1 Divisions of Nephrology and Molecular Medicine, Oregon Health Sciences University, and Portland Veterans Affairs Medical Center, Portland, Oregon 97201; and 2 Department of Medicine, University of California, San Diego, La Jolla, California 92093
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ABSTRACT |
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The small guanine nucleotide-binding protein Ras, activated by peptide mitogens and other stimuli, regulates downstream signaling events to influence transcription. The role of Ras in solute signaling to gene regulation was investigated in the murine inner medullary collecting duct (mIMCD3) cell line. Urea treatment (100-200 mM), but not sham treatment, increased Ras activation 124% at 2 min; the effect of NaCl did not achieve statistical significance. To determine the contribution of Ras activation to urea-inducible signal transduction, mIMCD3 cells were stably transfected with an expression plasmid encoding a dominant negative-acting N17Ras mutant driven by a dexamethasone-inducible (murine mammary tumor virus) promoter. After 24 h of induction, selected cell lines exhibited sufficient N17Ras overexpression to abolish epidermal growth factor- and hypotonicity-mediated signaling to extracellular signal-regulated kinase (ERK) phosphorylation, as determined by immunoblotting. Conditional N17Ras overexpression inhibited urea- and NaCl-inducible ERK phosphorylation by 40-50%, but only at 15 min, and not 5 min, of treatment. N17Ras induction, however, almost completely inhibited urea-inducible Egr-1 transcription, as quantitated by luciferase reporter gene assay, but failed to influence tonicity-inducible (TonE-mediated) transcription. N17Ras overexpression also blocked urea-inducible expression of the transcription factor Gadd153 but did not influence osmotic or urea-inducible apoptosis. In addition, urea treatment induced recruitment of the Ras activator Sos to the plasma membrane. Taken together, these observations suggest a role for Ras signaling in the IMCD cell response to urea stress.
hypertonicity; cell volume regulation, Gadd153; extracellular signal-regulated kinase; mitogen-activated protein kinase
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INTRODUCTION |
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CELLS OF THE RENAL medulla are uniquely exposed to elevated concentrations of urea and NaCl as a consequence of the renal concentrating mechanism. Hypertonicity per se initiates a characteristic signaling pathway in yeast and higher eukaryotes, culminating in activation of mitogen-activated protein kinases (MAPKs). In renal epithelial cells, NaCl (16, 20, 30, 36) and urea (6) activate extracellular signal-regulated kinases (ERK) as well as their upstream activators; however, significant differences exist in the magnitude, kinetics, and MAPK/ERK kinase inhibitor sensitivity in response to each of these solutes (35). Multiple parallel signaling events converge on ERK activation; among the best characterized is the Ras-Raf-MEK pathway operative principally in response to mitogenic stimuli.
In addition to ERK activation, multiple signaling events are regulated
by urea in the renal medullary cell model. Although urea is not
mitogenic for murine inner medullary collecting duct (mIMCD3) cells,
the urea response exhibits hallmarks of a receptor tyrosine
kinase-mediated pathway, including activation of phospholipase C-
(9), activation of phosphatidylinositol 3-kinase (PI3K) and its
effectors Akt and p70 S6 kinase (38), activation of Shc with
recruitment of Grb2 (38), and induction of immediate-early genes (7,
10). Urea also exerts a prooxidant effect and increases expression of
the stress-responsive Gadd153 gene in an oxidative stress-dependent
fashion (37).
We sought to determine whether the ability of urea to activate ERK signaling and, thereby, regulate urea-inducible gene transcription was mediated in part by Ras. Ras activation is regulated positively by guanine nucleotide exchange factors (GEFs), which catalyze the conversion of inactive GDP-bound Ras to active GTP-bound Ras, and negatively by GTPase-activating proteins (GAPs), which enhance the intrinsic GTP hydrolytic activity of Ras. Activated Ras exhibits diverse effects at the cellular level, including regulation of proliferation, differentiation, metabolism, and apoptosis (25); at the systemic and organismal level, it has been implicated in malignant transformation (17). Although pharmacological suppression of Ras signaling has remained problematic, molecular dissection of Ras-dependent signal transduction events and their physiological consequences has been facilitated by the identification of Ras mutants, exhibiting dominant inhibitory actions when overexpressed (5, 14). After first demonstrating the ability of urea to activate Ras, we sought to explore the contribution of this event to biochemical and physiological aspects of urea signaling in the renal medullary mIMCD3 cell line.
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METHODS |
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Cell culture and solute treatment. mIMCD3 cells (American Type Culture Collection) were maintained and passaged as previously described (7). 3T3 cells were obtained from the Vollum Institute for Advanced Biomedical Research at Oregon Health Sciences University and were maintained in DMEM-F-12 (JRH, Lenexa, KS) supplemented with 10% fetal bovine serum (JRH). Before each experiment, cells were placed in serum-free medium for 24 h. Solute treatment consisted of gentle, dropwise addition to mIMCD3 monolayers of an aliquot of concentrated urea (4.2 M) or NaCl (2.25 M) in sterile water or an equal volume of NaCl (150 mM) in sterile water (sham treatment).
Nonisotopic Ras GTP loading assay. Ras activation is defined as the percentage of Ras molecules in the active GTP-bound state, i.e., Ras-bound GTP/(Ras-bound GDP + Ras-bound GTP) × 100, and was measured using a recent modification (28) of our original protocol (27). Briefly, cells were lysed in HEPES-based buffer containing 1% Nonidet P-40, and protein G-agarose beads preincubated with the rat monoclonal pan-Ras antibody Y13-259 (Zymed) and a rabbit anti-rat IgG-Fc secondary antibody (experimental sample) were added to half of the lysate. Beads preincubated with (nonspecific) rat IgG and the rabbit anti-rat secondary antibody (control sample) were added to the other half of the sample. Samples were shaken for 1 h, which quantitatively immunoprecipitates Ras; Mg2+ and high salt concentration in the buffer inhibit Ras-directed GAP activity and GTP/GDP dissociation from Ras. In addition, Y13-259 is a Ras-neutralizing antibody that inhibits interaction of GEF and GAP with Ras. GDP and GTP were quantitatively eluted from the Ras immunoprecipitates by heating, a process that destroys <5% of these nucleotides. GTP was measured after conversion to ATP with use of the enzyme nucleoside diphosphate kinase. ATP was then measured by the luciferase-luciferin system (28). GDP was first converted to GTP by use of pyruvate kinase and phosphoenolpyruvate and was then quantitated as described above. Standard curves were prepared with each assay, and the amount of sample GDP and GTP was calculated as the difference between the experimental and control samples. Because the GDP assay measures GTP + GDP, the amount of GTP is subtracted from the sum to yield the amount of GDP. Both assays are sensitive to 1 fmol of nucleotide (28).
Immunoblot analysis. Detergent lysates were prepared from serum-deprived mIMCD3 cells, as previously described (7). Equal amounts of protein (µg) were loaded per lane and subjected to SDS-PAGE. After electrophoresis, proteins were subjected to semidry transfer to polyvinylidine difluoride membrane and then incubated with anti-Gadd153, anti-Ras, or anti-Sos (all from Santa Cruz Laboratories) or anti-P-ERK, anti-P-stress-activated protein kinase (SAPK), anti-P-p38, or anti-phospho-Akt (New England BioLabs) primary antibodies and appropriate horseradish peroxidase-linked secondary antibodies (according to the manufacturer's directions). Blots were visualized with enhanced chemiluminescence (Renaissance, DuPont) and then by fluorography. For membrane protein preparation, monolayers were washed twice with ice-cold PBS, scraped into 600 µl of lysis buffer [20 mM Tris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 20 µg/ml leupeptin, and 80 µg/ml aprotinin], and subjected to homogenization (30 strokes in a Dounce homogenizer) with a tight pestle. Lysates were centrifuged at 130,000 g for 60 min at 4°C. The resultant supernatant was retained as cytoplasmic lysate. The pellet, representing the membrane fraction, was resuspended by homogenization (Dounce homogenizer) with 300 µl of lysis buffer supplemented with 1.2% Triton X-100 and then clarified in a microcentrifuge at 12,000 rpm for 5 min at 4°C. Protein concentration in whole cell lysates and subcellular fractions was quantitated with the Bio-Rad protein assay kit according to the method of Bradford (2).
Transfection and reporter gene assays.
The construction of Egr-1-Luc, comprised of 1.2 kb of the murine Egr-1
5'-flanking sequence (including the minimal promoter) (31)
subcloned upstream of the promoterless luciferase reporter vector pXP2
(24), has previously been described (10). BGT-2X-Luc was prepared by
subcloning a double-stranded oligonucleotide encoding two tandem
repeats of the canine tonicity enhancer element (TonE) (23, 29)
upstream of the thymidine kinase promoter in
BamH I/Hind III-cleaved vector TK-Luc (24).
(The 5'-oligonucleotide sequence was GAT CCT ACT TGG TGG AAA AGT
CCA GTC GAT ACT TGG TGG AAA AGT CCA GA; the 3'-oligonucleotide
sequence was AGC TTC TGG ACT TTT CCA CCA AGT ATC GAC TGG ACT TTT CCA
CCA AGT AG.) Cells were transiently transfected with 10 µg of
Egr-1-Luc + 3 µg of cytomegalovirus-Gal per subconfluent 100-mm dish
via electroporation, as described elsewhere (10). pMMTV-Ras-Asn17
(N17Ras) expression vector was provided by G. Cooper (5). With use of
this vector, mIMCD3 cells were stably transfected via electroporation
(<1% transfection efficiency) and selected in G418-containing medium commencing 48 h after transfection. When individual colonies became visible (i.e., >1 mm diameter), culture medium was removed and cells
were aspirated with several microliters of medium with use of a
200-µl tip on a Gilson-style pipettor. Cells obtained from individual colonies were dispersed into labeled wells of 24-well plates
prefilled with prewarmed DMEM-F-12 + 10% fetal bovine serum. Wells achieved confluence in ~2 wk and were propagated for further study. Luciferase and -galactosidase activities in detergent lysates
were determined as previously described (10); the former was normalized
with respect to the latter. The duration of control and solute
treatments was 6 h (starting 48 h after transfection). Dexamethasone
treatment for 24 h decreased total protein content in cell monolayers
by ~5% in untransfected cells and by ~14% in N17Ras-B7 cells.
Caspase-3 assay. Caspase-3 (cpp32) microfluorescence assay was performed according to a modification of the method of Enari et al. (12). Briefly, cells were washed twice with ice-cold PBS, scraped into 50 µl of extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 µM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain, and 10 µg/ml chymopapain), and lysed with five freeze-thaw cycles. After centrifugation at 10,000 g for 12 min at 4°C, supernatants were assayed for protein concentration, as described above. Cell lysate (25 µg) was incubated in a reaction volume of 50 µl with fluorogenic substrate (N-acetyl-DEVD-MCA, 10 µM; BioMol), 100 mM HEPES-KOH, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM DTT, and 0.1 mg/ml ovalbumin for 60 min at 30°C in a 96-well microtiter plate (Falcon). Enzyme activity was detected by Cytofluor II (PerSeptive Biosystems, Framingham, MA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
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RESULTS |
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Urea increases Ras activation in mIMCD3 cells.
The ability of urea and NaCl to activate Ras was investigated using an
enzymatic method to measure Ras-GTP/GDP (27, 28). Under basal
conditions, ~5% of immunoprecipitable Ras was activated (GTP bound;
Fig. 1). After 2 min of urea
treatment (200 mM or 200 mosmol/kgH2O), Ras activation was
increased 124% (to 15.3%) and decreased promptly thereafter. At 200 mosmol/kgH2O, NaCl increased Ras
activation by only 38 and 44% at 5 and 10 min, respectively. The
effect of NaCl, however, did not achieve statistical significance. In
the nonrenal 3T3 cell line, urea exerted no effect on Ras activation (data not shown).
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Generation of mIMCD3 cell lines exhibiting high-level inducible
expression of the dominant negative-acting Ras isoform N17Ras.
Because of these biochemical data demonstrating urea-inducible Ras
activation, the role of this signaling event in the phenotype of the
urea-stressed cell was examined. mIMCD3 cells were stably transfected
with an expression vector encoding a dominant negative-acting mutant
(N17) Ras isoform under the control of the inducible
dexamethasone-responsive murine mammary tumor virus (MMTV) promoter
(kindly provided by G. Cooper) (5). Of ~50 clones isolated after
stable transfection and antibiotic selection, several exhibited marked
induction of the mutant Ras in response to dexamethasone treatment (300 nM for 24 h). Clone B7 (lane 7, Fig.
2) was selected for further study because
of its relatively low level of basal Ras expression and its marked
induction in response to dexamethasone treatment. To confirm that
overexpression of the inducible N17Ras-encoding plasmid permitted
inducible inhibition of Ras-dependent events, control experiments were
performed. Although there are no documented Ras-dependent events in
this cell line, the effects of hypotonicity and the peptide mitogen
epidermal growth factor on ERK activation (phosphorylation) have been
shown to be Ras dependent in other models (e.g., Ref. 32). In
untransfected mIMCD3 cells, both of these stimuli, as well as phorbol
ester (12-O-tetradecanoylphorbol-13-acetate) treatment, markedly increased ERK phosphorylation, as determined by anti-P-ERK immunoblotting (Fig. 3).
Undernone of these conditions, however, was ERK activation
substantially inhibited by dexamethasone pretreatment. In contrast, in
the N17Ras-B7 cell line, ERK phosphorylation in response
to hypotonic stress and treatment with epidermal growth factor and
12-O-tetradecanoylphorbol-13-acetate was markedly attenuated by dexamethasone pretreatment, implying a role for Ras in these activation events. These experiments appeared to validate the present
model for investigating the contribution of Ras signaling to the
urea-stressed phenotype.
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Effect of N17Ras overexpression on urea and NaCl signaling to ERK
activation.
In untransfected cells, urea and NaCl increased ERK phosphorylation at
5 and 15 min of treatment at 200 and 800 mosmol/kgH2O solute, consistent
with our prior observations and observations of others (1, 35, 36)
(Fig. 4). In parental mIMCD3 cells, dexamethasone exerted no effect on ERK phosphorylation under any experimental condition examined, nor did it have an effect on the level
of (endogenous native) Ras expression. In the N17Ras-B7 cells,
dexamethasone treatment produced a pronounced increase in Ras
immunoreactivity, as demonstrated by immunoblotting. At lower
concentrations of urea (200 mM), the effect of dexamethasone treatment
(i.e., N17Ras induction) relative to no treatment was demonstrable only
at 15 min. At 5 min of treatment, the effect did not achieve
statistical significance (Fig. 5). In the
absence of dexamethasone treatment, the effect of 200 mosmol/kgH2O urea and NaCl in the
N17Ras-B7 cell line was substantially less than that of untransfected
cells, suggesting a small amount of basal (constitutive) expression of
N17Ras. At high concentrations of solute (800 mosM), there was no
consistent effect of N17Ras overexpression on urea- or NaCl-inducible
ERK phosphorylation. Data from three separate experiments are combined
in Fig. 5. At 15 min of solute treatment in N17Ras-B7 cells,
dexamethasone treatment inhibited urea- and NaCl-inducible ERK
phosphorylation by 38 and 46%, respectively, whereas at 5 min of
treatment, there was no effect. Importantly, these results were
corroborated in a second N17Ras-overexpressing cell line, N17Ras-B9
(Fig. 2; data not shown).
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Effect of N17Ras overexpression on urea and NaCl signaling to other
MAPKs.
In light of the findings described above and the ability of urea and
NaCl to activate other members of the MAPK family, the effect of
dominant negative Ras expression on solute-inducible p38 and SAPK
activation was investigated. NaCl (400 mosM for 15 min) markedly
activated p38 and hypotonicity (50% medium dilution for 5 min)
minimally activated p38, consistent with prior observations of the
authors and others, whereas urea had no discernible effect (Fig.
6). Dexamethasone pretreatment had no
effect on either of these signaling events, implying specificity of the
ERK events. With respect to SAPK, hypertonicity, but not urea or
hypotonicity, induced activation (phosphorylation). Similar to the case
with p38, N17Ras overexpression failed to inhibit the effect, a finding previously noted by Kawasaki et al. (18).
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Overexpression of N17Ras abrogates transcriptional
regulation by urea but not by NaCl.
Because immediate-early gene transcription in response to urea
treatment is mediated via serum response element (SRE)/Ets motifs and
because this process is likely Ras dependent (4), the ability of
dominant negative-acting N17Ras to block the effect of urea on
transcription was examined. When N17Ras-B7 cells were transiently
transfected with a luciferase reporter gene driven by the proximal 1.2 kb of the murine Egr-1 5'-flanking sequence, urea increased
reporter gene activity ~19-fold, consistent with earlier observations
(10) (Fig. 7). Pretreatment with
dexamethasone inhibited basal Egr-1 transcription by only 22%, whereas
it inhibited urea-inducible transcription by fully 70%. Importantly,
in untransfected cells, the effect of dexamethasone pretreatment on
urea-inducible Egr-1 transcription was negligible (data not shown). For
comparison purposes, the effect of dominant negative N17Ras
overexpression on NaCl-inducible transcription mediated via the osmotic
response element (ORE) (15)/TonE (29) was examined in parallel. The BGT1 TonE is a well-characterized tonicity-responsive
cis-acting element; two tandem repeats
of this element were subcloned upstream of the thymidine kinase
promoter in a luciferase reporter vector. Consistent with others'
observations (29), hypertonicity increased reporter gene activity by
21-fold; however, dexamethasone pretreatment failed to influence this
effect. Urea (200 mM) fails to appreciably activate TonE-mediated
transcription (data not shown), and NaCl (200 mosmol/kgH2O) fails to exert a
substantial effect on Egr-1 transcription (10); therefore, the effect
of N17Ras expression on these events was not examined.
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Overexpression of N17Ras blocks urea-inducible
expression of Gadd153.
Stressors of the endoplasmic reticulum induce expression of the
transcription factor Gadd153. Although not a bona fide endoplasmic reticulum stressor, urea increases expression of Gadd153 at the protein
and mRNA levels (37). As an additional manifestation of the
urea-stressed phenotype, the effect of Ras inhibition on Gadd153
expression was examined. In control (untransfected) cells (Fig.
8), urea treatment (200 mM for 6 h)
increased Gadd153 protein expression by immunoblotting, as did the
potent positive controls (34) tunicamycin and cadmium chloride.
Dexamethasone pretreatment exerted no effect on the ability of any of
these stressors to activate Gadd153 expression. In the N17Ras-B7 cells,
all three stimuli increased Gadd153 protein abundance relative to
control; however, the effect of cadmium chloride was much more
pronounced than in untransfected cells. Importantly, N17Ras
overexpression virtually abolished the effect of urea on Gadd153
immunoreactivity and substantially inhibited the effects of tunicamycin
and cadmium chloride.
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Overexpression of N17Ras does not influence solute-inducible
apoptosis.
It has previously been shown that urea and NaCl may induce apoptosis in
mIMCD3 cells (26) and that interruption of urea- and NaCl-inducible
signaling events may markedly exacerbate this phenomenon (38).
Therefore, the effect of Ras inhibition on solute-inducible apoptosis
was examined using the sensitive cpp32 (caspase-3) assay (12, 38). In
the present model, 400 mM urea modestly increased apoptosis whereas 200 mosM NaCl markedly increased apoptosis (Fig.
9). (Urea at 200 mM, used in the present
and previous signaling studies, fails to increase caspase-3 activity.)
Pretreatment with dexamethasone to induce N17Ras expression neither
inhibited nor potentiated these effects. In addition, urea and NaCl
induce phosphorylation of Akt (38), an event correlated with apoptosis. Inducible overexpression of N17Ras had no effect on urea- or
NaCl-inducible Akt phosphorylation, as determined by anti-P-Akt
immunoblotting (data not shown).
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Solute-inducible Ras activation may be Sos mediated.
The guanine nucleotide exchange factor Sos mediates Ras activation in
response to mitogens in multiple cell culture models. Because Sos
activation of Ras requires membrane targeting, the ability of urea to
induce translocation of Sos to the plasma membrane was assessed through
immunoblot analysis of membrane preparations. Urea increased Sos
abundance in the plasma membrane by ~100% (Fig. 10).
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DISCUSSION |
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In this series of experiments it is shown that 1) urea activates Ras, 2) inducible overexpression of dominant negative-acting N17Ras inhibits urea-inducible ERK activation in a sharply time-dependent fashion, 3) dominant negative Ras markedly inhibits urea-inducible Egr-1 transcription without affecting hypertonicity-inducible transcription, 4) dominant negative Ras blocks urea-inducible Gadd153 expression, and 5) the ability of urea to activate Ras may be mediated through the GEF Sos.
Although the ability of urea to activate Ras has not previously been described in any context, Ras activation has been inferred in other anisotonic contexts. Although Ras activation was not directly observed, hypotonicity-inducible ERK activation was inhibitable by N17Ras expression in a human intestinal cell line (32). In similar fashion, Ras activation in response to hypertonic stress has been implicated through biochemical effects of overexpressing dominant negative-acting Ras isoforms (18). Terada et al. (30) demonstrated activation of the Ras effector Raf in hypertonically stressed Madin-Darby canine kidney cells. In aggregate, these data suggest a possible role for Ras activation in cell volume regulation, as was postulated by Lang et al. (22), in part on the basis of the perturbed volume set point and regulatory responses observed in cells stably transfected with a constitutively active (oncogenic) Ha-Ras isoform. The inability of moderate hypertonic stress in the present context (as a tonicity control for urea treatment) to activate Ras cannot exclude a modest role for Ras signaling, nor can it exclude Ras signaling at more pronounced degrees of osmotic stress or at later time points. Present data do, however, appear to eliminate an obligate role for Ras signaling in hypertonicity-inducible transcription through the ORE/TonE.
Ras dependence of Gadd153 expression has not previously been described. In the model of Fas- and ceramide-induced apoptosis in human leukemic cells, a Ras-dependent posttranslational modification (activation/phosphorylation) of Gadd153 was reported using a transfected dominant negative approach (3). Fan and Bertino (13) observed that overexpression of constitutively activated K-Ras may upregulate Gadd45, a related gene reported to be tonicity (but not urea) responsive (19).
The cell line(s) exhibiting inducible N17Ras overexpression identified through our selection process appears to be well suited to the present studies. Cell lines B7 (used for most studies) and B9 (used in corroboratory studies) exhibit a marked (>10-fold; see below) increase in N17Ras expression after 24 h of dexamethasone induction. In all studies the potentially confounding effect of dexamethasone was eliminated through parallel treatment of control cells. Under no circumstances did dexamethasone treatment alone induce any detectable phenotypic changes with respect to the outcome measures examined in these studies (see below). The degree of basal N17Ras expression (the "leakiness" of the inducible expression system) could not be reliably estimated owing to the comigration of N17Ras and native Ras. (The precise degree of N17Ras overexpression could also not be reliably estimated for similar reasons.) Nonetheless, the uninduced N17Ras-transfected cells resembled native mIMCD3 cells with respect to all experimental outcome measures reported here (e.g., urea-inducible ERK activation, Egr-1 reporter gene expression, and Gadd153 expression). The solitary exception, unrelated to the urea response, was the greater response of the uninduced N17Ras transfectants to the heavy metal and oxidative stressor cadmium chloride (Fig. 8). Qualitatively, the N17Ras-B7 cell line proliferated somewhat more slowly than did control cells and exhibited greater sensitivity to thawing after cryopreservation (data not shown). With respect to functional data, inducible overexpression of N17Ras effectively inhibited known Ras-dependent events such as mitogen- and hypotonicity-associated ERK activation (Fig. 3). Therefore, it constituted a valid model in which to explore Ras-dependent aspects of the urea-inducible phenotype.
The timing of Ras-dependent signaling to ERK activation warrants comment. In contrast to the 15-min time point, Ras inhibition failed to block solute signaling to ERK at the 5-min time point. In aggregate, these data would suggest that the Ras-dependent effect on ERK activation peaks at 15 min whereas the Ras-independent effect peaks at 5 min. In general, ERK activation in response to potentially Ras-dependent mitogenic stimuli occurs quite early (e.g., 5 min). It is conceivable that in the present context an early (5-min) Ras-dependent signaling event leading to ERK activation is masked by a superimposed and maximal, yet transient, Ras-independent phenomenon, although data in support of this possibility are lacking.
The modest ability of inducible expression of N17Ras to blunt solute-inducible ERK activation, as assessed by anti-P-ERK immunoblotting (40-50% and only at 15 min; Fig. 4), contrasts with the ability of this stimulus to markedly inhibit urea-inducible Egr-1 reporter gene activity (Fig. 7). We previously hypothesized that the ability of urea to activate ERK accounted for urea-inducible Egr-1 transcription in its entirety, primarily on the basis of studies with the MAPK kinase (MEK) inhibitors PD-98059 and U-0126. These prior observations are not inconsistent with the present data. It is likely that at least two primary stimuli for ERK activation are operative in response to urea stress: one Ras dependent and (at least) one Ras independent. Both of these stimuli would appear to be MEK dependent on the basis of earlier data. Given the potential pleiotropic actions of urea, its ability to signal through multiple parallel pathways should not be surprising.
Interestingly, urea- and NaCl-inducible ERK activation at 15 min are
approximately equivalently sensitive to Ras inhibition (Fig. 5). We
previously showed that the mechanisms of ERK activation in response to
NaCl (hypertonicity) and urea are distinct. The former is likely volume
mediated, in contrast to that of urea, and the sensitivity (i.e.,
inhibition constant) of these phenomena with respect to
MEK inhibitors differs by 1 log unit (35). Although it does not
conflict with earlier data, the finding that these two solute stimuli
should exhibit approximately equivalent sensitivity to Ras inhibition
is nonetheless unexpected. The ability of Ras inhibition to partially
block ERK activation in response to NaCl and the relative inability of
NaCl to demonstrably activate Ras appear inconsistent. A modest effect
of NaCl on Ras activation too subtle to achieve statistical
significance, however, cannot be excluded (Fig. 1), nor can a modest
effect of NaCl be excluded at a later time point.
Importantly, the effect of Ras inhibition on ERK activation may be more pronounced than indicated by Fig. 5. For rigorous analysis, statistical comparisons were only performed between different conditions in the same cell line (e.g., without and with dexamethasone in the N17Ras cells). Inspection of Fig. 4, however, and data from all similar experiments suggests that the ability of relatively low-dose urea and NaCl (200 mosmol/kgH2O) treatment to induce ERK activation is blunted, even in the absence of dexamethasone induction. We speculate that this is a function of modest leakiness of the inducible expression system (5), in which a basal level of N17Ras expression is constitutively present. At higher osmolarities, in contrast, the responses of the transfected and untransfected cell lines were virtually identical. It could further be speculated that these data suggest a greater Ras dependence in the response to low-dose osmotic and urea stress; alternatively, the differential effect could be a manifestation of degree of activation alone, with the basal leakiness of N17Ras expression providing insufficient inhibition to blunt the signal generated in response to a robust osmotic stress.
Inducible expression of dominant negative-acting N17Ras failed to sensitize cells to the proapoptotic effect of elevated urea and NaCl concentrations. For these analyses, a higher concentration of urea (400 mM) than has been used in our signaling studies was required, because urea (200 mM) fails to exert a proapoptotic effect in this model (data not shown). Although associated with apoptotic induction in other models, in the mIMCD3 cell line, dexamethasone alone (in untransfected cells) failed to exert a proapoptotic effect (data not shown); therefore, it was unlikely to confound the present analysis. Previously, we suggested that a different receptor tyrosine kinase effector pathway, PI3K activation, may confer an element of resistance to osmotic and urea stress (38). Urea treatment and, to a lesser degree, hypertonicity activated PI3K and a PI3K-dependent signaling cascade, albeit with dissimilar kinetics. Inhibition of PI3K action with wortmannin and LY-294002 resulted in a dramatic increase in caspase-3 activation, as well as an increase in annexin V binding (another biochemical marker of apoptosis), in response to urea and hypertonic stress (38).
Multiple signaling events converge on Ras activation (33). The nucleotide exchange factor Sos is operative in response to RTK-dependent pathways in other models, and urea signaling exhibits hallmarks of an RTK-like pathway (6, 9, 10). In addition, urea activates the Shc adapter protein and recruits to Shc another adapter, Grb2 (38). Grb2, in turn, recruits Sos in other models. For these reasons, Sos represented an attractive candidate upstream activator of Ras in the present model. Urea treatment induced the recruitment of immunoreactive Sos to the plasma membrane. These data implicate Sos in the urea-dependent activation of Ras but do not confirm a functional role. Similarly, involvement of other urea-dependent Ras activation events cannot be excluded. For example, urea exerts a prooxidant effect at the cellular level (37), and a direct activating redox effect on Ras has recently been described in other contexts (11, 21).
Finally, in the limited sampling of cell types undertaken here, the ability of urea to activate Ras was restricted to medullary cells and was absent from 3T3 cells. We have similarly observed other urea-inducible signaling events to be specific to cells of renal epithelial origin, including ERK activation (35), immediate-early gene expression (8), and Gadd153 expression (37). Whether the cell type-specific effect on Ras activation in the present context is a consequence of tissue-specific expression of an upstream urea-responsive activating kinase, as we have speculated previously (9), remains to be established.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52494 (to D. M. Cohen) and the National Kidney Foundation.
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FOOTNOTES |
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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 University, 3314 SW US Veterans Hospital Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).
Received 23 July 1999; accepted in final form 22 October 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berl, T.,
G. Siriwardana,
L. Ao,
L. M. Butterfield,
and
L. E. Heasley.
Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
272:
F305-F311,
1997
2.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[ISI][Medline].
3.
Brenner, B.,
U. Koppenhoefer,
C. Weinstock,
O. Linderkamp,
F. Lang,
and
E. Gulbins.
Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153.
J. Biol. Chem.
272:
22173-22181,
1997
4.
Cahill, M. A.,
R. Janknecht,
and
A. Nordheim.
Signalling pathways: jack of all cascades.
Curr. Biol.
6:
16-19,
1996[ISI][Medline].
5.
Cai, H.,
and
G. M. Cooper.
Inducible expression of ras N17 dominant negative inhibitory protein.
In: Small GTPases and Their Regulators. Ras Family, edited by W. E. Balch,
C. J. Der,
and A. Hall. New York: Academic, 1995, p. pt. A, p. 231-237.
6.
Cohen, D. M.
Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.
Proc. Natl. Acad. Sci. USA
93:
11242-11247,
1996
7.
Cohen, D. M.,
W. W. Chin,
and
S. R. Gullans.
Hyperosmotic urea increases transcription and synthesis of Egr-1 in murine inner medullary collecting duct (mIMCD3) cells.
J. Biol. Chem.
269:
25865-25870,
1994
8.
Cohen, D. M.,
and
S. R. Gullans.
Urea induces Egr-1 and c-fos expression in renal epithelial cells.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
264:
F593-F600,
1993
9.
Cohen, D. M.,
S. R. Gullans,
and
W. W. Chin.
Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase.
J. Clin. Invest.
97:
1884-1889,
1996
10.
Cohen, D. M.,
S. R. Gullans,
and
W. W. Chin.
Urea-inducibility of Egr-1 in murine inner medullary collecting duct cells is mediated by the serum response element and adjacent Ets motifs.
J. Biol. Chem.
271:
12903-12908,
1996
11.
Deora, A. A.,
T. Win,
B. Vanhaesebroeck,
and
H. M. Lander.
A redox-triggered Ras-effector interactionrecruitment of phosphatidylinositol 5'-kinase to Ras by redox stress.
J. Biol. Chem.
273:
29923-29928,
1998
12.
Enari, M.,
R. V. Talanian,
W. W. Wong,
and
S. Nagata.
Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.
Nature
380:
723-726,
1996[ISI][Medline].
13.
Fan, J.,
and
J. R. Bertino.
K-ras modulates the cell cycle via both positive and negative regulatory pathways.
Oncogene
14:
2595-2607,
1997[ISI][Medline].
14.
Feig, L. A.,
and
G. M. Cooper.
Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.
Mol. Cell Biol.
8:
3235-3243,
1988[ISI][Medline].
15.
Ferraris, J. D.,
C. K. Williams,
K. Y. Jung,
J. J. Bedford,
M. B. Burg,
and
A. Garcia-Perez.
ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress.
J. Biol. Chem.
271:
18318-18321,
1996
16.
Itoh, T.,
A. Yamauchi,
A. Miyai,
K. Yokoyama,
T. Kamada,
N. Ueda,
and
Y. Fujiwara.
Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells.
J. Clin. Invest.
93:
2387-2392,
1994[ISI][Medline].
17.
Joneson, T.,
and
D. Bar-Sagi.
Ras effectors and their role in mitogenesis and oncogenesis.
J. Mol. Med.
75:
587-593,
1997[ISI][Medline].
18.
Kawasaki, H.,
T. Moriguchi,
S. Matsuda,
H. Z. Li,
S. Nakamura,
S. Shimohama,
J. Kimura,
Y. Gotoh,
and
E. Nishida.
Ras-dependent and Ras-independent activation pathways for the stress-activated protein kinase cascade.
Eur. J. Biochem.
241:
315-321,
1996[Abstract].
19.
Kultz, D.,
S. Madhany,
and
M. B. Burg.
Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2.
J. Biol. Chem.
273:
13645-13651,
1998
20.
Kwon, H. M.,
T. Itoh,
J. S. Rim,
and
J. S. Handler.
The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes.
Biochem. Biophys. Res. Commun.
213:
975-979,
1995[ISI][Medline].
21.
Lander, H. M.,
D. P. Hajjar,
B. L. Hempstead,
U. A. Mirza,
B. T. Chait,
S. Campbell,
and
L. A. Quilliam.
A molecular redox switch on p21ras. Structural basis for the nitric oxide-p21ras interaction.
J. Biol. Chem.
272:
4323-4326,
1997
22.
Lang, F.,
M. Ritter,
E. Woll,
H. Weiss,
D. Haussinger,
J. Hoflacher,
K. Maly,
and
H. Grunicke.
Altered cell volume regulation in ras oncogene expressing NIH fibroblasts.
Pflügers Arch.
420:
424-427,
1992[ISI][Medline].
23.
Miyakawa, H.,
S. K. Woo,
C. P. Chen,
S. C. Dahl,
J. S. Handler,
and
H. M. Kwon.
Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity.
Am. J. Physiol. Renal Physiol.
274:
F753-F761,
1998
24.
Nordeen, S. K.
Luciferase reporter gene vectors for analysis of promoters and enhancers.
Biotechniques
6:
454-458,
1988[ISI][Medline].
25.
Rommel, C.,
and
E. Hafen.
Rasa versatile cellular switch.
Curr. Opin. Genet. Dev.
8:
412-418,
1998[ISI][Medline].
26.
Santos, B. C.,
A. Chevaile,
M. J. Hébert,
J. Zagajeski,
and
S. R. Gullans.
A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality.
Am. J. Physiol. Renal Physiol.
274:
F1167-F1173,
1998
27.
Scheele, J. S.,
J. M. Rhee,
and
G. R. Boss.
Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts.
Proc. Natl. Acad. Sci. USA
92:
1097-1100,
1995[Abstract].
28.
Sharma, P. M.,
K. Egawa,
Y. Huang,
J. L. Martin,
I. Huvar,
G. R. Boss,
and
J. M. Olefsky.
Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action.
J. Biol. Chem.
273:
18528-18537,
1998
29.
Takenaka, M.,
A. S. Preston,
H. M. Kwon,
and
J. S. Handler.
The tonicity-sensing element that mediates increased transcription of the betaine transporter gene in response to hyperosmotic stress.
J. Biol. Chem.
269:
29379-29381,
1994
30.
Terada, Y.,
K. Tomita,
M. K. Homma,
H. Nonoguchi,
T. Yang,
T. Yamada,
Y. Yuasa,
E. Krebs,
S. Sasaki,
and
F. Marumo.
Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.
J. Biol. Chem.
269:
31296-31301,
1994
31.
Tsai-Morris, C.,
X. Cao,
and
V. P. Sukhatme.
5'-Flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene.
Nucleic Acids Res.
16:
8835-8846,
1988[Abstract].
32.
Van der Wijk, T.,
J. Dorrestijn,
S. Narumiya,
J. A. Maassen,
H. R. De Jonge,
and
B. C. Tilly.
Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway.
Biochem. J.
331:
863-869,
1998[ISI][Medline].
33.
Vojtek, A. B.,
and
C. J. Der.
Increasing complexity of the Ras signaling pathway.
J. Biol. Chem.
273:
19925-19928,
1998
34.
Wang, X. Z.,
B. Lawson,
J. W. Brewer,
H. Zinszner,
A. Sanjay,
L. J. Mi,
R. Boorstein,
G. Kreibich,
L. M. Hendershot,
and
D. Ron.
Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153).
Mol. Cell Biol.
16:
4273-4280,
1996[Abstract].
35.
Yang, X.-Y.,
Z. Zhang,
and
D. M. Cohen.
ERK activation by urea in renal inner medullary cells.
Am. J. Physiol. Renal Physiol.
277:
F176-F185,
1999
36.
Zhang, Z.,
and
D. M. Cohen.
NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
271:
F1234-F1238,
1996
37.
Zhang, Z.,
X.-Y. Yang,
and
D. M. Cohen.
Urea-associated oxidative stress and Gadd153/CHOP induction.
Am. J. Physiol. Renal Physiol.
276:
F786-F793,
1999
38.
Zhang, Z.,
X.-Y. Yang,
S. P. Soltoff,
and
D. M. Cohen.
PI3K signaling in the murine kidney inner medullary cell response to urea.
Am. J. Physiol. Renal Physiol.
278:
F155-F164,
2000