Modulation of T1alpha expression with alveolar epithelial cell phenotype in vitro

Zea Borok, Spencer I. Danto, Richard L. Lubman, Yuxia Cao, Mary C. Williams, and Edward D. Crandall

Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033; and The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1alpha expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1 (DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1alpha expression was quantified by Northern and Western blotting, respectively. Expression of T1alpha progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1alpha expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1alpha on days 4 and 8. T1alpha expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1alpha expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

collagen gel; keratinocyte growth factor; rat serum; transdifferentiation; type I cell

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ALVEOLAR EPITHELIUM consists of two morphologically distinct cell types. Alveolar type II (AT2) cells are cuboidal in shape and reside primarily in alveolar corners, where they serve as the primary sites of surfactant synthesis and secretion. The thin, expansive processes of alveolar type I (AT1) cells, the functional properties of which remain largely unknown, form most of the gas-exchange surface of the lung. On the basis of studies in adult animals after acute lung injury, AT1 cells have been presumed to be terminally differentiated and incapable of self-renewal (1, 16). AT2 cells, which have the potential for both proliferation and differentiation into AT1 cells, are considered to be the progenitors of AT1 cells in the adult animal in vivo. Current evidence suggests that conversion from the AT2 to AT1 cell phenotype may occur by transdifferentiation, a process by which a fully differentiated cell changes phenotype without undergoing cell division (41).

AT2 cells cultivated on inflexible substrata lose their phenotypic hallmarks and acquire the morphological features of AT1 cells. Concurrent with the loss of lamellar bodies and other characteristics of differentiated AT2 cells, alveolar epithelial cells (AEC) in culture cease to express AT2 cell-specific genes and gradually express a number of recently described markers specific for AT1 cells in vivo (8, 11, 14, 27, 35). Data from several recent studies suggest that AT2 cells are capable of transdifferentiating toward an AT1 cell phenotype in vitro as they do in situ (10, 38).

Culture conditions have been established that profoundly influence the transitions between AT2 and AT1 cell phenotypes in vitro (4, 6, 10, 15, 38, 40). With the use of specific markers for both AT2 and AT1 cells, it has been demonstrated that AEC phenotype can be modulated in vitro by interactions with soluble factors. We have recently demonstrated that inclusion of autologous serum in culture media markedly inhibits the progression of rat AT2 cells toward the AT1 cell phenotype, as determined by morphology and binding of the AT1 cell-specific monoclonal antibody VIIIB2 (4). Keratinocyte growth factor (KGF), an epithelial mitogen that stimulates rat AT2 cell proliferation both in vitro and in vivo, has also recently been shown to influence AEC phenotype. KGF upregulates expression of surfactant apoproteins SP-A, -B, and -C, markers of the AT2 cell phenotype, while reciprocally inhibiting expression of aquaporin-5, a specific marker of AT1 cells in the adult lung (5, 40). Treatment with KGF also promotes AT2 cell morphology in cultured AEC, including the presence of lamellar bodies, even in cells that have partially undergone transition toward the AT1 cell phenotype.

Interactions with the extracellular matrix, as well as matrix-induced changes in cell shape, also influence AEC phenotype (15). Culture of AEC on detached collagen gels, which contract and thus limit cell spreading, results in either maintenance or reinduction of AT2s cell characteristics as a result of changes in cell shape (10, 38). Retention of aspects of the AT2 cell phenotype on detached gels is associated with a coordinate decrease in monoclonal antibody VIIIB2 binding (10), consistent with the concept that conditions that promote the AT2 cell phenotype reciprocally downregulate expression of AT1 cell phenotypic characteristics.

T1alpha is a recently identified gene that is expressed in choroid plexus, ciliary epithelium, and adult rat lung (45). Its expression is restricted specifically to the apical membrane of AT1 cells in the adult lung, making it a useful new marker for the AT1 cell phenotype. Expression of T1alpha is developmentally regulated, with a marked increase late in gestation concurrent with the appearance of AT1 cells in the fetal lung (35, 45). T1alpha expression in AEC grown on inflexible substrata increases with time in culture, consistent with the cells undergoing transition toward the AT1 cell phenotype (14). The specific factors that regulate T1alpha expression in vivo are currently unknown, although in vitro studies indicate that TGT3 (= hepatic nuclear factor-3), thyroid transcription factor 1, and Sp1 elements regulate transcriptional activity of the T1alpha promoter (34).

In this study, we evaluated the effects of modulating AEC phenotype in vitro on T1alpha expression using soluble factors and changes in cell shape that have previously been shown to profoundly influence AEC phenotype as determined by cell morphology and expression of AT1 and AT2 cell markers (4, 5, 10). Our results demonstrate that, similar to other markers of AT1 cell phenotype, expression of T1alpha can be regulated by both soluble factors and changes in cell shape. Expression of T1alpha remains low or is reduced by conditions that promote retention of the AT2 cell phenotype, whereas T1alpha expression is increased by conditions that promote transition toward the AT1 cell phenotype. Our results indicate that reversible changes in T1alpha expression occur during manipulation of AEC phenotype and provide additional support for the concept that progression toward the AT1 cell phenotype is at least partially reversible.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell Isolation and Culture

AT2 cells were isolated from adult male Sprague-Dawley rats by disaggregation with elastase (2.0-2.5 U/ml; Worthington Biochemical, Freehold, NJ) followed by panning on IgG-coated bacteriological plates (13). The enriched AT2 cells were resuspended in defined serum-free medium (MDSF) consisting of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma Chemical, St. Louis, MO) supplemented with 1.25 mg/ml bovine serum albumin (BSA; Collaborative Research, Bedford, MA), 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) or onto collagen gels according to the experimental design described below. Media were changed on the second day after plating and every other day thereafter. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. AT2 cell purity (>90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (25). Cell viability (>90%) was measured by trypan blue dye exclusion. All other chemicals were purchased from Sigma Chemical and were of the highest commercial quality available.

RNA Isolation and Northern Analysis

Total RNA was isolated by the acid phenol-guanidinium-chloroform method of Chomczynski and Sacchi (7). Equal amounts of RNA (5 or 10 µg) were denatured with formaldehyde, size fractionated by agarose gel electrophoresis under denaturing conditions, and transferred to nylon membranes (Hybond N+; Amersham Life Science, Cleveland, OH). RNA was immobilized by ultraviolet cross-linking. Blots were prehybridized for 2 h at 65°C in 1 M NaPO4 buffer (pH 7), 7% SDS, and 1% BSA. Hybridization was performed for 16 h at 65°C in the same buffer. Blots were probed with a T1alpha -specific cDNA probe (35) labeled with [alpha -32P]dCTP (Amersham) by the random-primer method using a commercially available kit (Boehringer Mannheim, Indianapolis, IN). Blots were washed at high stringency [0.5× SSC (75 mM NaCl-7.5 mM sodium citrate, pH 7.0) with 0.1% SDS at 55°C] and visualized by autoradiography. Differences in RNA loading were normalized using a 24-mer oligonucleotide probe for 18S rRNA end labeled with [32P]ATP. Relative amounts of T1alpha mRNA were determined using scanning densitometry.

Western Blotting

After washing two times in phosphate-buffered saline, pH 7.2, AEC were lysed at 4°C in equal volumes per monolayer of buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris · HCl, pH 8.0, to which was added phenylmethylsulfonyl fluoride (0.1 mM), leupeptin (0.1 µg/ml), and pepstatin (0.1 µg/ml). SDS-polyacrylamide gel electrophoresis was performed using standard methods before Western blotting. Equal amounts of protein were loaded onto each lane as determined by the Bradford assay (Bio-Rad, Hercules, CA). Gels were transferred to nitrocellulose membranes (Bio-Rad) by electrophoresis. After transfer, membranes were placed in Tris-buffered saline (TBS) containing 5% nonfat dry milk and gently agitated for 1 h at room temperature. Blots were then washed with TBS and incubated overnight at 4°C in a 1:1,000 dilution of hybridoma supernatant containing the anti-T1alpha monoclonal antibody as previously described (45). Blots were washed in TBS containing 0.05% Tween 10 (TBST) and incubated for 2 h at room temperature in alkaline phosphatase-labeled goat anti-mouse IgG (Cappel, Durham, NC) diluted to 1:7,000 in TBST. After extensive washing in TBST, color was developed by 30 min of incubation in the phosphatase substrate 5-bromo-4-chloro-3-indoloyl phosphate and nitro blue tetrazolium. As previously reported, only one immunoreactive band was detected in all samples tested (45). Secondary antibody controls do not show detectable bands at any molecular weight. Relative amounts of T1alpha protein were determined using scanning densitometry (45).

Within the range of protein concentrations observed, the Western blot signals were linear and reproducible. Samples were run at several different protein concentrations above and below those used for the final experiments reported herein to demonstrate for each cell sample that densitometric results were in the linear range. As a further control, a standard sample of whole rat lung protein was included in each blot to control for small blot-to-blot variations in the intensity of the alkaline phosphatase-catalyzed color reaction.

Experimental Design

Rat serum. To evaluate the effects of rat serum (RS) on T1alpha expression, freshly isolated AT2 cells were plated at a density of 1 × 106/cm2 on polycarbonate filters in MDSF or in MDSF supplemented with either 10% newborn bovine serum (BS) or 5% RS from day 0. RNA and protein were harvested from AEC on days 0, 1, and 8 in culture for analysis of T1alpha expression. To explore the potential for reversal of T1alpha expression on later days in culture, cells maintained in MDSF or MDSF + BS from the time of plating to day 4 (at which time cells already express abundant T1alpha mRNA and protein) were changed to MDSF + RS for an additional 4 days. Cells were harvested on day 8 for analysis of RNA and protein for T1alpha expression.

KGF. To evaluate the effects of KGF on T1alpha expression with time in culture, cells were plated at a density of 1 × 106/cm2 on polycarbonate filters from day 0 in either MDSF or MDSF supplemented with KGF (10 ng/ml; R&D Systems, Minneapolis, MN). Monolayers in MDSF ± KGF were harvested on days 0, 1, and 8 for analysis of mRNA and protein for T1alpha expression. To determine whether T1alpha expression could be reversed by addition of KGF at later times in culture, cells maintained in MDSF until day 4 were switched to media supplemented with KGF (10 ng/ml) for an additional 4 days. RNA and protein were isolated from control and KGF-treated monolayers on days 6 and 8 for analysis of T1alpha expression.

Collagen gels. Thick collagen gels were prepared using rat tail collagen (Collaborative Research) as previously described but without a fibroblast feeder layer (10). Briefly, 0.3 ml of neutralized collagen (3 mg/ml) were gelled in each well of 24-well cluster dishes. AT2 cells were then plated onto the gels in MDSF + 10% BS at a density of 5 × 105/cm2. Cells were cultivated on attached gels for 4 (AG4) or 8 (AG8) days. Under these conditions, AEC express high levels of T1alpha by day 4, consistent with the cells undergoing transition toward an AT1 cell phenotype. To test whether expression of T1alpha could be inhibited by changes in cell shape, gels were detached on day 1 (DG1; which permits the gels to contract, allowing the cells to assume a more cuboidal morphology) and maintained as floating gels until day 8. RNA and protein were harvested on days 1 and 8. To test for reversibility of T1alpha expression, gels were detached on day 4 (DG4) and maintained in culture until day 8. Cells were harvested on days 4 and 8 for analysis of T1alpha mRNA and protein.

Statistical Analysis

Results are expressed as means ± SE. Significance (P < 0.05) of differences was determined by Student's t-test or, where multiple time points were compared, by one-way analysis of variance.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Time Course of T1alpha Expression in AEC Monolayers

Expression of T1alpha mRNA and protein was analyzed by Northern and Western blotting, respectively, in freshly isolated AT2 cells and in AEC monolayers cultured on polycarbonate filters for 1 and 8 days in MDSF (Fig. 1, A and B). T1alpha mRNA was minimally detectable in AT2 cells and increased dramatically in cultured AEC from day 1 to day 8. T1alpha protein showed similar changes, with marked relative increases noted from day 1 to day 8.


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Fig. 1.   Time course of T1alpha expression in alveolar epithelial cells (AEC) cultured in defined serum-free medium (MDSF) on Nuclepore filters. A: as shown in this representative Northern blot, T1alpha mRNA levels are minimally detectable in freshly isolated alveolar type II (AT2) cells (d.0) and increase between day 1 (d.1) and day 8 (d.8). RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for freshly isolated AT2 cells (d.0 = 100%) indicates that no difference in T1alpha mRNA levels was present in AEC between day 0 and day 1, whereas an increase in T1alpha mRNA was present in AEC on day 8 relative to day 0 and day 1; n = 6 for all conditions. * Significantly different from d.0 and d.1. B: AEC grown in MDSF express increased T1alpha protein between days 1 (d.1) and 8 (d.8), as indicated by the single 36-kDa band of immunoreactive protein shown in this representative Western blot. T1alpha protein levels are minimally detectable in freshly isolated AT2 cells (d.0) and increase in parallel to mRNA levels for AEC between day 1 and day 8. Protein loading was approximately equal for all conditions. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for freshly isolated AT2 cells (d.0 = 100%) indicates that no difference in T1alpha protein abundance was present in AEC between day 0 and day 1, whereas an increase in T1alpha protein abundance was present in AEC on day 8 relative to day 0 and day 1; n = 3 for all conditions. * Significantly different from d.0 and d.1.

Effects of Serum on T1alpha Expression in AEC Monolayers

Expression of T1alpha mRNA and protein was analyzed by Northern and Western blotting, respectively, in AEC monolayers cultured on polycarbonate filters for 1 and 8 days in the presence or absence of either BS or RS. T1alpha mRNA expression increased dramatically in cultured AEC from day 1 to day 8 in either MDSF or MDSF + BS (Fig. 2A). In contrast, induction of T1alpha mRNA was prevented on day 8 when cells were grown in medium supplemented with RS from the time of plating, with levels of T1alpha on day 8 showing no increase relative to day 1. In experiments in which media were changed on day 4 from MDSF ± BS to MDSF + RS (Fig. 2B), T1alpha gene expression was markedly decreased on day 8 compared with monolayers maintained in MDSF until day 8. These effects on T1alpha mRNA were reflected by parallel changes in T1alpha protein (Fig. 3). Levels of T1alpha protein were reduced in AEC grown in MDSF + RS from day 1 through day 8 compared with AEC grown in MDSF ± BS for the same period. For AEC grown in MDSF + BS from the time of plating until day 4 and then in MDSF + RS from day 4 through day 8, levels of T1alpha protein were decreased on day 8 compared with monolayers maintained in MDSF + BS throughout the culture period.


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Fig. 2.   Effects of serum on T1alpha mRNA in cultured AEC. A: as shown in this representative Northern blot, AEC monolayers grown in MDSF ± bovine serum (BS) express increased levels of T1alpha mRNA on day 8 relative to day 1. When cells are plated in the presence of rat serum (RS), induction of T1alpha mRNA is markedly inhibited on day 8. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for AEC grown in MDSF on day 1 (d.1 MDSF = 100%) indicates that no differences in T1alpha mRNA were present in AEC cultivated in MDSF or MDSF + BS or MDSF + RS on day 1, or AEC grown in MDSF + RS on day 8, whereas a significant increase in T1alpha mRNA level was present on day 8 in AEC grown in either MDSF or MDSF + BS; n = 3 for all conditions. * Significantly different from d.1 MDSF, d.1 MDSF + BS, d.1 MDSF + RS, and d.8 RS. B: this representative Northern blot demonstrates that, when media are changed from MDSF to MDSF + RS on day 4 (d.4right-arrow8 MDSFright-arrowMDSF + RS), T1alpha gene expression is markedly decreased on day 8 compared with monolayers maintained from day 0 until day 8 in MDSF or MDSF + BS. These findings are consistent with at least partial reversal of AEC phenotype by RS. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for monolayers grown in MDSF from day 0 through day 8 (d.8 MDSF = 100%) indicates that no difference in T1alpha mRNA was present between AEC cultivated in MDSF or MDSF + BS from day 0 through day 8, whereas significant decreases in T1alpha mRNA levels were present in AEC grown in MDSF + RS from day 0 through day 8 or in MDSF from day 0 through day 4 followed by MDSF + RS from day 4 through day 8. No statistically significant difference was observed for monolayers grown in MDSF + BS from day 0 through day 4 followed by MDSF + RS from day 4 through day 8 (d.4right-arrow8 MDSF + BSright-arrowMDSF + RS) relative to other conditions; n = 5 for all conditions. * Significantly different from d.8 MDSF. ** Significantly different from d.8 MDSF and d.8 MDSF + BS.


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Fig. 3.   Effects of serum on T1alpha protein in cultured AEC. As illustrated in this representative Western blot, T1alpha protein abundance is decreased in AEC grown in MDSF + RS from day 0 through day 8 relative to AEC maintained for the same period in MDSF. T1alpha protein abundance was also decreased on day 8 in AEC grown from day 0 to day 8 in MDSF + RS or from day 0 through day 4 in MDSF + BS and from day 4 through day 8 in MDSF + RS (d.4right-arrow8 MDSF + BSright-arrowMDSF + RS) relative to AEC maintained in MDSF + BS from day 0 through day 8. Protein loading was approximately equal for all conditions. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha protein abundance in AEC grown in MDSF from day 0 through day 8 (d.8 MDSF = 100%) indicates that no difference in T1alpha protein abundance was present in AEC grown from day 0 through day 8 in MDSF vs. MDSF + BS, whereas a relative decrease was present in AEC grown in MDSF + RS from day 0 through day 8; n = 3 for all conditions. * Significantly different from d.8 MDSF+RS, d.4right-arrow8 MDSFright-arrowMDSF + RS, or d.4right-arrow8 MDSF + BSright-arrowMDSF + RS. ** Significantly different from d.8 MDSF and d.8 MDSF + BS.

Effects of KGF on T1alpha Expression

Cultivation of AEC in the presence of KGF (10 ng/ml) from the time of plating markedly decreased T1alpha mRNA on day 8 (Fig. 4A) compared with untreated monolayers maintained in MDSF. Abundance of T1alpha mRNA for cells grown in the presence of KGF was similar on day 8 to levels in untreated cells on day 1. Addition of KGF to AEC monolayers from day 4 resulted in a progressive decline in T1alpha mRNA on subsequent days in culture (Fig. 4B). T1alpha mRNA levels in AEC treated with KGF on day 4 were decreased by day 6 relative to monolayers maintained in MDSF without KGF, with a further decrease noted on day 8. Similar effects of KGF on T1alpha protein were noted (Fig. 5). After addition of KGF on day 4, levels of T1alpha protein on day 8 were reduced relative to untreated monolayers in MDSF, consistent with the ability of KGF to both inhibit and reverse expression of T1alpha in cultured AEC.


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Fig. 4.   Effects of keratinocyte growth factor (KGF) on T1alpha mRNA in cultured AEC. A: as indicated in this representative Northern blot, AEC monolayers grown in MDSF express increased levels of T1alpha mRNA on day 8 relative to day 1. Cultivation of AEC in the presence of KGF from the time of plating inhibits expression of T1alpha mRNA relative to monolayers in MDSF on day 8. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for monolayers grown in MDSF on day 1 (d.1 MDSF = 100%) indicates that no differences in T1alpha mRNA levels were present in AEC grown in MDSF or MDSF + KGF on day 1 or MDSF + KGF on day 8, whereas a significant increase in T1alpha mRNA level was present in AEC grown in MDSF on day 8; n >=  3 for all conditions. * Significantly different from all other conditions. B: as shown in this representative Northern blot, exposure to KGF from day 4 results in a decrease in AEC T1alpha mRNA levels on day 6 (MDSF + KGF d.4right-arrowd.6) and day 8 (MDSF + KGF d.4right-arrowd.8) compared with those in AEC maintained in MDSF through day 6 or day 8, respectively, consistent with at least partial reversal of the transition toward the alveolar type I (AT1) cell phenotype by KGF. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for monolayers grown in MDSF on the same day in culture indicates that T1alpha mRNA levels were relatively reduced in AEC grown in MDSF + KGF from day 4 through 6 compared with AEC grown in MDSF through day 6 and further reduced on day 8; n = 8 for all conditions. * Significantly different from MDSF d.6. ** Significantly different from MDSF d.8.


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Fig. 5.   Effects of KGF on T1alpha protein. As shown in this representative Western blot, exposure of AEC monolayers to KGF from day 4 results in a decrease in AEC T1alpha protein abundance on day 8 (MDSF + KGF d.4right-arrowd.8) compared with those maintained in MDSF on day 8, consistent with at least partial reversal of the transition toward the AT1 cell phenotype by KGF. Protein loading was approximately equal for all conditions. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha protein abundance for monolayers grown in MDSF on the same day in culture indicates that T1alpha mRNA levels were relatively reduced in AEC grown in MDSF + KGF from day 4 through 8 compared with AEC grown in MDSF through day 8; n = 2 for MDSF d.6 and MDSF + KGF d.4right-arrowd.6; n = 4 for MDSF d.8 and MDSF + KGF d.4right-arrowd.8. * Significantly different from MDSF d.8.

Modulation of T1alpha Expression in AEC Cultured on Detached Collagen Gels

AEC cultured on attached collagen gels for 4 (AG4) or 8 (AG8) days express high levels of T1alpha mRNA relative to cells harvested on day 1 (AG1; Fig. 6). When gels are detached on day 1, allowing the gels to contract, levels of T1alpha on day 8 are reduced relative to AG4 or AG8, consistent with inhibition of T1alpha induction under these conditions. Detachment of gels on day 4, once abundant levels of T1alpha mRNA are already expressed, results in decreased expression of T1alpha on day 8 relative to AG4 and AG8, indicating reversal of T1alpha expression by changes in cell shape. Similar effects are observed on T1alpha protein (Fig. 7), consistent with both inhibition and reversal of T1alpha expression when AEC are grown on detached collagen gels.


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Fig. 6.   Modulation of T1alpha mRNA expression in AEC cultured on detached collagen gels. AEC cultured on attached collagen gels for 4 (AG4) or 8 (AG8) days express increased levels of T1alpha mRNA relative to day 1 (AG1) by Northern analysis. As shown in this representative experiment, detachment of gels on day 1 (DG1) inhibits expression of T1alpha mRNA relative to AG4 or AG8 when DG are harvested on day 8 (DG1right-arrowD8). Cultures maintained initially as attached gels and detached on day 4 (DG4right-arrowD8) manifest a decrease in T1alpha expression on day 8 relative to AG4 and AG8, consistent with reversal of AT1 cell phenotype. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha mRNA levels for monolayers grown on attached gels on day 1 (AG1 = 100%) indicates that T1alpha mRNA levels were increased for AEC monolayers grown on attached gels on day 4 and day 8 and were relatively reduced for day 8 AEC monolayers grown on gels detached on either day 1 or day 4. DG1right-arrowD4, AEC grown on collagen gels detached on day 1 and harvested on day 4; n >=  2 for all conditions. * Significantly different from AG1, DG1right-arrowD8, and DG4right-arrowD8.


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Fig. 7.   Modulation of T1alpha protein in AEC cultured on detached collagen gels. As indicated in this representative Western blot, T1alpha protein is increased relative to day 1 (AG1) in cells grown on attached collagen gels for 4 (AG4) or 8 (AG8) days. Detachment of gels on day 1 results in a decrease in T1alpha protein abundance in AEC monolayers on day 4 (DG1right-arrowD4) or day 8 (DG1right-arrowD8) relative to that for AEC monolayers grown on attached gels for 8 days (AG8), consistent with inhibition of the transition toward the AT1 cell phenotype. Detachment of gels on day 4 results in a decrease in T1alpha protein on day 8 (DG4right-arrowD8) relative to AEC grown on attached gels for 8 days, in parallel with the changes in T1alpha mRNA. Protein loading was approximately equal for all conditions. Bar graph showing average relative densitometric values (± SE) normalized to T1alpha protein abundance for monolayers grown on attached gels on day 1 (AG1 = 100%) indicates that T1alpha protein abundance was increased for AEC monolayers grown on attached gels on day 4 and day 8. T1alpha protein abundance was relatively reduced for day 4 AEC monolayers grown on gels detached on day 1 and for day 8 AEC monolayers grown on gels detached on either day 1 or day 4; n = 2 for all conditions. * Significantly different from AG1. ** Significantly different from AG1, DG1right-arrowD4, DG1right-arrowD8, and DG4right-arrowD8.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

T1alpha is a novel gene that, in the adult rat lung, is expressed exclusively by AT1 cells. T1alpha is absent or expressed at low levels in populations of freshly isolated AT2 cells but is rapidly induced after cell isolation (14). Previous attempts to modulate T1alpha expression using soluble factors (e.g., phorbol esters and epidermal growth factor) in cultured AEC have been unsuccessful (35). We demonstrate in this study that AEC grown in either MDSF or medium supplemented with BS express increasing amounts of T1alpha mRNA and protein with time in culture (Figs. 1 and 2). In the presence of conditions previously shown to promote retention of AT2 cell phenotype [KGF or RS from the time of plating or culture on collagen gels that are detached within 24 h after plating (4, 5, 10, 38)], the increase in T1alpha expression on day 8 is prevented. Addition of KGF or detachment of the gels at later times in culture, after AT1 cell characteristics are already expressed, has been shown to induce reversion toward the AT2 cell phenotype (5, 10, 38). We demonstrate herein that these conditions also partially reverse the increase in T1alpha in a manner consistent with reversal of the transition from the AT2 toward the AT1 cell phenotype. Our results indicate that expression of T1alpha can be modulated by both specific extracellular soluble factors and alterations in cell shape, in parallel with previously described changes in AEC phenotype.

Although expression of T1alpha consistently follows changes in AEC phenotype, subtle differences in the degree to which this occurs are evident among the different sets of conditions investigated in this study. Growth of AEC monolayers in the presence of RS from day 0 through day 8 or day 4 through day 8 resulted in ~50 and ~75% reductions in T1alpha mRNA expression, respectively, compared with those grown in MDSF (Fig. 2B). Substantial decreases in T1alpha protein abundance, although somewhat smaller for AEC monolayers grown in RS from day 4 through day 8 compared with those grown in RS from day 0 through day 8, were seen under both conditions, as determined by immunoblot (Fig. 3). KGF had a qualitatively similar, although slightly less profound, effect than RS on T1alpha mRNA expression when present from either day 4 through day 6 (~30% decrease) or day 4 through day 8 (~50% decrease; Fig. 4), again with correspondingly smaller effects on protein abundance (Fig. 5). Cultivation of AEC on detached collagen gels resulted in marginally greater suppression of T1alpha mRNA expression compared with RS (~50% decrease from day 1 through day 8 for AEC grown on detached gels; Fig. 6) but caused the greatest reduction of T1alpha protein abundance of any of the three sets of conditions (Fig. 7).

Analysis of differences in the relative amounts of T1alpha mRNA and protein present may offer some insight into the levels at which regulation of T1alpha is affected under each condition. The relatively large effect of the soluble factors RS and KGF on reduction of T1alpha mRNA and the correspondingly smaller effect on protein abundance suggest that these factors may exert their effects primarily via transcriptional mechanisms of regulation. The minimal difference in T1alpha protein abundance between longer (day 0 through day 8) and shorter (day 4 through day 8) KGF exposures (Fig. 5) can most easily be explained by a decrease in mRNA transcription and a parallel decrease in protein synthesis, coupled with a long turnover time (>36 h) for the protein as seen in baseline studies (Williams, unpublished observations). Alternatively, although T1alpha mRNA levels are substantially reduced by growth on detached vs. attached collagen gels, protein levels are reduced to an even greater degree by these conditions. In contrast to the effects of KGF (and to a lesser extent RS) on protein abundance, AEC cultivated on detached gels show a correspondingly larger decrease in abundance of T1alpha protein relative to the decline in mRNA levels when grown in that fashion from days 1 through 8 or days 4 through 8 (Figs. 6 and 7). These data suggest that the effects on T1alpha expression of cultivation of AEC on detached collagen gels occur at both pre- and posttranslational levels, with increased protein turnover likely occurring in addition to effects on mRNA transcription and/or stability. Taken together, these data are most consistent with effects at both transcriptional and translational and/or posttranslational levels that are different for soluble factors vs. manipulation of cell shape.

Despite the apparent similarities of the effects of KGF, RS, and cultivation on detached collagen gels on AEC phenotype, our finding that they differ in the degree of their effects on T1alpha expression is consistent with the very different nature of each stimulus. KGF is a polypeptide growth factor whose effects are restricted to epithelial cells. KGF acts as a mitogen but also stimulates differentiation in alveolar epithelium and other epithelia (5, 17, 19, 30). Evidence for the role of KGF as a modulator of cell phenotype comes from both in vivo and in vitro data (30, 42). KGF has been shown to play a role in the development of epithelial tissues and in maintenance of epithelial integrity and also directly influences the morphological appearance and phenotypic characteristics of a number of epithelial cell types during development and adult life (31, 37). KGF has been shown to reduce or prevent lung damage due to various mediators of oxidant injury, both as a result of its effects on AT2 cell proliferation and, as recently demonstrated, via stabilization of the actin cytoskeleton (24, 29, 43). KGF is expressed in many tissues at increased levels after injury, consistent with a role for KGF in normal wound healing and repair, and is thought to play a critical role in normal lung growth and development (12, 26, 32, 33, 39, 44). In contrast, RS is a complex mixture of serum proteins, lipids, and carbohydrate moieties whose constituents remain incompletely characterized. It contains many different soluble factors, although KGF is not present in detectable quantities (data not shown). Previous studies have demonstrated that RS is able to preserve AT2 cell morphology and pattern of phospholipid synthesis, consistent with the ability of RS to promote retention of the AT2 cell phenotype (9, 42). The specific components in RS responsible for the effects on phenotype are not known and may be multiple and synergistic in their effects.

Changes in cell shape exert effects on cell-specific gene expression and differentiation via multiple signaling pathways, including activation of integrin receptors and direct effects on the actin cytoskeleton (21, 23, 36). In light of the highly complex nature of this growth condition, it is not surprising that T1alpha expression appears to be affected by changes in cell shape at multiple levels and in ways that are probably different from the effects of RS and KGF. Effects of changes in cell shape on gene expression have been demonstrated in a variety of cell types, including hepatocytes and mammary epithelial cells, independent of effects of particular extracellular matrix ligands (18, 20). In addition to those studies cited above (10, 38), effects of changes in AEC cell shape on gene expression have also been demonstrated for intercellular adhesion molecule-1, with cultivation on detached collagen gels suppressing expression in a manner similar to that described herein (28). Acute stretch of AT2 cells has been shown to effect mobilization of intracellular Ca2+ stores, leading to increased surfactant secretion (46). Such changes in intracellular ion concentration are but one of many possible mechanisms by which AEC phenotype and T1alpha expression could be modulated by conditions that influence cell shape.

Although our current data demonstrate subtle variations in regulation of T1alpha expression by different experimental conditions, neither these conditions nor any others have thus far been shown to alter T1alpha expression without also causing changes in AEC phenotype. T1alpha is present and undergoes developmental regulation in tissues other than lung epithelium, however, implying that its expression cannot be solely dependent on AEC phenotype. T1alpha mRNA is abundantly expressed during development in the brain, foregut, and kidney. In the adult, T1alpha protein becomes restricted to the lung epithelium, choroid plexus of the central nervous system, and ciliary epithelium of the eye (35, 45). T1alpha is initially expressed throughout embryonic lung during development, subsequently becoming restricted to the more distal regions of the lung destined to become alveoli. The pattern of expression of T1alpha during lung development suggests that postnatal restriction of expression of T1alpha to AT1 cells is likely the result of active inhibition of its expression in other cell types. How suppression of T1alpha expression occurs in other tissues is currently unknown. Nonetheless, further identification of specific factors that regulate T1alpha expression in cells other than AEC could provide valuable insights into regulation of both T1alpha expression in alveolar epithelium and AEC differentiation (34).

Although the functional properties of T1alpha remain unknown, its tissue distribution has led to speculation about possible involvement of T1alpha in fluid and ion transport (45). Recent preliminary data indicate that T1alpha does not serve as a water channel in alveolar epithelium (22). Nonetheless, the possibility remains that T1alpha is a protein directly involved in either transport or regulation of transport. Although cultivation on collagen gels has not been specifically studied for effects on transport or expression of transport proteins, RS has been shown to increase short-circuit current, an index of active ion transport, across AEC monolayers (4). KGF has also been shown to stimulate short-circuit current and upregulate sodium pump expression in AEC, independent of its effects on cell proliferation (2). Although the opposing effects of KGF on sodium pump and T1alpha expression do not suggest a direct involvement of T1alpha in active sodium transport, further studies are indicated to delineate the role of T1alpha , an apical membrane protein, in this and other transmembrane processes.

The results of the present study indicate that T1alpha expression appears to be modulated in a similar fashion to other previously described markers of the AT1 cell phenotype. Maintenance of AT2 cell phenotype by either soluble factors or changes in cell shape is associated with a reciprocal decrease in expression of T1alpha , whereas reinduction of the AT2 cell phenotype on later days in culture is accompanied by a reciprocal reduction in T1alpha expression. Although no effects on T1alpha expression independent of changes in AEC phenotype were observed, differences in the effects of RS, KGF, and cultivation on collagen gels on T1alpha mRNA and protein levels suggest different mechanisms of modulation. The physiological role of T1alpha , the mechanisms by which its expression is regulated, and the relationship between T1alpha expression and AEC differentiation are all subjects of great potential interest that merit additional study.

    ACKNOWLEDGEMENTS

We note with appreciation the expert technical support of Martha Jean Foster and Monica Flores. We thank Dr. Antoinette Wetterwald (University of Bern, Switzerland) for kindly providing the T1alpha monoclonal antibody.

    FOOTNOTES

This work was supported in part by the American Lung Association, the American Heart Association, the American Heart Association-Greater Los Angeles Affiliate, National Heart, Lung, and Blood Institute Grants HL-02836 (Z. Borok), HL-03609 and HL-51928 (R. L. Lubman), HL-38578 and HL-38621 (E. D. Crandall), and HL-47049 (M. C. Williams), and the Hastings Foundation. E. D. Crandall is Hastings Professor of Medicine and Kenneth T. Norris, Jr., Chair of Medicine.

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. §1734 solely to indicate this fact.

Address for reprint requests: Z. Borok, Div. of Pulmonary and Critical Care Medicine, Univ. of Southern California, GNH 11-900, 2025 Zonal Ave., Los Angeles, CA 90033.

Received 6 February 1998; accepted in final form 1 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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