1 Institut National de la Santé et de la Recherche Médicale U. 467, Faculté de Médecine Necker 75730 Paris Cedex 15; and 2 Centre National de la Recherche Scientifique Unité Propre de Recherche 1524, Hôpital St-Vincent de Paul 75674 Paris, France
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ABSTRACT |
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The almost ubiquitously expressed ClC-2 chloride channel is
activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein
tyrosine phosphorylation and activation of these channels. This study
examines the possibility that transforming growth factor- (TGF-
)
modulates ClC-2 activity in human colonic epithelial (T84) cells.
TGF-
(0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a
superimposed reversible activation of the current was observed at 8.3 nM TGF-
. Both effects required activation of the intrinsic epidermal
growth factor receptor (EGFR) tyrosine kinase activity, of
phosphoinositide 3-kinase, and of protein kinase C. With
microspectrofluorimetry of the pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-
was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-
-induced alkalinization activates
ClC-2 current. This study indicates that ClC-2 channels are
targets for EGFR signaling in epithelial cells.
hydrogen ion concentration; protein kinase C; signal transduction; patch-clamp techniques
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INTRODUCTION |
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MANY CELL ACTIVITIES, including mitogenesis, the induction of differentiation, enhanced cell migration, mucosa protection and wound repair, and the modulation of absorptive/secretory transport and membrane excitability, are activated by signals from the epidermal growth factor receptor (EGFR). Although the activation of EGFR and intracellular signaling have been extensively studied, relatively few studies have examined the acute regulation of ion channels by epidermal growth factor (EGF), and even these have been limited to cationic channels such as Ca2+-dependent K+ channels (40), voltage-dependent Kv1.2 (41, 54), Kv1.3 (5) and Kir2.1 K+ channels (63).
The ClC-2 gene belongs to a large family whose first member, ClC-0, was discovered by expression cloning in the Xenopus oocyte (24). There are at least nine ClC genes in mammals, most of which encode functional chloride channel proteins (23). The almost ubiquitous ClC-2 is activated by membrane hyperpolarization (50), a low extracellular pH (10, 25), and osmotic cell swelling (20). We have identified a chloride current with the same biophysical properties and sensitivity to osmotic changes in the T84 line of colon cells (14, 15).
The ClC-2 chloride current has been characterized in several cell types (22), but the physiological relevance of native ClC-2 channels has not yet been elucidated. Three proposals are presently considered. First, ClC-2 may play an important role in setting the intracellular chloride concentration of hippocampus neurons, thus influencing the direction of GABAA responses from excitatory to inhibitory (48, 49). Second, immunolocalization of ClC-2 at the apical membranes of lung and intestinal epithelial cells (36) has led to the hypothesis that pharmacological ClC-2 activation may provide an alternative pathway for chloride secretion across cystic fibrosis epithelia (42). Third, ClC-2 channels may be critical for some essential cellular activities associated with cell volume changes, as suggested by the sensitivity of mammalian ClC-2 to osmotic changes and by recent reports showing that ClC-2 helps to regulate cell volume when expressed in oocytes (17) and insect Sf9 cells (64). However, this last function is still debated, because ClC-2 was not found to contribute to regulatory volume decrease in another study (4).
The activation of ClC-2 channels in response to cell swelling and low extracellular pH involves the dissociation of an NH2-terminal region of ClC-2 from a docking site located in the cytosolic loop between transmembrane segments D7 and D8 (25). But dephosphorylation of serine/threonine residues in the channel or a related protein also appears to be necessary for current activation after cell swelling in T84 cells (15) as well as during the cell cycle for a ClC-2-like current in ascidian embryo cells (60).
Hypotonic cell swelling activates an outwardly rectifying anionic
conductance, with biophysical and pharmacological properties different
from those of ClC-2, in most of the cells studied to date
(38). Published data on these endogenous currents indicate that protein tyrosine phosphorylation is involved in the hypotonicity signal transduction pathway in some cell types (47, 53,
61) and that EGF potentiates the hypotonicity-induced anionic
efflux (53). Application of the tyrosine kinase
p56lck directly activates these swelling-sensitive chloride
channels in the absence of cell swelling or even during cell shrinkage in lymphocytes (30). This raises the question of whether a
similar mechanism is involved in the activation of ClC-2 current
(IClC-2). We have therefore investigated, using
the nystatin-perforated patch-clamp technique, the modulation of
IClC-2 by the physiologically relevant tyrosine
kinase activator, transforming growth factor (TGF)-. EGFR are
present on many types of cells, including those bearing ClC-2-like
channels. EGFR can be activated by several ligands, but TGF-
is
thought to be the most physiological ligand for EGFR in the nervous
system (26) and the gastrointestinal tract
(57) because of its abundance in these tissues.
We find that IClC-2 is modulated by TGF- by
mechanisms involving phosphorylation of the receptor on tyrosine
residues, activation of phosphoinositide (PI) 3-kinase and protein
kinase C (PKC), as well as by TGF-
-induced changes in intracellular
pH (pHi).
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MATERIALS AND METHODS |
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Cells. T84 cells were provided by the American Type Culture Collection (ATCC, Rockville, MD). Passages 46-56 were used. Cells were grown at 37°C in an atmosphere of 5% CO2-95% air in Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1) containing 10% FCS, 2 mM glutamine, 100 IU penicillin, and 170 µM streptomycin. Cells were grown in 25 cm2 plastic flasks until they reached about 90% confluence and were then subcultured once a week by trypsinization. For patch-clamp experiments, cells were plated at low density on 35-mm2 petri dishes (Corning) that were directly mounted on the stage of an inverted microscope (Zeiss IM35) 2-3 days after plating. To prevent space-clamp artifacts through cell junctions, we took all recordings from individual cells.
Current recording.
Patch-clamp experiments were performed at room temperature with an
Axopatch 200A amplifier controlled by a computer via a CED 1401 interface (CED, Cambridge, UK) (14). Currents were filtered at 100 Hz and then sampled on-line at 200 Hz. Pipettes were
pulled from hard glass (Kimax 51), coated with Sylgard, and fire
polished to a resistance of 1-3 M when filled with
intracellular solutions. Currents were recorded by applying regular
20-s voltage pulses of the desired amplitude from a holding potential
of 0 mV every 60 s. Current relaxation was measured as the
difference in current amplitudes between 20 s and 50 ms.
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pH Imaging. T84 cells cultured on thin glass coverslips for 48 h were loaded with 2 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetometoxy ester (BCECF-AM), diluted from a 2-mM stock solution in DMSO for 45 min at 37°C in 95% air-5% CO2 in a solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 25 sucrose, 10 HEPES-Na (pH 7.4). Cells were washed twice with the same solution to remove excess BCECF-AM and allowed to equilibrate for at least 20 min at 37°C. The coverslips were mounted on the stage of an inverted microscope (Olympus IX-70) in a homemade perfusion chamber and continuously perfused with solutions kept at 24°C. The cells were allowed to equilibrate for at least 10 min before experiments were begun. Cells were excited at wavelengths of 440 and 495 nm alternately, and the photometric data were recorded at a rate of four images/min at 530 nm via a V/ICCD camera (Princeton Instruments). All devices were controlled by a computer using the MetaFluor program (Universal Imaging), which was also used to analyze the data. The fluorescence ratios (495/440) calculated from these measurements were converted to pH at the end of each experiment by a two-point internal calibration method (51). The K+/H+ exchanger nigericin (final concentration 10 µM) was dissolved in a solution containing (in mM) 145 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 25 sucrose, 10 HEPES-N-methylglucamine (pH 6.9 or 7.6).
Drugs.
Stock solutions (16.7 µM) of TGF- (Sigma) were prepared in PBS
containing 1% BSA, aliquoted, and stored at
80°C. Stock solutions were diluted in the external bath solution to the desired concentration just before each experiment. Cells used in experiments designed to test
the effects of growth factors were placed in serum-free medium for
4-5 h before recordings were started. Longer periods of serum
deprivation (>12 h) weakened the cells and did not improve the responses.
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RESULTS |
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We previously described a hyperpolarization-activated
Cl current in T84 cells using the whole cell
configuration of the patch-clamp technique (14). This
current closely resembles the ClC-2 channel heterologously expressed in
Xenopus oocytes (50). Because human ClC-2 has
been cloned from T84 cells (9), this
hyperpolarization-activated Cl
current will be hereafter
referred to as the ClC-2 current, IClC-2. Similar whole cell chloride currents with slow activating kinetics and
strong inward rectification with a threshold potential of activation
close to
60 mV were recorded in the present work by the
nystatin-perforated patch-clamp technique (Fig.
1, A and B).
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After establishment of the whole cell recording, the IClC-2 amplitude increased at 5-10 min and then remained stable for tens of minutes (Fig. 1C).
Effect of low concentrations of TGF- on IClC-2.
All results were obtained once the recording had stabilized. Applying
0.17 nM (1 ng/ml) TGF-
resulted in a gradual and irreversible decrease in the amplitude of IClC-2 that
continued for several minutes after washout before inhibition was
maximal (Fig. 1D). The mean quasi-steady-state inhibition of
the current recorded at
120 mV was 46.0 ± 4.8%,
n = 11 (P < 0.0001). We did not
observe any reversibility, even at the lowest active concentration of TGF-
, 0.5 ng/ml (33 ± 3.5% inhibition, n = 5). A similar effect occurred at less hyperpolarized command potentials
(Fig. 1, D and E). TGF-
inhibited current
relaxation to the same extent at
80 and
120 mV (mean current
decrease at
80 mV was 55.8 ± 12.4% and 51.2 ± 17.5% at
120 mV; n = 5; P > 0.05), suggesting that the decrease in current amplitude did not result from a shift in
the voltage dependence of current activation. Such a shift would have
led to greater inhibition of the current around the threshold of
activation than at more negative potentials.
Additional effect of a high concentration of TGF- on
IClC-2.
Although circulating levels of EGF are low (34), the
gastrointestinal tract can be exposed to concentrations of EGF in
excess of 100 ng/ml (18, 55). High concentrations of
TGF-
may also be present in the microenvironment of EGFR, because
TGF-
is produced by colonic epithelial cells and cells of the lamina
propria (57 and references therein). Because EGF or TGF-
effects on several transport systems in the gastrointestinal tract have
been demonstrated at concentrations >1 ng/ml ranging from 30-200
ng/ml, we investigated the effect of a higher concentration of TGF-
(50 ng/ml) on IClC-2 amplitude. The effect was
different from that obtained with 0.17 nM TGF-
in most of the cells
tested (Fig.
2A).
TGF-
at 8.3 nM first caused a slight increase in current amplitude,
followed by a small, slow decrease. Importantly, the current amplitude decreased more rapidly to a new steady state after washout. This pattern of response was observed in 26 of 33 tested cells and may be
due to two simultaneous opposing effects, a reversible increase in
current amplitude and a sustained decrease. The TGF-
-induced increase in IClC-2 was reversed after washout,
unmasking the long-lasting decrease in IClC-2.
This resulted in the net current changes varying during TGF-
application, with larger or smaller increases in current amplitude or
even no apparent change. But the removal of TGF-
invariably resulted
in a rapid decrease in IClC-2 amplitude by
32.7 ± 11.5%, n = 26 (statistically different
from IClC-2 value before TGF-
application,
P < 0.001), whatever the response in this first phase.
Changes in current relaxation were not accompanied by changes in
current amplitude recorded at 0 mV or just after the voltage jump to
120 mV (see Fig. 2B) or at +40 mV (not shown). Thus both
increases and decreases in current were due to
IClC-2 and not to other chloride and/or distinct
ionic currents. The concomitant current increase was not detected in a
small number of cells (n = 7), and the basal amplitude
of IClC-2 was irreversibly reduced (by 36.0 ± 2.2%, Fig. 2, C and D), as was observed for 0.17 nM TGF-
. Figure 2E illustrates I-V
obtained during fast voltage ramps from
120 to +60 mV applied at the
end of the 20-s hyperpolarizing voltage jumps before and after
stimulation by TGF-
. Both curves reverse at the same potential
value,
22 mV, close to the expected reversal potential for chloride
ions as calculated by Nernst equation (
17.2 mV). These
current-voltage relations also show that open channels were equally
inhibited throughout the whole voltage range. The presence of the first phase (activation) did not depend on basal current values as shown in
Fig. 2F.
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Decreased basal IClC-2 amplitude due to phosphotyrosine
phosphatases inhibitors.
Phosphotyrosine phosphatase (PTP) inhibitors mimic the effects of
several growth factors and potentiate (53, 61) or decrease (11, 52) volume-regulated anion channels. Application of
100 µM pervanadate caused a gradual decrease in
IClC-2 by 60.5 ± 6.4%, n = 3 (Fig. 3, A and
B), and 20 µM dephostatin inhibited
IClC-2 by 52.3 ± 7.8%, n = 3 (not shown). The effects of both inhibitors were irreversible.
Unlike vanadate, pervanadate has been shown to be a stronger and
irreversible PTP inhibitor (21), and dephostatin may be
only poorly washed out of the cells (61). These inhibitory effects probably reflect the activity of endogenous protein tyrosine kinases (PTK) that are active at rest and further indicate that IClC-2 is negatively regulated by protein
tyrosine phosphorylation.
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Prevention of modulation of IClC-2 by TGF- by PI
3-kinase inhibitors.
One signaling pathway activated in response to EGF involves the
recruitment of PI 3-kinase via the Src homology 2 (SH2) domains of its
85-kDa regulatory subunit to tyrosine phosphorylated residues of
receptor proteins Erb3 (46) or p120cbl
(45). EGF also recruits the regulatory (85 kDa)
and catalytic (110 kDa) subunits of PI 3-kinase in T84 cells
(59) via the formation of the EGFR/ErbB2 heterodimer
(27). We looked at the influence of PI 3-kinase on the
action of TGF-
on IClC-2 by determining whether IClC-2 was modulated by TGF-
in the
presence of the PI 3-kinase inhibitors wortmannin (100 nM) or LY-294002
(20 µM). Cells were pretreated with either inhibitor and then
submitted to the higher concentration of TGF-
(8.3 nM) to assess the
possible involvement of PI 3-kinase in the stimulatory and inhibitory
effects of TGF-
. Because 8.3 nM TGF-
induces a biphasic response,
activation and inhibition, and because 0.17 nM is able to induce only
the inhibitory effect, we decided to test the various pharmacological agents at this higher concentration. There was neither an increase during TGF-
application nor a decrease during its washout in cells
treated with PI 3-kinase inhibitors (wortmannin, n = 10 or LY-294002, n = 4, Fig.
4, A and B).
TGF-
slightly decreased current amplitude by 18 and 20% in two
other cells pretreated with wortmannin, a residual effect not observed
with the use of LY-294002. These results thus indicate that PI 3-kinase
activation is involved in the transduction pathway leading to
IClC-2 modulation.
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The actions of TGF- require PKC activation.
As stated above, we previously reported that stimulation of PKC by PMA
strongly inhibits IClC-2. Because stimulation of
EGFR results in activation of the
-isoform of phospholipase C (PLC), leading to PKC recruitment to the membrane (56), we have
now examined the responses to TGF-
of cells treated for 2 h
with GF109203X, a selective PKC inhibitor, to determine whether
TGF-
-induced IClC-2 inhibition is mediated by
PKC activation. In contrast to our previous experiments performed with
the PKC inhibitors staurosporine and calphostin C (14),
GF-109203X did not change the kinetics of IClC-2
activation at rest. We therefore checked whether this inhibitor
prevented the PMA-induced decrease in IClC-2.
The amplitude of IClC-2 was irreversibly
decreased by 100 nM PMA (70 ± 3.3%, n = 5, not
shown) and 500 nM (89 ± 8%, n = 3, Fig.
5A). Preincubation of the
cells with 1 µM GF-109203X produced a marked attenuation of
inhibition of IClC-2 caused by 500 nM PMA
(n = 3, compare Fig. 5, A and B)
and completely prevented inhibition by 100 nM PMA (data not shown).
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Changes in cytosolic pH caused by TGF-.
EGF/TGF-
activates the isoform 1 of the
Na+/H+ exchanger (NHE) in several cell types by
mechanisms that may involve the activation of PKC (19,
62). This results in intracellular alkalinization in
bicarbonate-free solutions and the presence of an inwardly directed
sodium concentration gradient. We therefore postulated that at least
one type of the response of IClC-2 to 8.3 nM
TGF-
mediated by PKC was due to a change in pHi. Such a
pHi change could be expected, because it was not heavily
buffered during IClC-2 recording. Because we
have found no report of the regulation of pHi in T84 cells,
we first examined whether TGF-
induced a change in pHi
in these cells.
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TGF--induced IClC-2 increase depends on external
Na+.
From the above experiments, we postulated that the superimposed
activation of IClC-2 observed with high doses of
TGF-
was the result of a TGF-
-induced reversible cell
alkalinization. If TGF-
-induced activation of NHE and the resulting
cytosolic alkalinization contribute to the increase of
IClC-2 amplitude, this effect should be
prevented by blocking NHE, by, for example, replacing external
Na+ with tetraethylammonium (TEA+). We
previously observed that omission of external Na+ does not
affect the properties of IClC-2 per se with
conventional whole cell patch-clamp recordings
(14). The present study show that the basal
IClC-2 was reversibly diminished by 12 ± 7.6% (n = 7) when Na+ was removed (not
shown), probably because of the concomitant TEA+-induced
pHi decrease (shown in Fig. 6B) or because of
some degree of cell shrinkage. In the experiment illustrated in Fig.
7, the response of
IClC-2 to TGF-
was remarkably dependent on
external Na+. Although TGF-
did not result in a marked
current change in the presence of Na+, the amplitude of
IClC-2 decreased sharply when Na+
was removed but with TGF-
still present (compare with Fig.
2A). The same results were obtained in five cells, with a
mean decrease in IClC-2 amplitude of 63.7 ± 4.0%. The rapid, pronounced decrease in
IClC-2 after Na+ removal may involve
cytosolic acidification due to uncompensated metabolic acid production
and to NHE reversal. Because the mean decrease in
IClC-2 due to removal of external
Na+ before TGF-
application was only 12% and the
omission of Na+ from the perfusion solution during TGF-
application resulted in cell acidification of about 0.15 pH unit (Fig.
6B) compared with that before TGF-
application, it is
reasonable to assume that the major mechanism of
IClC-2 inhibition after Na+ removal
(Fig. 7) was not intracellular acidification. Thus the most likely
interpretation for the absence of the 8.3 nM TGF-
-induced IClC-2 decrease in the presence of
Na+ is that NHE was activated by TGF-
and that the
resulting cell alkalinization contributed to the increase in
IClC-2 and hampered the underlying decrease in
current.
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DISCUSSION |
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We find that TGF- has a dual effect on the endogenous
IClC-2 in T84 cells. It causes a long-lasting
decrease in current amplitude that is detectable over a wide range of
agonist concentrations (0.17-8.3 nM) and a superimposed reversible
increase in current amplitude in response to the highest concentration
used (8.3 nM). This latter finding implies that a divergent downstream
signaling pathway is initiated by 8.3 nM TGF-
to increase
IClC-2 activity. Although the results obtained
with pharmacological agents should be reinforced with biochemical
proofs, our data suggest that both the inhibition and the activation of
IClC-2 require the activation of PI 3-kinase and
PKC. However, only 8.3 nM TGF-
caused cytosolic alkalinization,
suggesting that the TGF-
-induced cytosolic alkalinization accounts
for the IClC-2 increase. The different pathways
presumably involved in the regulation of IClC-2
are depicted in Fig. 8.
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Direct activation of classic and novel PKC isoforms by phorbol esters
has been reported to inhibit IClC-2 in T84 cells
(14) and in neurons (33, 48). The need to
activate both PI 3-kinase and PKC for TGF- to modulate
IClC-2 is highlighted by the recent demonstration that the major lipid product of PI 3-kinase,
PtdIns(3,4,5)P3, binds to the
pleckstrin homology (12) and SH2 domains (1) of PLC-
, whose activity leads to PKC activation. Such
interactions seem to be required for the activation of PLC-
by
growth factors. Our finding that PI 3-kinase inhibitors are as potent
as PKC inhibitors in preventing the modulation of
IClC-2 by TGF-
supports this scheme and
points to the fact that full activation of PKC by TGF-
in T84 cells
requires PI 3-kinase product(s).
Our results also show that PI 3-kinase inhibitors reduce the basal amplitude of IClC-2. Three classes of PI 3-kinases, equally sensitive to wortmannin or LY-294002, have been identified in mammalian cells (16). The growth factor-independent class III PI 3-kinase product PtdIns(3)P is present in significant amounts in unstimulated T84 cells, and its level is rapidly decreased by treatment with wortmannin (59). The only function for PtdIns(3)P currently known concerns membrane trafficking and vesicle morphogenesis (43). Hence, the basal inhibition of IClC-2 by inhibitors of PI 3-kinases suggests that the number of active ClC-2 channels in the membrane might be regulated by vesicular transport, as for other transport proteins (6).
High concentrations of TGF- resulted in concomitant opposite effects
on IClC-2 that were both mediated by PKC
activation, emphasizing that PKC activated more than one downstream
pathway. We found that high concentrations of TGF-
modulate
pHi, suggesting that these variations in pHi
contribute to the modulation of IClC-2. This
hypothesis is supported by both earlier reports showing an inhibition
of IClC-2 by acidic internal pH in
Aplysia neuron (7) and by a more recent study
performed on rat astrocytes showing that cell acidification decreases
and cell alkalinization increases IClC-2
(37). TGF-
induced a cytosolic alkalinization, but
acidification also occurred either exclusively or before
alkalinization, as reported in other cell types (e.g., see Ref.
31). The mechanisms underlying the acidification remain
unclear, but one possibility is an increase in metabolic activity after
addition of TGF-
(19). Thus it is reasonable to assume
that the variation in pHi of a single cell may depend on
the relative contributions of acid loading and extruding mechanisms.
This assumption could explain the two types of
IClC-2 variation after 8.3 nM TGF-
addition
(Fig. 2, A and C). According to this
hypothesis, one can assume that cell acidification may have been
predominant in the cells that responded to 8.3 nM TGF-
by an
immediate decrease in IClC-2 (see Fig.
2C) and may have enhanced TGF-
-induced inhibition of
IClC-2 after Na+ removal (Fig. 7).
However, our results exclude acidification as the major cause of
current inhibition in response to TGF-
or after direct activation of
PKC by PMA, because application of 100 nM PMA or 0.17 nM TGF-
did
not acidify the cells but did strongly inhibit
IClC-2. Independently of cell acidification, inhibition of IClC-2 may be due to
phosphorylation of the ClC-2 protein, which contains 10 consensus PKC
phosphorylation sites (9). Our preliminary results
indicate that the ClC-2 protein can indeed be phosphorylated by PKC and
TGF-
, but the link between phosphorylation and activity remains to
be identified. Moreover, our results suggest that the negative
modulation of ClC-2 current by TGF-
may be overridden if the
concentration of TGF-
rises and induces a concomitant cell alkalinization.
We previously suggested that activation of
IClC-2 by hypotonicity implicates
serine/threonine dephosphorylation mechanisms (15). In
some cell types, PTK seem to be required for the activation of the
volume-sensitive chloride current (ICl,Swell)
(47, 53, 61). In our model, inhibition of
IClC-2 by PTP inhibitors and TGF- does not
support a role for PTK in activation of IClC-2 during cell swelling, but rather reminds one of the inhibitory effect
of PTP inhibitors on ICl,Swell in chromaffin and
fibroblastic cells (11, 52). In fact PTK activation might
be involved in the negative modulation of IClC-2
by hypotonicity observed in rat osteoblasts (8) and in
mouse mandibular cells (29).
The pleiotropic effects of EGFR on cellular processes and the absence
of knowledge of the precise function of IClC-2
make it difficult to evaluate the physiological significance of the modulation of IClC-2 by TGF-. However, some
hypothesis can be formulated on the basis of the currently proposed
functions for IClC-2 in transepithelial chloride
transport. It was recently shown that in polarized cell monolayers
derived from the Caco-2 cell line that models the human small
intestinal epithelium, ClC-2 channels located at the apical aspect of
the tight junction complex may participate in chloride secretion
(35). However, the same study, as well as our preliminary
observations (32), suggests that the location of ClC-2
channels differs between small intestinal and colonic cells. In the
colon, ClC-2 immunoreactivity is found in basolateral membranes of
luminal surface and crypt cells, implying that ClC-2 channels may
likely contribute to chloride absorption in the colon. On the other
hand, EGF and TGF-
have been shown to regulate Na+ or
Cl
transport in intestinal epithelia. EGF stimulates
small intestinal NaCl absorption through activation of apical NHE3
(28), whereas EGF and TGF-
inhibit
Ca2+-dependent chloride secretion in the colonic T84 cell
line (58). This latter effect probably involves inhibition
of basolateral K+ channels (2). Both effects
initiated after binding of growth factors to their basolateral
receptors are mediated primarily by activation of PI 3-kinase
(28, 59). Thus modulation of the opening of ClC-2 channels
may be another means for EGF/TGF-
to regulate chloride secretion or
absorption in the intestinal tract. In response to TGF-
, ClC-2
channels might be finely regulated, for example, preferentially closed
in the presence of low concentrations of TGF-
, whereas higher
concentrations along with alkalinization processes may limit the
closure of the channels.
The modulation of IClC-2 by TGF- may also be
of physiological significance in the nervous system where ClC-2
channels act as regulators of GABAA responses in neurons in
which both ClC-2 (44) and EGFR have been identified
(3, 13).
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ACKNOWLEDGEMENTS |
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The English text was edited by Owen Parkes.
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FOOTNOTES |
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This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Association Française de Lutte contre la Mucoviscidose. M. Bali was supported by a fellowship from Centre Volvic pour la Recherche sur les Oligo-éléments and Fondation pour la Recherche Médicale. J. Lipecka was supported by a fellowship from the French Ministry of Foreign Affairs and Association Française de Lutte contre la Mucoviscidose.
Address for reprint requests and other correspondence: A. Edelman, INSERM U. 467, Faculté de Médecine Necker, 156, rue de Vaugirard, 75015 Paris, France (E-mail: edelman{at}necker.fr).
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 31 August 2000; accepted in final form 16 January 2001.
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