pretreatment, 9.5-fold. In
contrast, induction by hypertonicity was only 4.6-fold in the presence
of urea pretreatment.

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Fig. 6.
Other cell activators fail to suppress tonicity-inducible
TonEBP-mediated transcription. Luc activity (normalized to
-galactosidase activity) in mIMCD-3 cells cotransfected with
BGT-2X-Luc and CMV-Gal and receiving the indicated pretreatment (200 mM
urea; 100 nM epidermal growth factor; 100 nM
12-O-tetradecanoyl phorbol-13-ester) 30 min before isotonic
(Control) or hypertonic (+100 mM NaCl) treatment × 6 h is
shown. Induction by NaCl [(+NaCl)/( NaCl)] is indicated numerically
for each pretreatment group. Data are normalized to control treatment
in the absence of pretreatment (first bar in graph) and represent the
means ± SE of 3 separate experiments, each with determinations
performed in triplicate.
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We next sought to determine whether the ability of urea to block
TonEBP-mediated transcription required pretreatment with urea or
whether concurrent administration would be suitable. Urea was added at
varying time points relative to the institution of hypertonic stress
(from 60 min before to 120 min after), and the effect on
tonicity-mediated transcription was determined (Fig. 7). NaCl treatment (200 mosmol/kgH2O × 6 h) increased reporter gene
activity ninefold. Urea was equally inhibitory when added 60 or 30 min
before, or simultaneously with NaCl. Even 2 h after NaCl
treatment, urea still partially abrogated the effect of hypertonicity.

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Fig. 7.
Influence of pretreatment interval on ability of urea to
protect from tonicity-inducible TonEBP-mediated transcription. Luc
activity (normalized to -galactosidase activity, and expressed
relative to Control) in mIMCD-3 cells cotransfected with BGT-2X-Luc and
CMV-Gal and treated with 200 mM urea at the indicated time relative to
treatment with 100 mM NaCl (e.g., " 30" indicates urea treatment
precedes NaCl treatment by 30 min is shown). The dashed line labeled
"+ NaCl Alone" indicates reporter gene activity in response to
treatment with NaCl without urea, where the shaded region corresponds
to ±SE; the dashed line labeled "No Treatment" indicates
reporter gene activity in the absence of urea or NaCl treatment (there
is no shaded region because SE = 0 following normalization to
Control). Data are normalized to control treatment in the absence of
pretreatment and represent the means ± SE of 3 separate
experiments, each with determinations performed in triplicate.
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Multiple signaling pathways have been shown to be activated by urea
treatment in the mIMCD-3 model. Extracellular signal-regulated kinase
signaling mediates, in part, the ability of urea to upregulate immediate-early gene expression (5). Phosphoinositide
3-kinase signaling is instrumental in renal epithelial cell survival of urea and hypertonic stress (35). Oxidative stress mediates
the ability of urea to activate transcription and expression of the stress-responsive transcription factor, Gadd153 (34). We
therefore examined the effect of pharmacological inhibitors of each of
these signaling events to assess whether the respective pathways
mediated the ability of urea to block signaling to TonEBP expression
and action. Although some of these compounds exhibited modest effects on basal and hypertonicity-inducible TonEBP action (i.e., reporter gene
activity), none significantly influenced the ability of urea to block
signaling by hypertonicity (Fig. 8).
Although urea has been shown by some to modestly activate p38, a
pharmacological inhibitor of this pathway was not examined in the
transient transfection assay (see Figs. 10 and 11) because it would be
expected to independently inhibit potentially p38-mediated
tonicity-inducible transcription (25), rendering the data
uninterpretable.

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Fig. 8.
Effect of inhibitors of urea signaling on urea-associated
inhibition of TonEBP signaling. Luc activity (normalized to
-galactosidase activity, and expressed relative to "Control"
with "No addition") in mIMCD-3 cells cotransfected with BGT-2X-Luc
and CMV-Gal and pretreated for 30 min with the indicated inhibitors of
known urea-inducible signaling events (50 µM PD98059; 10 µM
LY294002; or 30 mM NAC) before treatment with NaCl alone (100 mM × 6 h), or urea (200 mM × 30 min) followed by NaCl. Data
are normalized to control treatment in the absence of pretreatment
(first open bar in graph) and represent the means ± SE of 3 separate experiments, each with determinations performed in
triplicate.
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TonEBP-mediated transcription is blocked by pharmacological inhibitors
of proteasome function (29). It was therefore hypothesized that urea might act via inhibiting proteasome function. Consistent with
the findings of Woo et al. (29), proteasome inhibition with MG-132 pretreatment substantially inhibited TonE-dependent transcription of the BGT-2X-Luc reporter gene (Fig.
9A); the degree of inhibition
was comparable to that seen with urea pretreatment (e.g., Fig. 4).
MG-132 treatment also resulted in significant accumulation of high- and
low-molecular-weight ubiquitinated proteins (as determined via
anti-ubiquitin immunoblotting; Fig. 9B) as would be
anticipated with inhibition of proteasome-dependent catabolism; however, urea failed to mimic this effect. Therefore, urea does not
appear to influence TonEBP signaling via inhibition of proteasome function.

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Fig. 9.
The effect of urea on TonEBP signaling is not mediated by
inhibition of proteasome function. A: Luc activity
(normalized to -galactosidase activity, and expressed relative to
Control) in mIMCD-3 cells cotransfected with BGT-2X-Luc and CMV-Gal,
receiving no treatment (Control) or hypertonicity (+NaCl; 100 mM × 6 h) in the absence ( MG-132) or presence of 30-min
pretreatment with the proteasome inhibitor, MG-132. B:
effect of indicated pretreatment (None, 200 mM Urea × 30 min, 10 µM MG-132 × 30 min) on global protein ubiquitination following
6 h of control (C) or NaCl (N; 200 mosmol/kgH2O NaCl) treatment, as assessed by anti-ubiquitin
immunoblotting. One of two replicate experiments is depicted.
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Activation of the p38 mitogen-activated protein kinase (MAPK) has also
been variably implicated in tonicity-dependent gene regulation
(25). Therefore, a possible role for urea in inhibiting tonicity-inducible p38 activation was explored. In mIMCD-3 cells, hypertonic NaCl (200 mosmol/kgH2O) dramatically increased
p38 phosphorylation as determined by anti-P-p38 immunoblotting (Fig. 10), consistent with prior observations
(3, 32). Not only did urea pretreatment fail to inhibit
this effect, it exerted a modest potentiative influence. As a correlate
of these data, the ability of p38 to mediate the effect of NaCl on
TonEBP expression was examined using the RPA assay. Pretreatment of
mIMCD-3 cells with the p38 inhibitor, SB203580, failed to influence
NaCl-inducible TonEBP mRNA induction (Fig.
11). Specifically, SB203580 inhibited basal TonEBP mRNA expression by ~16% (n = 3;
P < 0.05). NaCl treatment increased TonEBP expression
by 150% (relative to control
SB203580) in the absence of
SB203580 and by 80% (relative to control + SB203580) in its
presence, but there was no statistically significant difference between
NaCl treatment in the presence or absence of the p38 inhibitor (n = 3; data not shown).

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Fig. 10.
Effect of urea on tonicity-inducible p38
phosphorylation. Effect of NaCl treatment (100 mM) for the indicated
duration (in h), in the presence (+Urea) and absence ( Urea) of
pretreatment with urea (200 mM × 30 min) on p38 phosphorylation
as determined by anti-P-p38 immunoblotting is shown. A:
representative immunoblot. B: means ± SE of
densitometric data from 3 independent experiments. P < 0.05 with respect to the absence of urea pretreatment.
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Fig. 11.
Effect of protein synthesis inhibition on expression of
TonEBP and aldose reductase. Effect of pretreatment with the indicated
concentration of cycloheximide (CHX; × 30 min) on control treatment
(C) and hypertonicity-inducible (H; 100 mM NaCl × 6 h)
expression of TonEBP (A) and the tonicity-responsive
effector gene, aldose reductase (B), as determined by RPA is
shown. One of two replicate experiments is depicted. The last 2 lanes
in A [excluding probe-only lane (P)] indicate the effect
on TonEBP expression of pretreatment with the p38 inhibitor, SB203580
(50 µM × 30 min). These latter data are representative of 3 such independent experiments.
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Because urea and hypertonicity may influence protein synthesis, we also
examined the dependence of NaCl-inducible TonEBP expression on protein
synthesis. The nonspecific inhibitor of protein synthesis, cycloheximide, failed to influence the ability of hypertonic NaCl to
increase TonEBP expression (Fig. 11). To confirm that an effective dose
of cycloheximide had been used, the effect of cycloheximide on
tonicity-inducible expression of the aldose reductase gene was examined
(Fig. 11B). Consistent with a requirement for a prior round
of protein synthesis (e.g., expression of the TonEBP protein), cycloheximide pretreatment dramatically inhibited the ability of
hypertonic NaCl to increase aldose reductase mRNA expression without
influencing the level of expression of the control actin gene.
Last, we sought to determine the functional consequence of the ability
of urea to downregulate TonEBP signaling. Because osmotically responsive genes such as aldose reductase and the betaine cotransporter are regulated at the transcriptional level by TonEBP and the tonicity enhancer element, the effect of urea on tonicity-inducible expression of such genes was investigated. As anticipated, aldose reductase expression was dramatically upregulated by hypertonic mannitol or NaCl,
whereas urea failed to increase expression (Fig.
12A). These changes occurred
in the absence of any effect on actin gene expression (Fig.
12A). Consistent with data concerning the effect of urea on
TonEBP expression and TonEBP-dependent reporter gene expression, urea
pretreatment substantially inhibited the effect of either NaCl or
mannitol on aldose reductase expression (Fig. 12B).

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Fig. 12.
Urea inhibits tonicity-inducible aldose reductase gene
expression. A: effect of urea (200 mM), mannitol (200 mM),
and NaCl (100 mM) on aldose reductase and actin mRNA expression by RPA
following 6 or 24 h of treatment in mIMCD-3 cells. B:
effect of urea pretreatment (200 mM × 30 min) on NaCl- (100 mM × 6 h) and mannitol-inducible (200 mM × 6 h)
TonEBP and actin expression by RPA. P indicates probe alone. For each
figure, 1 of 2 replicate experiments is depicted.
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DISCUSSION |
Urea inhibits the ability of hypertonicity to increase expression
of TonEBP, and thereby blocks downstream TonEBP-mediated transcription.
Because in the renal medulla, the only mammalian tissue routinely
subjected to marked hypertonicity, this stress coexists with an
elevated urea concentration, the inhibitory effect of urea may have
significant implications in vivo. Much emphasis in
hypertonicity-inducible gene regulation has focused on the role of
posttranslational modification of the TonEBP transcription factor
itself (15, 29); acute regulation of TonEBP mRNA or protein abundance has received less attention (e.g., Ref.
28). Unexpectedly, expression of TonEBP itself appears to
be regulated fairly early (2-6 h) following application of
hypertonicity. In corresponding fashion, TonEBP-dependent transcription
is also markedly upregulated by 6 h of hypertonic treatment.
Miyakawa et al. (15) and Woo et al. (29) had
earlier observed upregulated TonEBP expression at 18 h of
hypertonic stress in the MDCK model. A single recent observation
suggested upregulation in TonEBP mRNA abundance as early as 6 h;
however, this increase was very modest (~100% increase)
(28). Our finding that TonEBP expression and action are
upregulated relatively early in this model might suggest a greater role
for TonEBP abundance (in contrast to activation) in implementation of
the downstream effector response to hypertonicity. In further support
of this hypothesis is the observation that inhibition of TonEBP protein
synthesis with cycloheximide blocks the tonicity-inducible upregulation
in expression of the hypertonicity effector gene, aldose reductase
(Ref. 1 and data herein). Such data, obtained through the
use of a global inhibitor of protein synthesis such as cycloheximide,
do not definitively establish the inhibition of TonEBP protein
expression as the proximate cause of this effect. In contrast, the
ability of urea to specifically block expression of both TonEBP and
aldose reductase substantially strengthens this argument [i.e., urea
is not a global inhibitor of protein synthesis (6) and is
therefore a more specific inhibitor of TonEBP expression than is
cycloheximide]. In aggregate, however, these data do not establish
that upregulated TonEBP expression is sufficient for the
hypertonicity-inducible transcriptional response.
It is puzzling that urea, which is known to counteract some of the
adverse effects of NaCl exposure (33), should block the ability of hypertonicity to increase TonEBP expression or action. Such
data raise the provocative question of whether TonEBP expression is an
adaptive or maladaptive event in the hypertonic stress response. Because genes deemed essential for adaptation to hypertonic stress (e.g., aldose reductase, betaine cotransporter,
Na+-dependent myo-inositol cotransporter)
represent downstream targets of TonEBP, it should stand to reason that
suppression of TonEBP upregulation and action would sensitize cells to
hypertonicity-inducible cell death. In fact, however, a number of
studies have suggested a beneficial effect of combining urea and NaCl
(20, 24, 33). Because interruption of signaling and gene
expression events required for osmolyte accumulation are detrimental to
cell and organismic functioning, it is highly likely that an ancillary
non-TonEBP-dependent protective pathway is upregulated in the presence
of urea pretreatment, limiting the requirement for maximal TonEBP
expression in response to hypertonicity. The heat shock proteins may
directly influence MAPK (e.g., p38) signaling (2), which
has been implicated in TonEBP-mediated transcriptional regulation and
acquisition of the hypertonically-stressed phenotype (11, 25,
27). Most (e.g., Refs. 8 and 21), but not all
(13), studies have failed to demonstrate upregulated heat
shock protein (e.g., hsp27 or hsp70) expression in response to urea
stress; involvement of a subset of the heat shock proteins has,
however, been inferred by some (19, 22). It remains
possible that urea-inducible heat shock protein upregulation, or
another as yet undescribed mechanism, simultaneously protects from
hypertonic stress and abrogates TonEBP signaling.
The urea-initiated signaling events through which TonEBP expression is
inhibited are unclear. Several groups have examined the role of p38 in
tonicity-inducible transcriptional regulation and have arrived at
contradictory conclusions (10, 25). Proteasome processing
has also been recently implicated (29). Urea fails to
influence either p38 signaling or proteasome function. In addition, none of several previously identified urea-inducible signaling cascades
(31, 34, 35) appears to be involved.
The ability of urea to influence tonicity-inducible gene regulation may
explain previously recognized phenomena in other contexts. In renal
epithelial cells, urea treatment induces the accumulation of only
glycerophosphorylcholine (GPC), and this is a consequence of decreased
GPC degradation (reviewed in Ref. 9). Hypertonic NaCl treatment in
these models induces accumulation of betaine and
myo-inositol via increased transcription of the genes
encoding their principal Na+-dependent cotransporters
(reviewed in Ref. 4). Interestingly, treatment of renal epithelial
cells with urea resulted in a decrement in intracellular betaine
content in several models (16-18); this could
represent the consequence of an inhibitory effect of urea on
(basal) TonE-mediated transcription as is observed in the present data. Similarly, there has been a trend for treatment of cells with
urea plus NaCl to effect a more modest accumulation of betaine than is
observed in the presence of NaCl treatment alone
(16-18). Again, the ability of urea to abrogate
hypertonicity-inducible TonE-mediated transcription could account for
this finding. Because of the purportedly protective effect of betaine
with respect to urea stress (reviewed in Ref. 30), the inability of
urea-treated cells to appreciably accumulate betaine has proved
puzzling. A direct inhibitory effect of urea on the signaling events
leading to upregulated betaine transport offers a mechanistic (although not a teleological) explanation. Again, it would be anticipated that
such a potentially maladaptive event would exacerbate NaCl-inducible apoptosis, rather than inhibit it (33); however,
additional urea-responsive protective events are likely operative,
obviating the need for maximally upregulated TonEBP-dependent signaling.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institutes of Diabetes and
Digestive and Kidney Diseases Grant DK-52494, the American Heart
Association, and by the Department of Veterans Affairs.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. M. Cohen, Mailcode PP262, Oregon Health Sciences Univ., 3314 S.W. US
Veterans Hospital Rd., Portland, OR 97201 (E-mail:cohend{at}ohsu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 October 2000; accepted in final form 29 January 2001.
 |
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