ERK activation by urea in the renal inner medullary mIMCD3
cell line
Xiao-Yan
Yang,
Zheng
Zhang, and
David M.
Cohen
Divisions of Nephrology and Molecular Medicine, Oregon Health
Sciences University and the Portland Veterans Affairs Medical
Center, Portland, Oregon 97201
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ABSTRACT |
Urea- and NaCl-inducible extracellular signal-regulated kinase
(ERK) phosphorylation exhibited dissimilar kinetics. Among cell lines examined, the effect of urea was unique to mIMCD3 inner medullary collecting duct cells and MDCK cells.
Urea-inducible ERK activation was ~10-fold less sensitive to the MEK
inhibitor, PD-98059, than was that of NaCl. This difference did not
appear to be accounted for by differential activation of MEK isoforms. Interestingly, the inhibitor of p38 activation, SB-203580, abrogated the effect of both urea and NaCl upon both ERK and MEK activation; however, the former was much less sensitive to the inhibitor. Consistent with this observation, NaCl was much more effective than
urea at inducing p38 phosphorylation. The effect of hypertonic stress
(e.g., sorbitol 100 mM) could be blocked by appropriate medium dilution
such that isotonicity was maintained. In marked contrast, the effect of
hyperosmotic urea could not be blocked in this fashion, implying the
absence of dependence upon cell volume. Together, these data suggest
that cells of the renal inner medulla are potentially uniquely
responsive to urea and that urea and hypertonic stressors induce ERK
activation through distinct mechanisms.
mitogen-activated protein kinase; sodium chloride; p38; extracellular signal-regulated kinase
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INTRODUCTION |
CELLS OF THE RENAL INNER medulla are continually
exposed to an elevated concentration of both NaCl and urea in vivo.
NaCl (11, 13, 22) and urea (5) both activate the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs)
in renal epithelial cells, but in other respects the signaling pathways
activated by these stimuli are distinct. Hypertonicity, acting through
a putative tonicity-responsive transcription factor (17), activates
transcription of genes whose protein products are involved in the
synthesis or transport of organic osmolytes (reviewed in Ref. 3).
Although little is known about the signaling events involved, this
process has been reported to be either dependent on (9, 20) or
independent of (12) activation of the stress-responsive p38 MAPK in
different models. In contrast, urea, in cells of the renal inner
medulla, activates hallmarks of a receptor tyrosine kinase-mediated
pathway including transcription of immediate-early genes in an ERK- and
Elk-1-dependent fashion (5, 6, 8) and activation of phospholipase C-
with resultant generation of inositol 1,4,5-trisphosphate (7). The
ability of these two disparate signaling pathways to converge upon ERK
activation prompted us to investigate the mechanism of
urea- and hypertonicity-inducible ERK activation.
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METHODS |
Cell culture and treatment.
mIMCD3 inner medullary collecting duct cells were grown
and passaged as previously described. mDCT distal convoluted tubule
(kindly supplied by P. Friedman, Dartmouth Medical
School), MDCK, HEK293, COS-7, and NIH3T3 cells were
maintained in DMEM-F12 supplemented with 10% fetal bovine serum and
passaged after confluence. Supplementation of medium with
hyperosmotic stressors was performed in situ through addition of an
aliquot of concentrated solute in sterile water. Additional control
experiments were performed via complete medium change to medium that
had previously been supplemented with the relevant solute. For
experiments combining addition of solute with dilution of the
hyperosmotic medium (see Fig. 12), appropriate volumes of sterile water
were premixed with added solute (sorbitol or urea) prior to addition to
the well such that both stimuli were applied simultaneously.
Pretreatment with PD-98059 and SB-203580 (at the indicated
concentrations) was performed for 30 min.
Immunoblotting. Whole cell detergent
lysates were prepared and subjected to electrophoresis as previously
described (6). Anti-phospho-ERK, anti-phospho-p38,
anti-phospho-MEK1/2, anti-phospho-MKK4, anti-phospho-MKK3/6
(all from New England BioLabs), anti-MEK1 (Santa Cruz Laboratories),
and anti-MEK2 (Santa Cruz Laboratories) antibodies were used for
immunoblotting or immunoprecipitation according to the manufacturer's
directions. MEK1 and MEK2 were discriminated on the basis
of approximate molecular mass; however, control experiments were also
performed with immunoprecipitation with isoform-specific antibody
followed by phospho-specific antibody immunoblotting to confirm the
identity of the differentially migrating bands. In the short time
courses examined for the present study, there was no appreciable change
in total ERK or MAPK/ERK kinase (MEK) abundance as assessed by anti-ERK
and anti-MEK immunoblotting in the setting of urea or NaCl treatment.
On the basis of molecular mass standards, immunoreactive bands migrated
at the following approximate molecular masses: anti-P-ERK, 42 and 44 kDa; anti-P-MEK, doublet at 57 kDa; anti-P-p38, 40 kDa; anti-P-MKK4, 51 kDa; and anti-P-MKK3/MKK6, 43 and 45 kDa (identity of MKK3 and MKK6 was assigned on the basis of relative molecular mass). Unless indicated, depicted immunoblots are representative of at least two separate experiments.
Where data are depicted graphically, immunoblots from between three and
seven separate experiments were quantitated via scanning densitometry
using an Epson ES-1000C scanner and Image software (National Institutes
of Health). For experiments describing the effects of PD-98059 upon
urea- and NaCl-inducible ERK and MEK phosphorylation (see Figs. 4 and
7), 8 µg of protein from NaCl-treated cells and 4 µg of protein
from urea-treated cells was used in an effort to generate approximately
equivalent maximal signal intensity and thereby minimize confounding by
film sensitivity and linearity. For determination of MEK isoform
phosphorylation, area under the complex curve generated
densitometrically was mathematically reduced into peaks corresponding
to MEK1 and MEK2 phosphorylation. In all lanes examined, anti-P-MEK
immunoreactivity corresponding to MEK1 exceeded that of MEK2 (e.g.,
Fig. 7B). On the basis of approximate interpeak distance (distance between MEK1 and MEK2 arrowheads in Fig. 7, A and
B) and with the assumption of a
near-normal distribution for each peak, it was estimated that the
contribution of MEK2 anti-P-MEK immunoreactivity to the principal peak
(corresponding primarily to MEK1) was relatively small (see below).
Therefore, a vertical line was scribed from the apex of the MEK1 peak
(maximum y value) perpendicular to the
baseline (x-axis), separating the area
under the curve into components A and
B (see Fig.
7B). The degree of MEK1
phosphorylation was approximated as (2 × B) and that for MEK2
phosphorylation, A
B. Although this method potentially introduced a modest but consistent inaccuracy (a very minor component of the area of component B was
potentially contributed by MEK2), it was superior to arbitrarily
dividing the complex curve into components corresponding to the MEK1
peak and the MEK2 peak, particularly at relatively low levels of MEK2 phosphorylation.
MEK activity. MEK activity was
assessed via immune complex kinase assay as previously described (21).
Briefly, mIMCD3 monolayers were washed with ice-cold PBS, lysed in situ
with lysis buffer (in mM: 50 HEPES, 150 NaCl, 1.5 MgCl2, 1 EGTA, 10 NaF, pH 7.5, 800 µl/P100 dish) supplemented with 10% glycerol, 1%
Triton X-100, 1% phenylmethylsulfonyl fluoride, and 10 µg/ml
aprotinin for 15 min at 0°C. Lysate was scraped from the plate,
centrifuged at 4°C for 5 min in a microcentrifuge to pellet
insoluble debris, and subjected to immunoprecipitation with anti-MEK1
or -MEK2 (2 µg/sample) and Protein A/G beads (25 µl/sample).
Immunoprecipitates were washed twice with lysis buffer and once with
incomplete kinase buffer (in mM: 50 Tris, pH 7.4, 10 MgCl2, and 1 dithiothreitol), and
then resuspended with 20 µl of complete kinase buffer
[incomplete kinase buffer + 50 µM ATP + kinase-inactive ERK
substrate (Santa Cruz Laboratories; 1 µg/sample) + [
-32P]ATP (1/40
volume)]. Kinase reaction was performed at 30°C
for 40 min and terminated with one volume of 2× Laemmli sample
buffer. Radiolabeled immunoprecipitates were resolved via SDS-PAGE and exposed for autoradiography. Densitometry was quantitated as described above.
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RESULTS |
Urea activates ERKs more potently and earlier than
does NaCl in mIMCD3 cells. We have shown that urea
activates Egr-1 transcription in a largely ERK-dependent fashion in
renal medullary cells (5). To determine the relevance of this effect at
lower concentrations of urea, dose-response studies were performed.
Urea in concentrations as low as 20 mM activated ERK (p44 and p42), as
measured by phospho-specific antibody immunoblotting (Fig.
1). The half-maximal effect occurred at
~40 mM (data not shown), consistent with that observed in the context
of urea-inducible immediate early gene transcription (8). The effect of
urea (200 mM) upon ERK activation was maximal at 5 and 10 min and was
essentially undetectable by 60 min (Fig. 2). In contrast, treatment of cells with an
equiosmolar dose of NaCl (100 mM) revealed minimal activation at
5 min. NaCl exerted its maximal effect at 15 min of
treatment, and this degree of activation was considerably less than
that of urea. NaCl-inducible ERK activation was still evident at 120 min, the last time point examined.

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Fig. 1.
Dose response of urea effect upon extracellular signal-regulated kinase
(ERK) (p44 and p42) phosphorylation. Anti-phospho-ERK immunoblot of
detergent lysates of mIMCD3 inner medullary collecting duct cell
monolayers treated with the indicated concentration of urea for 10 min.
C, control.
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Fig. 2.
Time course of urea- and NaCl-inducible ERK phosphorylation.
Anti-phospho-ERK immunoblot of detergent lysates of mIMCD3 monolayers
treated for the indicated intervals with equiosmolar urea (200 mM) or
NaCl (100 mM).
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Urea-inducible ERK activation is not
universal. In marked contrast to observations with
mIMCD3 cells, urea and NaCl (each at 200 mosmol/kgH2O) failed
to activate ERK in two renal nonmedullary cell lines, HEK293 and COS-7
cells (Fig. 3). These cell lines were
originally examined because of the readiness with which they may be
transfected and with the expectation that they might provide a
tractable model for examining urea-inducible signaling. An extremely modest response to urea in the murine mDCT renal cell line could not be
excluded. Only in the MDCK cell line was a robust response to urea
observed. In the nonrenal NIH3T3 cell line, urea induction was absent.
In all cell lines examined except for mIMCD3 and MDCK, however, the
effect of phorbol ester (a positive control for ERK activation)
markedly exceeded that of urea (Fig. 3). Therefore, in only these two
lines was a potent activation in response to urea observed.

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Fig. 3.
Cell type specificity of urea effect. Effect of no treatment (C), urea
(U, 200 mM), NaCl (100 mM), or
12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) for 10 min upon ERK phosphorylation in mIMCD3, MDCK, mDCT, HEK293, COS-7, and
3T3 cells.
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Differential sensitivity of urea- and NaCl-inducible
ERK phosphorylation to PD-98059. Because of the
discrepant kinetics between urea- and NaCl-inducible ERK activation,
the role of upstream ERK activators was examined. It was observed that
NaCl was less potent than urea at activating ERK phosphorylation;
therefore, approximately equipotent doses of urea (200 mM) and NaCl
(200 mM/400 mosmol/kgH2O) were
established densitometrically and used for further comparison. At each
given time point of solute treatment, urea treatment was much less
sensitive to the MEK inhibitor PD-98059 than was NaCl treatment (Fig.
4). At 5 min of NaCl treatment, the
IC50 for PD-98059 was ~1.3 µM,
whereas at 5 min of urea treatment it was ~20 µM. Similarly, at 15 min of NaCl treatment, the IC50 for PD-98059 was ~8 µM, whereas the effect of even
100 µM PD-98059 upon urea-inducible ERK activation was modest (25%
inhibition). There was a tendency for very high concentrations of
PD-98059 (100 µM) to activate ERK in other treatment groups. This
differential PD-98059 sensitivity of urea- and NaCl-inducible ERK
phosphorylation was also evident when urea and NaCl of equivalent
osmolarity were applied (Fig. 4C).
Hypotonicity-inducible ERK phosphorylation was also highly sensitive to
PD-98059, analogous with hypertonicity (data not shown). There appeared
to be lot-to-lot variability in the potency of PD-98059; for each
experiment, the effects of PD-98059 upon both NaCl and urea were
evaluated in parallel.

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Fig. 4.
Differential sensitivity of urea- and NaCl-inducible ERK activation to
PD-98059. Inhibitory effect of 30-min pretreatment with increasing
concentration of PD-98059 upon ERK phosphorylation in response to urea
treatment (200 mM for 15 or 5 min) or NaCl treatment (200 mM/400
mosmol/kgH2O for 15 or 5 min) in
mIMCD3 cells. Representative anti-phospho-ERK immunoblots are depicted
(A), as are pooled densitometric
data from 4-7 independent experiments
(B). In
C, the effect of either 200 or 400 mosmol/kgH2O urea or NaCl (for 15 min), in presence (+) or absence ( ) of pretreatment with
PD-98059 (30 µM), is depicted with respect to ERK phosphorylation.
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Differential MEK activation. The only
PD-98059-sensitive mechanism of ERK activation described involves the
ERK activators, MEK1 and MEK2. Interestingly, MEK2 is reported to be
~25-fold less sensitive to PD-98059 than MEK1 (1). It was therefore initially hypothesized that urea-inducible ERK activation would be more
highly MEK2 dependent than NaCl-inducible ERK activation. To examine
this hypothesis, the ability of urea and NaCl to activate MEK isoforms
was evaluated via immune complex kinase assay. Anti-MEK1 or anti-MEK2
immunoprecipitates prepared from control and solute-treated mIMCD3
cells were assayed for ERK-directed (MEK) kinase activity. Although
abundant MEK activity was evident in anti-MEK1 immunoprecipitates, MEK1
activity was only increased by ~25% following both urea and NaCl
treatment (Fig.
5A).
This was a consequence of the high basal level of MEK-like kinase
activity evident in the anti-MEK1 immunoprecipitates under control
conditions (Fig. 5B), but not
evident in anti-P-MEK immunoblots (see below).
ERK-directed (MEK-like) kinase activity in the anti-MEK2
immunoprecipitates was in general less than that of the anti-MEK1
immunoprecipitates (Fig. 5B) but was
increased fourfold (relative to control) in response to urea treatment
and threefold in response to NaCl treatment. (Less constitutive
ERK-directed kinase activity was evident in the anti-MEK2
immunoprecipitates.) This modest differential effect between urea and
NaCl was preserved in all experiments; however, the magnitude of this
difference was highly variable between experiments and did not achieve
statistical significance. Although the differential PD-98059
sensitivity of urea- and NaCl-inducible ERK activation did not appear
to be a consequence of differential activation of MEK isoforms, the
relative activities of MEK2 with respect to MEK1 could not be
established in this native system because of possible differential
affinity of the respective antibodies influencing efficiency of
immunoprecipitation.

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Fig. 5.
MEK activation by urea and NaCl. A:
effect of urea (200 mM) and NaCl (200 mM/400
mosmol/kgH2O) upon MEK1 and MEK2
activity as quantitated by ERK-directed (MEK) kinase activity in immune
complex kinase assay following anti-MEK1 or anti-MEK2
immunoprecipitation; n = 4 separate
experiments. * P < 0.05 with
respect to control treatment for each isoform.
B: autoradiograph from representative
kinase assay of lysates prepared from control, urea-treated (U), and
NaCl-treated (N) cells and immunoprecipitated with anti-MEK1
(lanes 1-3) or anti-MEK2
(lanes 4-6). Solid arrowhead,
phosphorylated MEK substrate (kinase-inactive ERK).
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To address this issue, the relative efficacy of the anti-MEK antibodies
in immunoprecipitating their respective targets was evaluated. The
anti-P-MEK immunoblotting model system employed also provided
correlative data regarding the relative magnitudes of solute-inducible
MEK activation. Under control conditions, no phosphorylated MEK was
detectable in mIMCD3 cell lysates (Fig. 6,
lane 7). Following both urea and
NaCl treatment, an immunoreactive doublet was evident (Fig. 6,
lanes 8 and
9). The absence of detectable signal
under control conditions prevented determination of multifold inductions for both MEK1 and MEK2 phosphorylation.
Although it appeared that de novo MEK1 phosphorylation exceeded that of
MEK2 following treatment with either solute, equal avidity of the
antigen-antibody (anti-P-MEK) interaction for each isoform could not be
assumed. Lysates from mIMCD3 cells were then immunoprecipitated with
either anti-MEK1 (Fig. 6, top) or
anti-MEK2 (Fig. 6, bottom) antibody, and the resultant immunoprecipitates as well as the immunodepleted lysates were examined by anti-P-MEK immunoblotting and compared with
total (naive) lysate. Immunoprecipitation with anti-MEK1 substantially
immunodepleted the more rapidly migrating band of the doublet
and markedly enriched this species in the immunoprecipitate. Immunoprecipitation with anti-MEK2 antibody was relatively ineffective at immunodepleting the more slowly migrating band; however, it specifically resulted in the appearance of only this species in the
immunoprecipitates. It was concluded from these studies that: 1) urea and NaCl increased
phosphorylation of both MEK1 and MEK2 and to a greater extent than
would be anticipated by the immune complex kinase assay;
2) MEK1 represented the more rapidly
and MEK2 the more slowly migrating components of the doublet
(consistent with the slightly greater molecular mass of the latter);
and 3) anti-MEK1 immunoprecipitated
MEK1 specifically and almost quantitatively, whereas anti-MEK2
immunoprecipitated MEK2 specifically but not quantitatively.

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Fig. 6.
MEK phosphorylation. Anti-phospho-MEK1/2 immunoblot of whole cell
detergent lysates prepared from control-treated cells (C) or cells
treated with urea (U; 200 mM for 5 min) or NaCl (N; 200 mM/400
mosmol/kgH2O for 5 min). Replicate
treated wells were immunoprecipitated with anti-MEK1
(top) or anti-MEK2
(bottom) prior to resolution for
anti-phospho-MEK immunoblotting. Following immunoprecipitation,
immunodepleted (lanes 1-3) and
immunoprecipitated (lanes 4-6)
fractions from each condition were electrophoresed for comparison with
each other and total (naive) lysate (lanes
7-9). Equal microgram quantities of total lysate
and immunodepleted lysate were used for comparison; immunoprecipitates
could not be similarly controlled, because of enrichment of the
precipitating antibody. IP, antibody used for immunoprecipitation.
Solid arrowhead denotes immunoglobulin light chain (nonspecific band);
shaded arrowhead denotes MEK2; and open arrowhead denotes
MEK1.
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The ability of urea and NaCl to activate MEK isoforms was examined in
the presence of the MEK inhibitor, PD-98059. The specific mechanism of
action of PD-98059 has been the subject of controversy; although
described as a MEK inhibitor, other data have suggested that it may
function as a direct inhibitor of Raf action. We therefore sought to
determine whether the differential PD-98059 sensitivity observed with
ERK activation would be recapitulated at the level of MEK
phosphorylation. Phospho-MEK1 and phospho-MEK2 nearly comigrated, as is
evident in Fig. 6. When abundance of both phosphorylated isoforms was
considered in aggregate and integrated densitometrically, urea-inducible MEK phosphorylation was substantially less
PD-98059-sensitive than was NaCl-inducible MEK phosphorylation (e.g.,
Fig.
7A). We employed a simple deconvolution method to reduce the complex curve generated after densitometric scanning of the phospho-MEK doublet, thereby permitting evaluation of the effect of PD-98059 upon both MEK1
and MEK2 phosphorylation (Fig. 7B; see
METHODS). The identity of the upper
(more slowly migrating) band as MEK2 had previously been established
via immunoprecipitation and immunodepletion analysis (Fig. 6). Both
urea-inducible MEK1 (Fig. 7C) and
urea-inducible MEK2 (Fig. 7D)
phosphorylation were substantially less sensitive to PD-98059 than were
the corresponding phosphorylation events induced by hypertonicity
(NaCl). As little as 1 µM PD-98059 significantly reduced
NaCl-inducible MEK1 and MEK2 phosphorylation. This differential PD-98059 sensitivity paralleled that observed with ERK phosphorylation. In addition, neither MEK isoform was markedly inhibited by PD-98059 in
the context of urea treatment, mitigating against a contribution of
differential MEK isoform activation in response to urea vs. NaCl.

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Fig. 7.
Effect of PD-98059 upon MEK phosphorylation.
A: representative anti-P-MEK
immunoblot of mIMCD3 cells pretreated with PD-98059 (0-30 µM)
prior to urea (200 mM for 5 min) or NaCl (200 mM/400
mosmol/kgH2O for 5 min).
B: example of complex
("double-peaked") curve generated by densitometric analysis of
the anti-P-MEK immunoreactive doublet, comprised of superimposed P-MEK1
(open arrowhead) and P-MEK2 (shaded arrowhead) peaks. Vertical line,
scribed from the apex of the primary (MEK1) peak to the baseline,
divides the area under the curve into components
A and B, which are
used to estimate areas under the P-MEK1 and P-MEK2 curves according to
the indicated formulas (see
METHODS).
C and
D: effect of pretreatment with
indicated concentrations of PD-98059 upon urea-inducible and
NaCl-inducible phosphorylation of MEK1
(C) and MEK2
(D).
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To further substantiate this interpretation, the effect of the newly
described MEK inhibitor, U-0126 (10), was examined. U-0126 is at least
an order of magnitude more potent than PD-98059 and, more importantly,
does not appear to exhibit appreciable isoform selectivity with respect
to MEK1 and MEK2 [IC50 (in
vitro) of 0.07 and 0.06 µM, respectively] (10).
Similar to data acquired with PD-98059, urea-inducible ERK activation
was far less sensitive to this compound than was NaCl-inducible ERK
activation (Fig. 8). It therefore appeared
highly unlikely that the differential PD-98059 sensitivity of urea- and
NaCl-inducible ERK activation was a consequence of selective activation
of either MEK1 or MEK2. The ability of urea to activate other MAPK
kinases (MAPKKs) was then evaluated.

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Fig. 8.
Effect of the non-isoform-selective MEK inhibitor, U-0126, upon
solute-inducible ERK activation. Anti-P-ERK immunoblot of lysates
prepared from cells pretreated for 30 min with 0, 0.3, 1, or 10 µM
U-0126 prior to the indicated solute treatments, demonstrating
differential sensitivity.
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Phosphorylation of other MAPKKs.
MAPKKs for the non-ERK members of the MAPK family include MKK4/SEK1,
the principal SAPK/JNK activator, and MKK3 and MKK6, principal MAPKKs
for p38. The ability of urea and NaCl to induce phosphorylation of each
of these kinases was investigated as a correlate of the effect of these
solutes upon MEK1 and MEK2 activation. MKK6 activation was detectable under control conditions and was modestly activated by NaCl treatment (Fig. 9); activation by NaCl was more
evident at 30 min than at 5 min. The effect of urea, on an equiosmolar
basis, was less pronounced than that of NaCl. MKK3 phosphorylation was
absent under control conditions and, in general, paralleled that of
MKK6. Urea-inducible MKK3 phosphorylation could not be demonstrated.
MKK4, in contrast, exhibited readily detectable phosphorylation under
basal conditions and was not reproducibly activated in the present
context by any of these conditions. Treatment with higher urea
concentrations decreased MKK4 phosphorylation.

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Fig. 9.
Effect of urea and NaCl upon other MAPKKs. Anti-phospho-MKK3/MKK6 and
anti-phospho-MKK4 (SEK1) immunoblots of lysates prepared from mIMCD3
cells treated for the indicated durations with the indicated
concentrations of NaCl or urea.
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Effect of the p38 inhibitor,
SB-203580. A possible role for another
PD-98059-insensitive kinase in urea signaling to ERK activation was
suggested by the above data. Recently p38 has been implicated in stress
signaling to ERK activation (16), as well as stress signaling to
immediate-early gene expression (15). In addition, an interrelationship
between p38 signaling and JNK signaling has also been suggested (20).
Therefore, the ability of the p38 inhibitor, SB-203580 (14), to
abrogate the urea effect was examined using immunoblots detecting ERK
and MEK phosphorylation. SB-203580 (50 µM) substantially inhibited
the ability of NaCl to activate this signaling cascade (Fig.
10). This is within the dose range reported to be required for inhibition of p38 effect in other cells of
renal epithelial origin (20). In contrast, the effect of SB-203580 upon
urea signaling was much more modest at the levels of both MEK1/2 and
ERK phosphorylation. We and others have shown that the effect of urea
upon p38 activation is modest at best, compared with that of NaCl, in
in vitro kinase assay (2, 23). We examined p38 phosphorylation as a
potentially more sensitive index of activation and confirmed the
observations of Berl et al. (2). Specifically, NaCl markedly increased
p38 phosphorylation as early as 5 min and in concentrations as low as
100 mM (the lowest concentration examined; Fig.
11). The effect of NaCl persisted until
at least 30 min of treatment. For a given degree of hyperosmolarity, urea treatment exerted a much more modest effect than did NaCl upon p38
phosphorylation, and this effect was most pronounced at 5 min rather
than 30 min of treatment.

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Fig. 10.
Effect of SB-203580 upon ERK and MEK phosphorylation. Effect of urea
(200 mM for 5 and 15 min) and NaCl (200 mM/400
mosmol/kgH2O for 5 and 15 min) on
ERK and MEK phosphorylation as determined by anti-phospho-ERK and
anti-phospho-MEK immunoblotting, respectively, in absence or presence
of pretreatment with SB-203580 (50 µM for 30 min).
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Fig. 11.
Urea- and NaCl-inducible p38 phosphorylation. Effect of indicated
solute treatments on p38 phosphorylation in anti-phospho-p38
immunoblotting.
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Effect of urea upon ERK activation is likely volume
independent. Hypertonicity-inducible ERK activation is
presumed to be mediated through resultant cell shrinkage. To
investigate a role for cell volume in urea-inducible vs.
sorbitol-inducible ERK activation, mIMCD3 cells were exposed to 100 mM
solute for 10 min. In parallel wells, medium was diluted to achieve the
indicated intermediate osmolarities (375, 350, 325, and 300 mosmol/kgH2O; see Fig.
12), whereas concentration of the
supplemented solute was maintained at 100 mM. (Sorbitol rather than
NaCl was used as the hypertonic stressor because NaCl constituted the
principal osmotically active solute in the medium, and hence, dilution
of applied hypertonic NaCl would essentially restore the medium to near
its original composition.) As anticipated, hypertonic sorbitol (100 mM)
enhanced ERK phosphorylation at 10 min of treatment. This effect was
inhibited by normalization of final osmolality, consistent with an
effect dependent upon cell volume. In marked contrast, the effect of urea treatment was independent of final medium osmolality. Because urea
inclusion in isosmotic media can result in cell swelling, and because
we and others have shown that cell swelling can independently result in
ERK activation, as an additional control, we pharmacologically assessed
the potential contribution of cell swelling to the invariant ERK
activation observed in the presence of urea and the various final
medium osmolarities depicted in Fig. 10. As described above, cell
swelling-inducible ERK phosphorylation, like NaCl-inducible ERK
phosphorylation (Fig. 4), but in contrast to urea-inducible ERK
phosphorylation, is highly sensitive to PD-98059. The effect of
PD-98059 upon ERK activation in the presence of urea treatment was
independent of the degree of normalization of medium osmolarity. Under
all conditions, ERK phosphorylation was equivalently inhibited (data
not shown). Had urea-inducible ERK phosphorylation been mediated under
any of these conditions of diminishing total osmolarity by a component
of cell swelling, then these conditions should have exhibited a much
more pronounced sensitivity to PD-98059. Therefore, urea-inducible ERK
phosphorylation is likely not mediated by cell shrinkage, and the
inability of normalization of medium osmolarity to abrogate the effect
of urea is likely not attributable to a component of swelling-inducible
ERK activation.

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Fig. 12.
Isotonic exposure to solutes. Effect of medium supplementation (for 10 min) with sorbitol (100 mM) or urea (100 mM) in absence ("400")
or presence of medium dilution with sterile water to achieve the
indicated final osmolarity ("375," "350," "325,"
"300"). Final osmolarities (estimated in
mosmol/kgH2O) are depicted above the gel.
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DISCUSSION |
We sought to investigate the mechanism underlying urea-inducible ERK
activation. The unusual ability of cells of the inner medulla to
respond to urea treatment with ERK activation may reflect a uniquely
differentiated phenotype acquired by cells perpetually bathed in urea
in vivo. This effect of urea exceeded that of hypertonicity and even
the potent ERK activator and positive control, phorbol ester. Other
data have suggested the presence of a specific urea-inducible signaling
cascade in cells of this lineage including the ability of urea to
activate immediate-early gene transcription (6, 8) and hallmarks of
receptor tyrosine kinase activation (7).
An additional striking finding is the marked differential sensitivity
of urea- and NaCl-inducible ERK phosphorylation to the MEK inhibitor,
PD-98059. This differential sensitivity could be consistent with
differential activation of each of the two principal ERK kinases, MEK1
and MEK2, whose IC50 values with
respect to this inhibitor are reportedly 2 µM and 50 µM,
respectively (1). This possibility was investigated in detail. The
following lines of evidence mitigate against this explanation:
1) although both urea and NaCl
treatment increased MEK2 activity to a greater extent than MEK1
activity in immune complex kinase assays (as a consequence of the high
basal level of ERK-directed kinase activity in the anti-MEK1
immunoprecipitates), MEK1 was likely the principal operative kinase in
response to either solute in both immune complex kinase assay (Fig. 5)
and anti-P-MEK immunoblotting (Fig. 6);
2) phosphorylation of both MEK1 and
MEK2 was inhibited to a much lesser extent by PD-98059 in the context
of urea treatment than in the context of NaCl treatment (Fig. 7); and
3) urea-inducible ERK activation was
also substantially less sensitive to the non-isoform-specific MEK
inhibitor, U-0126, than was NaCl-inducible ERK activation (Fig. 8). Activation of a coimmunoprecipitating phosphatase activity (e.g., Ref. 4) or kinase activity could be differentially
regulated by the two solutes, influencing the results of the immune
complex kinase assay. Supporting this interpretation is the high basal level of ERK-directed kinase activity evident in anti-MEK1
immunoprecipitates prepared from control-treated cells (Fig. 5), when
contrasted with the complete absence of basal MEK1 or MEK2
phosphorylation observed in the setting of anti-P-MEK immunoblotting
(e.g., Fig. 6). Alternatively, a known or novel non-MEK1/2 upstream ERK
activator may be preferentially activated by urea. An examination of
the other principal MAPKK activities (MKK3, MKK4, and MKK6), however, failed to identify such a candidate. Regardless of the molecular mechanism, the significant differential PD-98059 (and U-0126) sensitivity may have implications for physiological responses to this
and related pharmacological agents and represents, to our knowledge,
the first report wherein relatively PD-98059-sensitive and -insensitive
ERK activators are described in parallel in the same model.
Data in Fig. 4 clearly indicate that, for a given time point,
urea-inducible ERK activation is considerably less sensitive to
PD-98059 than is NaCl-inducible ERK activation. Although the data in
Fig. 4 appear to depict dissimilar
IC50 values for PD-98059 for
different solute treatments, an alternative explanation may also be
operative. PD-98059 exhibits a reasonably "sigmoidal" inhibitory
dose-response in the contexts of urea treatment at 5 min and NaCl
treatment at both 5 and 15 min, therefore the apparent IC50 values (concentration of
PD-98059 corresponding to a 50% inhibition in ERK
activation/phosphorylation) may approximate the true
IC50 for this inhibitor. In the
case of urea treatment for 15 min, in contrast, no amount of PD-98059
inhibits ERK activation greater than 25%; therefore,
IC50 is difficult to interpret. If both PD-98059-sensitive and PD-98059-insensitive mechanisms of ERK
activation are operative simultaneously, then the
IC50 will be overestimated; no
amount of inhibitor will produce complete inhibition. Only the effect
of NaCl at 5 min of treatment is fully inhibitable by PD-98059. It also
remains possible that the relatively MEK-independent pathway of ERK
activation in response to urea is an alternate, or redundant, pathway
only operative in the context of MEK1 and MEK2 inhibition; this
possibility could not be experimentally addressed. Regardless of the
interpretation, however, these data suggest fundamental differences in
the mechanisms of urea and NaCl signaling to ERK activation. In
addition, in the case of curves U200/5' and N400/15' (Fig.
4B), the dose-response relationship may also be confounded by the potentially activating effect of high
doses (100 µM) of PD-98059. Interestingly, for a given solute stimulus, the PD-98059 sensitivity of solute-inducible ERK activation is time dependent (Fig. 4B). Whether
this reflects differential activation of upstream signaling events or,
less likely, inhibitor inactivation in solution or intracellularly
remains unclear.
The inability to quantitatively establish the relative contributions of
MEK1 and MEK2 in urea-inducible ERK activation in this native system
precipitated an investigation of other potential upstream activating
events. A recent report described the unexpected ability of p38 to
function upstream of ERK activation in a specific stress-responsive
model (16). Similarly, we show here that NaCl-inducible, but not
urea-inducible, ERK phosphorylation is highly sensitive to
pharmacological inhibition of p38. In contrast to the observations of
Ludwig et al. (16), however, p38-dependent ERK activation in the
present model required only 5 min, whereas it required at least 60 min
in the model of arsenite stress. In addition, a role for p38 has been
suggested in stress (e.g., arsenite) induction of immediate-early gene
expression, based largely upon inhibitor and overexpression data (15).
In this model, as well, however, stress-inducible p38 activation
required at least 15-30 min to become evident (15). For p38
activation to precede ERK activation in the model of urea stress, it
would need to be evident by 5 min of treatment, when ERK activation is
maximal. This is precisely what is observed in the present model (Fig.
11). Interestingly, p38 has also recently been implicated in some renal
models in the downstream events initiated by hypertonic (but not
urea-associated) stress including expression of genes encoding heat
shock proteins and an osmolyte transporter (9, 20). Originally, we
reported that urea treatment failed to appreciably induce p38
activation in mIMCD3 cells. Although modest upregulation was detectable
at only 5 min of urea treatment, this increase failed to achieve statistical significance (23). Berl et al. (2) reported that urea
modestly but reproducibly increased p38 activity, relative to NaCl in
this cell line. Using the more sensitive anti-phospho-p38 immunoblot
assay, we confirmed their observation. Nonetheless, the ability of urea
to activate this kinase is much more modest than that of hypertonic stressors.
We present evidence suggesting that cell shrinkage does not mediate the
effect of urea upon ERK activation. Whereas the ability of hypertonic
sorbitol to induce ERK phosphorylation could be completely abolished by
concomitant medium dilution, the inability of medium dilution to
abrogate the effect of hyperosmotic urea upon ERK activation argues
against a volume-mediated component. Cell swelling, like cell
shrinkage, may result in ERK activation (18, 19). Inclusion of urea in
isosmotic medium may induce cell swelling. Therefore, it could not be
excluded that a small component of the urea effect is attributable to a
change in volume. Nonetheless, among the broad range of medium
dilutions employed in the present study, none influenced the effect of
urea upon ERK activation. In addition, swelling-inducible ERK
activation, like that induced by NaCl, is far more sensitive to
PD-98059 than is urea-inducible ERK activation. At none of the
intermediate dilutions depicted in Fig. 12 was the degree of ERK
phosphorylation more sensitive to PD-98059 than was urea treatment
alone. Therefore, it is highly unlikely that swelling-associated ERK
phosphorylation accounted for the inability of medium dilution to
abrogate the effect of urea.
In aggregate, these studies indicate that among cell lines examined,
urea potently induces ERK phosphorylation only in the mIMCD3 and MDCK
cell lines. In addition, this effect is accompanied by enhanced MEK1
and MEK2 activation, and is likely independent of changes in cell
volume. The relative PD-98059 insensitivity of urea-inducible (in
contrast to NaCl-inducible) ERK activation is not a consequence of
differential MEK1/2 isoform activation.
 |
ACKNOWLEDGEMENTS |
These studies were supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-52494.
 |
FOOTNOTES |
X.-Y. Yang and Z. Zhang contributed equally to this work.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. M. Cohen,
PP262, Oregon Health Sciences Univ., 3314 S.W. US Veterans Hospital
Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).
Received 9 October 1998; accepted in final form 2 April 1999.
 |
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