Modulation of ion conductance and active transport by TGF-{beta}1 in alveolar epithelial cell monolayers

Brigham C. Willis,1 Kwang-Jin Kim,2 Xian Li,2 Janice Liebler,2 Edward D. Crandall,2 and Zea Borok2

1Department of Anesthesiology Critical Care Medicine, Childrens Hospital, Los Angeles, and 2Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033

Submitted 5 November 2002 ; accepted in final form 23 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Transforming growth factor-{beta}1 (TGF-{beta}1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-{beta}1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-{beta}1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance (Rt) and increase in short-circuit current (Isc). Apical amiloride or basolateral ouabain on day 6 inhibited Isc by 80 and 100%, respectively. Concurrent increases in expression of Na+-K+-ATPase {alpha}1- and {beta}1-subunits were observed in TGF-{beta}1-treated monolayers. No change in the {alpha}-subunit of the rat epithelial sodium channel ({alpha}-rENaC) was seen. Exposure of confluent monolayers to TGF-{beta}1 from day 4 resulted in an initial decrease in Rt within 6 h, followed by an increase in Isc over 72-96 h. These results demonstrate that TGF-{beta}1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased Na+-K+-ATPase, but without a change in {alpha}-rENaC.

transforming growth factor; alveolar epithelium; alveolar epithelial cells; epithelial sodium channel; sodium pump; acute lung injury


THE NORMAL ALVEOLAR EPITHELIUM forms a barrier to the passive movement of fluid and solute from the pulmonary capillaries and lung interstitium to the alveolar air spaces. In addition, alveolar epithelial cells (AEC) vectorially transport sodium via apical amiloride-sensitive epithelial sodium channels and basolateral sodium pumps (Na+-K+-ATPase), creating an osmotic gradient for fluid reabsorption that contributes to the maintenance of the relatively dry alveolar air spaces (8, 21, 40, 41). After lung injury, disruption of the alveolar epithelial barrier and an increase in permeability contribute to the development of alveolar edema. Upregulation of alveolar epithelial fluid transport after such injury, despite increased alveolar permeability to protein, may help ameliorate edema fluid accumulation (20, 40). Of note, certain growth factors, including epidermal growth factor (EGF) and keratinocyte growth factor (KGF), have been shown to play a role in this process, modulating alveolar epithelial permeability characteristics and concurrently enhancing active ion transport (2, 4, 12).

Transforming growth factor-{beta}1 (TGF-{beta}1) is one of more than 17 different TGF isoforms (14) and has been implicated as a mediator of acute lung injury (ALI) (26, 35, 44). This multifunctional polypeptide elicits a wide range of biological responses depending on cell and tissue type, including regulation of differentiation and cell cycle, induction of apoptosis, induction of extracellular matrix formation, and modulation of hematopoiesis, angiogenesis, chemotaxis, and immune function (18, 45). Originally identified by its ability to stimulate anchorage-independent proliferation of rat kidney and fibroblast cell lines, TGF-{beta}1 has since been found to enhance the growth of most cells of mesenchymal origin while inhibiting the growth and differentiation of epithelial cell types (14, 18, 45).

The effects of various TGF-{beta} isoforms on lung epithelium have been well studied with regard to their roles in the induction and propagation of fibrosis and late-stage fibroproliferative processes (46). In contrast, evaluation of effects on normal lung physiology or during the early stages of ALI has been more limited. TGF-{beta}1 has been shown to regulate alveolar and bronchial epithelial cell proliferation and differentiation in vitro and in vivo, particularly after lung injury (22, 28, 50). Multiple TGF-{beta}1-inducible genes, including procollagen III, an early predictor of the severity of ALI (9), are upregulated within 2 days after lung injury (26). A recent investigation (44) demonstrated that mice lacking the integrin {alpha}v{beta}6 (a critical mediator of TGF-{beta}1 activation in lung and skin) were completely protected from bleomycin-induced pulmonary edema. This suggests that TGF-{beta}1 is integral to the disruption of the alveolar epithelial barrier and that its actions may contribute to the impaired fluid and ion transport dynamics seen in ALI.

AEC in primary culture constitute a well characterized in vitro model with which to evaluate properties of the alveolar epithelium. When grown on semipermeable supports, isolated alveolar type II (ATII) cells form polarized high-resistance monolayers, acquire a type I (ATI) cell-like phenotype, and exhibit active sodium transport that occurs in a vectorial fashion, similar to that observed in the alveolar epithelium in vivo (7, 8, 10, 13). To investigate the role of TGF-{beta}1 on alveolar epithelial barrier properties, we evaluated effects of TGF-{beta}1 on transepithelial resistance (Rt, a measure of monolayer permeability) and short-circuit current (Isc, a measure of active ion transport) in isolated rat AEC grown as monolayers on polycarbonate filters. Effects of TGF-{beta}1 on monolayer bioelectric properties were correlated with changes in expression of apical Na+ channels and basolateral sodium pumps by Western analysis and immunofluorescence staining of AEC monolayers.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell isolation and preparation of rat AEC monolayers. ATII cells were isolated from adult male Sprague-Dawley rats (average weight ~125 g) by disaggregation with elastase (2.0-2.5 U/ml; Worthington Biochemical, Freehold, NJ), followed by differential adherence on IgG-coated bacteriological plates (3, 15, 16). All animals involved with the study were treated and handled according to protocols approved by the University of Southern California Institutional Animal Care and Use Committee. The enriched ATII cells were resuspended in minimal defined serum-free medium (MDSF) consisting of DMEM and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma Chemical, St. Louis, MO), supplemented with 1.25 mg/ml BSA (Sigma), 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin (3). Cells were seeded onto tissue culture-treated polycarbonate (Nuclepore) filter cups (Transwell; Corning Costar, Cambridge, MA) at a density of 1.0 x 106 cells/cm2. From the time of plating, or from day 4 to day 8 after being plated, cells were maintained either in MDSF or in MDSF supplemented with recombinant human TGF-{beta}1 (R&D Systems, Minneapolis, MN) at concentrations ranging from 0.1 to 200 pM (0.0025-5 ng/ml). Equivalent amounts of TGF-{beta}1 vehicle (4 mM HCl with 1 mg/ml of BSA) were added to unsupplemented monolayers. The maximum amount of vehicle or TGF-{beta}1 added to media was 2.5 µl/ml. The dosage range for TGF-{beta}1 was chosen on the basis of previous studies and known physiological levels (22, 44). Cultures were maintained in a humidified 5% CO2 incubator at 37°C. Media were changed every 2-3 days. ATII cell purity (>=90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (38). Viability (>95%) was measured by trypan blue dye exclusion.

Measurement of monolayer bioelectric properties. Rt (K{Omega} · cm2) and spontaneous potential difference (SPD; mV, apical side as reference) were measured using a rapid screening device (Millicell-ERS; Millipore, Bedford, MA). Equivalent Isc (µA/cm2) was calculated from the relationship Isc = SPD/Rt (Ohm's law), as previously described (2, 4). The effects of TGF-{beta}1 supplementation on bioelectric properties were evaluated on days 4 and 6 in culture for monolayers maintained with or without TGF-{beta}1 from the time of plating. In separate experiments, monolayers were allowed to reach confluence on day 4 and were subsequently maintained with or without TGF-{beta}1 from days 4-8.

Effects of sodium transport inhibitors on Isc. To elucidate the mechanisms underlying the effects of TGF-{beta}1 on Isc, bioelectric properties of AEC monolayers in MDSF with or without TGF-{beta}1 were measured on day 6 in the presence of apical amiloride (10 µM), an inhibitor of epithelial sodium channel activity, or basolateral ouabain (1 mM), an inhibitor of Na+-K+-ATPase activity. Measurements were made over 30 min after addition of the inhibitors. Control monolayers were treated with equivalent amounts of vehicle (DMSO for amiloride or PBS, pH 7.2, for ouabain).

Cell number determination. The number of adherent cells in monolayers maintained in MDSF with or without TGF-{beta}1 was determined on days 1, 4, and 6 after seeding to assess plating efficiency (day 1) and to normalize for subsequent cellular protein analysis. Filters were washed with cold PBS, cells were lysed, and nuclei were stained by incubating at 37°C overnight in a solution containing 0.1% Nonidet P-40, 0.1 M citric acid, and 0.1% crystal violet. Stained nuclei were counted using a hemacytometer (5).

Western analysis. AEC monolayers were lysed in 2% SDS sample buffer at 37°C for 15 min. Protein extracted from equivalent numbers of AEC from control and TGF-{beta}1-treated monolayers was resolved by SDS-PAGE under reducing conditions using the buffer system of Laemmli (31) and was electrophoretically blotted onto Immobilon-P nylon membranes (Millipore) using procedures modified from Towbin et al. (48). Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) with BSA as standard. Membranes were blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween (TBST) at pH 7.5. They were then incubated for 2 h at room temperature with a mouse monoclonal antibody (MAb) against the Na+-K+-ATPase {alpha}1-subunit (6H; M. Caplan, Yale Univ.) or a rabbit polyclonal antibody against the {alpha}-subunit of the rat epithelial sodium channel ({alpha}-rENaC; Alpha Diagnostic International, San Antonio, TX) in TBST. To assure specificity of the ENaC antibody, rat renal cortex membrane lysate was used as a positive control, and for some samples, anti-{alpha}-rENaC antibody was preincubated with a blocking peptide (Alpha Diagnostic). Finally, blots were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG conjugates for 1 h at room temperature, and antibody complexes were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). Blots were scanned using a Powerlook III color scanner (Umax, Fremont, CA) and Photoperfect 4.4 software (Binuscan, Monte Carlo, Monaco), and images were analyzed using Photoshop 5.5 (Adobe Systems, San Jose, CA) and Image-Pro Plus 4.1 (Media Cybernetics, Silver Spring, MD).

Immunofluorescence. On day 6, monolayers on filters maintained in MDSF with or without TGF-{beta}1 (50-100 pM) were rinsed once with ice-cold PBS and fixed with 100% methanol at 4°C for 10 min. After being rinsed in PBS and blocked with 5% BSA at room temperature for 1 h, monolayers were incubated with mouse anti-Na+-K+-ATPase {alpha}1- or {beta}1-subunit MAbs ({beta}1:IEC 1/48; A. Quaroni, Cornell Univ.) (36) overnight at 4°C. After being washed extensively, filters were incubated with appropriate secondary antibodies conjugated to FITC, rinsed again, and postfixed in 3.7% formalin. They were then treated with Vectashield antifade mounting medium with propidium iodide to stain nuclei (Vector Laboratories, Burlingame, CA) and viewed with an Olympus BX60 microscope equipped with epifluorescence optics. Images were captured with a cooled charge-coupled device camera (Magnafire; Olympus, Melville, NY) with a barrier filter equipped for simultaneous detection of FITC and propidium iodide. Captured images were imported into Adobe Photoshop (Adobe Systems, Mountain View, CA) as TIFF files.

Statistical analysis. Results are expressed as means ± SE. Significance (P < 0.05) of differences in Rt, Isc, cell number, and densitometric measurements of ENaC and Na+-K+-ATPase protein levels was determined by either unpaired Student's t-test or one-way ANOVA with Dunnett's multiple comparison test for multiple groups.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of TGF-{beta}1 on AEC monolayer bioelectric properties. AEC maintained in MDSF formed functional, confluent monolayers that actively transported sodium by day 4, with Rt = 3.11 ± 0.30 K{Omega} · cm2 and Isc = 2.20 ± 0.14 µA/cm2 on day 6. In the presence of TGF-{beta}1 from the time of plating (day 0), there was a concentration-dependent decrease in Rt (Fig. 1A) and increase in Isc (Fig. 1B) on day 6. Rt reached a nadir of 1.66 ± 0.30 K{Omega} · cm2 (55% of that in MDSF), and Isc increased to a maximum of 3.41 ± 0.09 µA/cm2 (155% of that in MDSF) with increasing concentrations of TGF-{beta}1. Maximal reductions in Rt and increases in Isc were seen at TGF-{beta}1 concentrations of 50 pM (1.25 ng/ml). Concentrations >200 pM resulted in a lack of formation of tight monolayers (data not shown).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Effects of transforming growth factor (TGF)-{beta}1 on alveolar epithelial cell (AEC) monolayer bioelectric properties on day 6. There was a dose-dependent reduction of transepithelial resistance (Rt; A) and increase of short-circuit current (Isc; B) in AEC monolayers treated with TGF-{beta}1 from day 0. EC50 was 5.5 pM and 10 pM, respectively. Each data point represents mean ± SE of measurements from 36-60 monolayers from >3 preparations. *Significantly different from minimal defined serum-free medium (MDSF).

 

Rt was lower on days 4 and 6 in TGF-{beta}1-treated monolayers compared with MDSF, and there was a progressive decrease in Rt between days 4 and 6 in TGF-{beta}1-treated monolayers, which reached statistical significance at 10 pM (data not shown). There was a trend toward an increase in Isc on day 4 after treatment with TGF-{beta}1 (data not shown). Isc was maintained at higher levels than in MDSF on day 6 in the presence of 50 and 100 pM TGF-{beta}1 (Fig. 1B).

Monolayers with or without TGF-{beta}1 from days 4-8 in culture demonstrated similar results, with significantly lower Rt (Fig. 2A) and higher Isc (Fig. 2B) in treated monolayers. Resistance decreased from 2.88 ± 0.15 K{Omega} · cm2 to an initial nadir of 1.60 ± 0.16 K{Omega} · cm2 (56% of baseline) within 6 h of addition of TGF-{beta}1. Isc did not increase significantly from a baseline level of 2.01 ± 0.05 µA/cm2 until 72 h after addition of TGF-{beta}1, reaching a maximum of 2.86 ± 0.19 µA/cm2 (136% of baseline) at 96 h.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Effects of TGF-{beta}1 (50 pM) on monolayer bioelectric properties after treatment on day 4 (d. 4) in culture. A: Rt decreased after 6 h of exposure to TGF-{beta}1on day 4 and was maintained significantly lower than monolayers in MDSF through 96 h. B: Isc increased after exposure to TGF-{beta}1 for 72 h and was maintained significantly higher than monolayers in MDSF through 96 h. *Significantly different from MDSF and time 0. Each data point represents mean ± SE of measurements from 36-48 monolayers from >3 preparations.

 

Effects of sodium transport inhibitors on Isc. The effects of amiloride and ouabain on Isc were evaluated on day 6 in the presence and absence of TGF-{beta}1 (50 pM). Apical (but not basolateral) amiloride (10 µM) immediately decreased Isc ~75-85% in MDSF with or without TGF-{beta}1 (Fig. 3A). The residual amiloride-insensitive current was similar in both TGF-{beta}1-treated monolayers and MDSF controls. Addition of basolateral ouabain (1 mM) completely inhibited Isc over 30 min in MDSF with or without TGF-{beta}1 (Fig. 3B). Ouabain- and amiloride-sensitive Isc were significantly greater in TGF-{beta}1-treated monolayers.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Effects of sodium transport inhibitors on Isc in the presence or absence of TGF-{beta}1. A: apical amiloride (Amil; 10 µM) rapidly decreased Isc ~75-85% in MDSF ± TGF-{beta}1 (50 pM). Amiloride-sensitive Isc was significantly greater in TGF-{beta}1-treated monolayers. B: basolateral ouabain (1 mM) completely inhibited Isc over 30 min in MDSF ± TGF-{beta}1 (50 pM). Ouabain-sensitive Isc was significantly greater in TGF-{beta}1-treated monolayers. Each data point represents mean ± SE for >=12 monolayers from 3 preparations.

 

Effect of TGF-{beta}1 on monolayer cell number. Adherent cell number was determined for AEC monolayers cultivated in MDSF with or without TGF-{beta}1 on days 1, 4, and 6. There were no significant differences in cell number in MDSF plus TGF-{beta}1 compared with MDSF on day 1 (Fig. 4). However, cell number was significantly decreased on day 4 (49 ± 18%) and day 6 (44 ± 12%) in TGF-{beta}1-treated monolayers compared with control monolayers.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4. Effects of TGF-{beta}1 on cell number. Addition of TGF-{beta}1 (100 pM) from day 0 significantly decreased adherent cell number on days 4 and 6. Cell number on day 1 was similar in the presence or absence of TGF-{beta}1. Each data point represents the mean ± SE for 8-12 monolayers from 3 preparations. *Significantly different from day 1 and MDSF on all days.

 

Effects of TGF-{beta}1 on Na+-K+-ATPase {alpha}1- and {beta}1-subunit expression. Protein was harvested from equivalent numbers of AEC from monolayers cultivated in MDSF with or without TGF-{beta}1. This protein was analyzed on day 6 for expression of the {alpha}1-subunit of Na+-K+-ATPase by Western blotting. As shown in Fig. 5, {alpha}1-subunit protein was significantly increased in AEC exposed to TGF-{beta}1 at all concentrations evaluated. Analysis of the {beta}1-subunit of Na+-K+-ATPase in AEC by Western blotting is not quantitative (51), as preparation of the protein requires initial deglycosylation, and thus was not performed.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Effects of TGF-{beta}1 on expression of Na+-K+-ATPase {alpha}1-subunit protein per cell. A: representative Western blot (n = 5) demonstrates a significant increase in expression of Na+-K+-ATPase {alpha}1-subunit protein per cell in AEC monolayers on day 6 after exposure to TGF-{beta}1 from day 0. Protein loading was normalized for cell number. B: bar graph summarizing average relative densitometric values (± SE) reveals significantly higher levels of Na+-K+-ATPase {alpha}1-subunit protein expression per cell after exposure to TGF-{beta}1. *Significantly different from MDSF.

 

Immunofluorescence was also used to compare cell surface-associated expression of {alpha}1- and {beta}1-subunit proteins in AEC monolayers in MDSF with or without TGF-{beta}1. As shown in Figs. 6 and 7, immunoreactivity for both {alpha}1- and {beta}1-subunits of Na+-K+-ATPase was increased in TGF-{beta}1-treated monolayers compared with untreated monolayers. No reactivity was seen with MF20, a mouse MAb that is reactive with chicken myosin (D. Fischman, Cornell Univ.).



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 6. Effects of TGF-{beta}1 (100 pM) on expression of Na+-K+-ATPase {alpha}1-subunit protein. Surface immunoreactivity for Na+-K+-ATPase {alpha}1-subunit (green) was assessed by immunofluorescence on day 6. A: monolayer in MDSF reacted with anti-Na+-K+-ATPase {alpha}1-subunit monoclonal antibody (MAb). B: monolayer in MDSF + TGF-{beta}1 reacted with anti-Na+-K+-ATPase {alpha}1-subunit MAb. C: monolayer in MDSF reacted with MF20 control antibody. D: monolayer in MDSF + TGF-{beta}1 reacted with MF20 control antibody. Red staining represents propidium iodide-stained nuclei. Photographs are representative of 16 monolayers from 4 separate experiments. Original magnification, x400.

 


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 7. Effects of TGF-{beta}1 (50 pM) on expression of Na+-K+-ATPase {beta}1-subunit protein. Surface immunoreactivity for Na+-K+-ATPase {beta}1-subunit (green) was assessed by immunofluorescence on day 6. A: monolayer in MDSF reacted with anti-Na+-K+-ATPase {beta}1-subunit MAb. B: monolayer in MDSF + TGF-{beta}1 reacted with anti-Na+-K+-ATPase {beta}1-subunit MAb. C: monolayer in MDSF reacted with MF20 control antibody. D: monolayer in MDSF + TGF-{beta}1 reacted with MF20 control antibody. Red staining represents propidium iodide-stained nuclei. Photographs are representative of 16 monolayers from 4 separate experiments. Original magnification, x400.

 

Effect of TGF-{beta}1 on {alpha}-rENaC expression. Protein from equivalent numbers of AEC from monolayers cultivated in MDSF with or without TGF-{beta}1 was analyzed on day 6 for {alpha}-rENaC protein expression by Western blot. As shown in Fig. 8, {alpha}-rENaC protein is detected as a major band of ~60 kDa and a fainter higher-molecular-weight band of ~80 kDa (32) in MDSF with or without TGF-{beta}1. Expression of {alpha}-rENaC was similar in 50 pM TGF-{beta}1-treated monolayers compared with control monolayers maintained in MDSF. In one pilot experiment, monolayers exposed to 100 pM TGF-{beta}1 had equivalent {alpha}-rENaC expression compared with MDSF or 50 pM TGF-{beta}1 (data not shown). Rat renal cortex was used as a positive control. To assure specificity of the antibody, in some experiments the anti-{alpha}-rENaC antibody was first adsorbed with an {alpha}-rENaC-specific control peptide with consequent disappearance of the prominent bands (data not shown).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 8. Effects of TGF-{beta}1 on expression of {alpha}-subunit of rat epithelial sodium channel ({alpha}-rENaC) protein per cell. A: representative Western blot (n = 3) demonstrates no change in expression of {alpha}-rENaC protein in AEC monolayers on day 6 after exposure to TGF-{beta}1 from day 0. Protein loading was normalized for cell number. Rat renal cortex protein is shown as a positive control (C). B: bar graph summarizing average relative densitometric values (± SE) reveals no significant difference in {alpha}-rENaC protein expression after exposure to TGF-{beta}1.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Exposure of rat AEC to TGF-{beta}1 from the time of plating resulted in dose- and time-dependent decreases in Rt after 4 and 6 days of exposure, indicating an increase in monolayer ion conductivity. Concurrently, TGF-{beta}1 induced dose- and time-dependent increases in Isc, reflecting upregulation of transepithelial active ion transport. Addition of TGF-{beta}1 after monolayers reached confluence revealed a rapid decrease in Rt (within 6 h), followed by an increase in Isc after 72-96 h. Studies in the presence and absence of inhibitors of apical Na+ channels (amiloride) and basolateral sodium pumps (ouabain) indicate that the observed increases in Isc in the presence of TGF-{beta}1 are accounted for by an increase in transepithelial Na+ transport. Analysis of cellular protein expression revealed that concurrent with its effects on Isc, TGF-{beta}1 increased expression of both {alpha}1- and {beta}1-subunits of Na+-K+-ATPase, whereas levels of {alpha}-rENaC remained unchanged.

Similar effects of TGF-{beta} isoforms on monolayer permeability and junctional integrity have been observed by other investigators both in AEC and in other cell types. Bovine endothelial cells have been shown to undergo tight junctional disassembly and cell separation after exposure to TGF-{beta}1, possibly due to activation of a myosin light chain kinase (MLCK)-dependent signaling cascade (23). TGF-{beta}3 disrupted Sertoli cell tight junction function and formation in rats accompanied by reductions in expression of several tight junction-associated proteins, including claudin-11, occludin, and zonula occludens-1 (ZO-1) (34). Treatment of cultured human proximal tubular epithelial cells with TGF-{beta}1 resulted in adherens and tight junctional complex disassembly, loss of adherens junction attachment to the cytoskeleton, and reductions in expression of occludin and ZO-1 (47). These effects appeared to be associated with colocalization of the TGF-{beta}1 type II receptor with the adherens junction and association of the TGF-{beta}1 signaling intermediates Smad3 and Smad4 with {beta}-catenin after dissociation of {beta}-catenin from the adherens junction complex.

Rt was recently reported to decrease in response to TGF-{beta}1 in rat AEC monolayers, possibly secondary to depletion of intracellular glutathione (44). Absolute resistance was not reported, and Rt was measured 48 h after cell plating. In our experiments, Rt was extremely low or immeasurable at that time, since confluent monolayers with high resistance usually become established only by days 3-4 in culture, concurrent with the acquisition of characteristics associated with the ATI cell phenotype. For these reasons, we measured Rt from day 4 onward and showed that TGF-{beta}1 decreases resistance by nearly 50% in AEC monolayers. The mechanism underlying the observed increase in passive ion conductance is not known. Although depletion of intracellular glutathione could potentially play a role as suggested above, how this translates into disruption of alveolar epithelial barrier properties is currently unknown. Possible effects of TGF-{beta}1 on tight junction-associated proteins have not been evaluated to date in AEC, and further study will be necessary to delineate the mechanism of our findings in AEC. However, a mechanism distinct from the MLCK-mediated cell contraction pathway seen in bovine endothelial cells (23) seems likely. AEC monolayers treated with TGF-{beta}1 retain relatively high Rt, appear confluent morphologically with intact cell-cell borders, and have no obvious cell separation by immunofluorescence microscopy.

Intact alveolar epithelial barrier function is essential to maintenance of the relatively dry condition of the alveolar air spaces, and mortality in ALI may be related to the loss of epithelial barrier function (41). Bleomycin-induced lung injury resulted in an increase in alveolar permeability to protein with a concomitant increase in net alveolar fluid clearance (20). Increased alveolar fluid clearance was attributed to an increase in transport capacity of the alveolar epithelium due to proliferation of ATII cells after ALI, since increased 22Na+ uptake per cell could not be demonstrated in ATII cells isolated from these animals. Effects on ATI or ATI-like cells were not evaluated. The complete protection from bleomycin-induced ALI observed in mice unable to activate TGF-{beta}1 suggests that TGF-{beta}1 is a critical mediator of the effects of bleomycin on alveolar epithelial barrier function (44). In this study, we demonstrate that, in AEC monolayers that have acquired the phenotypic characteristics of ATI cells, TGF-{beta}1 directly upregulated Isc and active transepithelial sodium transport. Concurrent with the increase in Isc, we demonstrated upregulation of both the {alpha}1-and {beta}1-subunits of the Na+-K+-ATPase but not of {alpha}-rENaC. This suggests that the effects of TGF-{beta}1 were due, at least in part, to an increase in membrane-associated as well as total cellular Na+-K+-ATPase expression. In addition, despite the observed increase in Isc between 0.1 and 100 pM TGF-{beta}1-treated monolayers, levels of total sodium pump protein were similar at all concentrations of TGF-{beta}1. This suggests that some other regulatory mechanism may be involved in full upregulation of sodium pump activity.

Regulation of Na+-K+-ATPase activity occurs at multiple levels, but, most commonly, short-term regulation is effected as a result of changes in the number of pumps inserted into the plasma membrane, changes in the phosphorylation state of the protein, and relative cellular ATP levels (1, 17, 33). Sustained regulation occurs both by changes in intracellular trafficking and total protein expression (2, 4, 17, 33). Our findings indicate an overall increase in sodium pump expression with chronic exposure to TGF-{beta}1, consistent with a long-term regulatory mechanism. We also found a marked increase in membrane-associated protein (Figs. 6 and 7), which may indicate increased membrane insertion as well. Whether changes in pump activity through modification of its phosphorylation state or ATP levels occur with TGF-{beta}1 exposure will require further investigation (1, 29).

Despite the effects of TGF-{beta}1 on Isc and an increase in amiloride-sensitive sodium transport, {alpha}-rENaC levels did not increase with TGF-{beta}1 exposure. ENaC regulation and expression are complex. ENaC is a heteromultimeric protein formed by three subunits, {alpha}-, {beta}-, and {gamma}-, that associate in a complex of varying stoichiometry (49). Surface expression of ENaC is only a small fraction of total cellular expression, and maturation and assembly of the subunits and subsequent membrane insertion seem to be the limiting steps for expression of functional complexes (19, 37, 49). All three ENaC subunits have been identified in the alveolar epithelium, although the relative contribution of ENaC subunits to amiloride-sensitive sodium transport remains controversial. In the lung, stimulation of transepithelial ion transport by various agents is not always associated with predictable changes in ENaC expression. For example, glucocorticoids have been shown to stimulate alveolar transepithelial sodium transport via increased ENaC mRNA and protein synthesis (11, 24). Similarly, {beta}-adrenergic agents stimulated transepithelial sodium transport and increased ENaC mRNA expression in ATII cells (42). In contrast, chronic exposure to KGF decreased mRNA expression of all three subunits of ENaC in AEC, despite inducing upregulation of transepithelial ion transport and increasing Na+-K+-ATPase expression (2). In addition, it appears that alternative splicing of ENaC transcripts can result in channel variants with different functional characteristics (32). An alternate, truncated transcript of {alpha}-rENaC resulted from a premature stop codon and deletion of 199 amino acids, and the product of this transcript was a 57-kDa protein, smaller than the expected 79-kDa {alpha}-ENaC protein. The smaller product, which lacked the second transmembrane domain, did not produce an amiloride-sensitive current (32).

In this study, we identified two {alpha}-rENaC bands by Western blotting in AEC, one of just under 60 kDa (which predominated) and one of ~80 kDa. The overall intensity of both bands did not increase despite the observed increase in amiloride-sensitive transepithelial sodium transport. Similar to our previous findings with both EGF and KGF, the increase in transepithelial transport in the presence of TGF-{beta}1 was associated with an increase in amiloride-sensitive current without an increase in total {alpha}-rENaC protein (2, 6, 27). Possible explanations for the observed increase in amiloride-sensitive current in this study include an increase in the open probability of the channel, an increase in the probability of ENaC multimer formation, posttranslational modification of the ENaC multimer leading to increased activity, increased current through channels previously operating below maximal capacity in response to an increased gradient for sodium entry created by an increase in sodium pump expression, or upregulation of another type of cation channel by TGF-{beta}1 (2, 19, 25, 37). Several studies have suggested that the predominant AEC Na+-conducting channel may be a nonselective cation channel other than ENaC (2, 19, 25, 27, 37, 39, 43). Consistent with this, we previously demonstrated that the EGF-induced increase in Isc in rat AEC monolayers occurred via an increase in activity of nonselective amiloride-sensitive cation channels, without effecting a change in ENaC mRNA or activity (12, 27). Accordingly, although the mechanism for increased cell sodium entry rate across the apical cell membrane (leading to increased transepithelial sodium transport) seen in response to TGF-{beta}1 exposure is unclear, it may involve an increase in expression or function of nonselective cation channels and/or increased membrane insertion or function of ENaC.

The reduction in cell number accompanying the increase in Isc due to TGF-{beta}1 exposure indicates a dramatic increase in the transport capacity of individual AEC within the monolayer. The observed 50% reduction in adherent cells that occurred between days 1 and 4 could be the result of increased cell spreading, a direct toxic effect, or induction of apoptosis. It is unlikely to represent a toxic effect, since the remaining cells formed confluent, functional monolayers with appreciable resistance and actively transported sodium. Although TGF-{beta}1 is known to enhance or induce apoptosis in many cell types, including gastric carcinoma cells, primary hepatocytes, and lung bronchial epithelial cells (22, 30, 45), it remains to be determined whether TGF-{beta}1-induced apoptosis contributes to the observed reduction in monolayer cell number. In addition, TGF-{beta}1 clearly changes AEC morphology in cultured monolayers. Alterations in total membrane surface area may have contributed to the increase in sodium pump protein per cell, contributing to the overall increase in Isc. Further study will be needed to fully describe the morphological changes induced by TGF-{beta}1.

In summary, we have demonstrated that TGF-{beta}1 increases rat AEC monolayer ion conductance, accompanied by an increase in active transepithelial sodium transport, in a dose- and time-dependent fashion. The increased active sodium transport was ouabain and amiloride sensitive, and expression of Na+-K+-ATPase was increased, whereas expression of {alpha}-ENaC was not. Consistent with the complexity of its effects in other cell types, TGF-{beta}1 appears to induce distinct effects on the active transport and ion conductance properties of AEC that may have opposing effects on alveolar fluid balance in ALI.


    DISCLOSURES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Grants HL-38578, HL-38621, HL-38658, HL-62569, HL-64365, and HL-72231, the American Heart Association Grant-in-Aid 9950442N, and the Hastings Foundation.


    ACKNOWLEDGMENTS
 
We note with appreciation the expert technical assistance of Zerlinde Balverde, Susie Parra, and Juan Ramon Alvarez.

E. D. Crandall is Hastings Professor and Kenneth T. Norris Jr. Chair of Medicine.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Borok, Division of Pulmonary and Critical Care Medicine, Univ. of Southern California, IRD 620, 2020 Zonal Ave., Los Angeles, CA 90033 (E-mail: zborok{at}usc.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
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Bertorello AM and Katz AI. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am J Physiol Renal Fluid Electrolyte Physiol 265: F743-F755, 1993.[Abstract/Free Full Text]
  2. Borok Z, Danto SI, Dimen LL, Zhang XL, and Lubman RL. Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF. Am J Physiol Lung Cell Mol Physiol 274: L149-L158, 1998.[Abstract/Free Full Text]
  3. Borok Z, Danto SI, Zabski SM, and Crandall ED. Defined medium for primary culture de novo of rat alveolar epithelial cells. In Vitro Cell Dev Biol 30A: 99-104, 1994.
  4. Borok Z, Hami A, Danto SI, Lubman RL, Kim KJ, and Crandall ED. Effects of EGF on alveolar epithelial junctional permeability and active sodium transport. Am J Physiol Lung Cell Mol Physiol 270: L559-L565, 1996.[Abstract/Free Full Text]
  5. Borok Z, Hami A, Danto SI, Zabski SM, and Crandall ED. Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro. Am J Respir Cell Mol Biol 12: 50-55, 1995.[Abstract]
  6. Borok Z, Mihyu S, Fernandes VFJ, Zhang XL, Kim KJ, and Lubman RL. KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells. Am J Physiol Cell Physiol 276: C1352-C1360, 1999.[Abstract/Free Full Text]
  7. Cheek JM, Evans MJ, and Crandall ED. Type I cell-like morphology in tight alveolar epithelial monolayers. Exp Cell Res 184: 375-387, 1989.[ISI][Medline]
  8. Cheek JM, Kim KJ, and Crandall ED. Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am J Physiol Cell Physiol 256: C688-C693, 1989.[Abstract/Free Full Text]
  9. Chestnutt AN, Matthay M, Tibayan FA, and Clark JG. Early detection of type III procollagen peptide in acute lung injury. Pathogenetic and prognostic significance. Am J Respir Crit Care Med 156: 840-845, 1997.[Abstract/Free Full Text]
  10. Christensen PJ, Kim S, Simon RH, Toews GB, and Paine RD. Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells. Am J Respir Cell Mol Biol 8: 9-15, 1993.[ISI][Medline]
  11. Dagenais A, Denis C, Vives MF, Girouard S, Masse C, Nguyen T, Yamagata T, Grygorczyk C, Rashmi K, and Berthiaume Y. Modulation of {alpha}-ENaC and {alpha}1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 281: L217-L230, 2001.[Abstract/Free Full Text]
  12. Danto SI, Borok Z, Zhang XL, Lopez MZ, Patel P, Crandall ED, and Lubman RL. Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells. Am J Physiol Cell Physiol 275: C82-C92, 1998.[Abstract/Free Full Text]
  13. Danto SI, Zabski SM, and Crandall ED. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies. Am J Respir Cell Mol Biol 6: 296-306, 1992.[ISI][Medline]
  14. Dennler S, Goumans MJ, and ten Dijke P. Transforming growth factor {beta} signal transduction. J Leukoc Biol 71: 731-740, 2002.[Abstract/Free Full Text]
  15. Dobbs L. Isolation and culture of alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 258: L134-L147, 1990.[Abstract/Free Full Text]
  16. Dobbs L, Gonzalez R, and Williams MC. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134: 141-145, 1986.[ISI][Medline]
  17. Dunbar L and Caplan M. The cell biology of ion pumps: sorting and regulation. Eur J Cell Biol 79: 557-563, 2000.[ISI][Medline]
  18. Dunker N and Krieglstein K. Targeted mutations of transforming growth factor-{beta} genes reveal important roles in mouse development and adult homeostasis. Eur J Biochem 267: 6982-6988, 2000.[Abstract/Free Full Text]
  19. Ecelbarger CA, Kim GH, Terris J, Masilamani S, Mitchell C, Reyes I, Verbalis JG, and Knepper MA. Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney. Am J Physiol Renal Physiol 279: F46-F53, 2000.[Abstract/Free Full Text]
  20. Folkesson HG, Nitenberg G, Oliver BL, Jayr C, Albertine KH, and Matthay M. Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin. Am J Physiol Lung Cell Mol Physiol 275: L478-L490, 1998.[Abstract/Free Full Text]
  21. Goodman BE, Kim KJ, and Crandall ED. Evidence for active sodium transport across alveolar epithelium of isolated rat lung. J Appl Physiol 62: 2460-2477, 1987.[Abstract/Free Full Text]
  22. Hagimoto N, Kuwano K, Inoshima I, Yoshimi M, Nakamura N, Fujita M, Maeyama T, and Hara N. TGF-{beta}1 as an enhancer of fas-mediated apoptosis of lung epithelial cells. J Immunol 168: 6470-6478, 2002.[Abstract/Free Full Text]
  23. Hurst IVV, Goldberg PL, Minnear FL, Heimark RL, and Vincent PA. Rearrangement of adherens junctions by transforming growth factor-{beta}1: role of contraction. Am J Physiol Lung Cell Mol Physiol 276: L582-L595, 1999.[Abstract/Free Full Text]
  24. Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, and Thomas CP. Glucocorticoid-stimulated lung epithelial Na+ transport is associated with regulated ENaC and sgk1 expression. Am J Physiol Lung Cell Mol Physiol 282: L631-L641, 2002.[Abstract/Free Full Text]
  25. Jain L, Chen XJ, Ramosevac S, Brown LAS, and Eaton DC. Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. Am J Physiol Lung Cell Mol Physiol 280: L646-L658, 2001.[Abstract/Free Full Text]
  26. Kaminski N, Allard JD, Pittet JF, Griffiths MJD, Morris D, Huang X, Sheppard D, and Heller RA. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci USA 97: 1778-1783, 2000.[Abstract/Free Full Text]
  27. Kemp PJ, Borok Z, Kim KJ, Lubman RL, Danto SI, and Crandall ED. Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels. Am J Physiol Cell Physiol 277: C1058-C1065, 1999.[Abstract/Free Full Text]
  28. Khalil N, Parekh TV, O'Connor RN, and Gold LI. Differential expression of transforming growth factor-{beta} type I and II receptors by pulmonary cells in bleomycin-induced lung injury: correlation with repair and fibrosis. Exp Lung Res 27: 233-250, 2002.
  29. Klip A and Ewart SH. Hormonal regulation of the Na+-K+-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am J Physiol Cell Physiol 269: C295-C311, 1995.[Abstract/Free Full Text]
  30. Kuwano K, Kunitake R, Maeyama T, Hagimoto N, Kawasaki M, Matsuba T, Yoshimi M, Inoshima I, Yoshida K, and Hara N. Attenuation of bleomycin-induced pneumopathy in mice by a caspase inhibitor. Am J Physiol Lung Cell Mol Physiol 280: L316-L325, 2001.[Abstract/Free Full Text]
  31. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.[ISI][Medline]
  32. Li XJ, Xu RH, Guggino WB, and Snyder SH. Alternatively spliced forms of the {alpha} subunit of the epithelial sodium channel: distinct sites for amiloride binding and channel pore. Mol Pharmacol 47: 1133-1140, 1995.[Abstract]
  33. Lopina OD. Na+-K+-ATPase: structure, mechanism, and regulation. Membr Cell Biol 13: 721-744, 2000.[Medline]
  34. Lui WY, Lee WM, and Cheng CY. Transforming growth factor-{beta}3 perturbs the inter-Sertoli tight junction permeability barrier in vitro, possibly mediated via its effects on occludin, zonula occludens, and claudin-11. Endocrinology 142: 1865-1877, 2001.[Abstract/Free Full Text]
  35. Maniscalco WM, Sinkin RA, Watkins RH, and Campbell MH. Transforming growth factor-{beta}1 modulates type II cell fibronectin and surfactant protein C expression. Am J Physiol Lung Cell Mol Physiol 267: L569-L577, 1994.[Abstract/Free Full Text]
  36. Marxer A, Stieger B, Quaroni A, Kashgarian M, and Hauri HP. Na+-K+-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to {beta} subunit. J Cell Biol 109: 1057-1068, 1989.[Abstract]
  37. Masilamani S, Kim GH, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC {alpha}, {beta}, and {gamma} subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999.[ISI][Medline]
  38. Mason RJ, Walker SR, Shields A, and Henson JE. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131: 786-788, 1985.[ISI][Medline]
  39. Matalon S and O'Brodovich H. Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annu Rev Physiol 61: 627-661, 1999.[ISI][Medline]
  40. Matthay M, Folkesson HG, and Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82: 569-600, 2002.[Abstract/Free Full Text]
  41. Matthay M and Wiener-Kronish J. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am Rev Respir Dis 142: 1250-1257, 1990.[ISI][Medline]
  42. Minakata Y, Suzuki S, Grygorczyk C, Dagenais A, and Berthiaume Y. Impact of {beta}-adrenergic agonist on Na+-channel and Na+-K+-ATPase expression in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 275: L414-L422, 1998.[Abstract/Free Full Text]
  43. Palmer LG. Epithelial Na channels: function and diversity. Annu Rev Physiol 54: 51-66, 1992.[ISI][Medline]
  44. Pittet JF, Griffiths MJD, Geiser T, Kaminski N, Dalton SL, Huang X, Brown LAS, Gotwals PJ, Koteliansky VE, Matthay M, and Sheppard D. TGF-{beta} is a critical mediator of acute lung injury. J Clin Invest 107: 1537-1544, 2001.[Abstract/Free Full Text]
  45. Schuster N and Krieglstein K. Mechanisms of TGF-{beta}-mediated apoptosis. Cell Tissue Res 307: 1-14, 2001.[ISI]
  46. Sime PJ and O'Reilly KMA. Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment. Clin Immunol Immunopathol 99: 308-319, 2001.
  47. Tian YC and Phillips AO. Interaction between the transforming growth factor-{beta} type II receptor/Smad pathway and {beta}-catenin during transforming growth factor-{beta}1-mediated adherens junction disassembly. Am J Pathol 160: 1619-1628, 2002.[Abstract/Free Full Text]
  48. Towbin HH, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 76: 43-50, 1979.
  49. Valentijn JA, Fyfe GK, and Canessa CM. Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes. J Biol Chem 273: 30344-30351, 1998.[Abstract/Free Full Text]
  50. Zeng X, Gray M, Stahlman MT, and Whitsett JA. TGF-{beta}1 perturbs vascular development and inhibits epithelial differentiation in fetal lung in vivo. Dev Dyn 221: 289-301, 2001.[ISI][Medline]
  51. Zhang XL, Danto SI, Borok Z, Eber JT, Martin-Vasallo P, and Lubman RL. Identification of Na+-K+-ATPase {beta} subunit in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 272: L85-L94, 1997.[Abstract/Free Full Text]