Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro

Jorge A. Gutierrez1, Robert F. Gonzalez2, and Leland G. Dobbs2,3

1 Department of Pediatrics, 2 Cardiovascular Research Institute, and 3 Department of Medicine, University of California, San Francisco, California 94143-0106

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The pulmonary alveolar epithelium is composed of two distinct types of cells, type I and type II cells, both of which are critical for normal lung function. On the basis of experiments of both nature and in vivo studies, it has been hypothesized that expression of the type I or type II phenotype is influenced by mechanical factors. We have investigated the effects of mechanical distension on the expression of specific markers for the type I and type II cell phenotypes in cultured alveolar type II cells. Rat alveolar type II cells were tonically mechanically distended in culture. Cells were analyzed for a marker for the type I phenotype (rTI40, an integral membrane protein specific for type I cells) and for markers for the type II phenotype [surfactant protein (SP) A, SP-B, and SP-C] as well as for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mechanical distension caused a 68 ± 25% (n = 3) increase in mRNA content of rTI40 relative to undistended controls. In contrast, mechanical distension resulted in a decrease in mRNA content of SP-B to 35 ± 19% (n = 3) and of SP-C to 20 ± 6.7% (n = 3) of undistended controls. There was no effect on mRNA content of SP-A or GAPDH. The differences in mRNA content of SP-B and SP-C were found to be primarily due to changes at the transcriptional level by nuclear run-on assays. The effects on rTI40 appear to be due to posttranscriptional events. These data show that mechanical distension influences alveolar epithelial phenotypic expression in vitro, at least in part, at the transcriptional level.

type II cell; type I cell; mechanical stretch; gene expression

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ALVEOLAR EPITHELIUM is composed of two morphologically distinct types of cells. Type I cells cover >95% of the alveolar surface, providing both the tight barrier and the short diffusion pathway between the air and blood components that are essential for efficient gas exchange. Type II cells synthesize, secrete (8), and recycle (39) surfactant components, which are responsible for lowering surface tension at the alveolar air-liquid interface, preventing alveolar collapse. Type II cells also produce immune effector molecules (36), transport ions (21), and act as stem cells in alveolar repair after injury (12).

The development and maintenance of normal alveolar epithelial phenotype are felt to be critical for normal lung function. Factors that may be involved in the regulation of expression of differentiated alveolar epithelial phenotypes include cell shape (34, 35), extracellular matrix (35), cell-to-cell interactions (35), growth factors (2), and soluble factors (29). It has been suggested also that mechanical factors may be important in modulating alveolar epithelial phenotypic expression. Experiments with fetal model systems have shown that mechanical forces play an important role in regulating lung growth (24) and suggested that these forces may be involved in determining alveolar epithelial phenotype (1). Mechanical factors also affect alveolar epithelial cells in the mature lung (22). Mechanical distension is felt to be critical for compensatory lung growth after a partial pneumonectomy; stretch-induced changes during this process result in an increase in the cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) and activation of cAMP-dependent protein kinase in vivo (33).

Despite in vivo evidence that mechanical forces play an important role in both the developing as well as the mature lung (1, 22, 33), there is little information about how mechanical factors affect alveolar epithelial phenotypic expression in vitro. This report represents the first description of the effects of mechanical distension on the expression of molecular markers for the type I and type II cell phenotypes in vitro. Our results suggest that, in our model system, mechanical forces are potent regulators of alveolar epithelial phenotypic expression.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolation of alveolar type II cells. Alveolar type II cells were isolated from the lungs of pathogen-free adult male Sprague-Dawley rats weighing 180-200 g (Charles River, Hollister, CA) by previously described methods (7). Porcine pancreatic elastase was purchased from Boehringer Mannheim (Indianapolis, IN). We found that with some lots of elastase, preparations of type II cells contained 1-10% type I cells as contaminants. To remove type I cells from type II cell preparations, we used a depletion strategy utilizing a monoclonal antibody against rTI40 (11), an apical membrane protein of the type I cell, and goat anti-mouse immunoglobulin G coupled to magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were separated by sorting through magnetic columns. This method yielded populations of cells that were 92 ± 2% (n = 6) type II cells, containing <0.5% type I cells by indirect immunofluorescence. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis for rTI40 in preparations depleted of type I cells were negative.

Cell culture and mechanical distension. Cells were cultured on elastic membranes and distended by previously described methods (38). Briefly, type II cells were cultured for 20-24 h on circular silicone membrane dishes coated with fibronectin in Dulbecco's modified Eagle's medium (GIBCO BRL, Gaithersburg, MD) containing 5% fetal bovine serum, 100 U penicillin/ml, and 10 µg gentamicin sulfate/ml (all from Univ. of California, San Francisco, Cell Culture Facility). Membranes were placed in stretching devices that provided space for two groups of three membranes per device. Each group of three membranes can be stretched independently, allowing three stretched membranes to be compared with three unstretched membranes under equivalent conditions in the same stretching device. Membranes were placed onto a porous base overlying a fluid-filled chamber. Membranes were held in place by an acrylic top plate with round borings forming the wells. One-half of the membranes in each chamber were distended by applying hydrostatic pressure beneath the membranes (Fig. 1). The amount of distension was controlled by the volume of fluid added. Changes in two-dimensional cellular surface area previously had been correlated with volume of fluid added to the same system (38). In the current experiment, we used a distending volume that resulted in a 21% increase in cellular surface area. Membranes were maintained in the distended state for 18 h. Maintenance of distension was assessed by evaluating the devices for leaks as well as the amount of fluid removal required to return the membranes to their relaxed state. After the 18-h experimental period, tissue culture medium was removed and membranes were washed twice with sterile phosphate-buffered saline at 4°C. Cells were harvested and samples were processed as described below.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Stretch device. Type II cells were cultured on circular silicone membranes. Membranes were placed in stretch devices onto a porous base overlying a fluid-filled chamber. Membranes were held in place by an acrylic top with round borings forming wells. Membranes were distended by applying hydrostatic pressure beneath membranes. Amount of distension was controlled by volume of fluid added.

Preparation of RNA, RT-PCR, Southern blotting, and hybridization. Total cellular RNA was extracted and isolated using RNA-STAT (Tel-Test, Friendswood, TX). To obtain sufficient amounts of RNA for accurate quantitation and analysis of four genes by RT-PCR, in each experiment we pooled cells from three membranes. The amount of total recovered RNA varied <15% from sample to sample and between groups. The concentration of each sample was adjusted to 0.125 µg RNA/µl with diethyl pyrocarbonate-treated water, 0.5 µg of RNA was loaded onto a 1% agarose gel, and the RNA was fractionated by electrophoresis. Ethidium bromide staining showed that lanes contained bands of comparable intensity for 18S and 28S RNA (Fig. 2). RNA was then transferred to Nytran filters (Schleicher and Schuell, Keene, NH) by positive pressure (Posiblotter; Stratagene, La Jolla, CA), and filters were probed with an alpha -32P-labeled sheep partial 18S cDNA probe with the identical sequence to rat 18S RNA (Stephen Black, Dept. of Pediatrics, Univ. of California, San Francisco), thereby confirming quantitation and equal loading as well as documenting the integrity of the RNA samples (Fig. 2).


View larger version (104K):
[in this window]
[in a new window]
 
Fig. 2.   Evaluation of total RNA. Total cellular RNA was extracted as described in METHODS. RNA (0.5 µg) from each sample was size fractionated by electrophoresis through 1% agarose gels and stained with ethidium bromide, and the 18S RNA was visualized with ultraviolet light (A). RNA was transferred to Nytran filters. Filters were probed with [32P]dCTP-labeled cDNA for 18S RNA before undergoing autoradiography (B). In each, first 2 lanes represent control samples and second 2 lanes represent stretch samples.

Aliquots of RNA were then subjected to RT-PCR for rTI40, surfactant protein (SP) A, SP-B, SP-C, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using RT-avian myeloblastosis virus (Boehringer Mannheim) in the presence of 10× PCR buffer (Perkin-Elmer, Branchburg, NJ) and all four deoxyribonucleotides to convert mRNA to cDNA. The cDNA was then amplified using Taq polymerase (Perkin-Elmer) in the presence of the above reaction mixture. The following specific oligonucleotide PCR primers were used to amplify the genes of interest (Biomolecular Resource Center, San Francisco, CA): rTI40: 5'-GCC ATC GGT GCG CTA GAA GAT GAT CTT-3' (identical to bases 53-80), 5'-GTG ATC GTG GTC GGA GGT TCC TGA GGT-3' (complementary to bases 201-257); SP-A: 5'-TTT CCA GCT TAC CTG GAT GAG G-3' (identical to bases 13-25), 5'-GGA GTC TGG TCT TCA ATC ATG C-3' (complementary to bases 301-323); SP-B: 5'-AAT GAC CTG TGC CAA GAG TGT G-3' (identical to bases 196-218), 5'-AGG ACC AGC TTG TTC AGC AGA G-3' (complementary to bases 509-531); SP-C: 5'-GTG GTT GTG GTG GTA GTC CTT G-3' (identical to bases 127-149), 5'-TAG CAG TAG GTT CCT GGA GCA GCT G-3' (complementary to bases 380-402); GAPDH: 5'-GAC AAG ATG GTG AAG GTC GG-3' (identical to bases 25-44), 5'-CAT GGA CTG TGG TCA TGA GC-3' (complementary to bases 543-562). The number of amplification cycles was determined by evaluating samples subjected to serial amplifications to determine the linear range for each target cDNA and then choosing an amplification number within the linear range (12 for rTI40; 24 for SP-A, SP-B, and GAPDH; and 30 for SP-C).

PCR products were separated by electrophoresis through 2% agarose gels, stained with ethidium bromide, and visualized with ultraviolet light. The cDNA was then transferred to Nytran filters by capillary action. Filters were probed with the corresponding full-length cDNAs for rat rTI40, SP-A, SP-B, SP-C, or GAPDH. All cDNA inserts were excised intact from their vectors with an appropriate restriction enzyme (Boehringer Mannheim), purified by electrophoresis through an agarose gel, and then labeled with [alpha -32P]dCTP (NEN Research Products, Boston, MA) by random-primer second-strand synthesis using Random Primer Labeling Kit (GIBCO BRL). Unincorporated nucleotides were removed using a NucTrap Probe Purification column (Stratagene). Filters were prehybridized for 10 min in QuikHybe hybridization solution (Stratagene) at 68°C. Hybridization was performed in 10 ml of QuikHybe solution containing 1.25 × 106 disintegrations · min-1 (dpm) · ml-1 for 18 h at 68°C. Hybridized filters were washed three times with a solution of 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% sodium dodecyl sulfate (SDS) at 25°C for 15 min and once with a solution of 0.1× SSC-0.1% SDS at 60°C for 30 min. Filters were subjected to autoradiography using Hyperfilm (Amersham) before quantification of radiolabeled bands by volume integration of pixels measured by phosphorimage analysis (Imagequant; Molecular Dynamics, Sunnyvale, CA.).

Isolation of nuclei and run-on transcription assay. Cells were removed from membranes by scraping membranes twice in phosphate-buffered saline; pelleted by centrifugation in a table-top centrifuge at 500 g at 4°C for 10 min; and then lysed in 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris · HCl), 10 mM NaCl, 3 mM MgCl2, and 0.1% Nonidet P-40 (Pharmacia). Nuclei were pelleted by centrifugation at 500 g for 5 min at 4°C. Pellets containing nuclei were suspended in a solution of 50 mM Tris · HCl, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA (all purchased from Sigma). Nuclei were centrifuged again at 500 g at 4°C and resuspended in 100 µl of the same buffer. Nuclei were used immediately. Extracts from twelve cultured membranes yielding a total of 10-12 × 106 nuclei were processed for each condition in the run-on transcription assays.

Nuclei were added to 100 µl of a reaction buffer containing 10 mM Tris · HCl, 5 mM MgCl2, 300 mM KCl (all from Sigma), 10 mM ATP, 10 mM CTP, 10 mM GTP, 0.1 pM cold UTP (all from Boehringer Mannheim), 10 mM dithiothreitol (Promega), 10 U RNasin (GIBCO BRL), and 200 µCi of [alpha -32P]UTP (NEN). After incubation at 25°C for 20 min, nuclei were placed at 4°C, and 5 units of ribonuclease-free deoxyribonuclease I (GIBCO BRL) and 10 µl of 20 mM CaCl2 were added to the mixture. Samples were mixed by gentle shaking and incubated at 37°C for 30 min. Forty units of proteinase K (Boehringer Mannheim) were added, and nuclei were lysed in 5% SDS, 50 mM EDTA, and 100 mM Tris · HCl. Fifty micrograms of yeast tRNA (GIBCO BRL) were added to the lysate as a carrier, and samples were mixed and allowed to incubate at 37°C for 30 min. RNA was extracted by incubating samples in 5% beta -mercaptoethanol, 0.2% sarcosyl, 2 M sodium acetate, and acidic phenol and chloroform-isoamyl alcohol (24:1) on ice for 15 min before centrifugation at 14,000 revolutions/min for 5 min. RNA was extracted a second time with phenol-chloroform-isoamyl alcohol. Unincorporated nucleotides were removed using a Centricon 100 concentrator (Amicon, Beverly, MA).

Equal amounts of radioactive elongated RNA (2-5 × 106 dpm) were hybridized to 5 µg of linearized, denatured plasmids containing cDNA inserts for rTI40, SP-A, SP-B, or SP-C or control plasmid DNA with no insert that had been spotted onto Nytran filters (Schleicher and Schuell), using a vacuum slot blotter (Schleicher and Schuell). Filters were prehybridized for 2 h at 65°C in a 15-ml centrifuge tube (Corning) containing 5 ml of a solution of 10× Denhardt's solution (1× Denhardt's is 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 6× SSPE (1× SSPE is 0.15 M NaCl, 0.01 M NaHPO4, and 0.001 M EDTA, pH 7.4), 1% SDS, and 10 µg salmon sperm DNA/ml. Hybridization was performed using the same solution with the addition of 5% dextran sulfate containing labeled RNA for 20 h at 65°C. Hybridized filters were washed once with 0.1% SSPE-1% SDS for 15 min at 25°C and then twice with the same solution at 65°C. Filters were then subjected to autoradiography as described above; quantitation of radioactivity was performed by volume integration, using phosphorimage analysis (Imagequant, Molecular Dynamics).

Statistical analysis. Results are expressed as the percent change from unstretched controls, means ± SD of 3 experiments, each with a different preparation of cells. The effects of mechanical distension on type II cells were compared with those of undistended controls by the Wilcoxon signed rank test. A value of P < 0.05 was considered statistically significant. Controls varied from experiment to experiment by <25%.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Distension of type II cells causes an increase in the mRNA content of rTI40. RNA isolated from cells undergoing mechanical distension and subjected to RT-PCR for rTI40 yielded a 68 ± 25% (n = 3, P < 0.05) increase in mRNA for rTI40 compared with mRNA obtained from control cells cultured in parallel in the same device but not subjected to mechanical distension (Fig. 3). The content of mRNA was quantitated by Southern blot and phosphorimage analyses of RT-PCR products. These findings support the hypothesis that mechanical distension favors expression of the type I phenotype.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of mechanical distension of type II cells on content of mRNA for rTI40. Southern blots were performed from RNA obtained from unstretched control (C) and stretched (S) cells that was subjected to reverse transcription-polymerase chain reaction (RT-PCR) for rTI40 as described in METHODS. Autoradiograms (shown above columns) were obtained before quantification of radioactivity by volume integration of pixel values as measured by phosphorimage analysis; first 2 lanes of each represent duplicate control samples and next 2 lanes represent duplicate stretch samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to ensure quantitative recovery of cDNA after RT. Results are expressed as percent change from unstretched controls, means ± SD of 3 experiments, each with a different preparation of cells. * P < 0.05 compared with respective control cells.

Distension of type II cells causes a decrease in the mRNA content of SP-B and SP-C. In contrast to the effects of mechanical distension on rTI40 mRNA, mechanical distension caused a reduction in the expression of mRNA for two of the markers of the type II phenotype. Southern blot and phosphorimage analyses of mRNA subjected to 24 cycles of amplification by RT-PCR for SP-B showed that mechanical distension resulted in a decrease in mRNA content for SP-B to 35 ± 19% (n = 3, P < 0.005) of control (undistended) cells cultured in parallel chambers of the same device. Similar analysis of RT-PCR products after 30 cycles of amplification for SP-C revealed a decrease in mRNA content for SP-C to 20 ± 6.7% (n = 3, P < 0.005) of control (undistended) cells (Fig. 4).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of mechanical distension of type II cells on content of mRNA for surfactant protein (SP) A, SP-B, and SP-C. Southern blots were performed from RNA obtained from unstretched control (C) and stretched (S) cells that was subjected to RT-PCR as described in METHODS. Filters were subjected to autoradiography before radioactivity was quantitated by volume integration of pixel density by phosphorimaging. Autoradiograms, each representative of 3 experiments, are shown above columns; in each, first 2 lanes represent duplicate control samples and next 2 lanes represent duplicate stretch samples. GAPDH was used as a control to ensure quantitative recovery of cDNA after RT. Results are expressed as percent change from unstretched controls, means ± SD of 3 experiments, each with a different preparation of cells. * P < 0.05 compared with respective control cells.

Distension of type II cells has no effect on the mRNA content of SP-A and GAPDH. A third marker of the type II phenotype, SP-A, was unaffected by mechanical distension. The content of SP-A mRNA after 24 cycles of RT-PCR was not significantly different when RNA prepared from stretched cells was used in comparison with RNA from unstretched control cells (Fig. 4). Although all conditions of RT-PCR were selected to be in the linear range, to be sure that differences between groups were not obscured by overamplification, we amplified samples for 6, 12, 18, 24, and 30 cycles; there were no differences in SP-A mRNA content between distended or unstretched controls under any of these RT-PCR conditions (data not shown).

Aliquots of RNA were also amplified for GAPDH. Mechanical distension did not result in a significant change in the mRNA content of GAPDH, and it appears that GAPDH is constitutively expressed under both stretched and control conditions. GAPDH was therefore used as a control to ensure quantitative recovery of cDNA following reverse transcription.

Nuclear run-on transcription assays. To determine whether the effects of mechanical distension on the content of specific mRNAs were due to transcriptional or posttranscriptional changes, we performed nuclear run-on assays. The results are shown in Fig. 5, which summarizes the results and shows the amount of radioactivity for each condition as quantitated by phosphorimage analysis. Mechanical distension caused a very small (3.3%) difference in the level of transcription for rTI40; this difference is not statistically significantly different from that of unstretched control cells. In contrast, there was a marked decrease in the transcription of SP-B and SP-C. The level of transcription of SP-B was decreased to 47 ± 6% (n = 3, P < 0.05) of that of control unstretched cells, and the level of transcription of SP-C was decreased to 25 ± 13% (n = 3, P < 0.05) of that of controls. There were no observed differences in the levels of transcription of SP-A between nuclei obtained from cells subjected to distension relative to nuclei obtained from control cells (data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Nuclear run-on transcription assays were performed on nuclei prepared from cells maintained in distended state for 18 h (S) and from control unstretched cells (C) cultured in same device. Freshly prepared nuclei were incubated with [32P]UTP. Radiolabeled RNA was isolated and hybridized to denatured linearized cDNAs for rTI40, SP-B, and SP-C. Denatured linearized plasmid without an insert (pGEM) was used as a negative control. Filters were subjected to autoradiography before radioactivity was quantitated by volume integration of pixel density by phosphorimaging. Autoradiograms, each representative of 3 experiments, are shown above columns, which express data (means ± SD) from the 3 experiments, each with a different preparation of type II cells. * P < 0.05 compared with respective control cells.

Together, these data show that the decrease in mRNA content of SP-B and SP-C may be due to changes at the transcriptional level. It is likely that at least some of the changes in the mRNA content of rTI40 are due to posttranscriptional events.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The lung is a dynamic organ in which volume changes regularly, both with normal tidal breathing and with periodic larger breaths (sighs). Although it remains controversial whether individual alveolar cells are stretched or unfolded in vivo, it has been suggested that mechanical factors may be important in modulating alveolar epithelial phenotypic expression. Information regarding the relationship between mechanical factors and alveolar epithelial phenotypic expression has been largely derived from studies with fetal model systems (1). These experiments support the concept that mechanical forces influence expression of alveolar cell phenotype during lung development. In studies performed on fetal sheep lungs in utero, maintaining the fetal lung in an overdistended state by tracheal ligation subjectively favored the expression of the type I phenotype while inhibiting the expression of the type II phenotype; underdistension of fetal lung in utero by chronic tracheal drainage has the opposite effect (1). However, none of these reports provided quantitative data regarding numbers of type I and type II cells or biochemical data supporting these subjective conclusions. The effects of mechanical forces on alveolar phenotypic expression in adult lung remain unexplored.

In the present study, cultures of type II cells were subjected to 18 h of tonic mechanical distension. Cells were distended after only 20-24 h in culture to allow adherence to the membranes while minimizing the loss of differentiated function known to occur when type II cells are cultured on a substratum that promotes spreading. The amount of distension chosen for this study results in an increase in cell surface area of 21% and is known to stimulate calcium mobilization and surfactant secretion in type II cells in vitro (38). We used rTI40, a gene that encodes a protein that is localized by immunocytochemical methods to the apical plasma membrane of type I cells (11), as a marker for the differentiated type I cell phenotype. This gene is identical to OTS-8, a sequence isolated from transformed mouse osteoblastic cells (26), and has also been called T1alpha after its cloning and characterization from rat lung (32). Although the function of the protein encoded by this gene remains unknown, rTI40 has proven to be a useful marker of the extent of injury to the alveolar epithelium (23). SP-A, -B, and -C were used as markers of the differentiated type II cell phenotype. Mechanical distension of type II cells for 18 h resulted in a 68% increase in the mRNA content of a marker of the type I phenotype and a decrease in the mRNA content of two of the markers of the type II phenotype to 20% (SP-C) and 35% (SP-B) of control. Interestingly, there was no observed effect on mRNA content of SP-A, another, although less specific than SP-C, marker of the type II phenotype. This apparent coregulation of SP-B and SP-C expression, both different from SP-A expression, has been observed previously. For example, in the developing lung, agents that increase intracellular cAMP concentration cause an increase in SP-A mRNA but have only a modest effect on mRNA content of SP-B and SP-C (18, 27). In human fetal lung tissue in vitro, glucocorticoids exert a marked stimulatory effect on the levels of SP-B and SP-C mRNAs (28, 37) while exerting both stimulatory (at low concentrations) and inhibitory (at high concentrations) effects on the levels of SP-A mRNA (2). In our system, mechanical distension favors the type I phenotype while inhibiting two of the markers of the type II phenotype. These findings are consistent with studies with other model systems (34, 35) in which there is inverse coregulation of the expression of the type I and type II phenotypes (3). We do not know whether the observed changes reflect alterations in cells already expressing the mRNAs of interest and/or reflect a change in the percentage of cells expressing these mRNAs.

The observed differences in mRNAs appear to be due to changes at both the transcriptional and posttranscriptional levels. Mechanical distension resulted in a decrease in newly transcribed mRNA for SP-B to 45% of control and a decrease for SP-C to 25% of control values by nuclear run-on transcription assays. Although we have not directly measured mRNA stability, the differences in transcriptional rates of SP-B and SP-C suggest that the observed differences are regulated by transcriptional events. There was a small increase observed in the transcriptional rate of rTI40. The differences in mRNA content for rTI40 may therefore occur primarily at the posttranscriptional level, although larger initial changes in transcription, as well as in mRNA content, may have been missed by assessing only transcriptional rates at the end of the study period.

These data demonstrate that mechanical distension influences alveolar epithelial phenotypic expression in vitro. Previously, the expression of markers of alveolar epithelial phenotype was felt to be influenced primarily by cell shape (34, 35), extracellular matrix (35), and hormones (2, 29). Type II cells cultured on plastic and allowed to flatten and spread in some aspects morphologically resemble type I cells. We have previously reported that type II cells cultured on tissue culture plastic for 4 days express increasing amounts of rTI40 (11). In contrast, these cells contain decreased content of mRNAs for SPs (35) and have a diminished capability to synthesize phospholipids (20). Type II cells cultured on an Engelbreth-Holm-Swarm tumor basement membrane and floating collagen gels remain cuboidal and retain more characteristics of differentiated type II cells (34). In a previous study, we demonstrated that both single and multiple mechanical stretches-relaxations applied to cultured type II cells first stimulate calcium mobilization and then surfactant secretion (38), providing direct evidence that type II cells in culture respond to mechanical stimuli. In the present study, we have provided evidence of a direct link between mechanical forces and regulation of the genes for the markers of differentiated alveolar cell phenotype.

These findings provide another example of the important role mechanical forces play in modulating gene expression in various biological systems. One of the best studied systems of physical forces and cell phenotypic expression is that of skeletal muscle (14). Both gene expression and muscle fiber type appear to be markedly affected by stretch and force generation. Mechanical loads on cultured cells can have dramatic effects on gene regulation. Shear stress stimulates the expression on mRNAs for platelet-derived growth factor A and platelet-derived growth factor B (30, 17), tissue plasminogen activator (5), and intracellular adhesion molecule-1 in human vascular endothelial cells (25). Mechanical stretching also causes an increase in the amount of mRNA for atrial natriuretic factor in neonatal rat cardiac atriocytes (13). The recent identification of stress-responsive elements in the promoter regions of some genes has provided a possible direct link between physical force and gene expression (31). In endothelial cells, one pathway by which shear stress regulates gene expression is the binding of transcription factors to a specific 6- to 12-base pair "shear-stress response element" (SSRE) found upstream from the start site of shear-stress-sensitive genes (30). The 6-base pair SSRE occurs four times in the 5' flanking region of rTI40 and once in the 5' flanking region of SP-C, suggesting that good candidates for mechanosensitive response elements exist in both of these genes. The SSRE is not, however, involved in the regulation of some mechanically sensitive genes (16), suggesting that other mechanisms also may exist.

The objective of the present study was to determine the effects of mechanical distension on the phenotypic expression of markers of pulmonary alveolar cell differentiation. Although prior studies of fetal lung development in vivo support the concept that mechanical factors affect both growth and phenotypic expression of alveolar epithelium, the relevance of our observations in vitro to lung development is uncertain because of the limitations of our model system. For the current studies, we used type II cells in primary culture, an accepted model for many studies of type II cell functions such as surfactant synthesis, secretion, and reuptake (39) and ion transport (21, 15). We used type II cells cultured for 40 h in these studies. Type II cells in primary culture gradually (over days) cease expressing markers of the type II cell phenotype (6, 19) and begin to express markers of the type I phenotype (4, 10). It is not currently known whether these cells in transition are phenotypically more similar to type II cells, type I cells, an intermediate II-I cell type, or a putative alveolar epithelial stem cell. Although the type II phenotype can be better preserved by culturing type II cells on different matrices or with different apical surface conditions (9, 34), we have found that we cannot apply mechanical forces to cells cultured in this fashion. Despite these considerations, the data presented in this report support the hypothesis that mechanical distension influences alveolar epithelial phenotypic expression in vitro. Both transcriptional and posttranscriptional mechanisms appear to be involved. These findings provide the first biochemical support linking mechanical forces and the regulation of alveolar epithelial phenotypic expression.

    ACKNOWLEDGEMENTS

We thank Lennell Allen for assistance in the preparation of this manuscript and Wen Zhou for technical assistance.

    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-24075 and R01-HL-57426.

Address for reprint requests: J. A. Gutierrez, Dept. of Pediatrics, Univ. of California, San Francisco, 505 Parnassus Ave., Moffitt 680, Box 0106, San Francisco, CA 94143-0106.

Received 28 April 1997; accepted in final form 22 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alcorn, D., T. M. Anderson, T. F. Lambert, J. E. Maloney, B. C. Ritchie, and P. M. Robinson. Morphologic effects of chronic tracheal ligation and drainage in the fetal lamb lung. J. Anat. 123: 649-660, 1977[Medline].

2.   Boggaram, V., M. E. Smith, and C. R. Mendelson. Regulation of expression of the gene encoding the major surfactant protein (SP-A) in human fetal lung in vitro. Disparate effects of glucocorticoids on transcription and on mRNA stability. J. Biol. Chem. 264: 11421-11427, 1989[Abstract/Free Full Text].

3.   Danto, S. I., J. M. Shannon, Z. Borok, S. M. Zabski, and E. D. Crandall. Reversible transdifferentiation of alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 12: 497-502, 1995[Abstract].

4.   Danto, S. I., S. M. Zabski, and E. D. Crandall. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies. Am. J. Respir. Cell Mol. Biol. 6: 296-306, 1992[Medline].

5.   Diamond, S. L., J. B. Sharefkin, C. Dieffenbach, K. L. Frasier-Scott, V. McIntire, and S. G. Eskin. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J. Cell. Physiol. 143: 364-371, 1990[Medline].

6.   Diglio, C. A., and Y. Kikkawa. The type II epithelial cells of the lung. IV. Adaptation and behavior of isolated type II cells in culture. Lab. Invest. 37: 622-631, 1977[Medline].

7.   Dobbs, L. G., R. Gonzalez, and M. C. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986[Medline].

8.   Dobbs, L. G., R. J. Mason, M. C. Williams, B. J. Benson, and K. Sueishi. Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats. Biochim. Biophys. Acta 713: 118-127, 1982[Medline].

9.   Dobbs, L. G., M. Pian, S. Dumars, M. Maglio, and L. Allen. Maintenance of the differentiated type II cell phenotype by culture with an apical air surface. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L347-L354, 1997[Abstract/Free Full Text].

10.   Dobbs, L. G., M. C. Williams, and A. E. Brandt. Changes in biochemical characteristics and pattern of lectin binding of alveolar type II cells with time in culture. Biochim. Biophys. Acta 846: 155-156, 1985[Medline].

11.   Dobbs, L. G., M. C. Williams, and R. Gonzalez. Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochim. Biophys. Acta 970: 146-156, 1988[Medline].

12.   Evans, M. J., L. J. Cabral, R. J. Stephens, and G. Freeman. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp. Mol. Pathol. 22: 142-150, 1975[Medline].

13.   Gardner, D. G., H. R. Wirtz, and L. G. Dobbs. Stretch-dependent regulation of atrial peptide synthesis and secretion in cultured atrial cardiocytes. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E239-E244, 1992[Abstract/Free Full Text].

14.   Goldspink, G., A. Scutt, P. T. Loughna, D. J. Wells, T. Jaenicke, and G. F. Gerlach. Gene expression in skeletal muscle in response to stretch and force generation. Am. J. Physiol. 262 (Regulatory Integrative Comp. Physiol. 31): R356-R363, 1992[Abstract/Free Full Text].

15.   Goodman, B. E., and E. D. Crandall. Dome formation in primary cultured monolayers of alveolar epithelial cells. Am. J. Physiol. 243 (Cell Physiol. 12): C96-C100, 1982[Abstract/Free Full Text].

16.   Hanlon, N. J., T. Collins, M. A. Gimbrone, and N. Resnick. Regulation of the endothelial PDGF-A gene by shear stress (Abstract). Circulation 90: 0468, 1994.

17.   Hseih, H. J., N. Q. Li, and J. A. Frangos. Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H642-H646, 1991[Abstract/Free Full Text].

18.   Liley, H. G., R. T. White, R. G. Warr, B. J. Benson, S. Hawgood, and P. L. Ballard. Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung. J. Clin. Invest. 83: 1191-1197, 1989[Medline].

19.   Mason, R. J., and L. G. Dobbs. Synthesis of phosphatidylcholine and phosphatidylglycerol by alveolar type II cells in primary culture. J. Biol. Chem. 255: 5101-5107, 1980[Abstract/Free Full Text].

20.   Mason, R. J., L. G. Dobbs, R. D. Greenleaf, and M. C. Williams. Alveolar type II cells. Federation Proc. 36: 2697-2702, 1977[Medline].

21.   Mason, R. J., M. C. Williams, J. H. Widdicombe, M. J. Sanders, D. S. Misfeld, and L. C. Berry. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc. Natl. Acad. Sci. USA 79: 6033-6037, 1982[Abstract].

22.   Massaro, G. D., and D. Massaro. Morphologic evidence that large inflations of the lung stimulate secretion of surfactant. Am. Rev. Respir. Dis. 127: 235-236, 1983[Medline].

23.   McElroy, M. C., J. F. Pittet, S. Hashimoto, L. Allen, J. P. Wiener-Kronish, and L. G. Dobbs. A type I cell-specific protein is a biochemical marker of epithelial injury in a rat model of pneumonia. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L181-L186, 1995[Abstract/Free Full Text].

24.   Moessinger, A. C., R. Harding, T. M. Adamson, M. Singh, and G. T. Klu. Role of lung fluid in growth and maturation of the fetal sheep lung. J. Clin. Invest. 86: 1270-1277, 1990[Medline].

25.   Nagel, T., N. Resnick, W. J. Atkinson, C. F. Dewey, and M. A. Gimbrone. Shear stress selectively upregulates intracellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J. Clin. Invest. 94: 885-891, 1994[Medline].

26.   Nose, K., H. Saito, and T. Kuroki. Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells. Cell Growth Differ. 1: 511-518, 1990[Abstract].

27.   Odom, M. J., J. M. Snyder, and C. R. Mendelson. Adenosine 3'5'-monophosphate analogs and beta-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro. Endocrinology 121: 1155-1163, 1987[Abstract].

28.   O'Reilly, M. A., J. C. Clark, and J. A. Whitsett. Glucocorticoid enhances pulmonary surfactant B gene transcription. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L37-L43, 1991[Abstract/Free Full Text].

29.   Rannels, S. R., R. N. Grove, and E. Rannels. Matrix-derived soluble components influence type II pneumocytes in primary culture. Am. J. Physiol. 256 (Cell Physiol. 25): C621-C624, 1989[Abstract/Free Full Text].

30.   Resnick, N., T. Collins, W. Atkinson, D. T. Bonthron, C. F. J. Dewey, and M. A. Gimbrone. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc. Natl. Acad. Sci. USA 90: 4591-4595, 1993[Abstract].

31.   Resnick, N., and M. A. Gimbrone. Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 9: 874-882, 1995[Abstract/Free Full Text].

32.   Rishi, A. K., M. Joyce-Brady, J. Fisher, L. G. Dobbs, J. Floros, J. Vanderspek, J. S. Brody, and M. C. Williams. Cloning, characterization, and developmental expression of a rat alveolar type I cell gene in embryonic endodermal and neural derivatives. Dev. Biol. 167: 294-306, 1995[Medline].

33.   Russo, L. A., S. R. Rannels, K. S. Laslow, and D. E. Rannels. Stretch-related changes in lung cAMP after partial pneumonectomy. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E261-E268, 1989[Abstract/Free Full Text].

34.   Shannon, J. M., S. D. Jennings, and L. D. Nielson. Modulation of alveolar type II cell differentiated function in vitro. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L427-L436, 1992[Abstract/Free Full Text].

35.   Shannon, J. M., R. J. Mason, and S. D. Jennings. Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions. Biochim. Biophys. Acta 931: 143-156, 1987[Medline].

36.   Strunk, R. C., D. M. Eidlen, and R. J. Mason. Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J. Clin. Invest. 81: 1419-1426, 1988[Medline].

37.   Venkatesh, V. C., D. M. Ianuzzi, R. Ertsy, and P. L. Ballard. Differential glucocorticoid regulation of the pulmonary hydrophobic surfactant proteins SP-B and SP-C. Am. J. Respir. Cell Mol. Biol. 8: 222-228, 1993[Medline].

38.   Wirtz, H. R., and L. G. Dobbs. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250: 1266-1269, 1990[Medline].

39.   Wright, J. R., and L. G. Dobbs. Regulation of pulmonary surfactant secretion and clearance. Annu. Rev. Physiol. 53: 395-414, 1991[Medline].


AJP Lung Cell Mol Physiol 274(2):L196-L202
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society