Influence of the cytoskeleton on surfactant protein gene
expression in cultured rat alveolar type II cells
John M.
Shannon,
Tianli
Pan,
Karen E.
Edeen, and
Larry D.
Nielsen
Department of Medicine, National Jewish Medical and Research Center,
and Division of Pulmonary Sciences and Critical Care Medicine,
University of Colorado Health Sciences Center, Denver, Colorado
80206
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ABSTRACT |
We have investigated
the role of the cytoskeleton in surfactant protein gene expression.
Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested
on primary cultures of adult rat alveolar type II cells. Treatment with
any of the drugs did not result in dramatic cell shape changes, but
ultrastructural examination revealed that the cytoplasm of cells
treated with CD was markedly disorganized; cells treated with Col did
not exhibit such changes. Treatment with any of the three drugs
resulted in a reduction in surfactant protein (SP) mRNAs. These
decreases were not the result of cell toxicity, since overall protein
synthesis was unimpaired by drug treatment. Washing the cells followed
by an additional 2 days of culture resulted in a reaccumulation of SP
mRNAs in CD-treated cells but not in Col-treated cells. Washing of
Noco-treated cultures resulted in partial recovery. SP mRNA stability
was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules
significantly affected the half-lives of mRNAs for SP-A, SP-B, and
SP-C. These data support a role for the cytoskeleton in the maintenance
of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.
lung; pulmonary surfactant
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INTRODUCTION |
A PRIMARY FUNCTION of alveolar type II cells is the
synthesis and secretion of pulmonary surfactant, which is a complex of phospholipids and the lung-specific surfactant protein (SP)-A, SP-B,
and SP-C (18). Because of the heterogeneity of cell types in the lung,
many studies on the regulation of type II cell differentiated function
have been carried out on purified type II cell populations in vitro.
One consistent result from these studies has been that type II cells
maintained on a tissue culture plastic substratum rapidly lose most
markers of differentiated function. A number of investigators have
attempted to improve maintenance of type II cell differentiation by
culturing cells on more biological substrata, with varying degrees of
success (24). Previous work from our laboratory has shown that type II
cells cultured on a reconstituted basement membrane gel derived from
the Engelbreth-Holm-Swarm (EHS) tumor, or in association with fetal rat
lung fibroblasts on floating collagen gels, exhibit significantly
improved maintenance of differentiation as gauged by morphology (14,
27, 29), patterns of phospholipid biosynthesis (14, 27, 29), production of SP-A protein (27), accumulation of mRNAs for SP-A, SP-B, and SP-C
(27, 28), and the ability to respond to hormones (14). We have
attributed the beneficial effects of these culture systems to the
establishment of requisite cell-extracellular matrix interactions and
cell-cell interactions, as well as to the maintenance of native type II
cuboidal cell shape.
The importance of normal cell shape to type II cell function is
underscored by our demonstration that known markers of type II cell
differentiation (morphology, patterns of phospholipid biosynthesis, and
accumulation of SP mRNAs) modulate with changes in cell shape (28).
Because cell shape reflects intracellular cytoskeletal organization, we
hypothesized that perturbations to cytoskeletal elements would result
in altered lung-specific gene expression. In this paper, we demonstrate
that disruption of either microfilaments or microtubules leads to the
depletion of mRNAs for SP-A, SP-B, and SP-C and that this results at
least in part from decreased mRNA stability.
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MATERIALS AND METHODS |
Isolation of alveolar type II cells.
Alveolar type II cells were dissociated from the lungs of specific
pathogen-free adult male Sprague-Dawley rats (Bantin Kingman, Fremont,
CA) with porcine pancreatic elastase (Worthington Biochemicals,
Freehold, NJ). Type II cells were further purified by the
immunoglobulin G panning method (9) and then resuspended in culture
medium, which was Dulbecco's modified Eagle's medium (DMEM; GIBCO
BRL, Gaithersburg, MD) containing 5% whole rat serum (Pel Freeze,
Rogers, AK), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml
amphotericin B (all from GIBCO BRL), and 10 µg/ml gentamicin sulfate
(Sigma Chemical, St. Louis, MO).
Cell culture method and drug
treatments. Two culture systems, both of which have
been shown to support improved type II cell differentiation, were used
in these experiments. The first system involves plating type II cells
onto a feeder layer of lethally irradiated fetal rat lung fibroblasts
grown on a rat tail collagen gel (29). Type II cells suspended in
culture medium were seeded onto irradiated day
19 fetal rat lung fibroblasts feeder layers at a
concentration of 5 × 105
cells/cm2; the day of seeding was
considered day 0 of culture. On
day 1 of culture, the medium was
changed to remove nonadherent type II cells, and then the
gel-fibroblast-type II cell complexes were detached from the culture
dish and allowed to float free in the medium (hereafter floating gel
cultures). In the second system, type II cells were seeded at a density
of 5 × 105
cells/cm2 into six-well cluster
dishes that contained 1 ml Engelbreth-Holm-Swarm (EHS) matrix
(Collaborative Biomedical Products, Waltham, MA). Depending on the
experimental design, cultures were maintained for 8-10 days in
both systems, with medium changes on alternate days.
Treatment with cytoskeleton-disrupting drugs was initiated on
day 7 of culture and was continued for
24 h. Microfilaments were disrupted with 5 µM cytochalasin D (CD;
Sigma) in dimethyl sulfoxide (Sigma; final concentration 0.1%).
Microtubules were disrupted with either 2.5 µM colchicine (Col;
Sigma), or 10 µM nocodazole (Noco; Sigma) dissolved in sterile
phosphate-buffered saline (PBS, pH 7.4). These concentrations have been
shown to be effective in disrupting cytoskeletal elements in other
systems (1-5, 11, 15, 33), including type II cells (7, 10). Medium
with no additions and vehicle-only additions served as controls. In
experiments designed to determine if the effects of the
cytoskeleton-disrupting drugs were reversible, treated cultures were
washed four times for 15 min with 5 ml of DMEM. Fresh culture medium
was then added, and the cultures were maintained an additional 2 days
until harvest.
Isolation of RNA and Northern blot
analysis. For RNA extraction, cultured cells were lysed
in 4 M guanidinium isothiocyanate, 0.5% laurylsarcosine, and 0.1 M
-mercaptoethanol in 25 mM sodium citrate buffer (GITC). RNA was
isolated by ultracentrifugation over a CsCl cushion, size fractionated
by electrophoresis through a denaturing 1% agarose gel, and
transferred to Nytran (Schleicher and Schuell, Keene, NH) by
capillary action. Northern blots were probed with cDNAs for rat SP-A,
SP-B, SP-C, human
-actin, and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) that had been radiolabeled to high specific
activity with
[
-32P]dCTP (ICN
Pharmaceuticals, Costa Mesa, CA) by random-primed second-strand
synthesis using a commercially available kit (GIBCO BRL). Hybridization
and washing of blots were performed as previously described (28).
Determination of mRNA stability. The
stability of mRNAs for SP-A, SP-B, SP-C, and GAPDH in the presence and
absence of cytoskeleton-inhibiting drugs was determined by measuring
their rate of degradation after inhibition of RNA transcription. These
experiments were only done using cells cultured on EHS gels. RNA
synthesis was inhibited by addition of actinomycin D (Act D; 10 mg/ml;
Calbiochem, La Jolla, CA); this concentration of Act D inhibited the
incorporation of radiolabeled uridine into type II cell RNA over a 12-h
period by 98% (data not shown). On day
8 of culture, cells were treated with Act D alone, Act
D plus CD, or Act D plus Col. Cells were harvested into GITC 3, 6, 9, and 12 h after addition of the drugs. Cultures treated with CD or Col
alone, their appropriate vehicle controls, or no additions served as
controls. RNA was isolated, and Northern blots were prepared. Direct
quantitation of hybridized signal was done using a phosphorus screen
and ImageQuant software version 3.3 (Molecular Dynamics, Sunnyvale,
CA).
Protein synthesis. After 20 h of
exposure to either CD, Col, Noco, or the appropriate vehicle control
beginning on day 7 of culture, cells
were incubated for 4 h in culture medium containing 25 µCi/ml
[35S]methionine
(Tran35S-Label; ICN). At the end
of the incubation period, floating gel cultures were washed two times
with PBS and then transferred to 9 ml of DMEM containing 10% fetal
bovine serum (GIBCO BRL) and 0.1% collagenase (CLS I; Worthington) and
were incubated at 37°C for 1-1.5 h to digest the gel. The
cells were collected by centrifugation at 200 g for 10 min, washed two times in PBS,
resuspended in 1 ml of PBS containing 1 mM phenylmethylsulfonyl
fluoride, 5 mM N-ethylmaleimide, and 5 mM EDTA (all from Sigma), and then stored at
20°C until
analysis. After being washed with PBS, the cells in EHS cultures were
digested free of the substratum by incubation with 2 ml of dispase
(Collaborative Biomedical) for 1-1.5 h at 37°C. The dispersed
cell aggregates were then collected, washed, and stored as described
for floating gel cultures.
Cell pellets were thawed on ice and then sonicated with two 30-s bursts
from a Branson 200 Sonifier equipped with a microtip. Duplicate
aliquots of lysate were precipitated with trichloroacetic acid (10%
final concentration) and collected on glass fiber filters (Whatman
GF/C, Hillsboro, OR), and then incorporated counts were determined by
scintillation counting and normalized to DNA content.
Morphology. Treated and untreated
cells cultured in the floating gel system were fixed in 2%
glutaraldehyde-4% paraformaldehyde, postfixed in 1.5% osmium
tetroxide, stained en bloc with uranyl acetate, and embedded in Polybed
812 (Polysciences, Warrington, PA). Blocks were sectioned perpendicular
to the plane of the culture substratum and stained as described
previously (29).
Statistics. Data were analyzed for
statistical significance by two-way analysis of variance (ANOVA) using
the JMP 3.0.2 computer software package (SAS Institute, Cary, NC). For
each condition, individual 95% confidence intervals were constructed
using a pooled estimate of the standard error obtained from the ANOVA
table. A value of P < 0.05 was
considered statistically significant.
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RESULTS |
Morphology. Treatment with either CD,
Col, or Noco had no effect on the gross morphology of cultured type II
cells when viewed by light microscopy. As shown in Fig.
1, cross sections of type II cells cultured
on floating gels demonstrated that the cells retained a cuboidal
morphology in all treatment groups. When viewed in the electron
microscope, however, clear differences were observed. Untreated type II
cells cultured on floating collagen gels appeared as previously
described (28, 29). The cells had a cuboidal shape and contained
abundant osmiophilic lamellar bodies (Fig. 2). The apical cell surfaces, although
lined with many microvilli, appeared colinear. In contrast, CD caused a
disorganization of the cytoplasm that was particularly evident in cell
apexes (Fig. 3). The majority of the
microvilli had disappeared, and the apical portion of the cells, which
protruded prominently into the luminal (medium) space, appeared to
undulate from cell to cell. In many of these cells, the apical
cytoplasm appeared to be nearly devoid of organelles. Cells treated
with Col did not exhibit the drastic changes in morphology seen with CD
(Fig. 4). Although there was some
protuberance of the apical portions of the cells into the medium, it
was not as pronounced as that seen with CD; furthermore, apical
microvilli appeared to be unaffected. The overall intracellular organization of the cells did not appear to be obviously disrupted to
the extent that it was difficult to distinguish Col-treated cells from
untreated cells.

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Fig. 1.
Morphology of type II cells cultured in the presence or absence of
cytoskeleton-disrupting drugs. All light micrographs are cross sections
photographed at the same original magnification (×625); bar = 20 µm. A: type II cells
cultured on floating gels for 8 days with no additions to the medium.
B: type II cells cultured on floating
gels for 8 days in the presence of 5 µM cytochalasin D during the
final 24 h. C: type II cells cultured
on floating gels for 8 days in the presence of 2.5 µM colchicine for
the final 24 h. Note that, under all conditions, the cells appear
cuboidal in cross section and that the cells contain numerous darkly
stained inclusions.
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Fig. 2.
Ultrastructure of type II cells cultured on floating gels for 8 days
with no additions to the culture medium. Cells exhibit numerous apical
microvilli, and the cytoplasm contains many osmiophilic lamellar
bodies. Original magnification ×7,000; bar = 2 µm.
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Fig. 3.
Ultrastructure of type II cells cultured on floating gels for 8 days,
with 5 µM cytochalasin D (CD) present for the last 24 h of culture.
CD treatment results in the loss of microvilli on the apical cell
surfaces, and those that remain appear blunted. Apical cytoplasm
appears disorganized and contains few organelles. Original
magnification ×7,000; bar = 2 µm.
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Fig. 4.
Ultrastructure of type II cells cultured on floating gels for 8 days
with 2.5 µM colchicine (Col) present for the last 24 h. In contrast
to cells treated with CD, those treated with Col do not exhibit
ultrastructural characteristics obviously different from those in
control cultures. Original magnification ×7,000; bar = 2 µm.
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SP gene expression. Disruption of
microfilaments with CD and microtubules with Col had significant
effects on the accumulation of mRNAs for SP-A, SP-B, and SP-C. In both
the floating gel (Fig. 5) and the EHS gel
(Fig. 6) culture systems, treatment with
either CD or Col for the 24 h between days
7 and 8 resulted in
pronounced decreases in the steady-state levels of all three SP mRNAs
to the point that they were barely detectable. In contrast, the
expression of GAPDH, which was used as a constitutive marker, was
essentially unaffected. Expression of
-actin mRNA was unaffected by
treatment with Col but was significantly increased by CD, which may be
due to an increase in gene transcription (32).

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Fig. 5.
Northern blot analysis of surfactant protein (SP) gene expression in
type II cells cultured for 8 days on floating gels in the presence of
no additions (+0), 5 µM CD, or 2.5 µM Col for the last 24 h of
culture. RNA was isolated by the guanidinium-CsCl method,
electrophoresed under denaturing conditions, blotted, and sequentially
probed with radiolabeled cDNAs for rat SP-A, SP-B, SP-C, human
-actin, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Each lane was loaded with 5 µg of total RNA. Treatment of type II
cells with either CD or Col results in a dramatic reduction in SP mRNA
levels. In contrast, GAPDH mRNA levels are essentially unaffected.
Treatment with Col has no effect on -actin mRNA levels, whereas CD
treatment causes an increase in -actin mRNA. Blot is representative
of 4 independent experiments.
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Fig. 6.
Effect of CD and Col on SP gene expression in type II cells cultured on
Engelbreth-Holm-Swarm (EHS) gels. Type II cells were cultured for 8 days with CD, Col, or CD + Col present for the final 24 h. Dimethyl
sulfoxide (DMSO) treatment served as a vehicle control for CD
treatment. Each lane was loaded with 5 µg of total RNA. Northern blot
demonstrates that, consistent with results seen on floating gels, CD or
Col markedly reduces steady-state levels of SP mRNAs. The combination
of CD + Col does not result in any further decrease. CD causes an
increase in -actin mRNA either by itself or in combination with Col.
Blot is representative of 3 independent experiments.
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To determine if the observed effects were due to cell toxicity, we
measured overall protein synthesis in treated and untreated cultures
(Table 1). The results showed
that neither CD nor Col inhibited protein synthesis; in contrast, each
drug significantly stimulated protein synthesis compared with controls.
Noco, another microtuble-disrupting drug, also appeared to stimulate
protein synthesis, but the differences were not statistically
significant. Importantly, treatment with any of the drugs had no effect
on the amount of DNA per culture (data not shown), also indicating that
the drugs were not toxic.
We next determined if the effects of the cytoskeleton-disrupting drugs
were reversible. Cells in both culture systems were maintained for 7 days and then were exposed to the cytoskeleton-disrupting drugs for 24 h. At this point, the cultures were repeatedly washed with drug-free
medium, given fresh medium, and then cultured for an additional 2 days
in the absence of drugs before analysis. The data are presented in
Figs. 7 and 8.
As expected, in both culture systems, treatment with CD, Col, and Noco
for 24 h resulted in a marked decrease in SP mRNAs on
day 8. In the floating gel system
(Fig. 7), washing out CD resulted in increased accumulation of all
three SP mRNAs on day 10. Washing
CD-treated floating gel cultures also resulted in a restoration of
intracellular organization when viewed by electron microscopy (data not
shown). The results of washing out CD in the EHS gel system also
resulted in increased SP gene expression (Fig. 8) but to a lesser
extent than that seen in floating gel cultures; whereas SP mRNAs in
washed CD-treated floating gel cultures recovered to levels at or
exceeding those seen in untreated day
10 cultures, SP mRNA levels in washed CD-treated EHS
gel cultures did not recover to those seen in untreated
day 10 cultures. Compared with
CD-treated cells on both substrata, levels of
-actin were decreased
in washed CD-treated cultures but still remained above those seen in
untreated controls. Levels of GAPDH mRNA remained relatively constant
over the course of the entire experiment regardless of substratum.

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Fig. 7.
Reversibility of effects of cytoskeleton-disrupting drugs on SP gene
expression in cultured type II cells. Type II cells were cultured for 8 days on floating gels. CD, Col, 10 µM nocodazole (Noco), or no
additions were present for the final 24 h. RNA was extracted from
one-half of the cultures. The remaining cultures were washed with
repeated changes of drug-free medium, given fresh medium, cultured an
additional 2 days in the absence of drugs, and then processed for RNA
extraction and Northern blot analysis. Each lane was loaded with 5 µg
of total RNA. Results show that treatment with Noco, like CD and Col,
causes a dramatic reduction in steady-state levels of SP mRNAs.
Expression of GAPDH mRNA is unaffected, as is the expression of
-actin, except in the case of CD treatment, where it is increased.
Washing CD or Noco from the cultures, followed by 2 days of recovery,
results in the reaccumulation of SP mRNAs. The extent of recovery after
removal of CD and Noco from the cultures is nearly complete. Washing CD
from the culture also reduces the expression of -actin mRNA. Col is
not effectively washed from the cultures, probably because it
irreversibly binds tubulin monomers. Blot is representative of 3 independent experiments.
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Fig. 8.
Reversibility of effects of cytoskeleton-disrupting drugs in type II
cells cultured on EHS gels. Type II cells were cultured for 8 days with
either no additions, CD, Col, or Noco present for the last 24 h.
One-half of the cultures were processed for RNA isolation on
day 8. The remaining cultures were
washed by repeated changes of drug-free medium, cultured for an
additional 2 days, and then processed for RNA extraction and Northern
blot analysis. Each lane was loaded with 5 µg of total RNA. Northern
blot shows that washing CD- or Noco-treated type II cells cultured on
EHS gels results in recovery of SP gene expression but that the
recovery is incomplete. This is in contrast to the results seen in Fig.
7 for floating gel cultures. Blot is representative of 3 independent
experiments.
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Washing Col-treated cultures did not restore SP mRNAs, most likely
because Col irreversibly binds tubulin monomers. We therefore tested
Noco, which is more reversible than Col. Washing of Noco-treated floating gel cultures resulted in recovery of mRNA levels for all three
SPs to levels similar to those seen in untreated day 10 cultures. Washing of Noco-treated EHS gel cultures,
however, was ineffective in restoring SP mRNA levels.
Effect of CD and Col on SP mRNA
stability. The effect of CD and Col on the stability of
mRNAs for SP-A, SP-B, SP-C, and GAPDH was assessed by using Act D to
inhibit RNA synthesis. Cultures were treated with Act D, Act D plus CD,
and Act D plus Col for up to 12 h, with samples taken every 3 h for
Northern analysis. Because of the potential toxic effects of long-term
treatment with Act D, we did not examine time points beyond 12 h.
Representative Northern blots of the effects of CD and Col treatment
are presented in Figs. 9 and
10, respectively; summary data from four
to five independent experiments are shown in Fig.
11. Despite the fact that the
concentration of Act D used in these experiments (10 µg/ml) inhibited
RNA synthesis by 98%, the steady-state levels of mRNAs for all three
SPs in the presence of Act D alone did not decline appreciably over the
12-h test period. These data suggest that SP mRNAs have a relatively
long half-life in the EHS culture system. Treatment with CD
significantly destabilized all three SP mRNAs, with the effect on SP-A
and SP-B mRNAs being slightly greater than that seen for SP-C. In
contrast, GAPDH mRNA levels, which declined slightly in the presence of
Act D alone, were not significantly affected by the addition of CD.
Treatment with Col also resulted in destabilization of the SP mRNAs,
but the effects were not as pronounced as those seen with CD. GAPDH mRNA levels in the presence of Act D plus Col were not significantly lower than those seen with Act D alone.

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Fig. 9.
Effect of CD on SP mRNA stability in cultured type II cells. Type II
cells cultured on EHS gels were treated with CD + 10 µg/ml
actinomycin D (Act D) or Act D alone beginning on day
8 of culture. Cultures were processed for RNA isolation
3, 6, 9, and 12 h after addition of drugs. Cultures given CD alone,
DMSO vehicle alone, or no additions for 12 h served as controls.
Northern blots were prepared using 5 µg of total RNA per lane and
sequentially probed for SP-A, SP-B, SP-C, and GAPDH. Representative
Northern blot shows that Act D by itself had little effect on the
steady-state level of any of the SP mRNAs over the time period tested.
In contrast, CD + Act D caused a precipitous drop in the levels of all
3 SP mRNAs over 12 h, indicating that depolymerization of the actin
cytoskeleton significantly compromised their stabilities. Stability of
GAPDH mRNA was not substantially affected by CD + Act D.
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Fig. 10.
Effect of Col on SP mRNA stability in cultured type II cells. Type II
cells cultured on EHS gels were treated with Col + 10 µg/ml Act D or
Act D alone beginning on day 8 of
culture. RNA was isolated from the cells 3, 6, 9, and 12 h after
addition of drugs. Cells treated for 12 h with Col alone,
phosphate-buffered saline (PBS) vehicle alone, or no additions served
as controls. Northern blots were loaded with 5 µg of total RNA per
lane and sequentially probed for SP-A, SP-B, SP-C, and GAPDH. This
representative Northern blot demonstrates that Col + Act D results in
decreased steady-state levels of mRNAs for all three SP over 12 h,
whereas Act D by itself had only a small effect. GAPDH mRNA was
minimally affected in either condition. This indicates that
stabilization of mRNAs for SP-A, SP-B, and SP-C requires an intact
microtubule cytoskeleton.
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Fig. 11.
Summary data of effects of CD and Col on SP mRNA stability in cultured
type II cells. Type II cells were cultured on EHS gels and treated with
Act D or Act D in combination with CD or Col as described in Figs. 9
and 10. Northern blots were prepared and sequentially probed with cDNAs
for SP-A (A), SP-B
(B), SP-C
(C), and GAPDH
(D). Direct quantitation of
radioactivity was determined by phosphorimaging. Data are presented as
the percentage of mRNA remaining compared with cells cultured for 12 h
in the presence of the appropriate vehicle. , Act D; , Act
D + CD; , Act D + Col. Error bars = SE;
* P < 0.05 vs. with Act D
only. Data show that Act D by itself had little effect on stability of
mRNAs for any of the SPs or GAPDH over the time period examined.
Treatment with CD or Col, however, resulted in a decrease in the
stability of SP-A (n = 5), SP-B
(n = 4), and SP-C
(n = 4) mRNAs that began to be
apparent by 6 h. Effects of CD on mRNA levels for all 3 SPs were
greater than those of Col. Treatment with either CD or Col had
essentially no effect on GAPDH mRNA stability
(n = 5).
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DISCUSSION |
In these experiments, we have investigated the role of the cytoskeleton
in the maintenance of SP gene expression in primary cultures of rat
type II cells. The data show that disruption of either microfilaments
or microtubules causes a markedly reduced accumulation of mRNAs for
SP-A, SP-B, and SP-C and that this results at least in part from
decreases in the stability of these mRNAs. Under the same conditions,
GAPDH mRNA was essentially unaffected. This lack of an effect of CD or
Col on GAPDH mRNA stability indicates that not all mRNAs require
association with an intact cytoskeleton for stabilization (see below).
A recent study by Dobbs and co-workers (10) has shown that long-term
cultures of rat type II cells treated with Noco have reduced levels of
SP-A, SP-B, and SP-C mRNAs, whereas cells treated with CD were
unaffected. The disparity between these data and the results reported
here could be due to differences in the concentration of CD used. Dobbs
et al. used CD at concentrations of 1 and 100 nM, whereas the
concentration used in our experiments was 5 µM, which was within the
concentration range used in other studies (1-5, 7, 10, 11, 15,
33). Notable among these are the results of Beresford and Agius (3), who observed that glucokinase mRNA expression was stimulated by 100 nM
CD but was depressed by concentrations of 2-20 µM.
Because we observed little decline in the levels for all three SP mRNAs
using a concentration of Act D that inhibited RNA synthesis by 98%
over 12 h, our data indicate that mRNAs for SP-A, SP-B, and SP-C are
relatively long lived in our primary cultures of adult type II cells.
Using cultured human fetal lung explants, Venkatesh et al. (34)
determined the half-lives for SP-B and SP-C mRNAs in the presence of
dexamethasone to be 16 and 19 h, respectively. Treatment of cultured
fetal rabbit lung explants with dexamethasone increased the half-life
of SP-C mRNA to 30 h (6). The long half-lives that we observed for SP-B
and SP-C are consistent with these observations, since our cells were
cultured in the presence of whole rat serum, which most likely contains corticosteroids. Our data also suggest that the half-life of SP-A mRNA
in our cultures of primary type II cells is somewhat longer than that
observed in human fetal lung explants (6, 13) in which
10
7 M dexamethasone
decreased SP-A mRNA half-life. The possible effects of serum
glucocorticoids in our cultures are difficult to estimate, since SP-A
mRNA and protein expression can vary in response to both the
concentration of glucocorticoid and the length of exposure (13, 17,
23). We also do not know if the differences in SP-A mRNA half-life that
we observed are due species differences, to the behavior of isolated
type II cells in culture, or to some other unknown factor(s).
Earlier studies from our laboratory indicated that type II cell
differentiation was improved when the cells were allowed to assume
their native cuboidal shape (27-29). Because CD- and Col-treated cells appear cuboidal in cross section, cell shape per se may not be as
important to type II cell differentiation as how shape reflects
intracellular structural organization, which is predominantly determined by the cytoskeleton. The data presented here demonstrate that perturbing cytoskeletal organization has a negative impact on
normal cell function. Support for this concept is suggested by our
previous work (28) in which type II cells were cultured on both
attached and floating collagen gels. SP mRNAs did not accumulate in the
attenuated cells in attached gel cultures, even though nuclear run-on
assays demonstrated that nuclei from these cells were transcribing
mRNAs for SP-A, SP-B, and SP-C. The lack of accumulation of any of the
SP mRNAs suggests that these mRNAs were destabilized in cells on
attached gels. Ultrastructural examination of type II cells cultured on
attached and floating collagen gels revealed differences in
cytoskeletal organization, the most notable being the presence of dense
accumulations of microfilaments in the basal region of cells on
attached gels. Releasing the collagen gels from the culture dish
resulted in cell shape changes that were accompanied by changes in
cytoskeletal organization, since these prominent microfilament bundles
disappeared. Significantly, detaching the gels also resulted in the
cells reaccumulating mRNAs for SP-A, SP-B, and SP-C to readily
detectable levels as soon as 24 h after detachment. Combining these
data with the observations in the present study, we hypothesize that
type II cells on attached gels transcribe SP mRNAs but that
cytoskeletal changes imposed by the pronounced spreading of the cells
over the substratum do not allow these mRNAs to associate with the
cytoskeleton in a manner that confers stability, and thus the mRNAs are
degraded. Detaching the gels results in the reorganization of the
cytoskeleton to a more normal configuration that allows association and
stabilization of the SP mRNAs and hence their reaccumulation.
Our observations on the effects of disrupting the cytoskeleton on cell
differentiation are also not unique among epithelial cells. Using
primary cultures of rat mammary epithelial cells maintained on a
laminin substratum, Blum et al. (4) demonstrated that treatment with
either CD or Col for 24 h resulted in a marked decrease in the
accumulation of the milk proteins
-casein, transferrin, and
-lactalbumin. This resulted from decreases in the mRNAs for these
proteins, which were in turn shown to be destabilized by treatment with
the drugs (5). Treatment of primary cultures of hepatocytes with CD
resulted in decreased expression of P-glycoprotein, which resulted from
rapid destabilization of P-glycoprotein mRNA in the absence of any
change in its rate of transcription (15). Similarly, incubation
of insulin-treated hepatocytes with CD at the same concentration
used in our experiments resulted in increased degradation of
glucokinase mRNA (3).
The role of the cytoskeleton in maintenance of gene expression is not
as simple as the data discussed above would imply. Because overall
protein synthesis is actually increased in type II cell cultures
treated with CD or Col, disruption of the cytoskeleton cannot lead to
the depletion of all mRNAs. Treatment with CD and Col may, in fact,
cause the upregulation of certain genes. This possibility is supported
by evidence from cultures of other cell types. Fibroblasts treated with
either CD, Col, or Noco respond with major increases in urokinase
plasminogen activator receptor mRNA and protein (2). Human dermal
fibroblasts treated with CD exhibit a shape-related induction in
expression of transforming growth factor-
mRNA and protein (33).
L929 cells treated with Col, Noco, or vinblastine to disrupt
microtubules show increased expression of nerve growth factor mRNA and
protein, whereas GAPDH mRNA is unaffected; CD had no effect on the
cells (1). The sum of the data detailing the positive and negative
effects of CD and Col on gene expression is that the effects of
disrupting the cytoskeleton on any given gene must be determined
empirically.
How does the cytoskeleton affect gene expression? Several studies have
demonstrated that polysomes bind to the cytoskeleton (11) and that
these polysomes contain specific mRNAs that are associated with the
cytoskeleton by their poly(A) tails. How this association confers
stability on the mRNAs is not understood. A growing literature also
supports the hypothesis that specific mRNAs are transported and
localized to very specific locations within the cell and that
cytoskeletal elements are involved both in targeting and anchoring
these mRNAs. The structural elements that distinguish
cytoskeleton-bound polysomes from either free polysomes or polysomes
associated with the endoplasmic reticulum, however, are not well
defined. All available evidence suggests that subcellular targeting of
specific mRNAs is mediated by sequences in the 3' untranslated
region of these mRNAs (for review see Ref. 12 and references therein).
The cytoskeleton may also be involved in supporting functional
differentiation by mechanisms other than stabilizing particular mRNAs.
Cultured mammary epithelial cells, like alveolar type II cells, require
an interaction with the extracellular matrix to sustain tissue-specific
gene expression in the presence of lactogenic hormones (16, 30). This
requirement has been more precisely defined to be for laminin (31).
Type II cells cultured on EHS tumor matrix, which is composed
predominantly of laminin, form alveolus-like structures (29).
Alveolus-like structure formation is dependent on laminin (19) as well
as on an uncharacterized activity of <10-kDa molecular mass (20).
Laminin binds to cells via both integrin and non-integrin receptors
(21). The morphological and biochemical differentiation induced in
mammary epithelial cells by laminin requires a functional
1-integrin receptor (16, 25).
This observation is particularly interesting in light of the
accumulating body of evidence that many of the well-characterized signal transduction pathways for growth factors and cytokines are also
activated by integrins (26, 35). Such activation may involve elements
of the cytoskeleton, since, in several systems, CD will disrupt
integrin-mediated signaling occurring via tyrosine kinases (8, 22).
Thus, although our data suggest an important role for the cytoskeleton
in the stabilization of SP mRNAs, we cannot rule out the possibility
that disruption of the actin cytoskeleton with CD also inhibits
transcription of these genes. This possibility is suggested by our
earlier observations on transcription of SP mRNAs by type II cells
cultured on attached versus floating collagen gels (28). As discussed
above, type II cells on attached gels transcribed SP mRNAs but did not
accumulate them, which we hypothesize is due to mRNA instability
resulting from cytoskeletal reorganization. It should be noted,
however, that the transcription of SP mRNAs in nuclei from cells on
attached gels was somewhat less than that seen in nuclei from cells on
floating gels. Thus it is possible that normal cytoskeletal
organization in the type II cell may be serving as a chemomechanical
bridge between the extracellular matrix and the nucleus and that
perturbations resulting from cell flattening may effect a
downregulation of SP gene transcription.
In summary, expression of mRNAs for SP-A, SP-B, and SP-C in cultured
type II cells is greatly reduced by disruption of either microfilaments
or microtubules, which act to stabilize these mRNAs. These results
demonstrate that an intact cytoskeleton is required to maintain this
aspect of type II cell differentiation, even when the cells exhibit
their normal cuboidal shape. These data, when considered with the
changes in cytoskeletal organization that must occur when type II cells
are cultured on inflexible substrata such as tissue culture plastic,
suggest a possible explantation for the rapid loss of SP gene
expression under those conditions.
 |
ACKNOWLEDGEMENTS |
This work was performed in the Lord and Taylor Laboratory for Lung
Biochemistry and the Anna Perahia Adatto Clinical Research Center. We
thank Janet Leiber for performing the electron microscopy and Leigh
Landskroner and Barry Silverstein for preparing the figures. We also
thank Kathy Ryan Morgan for excellent assistance in preparing the
manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Specialized Center for Research Grants HL-27353 and HL-56556.
Address for reprint requests: J. M. Shannon, National Jewish Medical
and Research Center, 1400 Jackson St., Denver, CO 80206.
Received 16 June 1997; accepted in final form 25 September 1997.
 |
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