Introduction

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The developing mammalian lung is a fluid secretory organ (1). Fetal lung fluid production and distention of the airways are required for normal lung morphogenesis. Retention of fetal lung fluid by experimental tracheal ligation in fetal sheep results in overdistension and early cellular differentiation, whereas chronic drainage of fetal lung fluid by exteriorized tracheal catheterization results in delayed development (4). This active fluid secretion in the fetal lung is dependent on chloride transport across the airway epithelium (5, 6).

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) (7). KGF is active predominantly on epithelial cells stimulating proliferation, migration, and morphogenesis (8). It is thought that KGF functions as a paracrine mediator because KGF is expressed by mesenchymal cells and KGF receptors are uniformly distributed on the basolateral membrane of epithelial cells in developing lung buds (8, 9, 13, 14). KGF is expressed as early as 14.5 d gestation in the embryonic mouse lung (13). In lung explants, KGF stimulates chloride and fluid secretion through cystic fibrosis (CF) transmembrane conductance regulator (CFTR)- independent chloride secretory pathways (15). Severe abnormalities of lung morphogenesis occur as a result of targeted expression of a dominant-negative KGF receptor in transgenic mice (16). Transgenic mice that overexpress KGF in respiratory epithelial cells develop pulmonary cystadenomas (17), consistent with the hypothesis that KGF stimulates fluid production in the developing lung.

Because KGF acts through non-CFTR channels, we chose to investigate the effects of KGF on an alternative chloride channel, CLC-2, which is expressed in airway epithelium. We have shown that CLC-2 is a fetal lung chloride channel that is concentrated along the apical surface of the prenatal lung and downregulated after birth (18). The single-channel properties of CLC-2 have recently been described in a CF cell line that overexpresses CLC-2 (19). It is a hyperpolarization-activated, pH-regulated chloride channel that can be inhibited by cadmium chloride. We speculate that the CFTR-independent chloride secretion induced by KGF in lung explants could be mediated through increased levels and activity of CLC-2. The hypothesis of this study is that KGF stimulates CLC-2 chloride channel expression in fetal lung epithelium.

In this study, the effect of KGF on CLC-2 expression is examined in primary fetal distal lung epithelial (FDLE) cells cultured from 18-d-gestation rat, which corresponds to the canalicular phase of lung development. The effects of short-term exposure to KGF in defined serum-free, hormone-supplemented medium were assessed at the messenger RNA (mRNA) and protein levels. We found that CLC-2 protein and mRNA are increased by KGF. This effect is not mediated through the CLC-2 promoter, but occurs through prolongation of the half-life of the CLC-2 mRNA transcript. Our results led us to speculate that KGF may positively influence pulmonary chloride and fluid secretion by a secondary pathway affecting CLC-2 mRNA degradation.

	Materials and Methods

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Primary FDLE Cell Culture

Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Eighteen-day-gestation, timed-pregnant dams were killed with carbon dioxide, and the fetuses were recovered by hysterectomy. Lungs from a single litter were pooled and placed in ice-cold Hanks' balanced salt solution (HBSS). Primary cells were isolated using a protocol modified from O'Brodovich and colleagues (20). Tissue was rinsed three times with cold HBSS, the trachea and bronchi were removed, and whole-lung segments were minced to 1-mm³ pieces. Tissue was digested with 0.125% trypsin (Gibco, Gaithersburg, MD) and 20 µg/ml deoxyribonuclease (DNase) (Worthington Biochemical Corp., Freehold, NJ) in HBSS at 37°C, filtered through a 70-µM nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ), and centrifuged to collect dissociated cells. Cells were resuspended and incubated in 0.1% collagenase and 20 µg/ ml DNase in Ham's F12 (Mediatech, Herndon, VA) for 20 min at 37°C, and then pelleted. Isolated cells were resuspended in Ham's F12 containing 10% fetal calf serum, penicillin, streptomycin, and fungizone, and grown on an uncoated flask for 30 min and again for 1 h to enrich for epithelial cells. Cell viability was assessed by trypan blue exclusion using an inverted Olympus microscope, and was greater than 90%. Primary epithelial cells were seeded at 0.2 to 0.3 × 10⁶ cells/cm² and grown at 37°C in defined fetal epithelial cell medium consisting of Ham's F12, 7.5 µg/ ml endothelial cell growth supplement, 5 µg/ml insulin, 7.5 ng/ml transferrin, 0.1 µM hydrocortisone, 15 µg/ml bovine pituitary extract (Collaborative Biomedical, Bedford, MA), and 20 ng/ml cholera toxin (List Biological Laboratories, Inc., Campbell, CA), as previously described (21). KGF (Sigma, St. Louis, MO) was added in the amount of 10 ng/ml unless otherwise indicated. Primary cells were fed the next morning and harvested at 48 h, when the monolayers reached confluence, for RNA or protein experiments.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Immunoblotting

Whole-cell lysates were prepared using hot 2% sodium dodecyl sulfate (SDS) (T > 65°C) on FDLE monolayers. Protein content was measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). For electrophoresis, 10 to 15 µg of lysate were separated on a 6% SDS-polyacrylamide system. Denatured protein was transferred to Protran nitrocellulose (Schleicher & Schuell, Keene, NH) in ice-cold Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS [pH 8.2]). The blot was blocked in 1% bovine serum albumin (BSA) and 5% dried milk in buffer containing 50 mM NaPO₄ (pH 7.4), 150 mM NaCl, and 0.05% Tween for 1 h at room temperature. A polyclonal chicken antiserum against the COOH-terminal region of CLC-2 (18) was incubated with the membrane overnight at 4°C at a 1:200 dilution in 1% BSA-5% milk blocking solution. Chicken antibody was amplified using sequential incubation in biotinylated antichicken immunoglobulin G (1:1,000) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and horseradish peroxidase-linked streptavidin (1:1,000) (Amersham Life Sciences, Arlington Heights, IL). The enhanced chemiluminescent reaction (Amersham) was used to detect antibody binding, and the blot was exposed to Hyperfilm-MP (Amersham). The specificity of this antibody has been reported previously (18). Controls consisted of monoclonal anti- -actin mouse ascites fluid (Sigma) at 1:1,000.

Northern Blot Analysis

Total RNA was isolated from FDLE cultures using the Trizol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. Quantity and quality of isolated RNA were determined by spectrophotometric measurements at optical density (OD) 260 and OD280, and by examination on an ethidium bromide-stained agarose gel. Total RNA, 10 to 15 µg, were electrophoresed on a 1% agarose gel containing 5% formaldehyde, 0.1 M 3-(N-Morpholino)propanesulfonic acid, 0.5 M sodium acetate, and 5 mM ethylenediaminetetraacetic acid (EDTA). RNA was transferred to Nytran Plus nylon membrane using the Turboblotter system. Filters were probed using rat CLC-2, -actin, and 18S ribosomal RNA riboprobes labeled with ³²P as previously described (18, 22) using the Maxiscript (Ambion, Austin, TX) transcription method. The membranes were hybridized using 1 to 3 × 10⁵ cpm/ml riboprobe in 0.5 M NaPhos, 7% SDS, and 1 mM EDTA overnight at 65°C; and washed with 40 mM NaPhos, 1% SDS, and 1 mM EDTA twice for 15 min, and 0.1% SDS/0.2× saline sodium citrate twice for 5 min at 65°C.

Densitometry and Statistical Analysis

Quantitation of protein and transcript levels was performed using an Ultralum image-grabbing system (Ultralum, Fullerton, CA) and two-dimensional Main software (Advanced American Biotechnology, Fullerton, CA). Peak absorbances of CLC-2 and -actin were determined for independent experiments. Data were expressed as the fold increase of CLC-2 band intensity in KGF versus control lanes, correcting for -actin in five independent litters. Paired samples were tested for significance (P < 0.05) by the Wilcoxon matched-pairs ranked-sum test. This test compares the rank order of paired samples and does not compare means or standard errors; thus error limits are not generated.

Time Course and Dose Response of KGF Stimulation of CLC-2 mRNA

Primary FDLE cells were grown in the presence or absence of 10 ng/ml KGF for 48 h. Time course for stimulation of CLC-2 was determined by adding 10 ng/ml KGF for 48, 24, 10, 5, 2.5, or 0 h before harvesting cells for total RNA. A dose-response curve for CLC-2 stimulation was determined using 0, 1, 10, 50, and 100 ng/ml KGF in culture for 48 h.

Promoter-Luciferase Plasmid Construction

The pGL-3 basic vector (Promega, Madison, WI) containing a luciferase gene and lacking a promoter was used to construct rat promoter clones of 1.6 kb, 990 base pairs (bp), 428 bp, 406 bp, 170 bp, and 108 bp upstream from the transcription start site of CLC-2. The CLC-2 promoter is GC rich; has no TATA box; and contains three CAAT boxes, four GC boxes (Sp1, Sp3 binding sites), and two stem-loop structures, which are highly conserved in human CLC-2 (23). There is an open reading frame corresponding to the human RNA polymerase II subunit hRPB17, which is 1,930 bp upstream from the CLC-2 coding sequence in the opposite direction (Chu and colleagues, manuscript submitted).

Luciferase Assays

Freshly isolated primary FDLE cells were incubated overnight in KGF +/ FDLE medium. The following day FDLE monolayers were cotransfected with CLC-2 promoter-luciferase gene constructs and with the -galactosidase expression plasmid (pCMV-Sport--gal; GIBCO BRL, Gaithersburg, MD) for calibration of transfection efficiency. A total of 2 µg of each plasmid and 20 µl of lipofectamine (GIBCO BRL) were mixed in 0.5 ml of FDLE medium and incubated at room temperature for 30 min. Each 35-mm dish was washed with 1 ml of serum-free medium before a mix of 0.5 ml of FDLE medium and 0.5 ml of DNA-lipofectamine was added and the cells were returned to the CO₂ incubator for 4 h. The transfection medium was removed, and cells were fed with FDLE medium with or without 10 ng/ml KGF and harvested 24 to 48 h later. Cells were washed with phosphate-buffered saline once and incubated with 250 µl Reporter Lysis Buffer (Promega) for 15 min at room temperature. The cells were then scraped and collected in microcentrifuge tubes on ice. After vortexing for 15 s, the cell suspensions were centrifuged at top speed (16,000 × g) on a microcentrifuge for 2 min at 4°C. The supernatants were collected and stored at 80°C until -galactosidase and luciferase activities were measured.

The -galactosidase Enzyme Assay System (Promega) was used for -galactosidase measurement according to the manufacturer's protocol. The Luciferase Assay System (Promega) was used for luciferase measurement. Readings were recorded on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). For each dish, duplicate measurements were taken, 20 s each, and an average was calculated. This average was then divided by the -galactosidase activity of the same dish, yielding the relative value of luciferase activity.

Actinomycin Inhibition of CLC-2 mRNA

To examine the effect of KGF on the half-life of CLC-2 mRNA, freshly isolated FDLE cells were grown overnight in the presence or absence of 10 ng/ml KGF. Cells were fed with fresh medium the following day and cultured for 24 h more with or without KGF. At 24, 18, and 12 h before cell harvest, 10 µg/ml actinomycin D were added to the culture medium.

	Results

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KGF Increases CLC-2 Protein by Western Blot Analysis

Cell density and morphology in KGF-treated and untreated cultures were indistinguishable at 48 h (data not shown). Control lysates prepared after 48 h in standard culture conditions expressed a 99-kD protein corresponding to CLC-2. This protein band visibly increased in intensity (Figure 1A) in KGF-treated cells (10 ng/ml for 48 h) compared with controls. Quantitative analysis was performed on cultures from five independent litters. To confirm comparable protein loads in each lane, the blot was cut below the 69-kD protein and simultaneously immunoblotted with an anti--actin monoclonal antibody. To compare protein expression of CLC-2, mean ratios of CLC-2 band intensity by densitometry were calculated in KGF versus control lanes, correcting for -actin. CLC-2 protein was 1.2-fold higher after KGF treatment, compared with untreated controls (Figure 1B). This was significant by the Wilcoxon matched-pairs ranked-sum test, P = 0.0051 (standard error bars are not used with this test).

View larger version (45K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 1.   (A) Western blot analysis of 18-d FDLE cell lysates using a polyclonal anti-C-terminal domain CLC-2 chicken serum. KGF increased the intensity of a 99-kD band compared with control. -actin was detected with a monoclonal antibody on the same blot to examine comparability of total protein loaded in each lane. (B) Densitometric analysis of immunoblots, corrected for -actin, revealed that KGF increased CLC-2 protein 1.2 times control. Significant by the Wilcoxon matched-pairs ranked-sum test, P = 0.0051 (n = 5 independent litters).

KGF Increases CLC-2 mRNA by Northern Blot Analysis

To determine whether CLC-2 regulation occurs at the transcriptional or translational level, or both, we constructed a dose-response curve of CLC-2 mRNA levels in FDLE cells grown with 0 to 100 ng/ml KGF for 48 h by Northern blot analysis. Increases in band intensity of CLC-2 mRNA were detectable at doses of at least 10 ng/ ml KGF (data not shown). Because this is the dose used to demonstrate increased cyst development in lung explants, we used this dose for further studies (15). A time course for CLC-2 mRNA upregulation during 48 h in culture demonstrated the expected 4-kb transcript (24) using a ³²P-labeled CLC-2 riboprobe (Figure 2A). A ³²P-labelled -actin riboprobe hybridized to the same Northern blot was used to ensure that comparable quality and quantity of total RNA was blotted. Densitometric measurements correcting for -actin in five independent litters revealed that exposure to 10 ng/ml KGF for 48 h before harvest increased CLC-2 mRNA 1.7-fold compared with untreated controls (Figure 2B, n = 3). This was significant by the Wilcoxon matched-pairs ranked-sum test, P = 0.0051. 

View larger version (65K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   Figure 2.   (A) Northern blot analysis demonstrating the time course of CLC-2 mRNA expression in the presence of 10 ng/ml KGF for 0, 2.5, 5, 10, 24, and 48 h before lysis of FDLE cells. All cells were cultured for a total of 48 h. -actin was used as a control for equivalent mRNA bound to nylon membrane. (B) Densitometric analysis of time course of CLC-2 mRNA stimulation by KGF corrected for -actin. KGF increased CLC-2 mRNA 1.7 times control at 48 h, significant by the Wilcoxon matched-pairs ranked-sum test, P = 0.0051 (n = 5 independent litters).

KGF Does Not Affect CLC-2 Promoter Activity

CLC-2 mRNA may be regulated by enhanced production or decreased degradation of transcript. Regulation of mRNA transcription by KGF was examined using CLC-2 promoter constructs in luciferase assays (Figure 3). These demonstrated that the highest level of promoter activity was conferred by the 237 bases upstream from the transcription initiation site. This region contains three Sp1/Sp3 binding sites. Relative luciferase activity of the 237 construct was nearly 8,000 times greater than constructs containing only the 108 or 170 bp upstream. The addition of KGF to the FDLE growth medium had no affect on promoter activity in any construct evaluated (Figure 3, P > 0.05, paired t test), suggesting that KGF does not influence mRNA expression at the level of gene transcription.

View larger version (25K): [in this window] [in a new window]   Figure 3.   Luciferase assay of CLC-2 promoter activity in the presence and absence of KGF. There is no enhancement of the CLC-2 promoter by KGF in any of the constructs tested (P > 0.05, paired t test, n = 4).

KGF Stabilizes CLC-2 mRNA

Because KGF does not activate the CLC-2 promoter directly, we next investigated the effect of KGF on CLC-2 half-life. mRNA transcription was inhibited by 10 µg/ml actinomycin D, and total RNA from cell lysates was electrophoresed, transferred to nylon membrane, and hybridized with CLC-2 and 18S ribosomal RNA riboprobes. CLC-2 bands decreased after 18 and 24 h of exposure to actinomycin D in primary cells grown in the absence of KGF, but did not decrease as rapidly in KGF-treated cells (Figure 4, n = 3). These findings are consistent with stabilization of CLC-2 transcripts, or a reduction in degradation. KGF did not stabilize CLC-2 protein; inhibition of protein translation with 150 µg/ml cycloheximide had no effect on CLC-2 protein half-life in the presence of KGF (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 4.   Northern blot analysis of 18-d FDLE cell mRNA exposed to actinomycin D for 0, 12, 18, or 24 h before harvest. KGF added to the culture medium increased CLC-2 mRNA at 18 and 24 h compared with untreated controls, suggesting that KGF stimulation of CLC-2 mRNA results from stabilization of transcripts.

	Discussion

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An in vitro primary fetal distal lung epithelial cell culture system was used to assess the role of KGF on the expression of the fetal chloride channel CLC-2. KGF enhanced expression of CLC-2 protein and mRNA in a dose- and time-dependent fashion. Our data suggest that KGF stimulates CLC-2 expression by stabilizing mRNA transcripts, because the degradation of CLC-2 mRNA was inhibited in the presence of KGF. Consistent with this mechanism, transcriptional upregulation of CLC-2 mRNA does not occur, as demonstrated by the CLC-2 promoter luciferase assay.

Fluid expansion of the developing lung occurs by accumulation of chloride and water in the airways (4, 5). KGF leads to fluid accumulation in fetal mouse lung explants (15). The inhibition of luminal dilation with bumetanide demonstrated that this process is mediated by chloride secretion. The authors determined that the KGF-sensitive chloride secretory pathway is independent of CFTR, because the same effect of KGF on luminal dilation was demonstrated in lung explants from CFTR knockout mice.

Non-CFTR chloride secretion must exist in the developing fetal lungs because CFTR is not essential for normal lung morphogenesis to occur. Normal and CF fetal lung explants expand at similar rates when grown in culture (25), and individuals born with CF have normal lung morphology at birth (26), suggesting that other chloride secretory pathways play a greater role than CFTR in fetal lung development. We have previously reported that the epithelial CLC-2 chloride channel is more abundantly expressed in utero (18, 22). At birth, the chloride-secreting fetal lung switches to a predominantly sodium-absorbing postnatal lung in preparation for air breathing. Coincident with this switch, CLC-2 mRNA and protein are dramatically downregulated. In addition, CLC-2 protein is expressed in the apical membrane of fetal lung epithelia (18, 22). This site of expression is consistent with the location expected for a fetal lung chloride channel to secrete chloride ions into the lumen.

KGF mediates multiple effects in the lung and has been implicated as an important mediator of epithelial cell growth (27). KGF is produced by cells of mesenchymal origin by 14.5 d gestation in the developing mouse (11). KGF binds to and activates the KGF receptor (KGFR), which is expressed in the developing respiratory epithelium by 12 d gestation in the mouse embryo (9). Epithelial cell differentiation is blocked in transgenic mice expressing a dominant-negative form of the FGF receptor-2 (FGFR2) (KGF) receptor under the control of the human lung surfactant protein (SP)-C promoter (16). Overexpression of KGF in transgenic mice disrupts normal lung morphogenesis and results in cystic dilatation of the lungs, mimicking pulmonary cystadenomas in humans. KGF in these transgenics did not promote maturation of the fetal respiratory epithelium (17), though Shiratori and associates found in rat embryonic lung rudiments that KGF stimulated proliferation and differentiation of alveolar epithelial type II cells (11).

The dose and time intervals for KGF exposure in this study are similar to those used by Zhou and coworkers (15). Within 5 h, mRNA transcripts were increased above control, reaching maximal intensity between 24 and 48 h. CLC-2 mRNA signal by Northern blot analysis was increased when 10 ng/ml KGF or more was added to primary cultures for 48 h. Although KGF does not influence synthesis of CLC-2 mRNA, we found that the half-life of CLC-2 mRNA was longer in primary rat FDLE cells exposed to KGF. This suggests that KGF influences processes that inhibit the degradation of CLC-2 transcripts. A similar effect on stabilization of CLC-2 protein was not demonstrated. Further investigation is required to elucidate this mechanism fully.

KGF is a potent mitogen for the developing respiratory epithelium. The onset of its expression and that of its receptor, KGFR, in the embryonic lung suggests an important paracrine mechanism for the developing lung (9, 14). The stimulation of CLC-2 expression by the developmental growth factor KGF in fetal respiratory epithelium suggests a candidate, non-CFTR chloride channel in the fetal lung, which we propose may contribute to fetal lung fluid production. We speculate that aberrant signaling of the KGF paracrine system or altered regulation of CLC-2 could result in congenital malformations of the lung in newborns by affecting pulmonary fluid distension. Cystadenomas are characterized by dilated cysts in lung parenchyma, and pulmonary hypoplasia is due to inadequate growth of the distal lung units. Further investigations in the human lung should be pursued to determine the role KGF and CLC-2 play in these congenital defects. In addition, elucidation of regulatory pathways for CLC-2 expression and chloride channel activation will be important for developing alternative therapies to bypass the defects in CFTR that utilize this endogenously expressed chloride channel in the human lung.