1 Divisions of Nephrology and Molecular Medicine, Oregon Health Sciences University and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201; and 2 Division of Signal Transduction, Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
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ABSTRACT |
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Growth factors and other stimuli increase the activity of phosphatidylinositol-3 kinase (PI3K), an SH2 domain-containing lipid kinase. In the murine kidney inner medullary mIMCD3 cell line, urea (200 mM) increased PI3K activity in a time-dependent fashion as measured by immune complex kinase assay. The PI3K effector, Akt, was also activated by urea as measured by anti-phospho-Akt immunoblotting. In addition, the Akt (and PI3K) effector, p70 S6 kinase, was activated by urea treatment in a PI3K-dependent fashion. PI3K inhibition potentiated the proapoptotic effect of hypertonic and urea stress. Urea treatment also induced the tyrosine phosphorylation of Shc and the recruitment to Shc of Grb2. Coexistence of activated Shc and PI3K in a macromolecular complex was suggested by the increase in PI3K activity evident in anti-Shc immunoprecipitates prepared from urea-treated cells. Taken together, these data suggest that PI3K may regulate physiological events in the renal medullary cell response to urea stress and that an upstream tyrosine kinase conferring activation of both PI3K and Shc may govern urea signaling in these cells.
sodium chloride; hypertonicity; mIMCD3 cells; apoptosis; stress
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INTRODUCTION |
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CELLS OF THE MAMMALIAN RENAL medulla are uniquely
exposed to an elevated and rapidly fluctuating urea concentration as a
consequence of the renal concentrating mechanism. In a murine cell line
derived from the renal inner medulla, urea in concentrations
physiologically unique to the renal medulla (200 mM) induces activation
of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) (7); activation of the ERK
substrate, Elk-1 (7); the generation of inositol 1,4,5-trisphosphate
(IP3) (11); activation of the receptor
tyrosine kinase (RTK)-specific phospholipase C (PLC) isoform, PLC-
(11); and transcription and translation of the immediate-early gene
transcription factor, Egr-1, in a serum response element (SRE)- and Ets
motif-dependent fashion (8, 12). These signaling events are
characteristic of those initiated by a RTK and have
suggested to some the presence of an upstream urea-sensing tyrosine
kinase. To date, none has been identified. Specific sensors of ambient
tonicity have been described in yeast (33, 34), although not in higher
eukaryotes. Urea does not function as a hypertonic stressor, per se; it
is a readily membrane-permeant solute that is unlikely to engender chronic changes in cell volume (23), in contrast to the functionally impermeant solute and other principal constituent of medullary osmolarity, NaCl. Accordingly, whereas NaCl exhibits a robust ability
to activate the tonicity-responsive kinases p38 and SAPK/JNK in murine
kidney inner medullary mIMCD3 cells, urea exerts a much more modest or even negligible effect (2, 60).
Phosphatidylinositol-3 kinase (PI3K), a phosphoinositide kinase, is a heterodimer comprising regulatory (p85) and catalytic (p110) subunits (6); the former contains two SH2 domains that mediate its interaction with diverse activated RTKs. PI3K is instrumental in regulating mitogenic signaling, apoptosis, and matrix and cytoskeletal organization (6); all three of these functions have been postulated to play a role in the cellular response to urea. Potential physiological effectors of PI3K include Ras (25), protein kinase C (PKC; 54), Akt (5, 22), and p70 S6 kinase (57).
The ability of urea to activate PI3K and potential PI3K effectors in renal medullary epithelial cells was therefore investigated as a potential physiological context for this signaling pathway. Urea potently activated PI3K in a time-dependent fashion and activated Akt and p70 S6 kinase in a PI3K-dependent fashion. Urea treatment of mIMCD3 cells also activated Shc, induced Grb2 recruitment, and increased PI3K activity in anti-Shc immunoprecipitates. In addition, PI3K inhibition sensitized cells to the proapoptotic effect of urea and hypertonic stress in the mIMCD3 cell line. In aggregate, these observations support a role for PI3K signaling in urea (and potentially hypertonic) signaling, as well as the existence of an upstream receptor or non-receptor tyrosine kinase responsive to ambient urea concentration.
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METHODS |
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Cell culture and solute treatment. mIMCD3 cells were maintained and passaged as previously described (8). Prior to each experiment, cells were placed in serum-free medium for 24 h. Solute treatment consisted of the 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).
PI3K activity. Anti-PY immunoprecipitates (using
antibody PY20) were assayed for PI3K activity as previously described
(50, 58). Briefly, lipids and kinase reaction buffer (in final
concentrations: ATP, 50 µM; MgCl2, 5 mM; HEPES, 1 mM; [-32P]ATP, 10 µCi) were added to
the immunoprecipitates and incubated for 10 min at room temperature.
Phosphatidylinositol (PI) was added to final concentration of 0.2 mg/ml. In experiments in which phosphatidylinositol 3,4,5-trisphosphate
[PI(3,4,5)P3] production was
assayed, PI(4,5)P2 and phosphatidylserine (in a 1:2 ratio) were added to 0.2 mg/ml final concentration of the combined lipids. Lipids were sonicated prior to use. Where indicated, immunoprecipitates were exposed to wortmannin (10 nM) for 10 min prior to the addition of
lipid and kinase buffer. The lipid kinase assay was stopped by the
addition of 80 µl of HCl (1 M) and 160 µl of methanol:chloroform (1:1 mixture). The lipid-containing organic phase was resolved on
oxalate-coated thin-layer chromatography plates (Silica
gel 60, MCB reagents; Merck, Rahway, NJ) developed in
chloroform:methanol:water:ammonium hydroxide (60:40:11.3:2), or in
n-propanol:2 M acetic acid (65:35) to quantify PI(3)P or
PI(3,4,5)P3 production, respectively. Radiolabeled spots
corresponding to PI(3)P or PI(3,4,5)P3 were quantified
using a Bio-Rad Phosphoimager. In these and subsequent graphically
depicted experiments, data from at least three separate experiments
(each consisting of 2-4 replicates per experimental condition per
experiment) were pooled, expressed as means ± SE, and then compared
via t-test (Microsoft Excel software).
Immunoprecipitation and immunoblot analyses. For anti-Shc immunoprecipitates, mIMCD3 cell monolayers were lysed in situ with Shc lysis buffer (45) composed of (in mM)30 Tris (pH 7.4); 150 NaCl, 10 EDTA, 1 PMSF, 1 sodium orthovanadate, and supplemented with 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 nM aprotinin, and 1 µM leupeptin, and immunoprecipitated with monoclonal anti-Shc antibody (0.5 µg/ml; Transduction Laboratories) and Protein G beads (20 µl/sample; Pharmacia). Following extensive washing with both Shc lysis buffer and then Tris-buffered saline, immunoprecipitates were eluted from agarose beads by boiling in 1× Laemmli sample buffer (43) prior to SDS-PAGE analysis. Alternatively, for p85 immunoblot analysis, anti-PY immunoprecipitates from RIPA-lysed cells were analyzed in similar fashion. Following electrophoresis, proteins were subjected to semi-dry transfer to polyvinylidene difluoride (PVDF) membrane and then incubated with anti-p85, anti-PY, anti-Grb2 (Santa Cruz Laboratories), 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) followed by autoradiography.
Transfection and reporter gene assays. The construction of
Egr-1-Luc, composed of 1.2 kb of the murine Egr-1 5' flanking
sequence (including the minimal promoter; Ref. 55) subcloned upstream of the promoterless luciferase reporter vector, pXP2 (37), has previously been described (12). BGT2X-Luc was prepared by subcloning a
double-stranded oligonucleotide encoding two tandem repeats of the
canine tonicity enhancer element [TonE: Takenaka et al. (51),
Miyakawa et al. (36)] upstream of the thymidine
kinase promoter in BamH I/Hind III-cleaved vector
TK-Luc (37). (5'Oligonucleotide sequence was gat cct act tgg tgg
aaa agt cca gtc gat act tgg tgg aaa agt cca ga; 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 Egr-1-Luc + 3 µg CMV-Gal per subconfluent 100-mm dish via
electroporation as described (12). Luciferase and -galactosidase
activities in detergent lysates were determined as previously described
(12); the former was normalized with respect to the latter (Luc/gal).
Control and solute treatments were for 6 h (starting 48 h after
transfection); inhibitors (wortmannin and LY-294002) were added 30 min
prior to solute addition unless otherwise indicated.
p70 S6 kinase assay. Control- and solute-treated mIMCD3
monolayers were lysed with 1 ml of lysis buffer [20 mM Tris (pH
7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml
leupeptin; Ref. 17], then immunoprecipitated with anti-p70 S6
kinase COOH-terminal antibody (Santa Cruz Laboratories) for 60 min at
4°C. Immunoprecipitates were washed twice with lysis buffer, twice
with kinase buffer [20 mM MOPS (pH 7.2), 25 mM
-glycerophosphate, 1.25 mM EGTA, 200 µM sodium orthovanadate, and
1 mM dithiothreitol (DTT)], and then subjected to an in vitro
kinase assay using an S6 kinase-specific peptide substrate in the
presence of inhibitors of other kinases, according to the
manufacturer's directions (Upstate Biotechnology). Incubation was
performed for 10 min at 30°C, after which the reactions were
spotted on phosphocellulose paper, washed extensively with 0.75%
phosphoric acid, and then washed with acetone prior to drying and
scintillation counting. Kinase activity was expressed in raw counts per
100-mm dish. Data are presented as means ± SE of at least three
separate experiments.
Cellular ATP content. An assay of intracellular ATP content was used as an index of cellular metabolic stress (29), as previously applied to renal cells (e.g., Ref. 48). This method was selected because other methods (e.g., propidium iodide exclusion and fluorescein diacetate conversion) were confounded by fluorescence of the vehicles and inhibitors used in the present study. mIMCD3 monolayers (in 24-well dishes and maintained in serum-free medium for 24 h) were treated for the indicated times with the indicated stressor for an additional 24 h. Cells were washed with ice-cold PBS and lysed in situ with 500 µl 0.5% Triton X-100. Plates were spun at 1,000 g for 10 min at 4°C. ATP content in 5 µl of lysate supernatant was quantitated using the ATP Determination Kit (Molecular Probes, Eugene, OR) by addition to 200 µl of complete assay buffer containing 1× reaction buffer [25 mM Tricine (pH 7.8), 5 mM MgSO4, 0.1 mM EDTA, and 0.1 mM sodium azide], 1.5 mM DTT, and 1.875 µg/ml luciferase. Reaction was initiated by injection of 100 µl of luciferin stock (0.5 mM luciferin in 1× reaction buffer) in a Berthold Lumat LB-9501 luminometer. Data were normalized to protein content per well, as determined by the DC Protein Assay (Bio-Rad) according to the manufacturer's directions.
Apoptosis assays. Caspase-3 (cpp32) microfluorescence assay was performed according to a modification of the method of Enari et al. (20). 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 PMSF, 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. Following centrifugation at 10,000 g for 12 min at 4°C, supernatants were assayed for protein concentration as 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% CHAPS, 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. Annexin binding was determined using the ApoAlert Annexin V Apoptosis Kit (Clontech) in accordance with the manufacturer's directions. Cells were stained with propidium iodide and Annexin V-FITC as directed, then sorted (FL1 for Annexin V-FITC and FL2 for propidium iodide) on a Becton-Dickinson Calibur instrument, following gating upon a representative population of cells established by forward- and side-angle light scatter. Apoptosis (early) was ascribed to cells exhibiting Annexin staining >40 fluorescence units (FL1) and PI staining <300 fluorescence units (FL2).
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RESULTS |
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Urea activates PI3K in a time-dependent fashion. In confluent,
serum-deprived mIMCD3 cells, urea increased PI3K activity 3.2-fold [relative to basal (time 0)] at 1 min of treatment
and 2.5-fold at 5 min of treatment (Fig.
1A). By 15 min, PI3K activity had returned to baseline. Sham treatment (treatment with isosmotic NaCl)
exerted no effect upon PI3K activity at any time point examined (data
not shown). In contrast to treatment with urea, NaCl treatment (200 mosmol/kgH2O) exhibited no effect at 1 min (Fig.
1A). PI3K activity was, however, increased by a factor of 3.1 at 5 min of NaCl treatment, and by a factor of 2.3 at 15 min of NaCl
treatment. Although there was considerable variation among experiments
in the magnitude of the PI3K activation by urea, the effect was
observed in each of four separate experiments with peak activation
ranging from 2.2- to 5.9-fold. To confirm that the immune complex
kinase assay was indeed measuring PI3K activity and not that of a
related lipid kinase, additional experiments were performed wherein
PI(4,5)P2 was employed as the substrate. Results with
PI(4,5)P2 precisely paralleled those observed using PI
(data not shown). In addition, the effect of wortmannin, a
pharmacological inhibitor of PI3K, was also examined. The PI3K activity
in anti-PY immunoprecipitates from control and urea-treated cells was
inhibited by 90% by wortmannin (10 nM) in vitro (data not shown).
The effect of increasing concentrations of urea upon PI3K activity was
next assessed. The 5-min rather than 1-min time point was selected for
ease of reproducibility. The ability of urea to activate PI3K activity
was not demonstrably dose-dependent within the range of 200-800 mM
(Fig. 1B), although PI3K activity was increased relative to
control at each of these concentrations.
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In correlative fashion, the effect of solute treatment upon abundance
of the SH2 domain-containing PI3K subunit, p85, in the anti-PY
immunoprecipitates used for measuring PI3K activity was determined via
immunoblot analysis. Consistent with the data presented in Fig.
1A, the abundance of p85 in anti-PY immunoprecipitates (Fig.
2) was maximal following 1 min of urea
treatment and was at least twofold greater than at time 0.
Thus, the urea-promoted increase in PI3K activity (Fig. 1A) was
due to an increase in the association of PI3K with anti-PY
immunoprecipitates. In similar fashion, p85 abundance in the anti-PY
immunoprecipitates from NaCl-treated cells was increased at the 5- and
15-min time points, but not at 1 min of treatment (Fig. 2).
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Effectors of PI3K in urea stress. Urea activates transcription
of the immediate-early gene, Egr-1 (8), through a signaling pathway
bearing hallmarks of a PKC- (11) and Ras-mediated event. Because Ras
represents a potential upstream activator of both PI3K activation and
Egr-1 transcription, we hypothesized that PI3K activation
may play a role in urea-inducible Egr-1 transcription. mIMCD3 cells
were transiently transfected with a luciferase reporter construct
driven by 1.2 kb of the murine Egr-1 proximal 5'flanking sequence
(diagrammed in Fig. 3A). As
previously reported (12), urea (200 mM) increased Egr-1 transcription
in luciferase reporter gene assay by sevenfold (Fig. 3A). The
pharmacological inhibitor of PI3K action, wortmannin (10 nM), failed to
inhibit urea-inducible transcription (Fig. 3A). Higher
concentrations of wortmannin (100 nM and 1 µM) and another PI3K
inhibitor, LY-294002 (10 and 30 µM), also failed to inhibit this
effect (data not shown). Because of the ability of hypertonic stress to
activate PI3K, albeit at later time points than urea, and the
incompletely understood nature of hypertonicity-inducible gene
regulation, the role of PI3K activation in a model of
hypertonicity-inducible gene transcription was examined in parallel.
Tandem repeats of the tonicity-responsive element (TonE) of the betaine
transporter gene (BGT1; Ref. 51) were subcloned upstream of the
thymidine kinase (TK) promoter and luciferase reporter
gene (Fig. 3B). Consistent with the observations of others (36,
51), hypertonic NaCl (200 mosmol/kgH2O) increased reporter gene activity 10-fold. Pretreatment with wortmannin (10 nM to 1 µM)
failed to abrogate NaCl-inducible transcription.
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p70 S6 kinase is a potential physiological effector of PI3K (53). Urea
(200 mM) significantly increased p70 S6 kinase activity in a
time-dependent fashion (Fig. 4A);
the effect was detectable as early as 1 min of treatment (2-fold
increase) and remained evident at 15 min of treatment (3-fold
increase). To bolster the argument that urea-inducible p70 S6 kinase
activity was a consequence of urea-inducible PI3K activity, the effect
of wortmannin and LY-294002 pretreatment upon urea-inducible p70 S6
kinase activity was examined. At 5 min of treatment, urea increased p70
S6 kinase activity by 75% (Fig. 4B). Pretreatment with either
wortmannin (10 nM) or LY-294002 (10 µM) exerted no statistically
significant effect upon basal p70 S6 kinase activity but significantly
inhibited the urea-inducible increment in S6 kinase activity by 55%
and 100%, respectively.
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Recent data suggest that the protein kinase Akt mediates the effects of
PI3K upon p70 S6 kinase activation (reviewed in Ref. 21). Urea
treatment (200 mM) increased phosphorylation of Akt as detected by
anti-phospho-Akt immunoblotting (Fig.
5A). The effect of NaCl was
consistently less pronounced. Both wortmannin (10 and 100 nM) and
LY-294002 (10 and 30 µM) inhibited the effect of urea upon Akt
phosphorylation in a dose-dependent fashion (Fig. 5B),
suggesting a role for PI3K activation in urea-inducible Akt activation.
The ability of both urea and NaCl to increase Akt phosphorylation was
steeply dose-dependent (Fig. 6) at the time points correlated with maximal induction (5 min for urea and 15 min for
NaCl). At 100 and 200 mosmol/kgH2O solute, the urea effect exceeded that of NaCl.
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We have previously shown that urea treatment results in the activation
of the SH2 domain-containing phospholipase, PLC-. We hypothesized
that a urea-activable (tyrosine-phosphorylated) upstream receptor or
non-receptor tyrosine kinase recruited and activated PLC-
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present data imply activation of another tyrosine kinase effector,
PI3K. To further implicate an upstream activating kinase, the ability
of urea to activate (induce the tyrosine phosphorylation of) the RTK
effector and adapter molecule, Shc, was examined. Under control
(sham-treated) conditions, immunoprecipitated Shc exhibited
demonstrable tyrosine phosphorylation by anti-PY immunoblotting (Fig.
7; left). Following urea treatment,
but not NaCl treatment, the degree of tyrosine phosphorylation was
markedly upregulated. In addition, the abundance of the adapter
molecule and Shc interaction partner, Grb2, was also markedly increased in anti-Shc immunoprecipitates from urea-treated cells (Fig. 7; right). The ability of urea to activate PI3K and Shc and to
recruit Grb2 strongly suggested the presence of a
phosphotyrosine-bearing upstream activator. It was hypothesized that
such an activator, as has been observed in other models, would serve as
a molecular "scaffold" for recruitment of multiple effectors.
Therefore, the ability of anti-Shc immunoprecipitates from urea-treated
cells to exhibit increased PI3K activity (diagrammed in Fig.
8A) was examined. Consistent with
this model, urea treatment of cells reproducibly increased PI3K
activity in anti-Shc immunoprecipitates (Fig. 8B).
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Physiological consequences of PI3K inhibition. To determine the
possible contributions of PI3K action to a physiological consequence of
hypertonic stress and elevated urea concentration, mIMCD3 cells were
treated with PI3K inhibitors (LY-294002, 30 µM; or wortmannin, 100 nM) and evaluated with respect to intracellular ATP content as an index
of cellular metabolic stress (29), as previously applied to renal cells
(e.g., Ref. 48). The effects of the two inhibitors were comparable.
Pretreatment with LY-294002 did not influence relative ATP content
(normalized to cell protein) under basal conditions in mIMCD3 cells
[n = 3 separate experiments, with 3-6 individual
determinations (replicates) per experimental condition; data not
shown]. Urea decreased cell ATP content by only 5% in the
absence of PI3K inhibition but by 18% in the presence of the inhibitor
(P < 0.05; LY-294002 vs. +LY-294002). Similarly, NaCl
decreased cellular ATP content by 18% in the absence of PI3K inhibition and by 29% in its presence (P < 0.05;
LY-294002 vs. +LY-294002). These data suggested that whereas
PI3K inhibition in isolation produced no adverse effect, its
superimposition upon osmotic and urea stress exacerbated an index of
metabolic stress.
Because others have shown that hyperosmotic solutes, including NaCl and
urea (44), may induce apoptosis and because PI3K activation has been
implicated in this process, a possible protective role of PI3K
signaling was evaluated in this context. Specifically, the ability of
PI3K inhibition by wortmannin and LY-294002 to potentiate urea- and
NaCl-inducible apoptosis was investigated. Caspase-3 activation has
been described as a "rubicon" of apoptosis, and
correlates closely with this phenomenon in vitro and in vivo (56).
Wortmannin treatment increased caspase-3 activity in mIMCD3 cells by
68% in the absence of hypertonic or urea stress (Fig. 9); however, this was not significantly
different from treatment with vehicle alone. Urea (200 mM) exerted no
adverse effect upon caspase activity, consistent with prior
observations concerning the ability of the mIMCD3 cell line to tolerate
this solute; however, a modest (30%) protective effect could not be
excluded. In the presence of wortmannin pretreatment, urea-associated
apoptosis was increased 171%. Urea at 400 mM and NaCl at 200 mosmol/kgH2O (100 mM) substantially increased caspase-3
activity. The effect of these solutes was increased by 266% and 185%,
respectively, in the presence of wortmannin pretreatment. Similar
findings were observed when cells were pretreated with LY-294002 (data
not shown). Of note, NaCl at 400 mosmol/kgH2O maximally
increased caspase-3 activity (mean = 24-fold, relative to control), and
a further increment could not be observed in the presence of wortmannin pretreatment (data not shown). In contrast, when cells were pretreated with the phospholipase A2 inhibitor, quinacrine, or the PLC
inhibitor, U-73122, there was only a modest effect (40% increase)
upon NaCl- and urea-inducible apoptosis (as measured via caspase
activation), which was indistinguishable from the effect of these
inhibitors upon control-treated cells (data not shown).
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To further corroborate these findings, the effect of PI3K inhibition
upon urea and osmotic tolerance was examined using another index of
apoptosis. Initiation of apoptosis is followed by translocation of
phosphatidylserine from the inner to the outer leaflet of the cell
membrane, a phenomenon detectable by cell staining with
fluorophore-conjugated annexin V. Following gating upon a homogeneous
population of FACS-sorted cells with respect to forward-
and side-angle light scatter (see METHODS), apoptosis
(early) was assigned to cells exhibiting PI staining <30 U and
annexin staining >40 U. Under control conditions, wortmannin
pretreatment increased the percentage of annexin V-positive cells by only 44.6% (Table 1). In the
presence of urea (400 mM) or NaCl (200 mM), in contrast, wortmannin
pretreatment increased the percentage of annexin
V-positive (apoptotic) cells by 178% and 91%, respectively.
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DISCUSSION |
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These data indicate that urea, in concentrations physiologically
relevant to the renal medulla in vivo, activated the lipid kinase and
RTK effector, PI3K, as well as the potential physiological PI3K
effectors, Akt and p70 S6 kinase. Inhibition of this pathway appeared
to modestly impair urea tolerance and enhance urea- and hypertonicity-associated apoptosis. Urea also induced tyrosine phosphorylation of Shc and recruitment to Shc of Grb2. Furthermore, PI3K activity was increased in anti-Shc immunoprecipitates prepared from urea-treated cells. In aggregate, these findings appear to support
the previously proposed model of receptor or non-receptor tyrosine
kinase in the initiation of urea signaling in cells of the renal medulla (11). Activation of membrane-associated kinases has
been described in hypertonic signaling in yeast (33, 34) and recently
in higher eukaryotes (41) but not in the setting of elevated urea
concentrations. In light of accumulating evidence including
urea-inducible immediate-early gene transcription (8), ERK and Elk-1
activation (7), IP3 generation (11), and activation of
PLC-(11), the involvement of an RTK-mediated signaling pathway appears increasingly likely in urea stress.
To our knowledge, these are the first studies to directly measure PI3K activity in response to hypertonic or urea stress and to implicate this pathway in osmotic and urea tolerance. Activation of PI3K has previously been suggested in other related contexts through the use of inhibitor and dominant negative-acting expression constructs. Activation of PI3K in response to elevated glucose concentrations in hepatocytes has been inferred through the use of dominant negative PI3K subunits (1). Inhibitor studies have implicated a role for PI3K in the hyperosmotic shrinkage-induced Na+-dependent glutamine uptake in muscle cells (32). Cell swelling in the setting of hypotonic stress has been associated with PI3K activation in one nonrenal model (28). However, in marked contrast to urea stress (7) and hypotonicity in renal (61) and other cells (42, 46, 47), swelling in this hepatic model was not associated with ERK activation (28). In the C6 glioma cell line, PI3K inhibitors failed to influence hypotonicity-inducible ERK activation (49). In addition to IP3, as discussed above, and PI(3,4,5)P3 (the lipid product of PI3K action), the role of another membrane-derived phospholipid has recently been described in osmotic stress signaling. PI(3,5)P2 is produced upon osmotic shock of yeast, presumably via a PI5K activity (18). Interestingly, osmotic stress in mammalian cells decreases abundance of this lipid (18).
Activations of PI3K and of Akt have been implicated in the prevention of apoptosis (21), and apoptosis has previously been demonstrated in renal medullary cells in response to both elevated urea concentrations and hypertonicity (44). In the present study, pharmacological inhibition of PI3K action potentiated the proapoptotic effect of urea and hypertonicity as determined by both a sensitive assay of caspase-3 activation and by an annexin V binding assay. It is therefore likely that PI3K action is essential for maximal osmotic and urea tolerance in these cells. This possibility is underscored by the decrement in cell ATP content associated with PI3K inhibition in the presence of hypertonic or urea treatment but not control treatment. The possible involvement of a wortmannin- and LY-294002-sensitive non-PI3K signaling intermediate cannot be excluded at present. The precise nature of this potential PI3K-associated protective (anti-apoptotic) pathway in the present context remains speculative. Such a protective effect would be of limited significance at solute concentrations not associated with a proapoptotic effect in this model (e.g., urea 200 mM). Pronounced activation of PI3K (Fig. 1) and PI3K effectors (e.g., Fig. 6), however, is observed under such conditions, raising the possibility of an additional role for PI3K-mediated signaling in the medullary cell response to urea and/or hypertonicity.
A second potential function of PI3K concerns mitogenic signaling; however, this would appear unlikely in the present model. Although urea activates DNA synthesis in canine renal MDCK and porcine renal LLC-PK1 cells without increasing cell number (10), no such effect is evident in mIMCD3 cells (8). A role for PI3K in regulating cytoskeletal dynamics and cell adhesion properties has been demonstrated in other models; a similar function can be envisioned in the context of urea (or hypertonic) stress. Hypertonic stress activated p125FAK, a component of focal adhesion complexes (59), and PI3K activation was required for p125FAK phosphorylation in the platelet-derived growth factor response (40). Hypertonic stress also influences actin expression and polymerization (3, 24, 52); the effect of urea upon these phenomena has not been examined. Interestingly, members of the Rho family of small GTP-binding proteins (including Rho, Rac, and Cdc42), which regulate focal adhesion formation and actin cytoskeletal rearrangement (39), also regulate activation of the tonicity (NaCl)-responsive MAPK, SAPK/JNK (14, 35, 38).
Urea-inducible activation of p70 S6 kinase appears to be a consequence of PI3K activation. Potential effectors of p70 S6 kinase are few; until recently, the only known physiological substrate of p70 S6 kinase was the S6 protein of the ribosomal 40S subunit. Phosphorylation of S6 is essential for cell proliferation and for the increment in protein synthesis that accompanies the G0/G1 cell cycle transition (19, 27). Interestingly, NaCl actually inhibits protein synthesis (e.g., Ref. 13, and references therein) but urea fails to do so (9). In addition, inhibitors of protein synthesis themselves may activate p70 S6 kinase (4), suggesting that the NaCl (but not the urea) effect may be a consequence of this phenomenon. p70 S6 kinase may also activate the nuclear protein and cAMP-dependent protein kinase A effector, cAMP-responsive element mediator (CREM) (15, 16). Urea, however, failed to activate transcription through tandem copies of the cAMP-responsive element in reporter gene experiments (Zhang and Cohen, unpublished observation).
The ability of urea to activate PLC- (11), PI3K, and Shc and the
association of PI3K activity with anti-Shc immunoprecipitates suggests
the presence of a urea-activable upstream receptor or non-receptor
tyrosine kinase. In model systems unrelated to the kidney
medulla, severe hypertonic stress induced the activation of the
receptors for epidermal growth factor (EGF), tumor necrosis growth
factor (TNF), and interleukin-1 (IL-1) (41), a process potentially related to an inhibition in receptor-directed phosphatase activity (26). Indiscriminate activation of membrane-associated RTKs
[e.g., EGF receptor (EGFR), hepatocyte growth factor receptor (HGFR), etc.], in contrast, is not observed in the
present model (data not shown). Accumulating data indicate that
signaling events engendered by urea treatment, despite some overlap,
are largely dissimilar from those activated by hypertonicity with
respect to MAPK isoforms activated and, ultimately, genes expressed
(reviewed in Refs. 30 and 31). In addition, whereas the response to hypertonicity appears to be universal among cell lines examined, the
response to urea has been restricted to a small subset of renal
epithelial cells including MDCK and mIMCD3 cells. Nonetheless, in a
fashion potentially similar to that of hypertonicity, the membrane-permeant solute urea likely influences activation of an
upstream receptor or non-receptor tyrosine kinase, the
identity of which remains obscure.
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ACKNOWLEDGEMENTS |
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We thank A. Bakke (Oregon Health Sciences University) for assistance with FACS analysis and R. K. Rathbun, P. S. Koh, T. A. Christianson, and G. C. Bagby (Oregon Health Sciences University) for assistance with apoptosis assays.
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FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants DK-52494 (to D. M. Cohen) and DE-10877 (to S. P. Soltoff) and the National Kidney Foundation (to D. M. Cohen).
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 S.W. US Veterans Hospital Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).
Received 3 February 1999; accepted in final form 22 July 1999.
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