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Involvement of chloride channels in TGF-1-induced apoptosis of human bronchial epithelial cells

Departments of ¹Biomedical Sciences, ²Internal Medicine, and ³Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska

Submitted 27 March 2007 ; accepted in final form 11 September 2007

	ABSTRACT

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Widespread damage of airway epithelium and defective epithelial repair are hallmarks of chronic asthma. Growth factors and cytokines spatially and temporally regulate epithelial shedding and repair. Within this context, a key function is exerted by transforming growth factor (TGF)-. Recent growing evidence suggests that chloride (Cl^–) channels are critical to cell apoptosis. We examined the effects of TGF-1 on Cl^– channel expression and activity and its relationship with apoptosis in human bronchial epithelial cells (HBECs). The small interfering RNA (siRNA) approach was used to investigate the potential role of CLC-3, a member of the volume-regulated Cl^– channel family, in apoptosis of HBECs. TGF-1 significantly induced HBEC apoptosis, which paralleled to a significant decrease in the endogenous expression of CLC-3 protein and mRNA transcripts. Outward rectifying and voltage-dependent CLC-3-like Cl^– currents in HBECs were diminished by TGF-1. siRNA for CLC-3 abolished Cl^– current and enhanced TGF-1-induced cell apoptosis. Overexpression of CLC-3 in HBECs inhibited TGF-1-induced cell apoptosis. Bcl-2 was also downregulated after TGF- stimulation. TGF-1-induced cell apoptosis was suppressed in Bcl-2-transfected HBECs. Our data demonstrate that CLC-3-like voltage-gated chloride channels play a critical role in TGF--induced apoptosis of human airway epithelial cells.

airway epithelial cells; Bcl-2; chronic asthma

It has been reported that CFTR and calcium-activated chloride channel are expressed in the airway epithelial cells (12, 26). Volume-regulated Cl^– channel (CLC) channels are expressed ubiquitously, and a role of these channels in proliferation and apoptosis has been suggested in several reports. It has been recognized that some of the Cl^– channel blockers affect the apoptosis of a variety of cell types such as endothelial cells, glioma cells, intestinal enterocytes, hepatocytes, and peripheral T lymphocytes (14, 27, 24, 32). Proliferating epithelial cells have high rates of mitosis and migration, both of which require change in cell volume and shape. Therefore, it is reasonable to speculate that volume-regulated Cl^– channels might play an important role in the proliferation of airway epithelial cells. TGF-1 participates in a variety of biological processes, including cell differentiation, proliferation, and apoptosis. We hypothesized and examined whether volume-regulated chloride channels contribute to TGF-1-induced apoptosis of bronchial epithelial cells. Our findings demonstrate that TGF-1 induces apoptosis of human bronchial epithelial cells (HBECs) via inhibition of CLC-3-like Cl^– currents in bronchial epithelial cells. Bcl-2 was downregulated after TGF-1 stimulation, and Bcl-2 overexpression in epithelial cells reversed TGF-1-induced cell apoptosis. These findings have major implications for linking CLC-3-like Cl^– channel to apoptosis of bronchial epithelial cells.

	MATERIALS AND METHODS

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Cell culture. We used primary human bronchial epithelial cells (Cambrex BioScience, Rutherford, NJ) and the BEAS-2B cell line, which was derived from human bronchial epithelium transformed by an adenovirus 12-SV40 hybrid virus (ATCC, Manassas, VA). Cells were seeded in vented T75 tissue culture flasks coated with type I collagen and grown in bronchial epithelial cell basal medium (Cambrex) as described by Krunkosky and colleagues (17) and Park et al. (25). Briefly, the expansion medium contained hrEGF (25 ng/ml), bovine pituitary extract (65 ng/ml), 50 nM all trans-retinoic acid, BSA (1.5 µg/ml), nystatin (20 IU/ml, GIBCO), hydrocortisone (0.5 µg/ml), insulin (5 µg/ml), transferrin (10 µg/ml), epinephrine (0.5 µg/ml), triiodothyronine (6.5 ng/ml), gentamicin (50 µg/ml), and 50 µg/ml amphotericin B (Cambrex). Upon 90% confluency, cells were dissociated with trypsin-EDTA (2nd passage), passed onto collagen-coated polyester Transwell inserts at 400,000 cells/insert, and cultured in an immersed condition until 90–100% confluent (4–5 days). The air-liquid interface was created by removing the apical medium and exposing cells only to bronchial epithelial cell growth medium (Cambrex):DMEM (Invitrogen) (1:1 ratio) with high glucose containing the same supplements as described above except EGF (BEGM:DMEM-H) on their basal surface and culturing the cells in a humidified environment with 97% air/3% CO₂ at 37°C. The medium beneath the cells was changed every day. Cells were used for the experiments when they were 90% confluent. Cells were cultured in the medium free of EGF and bovine pituitary extract for 24 or 48 h for synchronization and then exposed to cytokine or other mediators from the basolateral surface for the indicated times.

Electrophysiological recordings. The whole cell patch-clamp technique was employed to record Cl^– currents in HBECs. Whole cell recordings were performed using an Axopatch 200B patch-clamp amplifier, as described previously from our laboratory (18). The data were analyzed with pClamp 8.2 software and a Digidata 1322A interface (Axon Instruments, Foster City, CA). The whole cell recordings frequency response was set at 5 kHz with the 8-pole Axopatch amplifier Bessel filter. The series resistance and cell capacitance were compensated for through the use of the internal circuit of the amplifier. Ag-AgCl wires were immersed in the bath as well as the pipette solutions and then connected to the patch-clamp amplifier. The bath and the Ag-AgCl reference electrodes were used to minimize changes in liquid junction potential during some experiments. To obtain Cl^– voltage-current correlation, whole cell currents were recorded during voltage pulses, 200 ms in length, that were applied at 20-mV increments from –60 mV to +140 mV. Potential values were corrected for the liquid-junctional potential between the bath and the patch pipette solutions. Electrophysiological experiments were performed at room temperature (20–25°C). The patch pipettes were pulled on a Brown-Flaming P-97 puller (Sutter Instrument, Navato, CA). Patch pipettes were created from thick borosilicate glass capillary tubes (Harvard Apparatus, Kent, UK) and were fire polished. The electrical resistances of the pipettes, when filled with pipette solution, ranged from 3.0 to 4.5 M.

For the whole cell mode of patch clamp, the external bathing medium was the CsCl saline solution consisting of (in mM): 140 CsCl, 1 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4). The pipette solution contained the CsCl-saline solution consisting of (in mM): 140 CsCl, 1 EGTA, 0.1 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4). All reagents were purchased from Sigma Chemical (St. Louis, MO). Osmolality was set to 295 ± 5 mOsmol with sucrose. For the whole cell recording, solution osmolality was monitored using a freezing point osmometer (Microosmette; Precision Systems, Natick, MA). The bathing medium was exchanged by continuous perfusion.

Flow cytometry assay. Annexin V binding was performed on primary HBECs and BEAS-2B cells using a standard kit from BD Pharmingen to measure apoptosis (1, 2). Briefly, after rinsing cells twice with PBS, cells were resuspended in 100 µl of 1x binding buffer in a flow cytometry tube, to which 5 µl of Annexin V^FITC and 5 µl of propidium iodide were added and mixed well. After a 30-min incubation period at room temperature in the dark, 400 µl of 1x binding buffer was added, and flow cytometry was performed within 15 min.

RT-PCR analysis. Total RNA was extracted from primary HBECs or BEAS-2B cells using TRIzol (Invitrogen) and treatment with DNase (Ambion) according to the manufacturers' protocol. The total RNA was reverse transcribed with an OmniScript RT-PCR System (Qiagen). PCR was performed with a thermal cycler (PE2400, Applied Biosystems) under the following conditions: 1 cycle at 94°C for 5 min, followed by 30–36 cycles consisting of 1-min denaturation at 94°C, 1-min annealing at 60°C, and 2-min extension at 72°C. The last cycle was extended to 5 min at 72°C. Products were analyzed by 2% (wt/vol) agarose gel electrophoresis. The sequences of the 5' sense primers and the 3' antisense primers used in this study were as follows: CIC-2, sense, 5'-AGCAG CAACTAGATGAGCCTGTCA-3' and antisense, 5'-TCGATAGCTTTCCGGAGCTCTTT-3' (product size, 363 bp); CLC-3, sense, 5'-CATGTCAATGGGGAGG-3' and antisense, 5'-GCAAGAAAGGCAAAACT-3' (product size, 423 bp); and CLC-5, sense, 5'-ACCTAAGCTGCTCCAACCTCCTTT-3' and antisense, 5'-ACAAGAGATACGGC AAGGAAGGCA-3' (product size, 668 bp).

Real-time PCR analysis. Reverse transcription was performed using 1 µg of total RNA and oligo(dT) primers in a 20-µl reaction according to the manufacturer's protocol (PE Applied Biosystems, Foster City, CA). Primers for CLC-2, CLC-3, CLC-5, and GAPDH were designed using Primer Express software (PE Applied Biosystems). The following are sequences: for CLC-2 forward primer, 5'-AGCAGCAACTAGATGAGCCTGTCA-3' and reverse primer, 5'-TGACATAAGCATGGTCC ACTCCCA-3'; for CLC-3 forward primer, 5'-ATCGTCCAGCAGGCATTGGAGTAT-3' and reverse primer, 5'-GCCTGATGGAACCTTGATGCCAAA-3'; for CLC-5 forward primer, 5'-T GGTCCCAGCTTATCATCAGCACA-3' and reverse primer, 5'-AAGGAAGGCAAATAGGA GAGCCCA-3'; and for GAPDH forward primer, 5'-AGGTCGGAGTGAACGGATTTGG-3' and reverse primer, 5'-TCGCTCCTGGAAGATG GTGATG-3'. Real-time PCR was performed on the ABI Prism 7000 sequence detection system (PE Applied Biosystems) by using SYBR green (Applied Biosystems) as a dsDNA-specific binding dye. The cDNA were cycled 40 times after initial denaturation (95°C, 2 min) with the following parameters: denaturation, 95°C, 15 s, and annealing and extension, 60°C, 1 min. The threshold cycle (CT) was recorded for each sample to reflect the mRNA expression level. A validation experiment proved the linear dependency of the CT value for CLC-2, CLC-3, CLC-5, and GAPDH concentration and consistency of CT (CLC-2, CLC-3, and CLC-5 average CT minus GAPDH average CT) in a given sample at different RNA concentration. Therefore, CT was used to reflect the relative CLC-2, CLC-3, and CLC-5 expression levels. To determine the effects of different stimuli on CLC-2, CLC-3, and CLC-5 gene expression compared with unstimulated cells, CT was calculated (CT = CT stimulus – CT nonstimulated cells). CLC-2, CLC-3, and CLC-5 mRNA was indexed to the GAPDH using the following formula: 1/(2^CT) x 100%. The value of 2^CT was calculated to demonstrate the fold changes of CLC-2, CLC-3, and CLC-5 gene expression in stimulated cells compared with unstimulated cells.

Western immunoblots. Cells were lysed using eukaryotic membrane protein extraction reagent kit for 30 min supplemented with protease inhibitor mixture. Homogenates were centrifuged for 10 min at 12,000 g at 4°C. Protein quantification was performed on the supernatant using a DC protein assay kit from Bio-Rad (Hercules). Protein was boiled for 5 min in Laemmli-SDS sample buffer containing 600 mM -mercaptoethanol. Equal amounts of protein were loaded into each lane of acrylamide SDS-PAGE gel (Bio-Rad) and resolved at 120 V constant. Gels were transferred onto PVDF paper (Bio-Rad) at 400 mA constant for 1 h at room temperature, and membranes were blocked in blocking buffer. Blots were incubated in primary antibodies (CLC-2 1x 200 dilution, CLC-3 1x 500 dilution, CLC-5 1x 500 dilution) and rinsed three times followed by incubation with HRP-conjugated secondary antibodies (1x 2,000 dilution) for 90 min. After being washed, blots were developed with enhanced chemiluminescence on Hyperfilm (Amersham Biosciences, Arlington Heights, IL). CLC-2, CLC-3, CLC-5, and secondary HRP-conjugated antibodies were obtained from Santa Cruz Biotechnology. GAPDH antibody was purchased from Novus Biologicals. Densitometric analysis was performed directly from the blotted membrane using a Bio-Rad Molecular Imager system.

hCLC-3 and hBcl-2 cDNA transfection. All transfections of human airway epithelial cells (BEAS-2B) were done with a Nucleofector device and corresponding kits (Amaxa, Cologne, Germany) in six-well plates. Transfection protocols were performed following the manufacturer's instructions. Briefly, cells were plated in six-well Corning tissue culture plates. Twenty-four hours later, cells were transfected with 3 µg/ml hCLC-3/pc DNA 3.1 plasmid, which contains a full-length hCLC-3 cDNA, and pcDNA3.1 vector (hCLC-3/pcDNA3.1 was kindly provided by Dr. D. J. Nelson, Univ. of Chicago, Chicago, IL) or pORF-hBcl-2 (Invivogen, San Diego, CA). Analysis of apoptosis was done 48 h after transfection. The expression of CLC-3 and Bcl-2 protein was detected by Western blot analysis. The transfection efficiency was 60%-70%. BEAS-2B cells were also overexpressed with hCLC-3/pc DNA 3.1 plasmid and studied for apoptosis.

RNA interference and cell transfection. We have used the small interfering RNA (siRNA) approach, an effective tool to downregulate the expression of target genes in cultured mammalian cells. Two different high-performance purity grade siRNA to knock down CLC-3 were synthesized by Ambion One of the siRNA sequences. They were ^5'GGGCCCUUUUGUGCAUAUCtt^3' and its corresponding complementary strand ^5'GAUAUGCACAAAAGGGCCCtc^3'. Another was ^5'GCCAUUACUGCUGUGAUAGtt^3' and its corresponding complementary strand ^5'CUAUCAGCAGUAAUGG Ctg^3'. A nonsilencing oligonucleotide sequence (nonsilencing siRNA) that does not recognize any known homology to mammalian genes was also generated as a negative control. Lipofectamine 2000 reagent (Invitrogen) was used for transient transfection of BEAS-2B cells. Briefly, 4 x 10⁵ cells were seeded into each well of a six-well plate and cultured to 40–50% confluency. siRNA were diluted in RNase-free water to a final concentration of 20 µM (20 pmol/µl). For each well, siRNA stock was mixed with 5 µl Lipofectamine 2000 in F-12/DMEM medium (serum- and antibiotic-free) to a final volume of 800 µl. After 30 min, the siRNA (200 nm)/Lipofectamine 2000 complex was added to the well. Forty-eight hours after transfection, gene silencing was monitored at the mRNA and protein levels by RT-PCR and Western blotting, respectively. Cell proliferation assay was also repeated in these transfected cells.

Statistical analysis. Values for all measurements are expressed as means ± SE. One-way ANOVA was used to determine the difference among various experimental groups. Statistical difference between two groups was performed by the Student's t-test. Values of P < 0.05 were considered statistically significant.

	RESULTS

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Effect of TGF-1 on apoptosis of HBECs. Primary HBECs were cultured in serum-free media in an air-liquid interface and then exposed to TGF-1 (1–100 ng/ml) or the culture medium (control) for 48 h. TGF-1 induced apoptosis of HBECs in a dose-dependent manner (Fig. 1A). There was no difference in the effect of TGF-1 on the apoptosis of HBECs whether the cells were cultured under submerged conditions or under air-liquid interface conditions. Also, the effect of TGF-1 on the induction of apoptosis in BEAS-2B cells was similar to that in HBECs (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Effect of transforming growth factor (TGF)- on the apoptosis of primary human bronchial epithelial cells (HBECs). Cells were stimulated with TGF-1 (1–100 ng/ml). Apoptosis was measured by annexin V/PI labeling, and cells positive to annexin V were calculated using flow cytometer. A: there was a significantly increased number of apoptotic epithelial cells treated with TGF-1. B: epithelial cells were incubated with Cl– channel blockers (DIDS, NPPB, IAA94) in serum-free media for 48 h followed by measurement of apoptosis by annexin V labeling. Data represent means ± SE of n = 4. #P < 0.05 vs. control.

Chloride channel expression in HBECs. We examined mRNA transcript expression of the volume-regulated chloride channels (CLC-2, CLC-3, and CLC-5) in HBECs. HBECs expressed mRNA of all three voltage-gated chloride channels, CLC-2, CLC-3, and CLC-5 (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2. RT-PCR analysis of human CLC mRNA expression in primary human airway epithelial cells. A representative electrophoresis shows the expression of CLC-2, CLC-3, and CLC-5 in HBECs. M, a DNA marker (100 bp DNA ladder).

Selective decrease in total protein and mRNA transcript of CLC-3 chloride channels by TGF-1 in HBECs.

View larger version (14K): [in this window] [in a new window]   Fig. 3. qRT-PCR and Western blot analyses of CLC expression in primary HBECs. A: qRT-PCR analysis of CLC-2, CLC-3, and CLC-5 mRNA expression after 24-h stimulation by TGF-1 reported in relative quantitative expression. B: total protein lysates from airway epithelial cells were prepared for immunoblotting. Membranes were probed with anti-CLC-2, anti-CLC-3, and anti-CLC-5 antibody and reprobed with anti-GAPDH antibody. Data represent means ± SE of n = 3. #P < 0.05 vs. control.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Whole cell Cl– currents were recorded in control HBECs (A) and after treatment of the cells with 10 ng/ml TGF-1 (B). The bathing medium contained 140 mM CsCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH 7.4), and the pipette solution contained 140 mM CsCl, 1 mM EGTA, 0.1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH 7.4). Osmolality was set to 300 mOsmol with sucrose. Holding potential was –40 mV. Voltage pulses, 200 ms in duration, were applied at 20-mV increments from –60 to +140 mV. Outward Cl– current was 510 pA (A) and 44 pA (B) after treatment of the cells with TGF-1. C shows TGF-1-sensitive Cl– current (calculated by subtracting current in the presence of TGF-1 from the baseline current). This is a representative tracing of 5–7 individual cells.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Whole cell Cl– currents were recorded in control HBECs in the presence of 140 mM CsCl in the bath solution (A) and under low Cl– bath solution (B). Experimental conditions were similar to those described in Fig. 4 legend. This is a representative tracing of 5–7 individual cells.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Whole cell Cl– currents were recorded in control HBECs. A: experimental conditions were similar to those described in Fig. 4 legend. B: Current-voltage relationship (I/V) of the data is plotted. This is a representative tracing of 5 individual cells.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Whole cell Cl– currents were recorded in control HBECs (A) and after 2 min (B) and 5 min (C) of adding 10 µM DIDS to the cells. Experimental conditions were similar to those described in Fig. 8 legend. DIDS attenuated Cl– current in a time-dependent manner. This is a representative tracing of 5 individual cells.

Overexpression of CLC-3 Cl^– channel prevents TGF-1-induced apoptosis of HBECs.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Overexpression of CLC-3 in HBEC prevented TGF-1-induced apoptosis. A: Western blotting showed a dramatic increase in CLC-3 protein expression after CLC-3 cDNA transfection, whereas the level of CLC-3 protein in control cells was not changed. TGF-1 (100 ng/ml)-induced downregulation of CLC-3 was also reversed by CLC-3 overexpression. The involvement of CLC-3 in TGF-1-stimulated epithelial cell apoptosis was examined (B). TGF- (100 ng/ml)-induced apoptosis was significantly inhibited in CLC-3 overexpression cells. #P < 0.05 vs. control cells.

Effect of CLC-3 siRNA transfection on TGF-1-induced apoptosis of HBECs.

View larger version (14K): [in this window] [in a new window]   Fig. 9. CLC-3 small interfering RNA (siRNA) downregulated CLC-3 expression in BEAS-2B cells. A: CLC-3 mRNA expression and protein abundance were assessed by RT-PCR (top) and Western blotting (bottom), respectively, in epithelial cells transfected with CLC-3 siRNA or nonsilencing siRNA (NS siRNA). B: effect of TGF-1 (100 ng/ml) on the induction of apoptosis in BEAS-2B cells transfected with NS siRNA and silencing siRNAs (siRNA1 and siRNA2). #P < 0.05 vs. control.

Cl^– currents in CLC-3 siRNA-transfected BEAS-2B cells. Transfection of the BEAS-2B cells with CLC-3 siRNA significantly attenuated Cl^– currents. However, there was no effect of nonsilencing siRNA (Fig. 10).

View larger version (18K): [in this window] [in a new window]   Fig. 10. Whole cell Cl– currents were recorded in control BEAS-2B cells (A) and cells transfected with either CLC-3 siRNA (B) or NS siRNA (C). The bathing medium and pipette solution were same as in Fig. 4. The maximum current was 540 pA in control (A), 28.6 pA in CLC-3 siRNA (B), and 588 pA in NS siRNA (C) groups. This is a representative tracing of 3 individual cells.

Effect of TGF-1 on pro- and antiapoptotic protein in HBECs.

View larger version (10K): [in this window] [in a new window]   Fig. 11. Representative immunoblot showing the dose-dependent effect of TGF-1 on the expression of Bax and Bcl-2 in primary HBECs. An equal amount of protein (20 µg) was loaded in each lane, and proteins were resolved in SDS-PAGE followed by immunoblotting with monoclonal antibodies. GAPDH was run as a control.

View larger version (17K): [in this window] [in a new window]   Fig. 12. Overexpression of Bcl-2 upregulated CLC-3 expression in HBEC. A: increase in Bcl-2 protein was assayed by Western blot analysis. Bcl-2 overexpression also enhanced CLC-3 levels in airway epithelial cells. The inhibitory effect of TGF- on Bcl-2 was also reversed by overexpression of Bcl-2. CLC-2 and CLC-5 levels were not changed after Bcl-2 overexpression. TGF- (100 ng/ml)-induced apoptosis was dramatically decreased in Bcl-2 overexpressed cells. #P < 0.05 vs. control cells.

	DISCUSSION

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Apoptosis represents a model of genetically programmed cell death and describes a major mechanism by which tissue removes unwanted, aged, or damaged cells. Although cells of mammalian tissues consist of a broad diversity of phenotypes and genotypes, during the development of apoptosis, all cell types undergo similar morphological alterations including chromatin compaction and margination, nuclear condensation and fragmentation, and cell body shrinkage and blebbing (7, 13, 28). Characteristic apoptotic morphology reflects a drastic self-destruction of cytoskeleton and a catabolism of intracellular macromolecules. Complex interactions between extracellular microenvironment factors and internal gene expression occur before the initiation of apoptosis. Once activated, the apoptotic process can progress in the absence of extracellular insults. This suicidal feature of apoptosis has been well documented during embryonic development and morphogenesis as well as in adult tissue turnover.

Airway epithelial cells are exposed to a broad variety of biologically active environmental factors such as mechanical force, oxidative stress, radiation, heat, free radicals, virus and bacterial products, and inflammatory cytokines produced by activated immune cells. Induction of apoptosis has been reported in airway epithelial cells treated by some of the extracellular environmental and immunological factors. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in allergic airway inflammation and mediate apoptosis of airway epithelial cells during the allergen exposure (8, 33). Abnormal expression and dysfunction of these apoptosis-regulating genes may attenuate or accelerate vascular cell apoptosis and affect the integrity and stability of airway epithelial cells. In our study, we found decreased Bcl-2 levels and increased Bax levels after TGF-1 stimulation. Overexpression of Bcl-2 inhibited TGF-1-induced cell apoptosis. CLC-3 expression was also upregulated in Bcl-2 transfected cells. A number of reports recently have appeared claiming the additional involvement of Bcl-2 in the regulation of several types of plasma membrane ion channels. In particular, overexpression of Bcl-2 has been shown to exert cell type-specific inhibition (32) or enhancement of transmembrane capacitative Ca²⁺ entry (9, 32) to inhibit voltage-gated K⁺ channels in vascular smooth muscle cells (8). We also found that chloride current was inhibited by TGF-1. It seems that there is a link between Bcl-2 and CLC-3. Clarification of the regulation of Bcl-2 in CLC-3 in airway epithelial cell apoptosis may help design a new strategy for treatment of chronic asthma and its major complications, the subepithelial fibrosis, and airway remodeling.

TGF-1 functions in many cells and tissues that are involved in the cell cycle control, extracellular matrix, immune functions, and induction of apoptosis (19, 7). TGF-1 is involved with the intrinsic pathway because its receptor is not associated with a death domain. TGF-1 binds to the heteromeric complex of transmembrane serine/threonine kinase receptors, TGF- RI/RII, which induces phosphorylation of Smads. The Smad family consists of a common pathway Smad, receptor-regulated Smads, and inhibitory Smads. TGF-RI activates receptor-regulated Smads, Smad2 and Smad3. Activated Smad2 and Smad3 form a hetero-oligomeric complex with Smad4. The Smad complex translocates to the nucleus and regulates transcription. Smad6 and Smad7, inhibitory Smads, block the phosphorylation of Smad signaling (36). Thus, Smad7 can negatively regulate TGF--induced apoptosis of airway epithelial cells. The essential role of the Smad family in CLC-3 regulation is still unclear. Further study to identify the key function of Smad3 and Smad7 in chloride channel activation is warranted.

It is of interest that Cl^– channels involved in epithelial cell apoptosis may be stimulus-specific. A previous study has shown that both TNF- and IFN- can induce apoptosis in airway epithelial cells (35). There is no report that addresses direct connection between TNF- or IFN- and CLC-3 in any cell. However, antiapoptotic effect of CLC-3 has been studied in different cell types. EGF upregulated CLC-3 expression in prostate cancer cell line and prevented cells undergoing apoptosis (20). PDGF increased CLC-3 expression, and the overexpression of CLC-3 improved cell viability in cultured canine pulmonary artery smooth muscle cells (6). These results provide strong support to our hypothesis that CLC-3 play an important role in the apoptosis of airway epithelial cells. It is, however, unclear whether or not antiapoptotic effect of CLC-3 is limited to the apoptotic response of TGF-1. Studies to compare the effect of TGF-1 on CLC-3 to other apoptotic agents are clearly warranted to clarify this question.

In the present study, we recorded outward voltage-dependent Cl^– currents, and this current was DIDS sensitive. At the same time, this outward Cl^– current was also abolished by siRNA specially targeted to CLC-3. These data indicate that CLC-3 may be a dominant chloride channel in human airway epithelial cells. In response to TGF-1, CLC-3-like channel activity was significantly inhibited. Our results also showed that TGF-1 decreased CLC-3 protein expression. Hence, the activation of TGF- might be decreasing the number of CLC-3 channels. The lack of knowledge of the precise function of CLC-3 in airway epithelial cells makes it difficult to evaluate the physiological significance of the modulation of CLC-3 by TGF-1. However, Bcl-2 overexpression in prostate cancer epithelial cells significantly increased Cl^– current, and this was due to enhanced expression of CLC-3 (20). We also showed that TGF-1 downregulated Bcl-2 expression in airway epithelial cells. Overexpression of Bcl-2 increased CLC-3 expression. This suggests that the sensitivity to TGF--induced apoptosis in airway epithelial cells is mediated by increased proapoptotic protein and their effect on CLC-3 channels in human airway epithelial cells.

Two events closely associated with intracellular K⁺ and Cl^– efflux and water efflux/cell shrinkage have also been implicated in the regulation of apoptosis (34). Cell volume reduction may play a mechanistic role in apoptosis, since hypertonic medium induces or potentiates apoptosis in some studies, and regulatory volume decrease responses are enhanced in HeLa, U-937, PC-12 and NG108-15 cells undergoing apoptosis (22).

The mechanism by which chloride channel inhibition leads to an increased apoptotic activity remains unknown in this study. However, an increasing amount of evidence favors the concept that volume-regulated chloride channels are closely related to apoptosis. It has been reported that the blockade of volume-regulated chloride channel interferes with the mechanism of cell death through alteration of regulation of cardiomyocyte volume, and it has been suggested that the cellular function of Bcl-2, a key regulator of apoptosis, could be modulated through the activation of volume-regulated chloride channel and change in cell volume (29). Souktani and colleagues (30) found that both NPPB and IAA94 increased apoptosis in rabbit myocardium. In our study, NPPB, but not IAA94, could induce HBEC apoptosis. Several studies have demonstrated that selectivity of NPPB to chloride channel in different cell types such as portal vein and cerebral arteries is concentration dependent. Also, IAA94 is effective in substantially inhibiting regulatory volume decrease in cardiomyocytes. There are several NPPB- and IAA94-sensitive Cl^– channels including CLC-2, CLC-3, volume-regulated anion channel, Ca²⁺-activated Cl^– channel, and maxi-Cl^– channel. One of the possibilities is that NPPB is more sensitive to CLC-3 than IAA94. Overexpression of CLC-3 preventing TGF--promoted apoptosis supports the notion of the direct relationship between TGF- and CLC-3 in the induction of apoptosis. The results suggest that CLC-3 activation may be a key step in the chain of events associated with apoptosis.

In summary, TGF-1 induced apoptosis in airway epithelial cell and inhibited the expression of CLC-3 channels. Cl^– currents were downregulated by TGF- stimulation. Although the exact cellular and molecular mechanisms as to how the CLC-3 channels are involved in epithelial cell apoptosis remain unknown, our findings suggest that CLC-3 channels are important modulators of cell apoptosis in human airway epithelial cells and may play an important role in pathological processes including epithelial cell sloughing. Moreover, TGF--induced cell apoptosis could be important in widespread damage to bronchial epithelium, which might lead to subepithelial fibrosis and other hallmarks of chronic asthma. Clearly, further studies are necessary to elucidate the role of Cl^– channels in cell apoptosis in general and in the pathogenesis of chronic asthma. Clarification of the molecular mechanism of CLC-3 in asthma may lead to the development of therapeutic strategies to control this disease.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-070885 and R01-HL-073349 (both to D. K. Agrawal).

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. K. Agrawal, Creighton Univ. School of Medicine, CRISS II Rm. 510, 2500 California Plaza, Omaha, NE 68178 (e-mail: dkagr{at}creighton.edu)

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.

	REFERENCES

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1. Allen RT, Hunter WJ, Agrawal DK. Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-Myc and E1A. Scanning 20: 577–586, 2003.
2. Allen RT, Krueger KD, Dhume AS, Agrawal DK. Sustained Akt/PKB activation and transient attenuation of c-Jun N-terminal kinase in the inhibition of apoptosis by IGF-1 in vascular smooth muscle cells. Apoptosis 10: 525–535, 2005.[CrossRef][ISI][Medline]
3. Batra V, Musani AI, Hastie AT, Khurana S, Carpenter KA, Zangrilli JG, Peters SP. Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts. Clin Exp Allergy 34: 437–444, 2004.[CrossRef][ISI][Medline]
4. Cohn L, Elias JA, Chupp GL. Asthma: mechanisms of disease persistence and progression. Annu Rev Immunol 22: 789–815, 2004.[CrossRef][ISI][Medline]
5. Chakir J, Shannon J, Molet S, Fukakusa M, Elias J, Laviolette M, Boulet LP, Hamid Q. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-beta, IL-11, IL-17, and type I and type III collagen expression. J Allergy Clin Immunol 111: 1293–1298, 2003.[CrossRef][ISI][Medline]
6. Dai YP, Bongalon S, Hatton WJ, Hume JR, Yamboliev IA. CLC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery. Br J Pharmacol 145: 5–14, 2005.[CrossRef][ISI][Medline]
7. Druilhe A, Letuve S, Pretolani M. Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action. Biochim Biophys Acta 8: 481–495, 2003.
8. De Rose V, Cappello P, Sorbello V, Ceccarini B, Gani F, Bosticardo M, Fassio S, Novelli F. IFN-gamma inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis. J Leukoc Biol 8: 423–432, 2004.
9. Danielpour D. Functions and regulation of transforming growth factor-beta (TGF-beta) in the prostate. Effect of specific ion channel blockers on cultured Schwann cell proliferation. Eur J Cancer 41: 846–857, 2005.[CrossRef][ISI][Medline]
10. Ekhterae D, Platoshyn O, Krick S, Yu Y, McDaniel SS, Yuan JX. Bcl-2 decreases voltage-gated K⁺ channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C157–C165, 2001.[Abstract/Free Full Text]
11. Fürst J, Gschwentner M, Ritter M, Bottà G, Jakab M, Mayer M, Garavaglia L, Bazzini C, Rodighiero S, Meyer G, Eichmüller S, Wöll E, Paulmichl M. Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells. Pflügers Arch 444: 1–25, 2002.[CrossRef][ISI][Medline]
12. Hallows KR, Fitch AC, Richardson CA, Reynolds PR, Clancy JP, Dagher PC, Witters LA, Kolls JK, Pilewski JM. Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation. J Biol Chem 281: 4231–4241, 2006.[Abstract/Free Full Text]
13. Joseph J, Benedict S, Badrinath P, Wassef S, Joseph M, Abdulkhalik S, Nicholls MG. Elevation of plasma transforming growth factor beta1 levels in stable nonatopic asthma. Ann Allergy Asthma Immunol 91: 472–476, 2003.[ISI][Medline]
14. Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503–568, 2002.[Abstract/Free Full Text]
15. Kelly FJ, Dunster C, Mudway I. Air pollution and the elderly: oxidant/antioxidant issues worth consideration. Eur Respir J 40: 70s–75s, 2003.
16. Kay AB. Asthma and inflammation. J Allergy Clin Immunol 87: 893–910, 1991.[CrossRef][ISI][Medline]
17. Krunkosky TM, Fischer BM, Martin LD, Jones N, Akley NJ, Adler KB. Effects of TNF- on expression of ICAM-1 in human airway epithelial cells in vitro: signaling pathways controlling surface and gene expression. Am J Respir Cell Mol Biol 22: 685–692, 2000.[Abstract/Free Full Text]
18. Kim MJ, Cheng G, Agrawal DK. Chloride channels are expressed in human blood monocytes: a functional role in migration, adhesion, and volume change. Clinical Exp Immunol 138: 453–459, 2004.[CrossRef]
19. Lee KY, Bae SC. TGF-beta-dependent cell growth arrest and apoptosis. J Biochem Mol Biol 35: 47–53, 2002.[ISI][Medline]
20. Lemonnier L, Shuba Y, Crepin A, Roudbaraki M, Slomianny C, Mauroy B, Nilius B, Prevarskaya N, Skryma R. Bcl-2-dependent modulation of swelling-activated Cl^– current and CLC-3 expression in human prostate cancer epithelial cells. Cancer Res 64: 4841–4848, 2004.[Abstract/Free Full Text]
21. Miller M, Cho JY, McElwain K, McElwain S, Shim JY, Manni M, Baek JS, Broide DH. Corticosteroids prevent myofibroblast accumulation and airway remodeling in mice. Am J Physiol Lung Cell Mol Physiol 290: L162–L169, 2006.[Abstract/Free Full Text]
22. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487–9492, 2000.[Abstract/Free Full Text]
23. Nakao A, Sagara H, Setoguchi Y, Okada T, Okumura K, Ogawa H, Fukuda T. Expression of Smad7 in bronchial epithelial cells is inversely correlated to basement membrane thickness and airway hyperresponsiveness in patients with asthma. J Allergy Clin Immunol 110: 873–878, 2002.[CrossRef][ISI][Medline]
24. O'Loughlin EV, Pang GP, Noltorp R, Koina C, Batey R, Clancy R. Interleukin 2 modulates ion secretion and cell proliferation in cultured human small intestinal enterocytes. Gut 49: 636–643, 2001.[Abstract/Free Full Text]
25. Park JA, He F, Martin LD, Li Y, Chorley BN, Adler KB. Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C-delta-mediated mechanism. Am J Pathol 167: 651–661, 2005.[Abstract/Free Full Text]
26. Robichaud A, Tuck SA, Kargman S, Tam J, Wong E, Abramovitz M, Mortimer JR, Burston HE, Masson P, Hirota J, Slipetz D, Kennedy B, O'Neill G, Xanthoudakis S. Gob-5 is not essential for mucus overproduction in preclinical murine models of allergic asthma. Am J Respir Cell Mol Biol 33: 303–314, 2005.[Abstract/Free Full Text]
27. Rouzaire-Dubois B, Milandri JB, Bostel S, Dubois JM. Control of cell proliferation by cell volume alterations in rat C6 glioma cells. Pflügers Arch 440: 881–888, 2000.[CrossRef][ISI][Medline]
28. Robertson NM, Rosemiller M, Lindemeyer RG, Steplewski A, Zangrilli JG, Litwack G. TRAIL in the airways. Vitam Horm 67: 149–167, 2004.[Medline]
29. Shen RJ, Yang TP, Tang MJ. A novel function of BCL-2 overexpression in regulatory volume decrease. Enhancing swelling-activated Ca entry and Cl channel activity. J Biol Chem 277: 15592–15599, 2002.[Abstract/Free Full Text]
30. Souktani R, Ghaleh B, Tissier R, d'Anglemont de Tassigny A, Aouam K, Bedossa P, Charlemagne D, Samuel J, Henry P, Berdeaux A. Inhibitors of swelling-activated chloride channels increase infarct size and apoptosis in rabbit myocardium. Fundam Clin Pharmacol 17: 555–561, 2003.[CrossRef][ISI][Medline]
31. Wondergem R, Gong W, Monen SH, Dooley SN, Gonce JL, Conner TD, Houser M, Ecay TW, Ferslew KE. Blocking swelling-activated chloride current inhibits mouse liver cell proliferation. J Physiol 532: 661–672, 2001.[Abstract/Free Full Text]
32. Williams SS, French JN, Gilbert M. Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis. Cancer Res 60: 4358–4361, 2000.[Abstract/Free Full Text]
33. Ying S, Khan LN, Meng Q, Barnes NC, Kay AB. Cyclosporin A, apoptosis of BAL T-cells and expression of Bcl-2 in asthmatics. Eur Respir J 22: 207–212, 2003.[Abstract/Free Full Text]
34. Yu SP, Choi DW. Ions, cell volume and apoptosis. Proc Natl Acad Sci USA 97: 9360–9362, 2000.[Free Full Text]
35. Yohannes Tesfaigzi. Role of apoptosis in airway epithelia. Am J Respir Cell Mol Biol 34: 537–547, 2006.[Abstract/Free Full Text]
36. Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24: 5764–5774, 2006.[ISI]

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