INTRODUCTION

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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.

	MATERIALS AND METHODS

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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.

	RESULTS

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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.

View larger version (101K): [in this window] [in a new window]   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.

View larger version (125K): [in this window] [in a new window]   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.

View larger version (134K): [in this window] [in a new window]   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.

View larger version (142K): [in this window] [in a new window]   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.

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).

View larger version (48K): [in this window] [in a new window]   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.

View larger version (74K): [in this window] [in a new window]   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.

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.

View larger version (81K): [in this window] [in a new window]   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.

View larger version (88K): [in this window] [in a new window]   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.

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.

View larger version (77K): [in this window] [in a new window]   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.

View larger version (80K): [in this window] [in a new window]   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.

View larger version (18K): [in this window] [in a new window]   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).

	DISCUSSION

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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 10⁷ 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.