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
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ABSTRACT |
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T1 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 T1
expression using
either soluble factors or changes in cell shape to influence phenotype.
For studies on the effects of soluble factors on T1
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 T1
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 T1
expression was quantified by Northern and Western blotting, respectively. Expression of T1
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 T1
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 T1
on
days 4 and
8. T1
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 T1
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
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INTRODUCTION |
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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.
T1 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 T1
is developmentally
regulated, with a marked increase late in gestation concurrent with the
appearance of AT1 cells in the fetal lung (35, 45). T1
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 T1
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 T1
promoter
(34).
In this study, we evaluated the effects of modulating AEC
phenotype in vitro on T1 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 T1
can
be regulated by both soluble factors and changes in cell shape.
Expression of T1
remains low or is reduced by conditions that
promote retention of the AT2 cell phenotype, whereas T1
expression
is increased by conditions that promote transition toward the AT1 cell
phenotype. Our results indicate that reversible changes in T1
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.
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METHODS |
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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 T1Western 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-T1Within 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 T1KGF. To evaluate the effects of KGF on
T1 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
T1
expression. To determine whether T1
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 T1
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 T1 by day 4, consistent with the cells
undergoing transition toward an AT1 cell phenotype. To test whether
expression of T1
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 T1
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 T1
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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Time Course of T1 Expression in AEC Monolayers
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Effects of Serum on T1 Expression in AEC
Monolayers
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Effects of KGF on T1 Expression
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Modulation of T1 Expression in AEC Cultured on
Detached Collagen Gels
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DISCUSSION |
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T1 is a novel gene that, in the
adult rat lung, is expressed exclusively by AT1 cells. T1
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 T1
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 T1
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 T1
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 T1
in a manner consistent with reversal of the
transition from the AT2 toward the AT1 cell phenotype. Our results
indicate that expression of T1
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 T1 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 T1
mRNA expression, respectively, compared with those
grown in MDSF (Fig. 2B). Substantial
decreases in T1
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 T1
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 T1
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 T1
protein abundance of
any of the three sets of conditions (Fig. 7).
Analysis of differences in the relative amounts of T1 mRNA and
protein present may offer some insight into the levels at which
regulation of T1
is affected under each condition. The relatively
large effect of the soluble factors RS and KGF on reduction of T1
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
T1
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 T1
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 T1
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 T1
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 T1 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 T1 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 T1
expression could be modulated by
conditions that influence cell shape.
Although our current data demonstrate subtle variations in regulation
of T1 expression by different experimental conditions, neither these
conditions nor any others have thus far been shown to alter T1
expression without also causing changes in AEC phenotype. T1
is
present and undergoes developmental regulation in tissues other than
lung epithelium, however, implying that its expression cannot be solely
dependent on AEC phenotype. T1
mRNA is abundantly expressed during
development in the brain, foregut, and kidney. In the adult, T1
protein becomes restricted to the lung epithelium, choroid plexus of
the central nervous system, and ciliary epithelium of the eye (35, 45).
T1
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 T1
during lung development suggests that postnatal
restriction of expression of T1
to AT1 cells is likely the result of
active inhibition of its expression in other cell types. How
suppression of T1
expression occurs in other tissues is currently
unknown. Nonetheless, further identification of specific factors that
regulate T1
expression in cells other than AEC could provide
valuable insights into regulation of both T1
expression in alveolar
epithelium and AEC differentiation (34).
Although the functional properties of T1 remain unknown, its tissue
distribution has led to speculation about possible involvement of T1
in fluid and ion transport (45). Recent preliminary data indicate that
T1
does not serve as a water channel in alveolar epithelium (22).
Nonetheless, the possibility remains that T1
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 T1
expression do not
suggest a direct involvement of T1
in active sodium transport,
further studies are indicated to delineate the role of T1
, an apical
membrane protein, in this and other transmembrane processes.
The results of the present study indicate that T1 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 T1
, whereas reinduction of the
AT2 cell phenotype on later days in culture is accompanied by a
reciprocal reduction in T1
expression. Although no effects on T1
expression independent of changes in AEC phenotype were observed,
differences in the effects of RS, KGF, and cultivation on collagen gels
on T1
mRNA and protein levels suggest different mechanisms of
modulation. The physiological role of T1
, the mechanisms by which
its expression is regulated, and the relationship between T1
expression and AEC differentiation are all subjects of great potential
interest that merit additional study.
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ACKNOWLEDGEMENTS |
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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 T1 monoclonal antibody.
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FOOTNOTES |
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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.
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