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Unité Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To study the potential
influence of cystic fibrosis conductance regulator (CFTR) on
intracellular pH regulation during apoptosis induction, we used
PS120 Chinese hamster lung fibroblasts devoid of the
Na+/H+ exchanger (NHE1 isoform) transfected
with constructs, allowing the expression of CFTR and/or NHE1. Kinetics
of lovastatin-induced apoptosis were measured by orcein
staining, double staining with Hoechst-33258, propidium iodide, DNA
fragmentation, and annexin V labeling. In PS120 control cells, the
percentage of apoptotic cells after 40 h of lovastatin
treatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells,
this percentage was 40 ± 4%. In PS120 NHE1 cells, the
transfection with CFTR did not modify the percentage of apoptotic
cells after 40 h (control: 19 ± 3%, n = 8;
CFTR: 17 ± 1%, n = 8), indicating that blocking
intracellular acidification by overexpressing the
Na+/H+ exchanger inhibited the enhancement of
apoptosis induced by CFTR. In all cell lines, the initial pH
values were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification.
However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of this
increased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitable
Cl
/HCO
/HCO/HCO
Cl/HCO
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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APOPTOSIS IS A GENETICALLY DETERMINED physiological process that leads to defensive cell death, allowing the elimination of damaged or old cells from the organism. It is now well established that alterations in apoptosis contribute to the pathogenesis of several human diseases (45). Of these diseases, cystic fibrosis (CF), which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, may involve perturbation of epithelial cell apoptosis. A role of CFTR in apoptosis was first suggested based on the observation that CF patients exhibit high-molecular-weight DNA in the viscous mucus secretions of airway epithelia. The presence of this high-molecular-weight DNA suggests that it has been released in the extracellular spaces by necrotic cells that are unable to achieve DNA fragmentation and condensation. In normal secretory epithelia, programmed cell death may induce cytoplasmic acidification, which activates an acidic endonuclease allowing the cleavage, condensation, and packaging of DNA into apoptotic bodies that would be phagocytised. In contrast, in CF epithelia the impairment of this process leads to the release of large DNA fragments by senescent cells that are not phagocytised, and, therefore, enhances the viscosity of the mucus (14). This hypothesis is supported by the beneficial effect of inhaled DNase I treatment that improved the respiratory state of CF patients (37). The release of high-molecular-weight DNA fragments could be the consequence of apoptosis alteration in CF. Several authors have postulated that in CF, defective apoptosis arises from the inability of the cells to achieve the intracellular acidification necessary to activate acid endonucleases (4, 14, 26). On the other hand, different reports have described a decrease in intracellular pH (pHi) when the cells were treated with apoptosis inducers (7, 15, 24, 32). A possible hypothesis that could reconcile these findings is that an optimal apoptotic process can be linked to the magnitude of the intracellular acidification achieved during apoptosis.
It has been demonstrated that CFTR functionally interacts with the
Cl/HCO/HCO/HCO
To examine the role of CFTR on programmed cell death, both cell lines were stably transfected with a human cDNA CFTR, and lovastatin-induced apoptosis was determined using different technical approaches. On the other hand, the NHE1 isoform of the Na+/H+ exchanger can be over expressed in PS120 cells, with or without CFTR transfection, making it possible to study how blocking cytosolic acidification affects apoptosis induction.
The results presented in this article show that controlling pH through
the Cl/HCO
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell Culture and Transfection
The original CCL-39 Chinese hamster fibroblasts and the derived PS120 cell lines were grown in Dulbecco's modified Eagle's medium containing 15 mM NaHCO3, supplemented with 50 mg/ml streptomycin, 50 U/ml penicillin, and 7.5% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2-95% air.PS120 cell line. This cell line is a mutant Chinese hamster fibroblast cell line that lacks Na+/H+ exchange activity (36).
PS120 NHE1 cell line.
This cell line was obtained by transfection of PS120 cells with a
plasmid encoding for the NHE1 isoform of the human
Na+/H+ antiporter. Transfected PS120
fibroblasts were submitted to 1-h-long 50 mM NH
CCL-39 CFTR, PS120 CFTR, and PS120 NHE1 CFTR cell lines. CCL-39, PS120, and PS120 NHE1 cell lines stably expressing CFTR were generated by Lipofectamine-mediated transfection with constructs containing the full-length cDNA encoding human wild-type CFTR. These constructs were obtained by transferring the 4.5-kb CFTR cDNA excised from the pTG5960 plasmid (Transgène) in the polycloning site of the eukaryote expression vector pCB6. pCB6 is a 6.2-kb vector that possesses the neomycin resistance gene and a cloning site that is under the control of the cytomegalovirus promoter. To facilitate the introduction of the insert in the MluI cloning site of the pCB6 vector, the unique restriction sites SacI and PstI flanking the coding sequence of CFTR were transformed in the MluI site. The resulting pCB6 CFTR plasmid was transfected into PS120 and PS120 NHE1 cells using Lipofectamine according to the protocol provided by the manufacturer (Life Technologies, Cergy Pontoise, France). CCL-39 CFTR, PS120 CFTR, and PS120 NHE1 CFTR transfectants were isolated by growth in media containing G418. CCL-39 mock, PS120 mock, and PS120 NHE1 mock are cell lines that were stably transfected with the empty expression vector.
Rapid Screening of the Transfectants
The G418 selection procedure enabled us to obtain nine CCL-39 clones, 11 PS120 CFTR clones, and 15 PS120 NHE1 CFTR clones. To rapidly select the clones that exhibited the best expression of CFTR, a screening procedure based on the fluorimetric measurement of membrane potential was used. Changes in membrane potential of different cells were monitored with the fluorescent dye bis(1,3-diethylthiobarbiturate)-trimethineoxonol (Bisoxonol; Molecular Probes). Fluorescence emission of this dye increases with the membrane depolarization. All the experiments were performed in a Perkin Elmer LS-5 spectrofluorimeter connected to a recorder. Buffer (1.5 ml) was added to a quartz cuvette maintained at 37°C. Bisoxonol was prepared from a stock solution (15 mM in ethanol) at a concentration of 0.15 mM in H2O and added to the cuvette to give a final concentration of 1.5 µM. For each measurement, 106 cells were added and continuously stirred with a magnetic stirrer. The fluorescence signal was recorded with excitation at 540 nm (5-nm slit width) and emission at 580 nm (10-nm slit width). Changes in fluorescence were expressed as a percentage of the control value. The clones that exhibited a significant depolarization after the addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) were considered CFTR positive and were used for further molecular and functional characterizations.Molecular Identification of CFTR in Transfected Cell Lines
Reverse transcription and PCR amplification were performed using standard protocols in a thermal cycler (Techne). Total RNA was prepared from PS120 mock, PS120 CFTR, PS120 NHE1 mock, and PS120 NHE1 CFTR cells (2 × 106 cells) by using a micro RNA isolation kit (Tri InstaPur, Eurogentec) according to the manufacturer's directions. Primers were chosen to amplify a sequence of 297 bp localized between exon 10 and exon 12 of human CFTR. Reverse transcription was accomplished with recombinant Moloney murine leukemia virus reverse transcriptase (RT-MMLV, Life Technologies). Briefly, 15 µg of RNA were dissolved in 6 µl of water and mixed with 0.25 µM oligo 5'-CCATGAGTTTTGAGCTAAAGTCTGGC-3' (oligo A). The mixture was heated for 2 min at 70°C, chilled on ice, and completed to a volume of 20 µl to obtain a final reaction mixture containing 50 mM Tris · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.5 mM dNTP, and 10 mM dithiothreitol. The solution was incubated for 90 min at 37°C after addition of 400 units of reverse transcriptase. Five microliters of the reaction was then added to a 100-µl volume of the PCR mixture containing 20 mM Tris · HCl, pH 8.4, 50 mM KCl, 1 mM MgCl2, 0.025 µM oligo 5'-GTTCTTGGAGAAGGTGGAATCACA-3' (oligo B), and 0.025 µM oligo A (see above). The mixture was incubated for 4 min at 94°C, and 5 units of Taq polymerase (Life Technologies) was added. The conditions for amplification were as follows: each cycle consisted of incubation at 94°C for 2 s, 60°C for 2 s, and 72°C for 10 s, for a total of 30 cycles. At the end of this series, the reaction was incubated at 72°C for 10 min. Controls were performed without RT-MMLV and also without RNA. All buffers were prepared in diethyl pyrocarbonate-treated water. After RT-PCR, 10-20 µl of each reaction mixture was subjected to electrophoresis on a 0.8% agarose gel to size fractionate the RT-PCR products.The PCR-amplified fragments were subsequently cloned in the pGEM vector using Promega pGEM-T easy cloning kit. Plasmid DNA containing the 297-bp insert was then sequenced using oligonucleotides A and B (see above) as sequencing primers.
Functional Identification of CFTR in Transfected Cell Lines
Intracellular Cl measurements.
cAMP-dependent Cl fluxes were assessed using the
halide-sensitive fluorescent probe
6-methoxy-N-ethylquinolinium chloride (MEQ) according to the
protocol developed by Biwersi and Verkman (6). Transfected
cells (24-h-old) grown in petri dishes were loaded for 10 min at 37°C
with 5 mM 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) added to the culture medium. Dishes were carefully rinsed with NaCl solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, 5 glucose, and 20 HEPES, pH
7.4, and were then placed on an inverted microscope stage. Quantitative
measurements of MEQ fluorescence were made with an optical system
composed of a Zeiss ICM-405 inverted microscope with a Zeiss 40 objective (achromat oil 40/0.85). Fluorescence excitation was provided
by a 75-W xenon lamp (Osram) and was regulated by a computer-controlled shutter (Uniblitz). The excitation beam was filtered through narrow band filters (350 nm; Oriel) mounted in a motorized wheel (Lambda 10-2; Sutter Instrument) equipped with a shutter to control the exposure times. The incident and the emitted fluorescence radiation beams were separated through a Zeiss chromatic beam splitter. Fluorescence emission was selected through a 490-nm narrow band filter
(Oriel). The transmitted light images were viewed by an intensified
camera (Extended-ISIS; Photonic Science, Sussex, UK). The eight-bit
Extended-ISIS camera was equipped with an integration module to
maximize the signal-to-noise ratio. The video signal from the camera
proceeded to an image processor integrated in a DT-2867 image card
(Data Translation) installed in a Pentium 100 PC. The processor
converts the video signal into 512 lines by 768 square pixels/line by
eight bits per pixel. The eight-bit information for each pixel
represents one of the 256 possible gray levels, ranging from 0 (for
black) to 255 (for white). Image acquisition and analysis were
performed by the 2.1 version of AIW software (Axon Instruments). The
final calculations were made using Excel software (Microsoft).
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, and F0
is MEQ fluorescence in the presence of 140 mM potassium thiocyanate (KSCN).
Cl efflux was induced by isosmotic replacement of
the NaCl solution by NaNO3 solution containing (in mM) 140 NaNO3, 5 KNO3, 3 calcium gluconate, 1 MgSO4, 5 glucose, and 20 HEPES, pH 7.4. To determine the
fluorescent background, at the end of each experiment, PS120
transfected cells were incubated in 140 mM KSCN, which rapidly quenched the MEQ fluorescence.
Whole cell experiments.
Whole cell currents were recorded from 24-h-old PS120 transfected cells
grown in petri dishes maintained at 35°C for the duration of the
experiments. The ruptured-patch whole cell configuration of the
patch-clamp technique was used. Patch pipettes (resistance 2-3
M
) were made from borosilicate capillary tubes (1.5-mm outer diameter, 1.1-mm inner diameter; Clay Adams) using a two-stage vertical
puller (PP 83; Narishige, Tokyo, Japan) and filled with a solution
containing (in mM) 140 N-methyl-D-glucamine
(NMDG) Cl, 5 EGTA, 5 ATP, and 10 HEPES, pH 7.4. The bath
solution contained in (mM) 140 NaCl, 1 CaCl2, 60 mannitol, and 10 HEPES, pH 7.4. Cells were observed by using an inverted microscope, the
stage of which was equipped with a water robot micromanipulator (WR 89, Narishige). The patch pipette was connected via an Ag-AgCl wire to the
headstage of an RK-400 patch amplifier (Biologic). After formation of a
gigaseal, the fast compensation system of the amplifier was used to
compensate for the headstage intrinsic input capacitance and the
pipette capacitance. The membrane was ruptured by additional suction to
achieve the conventional whole cell configuration. At this stage, the
cell capacitance was compensated for by using settings available on the
RK-400 amplifier. No series resistance compensation was applied, but
experiments in which the series resistance was higher than 20 M were
discarded. Solutions were perfused in the extracellular bath by using a
four-channel glass pipette, the tip of which was placed as close as
possible to the clamped cell.
50 mV, and 400-ms pulses from 100 to +120 mV
were applied with increments of 20 mV every 2 s.
In Situ Apoptosis Evaluation
Apoptosis induction. Lovastatin-induced apoptosis was studied in CCL-39 mock, CCL-39 CFTR, PS120 mock, PS120 CFTR, PS120 NHE1 mock, and PS120 NHE1 CFTR cell lines. Lovastatin was dissolved in DMSO and kept in a stock solution of 4 mg/ml. The quantity of DMSO added to the incubation solutions never exceeded 0.01%. For kinetics studies, cell lines grown in petri dishes were incubated for 12, 16, 20, 30, or 40 h with 10 µM lovastatin added in the culture medium containing 15 mM NaHCO3. Parallel control experiments were performed by incubating the cells with 0.01% DMSO only instead of lovastatin. In another series of experiments, the external pH of the culture medium was increased by adding 20 mM HEPES buffered at pH 8.0, and the cells were incubated in this medium for 20, 30, and 40 h with 10 µM lovastatin.
Morphological counting of apoptotic cells. CCL-39 and PS120 transfected cells were grown in 35-mm petri dishes as described above. After the appropriate time of incubation with the apoptosis inductor (lovastatin), living cells were carefully washed with fresh culture medium and incubated 10 min in the presence of 100 µM Hoechst-33258 and propidium iodide. Nuclei were visualized with a fluorescence microscope using excitation 348 nm/emission 480 nm wavelength for Hoechst-33258 and excitation 500 nm/emission 640 nm wavelength for propidium iodide. Micrographs (color slides) were taken at each wavelength. Afterward, the preparation was washed and stained with 10 µl of orcein solution (1 g orcein, 10 ml 70% ethanol, 600 µl, and 12 N HCl). Micrographs of orcein-stained cells were then taken. Thus in a given culture, the same zone was visualized after staining with Hoechst-33258 and propidium iodide and after staining with orcein. Apoptotic cells were counted by comparing the three stainings. A cell was considered apoptotic only if the nuclei were not stained by propidium iodide and presented chromatin condensation with visible apoptotic bodies. The counts of apoptotic nuclei were performed directly on the micrographs. Between 100 and 200 cells were scored by four different observers who were blinded to the culture conditions. The number of cells with DNA condensation was expressed as the percentage of total cells.
Annexin V labeling. Apoptosis was also detected using the Apoalert annexin V apoptosis kit (Clontech kit K2025). For this purpose, PS120 cells were grown in 35-mm petri dishes. After lovastatin treatment, plated cells were rinsed with the binding buffer and incubated for 15 min in the dark at room temperature with annexin V-FITC conjugate (final concentration: 0.8 µg/ml) and propidium iodide (final concentration: 0.5 µg/ml). The cells were observed under a fluorescence microscope using a dual filter set for FITC and rhodamine (Olympus BH T2). Cells that had bound annexin V showed green staining in the plasma membrane. Cells that had lost membrane integrity exhibited red staining throughout the cytoplasm and diffuse green staining on the cell surface. Red- and green-stained cells were counted on micrographs. The percentage of apoptotic cells was calculated by comparing the number of pure green-stained cells to the total number of cells counted on phase-contrast micrographs. The counts were performed in a blind manner as described above.
Quantitative Analysis of Fragmented DNA
DNA fragmentation was assayed as previously reported (11, 18). After lovastatin treatment, cells grown in petri dishes were lysed by the addition of 500 µl of cold lysis buffer (7.5 mM Tris · HCl, 1.5 mM EDTA, and 0.25% Triton X-100, pH 8.0) for 30 min at 4°C. The lysates were centrifuged at 13,000 g for 30 min at 4°C to separate intact chromatin (pellet) from DNA fragments (supernatants). Pellets were resuspended in 500 µl of TE buffer (10 µM Tris · HCl and 1 mM EDTA, pH 7.5), and the DNA contents of pellets and supernatants were measured by a modification of the fluorimetric micromethod of Switzer and Summer (44) or by the PicoGreen DNA quantitation reagent (Molecular Probes kit P-7581). The percentage of DNA fragmentation was calculated by the ratio of the fragmented DNA in the supernatant over the total DNA.Analysis of DNA Fragmentation in Agarose Gel
PS120 transfected cells were grown in 100-mm petri dishes and treated with lovastatin (40 µg/ml) for 40 h. At the end of the treatment, cells were lysed at 4°C for 30 min in buffer containing 10 mM Tris-EDTA and 0.2% Triton X-100, pH 8.0. After RNase treatment (100 µg/ml) for 30 min at 37°C, the lysate was incubated in proteinase K (100 µg/ml) for 30 min at 37°C. DNA was precipitated with 0.5 M NaCl in isopropanol overnight at20°C. The precipitate was centrifuged at 13,000 g for 10 min, washed with 70% ethanol
solution, and allowed to dry. DNA was dissolved in 50 µl of Tris-EDTA
buffer. DNA samples were loaded onto 1% agarose gels and run
at 100 V for 1 h.
pHi Measurements
The fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure cytosolic pH, as described in detail previously (5). Quantitative digital measurements were made as described earlier.PS120 transfected cells grown in 35-mm petri dishes were incubated with
4 µM BCECF-AM at 37°C for 15 min in a humidified atmosphere of 5%
CO2-95% air. Loaded cells were carefully rinsed and placed on the stage of an inverted microscope. The cells were excited successively at 490 and 450 nm, and each pair of images was digitized and stored on the hard disk of a computer. The basal pHi
was determined on 10 images recovered every 20 s in the control
solution. Cells were then perfused with the different test solutions,
and images were successively recorded every 20 s. At the end of
each experiment, the fluorescence signals relating to pHi
changes were calibrated using the K+/H+
exchange ionophore nigericin. For this purpose, the cells were perfused
with KCl solutions (in mM) that comprised 140 KCl, 1 CaCl2,
20 HEPES, and 10 µM nigericin, the pH of which was adjusted to 8.0, 7.5, 7.0, and 6.5, respectively, with Tris. The initial rate of change
in pHi (
pHi/min) was measured with Microsoft
Excel software, using linear regression analysis on the traces. To
ensure an adequate renewal of the medium, the solutions were perfused at a rate of 2 ml/min.
To determine the activity of the
Cl/HCO-free solution containing (in
mM) 125 sodium gluconate, 15 NaHCO3, 5 potassium gluconate,
3 calcium gluconate, 5 glucose, and 20 HEPES, pH 7.4. Under these
conditions, Cl leaves the cell in exchange for
extracellular HCO
Chemical Compounds
Cell culture media and nutrients were obtained from GIBCO BRL (Basel, Switzerland). Lovastatin and Hoechst-33258 were obtained from Calbiochem (France Biochem, Meudon, France). Orcein, forskolin, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), DIDS, and 8-Br-cAMP were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Apoalert annexin V was obtained from Clontech Ozyme (Montigny le Bretonneux, France). PicoGreen and BCECF were obtained from Molecular Probes (Leiden, Netherlands). Cariporide (HOE-642) was a gift of Adventis (France).| |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Biochemical and Functional Characterization of PS120 Cells Expressing CFTR
With the use of pCB6-CFTR constructs, several antibiotic-resistant clones of PS120 and PS120 NHE1 cells were obtained. To select the clones that expressed CFTR, we used the oxonol technique to measure the relative changes of membrane potential induced by cAMP. Thus we selected one PS120 and one PS120 NHE1 clone that exhibited a stronger depolarization after 8-Br-cAMP application. These clones were named PS120 CFTR and PS120 NHE1 CFTR and were analyzed further for CFTR biochemical and functional expression.PS120 CFTR and PS120 NHE1 CFTR total RNA was reverse transcribed and
amplified by PCR using A and B primers. These primers amplify a 297-bp
stretch of sequence situated between exon 10 and exon 12 of the human
CFTR gene. An analysis of the RT-PCR products by electrophoresis on
agarose gels stained with ethidium bromide revealed only one product of
~300 bp (Fig. 1) in RNA
extracts. An identical analysis without prior reverse
transcription of the RNA sample revealed no amplification of any
product. The PCR products obtained from transfected cells were
sequenced and found to share 100% identity with the appropriate region
on the human CFTR mRNA. This indicated that the CFTR gene was stably
transfected and transcribed in these clones.
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To confirm CFTR functional expression, we used a cell membrane
halide-permeability assay with the Cl indicator
fluorescent dye diH-MEQ. The Cl permeabilities of cell
membranes were estimated by measuring intracellular MEQ fluorescence
using video microscopy. The flow of Cl across the
membrane was assessed by the addition or removal of Cl
from the bathing solutions. Initial relative Cl efflux
and influx rates are given in Fig. 2. The
application of 8-Br-cAMP (1 mM) significantly increased the initial
Cl efflux and influx only in CFTR-transfected PS120 NHE1
and PS120 cells. In contrast, the nucleotide did not modify
Cl fluxes in PS120 mock-transfected cells. Hence,
expression of CFTR in PS120 cells induced cAMP-activated
Cl permeability.
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To ensure that this increase in Cl fluxes was due to the
activation of Cl conductance, whole cell clamp
experiments were also performed. Whole cell currents were recorded with
Ca2+-free pipette solutions containing 140 mM NMDG
Cl, whereas hyperosmotic extracellular solutions
contained 140 mM NaCl and 50 mM mannitol. In PS120 mock- and PS120
CFTR-transfected cells, the voltage step protocol elicited small
currents (Fig. 3A) that
changed linearly with the membrane voltage. By contrast, only PS120
CFTR cells exposed to 1 mM 8-Br-cAMP exhibited an increase in membrane
currents (Fig. 3B), with the maximum increase obtained 3-4 min after the onset of perfusion. Figure 3E shows
that the activated currents presented a linear I-V
relationship that reversed close to 0 mV. Under these conditions, the
current amplitude at +100 mV reached 463 ± 53 pA, and the mean
conductance was 5.1 ± 0.6 nS (n = 3 cell
cultures).
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The experiments yielding these data were performed in symmetrical
Cl concentrations in the presence of EGTA in the pipette
to avoid involvement of intracellular Ca2+ and in
hyperosmotic bath solutions to block swelling-activated currents. The
reversal potential was very close to that of Cl, and, in
the absence of permeable cations in the pipette, indicated that the
current was carried by Cl.
Overall, these results strongly suggest CFTR functional expression. To
confirm this, we investigated the ion selectivity of the cAMP-induced
Cl conductance. For this purpose, all except 2 mM of the
Cl in the bath solution was replaced with
I. Figure 3C shows typical recordings of the
currents obtained in the presence of I, and Fig.
3E shows I-V relations for this current carrier.
Replacing external Cl with I strongly
decreased both inward and outward currents (currents at +100 mV = 95 ± 2 pA, conductance = 1.5 ± 0.2 nS,
n = 3) and shifted the reversal potential toward more
positive potentials (Erev = +10.2 ± 2.0 mV). The relative permeability
P
was reversible (currents at +100
mV = 300 ± 6 pA, conductance = 3.7 ± 0.3 nS,
n = 3). To further characterize the Cl
current, we tested the effect of the anion channel blocker NPPB that
was added to the bathing solution. As illustrated in Fig. 3E, the addition of 0.1 mM NPPB inhibited the whole cell
Cl currents within 2 min. The effect of this blocker was
reversible upon washing (data not given). The I-V
relationship is given in Fig. 3. Overall, 0.1 mM NPPB inhibited
reversibly both inward and outward currents.
The experiments illustrated in Fig. 3 were carried out on PS120
CFTR-transfected cells. Identical results were obtained with the clone
PS120 NHE1 CFTR (data not shown). The control mock-transfected cells
did not present any Cl conductance induced by cAMP, and
no PCR amplification product could be detected.
Time Course of Lovastatin-Induced Apoptosis in Different Clones
Cell counting experiments.
Figure 4A shows the
Hoechst-33258 and propidium iodide stainings of PS120 CFTR
cells before the addition of lovastatin, an efficient
apoptosis inducer in fibroblasts. The nuclei excluded propidium
iodide and exhibited a normal morphology with Hoechst-33258 labeling
with a diffuse staining of the normal chromatin. This pattern was also
observed in the other cell lines, i.e., PS120 mock-, PS120 NHE1 mock-,
and PS 120 NHE1 CFTR-transfected cells. After the addition of 10 µM
lovastatin for 30 h, the Hoechst-33258 staining revealed that
although several nuclei still displayed a normal morphology, other
cells exhibited very intense staining of condensed and fragmented
chromatin (Fig. 4C). Both types of cells were not stained by
propidium iodide, indicating preservation of the plasma membrane (Fig.
4C). In addition, dead cells possessing propidium
iodide-labeled nuclei were also detected in this preparation (Fig.
4E). The condensation and the fragmentation of DNA clearly show that lovastatin induces programmed cell death in live PS120 fibroblasts. These characteristic properties could also be observed independently with orcein staining. As shown with Hoechst-33258 staining, control cells that were not submitted to lovastatin treatment
did not exhibit chromatin condensation (Fig. 4B). By contrast, a dense and thin crown of nuclear coloration, typical of
chromatin condensation, could be observed in the lovastatin-treated cells (Fig. 4D).
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DNA fragmentation measurements.
To further demonstrate lovastatin induced-apoptosis in PS120
transfected cells, fragmentation of DNA was studied. Figure
6 illustrates the DNA fragmentation
patterns on an ethidium bromide-stained agarose gel produced 40 h
after exposure of PS120 CFTR cells to 10 µM lovastatin. The
characteristic ladder of DNA fragmentation, indicative of
internucleosomal DNA cleavage, was observed only in cells incubated
with lovastatin.
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pHi During Lovastatin-Induced Apoptosis
To determine the key proteins implicated in the control of the pH decrease during apoptosis, we then monitored the pH changes in our various cell lines after addition of lovastatin. The results are illustrated in Fig. 8, A and B. In the absence of lovastatin, pHi did not differ between one cell line to another and did not significantly vary with time (0 min: pHi = 7.38 ± 0.10; 40 h: pHi = 7.37 ± 0.09; n = 7; Fig. 8). After 20 h of incubation with 10 µM lovastatin, a decrease of ~0.25 pH units was observed in all cell lines. After 40 h of incubation, the pHi of PS120 mock, PS120 NHE1 mock, and PS120 NHE1 CFTR was not significantly different from that measured at 20 h. By contrast, treatment of PS120 CFTR cells with lovastatin for 40 h caused the pHi to strongly drop below the pHi value determined at 20 h (20 h: pHi = 7.30 ± 0.04; 40 h: pHi = 6.85 ± 0.10; n = 5, P < 0.001). As illustrated in Fig. 8A, this significant decrease of pHi was completely impaired in PS120 CFTR cells incubated concomitantly with 10 µM lovastatin and 0.1 mM DIDS (20 h: pHi = 7.18 ± 0.07; 40 h: pHi = 7.24 ± 0.09; n = 10, not significant). Moreover, increasing the pH of the culture medium to a value of 8.0 also prevented the decrease of cytoplasmic pH during lovastatin treatment in both PS120 mock- and PS120 CFTR-transfected cells (Fig. 8A).
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In another experimental series, the pHi of PS120 NHE1 mock and PS120 NHE1 CFTR cells was also measured during lovastatin treatment but in the presence of cariporide (30 µM), a potent blocker of the NHE1 isoform of the Na+/H+ antiporter (39). Under these conditions, control PS120 NHE1 mock and PS120 NHE1 CFTR cells maintained a pHi of 7.19 ± 0.10 and 7.33 ± 0.02 (n = 3), respectively. These values were significantly lower than those determined in the absence of cariporide (pHi = 7.54 ± 0.07, n = 5). Incubation of the cells with lovastatin for 20 h induced a significant pHi decrease (pHi of PS120 NHE1 mock = 6.90 ± 0.04; pHi of PS120 NHE1 CFTR = 6.91 ± 0.05; n = 3). At 40 h, an additional drop of pHi was observed in both cell lines (pHi of PS120 NHE1 mock = 6.72 ± 0.06; pHi of PS120 NHE1 CFTR = 6.74 ± 0.02; n = 3; Fig. 8B).
Figure 9 shows the relationship between
pHi and the percentage of apoptotic cells 40 h
after lovastatin addition. As observed, there is a significant
correlation between intracellular acidification and apoptosis
(correlation data: y = 56 × 427,
r = 0.95, P < 0.001).
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Cl/HCO
/HCO was
replaced by gluconate, causing the pHi to increase over a 6-min period. This effect was reversed when the gluconate was rinsed
away with a NaCl solution.
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In the second part of the experiment, the effect of removal and
addition of Cl to the incubation medium was studied in
the presence of 8-Br-cAMP (1 mM). Under these conditions, the initial
rate of pHi recovery was 2.3-fold more rapid in PS120 CFTR-
than in PS120 mock-transfected cells (Fig. 10, A and
B). This increase of pHi recovery induced by
cAMP in PS120 CFTR cells was not observed when the cells were also
incubated with 0.1 mM glibenclamide, and it was not modified in the
presence of 0.1 mM NPPB (Fig. 10B). On the other hand, the application of 0.1 mM DIDS completely inhibited the intracellular alkalinization induced by Cl removal in both cell lines
(Fig. 10, A and B). To further examine the nature
of this Cl/HCO was identical to that observed in the presence of
Na+. Moreover, removal of Na+ did not
impair the cAMP-induced alkalinization in PS120 CFTR cells (pH/min:
control = 0.40 ± 0.03; without Na+ = 0.38 ± 0.03; n = 3).
Role of Different Drugs on Lovastatin-Induced Apoptosis in PS120 Cells
The above results strongly suggest a role of CFTR in controlling apoptosis in lovastatin-treated PS120 cells. To further investigate this role, the actions of several modulators of Cl permeability were studied. These products were tested
in combination with lovastatin, and the percentage of apoptotic
cells was determined after 40 h of treatment. The histograms of
Fig. 11 compare the percentage of
apoptotic cells in PS120 mock-transfected or PS120 CFTR-transfected
cells with or without lovastatin treatment. We first investigated the
effect of DIDS (0.1 mM) on lovastatin-induced apoptosis. As
illustrated in Figs. 5A and 11, this stilbene derivative was
a very efficient inhibitor of apoptosis in both PS120 mock- and
PS120 CFTR-transfected cell lines. Together, our results imply that
CFTR is involved in the control of apoptosis, possibly through its action on the Cl/HCO conductive function of CFTR, we then studied the
effect of the Cl channel blocker NPPB. Figure 11 clearly
shows that NPPB (0.1 mM) did not decrease the apoptosis levels
in both cell lines. We also studied the effect of a sulfonylurea
derivative, glibenclamide, which has been shown to block CFTR. In our
experiments, glibenclamide (0.1 mM) application strongly reduced the
lovastatin-induced apoptosis in PS120 CFTR-transfected cells
only (Fig. 11).
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In the last series of experiments, we examined the effect of a cAMP-elevating agent. Incubation of the cells with 8-Br-cAMP (1 mM) increased lovastatin-induced apoptosis in PS120 mock- but not in PS120 CFTR-transfected cells (Fig. 11).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The aim of this work was to investigate the putative role of CFTR
in the control of apoptosis via pHi regulatory
mechanisms. For this purpose, PS120 cells devoid of
Na+/H+ antiporter, CCL-39 cells expressing
physiological levels of NHE1, and PS120 NHE1 cells overexpressing the
NHE1 isoform of the Na+/H+ antiporter were
stably transfected with a cDNA encoding for the human CFTR. After
selection, the clones were characterized to ensure that they expressed
a normal CFTR-mediated Cl conductance.
To induce apoptosis, we chose lovastatin. The mechanism by
which lovastatin induces apoptosis is related to the inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A reductase. This enzyme is
the rate-limiting enzyme of cholesterol biosynthesis. It generates mevalonate, resulting in the synthesis of the noncholesterol
metabolites required for cell survival. Thus the depletion of these
metabolites induced by lovastatin leads to programmed cell death.
Lovastatin has been shown to be an efficient apoptosis inducer
in different cell types such as HL-60 leukemic cells (32)
and C6 glial cells (8). However, one potential problem
with this drug is that it might decrease CFTR function. In fact, Shen
et al. (42) have suggested that 50 µM lovastatin could
reduce the number of cAMP-dependent Cl channels in the
apical membrane of epithelial cells. To circumvent this potential
problem, all experiments were carried out with a fivefold lower
lovastatin concentration (10 µM), which was sufficient to induce
apoptosis with a high efficiency. In addition, we verified that
there was no significant difference between lovastatin-treated and
lovastatin-untreated cells when measuring cAMP-induced Cl
currents. Considering that cAMP Cl permeability reflects
CFTR function, it could be reasonably concluded that under our
experimental conditions, lovastatin did not interfere significantly
with CFTR activity.
As expected, lovastatin treatment in all cell lines induced apoptosis in a time-dependent manner. To ensure a proper detection of the apoptotic process, we based our analysis on different criteria including nuclear staining, annexin V labeling, and DNA fragmentation. To optically separate apoptotic and necrotic cells, we used double staining with Hoechst-33258 and propidium iodide. Apoptotic cells excluded propidium iodide and presented visible condensed nuclei that appeared brightly stained by Hoechst-33258. Further labeling of these cells with orcein confirmed chromatin margination with the presence of apoptotic bodies (2). Annexin V-FITC binding was also used to detect apoptosis. Positive labeling correlates with the appearance of nuclear fragmentation and reflects a later phase of apoptosis (13). The use of all these techniques led to the observation that the CCL-39 and PS120 cells expressing CFTR underwent more apoptosis after lovastatin treatment than the cells not expressing CFTR. The number of apoptotic cells began to diverge significantly only after 20 h, being approximately twofold higher in CFTR-expressing cells after 40 h. The DNA fragmentation was also greater in CFTR-transfected cells, but this phenomenon occurred earlier and preceded the appearance of the first apoptosis images. This last observation is in accordance with published data that indicate that DNA fragmentation occurs before the formation of apoptotic bodies (10).
The participation of CFTR in the control of apoptosis has
already been suggested by several works but remains controversial. Contradictory results have been reported with some studies indicating that CFTR could increase apoptosis, whereas other works suggest a reverse effect. Interestingly, the study of phenotypic
abnormalities in CF epithelia has indicated the presence of
inappropriately high-molecular-weight DNA fragments in lung mucous
secretions, suggesting inefficient apoptosis (26).
Moreover, in C127 mammary epithelial cells expressing the F508 CFTR,
cycloheximide-induced apoptosis was clearly decreased
(15). On the contrary, a recent study performed in Hep G2
human cells indicates that CFTR inhibition induced apoptosis
(21).
The present study reveals more direct evidence that CFTR increases lovastatin-induced apoptosis in our cell systems and provides us with a potential mechanism for this effect. The examination of pHi revealed that the cell lines underwent intracellular acidification during lovastatin treatment. Moreover, there is a highly significant correlation between intracellular acidification and the percentage of apoptotic cells. The most significant observation is that this acidification was stronger in PS120 CFTR cells than in all other cell lines. According to previously published data and our results, it is very likely that the increase in apoptosis levels can be correlated with this drop of pHi (3, 14, 20, 24). In fact, this acidification is probably essential to allow the cells to enter into the apoptotic cycle, and it is possible that any means of preventing cytosolic acidification strongly decreases lovastatin-induced apoptosis. To challenge this hypothesis, we decided to block this acidification by using different experimental approaches. First, we increased the pH of the culture medium, and second, we overexpressed an extremely potent acid extruder, the Na+/H+ exchanger (NHE1 isoform). Under both experimental conditions, lovastatin slightly induced apoptosis in PS120 cells, but interestingly, CFTR was unable to increase the number of apoptotic cells. Thus clamping the pH by overexpressing the Na+/H+ exchanger or increasing external pH totally abolishes this acidification and impairs the ability of CFTR to enhance lovastatin-induced apoptosis. Moreover, in PS120 cells overexpressing NHE1, the ability of cariporide to enhance lovastatin-induced apoptosis and to concomitantly decrease pHi is consistent with this hypothesis. As a corollary, it has been demonstrated that an activation of the Na+/H+ antiporter activity suppressed lovastatin-induced apoptosis in HL-60 cells (32).
To have a physiological relevance, the mechanism that we propose needs to be functional in the presence of normal levels of NHE1. Therefore, we decided to study the effect of CFTR on apoptosis in a cell line expressing physiological levels of the Na+/H+ antiporter: the CCL-39 Chinese hamster fibroblasts. In these cells, lovastatin induced apoptosis, as it was also observed in the PS120 cell lines. When compared with the PS120 mock-transfected cells, the CCL-39 mock cells exhibited a significantly lower percentage of apoptotic cells, especially at 30 and 40 h. This result suggests that in the absence of CFTR, the basal level of NHE1 can limit the apoptosis induction. By contrast, the expression of CFTR in CCL-39 cells greatly enhanced the time course of apoptosis. This result confirms our finding that CFTR can positively regulate apoptosis in a physiologically relevant cell system and suggests a negative control of CFTR on NHE1 during apoptosis induction. We propose that the expression of CFTR is sufficient to modulate NHE1 when the protein is present in physiological quantities (CCL-39 CFTR cell lines) and that there are insufficient CFTR protein molecules to interact with NHE1 when NHE1 is overexpressed at the plasma membrane (PS120 NHE1 CFTR cell line). This would render CFTR inefficient at decreasing pHi in the cells overexpressing NHE1, resulting in a defective apoptosis process for the PS120 NHE1 CFTR cells.
Of the mechanisms that participate in the regulation of
pHi, the Cl/HCO removal in an HCO/HCO/HCO/HCO channels are
quite insensitive to stilbene derivatives (17); therefore,
the acidification is mediated by the
Cl/HCO/HCO channel blocker NPPB
does not modify the cAMP-sensitive acidification, showing that the
action of CFTR on the Cl/HCO translocation through the CFTR
channel. By contrast, the CFTR Cl channel blocker
glibenclamide completely prevented cAMP-dependent alkalinization. In
the absence of external Cl, this action could not be the
direct consequence of a decrease of Cl-conductive efflux
because such a diminution would increase the outward-directed
Cl gradient, allowing a better HCO channel but also as a channel regulator
(41) and that blocking the Cl channel
activity does not necessary impair the regulatory role of the protein.
It is possible that glibenclamide also blocks the regulatory function
of CFTR. Such a blockade could result in the loss of the ability of
CFTR to regulate the Cl/HCO channel activity was not necessarily involved.
The present data clearly indicate that the increase of
lovastatin-induced apoptosis observed in PS120 CFTR-transfected
cells was related to the activity of the
Cl/HCO/HCO/HCO
