Urea signaling to ERK phosphorylation in renal
medullary cells requires extracellular calcium but not calcium
entry
Xiao-Yan
Yang1,3,4,
Hongyu
Zhao1,3,4,
Zheng
Zhang1,3,4,
Karin D.
Rodland2,
Jean-Baptiste
Roullet1, and
David M.
Cohen1,2,3,4
1 Divisions of Nephrology and 3 Molecular Medicine,
and 2 Department of Cell and Developmental Biology, Oregon
Health Sciences University and the 4 Portland Veterans
Affairs Medical Center, Portland, Oregon 97201
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ABSTRACT |
The
renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of
the extracellular signal-regulated kinase (ERK) 1 and 2 in response to
urea treatment (200 mM for 5 min). Previous data have suggested
the involvement of a classical protein kinase C (cPKC)-dependent
pathway in downstream events related to urea signaling. We now show
that urea-inducible ERK activation requires extracellular calcium;
unexpectedly, it occurs independently of activation of cPKC isoforms.
Pharmacological inhibitors of known intracellular calcium release
pathways and extracellular calcium entry pathways fail to inhibit ERK
activation by urea. Fura 2 ratiometry was used to assess the effect of
urea treatment on intracellular calcium mobilization. In single-cell
analyses using subconfluent monolayers and in population-wide analyses
using both confluent monolayers and cells in suspension, urea failed to
increase intracellular calcium concentration. Taken together, these
data indicate that urea-inducible ERK activation requires calcium
action but not calcium entry. Although direct evidence is lacking, one
possible explanation could include involvement of a calcium-dependent
extracellular moiety of a cell surface-associated protein.
fura 2; inner medullary collecting duct; Madin-Darby canine kidney; protein kinase C; hypotonicity
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INTRODUCTION |
CELLS OF THE MAMMALIAN
KIDNEY medulla are subjected to elevated urea concentrations in
vivo. In cultured epithelial cells derived from this tissue, but not in
other cell types, urea activates a characteristic subset of signaling
events culminating in transcription and expression of immediate early
genes encoding transcription factors such as c-fos and Egr-1. Signaling
events implicated in the initiation of this downstream program include
activation of the Ras/Raf/mitogen-activated protein kinase (MAPK)/ERK
(MEK)-extracellular signal-regulated kinase (ERK) pathway (9, 40,
42) and phosphatidylinositol-3-kinase p70 S6 kinase-Akt pathway
(44). A similar profile of signaling events is
seen on engagement of receptors for growth factors and cytokines. Other
features of the urea response suggestive of a potentially
receptor-mediated phenomenon include activation and/or tyrosine
phosphorylation of RAFTK/PYK2 (43), phospholipase C (PLC)-
(11), and the adapter protein Shc, as well as
the recruitment to Shc of the adapter protein Grb2 (44).
Although the ability of urea to activate ERK was dependent on
activation of MEK1 and MEK2 (42), it was unexpectedly only
partially dependent on activation of the small GTP-binding protein Ras
(40). Because calcium-mediated signaling is an effector of
PLC-
and inositol trisphosphate (IP3) action, the role
of these events was explored in the cultured mIMCD3 cell model.
Urea-inducible ERK activation required the presence of
extracellular calcium, yet was independent of activation of the
calcium-dependent protein kinase C (PKC) isoforms, activation of
calmodulin and calmodulin-dependent protein kinases, and independent of
the voltage-gated calcium channel. Unexpectedly, the calcium
requirement was independent of intracellular calcium release or entry
of extracellular calcium, as evidenced by fura 2 fluorescence
ratiometry in cells in suspension or in confluent or subconfluent
monolayer culture. These data suggest the involvement of
calcium-dependent elements of the extracellular environment or matrix
in urea signaling in cells derived from the renal medulla.
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METHODS |
Cell culture, experimental manipulations, and materials.
Cells (mIMCD3) were grown in DMEM-F-12 in monolayers
(32), and on achieving confluence they were serum deprived
for 24 h before experimental manipulation unless otherwise noted.
Cells were treated with the gentle dropwise addition of concentrated stocks of urea or NaCl to achieve the desired final osmolarity. Downregulation of classic PKC (cPKC) isoforms was achieved through 6- or 18-h pretreatment with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA). For depletion of intra- and extracellular
calcium, monolayers were taken out of serum for 24 h and treated
with 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic
acid-acetomethoxy ester (BAPTA-AM; 75 µM for 30 min), after which
medium was washed twice with Hanks' balanced salt solution (HBSS; 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.4, 10 mM glucose) or calcium-free HBSS (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES, pH 7.4, 10 mM glucose, 10 mM EDTA) and cells were incubated in either HBSS or calcium-free HBSS
for 30 min before treatment with urea (200 mM for 5 min) or TPA (100 nM
for 5 min). Inhibitors and activators of calcium transport and
action (obtained from Calbiochem unless otherwise indicated) were used
as follows: KN-93, 20 µM for 30 min; A-23187, 10 µM for 5-30
min; thapsigargin, 10-100 nM times indicated duration; epidermal
growth factor (EGF, Sigma), 100 nM times indicated duration;
dantrolene, 100 µM for 30 min; xestospongin C, 10 µM for 30 min;
U-73122, 5-20 µM for 30 min; verapamil 10-100 µM for 30 min; nimodipine, 10 µM for 30 min; NiCl2 (Sigma),
1-3 mM for 30 min; CdCl2 (Sigma), 1-3 mM for 30 min; carboxyamido-triazole (CAI), 10 µM for 30 min;
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; Sigma), 2 µM for 30 min; caffeine, 10 mM for 5 min;
calmidazolium, 20 µM for 30 min; gadolinium chloride (Sigma), 20 µM-1 mM for 2-60 min; BAY K 8644, 10-100 µM for 30 min;
ATP 10 µM for indicated interval; and bradykinin 100 nM for indicated
interval. For titration of extracellular calcium (see Fig. 11),
BAPTA concentration ([BAPTA]) necessary to establish indicated free
calcium concentration (1.44, 2.33, and 5.43 mM BAPTA for 300, 100, and
30 nM free calcium, respectively) was estimated using MaxChelator
(3) shareware (www.stanford.edu/~cpatton/maxc.html). Immunoblot
experiments were performed at least twice with equivalent
results; calcium ratiometry was performed at least three times per
condition on 3 separate days with equivalent results.
Because it was observed that 30 min of calcium depletion approached the
threshold of cell tolerance in terms of adherence, an alternative
strategy was employed to partially dissociate the effects of adherence
and calcium depletion. Cells were grown on collagen-treated dishes
(BioCoat; Falcon/Becton-Dickinson 354400), which facilitate
integrin-mediated binding to the epithelial cell surface. Confluent
mIMCD3 or Madin-Darby canine kidney (MDCK) cells were washed three
times with minimal essential medium (MEM) (calcium-free) medium (Life
Technologies 11385) to deplete extracellular calcium and placed in
MEM. Cell adhesion of mIMCD3 was excellent; at 30 min, cells
were identical in appearance to control (calcium-replete) cells. At
2 h, nearly all cells remained adherent; however, by 6 h a
substantial percentage of cells (
50%) became nonadherent. Similarly, on collagen substrate, MDCK monolayers remained completely attached beyond 6 h of calcium depletion.
Subcellular fractionation.
Cells were lysed and scraped into PKC lysis buffer (18)
containing 20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 80 µg/ml
aprotinin, and 0.1% 2-mercaptoethanol; crude lysates were then
subjected to Dounce homogenization for 30 strokes with a tight pestle
and centrifuged at 60,000 g for 30 min. Supernatant
(cytosolic fraction) was carefully removed and aliquoted; pellet
(membrane fraction) was resuspended via Dounce homogenization in PKC
lysis buffer that had been supplemented with 1.2% Triton X-100 and
stored in aliquots after determination of protein concentration.
Quantitation of protein concentration and immunoblotting of equal
amounts (in µg) of whole cell lysates, and subcellular fractions were
performed as previously described (10) using the following
antibodies: anti-P-ERK (New England BioLabs); anti-PKC-
(Santa
Cruz); and anti-cPKC (MC5 monoclonal, Santa Cruz).
Intracellular calcium measurement.
For population-wide analysis of cytosolic calcium mobilization, mIMCD3
and MDCK cells were grown to dense confluence on glass coverslips in
DMEM-F-12 (Life Technologies). The cells were first rinsed twice with
HBSS (in mM; 130 NaCl, 4.7 KCl, 1.18 MgSO4, 15 HEPES, 1.25 CaCl2, 5 glucose, pH 7.4). It was confirmed in independent
experiments that this buffer was permissive for urea-inducible ERK
activation for at least 6 h (data not shown). Cells were then loaded with fura 2-AM (2 mM) for 30 min at 37°C, followed by a 45-min
washout in fura 2-free HBSS (37°C). Fluorescent emission was
monitored at 510 nm with alternate excitation at 340 nm
(F340) and 380 nm (F380) using a Hitachi F2000
spectrofluorometer (Hitachi Instruments, Naperville, IL); ratio (R) of
F340/F380 is depicted in the figures.
Alternatively, intracellular calcium concentration measurements were
conducted on cell suspensions obtained by trypsinization of confluent
cell monolayers. The suspended cells were loaded with fura 2 as
previously described, washed twice with fura 2-free HBSS, and assayed
for intracellular calcium concentration in a cuvette under constant,
gentle agitation (2-ml final volume). Calibration of the fura 2 signal
was performed using 20 µl of a 4:1 (vol/vol) mixture of Tris (pH 8.3, 1 M)-digitonin (2 g/l) and 20 µl EGTA (1 M) for Rmax and
Rmin, respectively, and a fura 2-calcium dissociation
constant of 224 (7, 35).
For single-cell assessment of intracellular calcium release, a
modification of the above protocol was used. mIMCD3 cells were grown to
desired degree of confluence (~70-100%) on disposable glass-bottom 35-mm dishes (MatTek) and placed in serum-free medium for
24 h before fura 2-AM loading. Cells were loaded with fura 2-AM
for 30 min and then placed in fura 2-AM-free calcium-containing HBSS
for 20 min to permit ester cleavage. Cells were treated with 1 volume
of 400 mM urea or with positive control (e.g., EGF 100 nM), and
intracellular calcium was determined as previously described (33). Approximately 20 cells/field were individually and
simultaneously analyzed per experiment.
Calmodulin-dependent protein kinase II assay.
Calmodulin-dependent protein kinase II assay (CaM) kinase activity was
measured in anti-CaM kinase II immunoprecipitates prepared from
detergent lysates of monolayers. Briefly, cells were lysed in
immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris,
pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5% NP-40) and immunoprecipitated with
2.5 µg of anti-CaM kinase II antibody (Transduction Laboratories) and
protein A/G-bound agarose beads (Pharmacia) at 4°C for 1 h. Immunoprecipitates were washed three times with assay dilution buffer
(20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM CaCl2) and
subjected to CaM kinase in vitro kinase assay in accordance with the
manufacturer's directions (Upstate Biotechnology, Lake Placid, NY;
17-135).
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RESULTS |
Urea signaling to ERK activation is calcium
dependent.
We sought to determine the role of extra- and intracellular calcium in
the ability of urea to induce ERK phosphorylation in epithelial cells
derived from the murine inner medulla. Confluent mIMCD3 cell monolayers
were pretreated with the cell membrane-permeant calcium chelator
BAPTA-AM and placed in calcium-free medium. After a preequilibration
period, cells were treated for 5 min with urea (200 mM) or the potent
ERK activator TPA (100 nM). Calcium depletion did not affect the basal
level of ERK phosphorylation or the level of ERK phosphorylation in
response to the potent cPKC activator TPA (Fig.
1); however, in contrast, ERK
phosphorylation in response to urea treatment was markedly inhibited by
calcium depletion, suggesting the presence of a calcium-dependent step
in urea signaling to this MAPK.

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Fig. 1.
Role of calcium in urea signaling to extracellular
signal-regulated kinase (ERK) activation. Effect of intracellular
1,2-bis(2-aminophenoxy)ethane
N,N,N',N'-tetraacetic
acid-acetomethoxy ester (BAPTA-AM) plus extracellular (EGTA) calcium
chelation on control, urea (200 mM for 5 min)- and phorbol ester
tetradecanoylphorbol 13-acetate (TPA; 100 nM for 5 min)-inducible ERK
phosphorylation, as determined by anti-phospho-ERK immunoblotting.
Solid arrowhead, migration of 43.5-kDa molecular mass standard; open
and shaded arrowheads, phospho-ERK1 and phospho-ERK2, respectively.
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Urea-inducible ERK activation is not mediated by
cPKC.
Because urea-inducible ERK activation was calcium dependent, because
urea treatment results in generation of IP3
(11), and because PKC has been implicated broadly in
downstream signaling events in response to urea (11), the
role of classic PKC (classic calcium-dependent) isoforms in this
phenomenon was investigated. Several complementary strategies were
devised to establish or exclude a role for cPKC. In preliminary
studies, the pharmacological PKC inhibitors staurosporine and
calphostin C failed to suppress urea-inducible ERK phosphorylation
(data not shown; concentrations and pretreatment intervals for these
and other inhibitors are described in METHODS). Both of
these inhibitors, however, induced ERK phosphorylation even in the
absence of solute treatment so that no conclusion with respect to PKC
dependence could be established. Protracted exposure to the PKC
activator TPA was then used to downregulate PKC. In previous studies,
6 h of exposure to 100 nM TPA had proved sufficient for this
purpose (11). Acute treatment with urea or TPA markedly
increased ERK phosphorylation (Fig. 2).
TPA pretreatment for 6 and for 18 h completely abolished the subsequent acute effect of TPA treatment but not that of urea treatment, implying the absence of dependence on cPKC
(calcium-dependent) isoforms.

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Fig. 2.
Role of protein kinase C (PKC) activation in
urea-inducible ERK signaling. Effect of downregulation of the classic
PKC (cPKC) isoforms with 6- and 18-h pretreatment with TPA (100 nM) on control (C)-, TPA (T)-, and urea (U)-inducible ERK
phosphorylation.
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Because of the striking nature of this finding, in light of previous
expectations with regard to urea signaling to ERK activation (11), translocation of calcium-dependent PKC isoforms was
further examined as an additional correlate of PKC activation. After
TPA or urea treatment, membrane and cytosolic fractions were prepared from mIMCD3 cells. Lysates were immunoblotted with antisera
specifically recognizing the abundant calcium-dependent PKC isoform,
PKC-
(anti-PKC-
), as well as an antibody broadly recognizing all
major cPKC isoforms (monoclonal MC5). PKC-
is thus far the only cPKC identified in rodent inner medullary collecting duct (IMCD)
(8). As anticipated, acute TPA treatment resulted in a
decrease in PKC-
immunoreactivity in the cytosolic fraction and a
corresponding increase in PKC-
immunoreactivity in the membrane
fraction (Fig. 3). Similarly, TPA
treatment resulted in a net decrease in cytosolic classical
(MC5-immunoreactive) PKC isoform abundance and a corresponding net
increase in the membrane fraction. This single band likely represented
only PKC-
. In contrast, urea (200 mM) treatment failed to induce
translocation of PKC-
or of any cPKC (MC5-immunoreactive) isoform.
These data, including the lack of effect of PKC inhibitors, the lack of
effect of PKC downregulation, and the absence of urea-inducible translocation of cPKC isoforms, strongly suggested that
calcium-dependent PKC isoforms did not mediate the calcium-dependent
effect of urea on ERK phosphorylation.

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Fig. 3.
cPKC translocation. Effect of control treatment (C),
treatment with urea (200 mM) for 5 or 30 min or with TPA (T; 100 nM for
5 min) on subcellular localization of cPKC isoforms. Top:
cytosolic and membrane fractions were subjected to immunoblotting with
anti-PKC- . Bottom: identical protein fractions were
subjected to immunoblotting with MC5 antibody, which recognizes all of
the cPKC isoforms (see text).
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Urea-inducible ERK phosphorylation is not mediated
by CaM kinase.
A second calcium-dependent pathway to ERK activation is mediated via
CaM kinase. Because CaM kinase activation has been shown to result in
calcium-dependent ERK activation in at least two other contexts
(12, 15), the role of this kinase in urea-inducible ERK
phosphorylation was next explored. CaM kinase operates in a
calmodulin-dependent fashion. The specific calmodulin inhibitor calmidazolium failed to block the effect of urea on ERK
phosphorylation. In fact, the inhibitor activated ERK phosphorylation
under both control and urea-treated conditions (data not shown). CaM
kinases are effectively and specifically inhibited by the compound
KN-93. This compound failed to inhibit urea-inducible ERK
phosphorylation and influenced neither basal nor treated level of
phosphorylation (data not shown). These data strongly suggested that
urea-inducible ERK phosphorylation was not CaM kinase dependent. To
corroborate these data, the effect of urea on CaM kinase activity was
examined via in vitro (immune complex) kinase assay. Urea treatment
failed to increase CAMKII activity (data not shown), whereas the effect of urea on the neural tissue-specific CAMKIV isoform was not examined in this renal epithelial cell line.
Effect of depletion of intracellular calcium stores.
Although extracellular or intracellular calcium was essential for the
ERK response to urea, as previously described, in some models calcium
entry is dependent on intracellular calcium release. In addition, urea
signaling results in activation of PLC-
and generation of
IP3 (11). Therefore, it remained possible that the inciting event was calcium mobilization from an intracellular IP3-sensitive calcium pool. Thapsigargin inhibits the
calcium ATPase (SERCA) in the endoplasmic reticulum and thereby
dissipates sequestered intracellular calcium stores. mIMCD3 cells were
pretreated with this compound for 30 min to deplete intracellular
calcium stores before exposure to urea treatment. Thapsigargin
pretreatment failed to block the ability of urea to increase ERK
phosphorylation (Fig. 4A). The
rapidity with which thapsigargin depletes intracellular calcium stores
is model dependent and is a function of the rate of diffusion of
calcium down its concentration gradient throughout the cytosol. To
confirm that thapsigargin was effective and to eliminate the
possibility that diffusion was too slow to result in depletion, an
additional series of experiments was performed. Cells were briefly
treated with thapsigargin (5 min); the ability of this short treatment
to induce ERK phosphorylation was consistent with acute calcium release
(Fig. 4B). EGF served as positive control for
agonist-inducible ERK activation. EGF-inducible and
thapsigargin-inducible calcium ERK phosphorylation were approximately
equivalent. Even EGF treatment (to acutely empty calcium stores) in the
presence of thapsigargin (to inhibit calcium reuptake) failed to
abrogate subsequent urea-inducible ERK phosphorylation. It was
therefore concluded that thapsigargin-sensitive intracellular calcium
stores were not integral to the urea response leading to ERK
activation. To further exclude this possibility, pharmacological
inhibitors of calcium release pathways were examined. The inhibitor of
IP3-mediated calcium release, xestospongin C
(16), failed to abrogate urea-inducible ERK
phosphorylation. Similarly, the phospholipase inhibitor (and consequent
inhibitor of IP3 generation), U-73122 (4),
also exerted no effect. Dantrolene and ruthenium red, inhibitors of the
ryanodine-sensitive intracellular calcium pool, also failed to
influence ERK phosphorylation in response to urea (data not shown).
Several of these inhibitors may also act on other pathways involved in
calcium metabolism (38); such considerations are only
crucial when inhibition is observed and are irrelevant here because
none of these inhibitors influenced urea signaling to ERK activation.

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Fig. 4.
Role of intracellular calcium release. A:
effect of thapsigargin (TG) pretreatment (10 or 100 nM for 30 min) on
ERK phosphorylation in response to control or urea treatment (200 mM
for 5 min). B: effect of control (C), thapsigargin (TG; 100 nM for 5 min), urea (U; 200 mM for 5 min), and epidermal growth factor
(EGF; 100 nM for 5 min) treatment on ERK phosphorylation, and effect of
thapsigargin treatment (100 nM for 30 min) and subsequent EGF treatment
(100 nM for 30 min) on control and urea (200 mM for 5 min)-inducible
ERK phosphorylation.
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Role of extracellular calcium.
To confirm that extracellular calcium was required for urea-inducible
ERK activation, the effect of calcium depletion in the absence of
intracelluar calcium chelation was examined. Cells were placed in
calcium-free medium and treated with urea or the calcium ionophore
A-23187. Depletion of extracellular calcium alone modestly decreased
the basal level of ERK phosphorylation and markedly abrogated the
ability of both urea and the calcium ionophore A-23187 to induce ERK
phosphorylation (Fig. 5A).
Depletion of extracellular calcium also blocked the ability of
hypotonicity and the calcium ionophore ionomycin to induce ERK
phosphorylation, but failed to inhibit the ability of EGF to activate
ERK (Fig. 5B). These data are consistent with a requirement
for extracellular calcium in the setting of ionophore and urea
treatment and intracellular calcium in response to EGF. This
distinction is significant because others have implicated activation of
the EGF receptor as a key mediator in osmotic signaling in nonrenal
cells (34). To further discriminate urea signaling from
EGF signaling, additional studies were performed. Pretreatment with EGF
downregulated EGF-inducible ERK activation without affecting
urea-inducible ERK activation (Fig.
6A). In addition, pretreatment
of cells with the tyrphostin inhibitor of EGF receptor signaling
AG-1478 markedly attenuated EGF-inducible ERK activation but had no
effect on urea-inducible ERK activation (Fig. 6B).

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Fig. 5.
Role of extracellular calcium entry. A: effect
of control treatment or treatment with urea (200 mM for 5 min), the
calcium ionophore A-23187 (10 µM for 5 min), or urea and A-23187 on
ERK phosphorylation, in the presence (+) or absence ( ) of
extracellular calcium (Cae2+). B: effect of
Cae2+ depletion ( ) or repletion (+) on ERK activation
at 5 min of treatment in response to EGF (10 nM), ionomycin (Iono, 4 µM), or hypotonic (Hypo) stress (50% medium dilution).
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Fig. 6.
Role of EGF in urea-inducible signaling to ERK
activation. A: anti-phospho-ERK immunoblot of mIMCD3 that
received no pretreatment or EGF pretreatment (100 nM for 2 h)
before treatment with control (C), urea (U; 200 mM for 5 min), or EGF
(100 nM for 5 min). B: anti-phospho-ERK immunoblot of cells
receiving no pretreatment or pretreatment with the EGF receptor
inhibitor AG-1478 (100 nM for 2 h) before treatment with control
(C), urea (U; 200 mM for 5 min), or EGF (100 nM for 5 min).
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Extracellular calcium may enter via 1) voltage-gated calcium
channels (including L-, T-, N-, P-, Q-, and R-types), 2)
ionotropic glutamate receptor channels [including the
N-methyl-D-aspartic acid (NMDA), kainate, and
DL-amino-3-hydroxy-5-methylisoxazole-4-propronic acid
(AMPA) receptors]; 3) stretch-activated cation channels
(including selective and nonselective types); and 4)
so-called store operated calcium channels (also known as calcium
release-activated channels), which respond to depletion of
intracellular calcium. In addition, calcium may enter via the
calcium exchanger, although it generally serves to export calcium
against its concentration gradient. Of these pathways, only L-type
calcium channels, stretch-activated calcium channels, store-operated
calcium channels, and the calcium exchanger have been observed in renal
epithelium. L-type calcium channels were implicated in calcium influx
in other renal models of cell volume regulation [e.g., in response to
cell swelling (23, 28) and hyperglycemia (13,
37)]. Verapamil, an inhibitor of L-type calcium channels,
failed to significantly influence urea-inducible ERK phosphorylation
(Fig. 7A). Other calcium
channel blockers, including nifedipine and nimodipine, similarly
exerted no effect (data not shown). BAY K 8644, an agonist of L-type
calcium channels, only modestly activated ERK in the present model and only at very high concentration (100 µM, Fig. 7B). With
respect to other calcium entry pathways, nickel, a blocker of T-type
calcium channels, as well as CAI, an inhibitor of store-operated
calcium channels, exerted no significant effect (data not shown). The inhibitor of stretch-activated calcium channels, gadolinium, failed to
inhibit urea-inducible ERK activation, even at concentrations in the
millimolar range (data not shown). Gadolinium or lanthanum also has
been used nonspecifically to inhibit diverse calcium entry pathways;
despite the conspicuous dependence of urea signaling on extracellular
calcium, neither metal in concentrations to 10 mM affected the urea
response with respect to ERK activation (data not shown).

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Fig. 7.
Effect of L-type calcium channel inhibition and
activation on urea-inducible ERK activation. A: verapamil
(Verap) at low (10 mM) and high (100 mM) doses fails to inhibit
urea-inducible ERK activation. B: BAY K 8644 fails to mimic
the effect of urea, although high concentration (100 µM) modestly
induces ERK phosphorylation.
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Because of our inability to establish the route of calcium entry in
response to urea, despite the conspicuous requirement for this ion, the
kinetics of this potentially novel phenomenon were examined in greater
detail. Interestingly, the ability of calcium depletion to abrogate
urea signaling was a function of the calcium-free pretreatment interval
(Fig. 8). Specifically, 5 min of calcium
depletion modestly inhibited urea signaling, whereas 15 min produced a
more profound inhibition. Essentially complete inhibition of urea
signaling required 30 min of calcium depletion. In similar fashion, the
ability of calcium repletion to restore urea responsiveness after
extracellular calcium depletion was investigated. Interestingly, and
consistent with data acquired through calcium depletion, calcium
repletion also exhibited a time-dependent effect. Specifically, 10 min
of calcium repletion exerted no effect, whereas at 1 and 4 h of
repletion there was restoration of the ability of urea to activate ERK
(Fig. 9). Based on these data, it was
initially surmised that calcium depletion might be disrupting cell
adhesion to substratum and that calcium repletion might gradually
permit re-establishment of normal architecture and hence calcium
responsiveness. If this hypothesis were correct, then nonadherent
mIMCD3 cells should fail to exhibit urea-inducible ERK activation.
Treatment of cells with urea in the nonadherent (suspension) but
calcium-replete state, however, did not appreciably influence the
ability of urea to activate ERK (Fig.
10A). Therefore, the ability
of calcium depletion to inhibit urea signaling was likely not a
consequence of disrupted cell matrix adherence. To further explore this
possibility, mIMCD3 cells were grown on a collagen substrate permitting
greater (integrin-dependent) adherence in the absence of extracellular
calcium. Cells grown under these conditions were substantially more
tolerant of absent extracellular calcium; cells remained largely
adherent beyond 6 h of calcium depletion. Even under these
conditions, however, urea signaling to ERK activation was dramatically
decreased at 0.5, 2, and 6 h of calcium depletion (Fig.
10B). Similar findings were observed with the renal
epithelial MDCK cell line, which also exhibits urea-inducible ERK
activation (42) and was used for single-cell calcium
analysis (see below).

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Fig. 8.
Time-dependent inhibition of urea-inducible ERK activation by
extracellular calcium depletion. Monolayers were subjected to
calcium-depleted medium ( ) or calcium-replete medium (+) for the
indicated interval before control treatment (C) or treatment with urea
(U; 200 mM for 5 min).
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Fig. 9.
Time-dependent rescue of urea responsiveness after calcium
depletion. Monolayers were subjected to calcium-depleted medium ( ) or
control calcium-containing medium (+) for 30 min before recovery in the
presence of calcium-replete medium for the indicated interval (0 min to
4 h). After recovery, cells received no treatment (C) or treatment
with urea (U; 200 mM for 5 min) or TPA (T; 100 nM for 5 min).
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Fig. 10.
Anchorage independence of urea-inducible ERK activation.
A: degree of ERK phosphorylation evident in lysates prepared
from mIMCD3 cells in suspension that were subjected to 5 or 15 min of
sham treatment or urea (200 mM). B: effect of protracted
calcium depletion (0-6 h) on ERK phosphorylation in response to
urea treatment (200 mM for 5 min) in mIMCD3 and Madin-Darby canine
kidney (MDCK) cell monolayers maintained on collagen-coated substrate
to maximize adherence. For uniformity, a single empty lane between what
are now lanes 2 and 3 of the MDCK blot has been
digitally removed without influencing relative signal intensity among
the remaining lanes.
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The extracellular calcium concentration permissive for urea signaling
to ERK was explored in greater detail. Specific reduction of
extracellular calcium concentration with BAPTA indicated that 300 nM
calcium was permissive for urea signaling (Fig.
11), whereas neither 30 nor 100 nM
calcium was sufficient. In addition, to control for potential chelation
of a noncalcium cation, pretreatment with the noncalcium-binding metal
chelator TPEN was used. In the presence of 1 mM extracellular calcium,
TPEN failed to abrogate urea signaling to ERK activation, underscoring
the dependence of this phenomenon on calcium per se and not on a
noncalcium (e.g., heavy metal) contaminant of the medium.

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Fig. 11.
Extracellular calcium concentration permissive for urea
signaling to ERK phosphorylation. Anti-phospho-ERK immunoblot of
lysates prepared from control-treated cells (C) or cells treated with
200 mM urea for 5 min (U) in standard medium (containing 1 mM free
calcium) or medium supplemented with BAPTA to achieve the indicated
final free calcium concentration. Cells were also pretreated with the
noncalcium-binding metal chelator TPEN (2 µM for 30 min; lanes
3 and 4), to control for nonspecific
(noncalcium-binding) effects of BAPTA treatment.
|
|
Effect of urea on intracellular calcium.
These data strongly implicated calcium signaling in the mIMCD3 cell
response to urea; however, inhibitor studies failed to identify a
potentially responsible extracellular entry or intracellular release
mechanism. Fura 2-based fluorescence ratiometric imaging was performed
to definitively establish whether urea treatment resulted in
intracellular calcium mobilization. Subconfluent monolayers of mIMCD3
cells were initially chosen for study because they are best suited to
single-cell analysis. In this model, in the presence of extracellular
calcium, EGF induced a marked increment in intracellular calcium (Fig.
12). Urea treatment, in marked
contrast, failed to increase intracellular calcium. In some experiments
(e.g., Fig. 12A), urea treatment actually suppressed
F340/F380, an effect potentially attributable
to volume-dependent quenching of the fura 2. Next, to complement
single-cell analyses, studies aimed at detecting population-wide
changes in intracellular calcium were performed. In the first model,
mIMCD3 cells were grown to dense confluence on glass coverslips before
loading with fura 2-AM. Under these conditions (and in the presence of
extracellular calcium), whereas the positive controls ATP
(14) and bradykinin rapidly mobilized intracellular
calcium, urea failed to do so (Fig. 13,
top). Because urea-inducible ERK activation also has been
demonstrated in MDCK cells (42), which have been widely
used for the study of agonist-inducible intracellular calcium release,
studies were performed in parallel with this cell line. Again, the
positive controls bradykinin (30) and ATP
(29) induced a marked increase in intracellular calcium release, whereas urea failed to produce an effect (Fig. 13,
bottom). In the second model of population-wide analysis of
intracellular calcium release or entry, trypsinized cells in suspension
were examined for a urea-inducible increment in intracellular calcium signaling in the presence of extracellular calcium. Again, whereas the
positive controls bradykinin and ATP promptly increased intracellular calcium in both mIMCD3 and MDCK cell lines, urea failed to do so (data
not shown). Data obtained from these three separate models strongly
suggest that intracellular calcium mobilization, either from an
extracellular source or intracellular stores, is not an element of
early urea signaling and is not responsible for the calcium-dependence
of urea-inducible ERK activation.

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Fig. 12.
Single-cell determination of intracellular calcium response to
urea in subconfluent mIMCD3 monolayers. A: individual
tracings of intracellular calcium concentration (as determined via
ratio of fura 2 emission at 510 nm in the presence of excitation at 340 and 380 nm) in each of ~20 cells in a subconfluent mIMCD3 monolayer
on a glass coverslip in response to urea (200 mM), PBS, and EGF (100 nM). B: data pooled from ~20 individual cells depicting
response to urea (200 mM), EGF (100 nM), and ionomycin (5 µM).
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Fig. 13.
Population-wide determination of intracellular calcium
response to urea in confluent mIMCD3 and MDCK monolayers. Ratio of fura
2 emission at 510 nm in the presence of excitation at 340 and 380 nm as
function of time in confluent monolayers of mIMCD3 (top) and
MDCK (bottom) cells treated with urea (200 mM), bradykinin
(100 nM), and ATP (10 µM).
|
|
 |
DISCUSSION |
These data indicate that although the ability of urea to activate
ERK in epithelial cells derived from the kidney medulla is dependent on
extracellular calcium, neither calcium entry nor intracellular release
of stored calcium is required for this effect. These findings raise
several questions with respect to prior observations in this and
related models and with respect to the mechanism of calcium action in
urea signaling. Calcium may influence cell signaling via one of three
general mechanisms: 1) intracellular release of stored
calcium, 2) entry of extracellular calcium, and
3) extracellular calcium effect without calcium entry or
release. These will be addessed in turn.
Calcium release.
Based on prior data it was initially hypothesized that intracellular
calcium release would mediate the effect of urea on ERK activation. Multiple calcium-dependent pathways of ERK
activation have been described; the best characterized proceed through
members of the Ras family of small G proteins (Ras and Rap) and include the following: 1) calcium-dependent activation of PYK2,
leading to activation of SOS, a Ras guanine nucleotide exchange factor (GEF); 2) calcium- and diacylglycerol-dependent activation
of the Ras-directed GEF, RasGRP; 3)
calcium-calmodulin-dependent activation of the Ras-directed GEF,
RasGRF; 4) CaM kinase-mediated inhibition of the
Ras-directed GTPase-activating protein (GAP), SynGAP, leading to Ras
activation; and 5) calcium-dependent activation of adenylate
cyclase leading to cAMP-mediated activation of the Rap-directed GEF,
cAMP-GEF (reviewed in Ref. 19). The first two
mechanisms are operative in response to activation of receptor tyrosine
kinases, events with which elements of urea signaling have been
compared (11). It was anticipated that calcium-dependent urea signaling would operate through the first pathway because urea-inducible PYK2 (43) and Ras (40)
activation have been shown previously in this model. Alternatively,
because PKC has been implicated in urea signaling and because urea
results in activation of PLC-
and release of IP3
(11), the second possibility also was considered. However,
consistent with the inability of a dominant negative-acting Ras mutant
to quantitatively inhibit urea-inducible ERK activation
(40), calcium-dependent Ras activation was not responsible
for urea signaling to ERK activation.
With respect to the role of PKC in urea signaling, further comment is
warranted The cPKC-independence of urea-inducible ERK activation [in
contrast to urea-inducible immediate-early gene transcription
(11)] was unexpected but convincingly demonstrated by
complementary techniques. PKC activation in response to urea stress was
suggested previously by the following observations: 1)
inhibitors of PKC action blocked the ability of urea to activate transcription of the Egr-1 gene; 2) downregulation of PKC
with chronic exposure to TPA blocked the ability of urea to activate Egr-1 transcription; and 3) urea treatment results in
activation of PLC-
and liberation of IP3, hallmarks of
production of the PKC activator diacylglycerol (11). The
present data, which examine an experimental end-point more proximal in
the urea signaling cascade, do not support the involvement of cPKC in
the early cell response to urea. Urea-inducible ERK activation was not
acutely influenced by PKC downregulation and did not induce
translocation of cPKC isoforms.
In contrast to urea treatment, the role of PKC in anisotonicity has
been explored previously in several contexts. Some (22, 25, 26,
36, 39) but not all (2, 17, 20) inhibitor-based and
PKC downregulation studies have implicated PKC activation in
hypertonicity-inducible signaling events and acquisition of the
hypertonically stressed phenotype. PKC activation also has been
directly observed in response to hypertonicity in cell culture models
(26, 36). With respect to hypertonicity-inducible ERK activation specifically in the renal epithelial cell model, a number of
investigators have indirectly implicated cPKC activation (22, 25,
39). Paradoxically, Preston et al. (31) showed that
pharmacological activation of PKC inhibits events essential for
acquisition of osmotic tolerance. Recently, Zhuang et al. (45) implicated both novel PKC and cPKC isoforms in ERK
activation in response to hypertonicity in the nonrenal 3T3 cell model
through a combination of inhibitor studies and direct assessment of PKC activation. In terms of ERK activation, however, 3T3 cells appear to be
unresponsive to urea treatment (42).
Calcium entry.
An extracellular, not intracellular, reservoir of calcium was essential
for urea-inducible ERK activation. Multiple calcium entry pathways
leading to ERK activation have been described, including L-type
(voltage-gated) calcium channels, the NMDA receptor, and the
Na+/Ca2+ exchanger. Based on models of
hyperglycemia (13, 37) and chronic renal failure with
attendant uremia (24), it was hypothesized that L-type
calcium channels may be responsible for the calcium-dependence of urea
signaling. Indirect evidence from both inhibitor and activator studies
strongly suggested that this pathway was not required for urea
signaling to ERK activation (Fig. 7). Furthermore, the inability of
urea to increase intracellular calcium as measured via fura 2 ratiometry virtually excluded this and other possible mechanisms of
calcium entry.
Direct effects of extracellular calcium.
Signaling events influenced by extracellular calcium in the absence of
calcium entry are less well established. The cell membrane-associated calcium sensor responds directly to changes in ambient calcium concentration (5). In contrast to the present model,
however, an effector arm of this G protein-coupled receptor influences intracellular calcium concentration (6). Cells interact
with matrix and substratum via cell adhesion molecules (CAMs) of the integrin and syndecan family, and with each other via CAMs of the
cadherin, selectin, and Ig-CAM families. Interactions mediated via
cadherins and selectins are calcium dependent; only the cadherins appear to be expressed to a significant degree on renal epithelium. Increasing evidence supports a role for CAM interactions in mediating or modulating intracellular signaling events in contrast to the purely
structural roles inferred earlier. Specifically, changes in cell
adhesion dramatically influences ERK signaling in some models, an
effect likely mediated at the level of MEK or Raf (1). This could account for the inability of Ras inhibition (upstream of
Raf) to substantially abrogate urea signaling to ERK in the present
model (40). Importantly, extracellular calcium depletion did not result in a global block to agonist-inducible ERK activation, as the ability of the peptide mitogen EGF to activate ERK was actually
enhanced after removal of extracellular calcium (Fig. 5). In related
fashion, calcium withdrawal induces loss of cell-cell contact,
presumably at the level of the tight junction (21, 27). A
consequence of this loss of cell-cell adhesion is a partial loss of
cell polarization with respect to apical and basolateral membrane
domains (41). Whether renal epithelial cell urea
responsiveness is dependent on either of these two phenomena-calcium
impingement on a CAM extracellular domain or on maintenance of cell
polarization remains speculative; however, both are intriguing possibilities.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-52494 (D. M. Cohen), as
well as by the American Heart Association and National Kidney Foundation.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. M. Cohen, Mailcode PP262, Oregon Health Sciences Univ., 3314 S.W. US
Veterans Hospital Rd., Portland, OR 97201 (E-mail:cohend{at}ohsu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 May 2000; accepted in final form 17 August 2000.
 |
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