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
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ABSTRACT |
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transforming growth factor; alveolar epithelium; alveolar epithelial cells; epithelial sodium channel; sodium pump; acute lung injury
Transforming growth factor-1 (TGF-
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-
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- 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-
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-
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
v
6 (a critical mediator of TGF-
1 activation in lung and skin) were completely protected from bleomycin-induced pulmonary edema. This suggests that TGF-
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-1 on alveolar epithelial barrier properties, we evaluated effects of TGF-
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-
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.
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METHODS |
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Measurement of monolayer bioelectric properties. Rt (K · 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-
1 supplementation on bioelectric properties were evaluated on days 4 and 6 in culture for monolayers maintained with or without TGF-
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-
1 from days 4-8.
Effects of sodium transport inhibitors on Isc. To elucidate the mechanisms underlying the effects of TGF-1 on Isc, bioelectric properties of AEC monolayers in MDSF with or without TGF-
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-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-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
1-subunit (6H; M. Caplan, Yale Univ.) or a rabbit polyclonal antibody against the
-subunit of the rat epithelial sodium channel (
-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-
-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-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
1- or
1-subunit MAbs (
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.
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RESULTS |
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Rt was lower on days 4 and 6 in TGF-1-treated monolayers compared with MDSF, and there was a progressive decrease in Rt between days 4 and 6 in TGF-
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-
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-
1 (Fig. 1B).
Monolayers with or without TGF-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
· cm2 to an initial nadir of 1.60 ± 0.16 K
· cm2 (56% of baseline) within 6 h of addition of TGF-
1. Isc did not increase significantly from a baseline level of 2.01 ± 0.05 µA/cm2 until 72 h after addition of TGF-
1, reaching a maximum of 2.86 ± 0.19 µA/cm2 (136% of baseline) at 96 h.
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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-1 (50 pM). Apical (but not basolateral) amiloride (10 µM) immediately decreased Isc
75-85% in MDSF with or without TGF-
1 (Fig. 3A). The residual amiloride-insensitive current was similar in both TGF-
1-treated monolayers and MDSF controls. Addition of basolateral ouabain (1 mM) completely inhibited Isc over 30 min in MDSF with or without TGF-
1 (Fig. 3B). Ouabain- and amiloride-sensitive Isc were significantly greater in TGF-
1-treated monolayers.
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Effect of TGF-1 on monolayer cell number. Adherent cell number was determined for AEC monolayers cultivated in MDSF with or without TGF-
1 on days 1, 4, and 6. There were no significant differences in cell number in MDSF plus TGF-
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-
1-treated monolayers compared with control monolayers.
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Effects of TGF-1 on Na+-K+-ATPase
1- and
1-subunit expression. Protein was harvested from equivalent numbers of AEC from monolayers cultivated in MDSF with or without TGF-
1. This protein was analyzed on day 6 for expression of the
1-subunit of Na+-K+-ATPase by Western blotting. As shown in Fig. 5,
1-subunit protein was significantly increased in AEC exposed to TGF-
1 at all concentrations evaluated. Analysis of the
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.
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Immunofluorescence was also used to compare cell surface-associated expression of 1- and
1-subunit proteins in AEC monolayers in MDSF with or without TGF-
1. As shown in Figs. 6 and 7, immunoreactivity for both
1- and
1-subunits of Na+-K+-ATPase was increased in TGF-
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.).
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Effect of TGF-1 on
-rENaC expression. Protein from equivalent numbers of AEC from monolayers cultivated in MDSF with or without TGF-
1 was analyzed on day 6 for
-rENaC protein expression by Western blot. As shown in Fig. 8,
-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-
1. Expression of
-rENaC was similar in 50 pM TGF-
1-treated monolayers compared with control monolayers maintained in MDSF. In one pilot experiment, monolayers exposed to 100 pM TGF-
1 had equivalent
-rENaC expression compared with MDSF or 50 pM TGF-
1 (data not shown). Rat renal cortex was used as a positive control. To assure specificity of the antibody, in some experiments the anti-
-rENaC antibody was first adsorbed with an
-rENaC-specific control peptide with consequent disappearance of the prominent bands (data not shown).
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DISCUSSION |
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Similar effects of TGF- 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-
1, possibly due to activation of a myosin light chain kinase (MLCK)-dependent signaling cascade (23). TGF-
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-
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-
1 type II receptor with the adherens junction and association of the TGF-
1 signaling intermediates Smad3 and Smad4 with
-catenin after dissociation of
-catenin from the adherens junction complex.
Rt was recently reported to decrease in response to TGF-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-
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-
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-
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-1 suggests that TGF-
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-
1 directly upregulated Isc and active transepithelial sodium transport. Concurrent with the increase in Isc, we demonstrated upregulation of both the
1-and
1-subunits of the Na+-K+-ATPase but not of
-rENaC. This suggests that the effects of TGF-
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-
1-treated monolayers, levels of total sodium pump protein were similar at all concentrations of TGF-
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-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-
1 exposure will require further investigation (1, 29).
Despite the effects of TGF-1 on Isc and an increase in amiloride-sensitive sodium transport,
-rENaC levels did not increase with TGF-
1 exposure. ENaC regulation and expression are complex. ENaC is a heteromultimeric protein formed by three subunits,
-,
-, and
-, 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,
-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
-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
-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 -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-
1 was associated with an increase in amiloride-sensitive current without an increase in total
-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-
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-
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-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-
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-
1-induced apoptosis contributes to the observed reduction in monolayer cell number. In addition, TGF-
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-
1.
In summary, we have demonstrated that TGF-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
-ENaC was not. Consistent with the complexity of its effects in other cell types, TGF-
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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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E. D. Crandall is Hastings Professor and Kenneth T. Norris Jr. Chair of Medicine.
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FOOTNOTES |
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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.
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REFERENCES |
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