From Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center and Department of Medicine, Division of Nephrology, Medical University of South Carolina, Charleston, South Carolina 29425
Received for publication, September 26, 2002, and in revised form, February 26, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The type 1 sodium-hydrogen exchanger
(NHE-1) is a ubiquitous electroneutral membrane transporter that is
activated by hypertonicity in many cells. NHE-1 may be an important
pathway for Na+ entry during volume restoration, yet
the molecular mechanisms underlying the osmotic regulation of NHE-1 are
poorly understood. In the present study we conducted a screen for
important signaling molecules that could be involved in
hypertonicity-induced activation of NHE-1 in CHO-K1 cells.
Hypertonicity rapidly activated NHE-1 in a
concentration-dependent manner as assessed by proton
microphysiometry and by measurements of intracellular pH on a
FLIPRTM (fluorometric imaging
plate reader). Inhibitors of
Ca2+/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated
this activation, whereas neither calcium chelation nor inhibitors of
protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and
Ca2+/calmodulin-dependent enzymes had
significant effects. Hypertonicity also resulted in the rapid tyrosine
phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and
CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an
inhibitor of Jak2. Immunoprecipitation studies showed that
hypertonicity stimulates the assembly of a signaling complex that
includes CaM, Jak2, and NHE-1. Formation of the complex could be
blocked by AG490. Thus, we propose that hypertonicity induces
activation of NHE-1 in CHO-K1 cells in large part through the following
pathway: hypertonicity The ubiquitous isoform of the Na+/H+
exchanger (NHE-1)1 is
essential for the regulation of cellular volume and intracellular pH.
NHE-1 is nearly quiescent in resting cells but is activated by a
variety of hormones and growth factors (1, 2). NHE-1 is also rapidly
activated by hypertonic stress in many cells (3), and this may be an
important pathway for Na+ entry during volume restoration.
Despite the potential importance of this process, the molecular
mechanisms underlying the regulation of NHE-1 by hypertonicity have not
been fully elucidated. The rapid activation of NHE-1 is often
associated with an increase in its phosphorylation (3). Kinases that
have been shown to directly phosphorylate NHE-1 include p90 S6 kinase
(4) and the Nck-interacting kinase (5). However, deletion of the major phosphorylation sites contained within residues 636-815 of NHE-1 only
reduces its response to growth factors by about 50% (6), suggesting
that mechanisms of regulation other than direct phosphorylation of
NHE-1 are also important.
Hypertonicity-induced shrinkage of mammalian cells is a powerful
stimulant for many protein kinases that could play important direct or
indirect roles in activating NHE-1. These include mitogen-activated protein kinases such as extracellular signal-regulated protein kinase
(ERK), stress-activated protein kinases (c-Jun N-terminal kinases)
(7-10), Src family tyrosine kinases p59fgr and
p56/59hck (11), protein kinase C (12-14), Janus kinase
(15), and phosphatidylinositol 3-kinase (13), although there is not a
consensus that any of those kinases mediate hypertonicity-induced
activation of NHE-1.
Hypertonicity induces rapid cellular shrinkage and activation of NHE-1
and, concomitantly, extensive tyrosine phosphorylation of members of
the Src family of tyrosine kinases p59fgr and
p56/59hck but not stress-activated protein kinase, p38, or
ERK1/2 (11). In that regard, ERK, stress-activated protein kinase, and
p38 do not appear to be involved in the activation of NHE-1 in human neutrophils or lung fibroblasts (9, 11). Cell shrinkage associated with
hypertonic exposure induces tyrosine phosphorylation of several proteins of ~ 40, 80-85, and 110-130 kDa in CHO cells
(16). The 40-kDa protein appears to be ERK2, although this enzyme was not shown to be involved in the shrinkage-induced stimulation of NHE-1
(16). The 80-85-kDa protein was later identified as cortactin
(cortical actin filament cross-linking
protein), a major target of the hypertonicity-stimulated Src family
kinase Fyn (17). However, Src family kinases did not appear to be
responsible for the hypertonic regulation of NHE-1 (17). The calmodulin
(CaM)-dependent myosin light chain kinase is involved in
shrinkage-induced activation of the NHE in astrocytes (18, 19). There
is contradictory evidence about a potential role for protein kinase C
(PKC) in hypertonicity-induced activation of NHE. In Ehrlich ascites
tumor cells, PKC inhibitors block hypertonicity-induced activation of NHE (12), whereas in lymphocytes (20) and in astrocytes (18), PKC-dependent pathways are not involved in this process.
The activation of NHE-1 by hypertonic challenge does not appear to
involve direct phosphorylation of the exchanger itself (3). This is
because NHE-1 is still activated in response to hypertonic stress in
cells transfected with truncated mutants of NHE-1 that lack all of its
putative phosphorylation sites (21). Thus, it is somewhat difficult to
reconcile that finding with other reports supporting a general role for
phosphorylation in the activation of NHE-1. A possible explanation that
could reconcile the differences in those reports would be
phosphorylation of ancillary regulatory proteins involved in the
hypertonicity-induced stimulation of NHE-1. One such protein is CaM,
which has been shown to be important in the activation of NHE-1 by
multiple distinct stimuli (22, 23). Deletion of a high affinity CaM
binding domain has been shown to inhibit hypertonicity-induced
activation of NHE-1 by up to 80%, suggesting that CaM is a key
regulator of shrinkage-induced activation of NHE (22). We recently
explored the interactions between CaM and phosphorylation reactions in
the process of activation of NHE-1 by the G protein- and phospholipase
C-linked bradykinin B2 receptor in cultured mIMCD-3 cells.
We proposed the existence of a novel pathway through which Jak2
(activated by the B2 receptor) indirectly increases the
activity of NHE-1 by inducing tyrosine phosphorylation of CaM, leading
to increased binding of CaM to NHE-1 (24). We hypothesized that this
new pathway might also mediate hypertonicity-induced activation of
NHE-1. This possibility is plausible in that hypertonicity has been
shown previously (15) to activate the Jak/STAT pathway. Thus, in the
current report, we studied the roles of CaM and Jak2 in the activation
of NHE-1 by hypertonicity in CHO-K1 cells. Our results demonstrate that this pathway may be a fundamental mechanism for the rapid regulation of
NHE-1 in multiple cell types.
Materials--
Fluo-3 and
2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein (BCECF) were
purchased from Molecular Probes (Eugene, OR).
5-(N-Ethyl-N-isopropyl)-amiloride was purchased
from RBI (Natick, MA). ET-18-OCH3, AG490, and D609 were
from Biomol (Plymouth Meeting, PA). Tetramethylammonium chloride, probenecid, phorbol 12-myristate 13-acetate, and various salts were
from Sigma.
1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), calmidazolium, fluphenazine, W-7, GF109203X, PD98059, and anti-phosphotyrosine monoclonal antibody were from Calbiochem. Anti-CaM monoclonal antibody,
anti-Jak2-agarose-conjugated antibody, and anti-phosphotyrosine
polyclonal antibody were from Upstate Biotechnology (Lake Placid, NY).
Anti-phosphospecific Jak2 antibody was from
BIOSOURCE International (Camarillo, CA) or QCB
(Hopkinton, MA). Anti-phosphospecific STAT antibody and immobilized
phosphotyrosine monoclonal antibody were from Cell Signaling Technology
(Beverly, MA). Anti-NHE-1 polyclonal antibody was from Chemicon
International (Temecula, CA). All cell culture media and supplements
were from Life Sciences (Grand Island, NY). CHO-K1 cells were purchased
from American Type Culture Collection (Manassas, VA). Polycarbonate
cell culture inserts for microphysiometry and black 96-well microtiter
plates needed for the FLIPRTM were from Corning Costar
(Cambridge, MA). Black pipette tips were from Molecular Devices
Corporation (Sunnyvale, CA).
DNA Constructs--
DNA constructs were obtained from the
following sources: minigene encoding the C-terminal residues of
Cell Culture--
CHO-K1 cells were maintained in Ham's F-12
medium supplemented with 10% fetal bovine serum, 10 mg/ml
streptomycin, 100 units/ml penicillin, and 400 µg/ml G-418 at
37 °C in a 5% CO2-enriched, humidified atmosphere.
Transfection of CHO-K1 cells--
Transient expression of the
cDNA constructs was achieved in CHO-K1 cells by transfection
with 1 µg of the various plasmids in the presence of 10-15 µl of
LipofectAMINETM (2 h in serum- and antibiotic-free medium) as described
previously (25). Transfection efficiency (>90%) was evaluated by
co-transfecting with 0.2 µg of cDNA encoding a green fluorescent
protein (Clontech) as a marker.
Microphysiometry--
The microphysiometer uses a light
addressable silicon sensor to detect extracellular protons (26). Each
of eight channels has two inlet ports for buffers, one of which usually
contains a vehicle control, and the other of which carries the test
substance. The cells are superfused with buffer, and valve switches and
stop-start cycles are totally controlled by a programmable computer.
Acidification rate data are transformed by a personal computer running
CytosoftTM version 2.0, and are presented as the extracellular
acidification rate (ECAR) in µV/s, which roughly correspond to
millipH (designation for pH unit/1000) units/min (Nernst
equation). To facilitate comparison of data between two channels,
values are typically expressed as a percentage of a baseline determined
by computerized analysis of the five data points prior to exposure of
the cell monolayers to a test substance.
For all of the experiments, CHO-K1 cells were plated onto polycarbonate
membranes (3-µm pore size, 12-mm size) at a density of 300,000 cells
per insert the night prior to experimentation. After cells were
attached to the membranes, they were growth-arrested in serum-free
culture medium for 20 h before the experiment. The day of the
study, cells were washed with serum-free, bicarbonate-free Ham's F-12
medium, placed into the microphysiometer chambers, and perfused at
37 °C with the same medium or balanced salt solutions. For most
studies, the pump cycle was set to perfuse cells for 60 s,
followed by a 30 s "pump-off" phase, during which
proton efflux was measured from the eighth through the twenty-eighth seconds. Cells were exposed to the test agent for three or four cycles
(270-360 s). Valve switches (to add or remove test agents) were
performed at the middle of the pump cycle. Data points were then
acquired every 90 s. The peak effect during stimulation was expressed as the percentage increase from baseline.
Measurement of Intracellular pH Using BCECF--
We used a
FLIPRTM fluorometric imaging plate reader system (27) to measure
intracellular pH in CHO-K1 cells. Cells were seeded (~ 50,000 cells/well) in 96-well clear bottom black microplates (Corning Costar
Corp., Cambridge, MA) and left overnight in CO2 incubator
at 37 °C. On the day of assay, cells were loaded with a dye (5 µM BCECF acetoxymethyl ester) in a loading buffer
(Hank's balanced salt solution, pH 7.4, containing 20 mM
HEPES, 2.5 mM probenecid) for 1 h at 37 °C. 20 min
before the end of the loading phase, 20 mM
NH4Cl was added to each well. Cells were then washed four
times with the loading buffer containing 20 mM
NH4Cl. In the FLIPRTM, cells were acid-loaded
by an ammonium chloride prepulse protocol. In this method, the
extracellular buffer contains NH4+ and
NH3 in an equilibrium that is essentially recapitulated in the cell interior. When the extracellular medium is changed to a buffer
lacking NH4Cl, intracellular NH3 diffuses
rapidly out of the cell, causing the cells to become acutely
loaded with protons donated from NH4+
(28). Some cells were pretreated with 1 µM EIPA for 10 min. Cells were allowed to recover from the acid load in the presence and absence of sodium and various test substances. Fluorescence excitation was obtained using an Argon laser (488 nm wavelength at 300 mW), and emission (~540 nm) was monitored kinetically (29).
We also performed experiments without the acid load. Cells in 96-well
plates were loaded with BCECF for 1 h. Negative control wells were
pretreated with 5 µM EIPA for 10-20 min. Plates were placed in the FLIPRTM, and fluorescence tracings were
recorded in the presence or absence of different concentrations of
sucrose. The tracings from at least six wells under the same conditions
were averaged, and tracings from the negative controls were subtracted.
The slopes of fluorescence changes were calculated as rate fluorescence
change (fluorescence counts per second).
Immunoprecipitation--
Quiescent CHO-K1 cell monolayers were
treated with hypertonic media or vehicle for 10 min and lysed in
1 ml/100-mm dish of radioimmune precipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Nonidet P-40, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin at 1 µg/ml of each). Cell lysates were pre-cleared by incubating with a
protein A-agarose bead slurry for 30 min at 4 °C. Pre-cleared lysates (1 µg/µl total cell protein) were incubated with
anti-Jak2/protein A-agarose, with anti-NHE-1 antibody or with
polyclonal anti-phosphotyrosine antibody overnight at 4 °C.
Phosphotyrosine and NHE-1 immunoprecipitates were captured by addition
of protein A-agarose. The agarose beads were collected by
centrifugation, washed three times with radioimmune precipitation assay
buffer, resuspended in 2× Laemmli sample buffer, boiled for 5 min, and
subjected to SDS-PAGE and subsequent immunoblot analysis with
monoclonal anti-CaM or anti-phosphotyrosine IgG. The same Western blots
were re-probed with the antibody used for immunoprecipitation to assure
that equal amounts of protein were loaded in each lane.
Jak2 Phosphorylation Assay--
Phosphorylation of Jak2 in
response to hypertonic medium was assessed using a Jak2 dual
phosphospecific antibody. Quiescent cells were treated with hypertonic
medium for 10 min and lysed in radioimmune precipitation assay buffer.
The lysates were subjected to SDS-PAGE under reducing conditions with
4-20% pre-cast gels (Novex, San Diego, CA). After semi-dry transfer
to polyvinylidene difluoride membranes, membranes were blocked with a
Blotto buffer and incubated with the phospho-Jak2 antibody (0.5 µg/ml). After incubation with alkaline phosphatase-linked secondary
antibodies immunoreactive bands were visualized by a chemiluminescent
method (CDP StarTM; New England Biolabs, Beverly, MA) using
pre-flashed Kodak X-AR film and quantified using a GS-670 densitometer
and Molecular Analyst software (Bio-Rad). Alternatively, bands were
visualized with a StormTM PhosphorImager (Amersham Biosciences). To
ensure that equal amounts of protein were loaded in each lane, blots were stripped and re-probed with control Jak2 antibody, which recognizes both phosphorylated and nonphosphorylated Jak2 equally.
STAT3 Assay--
STAT3 phosphorylation was assessed using a
phospho-specific STAT3 (Tyr705) antibody, which
specifically detects STAT3 only when activated by phosphorylation at
Tyr705. Cells were treated with NaCl or sucrose, harvested
in Laemmli buffer, and subjected to SDS-PAGE under reducing conditions
with 4-20% pre-cast gels (Novex). After semi-dry transfer to
polyvinylidene difluoride membranes, membranes were blocked with a
Blotto buffer and incubated with the phospho-STAT3 antibody at a 1:1000
dilution. After incubation with alkaline phosphatase-linked secondary
antibodies, immunoreactive bands were visualized using Vistra
ECF Western blotting system (Amersham Biosciences) and a StormTM
PhosphorImager (Amersham Biosciences). To ensure that equal amounts of
protein were loaded in each lane, blots were stripped and re-probed
with control STAT3 antibody, which recognizes total
(phosphorylation-state independent) levels of STAT3 protein.
Statistical Analysis--
Data were analyzed for repeated
measures by Student's t test for unpaired two-tailed
analysis. p values less than 0.05 were considered significant.
Hypertonicity Activates NHE-1 in CHO-K1 Cells--
We used a
CytosensorTM microphysiometer (Molecular Devices Corporation,
Sunnyvale, CA) (26) to measure proton efflux from intact monolayers of
CHO-K1 cells plated onto polycarbonate membranes. We have used the
microphysiometer previously to specifically study Na+/H+ exchange in fibroblasts (25, 30),
enterocytes (31), and polarized epithelial mIMCD-3 cells (24). Fig.
1A shows that cells treated
with media made hypertonic (450 mosmol/liter) by addition of
NaCl (closed circles) or sucrose (open circles) to isotonic medium had
rapid >70% increases in ECAR. Fig. 1B shows that
the stimulatory effect of sucrose occurred in sodium-containing balanced salt solution but not in a solution in which
tetramethylammonium was substituted for sodium. Pretreatment of cells
for 30 min with 1 µM of the NHE-1 inhibitor, EIPA, did
not affect the basal rate of extracellular acidification but greatly
attenuated the increase in ECAR caused by hypertonic treatment. Thus,
the increased ECAR induced by exposure to hypertonic media was
both sodium-dependent and inhibitable by an NHE inhibitor,
suggesting the involvement of NHE-1 (CHO-K1 cells only express
NHE-1). In some experiments, cells were perfused with a buffer in which
pyruvate was substituted for glucose to minimize the effects of
glycolysis on ECAR. Under these conditions, medium made hypertonic by
the addition of sucrose increased ECAR by ~85% (Fig.
1B).
To support our microphysiometry data, we used another method of
studying NHE activity by measuring intracellular pH using BCECF
fluorescence on a FLIPRTM. Cells were acid-loaded by the
ammonium chloride prepulse method as described under "Experimental
Procedures" and then were allowed to recover in the presence or
absence of different concentrations of sucrose with or without the
addition of EIPA. Those experiments showed that sucrose increases the
rate of recovery from acid load in a
concentration-dependent manner (not shown). We also
monitored NHE-1 activation on a FLIPRTM without an acid
load to more closely approximate the measurement conditions used in the
microphysiometry experiments. The results presented in Fig.
1C demonstrate a concentration-dependent increase in
the rate of fluorescence change after stimulation of cells with
media made hypertonic by addition of sucrose. Thus, media made
hypertonic by addition of NaCl or sucrose activate NHE-1 in CHO-K1
cells as measured by two independent assays. Further, hypertonicity-induced changes in ECAR are due nearly completely to the
activation of NHE-1 independent of glycolysis-mediated production of protons.
Lack of a Role for Classical Signaling Intermediates in
Hypertonicity-induced Activation of NHE-1 in CHO-K1 Cells--
Fig.
2 shows studies in which various
inhibitors were examined for effects on hypertonicity-stimulated ECAR
in CHO-K1 cells. Typically, cells were perfused for 15-30 min with
media containing chemical inhibitors of signaling molecules and then
exposed to hypertonic medium (400 mosmol/liter by addition of sucrose
or NaCl to isotonic medium) in the presence of inhibitors.
Control cells were pretreated only with vehicle. We also used cells
transiently transfected with inhibitory negative constructs of
potential signaling molecules, whereas cells transfected with an empty
vector served as a control.
Our previous work has shown a role for Gi
To test the possible involvement of PKC in NHE activation by
hypertonicity we used a PKC inhibitor (100 µM H-7), as
well as PKC depletion by prolonged exposure of cells to 160 nM phorbol 12-myristate 13-acetate (PMA). Those treatments
did not affect NHE activation by hypertonic solutions but were able to
inhibit PMA (1 µM)-elicited proton efflux, showing that
those maneuvers blocked PKC in our conditions (Fig. 2B).
Those results are consistent with the non-involvement of PKC in
hypertonicity-induced activation of NHE in CHO-K1 cells.
We next tested the hypothesis that Ras and/or its downstream effectors
are involved in hypertonicity-induced activation of NHE in CHO-K1
cells. Expression of inhibitory negative constructs of Ras (N17Ras) and
Raf (
Because of evidence that Src family kinases are volume-sensitive
enzymes (11), we tested the effects of Src inhibition on hypertonicity-induced activation of NHE. Neither transient transfection with Csk (a kinase that inactivates Src family tyrosine kinases) nor
incubation with PP1 (10 µM; a chemical inhibitor of Src)
had any effect on the activation of NHE by hypertonic medium (Fig. 2D) suggesting that Src family kinases are not involved in
the osmotic activation of NHE-1, which is in agreement with the study by Kapus et al. (17). Thus, many classical signaling
molecules do not appear to play a significant role in the
hypertonicity-induced activation of NHE-1 in CHO-K1 cells.
Inhibitor Studies Suggest the Involvement of CaM but Not
CaM-dependent Enzymes in Hypertonicity-induced Increases in
ECAR--
Taking into consideration that NHE-1 possesses CaM-binding
sites that are critical for its activity, we tested the effects of five
structurally distinct CaM inhibitors on hypertonicity-induced increases
in ECAR. All of the inhibitors were shown to significantly attenuate
the hypertonicity-induced activation of NHE (Fig.
3A). The inhibitors included
100 µM W-7 (~60% inhibition), 5 µM
calmidazolium (~90% inhibition), 10 µM fluphenazine
(~65% inhibition), 50 µM trifluoperazine (~55%
inhibition), and 1 µM ophiobolin A (~94% inhibition).
We also considered the possibility that the inhibitory effect was
because of a CaM-dependent enzyme. Thus, we tested specific
inhibitors of CaM kinases and myosin light chain kinase (10 µM each of KN-93, KT-5926, K-252a, and 50 µM ML-9), CaM-dependent cyclic nucleotide
phosphodiesterase (50 µM vinpocetine and 20 µM 8-methoxy-isobutylmethylxanthine), and calcineurin (1 µM cyclosporin A). None of those inhibitors had a
significant effect on the activation of NHE by medium made hypertonic
(450 mosmol/liter) by addition of sucrose (Fig. 3B). Thus,
these studies support a role for CaM (but not for
CaM-dependent enzymes) in the stimulatory effect of
hypertonicity on NHE-1 in CHO-K1 cells.
Role for Janus Kinase 2 in Hypertonicity-induced Activation of
NHE-1--
Because the Jak/STAT pathway is activated by hypertonicity
(15), and Jak2 is an indirect regulator of bradykinin-stimulated NHE-1
activity in mIMCD-3 cells (24), we hypothesized that the Jak/STAT
pathway could be involved in the hypertonicity-induced activation of
NHE in CHO-K1 cells. We tested this hypothesis in three separate sets
of experiments. First, pretreatment of cells for 30 min with 40 µM AG-490, a specific Jak2 inhibitor, effectively blocked
the stimulation of NHE by medium made hypertonic by addition of sucrose
or NaCl, suggesting the involvement of Jak2 in the hypertonicity-induced activation of NHE in CHO-K1 cells (Fig. 4A). To support the
microphysiometry data, we also monitored hypertonicity-induced NHE
activation in cells pretreated with AG-490 by measuring intracellular pH using BCECF fluorescence on a FLIPRTM without an acid
load. The results presented in Fig. 4B show that pretreatment of cells with 40 µM AG-490 clearly blocked
the hypertonicity-induced increase in the rate of fluorescence change
measured on a FLIPRTM. Thus, a Jak2 inhibitor was able to
block hypertonicity-induced activation of NHE-1 in CHO-K1 cells as
measured by two independent assays.
Second, we used Western blotting with a phosphospecific anti-Jak2
antibody to assess the phosphorylation state of Jak2. Exposure of cells
to medium made hypertonic (500 mosmol/liter by addition of sucrose) for
10 min induced an ~3-fold increase in the level of phosphorylation of
Jak2 (Fig. 4C). Those increases were because of increased
phosphorylation of Jak2, because we normalized the results by
re-probing the same blots with a control Jak2 antibody (which
recognizes Jak2 independent of its phosphorylation state) to confirm
that we had loaded equal amounts of Jak2 in the samples (Fig.
4D). Third, we tested the ability of hypertonicity to induce the phosphorylation of the STAT3 isoform (one direct target of Jak2).
Time- and concentration-dependent increases in STAT3
phosphorylation after stimulation cells with NaCl and sucrose were
assessed by Western blotting of cell lysates with a phosphospecific
STAT3 antibody, which specifically detects STAT3 only when activated by
phosphorylation at Tyr705. Fig.
5 demonstrates that treatment of cells
with both media made hypertonic by addition of either NaCl (Fig. 5,
A and B) or sucrose (Fig. 5, C and
D) caused time- and concentration-dependent increase in Tyr705 phosphorylation of STAT3. The increased
phosphorylation of STAT3 was inhibited by pretreatment of the cells
with AG-490 (40 µM for 30 min), supporting the
involvement of Jak2 in the activation of STAT3 by hypertonicity and
importantly confirming that hypertonicity activates Jak2. The results
were normalized by stripping and re-probing the same blots with a
control STAT3 antibody that detects total STAT3 independent of its
phosphorylation state. It is noteworthy that AG-490 almost entirely
inhibited Jak2 activity as suggested by the results of STAT3
phosphorylation but at the same time did not completely block
hypertonicity-induced activation of NHE-1. Therefore, it is possible
that there are additional Jak2-independent mechanisms of NHE-1
activation by hypertonicity.
Hypertonicity-induced Jak2 Phosphorylation Is Src
Family-independent--
Because of the evidence that Src family kinase
Fyn is an upstream regulator of Jak2 activation in fibroblasts during
oxidative stress (34) we studied the possible involvement of Src family kinases in hypertonicity-induced Jak2 phosphorylation. We pretreated cells for 30 min with 10 µM PP1 (Src family kinases
inhibitor) before stimulation for 10 min with medium made hypertonic
(500 mosmol/liter) by addition of sucrose to isotonic medium. Cell lysates were immunoprecipitated with an immobilized phosphotyrosine monoclonal antibody, followed by Western blotting with a Jak2 antibody.
Fig. 6 shows an ~2.5-fold increase in
the level of Jak2 phosphorylation after stimulation with hypertonic
medium. Pretreatment of cells with PP1 slightly decreased the basal
level of Jak2 phosphorylation indicating a possible role for Src family
kinases in Jak2 regulation. At the same time PP1 did not alter
significantly hypertonicity-induced Jak2 phosphorylation. Thus, these
results demonstrate that Src family kinases are not involved in
hypertonicity-induced Jak2 phosphorylation or in the activation of
NHE-1 by hypertonic medium (Fig. 2D).
Convergence of the CaM and Jak2 Signals--
In the next series of
experiments, we examined the possibility that hypertonicity induces a
physical interaction between Jak2 and CaM. This was accomplished by
using immunoprecipitation of lysates from cells treated with vehicle or
hypertonic medium with anti-Jak2/protein A-agarose,
followed by Western blotting with a CaM antibody. Fig.
7A shows that CaM and Jak2 are
co-precipitated and that this association can be increased ~2.5 times
by treatment of CHO-K1 cells for 10 min with medium made hypertonic
(500 mosmol/liter) by addition of sucrose. Pretreatment of the cells
with AG490 (50 µM for 30 min) significantly decreased the
amount of CaM in Jak2 immunoprecipitates, suggesting that
hypertonicity-induced Jak2 activity is necessary for the formation of
the complex that includes CaM and Jak2.
Because of our previous work on mIMCD-3 cells stimulated with
bradykinin, we considered the possibility that elevated
Ca2+ contributes to the activation of Jak2 (24). Therefore,
we studied a potential role for Ca2+ in the
hypertonicity-induced interaction between Jak2 and CaM. Elevation of
intracellular Ca2+ by incubation of CHO-K1 cells with a
calcium ionophore, A23187 (1 µM), caused an ~60%
increase in Jak2 phosphorylation in CHO-K1 cells (not shown), similar
to the effect in mIMCD-3 cells. Exposure of CHO-K1 cells to A23187 for
5 min also increased the amount of CaM in Jak2 immunoprecipitates by
~70% (Fig. 7B). Pretreatment of CHO-K1 cells for 30 min
with 50 µM BAPTA-AM, a cell-permeable Ca2+
sequestrant, did not decrease the amount of CaM in Jak2
immunoprecipitates from cells stimulated with medium made hypertonic
(500 mosmol/liter) by addition of sucrose, whereas it completely
abolished the increase of CaM in Jak2 immunoprecipitates caused by 1 µM A23187. Thus, although increased intracellular
Ca2+ increases Jak2 phosphorylation and induces formation
of a complex between CaM and Jak2, hypertonicity employs a
Ca2+-independent pathway of Jak2 activation, distinct from
the one used by the G protein-coupled bradykinin B2
receptor in mIMCD-3 cells (24). This notion is also supported by the
fact that medium made hypertonic (500 mosmol/liter) by addition of
sucrose causes only a very slight elevation of intracellular
Ca2+, as measured by Fluo-3 fluorescence in
FLIPRTM experiments (data not shown).
Hypertonicity Increases Tyrosine Phosphorylation of CaM in a
Jak2-dependent Manner--
We next tested the hypothesis
that hypertonicity stimulates tyrosine phosphorylation of CaM. Cells
were treated with medium made hypertonic (500 mosmol/liter) by addition
of sucrose or isotonic vehicle for 10 min. Cells were then lysed, and
the lysates were immunoprecipitated with a polyclonal phosphotyrosine
antibody as described under "Experimental Procedures." Subsequent
immunoblot analyses were performed with monoclonal anti-CaM antibodies.
The data presented in Fig. 7C demonstrate that CaM becomes
tyrosine-phosphorylated (~ 3.5-fold increase) in response to
hypertonicity and that pretreatment of the cells with AG490 (50 µM for 30 min) significantly decreases hypertonicity-induced tyrosine phosphorylation of CaM, suggesting that
the increased phosphorylation is induced by Jak2.
Presence of CaM in NHE-1 Immunoprecipitates--
We proposed
previously (24) that tyrosine phosphorylation of CaM induced by
bradykinin increases the binding of CaM to NHE-1 in mIMCD-3 cells. To
establish the presence of NHE-1 in the hypertonicity-induced CaM
signaling complex in CHO-K1 cells, we performed co-immunoprecipitation experiments in which NHE-1 was isolated from lysates of cells treated
with isotonic medium or medium made hypertonic (500 mosmol/liter) by
addition of sucrose. Immunoprecipitates were next probed with CaM
antibodies. Fig. 7D shows that CaM is present in NHE-1
immunoprecipitates and further that the amount of CaM complexed with
NHE-1 increases after treatment of CHO-K1 cells with hypertonic medium.
Pretreatment of the cells with AG490 (50 µM for 30 min)
significantly decreases the amount of CaM complexed with NHE-1
suggesting that Jak2-induced phosphorylation of CaM is essential for
the complex formation between NHE-1 and CaM. When the same blots were
stripped and re-probed with NHE-1 antibody, equal amounts of NHE-1
appeared in the samples from cells exposed to isotonic or hypertonic
media (data not shown).
In the current report we studied the regulation of
NHE-1 activity by hypertonicity in CHO-K1 cells. What is new about this work is that we have shown for the first time that CaM and Jak2, working in concert, are involved in hypertonicity-induced activation of
NHE-1. Specifically, we have shown that exposure of CHO-K1 cells to
hypertonic media phosphorylates and stimulates Jak2, increases
complexation of Jak2 with CaM, increases the Jak2-dependent tyrosine phosphorylation of CaM, and increases the activity of NHE-1 by
increasing complexation of CaM to NHE-1. These findings are important,
because in our previous work (24) we showed that Jak2 is involved in
the regulation of NHE-1 by bradykinin B2 receptors in
mIMCD-3 cells in a similar manner. Thus, this pathway of activating NHE-1 is not limited to G protein-coupled receptors or to polarized epithelial cells. We propose that it is a new fundamental mechanism for
the rapid regulation of NHE-1 by varied stimuli in multiple cell types.
The involvement of CaM in the hypertonicity-induced activation of NHE-1
is supported by the ability of five chemically distinct CaM inhibitors
to attenuate NHE-1 activation, the tyrosine phosphorylation of CaM, and
increased complexation of CaM to Jak2 and NHE-1 in cells exposed to
hypertonic media. Moreover, it appears that CaM itself, rather than a
CaM-dependent enzyme, is involved in the activation of
NHE-1 by hypertonicity in that multiple inhibitors of
CaM-dependent protein kinases, myosin light chain kinase,
CaM-dependent phosphodiesterase, and the
CaM-dependent phosphatase, calcineurin, are ineffective in
blocking the hypertonicity-induced activation of NHE-1. It is not
surprising that CaM plays a role in activating NHE in these cells, as
NHE-1 has two CaM binding sites that regulate its function (22). These
findings suggest that CaM can be induced to activate NHE-1 through
several upstream pathways, including increased intracellular calcium
and/or tyrosine phosphorylation of CaM. Our results should be
contrasted with two studies supporting a role for
CaM-dependent myosin light chain kinase activation and
phosphorylation of myosin light chains in shrinkage-induced activation
of NHE in astrocytes (18, 19). Thus, there remains the possibility that
CaM-dependent enzymes (as well as CaM itself) might also
play roles in activating NHE-1 in some cases.
CaM has been shown to be phosphorylated by both tyrosine and
serine-threonine kinases. Casein kinase II phosphorylates CaM on
multiple serine and threonine residues (Thr79,
Ser81, Ser101, and Thr117) (35,
36). Tyr99 is phosphorylated after activation of both the
insulin receptor (37, 38) and the epidermal growth factor receptor (39,
40) and Tyr138 after activation of the insulin receptor
(37, 38). There is also significant precedent for the idea that
tyrosine phosphorylation of CaM can alter its ability to interact with
and activate its downstream targets. Tyrosine phosphorylation of
Tyr99 of CaM generally increases the activity of its
various targets by either decreasing the concentration at which
half-maximal activation is attained (3'-5'-cyclic nucleotide
phosphodiesterase, plasma membrane Ca2+-ATPase, and
type II Ca2+-CaM-dependent protein kinase) or
by increasing the Vmax (calcineurin and neuronal
nitric-oxide synthase) (41, 42). However, phosphorylation of
Tyr99 of CaM has been variably reported to diminish its
activity (43) or to have no effect on the ability of CaM to activate
myosin light chain kinase (42) or type I cyclic nucleotide
phosphodiesterase (44). The current work supports the hypothesis that
tyrosine phosphorylation of CaM by Jak2, when induced by hypertonicity, results in activation of NHE-1.
The involvement of Jak2 in hypertonicity-induced activation of NHE-1 is
supported by the inhibition of hypertonicity-induced NHE-1 activity by
the Jak2 inhibitor AG-490, the tyrosine phosphorylation of CaM induced
by hypertonicity, the Jak2-dependent phosphorylation of
STAT3, and the disruption by AG-490 of the hypertonicity-induced tyrosine phosphorylation of CaM, the complexation of CaM with Jak2, and
the complexation of CaM with NHE-1. Because of incomplete inhibition of
hypertonicity-induced NHE-1 activity by the Jak2 inhibitor (Fig. 4,
A and B) we cannot rule out the existence of additional Jak2-independent mechanisms of hypertonicity-induced NHE-1
activation, such as a recently proposed mechanism of NHE-1 activation
through dynamic changes in the actin-based cytoskeleton (1). The role
for Jak2 in the regulation of NHE-1 activity is a relatively new finding.
The current results expand on our results (24) showing that G
protein-coupled receptors for bradykinin activate NHE-1 through a
pathway that involves Jak2 and CaM and Ca2+ mobilization.
In the current report, we have shown evidence that chelation of
Ca2+ with BAPTA-AM had little effect on the complex
formation between Jak2 and CaM and activation of NHE-1 by hypertonicity
(Fig. 7B). This supports an alternative mechanism that does
not require increases in intracellular calcium levels, such as the
tyrosine phosphorylation of CaM by Jak2, for inducing the complexation
of CaM with NHE-1 (Fig. 8). We showed
previously (24) that CaM could be a substrate for purified Jak2, so it
is reasonable that this reaction might also take place in intact cells.
We should note that angiotensin II appears to stimulate NHE-1 in
vascular smooth muscle cells through both calcium-dependent
and -independent pathways,2
so there is the possibility that G protein-coupled receptors might also
use the same calcium-independent pathway as hypertonicity to activate
NHE-1.
Jak2 phosphorylation and activation
tyrosine phosphorylation of CaM
association of CaM with NHE-1
NHE-1 activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptor kinase (
ARK-CT) and Csk were from Dr. R. Lefkowitz (Duke University), dominant negative Ras (N17Ras)
was from Drs. D. Aultschuler and M. Ostrowski (Columbus, OH),
and dominant negative Raf (
NRaf) was from Dr. L. T. Williams
(San Francisco, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Hypertonic medium stimulates NHE-1 activity
in CHO-K1 cells. Microphysiometry was performed on quiescent
CHO-K1 monolayers as described under "Experimental Procedures."
Panel A, medium made hypertonic (450 mosmol/liter) by the
addition of NaCl (closed circles) or by the addition of
sucrose (open circles) stimulates ECAR. Shaded
area represents time during which cells were exposed to NaCl or
sucrose. Panel B, ECAR stimulated by medium made hypertonic
(450 mosmol/liter) by addition of sucrose in various buffers, including
a balanced salt solution containing glucose and NaCl without and with 1 µM EIPA, a balanced salt solution containing
tetramethylammonium substituted mM per mM for
sodium, and sodium-replete buffer with pyruvate substituted for glucose
to minimize the glycolytic component of the acidification response. All
experiments were performed at least five times. Error bars
represent the S.E. (*, p < 0.01). Panel C,
the FLIPRTM was used to monitor NHE-1 activity by measuring
pHi. Cells were loaded with BCECF as described
under "Experimental Procedures." Negative control wells were
pretreated with 5 µM EIPA for 10-20 min. Plates were
placed in the FLIPRTM, and fluorescence tracings were
recorded in the presence or absence of different concentrations of
sucrose. The data were calculated as average fluorescence tracing from
at least six wells after subtraction of signals from negative control
wells. Rate of fluorescence change represents the slopes of the
fluorescence changes. Experiments were performed at least three times.
Data are presented as mean ± S.E.
View larger version (34K):
[in a new window]
Fig. 2.
Comparative effects of inhibitors of
candidate molecules upon activation of NHE by sucrose in CHO-K1.
ECAR measurements from CHO-K1 cell monolayers were obtained as
described under "Experimental Procedures." Panel A, lack
of involvement of pertussis-sensitive G protein subunits and G
protein
subunits. Pertussis toxin treatment (200 ng/ml for
18 h) had no effect on sucrose-stimulated activity of NHE-1 but
completely blocked 5-HT-increased ECAR (n = 6 for
each).
ARK-CT, a
-sequestering reagent, does not affect
sucrose-activated ECAR (n = 3). Panel B,
lack of involvement of PKC. Pretreatment of the cells with H-7 (100 µM) for 30 min or prolonged PMA treatment to induce PKC
depletion (160 nM PMA for 18 h) does not affect
sucrose-activated ECAR; both treatments abrogate PMA-increased ECAR
(n = 4). Panel C, lack of role for the
classical Ras-mitogen-activated protein kinase pathway. Expression of
inhibitory negative constructs of Ras (N17Ras) and Raf
(
NRaf) and a specific mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase-1 inhibitor (50 µM PD98059) do not affect NHE activation by sucrose
(n = 3). Panel D, lack of involvement of
Src. Expression of Csk and treatment with PP1 (10 µM) had
no effect on sucrose-stimulated ECAR (n = 3).
Transfections were performed as described under "Experimental
Procedures." Chemical inhibitors were added 30 min prior to addition
of sucrose (400 mosmol/liter). Error bars represent the mean
± S.E. *, p < 0.01 versus vehicle-treated
samples.
2
and Gi
3 in the G protein-coupled receptor
stimulation of NHE-1 in CHO-K1 cells (30). A role for G protein
subunits in the activation of an endogenous NHE in Xenopus
laevis oocytes has also been demonstrated by others (32).
Therefore, we tested a role for G protein
subunits and
Gi
subunits in hypertonicity-induced activation of NHE.
Pertussis toxin (200 ng/ml for 18 h) failed to inhibit NHE under
hypertonic conditions, suggesting that pertussis toxin-sensitive G
protein
subunits are not involved in hypertonicity-induced activation of NHE (Fig. 2A). Such treatment completely
blocked the increase in NHE activity induced by recombinant
5-hydroxytryptamine1A receptor, which activates
NHE-1 through pertussis toxin-sensitive Gi
2
and Gi
3 subunits (30). Likewise,
sequestration of G protein
subunits by transfecting cells with a
minigene construct that encodes
ARK-CT does not inhibit the NHE
activation by hypertonicity, showing a lack of involvement of G protein
subunits in this process (Fig. 2A). The expression of
ARK-CT in CHO-K1 cells was demonstrated by performing Western blots
on cell lysates with antibody specific for
ARK-1 (not shown). This
expressed protein was also functionally active, because it completely
prevented activation of ERK1/2 by the recombinant
5-hydroxytryptamine1A receptor in CHO-K1 cells (33).
NRaf) and a selective mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase-1 inhibitor (50 µM PD98059) had no effect on NHE activation by
hypertonicity (Fig. 2C). As a positive control, to ensure
that functional constructs were expressed, we measured the activity of
ERK1/2. All of those treatments blocked activation of ERK1/2 by the
recombinant 5-hydroxytryptamine1A receptor in CHO-K1
cells (25, 33). Thus, these studies do not support a role for the Ras
signaling pathway as an immediate conveyor of hypertonicity-induced
activation of NHE in CHO-K1 cells.
View larger version (35K):
[in a new window]
Fig. 3.
Effects of inhibitors of CaM and
CaM-dependent enzymes on sucrose-induced ECAR. ECAR
was measured by microphysiometry as described under "Experimental
Procedures." Cells were preincubated with inhibitors for 30 min prior
to stimulation with medium made hypertonic (450 mosmol/liter) by
addition of sucrose. W-7, fluphenazine, trifluoperazine, calmidazolium,
and ophiobolin A are inhibitors of CaM. KN-93 and KT-5926 are
inhibitors of CaM kinase, K-252a and ML-9 inhibit myosin light chain
kinase, vinpocetine and 8-methoxy-isobutylmethylxanthine are inhibitors
of CaM-dependent cyclic nucleotide phosphodiesterase, and
cyclosporin A inhibits calcineurin. All experiments were performed at
least four times. Error bars represent the mean ± S.E. ,
p < 0.05; *, p < 0.01;
,
p < 0.005 versus vehicle-treated
samples.
View larger version (41K):
[in a new window]
Fig. 4.
Involvement of Janus kinase 2 in
hypertonicity-induced NHE-1 activation. Panel A, ECAR
was measured by microphysiometry as described under "Experimental
Procedures." Cells were preincubated with 40 µM AG-490,
a selective Jak2 inhibitor, for 30 min prior to stimulation with
different concentrations of sucrose (330-600 mosmol/liter) or with
NaCl (450 mosmol/liter). Experiments were performed at least three
times in duplicate. Error bars represent the mean ± S.E.
, p < 0.05; *, p < 0.01 versus vehicle-treated samples. Panel B, NHE-1
activity was assessed by measuring pHi using the
FLIPRTM. Cells were loaded with BCECF as described under
"Experimental Procedures" and preincubated with 40 µM
AG-490 for the last 30 min of the loading phase. Negative control wells
were pretreated with 5 µM EIPA, an NHE-1 inhibitor, in
the presence or absence of AG-490 for 15 min. Plates were placed in the
FLIPRTM, and fluorescence tracings were recorded in the
presence or absence of sucrose. The data were calculated as average
fluorescence tracing from at least six wells after subtraction of
signals from negative control wells. Rate of fluorescence change
represents the slopes of the fluorescence changes. Experiments were
performed at least three times. Data are presented as mean ± S.E.
, p < 0.05; *, p < 0.01 versus vehicle-treated samples. Panel C, sucrose
induces tyrosine phosphorylation of Jak2. The phosphorylation state of
Jak2 was determined in whole cell lysates from CHO-K1 cells using a
phosphorylation state-specific antibody for Jak2 in an immunoblot as
described under "Experimental Procedures." Cells were treated with
medium made hypertonic (500 mosmol/liter) by addition of sucrose or
isotonic medium for 10 min, lysed, and subjected to immunoblot. The
inset is a representative immunoblot. The same blot was
stripped and re-probed with antibodies for total Jak2 that recognize
Jak2 independent of phosphorylation state (Panel D). The
experiment was repeated at least four times. Error bars
represent the mean ± S.E.
View larger version (35K):
[in a new window]
Fig. 5.
Effect of hypertonic media
on the phosphorylation of STAT3 protein in CHO-K1 cells.
Cells were treated with medium made hypertonic (500 mosmol/liter) by
addition of NaCl (Panel A) or sucrose (Panel C)
for various periods of time. The phosphorylation state of STAT3 was
determined in whole cell lysates from CHO-K1 cells using a
phosphorylation state-specific antibody for STAT3 in an immunoblot as
described under "Experimental Procedures." Panel B,
cells were pretreated for 1 h with 40 µM AG-490
(Jak2 inhibitor) and stimulated for 10 min with various concentrations
of NaCl. Panel D, the same conditions were used as in
Panel B, except that cells were stimulated with sucrose
rather than NaCl. All experiments were repeated at least four times.
Error bars represent the mean ± S.E. , p < 0.05; *, p < 0.01 versus vehicle-treated
samples.
View larger version (23K):
[in a new window]
Fig. 6.
Hypertonicity-induced Jak2 phosphorylation is
Src family-independent. Cells were preincubated with 10 µM PP1, an Src family kinases inhibitor, for 30 min prior
to stimulation with vehicle or with medium made hypertonic
(500 mosmol/liter) by addition of sucrose. Immunoprecipitations with an
immobilized phosphotyrosine monoclonal antibody followed by Western
blotting with a polyclonal anti-Jak2 antibody were performed as
described under "Experimental Procedures." Experiments were
performed at least three times. Error bars represent the
mean ± S.E. IP, immunoprecipitation; IB,
immunoblot.
View larger version (36K):
[in a new window]
Fig. 7.
Relationship between Jak2 and CaM in
sucrose-induced signaling complex. Panel A,
co-immunoprecipitations were performed as described under
"Experimental Procedures." In all panels, cells were
exposed to isotonic medium (Control) or medium made
hypertonic (500 mosmol/liter) by addition of sucrose. Panel
A, sucrose induces a complex that includes CaM and Jak2. Formation
of this complex is prevented by pre-incubation with AG490 (50 µM) for 30 min. Experiments were repeated at least four
times. Panel B, hypertonicity-induced complex formation
between Jak2 and CaM does not depend on Ca2+. Cells were
pretreated with vehicle or 50 µM BAPTA-AM and stimulated
with vehicle, a calcium ionophore (1 µM A23187), or with
sucrose. Cell lysates were immunoprecipitated with anti-Jak2/protein
A-agarose and immunoblotted with anti-CaM antibody as described under
"Experimental Procedures." Panel C, shows that treatment
with medium made hypertonic by addition of sucrose increases the amount
of CaM present in phosphotyrosine (PY) immunoprecipitates.
Pretreatment of the cells with AG490 decreases hypertonicity-induced
tyrosine phosphorylation of CaM. Experiments were repeated at least
four times. Panel D, co-immunoprecipitation experiments show
that treatment with hypertonic medium for 10 min increases the amount
of CaM in NHE-1 immunoprecipitates. Pretreatment of the cells with
AG490 decreases hypertonicity-induced complexation of CaM with NHE-1.
Experiments were repeated three times. Error bars represent
the mean ± S.E. for all panels , p < 0.05; *, p < 0.01 versus vehicle-treated
samples. IP, immunoprecipitation; IB,
immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
[in a new window]
Fig. 8.
Proposed pathway of hypertonicity-induced
activation of NHE-1 in CHO-K1 cells. This scheme is described
under "Discussion." PY indicates tyrosine
phosphorylation.
It is noteworthy that the pathway of activation of NHE-1 by
hypertonicity described within this report does not involve many classical signaling molecules that have been shown to activate NHE
under other conditions. These include pertussis toxin-sensitive G
protein subunits (45), G protein
subunits (32), PKC (12-14), the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase
kinase pathway (7-10), and Src family kinases (25). Although it has
been shown that members of the Src family are volume-sensitive enzymes
(11, 16, 17), our results do not support their role in the osmotic
activation of NHE-1, which is in agreement with the study by Kapus
et al. (17). Although Fyn (a member of Src family kinases)
is required for activation of Jak2 by oxidative stress (34), our data
suggest that hypertonicity-induced phosphorylation of Jak2 does not
depend on Src family kinases (Fig. 6). Thus, it is not clear at this
time how Jak2 is activated when CHO-K1 cells are exposed to hypertonicity.
In summary, we have shown for the first time that CaM and Jak2, working
in concert, are involved in hypertonicity-induced activation of NHE-1.
In CHO-K1 cells, exposure to hypertonic medium stimulates Jak2,
increases Jak2-dependent tyrosine phosphorylation of CaM,
increases complexation of Jak2 with CaM, as well as CaM with NHE-1, and
increases the activity of NHE-1 in a Jak2- and CaM-dependent manner. Moreover, this process does not
require the involvement of many classical signaling molecules. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells
in large part through the following pathway: hypertonicity Jak2
phosphorylation and activation
tyrosine phosphorylation of CaM
association of CaM with NHE-1
NHE-1 activation (Fig. 8). Further,
we suggest that this pathway is a fundamental mechanism for the rapid
regulation of NHE-1 by varied stimuli in multiple cell types.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Department of Veterans Affairs Merit Awards (to M. N. G. and J. R. R.) and a Research Enhancement Award program (to J. R. R., Y. V. M., and M. N. G.), by National Institutes of Health Grants DK52448 (to J. R. R.) and K01-DK02694 (to Y. V. M.), by laboratory endowments jointly supported by the Medical University of South Carolina (MUSC) Division of Nephrology and Dialysis Clinics, Incorporated (to J. R. R.), by an American Heart Association Fellowship Award (to Y. V. M.), and a MUSC University Research Foundation Award (to M. N. G.). The FLIPRTM is a shared MUSC resource obtained with Grant S10RR013005 from the Public Health Service. The microphysiometer is a shared Veterans Affairs resource obtained with a Veterans Affairs large equipment grant.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.
To whom correspondence should be addressed: Medical University of
South Carolina, 96 Jonathan Lucas St., Rm. 829 CSB, P. O. Box 250623, Charleston, SC 29425-2227. Tel.: 843-876-5128 or 843-789-6776; Fax:
843-876-5129 or 843-792-8399; E-mail: garnovsk@musc.edu.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M209883200
2 M. N. Garnovskaya, Y. V. Mukhin, J. H. Turner, T. M. Vlasova, M. E. Ullian, and J. R. Raymond, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NHE, Na+/H+ exchange;
NHE-1, type 1 sodium-proton
exchanger;
BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
BCECF, 2'-7'-bis[2-carboxymethyl]-5[6]-carboxyfluorescein;
CaM, calmodulin;
ECAR, extracellular acidification rate;
EIPA, 5-(N-ethyl-N-isopropyl)-amiloride;
ERK, extracellular signal-regulated protein kinase;
Jak2, Janus kinase 2;
mIMCD-3, mouse inner medullary collecting duct cell line;
PKC, protein
kinase C;
PMA, phorbol-12-myristate-13-acetate;
STAT, signal transducer
and activator of transcription;
CHO, Chinese hamster ovary;
ARK-CT, C-terminal
-adrenergic receptor kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Putney, L. K., Denker, S. P., and Barber, D. L. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 527-552[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Wakabayashi, S.,
Shigekawa, M.,
and Pouyssegur, J.
(1997)
Physiol. Rev.
77,
51-74 |
3. |
Grinstein, S.,
Woodside, M.,
Sardet, C.,
Pouyssegur, J.,
and Rotin, D.
(1992)
J. Biol. Chem.
267,
23823-23828 |
4. |
Takahashi, E.,
Abe, J.,
Gallis, B.,
Aebersold, R.,
Spring, D. J.,
Krebs, E. G.,
and Berk, B. C.
(1999)
J. Biol. Chem.
274,
20206-20214 |
5. |
Yan, W.,
Nehrke, K.,
Choi, J.,
and Barber, D. L.
(2001)
J. Biol. Chem.
276,
31349-31356 |
6. |
Wakabayashi, S.,
Bertrand, B.,
Shigekawa, M.,
Fafournoux, P.,
and Pouyssegur, J.
(1994)
J. Biol. Chem.
269,
5583-5588 |
7. |
Matsuda, S.,
Kawasaki, H.,
Moriguchi, T.,
Gotoh, Y.,
and Nishida, E.
(1995)
J. Biol. Chem.
270,
12781-12786 |
8. |
Aharonovitz, O.,
and Granot, Y.
(1996)
J. Biol. Chem.
271,
16494-16499 |
9. |
Bianchini, L.,
L'Allemain, G.,
and Pouyssegur, J.
(1997)
J. Biol. Chem.
272,
271-279 |
10. | Bouaboula, M., Bianchini, L., McKenzie, F. R., Pouyssegur, J., and Casellas, P. (1999) FEBS Lett. 449, 61-65[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Krump, E.,
Nikitas, K.,
and Grinstein, S.
(1997)
J. Biol. Chem.
272,
17303-17311 |
12. | Pedersen, S. F., Kramhoft, B., Jorgensen, N. K., and Hoffmann, E. K. (1996) J. Membr. Biol. 149, 141-159[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Sauvage, M.,
Maziere, P.,
Fathallah, H.,
and Giraud, F.
(2000)
Eur. J. Biochem.
267,
955-962 |
14. |
Snabaitis, A. K.,
Yokoyama, H.,
and Avkiran, M.
(2000)
Circ. Res.
86,
214-220 |
15. |
Gatsios, P.,
Terstegen, L.,
Schliess, F.,
Haussinger, D.,
Kerr, I. M.,
Heinrich, P. C.,
and Graeve, L.
(1998)
J. Biol. Chem.
273,
22962-22968 |
16. |
Szaszi, K.,
Buday, L.,
and Kapus, A.
(1997)
J. Biol. Chem.
272,
16670-16678 |
17. |
Kapus, A.,
Szaszi, K.,
Sun, J.,
Rizoli, S.,
and Rotstein, O. D.
(1999)
J. Biol. Chem.
274,
8093-8102 |
18. | Shrode, L. D., Klein, J. D., O'Neill, W. C., and Putnam, R. W. (1995) Am. J. Physiol. 269, C257-C266[Medline] [Order article via Infotrieve] |
19. | Shrode, L. D., Klein, J. D., Douglas, P. B., O'Neill, W. C., and Putnam, R. W. (1997) Am. J. Physiol. 272, C1968-C1979[Medline] [Order article via Infotrieve] |
20. | Grinstein, S., Rothstein, A., and Cohen, S. (1985) J. Gen. Physiol. 85, 765-787[Abstract] |
21. | Bianchini, L., Kapus, A., Lukacs, G., Wasan, S., Wakabayashi, S., Pouyssegur, J., Yu, F. H., Orlowski, J., and Grinstein, S. (1995) Am. J. Physiol. 269, C998-C1007[Medline] [Order article via Infotrieve] |
22. |
Bertrand, B.,
Wakabayashi, S.,
Ikeda, T.,
Pouyssegur, J.,
and Shigekawa, M.
(1994)
J. Biol. Chem.
269,
13703-13709 |
23. |
Wakabayashi, S.,
Bertrand, B.,
Ikeda, T.,
Pouyssegur, J.,
and Shigekawa, M.
(1994)
J. Biol. Chem.
269,
13710-13715 |
24. |
Mukhin, Y. V.,
Vlasova, T.,
Jaffa, A. A.,
Collinsworth, G.,
Bell, J. L.,
Tholanikunnel, B. G.,
Pettus, T.,
Fitzgibbon, W.,
Ploth, D. W.,
Raymond, J. R.,
and Garnovskaya, M. N.
(2001)
J. Biol. Chem.
276,
17339-17346 |
25. | Garnovskaya, M. N., Mukhin, Y., and Raymond, J. R. (1998) Biochem. J. 330, 489-495[Medline] [Order article via Infotrieve] |
26. | McConnell, H. M., Owicki, J. C., Parce, J. W., Miller, D. L., Baxter, G. T., Wada, H. G., and Pitchford, S. (1992) Science 257, 1906-1912[Medline] [Order article via Infotrieve] |
27. | Schroeder, K. S., and Neagle, B. D. (1996) J. Biomol. Screen. 1, 75-80 |
28. | Boron, W. F. (1977) Am. J. Physiol. 233, C61-C73[Medline] [Order article via Infotrieve] |
29. | Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979) Biochemistry 18, 2210-2218[Medline] [Order article via Infotrieve] |
30. |
Garnovskaya, M. N.,
Gettys, T. W.,
van Biesen, T.,
Prpic, V.,
Chuprun, J. K.,
and Raymond, J. R.
(1997)
J. Biol. Chem.
272,
7770-7776 |
31. | Prpic, V., Fitz, J. G., Wang, Y., Raymond, J. R., Garnovskaya, M. N., and Liddle, R. A. (1998) Am. J. Physiol. 275, G689-G695[Medline] [Order article via Infotrieve] |
32. |
Busch, S.,
Wieland, T.,
Esche, H.,
Jakobs, K. H.,
and Siffert, W.
(1995)
J. Biol. Chem.
270,
17898-17901 |
33. | Garnovskaya, M. N., van Biesen, T., Hawe, B., Casanas Ramos, S., Lefkowitz, R. J., and Raymond, J. R. (1996) Biochemistry 35, 13716-13722[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Abe, J.,
and Berk, B. C.
(1999)
J. Biol. Chem.
274,
21003-21010 |
35. | Sacks, D. B., Davis, H. W., Crimmins, D. L., Persechini, A., and McDonald, J. M. (1992) Biochem. Biophys. Res. Commun. 188, 754-759[Medline] [Order article via Infotrieve] |
36. | Sacks, D. B., Davis, H. W., Crimmins, D. L., and McDonald, J. M. (1992) Biochem. J. 286, 211-216[Medline] [Order article via Infotrieve] |
37. | Sacks, D. B., Fujita-Yamaguchi, Y., Gale, R. D., and McDonald, J. M. (1989) Biochem. J. 263, 803-812[Medline] [Order article via Infotrieve] |
38. | Joyal, J. L., Crimmins, D. L., Thoma, R. S., and Sacks, D. B. (1996) Biochemistry 35, 6267-6275[CrossRef][Medline] [Order article via Infotrieve] |
39. | De Frutos, T., Martin-Nieto, J., and Villalobo, A. (1997) Biol. Chem. 378, 31-37[Medline] [Order article via Infotrieve] |
40. | Benaim, G., Cervino, V., and Villalobo, A. (1998) Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 120, 57-65[CrossRef][Medline] [Order article via Infotrieve] |
41. | Sacks, D. B., Lopez, M. M., Li, Z., and Kosk-Kosicka, D. (1996) Eur. J. Biochem. 239, 98-104[Abstract] |
42. |
Corti, C.,
Leclerc L'Hostis, E.,
Quadroni, M.,
Schmid, H.,
Durussel, I.,
Cox, J.,
Dainese Hatt, P.,
James, P.,
and Carafoli, E.
(1999)
Eur. J. Biochem.
262,
790-802 |
43. | Fukami, Y., Nakamura, T., Nakayama, A., and Kanehisa, T. (1986) Proc. Natl. Acad. Sci. 83, 4190-4193[Abstract] |
44. | Saville, M. K., and Houslay, M. D. (1994) Biochem. J. 299, 863-868[Medline] [Order article via Infotrieve] |
45. | Voyno-Yasenetskaya, T. A. (1998) Biol. Signals Recept. 7, 118-124[Medline] [Order article via Infotrieve] |