Regulation of ClC-2 chloride channels in T84 cells by TGF-alpha

Moëz Bali1, Joanna Lipecka1, Aleksander Edelman1, and Janine Fritsch2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TGF-alpha ) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-alpha (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-alpha . 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-alpha was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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)-alpha . EGFR are present on many types of cells, including those bearing ClC-2-like channels. EGFR can be activated by several ligands, but TGF-alpha 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-alpha 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-alpha -induced changes in intracellular pH (pHi).


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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 MOmega 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.

The time course of IClC-2 activation was best approximated by single-exponential fits according to the equation
<IT>I</IT><SUB>ClC-2</SUB>(<IT>t</IT>)<IT>=A+B · exp</IT>(<IT>−t/&tgr;</IT>)
where IClC-2(t) is the amplitude of the current at time t, A and B are constants, and tau  is a time constant.

In some experiments, instantaneous current-voltage relationships (I-V) of activated channels were established using a fast voltage ramp (300 ms), applied immediately after 20-s activation of the channels at -120 mV.

The nystatin-perforated patch-clamp technique was used to provide measurement of whole cell chloride current while preventing the disappearance of diffusible components during cell dialysis. Nystatin was added to the standard solution that contained (in mM) 75 NaCl, 35 Na2SO4, 2 MgCl2, and 10 HEPES-Na (pH 7.3). The bath solution contained (in mM) 150 NaCl, 1 CaCl2, 1 MgCl2, 10 HEPES-Na (pH 7.4), 10 glucose, and 25 sucrose. These ionic concentrations and the amount of sucrose were determined experimentally to preserve osmotic equilibrium and avoid cell swelling under basal conditions. A stock solution of nystatin (54 mM) in DMSO was prepared daily and was diluted to 0.2 µM in the internal solution and sonicated for at least 30 s. Access resistance gradually declined after the formation of an on-cell patch as nystatin pores were inserted into the membrane. Current recordings started when the access resistances decreased to <20 MOmega (10-15 min after patch formation). Membrane capacitance and series resistances were routinely measured via the analog circuitry of the amplifier and compensated for before recording started. Cell capacitance ranged from 15-70 pF. The various compounds to be tested were applied when stable recordings were obtained.

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-alpha (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.

The specific inhibitor of EGFR-tyrosine kinase activity, PD-153035, was purchased from Tocris Cookson (Bristol, UK) and added to the external solution from a 1-mM stock in DMSO. Stock solutions of the PI 3-kinase inhibitors wortmannin, purchased from Tebu (1 mM in DMSO), and LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], purchased from Sigma (20 mM in ethanol), were prepared. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Stock solution of PMA at 500 µM was prepared in DMSO. The PKC inhibitor GF-109203X and the tyrosine phosphatase inhibitor dephostatin were purchased from Alexis (San Diego, CA), and 1-mM stock solutions were prepared in DMSO. Pervanadate was prepared by combining 0.1 mM orthovanadate and 1 mM H2O2. Excess H2O2 was eliminated with 100 IU/ml catalase (Sigma). The different vehicles used were without effect on the ClC-2 current.

Results are expressed as means ± SD. Difference between groups were tested using Mann-Whitney U-test for independent samples and Wilcoxon matched-pairs signed-rank tests for related samples, because the data are not normally distributed. The Spearman rank correlation coefficient was calculated to measure the degree to which a linear relationship occurs between two variables. Differences were considered significant at P < 0.05.


    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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Fig. 1.   Inhibition of ClC-2 current (IClC-2) by low concentrations of transforming growth factor (TGF)-alpha . Whole cell currents were recorded using the nystatin-perforated patch-clamp technique. A: current traces recorded during a series of 20-s voltage jumps delivered stepwise in 20-mV increments every 60 s from a holding potential of 0 mV from -120 to 0 mV and to +40 mV. The voltage jump protocol used to establish the current-voltage relationship (I-V) curve is shown above. Because ECl is different from 0 mV (= -21 mV), tail currents are recorded on back step to 0 mV. Zero current is indicated by dashed line. B: I-V curve corresponding to the current relaxations shown in A and measured as the difference between the current obtained at the end of the test pulse and the current recorded 50 ms after the onset of each voltage jump. C: IClC-2 amplitude recorded after establishment of the whole cell recording during successive voltage jumps from 0 to -120 mV in untreated cells. D: IClC-2 amplitude recorded during successive voltage jumps from 0 to -120 mV and -80 mV in the presence and absence of 0.17 nM TGF-alpha . TGF-alpha was added to the perfusion solution for 9 min (open bar), and the inhibitory effect was not reversible upon washout. E: corresponding current traces recorded at -120 mV (a, b) and -80 mV (c, d) before (a, c) and after (b, d) application of TGF-alpha . The voltage protocol is shown on top of the current traces. The exponential fits are shown and superimposed on the current traces. F: %decrease in IClC-2 induced by 0.17 nM TGF-alpha as a function of basal IClC-2 amplitude, which was normalized to cell membrane capacitance (pA/pF). The calculated Spearman correlation coefficient, rs = 0.163, indicates no correlation between these two parameters (P > 0.05).

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-alpha on IClC-2. All results were obtained once the recording had stabilized. Applying 0.17 nM (1 ng/ml) TGF-alpha 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-alpha , 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-alpha 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.

The activation kinetics of ClC-2 channels varies among the different cell types in which these channels have been studied (e.g., 8, 15, 39, 48, 50). In T84 cells, IClC-2 did not reach full activation even at the end of a 20-s hyperpolarizing pulse, a property similar to that observed after expression of ClC-2 channels in Xenopus oocytes (50). Steady-state IClC-2 amplitude at -120 mV was reached when hyperpolarization was prolonged for about 2 min (not shown). The time-dependent activation of the current recorded at -120 mV could be satisfactorily fitted in some cells by a single exponential. Figure 1E illustrates the current recordings and the best least-square fitting of the experiment shown in Fig. 1D. The mean values of tau  were not significantly changed before and after application of TGF-alpha (9.0 ± 2.0 before and 9.2 ± 2.4 s-1 after TGF-alpha , n = 6, P >0.05), whereas the calculated values of steady-state current amplitude at t = +infinity were diminished by TGF-alpha from 6.7 ± 2.2 to 3.7 ± 1.4 pA/pF (n = 6, P < 0.02). The fraction of TGF-alpha -induced decrease at t = +infinity was not statistically different from that measured at the end of the 20-s hyperpolarizing jumps (0.42 ± 0.07 vs. 0.43 ± 0.07 for six fitted recordings, P > 0.05). These results indicate that TGF-alpha decreased the amplitude of IClC-2 without changing its activation kinetics.

As already reported (14), the amplitude of IClC-2 varied from one cell to another. This dispersion in the normalized value of basal current amplitude may be attributable to variation of the expression of ClC-2 channels according to the proliferation and/or differentiation state of the cell. The magnitude of TGF-alpha -induced current decrease was independent of the basal IClC-2 amplitude (Fig. 1F; Spearman correlation coefficient, rs = 0.163; P > 0.05), indicating that the effect of TGF-alpha on IClC-2 could occur independently of the state of the cells.

Treating the cells for 1 h with 1 µM PD-153035, a potent, specific inhibitor of EGFR tyrosine kinase activity, completely prevented the inhibition of IClC-2 by TGF-alpha (n = 7, not shown), indicating that autophosphorylation of the EGFR was needed for the action of TGF-alpha .

Additional effect of a high concentration of TGF-alpha 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-alpha may also be present in the microenvironment of EGFR, because TGF-alpha is produced by colonic epithelial cells and cells of the lamina propria (57 and references therein). Because EGF or TGF-alpha 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-alpha (50 ng/ml) on IClC-2 amplitude. The effect was different from that obtained with 0.17 nM TGF-alpha in most of the cells tested (Fig. 2A). TGF-alpha 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-alpha -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-alpha application, with larger or smaller increases in current amplitude or even no apparent change. But the removal of TGF-alpha 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-alpha 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-alpha . 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-alpha . 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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Fig. 2.   Modulation of IClC-2 by high concentrations of TGF-alpha . A: representative plot of IClC-2 amplitude recorded during successive voltage jumps from the holding potential of 0 mV to -120 mV in a cell in which 8.3 nM TGF-alpha first activated IClC-2. Once current had stabilized (a), application of TGF-alpha induced a gradual increase that was maximal at 10 min (b). The current amplitude then returned to control level with TGF-alpha still present. There was a further current decrease to below control value upon TGF-alpha washout (c). Similar patterns of responses were obtained in 26 cells. B: examples of current traces recorded before (a), during (b), and after (c) TGF-alpha application shown in (A). Voltage protocol is shown on the top of current traces. C: representative plot of IClC-2 amplitude recorded as in A in a cell in which TGF-alpha produced an immediate, irreversible drop in current amplitude. This pattern of response occurred in 7 cells. D: corresponding current traces recorded before adding TGF-alpha (a) and after washout (b). At the end of the hyperpolarizing jumps, fast voltage ramps from -120 to +60 mV were applied. Voltage protocol is shown on the top of current traces. E: instantaneous I-V relationship established by a fast voltage ramps (300 ms) applied immediately after 20 s activation of the channels at -120 mV corresponding to the traces shown in (a) and (b) in B. F: %inhibition of IClC-2 induced by 8.3 nM TGF-alpha as a function of basal IClC-2 amplitude. Open symbols correspond to the cells that responded to TGF-alpha by an immediate decrease [calculated Spearman correlation coefficient rs = 0.43, P > 0.05, not significant]; , cells that showed a biphasic response (rs = 0.1, P > 0.05, NS). G: representative plot of IClC-2 amplitude recorded as in A in a cell preincubated with 1 µM of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor PD-153035 for 1 h. PD-153035 prevented the stimulatory as well as the inhibitory effects of 8.3 nM TGF-alpha . The same result was obtained in 10 cells.

Incubation of the cells with 1 µM of the EGFR inhibitor PD-153035 for 1 h prevented both the activation and the inhibition of the IClC-2 response to TGF-alpha (n = 10, Fig. 2G), indicating that autophosphorylation of the EGFR was needed for both actions of TGF-alpha and that the superimposed activation of TGF-alpha could not be attributed to a nonspecific effect resulting from the high concentration used here.

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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Fig. 3.   Inhibitory effect of the phosphotyrosine phosphatase inhibitor pervanadate (pV). A: representative plot of IClC-2 amplitude recorded as in Fig. 2A. pV at 0.1 mM irreversibly inhibited IClC-2 by 60.5 ± 6.4%, n = 3. B: corresponding current traces recorded before (a) and after (b) pV application.

Prevention of modulation of IClC-2 by TGF-alpha 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-alpha on IClC-2 by determining whether IClC-2 was modulated by TGF-alpha 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-alpha (8.3 nM) to assess the possible involvement of PI 3-kinase in the stimulatory and inhibitory effects of TGF-alpha . Because 8.3 nM TGF-alpha 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-alpha 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-alpha 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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Fig. 4.   Effects of the phosphoinositide (PI) 3-kinase inhibitor LY-294002. A: IClC-2 amplitude recorded during successive voltage jumps from 0 to -120 mV in the presence and absence of 8.3 nM of TGF-alpha in a T84 cell preincubated for 1 h with 20 µM LY-294002. IClC-2 amplitude was unchanged during application of TGF-alpha and after agonist removal. The same result was obtained in 4 cells. B: percentage inhibition of IClC-2 induced by TGF-alpha in the cells pretreated with PI 3-kinase inhibitors (100 nM wortmannin, n = 10 or 20 µM LY-294002, n = 4). C: IClC-2 amplitude recorded as in A. Acute application of 20 µM LY-294002 slowly decreased basal IClC-2 amplitude by 47.0 ± 4.2% (n = 3). D: IClC-2 amplitude recorded under the same conditions as in A, except that 500 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), was applied instead of TGF-alpha . PMA decreased the IClC-2 amplitude by 65%.

We also observed that cells pretreated with wortmannin or LY-294002 had currents smaller (4.0 ± 2.2 pA/pF, n = 16) than did untreated cells (5.7 ± 3.3 pA/pF, n = 46, P < 0.05). Acute application of either inhibitor slowly reduced the basal current amplitude, as illustrated in Fig. 4C for LY-294002. Wortmannin reduced IClC-2 amplitude by 30.2 ± 9.3% (n = 5) 25 min after application, and LY-294002 by 47.0 ± 4.2% (n = 3). The lack of TGF-alpha effect on cells treated with PI 3-kinase inhibitors may have resulted from the IClC-2 amplitude being too small, rather than from actual inhibition of PI 3-kinase activity. We checked this by testing the action of the PKC activator PMA a potent inhibitor of IClC-2 (14). The IClC-2 in three LY-294002-treated cells with a basal IClC-2 amplitude as small as 50 pA was still reduced by PMA (Fig. 4D).

The actions of TGF-alpha 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 gamma -isoform of phospholipase C (PLC), leading to PKC recruitment to the membrane (56), we have now examined the responses to TGF-alpha of cells treated for 2 h with GF109203X, a selective PKC inhibitor, to determine whether TGF-alpha -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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Fig. 5.   Blockade of TGF-alpha and PMA effects by the PKC inhibitor GF-109203X. A: PMA at 500 nM strongly and irreversibly reduced IClC-2 by 89 ± 8% (n = 3). Inset: current traces before (a) and after maximal current inhibition (b). Experimental protocol as in Fig. 2D. B: pretreatment of the cells for 2 h with 1 µM GF-109203X almost completely prevented inhibition by 500 nM PMA (n = 3). Inset: current traces before (a) and after (b) application of PMA. Experimental protocol as in Fig. 2D. C: the same pretreatment abolished inhibition by 0.17 nM TGF-alpha and mostly prevented the dual effects of 8.3 nM TGF-alpha (statistically different from TGF-alpha -induced IClC-2 inhibition in untreated cells, P < 0.001, n = 6) Inset: current traces before (a) and after (b) application of TGF-alpha . Experimental protocol as in Fig. 2B.

Incubation of the cells with 1 µM GF-109203X completely prevented inhibition by 0.17 nM TGF-alpha (n = 6) and produced a statistically significant attenuation of the responses to 8.3 nM TGF-alpha (P < 0.001, Fig. 5C). No response was detected in five cells during the application of 8.3 nM TGF-alpha or after its washout. Small inhibitory effects were seen in six other cells during the application of 8.3 nM TGF-alpha (by 18.3 ± 5.7%). These results thus indicate that TGF-alpha -induced decreases and increases are mediated by the activation of PKC.

Changes in cytosolic pH caused by TGF-alpha . EGF/TGF-alpha 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-alpha 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-alpha induced a change in pHi in these cells.

TGF-alpha (8.3 nM) reversibly increased the cytosol pH by 0.15 ± 0.10 pH units (n = 41) either immediately (n = 31) or after a transient drop (n = 10, Fig. 6A). However, the pHi did not increase in all the cells tested, because there was net acidification by 0.12 ± 0.05 pH unit in 14 cells and no change in 13 cells (not shown).


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Fig. 6.   Intracellular pH (pHi) variations in response to TGF-alpha and PMA. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded T84 cells were continuously perfused with HEPES-buffered saline solution. TGF-alpha was added to the perfusion solution as indicated. A: TGF-alpha first induced slight acidification and then alkalinization above control level. The alkalinization was reversed upon washout, and the new resting pHi stabilized below the initial pHi. This tracing is representative of 10 cells. B: replacement of external Na+ by tetraethylammonium (TEA+) reversibly reduced the steady-state pHi (n = 30). The same challenge acidified the cell to the same extent after TGF-alpha application. Reintroduction of Na+ caused the pHi to increase more rapidly toward a more alkaline value. C: changes in pHi in response to the sequential application of 2 concentrations of TGF-alpha . TGF-alpha at 8.3 nM caused cytosol alkalinization, but 0.17 nM did not. The same result was obtained in 17 cells. D: 100 nM PMA did not change pHi, whereas the subsequent addition of 500 nM PMA slowly increased pHi by 0.1 pH unit. The same result was obtained in 10 cells.

In the experiment shown in Fig. 6B, removal of external Na+ caused the pHi to decrease (by 0.15 ± 0.06 pH unit, n = 30), probably due to unmasking of background acid production that is balanced by NHE at steady-state pHi and/or to reversal of the exchanger. The pHi started to rise toward a new steady-state value after the application of TGF-alpha , and the removal of Na+ from the bath stopped this rise and acidified the cell as before TGF-alpha was added. The acidification was reversed by returning to the Na+-containing solution. The pHi increased rapidly during this phase and stabilized at a value above that before TGF-alpha application, indicating that TGF-alpha caused the pHi to increase by activating NHE. Such a result is best explained as a shift in the pHi dependence of NHE activation to more alkaline levels.

The changes in pHi depended on the concentrations of TGF-alpha (Fig. 6C). TGF-alpha at 0.17 nM did not change pHi, whereas a subsequent application of 8.3 nM TGF-alpha to the same cell raised the cytosol pH by 0.15 ± 0.05 pH unit (n = 17) or decreased pHi in a small number of cells by 0.13 ± 0.06 pH unit (n = 3, not shown).

EGF/TGF-alpha -induced cell alkalinization has been reported to require the activation of PKC in other cell types, because phorbol esters mimic the increase in pHi, whereas PKC inhibitors prevent the growth factor effect (19). One hundred nanomolar PMA had no effect on pHi (n = 34) in T84 cells, whereas the subsequent addition of 500 nM PMA to 10 cells increased the intracellular pH by 0.12 ± 0.06 pH units (Fig. 6D), a value not statistically different from the TGF-alpha -induced alkalinization (P > 0.1). The possibility that PKC-dependent processes are involved in modulation of pHi by TGF-alpha was further investigated by testing the effect of TGF-alpha on cells preincubated with 1 µM GF-109203X. Most of the 35 cells tested (26 cells) showed no change in pH, whereas the pH dropped in 9 cells, indicating that the activation of PKC mediated TGF-alpha -induced cell alkalinization but not acidification.

TGF-alpha -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-alpha was the result of a TGF-alpha -induced reversible cell alkalinization. If TGF-alpha -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-alpha was remarkably dependent on external Na+. Although TGF-alpha 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-alpha 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-alpha application was only 12% and the omission of Na+ from the perfusion solution during TGF-alpha application resulted in cell acidification of about 0.15 pH unit (Fig. 6B) compared with that before TGF-alpha 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-alpha -induced IClC-2 decrease in the presence of Na+ is that NHE was activated by TGF-alpha and that the resulting cell alkalinization contributed to the increase in IClC-2 and hampered the underlying decrease in current.


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Fig. 7.   Effects of 8.3 nM TGF-alpha in the presence and absence of external Na+. TGF-alpha did not greatly affect IClC-2 amplitude in the presence of external Na+, as in Fig. 2A. External Na+ was replaced by TEA+ at the time indicated in the continuous presence of TGF-alpha (open box). Omission of Na+ resulted in a rapid, pronounced inhibition of IClC-2. The same result was obtained in 6 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We find that TGF-alpha 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-alpha 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-alpha caused cytosolic alkalinization, suggesting that the TGF-alpha -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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Fig. 8.   Summary of the effects induced by TGF-alpha on IClC-2. The binding of TGF-alpha to EGFR stimulates a series of rapid responses, including phosphorylation (P) of tyrosine residues within the EGFR itself and within many other cellular proteins including PI 3-kinase and phospholipase C (PLC)-gamma . The major product of PI 3-kinase, phosphatidylinositol (3,4,5) triphosphate, leads to the enhancement of PLC-gamma membrane recruitment. The activity of the latter activates PKC, which in turn triggers 2 effects; it inhibits IClC-2, and it activates Na+/H+ exchanger (NHE), which alkalinizes the cell. The increase in pHi activates IClC-2 and competes with the PKC-induced IClC-2 inhibition.

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-alpha 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-gamma , whose activity leads to PKC activation. Such interactions seem to be required for the activation of PLC-gamma 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-alpha supports this scheme and points to the fact that full activation of PKC by TGF-alpha 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-alpha 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-alpha 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-alpha 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-alpha (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-alpha 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-alpha by an immediate decrease in IClC-2 (see Fig. 2C) and may have enhanced TGF-alpha -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-alpha or after direct activation of PKC by PMA, because application of 100 nM PMA or 0.17 nM TGF-alpha 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-alpha , 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-alpha may be overridden if the concentration of TGF-alpha 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-alpha 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-alpha . 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-alpha 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-alpha 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-alpha to regulate chloride secretion or absorption in the intestinal tract. In response to TGF-alpha , ClC-2 channels might be finely regulated, for example, preferentially closed in the presence of low concentrations of TGF-alpha , whereas higher concentrations along with alkalinization processes may limit the closure of the channels.

The modulation of IClC-2 by TGF-alpha 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).


    ACKNOWLEDGEMENTS

The English text was edited by Owen Parkes.


    FOOTNOTES

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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Am J Physiol Cell Physiol 280(6):C1588-C1598
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