PKC activation is required by EGF-stimulated
Na+-H+
exchanger in human pleural mesothelial cells
Yuang-Shuang
Liaw1,
Pan-Chyr
Yang1,
Chong-Jen
Yu1,
Sow-Hsong
Kuo2,
Kwen-Tay
Luh2,
Yuh-Jeng
Lin3, and
Mei-Lin
Wu3
1 Laboratory of Medicine,
2 Department of Internal Medicine, and
3 Institute of Physiology,
College of Medicine, National Taiwan University, Taipei 100, Taiwan,
Republic of China
 |
ABSTRACT |
Epidermal growth
factor (EGF) stimulates the
Na+-H+
exchanger, leading to enhanced cell proliferation. In human pleural
mesothelial cells (PMCs), the intracellular signaling mechanism
mediating the EGF-induced stimulation of the
Na+-H+
exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein,
to measure changes in intracellular pH
(pHi), we found that
1) EGF and
12-O-tetradecanoylphorbol 13-acetate
(TPA; a phorbol ester) both stimulate the ethylisopropyl amiloride-sensitive
Na+-H+
exchanger; 2) TPA-induced alkalosis
can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and
staurosporine) or by PKC downregulation, indicating that PKC activation
is involved in the stimulation of the
Na+-H+
exchanger. However, TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; and 3) the
stimulatory effect of EGF on the Na+-H+
exchanger acts via stimulation of tyrosine kinase-receptor activity because it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A, and herbimycin A). It also involves PKC activation because EGF-induced alkalosis was blocked by PKC inhibitors. These results suggest that PKC activation is one of the downstream signals for EGF-induced activation of the
Na+-H+
exchanger in primary cultures of human pleural mesothelial cells.
tyrosine kinase; epidermal growth factor; protein kinase C; sodium-hydrogen exchanger
 |
INTRODUCTION |
MESOTHELIAL CELLS, which cover the surface of the
parietal and visceral pleura, respond to both intrinsic stimuli, such
as growth factors, and extrinsic stimuli, such as asbestos fibers (2).
Some pleural diseases, including parapneumonic effusion, lupus,
pleurisy, and metastatic malignancy, result in the loss of mesothelial
integrity and the subsequent accumulation of pleural fluid, with an
increase in permeability (15). Cell proliferation in response to growth
factors contained in the pleural fluid may therefore be important in
restoring the normal mesothelium after pleural injury.
The housekeeping
Na+-H+
exchanger isoform 1 (NHE1) is amiloride and ethylisopropyl amiloride
(EIPA) sensitive and is expressed in most eukaryotic cells. It is
usually activated when the intracellular pH
(pHi) falls and exchanges
external Na+ for internal
H+. It appears to be involved in
multiple cellular functions including pHi regulation, transepithelial
transport, and cell volume control (13). Moreover, this exchanger can
be rapidly activated by various mitogenic factors such as growth
factors [e.g., epidermal growth factor (EGF)] and phorbol
esters [e.g., 12-O-tetradecanoylphorbol 13-acetate
(TPA)], resulting in DNA synthesis and cell proliferation (8, 13,
27, 35).
At least two major mechanisms have been proposed to be involved in the
activation of the
Na+-H+
exchanger by various stimulants. The first is a protein kinase C
(PKC)-dependent pathway involving either G protein-activated phospholipase C (PLC)-
1 or tyrosine kinase receptor-activated PLC-
1 (11, 19); activation of either system leads to the production
of inositol 1,4,5-trisphosphate and diacylglycerol (5), and the latter
then activates PKC, resulting in PKC translocation from the cytosol to
the membrane. This membrane association-activation event can be
mimicked by phorbol esters such as TPA, resulting in the irreversible
insertion of PKC into the lipid bilayer (24). In fibroblasts, it has
been shown that addition of phorbol esters or growth factors increases
the phosphorylation of the
Na+-H+
exchanger (31), thereby leading to an alkaline shift in the resting
pHi that can easily be detected
under nominally bicarbonate-free conditions. Recent studies (34, 35)
have also shown that deletion of all the major potential
phosphorylation sites only resulted in a 50% decrease in exchanger
activation in response to phorbol esters or growth factors, suggesting
that PKC and growth factors do not activate the exchanger exclusively
by direct phosphorylation (34, 35).
The second mechanism is a PKC-independent pathway in which the
Na+-H+
exchanger is activated either by growth factors that stimulate the
tyrosine kinase receptor (3, 10, 12, 35) or by
Ca2+- and/or
calmodulin-dependent disinhibition of the exchanger (6, 21, 33, 35).
There is evidence that, rather than directly phosphorylating the
Na+-H+
exchanger, tyrosine kinase phosphorylates an ancillary protein, and the
latter then activates the exchanger, resulting in the modulation of a
critical cytoplasmic region and the alteration of the "set point"
of the exchanger, leading to intracellular alkalosis (34, 35).
In a previous study (16), we showed the housekeeping
Na+-H+
exchanger to be one of the three major
pHi regulators in cultured human
pleural mesothelial cells (PMCs). Human PMCs possess EGF receptors
(28), but little is known about the cellular signaling pathway involved
in EGF-induced
Na+-H+
exchanger activation. Using
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
to continuously monitor pHi in
human PMCs, we have found that EGF-stimulated PKC activation,
presumably as a result of receptor-linked tyrosine kinase activity, is
required for activation of the exchanger.
 |
MATERIALS AND METHODS |
Chemicals and solutions. Unless
otherwise stated, all chemicals were purchased from Sigma (St. Louis,
MO) and all experiments were performed in HEPES-buffered solution
consisting of (in mM) 130 NaCl, 5.0 KCl, 1.0 MgCl2, 2.0 CaCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 at 37°C with NaOH. In
Na+-free Tyrode solution, NaCl was
totally replaced by 130 mM
N-methyl-D-glucamine (NMDG). EGF was purchased from Collaborative Biomedical Products (Bedford, MA); and herbimycin A, lavendustin A, and chelerythrine were
purchased from Research Biochemicals International (Natick, MA).
Isolation and cell culture of human
PMCs. Human PMCs were obtained from patients with
transudative pleural effusion, e.g., patients with liver cirrhosis or
heart failure, as described in our previous report (16). Briefly, after
removal of red blood cells from the pleural effusion by gradient
centrifugation, the pellet was washed once in RPMI 1640, resuspended in
the same medium to a volume of 8-10 ml, and seeded in RPMI 1640 containing 10% FCS, 100 U/ml of penicillin, and 100 µg/ml of
streptomycin in 75-cm2
vitrogen-coated tissue culture flasks. After 24 h, one-half of the
medium was replaced; thereafter, it was completely replaced every 3 days. The mesothelial cell cultures were maintained for 2-3
passages at 37°C in a humidified environment containing 5% CO2. Before
pHi measurement, cells were growth
arrested by transfer to low-serum medium (0.5% FCS) for another 24 h.
PMCs were identified by their characteristic morphological and
immunohistochemical features of positive staining for human cytokeratin
(types I and II) and vimentin and negative staining for CEA, desmin,
and von Willebrand's factor (18).
Measurement of pHi.
Measurement of pHi has been
described in detail in previous work by Wu and colleagues
(36, 37). Briefly, human PMCs, grown on a cover glass, were loaded with
5 µM BCECF-AM (Molecular Probes, Kent, OR) for 20 min at room
temperature in HEPES-buffered solution, then washed with HEPES-buffered
solution, and excited alternately by 490- and 440-nm wavelength light.
The excitation light was transmitted to the cell with a 510-nm dichroic
mirror under the microscope nosepiece, and the resulting fluorescence
was collected by a ×40 oil-immersion lens. The overall sampling
rate was 0.5 Hz. The 530-nm emission ratio (ratio of 490- to 440-nm
excitation) from the intracellular BCECF was calculated and converted
to a linear pH scale (see below) by in situ calibration at pH 4.5 and 9.5 at the end of the experiment using the nigericin technique (29).
The following equation was used to convert the fluorescence ratio into
pHi:
pHi = pK + log[(Rmax
R)/(R
Rmin)] + log(F440 min/F440 max), where pK is the negative log of dissociation constant for the dye
measured at pH 7.16; R is the ratio of the 530-nm fluorescence at
490-nm excitation to that at 440-nm excitation;
Rmax and
Rmin are the maximum and minimum
ratio values, respectively, from the data curve; and
F440 min and
F440 max are the minimum and
maximum fluorescence values, respectively, at 440-nm excitation.
Statistical method. All data are
expressed as the means ± SE of n
preparations. Statistical analysis was performed with the paired
Student's t-test or the Mann-Whitney
U-test. A value of P < 0.05 was considered
statistically significant.
 |
RESULTS |
EGF stimulates the
Na+-H+
exchanger in human PMCs.
Under nominally bicarbonate-free conditions (i.e., HEPES-buffered
medium), the addition of 100 ng/ml of EGF resulted in a slight initial
acidosis (0.02 ± 0.01 pH units; n = 6) of the resting pHi, followed
by a more marked pHi increase
(0.12 ± 0.05 pH units; n = 6 preparations; Fig.
1A,
Table 1) similar in magnitude to the
EGF-induced alkalosis seen in rat hepatocytes (~0.1 pH unit) (23).

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Fig. 1.
Epidermal growth factor (EGF) stimulates
Na+-H+
exchanger in human pleural mesothelial cells (PMCs).
A: initial transient acidosis
[as determined by intracellular pH
(pHi)] followed by
sustained alkalosis was induced by addition of EGF (arrowhead). Dotted
line, baseline pHi.
B: EGF (100 ng/ml) added (arrowhead)
in absence of external Na+
(Na+-free medium; NaCl was totally
substituted for with
N-methyl-D-glucamine).
C: EGF added in presence of
ethylisopropyl amiloride (EIPA; 10 µM; arrowhead). All experiments
were performed in nominally bicarbonate-free (i.e., HEPES-buffered)
medium at 37°C.
|
|
In HEPES-buffered medium, the
Na+-H+
exchanger is the main pHi
regulator in human PMCs (16). We therefore tested whether the EGF-induced alkalosis was due to activation of this exchanger. Figure
1B shows that a
pHi decrease occurs on transfer to
Na+-free medium, probably due to
inhibition of the exchanger, resulting in accumulation of metabolic
acid. Under these conditions, however, the initial EGF-induced acidosis
(Fig. 1B, arrowhead) was more marked
(Table 1) and the expected EGF-induced alkalosis (Fig. 1A) was completely abolished (Fig.
1B, Table 1). Similar results were
seen when the
Na+-H+
exchanger was inhibited by EIPA (10 µM; Fig.
1C, Table 1), indicating that the
EGF-induced alkalization seen in Fig.
1A was due to activation of the
Na+-H+
exchanger.
The statistical data for the above results are summarized in Table 1.
Tyrosine kinase-receptor activity is involved in the EGF-mediated
stimulation of the
Na+-H+
exchanger.
The EGF signaling pathway is known to act via activation of the
tyrosine kinase receptor (5), and the EGF receptor has recently been
found in human PMCs (28). We therefore tested whether tyrosine kinase
activation is involved in stimulation of the
Na+-H+
exchanger. Genistein and lavendustin A are both potent specific blockers of EGF-stimulated tyrosine kinase activity (1, 25). Figure
2, A and
B, shows that either 30 µM genistein
or 50 µM lavendustin A itself caused a decrease in
pHi (Table 1) by an unknown
mechanism. However, in the presence of either of these inhibitors, a
further decrease in pHi occurred
on addition of 100 ng/ml of EGF (Fig. 2,
A and
B, arrowheads; Table 1). Herbimycin A,
which binds to the thiol groups in tyrosine kinase, is another potent
blocker. After overnight pretreatment with herbimycin A (0.5 µM), the
EGF-mediated stimulatory effect was lost (Fig.
2C, arrowhead; Table 1). These results
all indicate that tyrosine kinase activation is involved in the
activation of the
Na+-H+
exchanger.

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Fig. 2.
Stimulatory effect of EGF on
Na+-H+
exchanger was completely blocked by tyrosine kinase inhibitors
genistein (30 µM; A), lavendustin
A (50 µM; B), and herbimycin A
(0.5 µM; C). Arrowheads, addition
of EGF (100 ng/ml).
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The statistical data for the above results are summarized in Table 1.
TPA, a potent phorbol ester, also stimulates the
Na+-H+
exchanger.
The membrane association-activation event during PKC activation can be
mimicked by TPA (24). Figure
3A shows
that, on addition of 1 µM TPA, a slight initial acidosis was seen
(0.02 ± 0.01 pH units; n = 8 preparations), followed by a significant increase in
pHi (0.11 ± 0.03 pH units;
n = 8 preparations; Table
2). On removal of all extracellular
Na+ (Fig.
3B) or on addition of EIPA (10 µM;
Fig. 3C), a
pHi decrease was seen due to
inhibition of the
Na+-H+
exchanger; however, the subsequent addition of TPA (1 µM) induced a
greater acidosis (Fig. 3, B and
C, arrowheads; Table 2) compared with
the control value (Fig. 3A), and the
subsequent alkalization was completely abolished (Fig. 3,
B and
C; Table 2), indicating that the
TPA-induced alkalization (Fig. 3A)
was due to stimulation of the
Na+-H+
exchanger.

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Fig. 3.
12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates
Na+-H+
exchanger. TPA (1 µM) was added (arrowheads) in control condition
(A),
Na+-free
(N-methyl-D-glucamine-substituted)
medium (B), and 10 µM EIPA medium
(C). Dotted line, baseline
pHi.
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Chelerythrine, staurosporine, and
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), potent PKC
blockers (14, 17, 30), were used to determine whether the TPA-induced
stimulation of the
Na+-H+
exchanger is mediated via activation of PKC. Figure 4,
A and B, shows that chelerythrine (7 µM)
or staurosporine (0.1 µM) completely abolished the TPA-induced
alkaline effect (Table 2). TPA caused irreversible insertion of PKC
into the lipid bilayer, resulting in cumulative, long-term PKC
stimulation (24), and overnight TPA treatment caused complete depletion
of endogenous PKC (i.e., downregulation) (38). After overnight
treatment with 1 µM TPA, the alkalization induced by the subsequent
addition of 1 µM TPA (Fig. 4C,
arrowhead) was abolished (Table 2). These results indicate that PKC
activation is indeed involved in the stimulation of the Na+-H+
exchanger in PMCs. After treatment with PKC blockers, the initial slight TPA-induced acidosis (Fig.
3A) became even larger (Fig. 4,
A-C,
arrowheads), although the difference did not always reach statistical
significance (Table 2). The mechanism responsible for the initial
intracellular acidosis induced by these PKC blockers is unknown.

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Fig. 4.
TPA-activated
Na+-H+
exchanger can be blocked by protein kinase C (PKC) inhibitors
chelerythrine (7 µM; A) and
staurosporine (0.1 µM; B) and by
PKC downregulation (1 µM TPA pretreatment for 12 h;
C). Arrowheads, addition of TPA (1 µM).
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|
The statistical data for the above results are summarized in Table
2.
PKC activation is required for EGF-mediated stimulation of the
Na+-H+
exchanger.
The above results indicate that activation of either tyrosine kinase or
PKC can result in stimulation of the
Na+-H+
exchanger (Figs. 1-4). PLC-
1 phosphorylation, resulting in PKC stimulation, is one of the downstream signaling pathways for tyrosine kinase-receptor activation (5). Thus the stimulatory effect of EGF on
the
Na+-H+
exchanger is probably due to PKC activation via tyrosine
kinase-receptor phosphorylation of PLC-
1. We used PKC blockers to
test this possibility (Fig. 5,
A-C).

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Fig. 5.
PKC activation is 1 of the downstream signaling pathways for
EGF-activated
Na+-H+
exchanger. A: chelerythrine (7 µM).
B: staurosporine (0.1 µM).
C: PKC downregulation (pretreatment
with 1 µM TPA for 12 h). D:
genistein (30 µM). E: lavendustin A
(50 µM).
A-C,
arrowheads: addition of EGF (100 ng/ml).
D and
E, arrowheads: addition of TPA (1 µM).
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|
Chelerythrine (7 µM; Fig. 5A),
staurosporine (0.1 µM; Fig. 5B),
or TPA (1 µM, overnight; Fig. 5C)
all completely inhibited EGF-induced
Na+-H+
exchanger activation (Fig. 5,
A-C,
arrowheads). In contrast, two potent tyrosine kinase blockers,
genistein (30 µM; Fig. 5D) and
lavendustin A (50 µM; Fig. 5E),
failed to inhibit the TPA-stimulated alkalization (Fig. 5,
D and
E, arrowheads). These results suggest that PKC activation is involved in the downstream signaling pathway of
EGF-induced
Na+-H+
exchanger activation.
The statistical data for the above results are summarized in Table
3.
 |
DISCUSSION |
Activation of the
Na+-H+
exchanger by growth factors, including EGF, was originally thought to
utilize signaling pathways acting directly through tyrosine kinase
because the receptor has intrinsic tyrosine kinase activity (3, 9, 10,
12). An earlier study by Moolenaar et al. (22), however, showed that
when the tyrosine-specific protein kinase was activated by monoclonal
antibodies, the
Na+-H+
exchanger could not be activated, indicating that the
pHi signal could be dissociated
from tyrosine kinase activity. At a later date, growth factor was
suggested to induce tyrosine phosphorylation of PLC-
1, thereby
activating the PKC-dependent pathway (20). Direct support for this
hypothesis has been provided by at least one recent related study by Ma
et al. (19), who used cells expressing platelet-derived growth factor
receptors that lacked the PKC- and phosphatidylinositol
3'-kinase-mediated signaling pathways and showed that both
pathways are required for platelet-derived growth factor-induced
activation of the NHE1 in transfected epithelial cells.
On the basis of the following evidence in human PMCs, we suggest that
PKC activation is also involved in EGF activation of the
Na+-H+
exchanger, possibly due to tyrosine kinase-receptor phosphorylation of
PLC-
1 (19, 20): 1) the
pHi increase induced by EGF or TPA
can be blocked by either EIPA or the use of
Na+-free conditions, indicating
that it is due to activation of the Na+-H+
exchanger by these two mitogens (Figs. 1 and 3, Tables 1 and 2);
2) the TPA-induced alkalosis can be
blocked by PKC inhibitors (chelerythrine and staurosporine) and PKC
downregulation (Fig. 4, Table 2), indicating that PKC is indeed
involved in the activation of the
Na+-H+
exchanger; and 3) the EGF-induced
alkalosis was blocked not only by tyrosine kinase inhibitors
(genistein, lavendustin A, and herbimycin A; Fig. 2, Table 1) but also
by PKC inhibitors (chelerythrine and staurosporine) and PKC
downregulation (Fig. 5,
A-C;
Table 3). Because the tyrosine kinase inhibitors did not block the TPA-mediated activation of the
Na+-H+
exchanger (Fig. 5, D and
E; Table 3), we cannot completely
exclude the possibility that they themselves did not induce an
alkalosis after extended treatment (Fig. 5,
D and
E). However, we found that once the
steady state of the pHi was
reached after addition of these blockers, the
pHi did not change to any great
extent until the end of the experiment (data not shown), suggesting
that the TPA-induced alkalosis seen in Fig. 5,
D and
E, is most probably due to
TPA-mediated activation of the exchanger and that PKC activation is one
of the downstream signals for EGF-mediated activation of the exchanger
in human PMCs, as shown in other cells (19, 20). In the present study,
however, we cannot completely rule out the possibilities that a mutual
effect of tyrosine kinase and PKC activation is simply expressed at the
EGF receptor or that multiple effects of various kinases and
phosphatases may also be involved in the EGF-induced
Na+-H+
exchanger. The upward shift in the resting
pHi produced by the addition of
EGF reflects a change in the set point, which determines the pHi sensitivity of the
exchanger. According to this model, the set point of the
H+ sensor is adjusted upward by
0.15-0.3 pH unit (13), the magnitude of the alkalosis recorded in
the present study.
The molecular mechanism involved in the PKC-mediated activation of the
Na+-H+
exchanger is still unclear. In fibroblasts, it has been shown that
phorbol esters increase NHE1 phosphorylation (31); therefore, it is
possible that PKC directly phosphorylates a serine residue and
activates the NHE1. However, with the use of deletion mutants expressed
in fibroblasts, a recent study (34) has shown that replacement of this
serine residue with alanine results in only a 50% reduction in phorbol
ester-induced alkalization, clearly indicating that PKC does not
activate the NHE1 by direct phosphorylation (35); therefore multiple
effects of various kinases and phosphatases may be involved.
It is interesting that, in the presence of
Na+-H+
exchanger blockers or tyrosine kinase and/or PKC inhibitors
(Figs. 1-5), although not always statistically significant, a
greater pHi decrease was normally
seen on addition of EGF and TPA (Tables 1 and 2). Because a small, but
consistent, initial pHi decrease
was seen on addition of these two mitogens (Figs.
1A and
3A), we suggest that the larger pHi decrease seen after abolition
of the alkalization (i.e., activation of the
Na+-H+
exchanger) was probably due to unmasking of the initial acidification induced by these two mitogens. However, the mechanism for this initial
small pHi decrease seen on the
addition of EGF or TPA (Figs. 1A and
3A) is unknown. One possibility is
that EGF and TPA cause transient increases in intracellular
Ca2+ levels, resulting in
displacement of H+ from common
buffering sites (32); another is overproduction of
H+ (e.g., via stimulation of the
metabolism) by the addition of EGF or TPA.
Recently, the EGF receptor has been shown to be present in human PMCs
(28) and rat mesothelial cells (15, 26), and its activation stimulates
cell proliferation (15, 26). However, the physiological role of
EGF-mediated stimulation of the
Na+-H+
exchanger, resulting in alkalosis in human PMCs, is not clear. Activation of the
Na+-H+
exchanger, resulting in intracellular alkalosis, is a common phenomenon
that stimulates cell proliferation in a variety of cell types (8, 13,
27), with small decreases in pHi
causing reduced cell proliferation (8) and a rise in
pHi being associated with
increased cell numbers (4). However, the role of the
Na+-H+
exchanger in stimulating cell growth as a result of intracellular alkalosis is still debatable (13) because some studies show that growth
factors or phorbol esters fail to induce a detectable rise in
pHi in physiological
bicarbonate-containing media (7, 13). In such cases,
pHi changes induced by the
Na+-H+
exchanger are clearly not of significance in the initiation of cell
growth. The possibility of the
Na+-H+
exchanger being involved in restoring normal human PMCs after pleural
injury requires further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Chiou-Mei Wu for excellent technical help. P.-C. Yang and
M.-L. Wu made equal contributions to this paper.
 |
FOOTNOTES |
This work was supported by National Taiwan University Hospital Grant
NTUH.S871008.
Address for reprint requests: M.-L. Wu, Dept. of Physiology, College of
Medicine, National Taiwan Univ., No. 1, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan, ROC.
Received 27 August 1997; accepted in final form 16 January 1998.
 |
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