Regulation of lung fibroblast tropoelastin expression by alveolar epithelial cells

Thomas J. Mariani, Sarah E. Dunsmore, Qinglang Li, Xueming Ye, and Richard A. Pierce

Department of Internal Medicine, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Epithelial-mesenchymal interactions are of critical importance during tissue morphogenesis and repair. Although the cellular and molecular aspects of many of these interactions are beginning to be understood, the ability of epithelial cells to regulate fibroblast interstitial matrix production has not been extensively studied. We report here that cultured alveolar epithelial cells are capable of modulating the expression of tropoelastin, the soluble precursor of the interstitial lung matrix component elastin, by lung fibroblasts. Phorbol ester-stimulated alveolar epithelial cells secrete a soluble factor that causes a time- and dose-dependent repression of lung fibroblast tropoelastin mRNA expression. This alveolar epithelial cell-mediated repressive activity is specific for tropoelastin, is effective on lung fibroblasts from multiple stages of development, and acts at the level of transcription. Partial characterization of the repressive activity indicates it is an acid-stable, pepsin-labile protein. Gel fractionation of alveolar epithelial cell conditioned medium revealed two peaks of activity with relative molecular masses of ~25 and 50 kDa. These data support a role for epithelial cells in the regulation of fibroblast interstitial matrix production.

extracellular matrix; epithelial-mesenchymal interactions; type II pneumocytes

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

THE CRITICAL ROLE of epithelial-mesenchymal interactions in the development of many complex tissues has been extensively studied. These interactions include mesenchyme-mediated modulation of epithelial cell growth and metabolism as well as epithelium-mediated regulation of mesenchymal cell growth and metabolism. Epithelial differentiation and tissue morphogenesis in the lung, kidney, and mammary glands are dependent on the underlying mesenchyme (5, 42). Mesenchymal cells produce soluble growth factors that can act directly on the epithelium (16, 26). In the developing lung, mesenchyme mediates branching morphogenesis (47), likely due to effects on epithelial cell proliferation and differentiation (2, 7, 46). Lung fibroblasts can also directly promote epithelial cell migration and/or invasiveness (21, 45) and can influence the production of pulmonary surfactant (46). Similar interactions also appear to be important in the maintenance and repair of adult tissues (11).

In addition to inducing epithelial morphogenesis, the mesenchyme is also responsible for the production of the interstitial matrix that determines the unique structural forms and functional properties of these tissues. For instance, the requisite property of tissue recoil in organs such as the lung, skin, and bladder is due to elastic fibers produced by cells of mesenchymal origin (fibroblasts, smooth muscle cells, etc.). Although numerous reports have described the regulation of tropoelastin gene expression by various cytokines (29), limited knowledge exists on the role of heterotypic cell-mediated tropoelastin regulation.

Several lines of evidence suggest that induction and modulation of mesenchymal cell proliferation and matrix production can be influenced by the epithelium. First, in the lung, fibrotic injury models characterized by epithelial damage suggest that normal, intact adult lung epithelium suppresses fibroblast proliferation and matrix production in vivo (1, 10). Second, during avian limb development, the specialized epithelial apical ectodermal ridge is essential for normal limb formation (41, 54). The ability of this epithelial structure to guide limb development is due, at least in part, to the production of fibroblast growth factor-4 (27) and may be mediated by transcription factor Msx-1 (39, 53). Furthermore, the limb bud ectoderm can influence mesenchymal cell chondrogenesis in vitro (48). Third, the thickened apical epithelial cap that forms during limb regeneration in the newt is necessary for proper regrowth and has been shown to have effects on mesenchymal extracellular matrix production (51) and turnover (52).

These data clearly indicate that epithelial-mesenchymal interactions can regulate the accumulation of interstitial matrix. However, direct characterization of the ability of the epithelium to affect mesenchymal cell extracellular matrix production has received little attention. We have utilized an in vitro model system to study the regulation of tropoelastin mRNA expression in rat pulmonary fibroblasts, a commonly used cell type for studying tropoelastin expression and the principal source of parenchymal lung elastin. We have assessed the ability of factors secreted by unstimulated or phorbol ester [phorbol 12-myristate 13-acetate (PMA)]-stimulated cultured alveolar epithelial cells to affect tropoelastin expression. PMA was used as an agonist of protein kinase C and effector of alveolar epithelial cell matrix accumulation. We report that primary cultures of lung alveolar epithelial cells elaborate factors that specifically regulate lung fibroblast tropoelastin gene expression. PMA-stimulated adult alveolar epithelial cells produce a soluble, protease-labile factor that transcriptionally represses lung fibroblast tropoelastin mRNA expression. This factor is active on lung fibroblasts from multiple stages of development and has been fractionated into two peaks at relative molecular masses of 25 and 50 kDa. These data demonstrate a role for alveolar epithelial cells in the regulation of lung interstitial matrix production.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials. Normal adult (250 g) Sprague-Dawley rats were purchased from Charles River Laboratories (Cambridge, MA). The fetal rat lung fibroblast cell line RFL-6 was obtained from the American Type Culture Collection (Rockville, MD). Calf serum was obtained from GIBCO BRL (Gaithersburg, MD). PMA and indomethacin were obtained from Sigma Chemical (St. Louis, MO).

Cell isolation. Type II pneumocytes were isolated from healthy adult rats essentially as described by Dobbs and Mason (14) and modified by Rannels and Rannels (37). Rats were killed with a lethal injection of pentobarbital sodium (60 mg/kg). After cannulation of the trachea, the pulmonary circulation was washed free of blood with 0.15 M NaCl. The lungs were removed from the thoracic cavity and were lavaged repeatedly with 0.15 M NaCl. Cells were liberated from the basement membrane by instillation of elastase (25 U/ml elastase and 0.05% BaSO4 in Joklik's minimal essential medium). The lungs were minced, and cells were separated from pieces of lung tissue by filtration through Nitex HC160 polyamide nylon mesh (Tetko, Elmsford, NY). Cells were centrifuged, resuspended, and then separated on a discontinuous Percoll density gradient. Epithelial cells were collected from the gradient, washed, and further purified by two 15-min intervals of differential adherence to tissue culture plastic. Nonadherent cells were plated at high density (2.08 × 105 cells/cm2) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum.

Adult rat lung fibroblasts (ALFs) were isolated from the minced lung pieces by digestion with collagenase and deoxyribonuclease (DNase; 0.5 mg/ml collagenase, 0.25 µg/ml DNase, and 0.5 mM EDTA in Hanks' balanced salt solution) as described by Dunsmore et al. (15). After digestion, fibroblasts were plated in DMEM supplemented with 10% calf serum and were grown to confluence at passage 3. Neonatal rat lung fibroblasts (NRLF) were isolated from 4-day neonatal rats as previously described (25). Neonatal rat aortic smooth muscle cells were isolated and cultured as previously described (3).

Isolation and application of conditioned medium. Freshly isolated type II pneumocytes were plated in 100-mm2 plates (1 × 107 cells) in DMEM plus 10% fetal calf serum. Twenty-four hours after plating (day 1), culture medium and nonadherent cells were aspirated, and attached cells were refed with DMEM plus 10% calf serum. One, three, or six days after isolation, cultures of alveolar epithelial cells were washed two times in phosphate-buffered saline (PBS) and received 10 ml of culture medium alone or were supplemented with 50 ng/ml PMA. The cells were then cultured for 24 h, at which time the conditioned medium (CM) was collected. CM was centrifuged at 2,000 g for 10 min at 4°C to remove cellular debris, transferred to a sterile container, and stored at 4°C. CM isolated from six independent preparations of type II epithelial cells was tested for effects on fibroblast tropoelastin expression.

Confluent 100-mm2 plates of lung fibroblasts were washed two times with PBS and were refed with nonconditioned culture medium or the same medium supplemented with type II cell CM that was filter sterilized through a 0.2-µm syringe filter (Gelman Sciences, Ann Arbor, MI) directly before application. CM supplementation was between 12.5 and 50%. Fibroblasts were incubated for 12-96 h with fresh medium given every 48 h and were harvested as described below. Replicate plates of fibroblasts were treated with culture medium supplemented with 50 ng/ml PMA or CM from confluent fibroblasts (at passage 3) incubated for 24 h in the absence or presence of 50 ng/ml PMA as controls.

For serum-free experiments, alveolar epithelial cells were grown in DMEM plus 10% calf serum to day 5, washed two times in PBS, and refed with serum-free DMEM with or without 50 ng/ml PMA. CM was harvested after 24 h. Fibroblasts were grown to confluence at passage 3 in DMEM plus 10% calf serum, washed two times in PBS, and treated with DMEM or the same medium supplemented with serum-free CM.

Northern blot analysis. Total RNA was isolated from cultured cells using a modification of the guanidine-phenol method (8) as described by Pierce et al. (32). Five micrograms of total RNA were denatured by incubation for 10 min at 68°C in 50% formamide, 1 M formaldehyde, and 50 ng/ml ethidium bromide and were immediately separated in a 1% agarose gel containing 1 M formaldehyde. RNA was transferred to Hybond-N+ (Amersham, Arlington Heights, IL) as previously described (33). Tropoelastin mRNA was detected using a 1.75-kb rat tropoelastin cDNA probe, pREL-115 (31). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was detected using a 1.3-kb rat GAPDH cDNA probe, pRGAPDH13 (32). T1alpha mRNA was detected using a 0.6-kb rat cDNA clone (38). Intercellular adhesion molecule (ICAM)-1 mRNA was detected using a 2.1-kb murine cDNA clone (20). Surfactant protein C mRNA was detected using a cDNA probe (19). Other probes used were Hf677 for alpha 1(I) procollagen (4), UMRc'ase54 for rat interstitial collagenase (36), and pFH6 for fibronectin (23). Transforming growth factor-beta 1 mRNA was detected using pmTGFb1-A, a 1-kb murine cDNA probe kindly provided by H. L. Moses (Vanderbilt University, Nashville, TN). mRNA signal intensity was determined by densitometry and was expressed relative to the level of GAPDH mRNA in the same sample.

In situ hybridization. For in situ hybridization analysis of tropoelastin mRNA expression, ALFs at passage 3 were plated in Lab-Tek chamber slides (Nunc, Naperville, IL) and were grown to confluence. Confluent cultures were incubated for 48 h in nonconditioned culture medium or the same medium supplemented to 50% with CM. After 48 h, the chamber and gasket were removed from the slide, and the cells were fixed immediately in 10% buffered Formalin for 10 min at 25°C. After fixation, slides were washed two times in diethyl pyrocarbonate-treated PBS for 5 min at 25°C, dehydrated in 70% ethanol, and stored at 4°C.

Hybridization was performed essentially as previously described (35). To begin the hybridization, slides were rehydrated in PBS, and nonspecific signal was blocked by treating the slides with 0.25% acetic anhydride in 0.1 M triethanolamine. The slides were covered with 50 µl of hybridization solution containing 2 × 104 counts · min-1 · µl-1 35S-labeled riboprobe, covered with Parafilm, and incubated at 55°C for 18 h in a humidified chamber. After hybridization, slides were washed stringently and were processed for autoradiography as previously described (40). Tropoelastin-specific riboprobes were derived from pREL-124DM as previously described (30).

Reverse transcription-polymerase chain reaction of tropoelastin pre-mRNA. Levels of tropoelastin pre-mRNA in fibroblast cultures were measured as an indication of the transcription rate for the tropoelastin gene according to the method of Swee et al. (49). Briefly, total RNA isolated for Northern analysis was digested with ribonuclease-free DNase to remove residual genomic DNA, reverse transcribed with random hexamers to make cDNA, and polymerase chain reaction (PCR) amplified using tropoelastin intron-specific primers (5'-GTCAGAGGTCAAGGTCTAGG-3' and 5'-TCAGTCTAGACATGCAACAC-3'). Amplification was optimized for template concentration and cycle number and was determined to be in a linear range. Amplification products were separated in a 1.2% agarose gel, blotted to Hybond-N+, and probed with an internal oligonucleotide primer (5'-GACATACCACCAGGTGGCGC-3'). Densitometry of autoradiographs was used to determine the relative level of tropoelastin pre-mRNA in the sample. Control reactions lacking reverse transcriptase were performed to rule out contamination of RNA by genomic DNA. Amplification of GAPDH mRNA from the same RNA samples was used to control for template concentration loading.

Partial characterization of CM. Effects of CM on cell proliferation and secreted protein synthesis were initially determined simultaneously. ALFs (2.5 × 105 cells /well) at passage 3 were plated in six-well culture dishes and were grown to confluence. Confluent cultures were washed and refed with nonconditioned culture medium or the same medium supplemented to 50% with CM. All cultures were supplemented with 50 µCi/ml [35S]methionine and 1 µCi/ml [3H]thymidine and were incubated for 48 h. The medium was harvested and adjusted to 1 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, and total protein synthesis was determined by quantitation of trichloroacetic acid (TCA)-precipitable 35S. The cell layer was lysed with 0.5 N NaOH and neutralized with 0.5 N HCl, and cell proliferation was determined by quantitation of TCA-precipitable 3H.

To further address the specificity of the activity, confluent cultures of ALFs were treated in serum-free medium supplemented with 50 µCi/ml [35S]methionine. After 48 h, the medium and cell layers were harvested and supplemented with protease inhibitors. Labeled proteins in fibroblast medium and cell layers were separated by 10% polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. Equal volumes of labeled medium or cell layer proteins were loaded for each condition. Acrylamide gels were dried and exposed to autoradiography film for 2-4 days.

To determine the nature of the regulatory factor in CM, serum-free CM was subjected to various treatments before addition to fibroblast cultures. Heat stability was assessed by heating the CM to 65°C for 30 min or boiling for 10 min. Protease sensitivity was determined by digestion with pepsin. CM was adjusted to pH 2.5 with 0.5 N HCl, digested with 0.2 mg/ml pepsin at 37°C for 2 h, and then neutralized with 0.5 N NaOH. Acid stability was assessed by adjusting the CM to pH 2.5 with 0.5 N HCl, incubating at 37°C for 2 h, and then neutralizing with 0.5 N NaOH. Lipid solubility was determined by three successive extractions with two volumes of ethyl acetate. After each treatment, the CM was dialyzed against serum-free medium and filter sterilized before it was added to fibroblast cultures.

Size fractionation of CM. Serum-free CM from PMA-treated alveolar epithelial cells was harvested and dialyzed against PBS at 4°C. Dialyzed CM was concentrated 10-fold by lyophilization and was separated by gel filtration through a Sephacryl S-100HR column (Pharmacia Biotech, Uppsala, Sweden) in PBS at 4°C. Molecular mass standards were used to determine the relative molecular mass of each fraction. One milliliter of concentrated CM was separated on a 120-ml bed volume column at a flow rate of 0.5 ml/min. After separation, fractions were dialyzed against serum-free culture medium and were stored at 4°C. Each fraction was tested for tropoelastin-repressing activity on RFL-6 cells. Confluent RFL-6 cells were treated for 48 h in 50% dialyzed alveolar epithelial cells treated with 50 ng/ml PMA (+PMA CM) from varying fractions and then were harvested for Northern blot analysis. The steady-state level of tropoelastin mRNA relative to GAPDH mRNA was determined for each fraction by densitometry as described above.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of cultured alveolar epithelial cell phenotype. In this study, we investigated the ability of lung epithelial cells to modulate the phenotype of lung interstitial cells. We chose to utilize epithelial cells isolated from the alveolar surface found in close association with lung fibroblasts of the alveolar interstitium. Primary cultures of rat type II pneumocytes have been used in numerous studies investigating the physiology of the alveolar epithelium and were, therefore, prime candidates for our studies. These cells have been extensively characterized and rapidly lose their differentiated phenotype in culture. Under the culture conditions we used, which promote attachment and spreading in the absence of proliferation, these cells have been reported to progressively acquire many characteristics of type I pneumocytes (12, 13). We investigated the expression of markers for type I and type II pneumocyte characteristics in our cultured alveolar epithelial cells (Fig. 1). Expression of the type II cell marker surfactant protein C was present in freshly isolated cells but was completely absent in cultured cells through day 6 and was unaffected by treatment with 50 ng/ml PMA (Fig. 1A). Expression of the type I cell-specific gene T1alpha has been reported to be induced in cultured type II cells (38). This gene was expressed at day 1 of culture in our epithelial cells and continued to be expressed at day 6 (Fig. 1B). Treatment of these cells with 50 ng/ml PMA repressed T1alpha steady-state mRNA levels at day 6. We also studied the expression of ICAM-1 in our alveolar epithelial cell cultures. ICAM-1 is expressed in the alveolar epithelium, primarily by type I pneumocytes, and has been shown to be induced in cultured type II pneumocytes (9). Similar to the expression of T1alpha , ICAM-1 was expressed early in culture and continued to be expressed at day 6 (Fig. 1C). Furthermore, ICAM-1 expression was also repressed by PMA treatment at day 6. These findings were consistent from three separate isolates of epithelial cells and indicate that our alveolar epithelial cells display many type I pneumocyte characteristics at day 6 in culture in the absence or presence of PMA.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Phenotypic analysis of cultured alveolar epithelial cells. Type II epithelial cells were isolated from normal adult rat lungs and were placed in culture for up to 6 days. Cultures were untreated or treated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for the final 24 h, and all cells were harvested for Northern blot analysis. A: representative Northern blot of alveolar epithelial cell RNA probed for surfactant protein C (SPC) mRNA. Lane 1, freshly isolated type II cells; lane 2, day 6 cells without PMA; lane 3, day 6 cells with PMA. B: representative Northern blot of alveolar epithelial cell RNA probed for T1alpha and intercellular adhesion molecule-1 mRNA. Lane 1, day 6 cells without PMA; lane 2, day 6 cells with PMA.

Alveolar epithelial cell CM regulates lung fibroblast tropoelastin expression. To determine whether alveolar epithelial cells can regulate lung fibroblast extracellular matrix production, we assayed the ability of cultured alveolar epithelial cell CM to alter tropoelastin gene expression from lung fibroblasts. We performed a dose-response experiment for the effect of alveolar epithelial cell CM on adult lung fibroblast tropoelastin mRNA levels (Fig. 2). CM from untreated epithelial cells (-PMA CM) had a modest stimulatory effect on lung fibroblast tropoelastin mRNA expression to a maximum of 132% of control levels. Conversely, +PMA CM at a concentration of 50% markedly repressed lung fibroblast tropoelastin mRNA expression to 11% of control levels. This repressive effect was seen for PMA-treated alveolar epithelial cells cultured on plastic for 1, 3, or 6 days and was greater for cells at day 6 than at day 1 (data not shown). Direct treatment of ALFs with 50 ng/ml PMA had only a minimal repressive effect (90% of control) on tropoelastin mRNA expression (Fig. 2). Due to the magnitude of the repressive effect, we chose to explore it in more detail than the potential stimulatory effect of untreated alveolar epithelial cell CM.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2.   Alveolar epithelial cell conditioned medium (CM) regulates adult lung fibroblast tropoelastin (Tropo) expression. Fibroblasts were treated with varying concentrations of alveolar epithelial cell CM for 48 h and then were harvested for Northern blot analysis. A: densitometric quantitation of autoradiographic signal expressing the ratio of Tropo mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in treated cultures as a percentage of the level in the control cultures that received nonconditioned medium. Values are means ± SD. NO CM, fibroblasts treated with nonconditioned medium; -PMA CM, fibroblasts receiving CM from untreated epithelial cells; +PMA CM, fibroblasts receiving CM from PMA-treated epithelial cells; PMA Direct, fibroblasts treated with nonconditioned medium supplemented with 50 ng/ml PMA. * P < 0.01 by t-test. B: representative Northern blot of fibroblast RNA probed for Tropo and GAPDH mRNA.

To determine the temporal pattern of the repressive effect of +PMA CM on lung fibroblast tropoelastin gene expression, we harvested fibroblasts at various intervals after treatment. As shown in Fig. 3, after 12 h in +PMA CM, adult lung fibroblast tropoelastin mRNA levels were decreased to ~50% of control levels. After 24-48 h in +PMA CM, fibroblast tropoelastin mRNA showed maximal repression to ~25% of control levels. To determine if the repression was due to a stable phenotypic change in our adult lung fibroblast cultures, after 48 h in +PMA CM, cultures were washed and refed with +PMA CM or nonconditioned culture medium. These cells were harvested 48 h later and showed continued repression of tropoelastin mRNA levels in the absence or presence of +PMA CM. This indicates that the repression caused by +PMA CM results in a sustained change in fibroblast gene expression.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Time course and stability of alveolar epithelial cell-mediated Tropo repression. Adult rat lung fibroblasts (ALFs) were treated with nonconditioned medium or 50% repressive alveolar epithelial cell CM (+PMA CM) for 12, 24, or 48 h and then were harvested for Northern blot analysis. Replicate cultures were also untreated for 96 h (96 h, C), treated with +PMA CM for 48 h, and then washed and refed with +PMA CM (96 h, CM) or nonconditioned medium (96 h, CM/C) for an additional 48 h. A: densitometric quantitation of autoradiographic signal expressing the ratio of Tropo mRNA to GAPDH mRNA in treated cultures as a percentage of the level in the control cultures that received nonconditioned medium. Values are means ± SD. Washout, removal of CM. * P < 0.01 by t-test. B: representative Northern blot of fibroblast RNA probed for Tropo and GAPDH mRNAs. C, fibroblasts treated with nonconditioned medium; CM, fibroblasts treated with +PMA CM.

Alveolar epithelial cell cultures contained minor contaminants (<10%) of primarily fibroblastic cells. To determine if the +PMA CM effect was due to factors produced by fibroblasts in the epithelial cell cultures, we assessed the ability of fibroblast CM to affect tropoelastin expression in similar fibroblast cultures. Confluent cultures of ALFs were allowed to condition medium for 24 h in the absence or presence of 50 ng/ml PMA. This CM was harvested and used to treat fresh cultures of ALFs in a manner similar to that described above for alveolar epithelial cell CM. CM from untreated cells or PMA-treated fibroblasts had no effect on fibroblast tropoelastin mRNA expression (data not shown).

Repression of tropoelastin expression is specific and occurs independent of effects on cell proliferation. Extracellular matrix gene expression can be affected by the proliferative state of the cell. To further characterize the repressive effect of +PMA CM, we assessed thymidine incorporation in our cultures of ALFs. Fibroblasts were grown to confluence and were treated with CM, essentially as described above for tropoelastin gene expression analysis, then were assayed for the incorporation of [3H]thymidine. Although cultures were confluent, mitotic cells were visible and thymidine incorporation was substantial and consistent, with ongoing cell proliferation. No differences in thymidine incorporation were observed for CM-treated lung fibroblasts compared with control cells treated with nonconditioned medium (Fig. 4A). This indicates that the effect of CM on fibroblast tropoelastin expression is not dependent on changes in fibroblast proliferative state.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   Specificity of Tropo repression by alveolar epithelial cell CM. A and B: confluent cultures of ALFs were treated with nonconditioned medium (control), untreated alveolar epithelial cell CM (-PMA CM), or PMA-treated alveolar epithelial cell CM (+PMA CM) in the presence of [35S]methionine or [3H]thymidine. A: mean level of trichloroacetic acid (TCA)-precipitable 35S in fibroblast medium (protein synthesis) and TCA-precipitable 3H in fibroblast cell layers (thymidine incorporation) from duplicate experiments expressed relative to control. B: representative SDS-polyacrylamide gel electrophoresis gel of 35S-labeled proteins recovered from fibroblast medium and cell layer. C: fibroblasts were untreated, treated with varying concentrations of +PMA CM, or treated with 50 ng/ml PMA for 48 h and harvested for Northern blot analysis. Shown is a representative blot probed for alpha 1(I) procollagen mRNA, fibronectin mRNA (FN), interstitial collagenase mRNA (C'ase), and GAPDH mRNA.

To determine if the repressive effect of alveolar epithelial cell CM was a generalized effect or was specific to tropoelastin, we assessed protein production by ALFs. Protein production was initially assayed as the accumulation of TCA-precipitable material in cell culture medium over a 24-h incubation in the presence of [35S]methionine. Fibroblasts treated with 50% -PMA CM showed no change in secreted protein synthesis compared with control cells treated with nonconditioned medium (Fig. 4A). Fibroblasts treated with 50% +PMA CM did show a modest decrease in protein synthesis, to ~75% of control (Fig. 4A). This level of repression was small relative to the level of repression of tropoelastin expression. Next, total protein production was assessed in untreated and CM-treated cultures labeled with [35S]methionine for 48 h. Labeled proteins secreted into the medium and total protein in the cell layer were assessed by denaturing sodium dodecyl sulfate (SDS)-PAGE (Fig. 4B). These results confirmed that there were no substantial changes in protein synthesis in CM-treated fibroblasts.

To directly determine the effects of alveolar epithelial cell CM on the expression of other extracellular matrix genes, ALFs treated with +PMA CM for 48 h were analyzed for expression of the genes encoding alpha 1(I) procollagen, fibronectin, and interstitial collagenase (Fig. 4C). The expression of all three of these genes was unaffected by +PMA CM. Direct treatment of rat lung fibroblasts with 50 ng/ml PMA, however, did modestly repress alpha 1(I) procollagen and fibronectin steady-state mRNA levels. Surprisingly, PMA alone did not induce rat lung fibroblast interstitial collagenase (MMP-13) gene expression.

To further address the specificity of this interaction, we assessed the ability of primary cultures of rat hepatocytes to secrete factors capable of regulating rat lung fibroblast tropoelastin gene expression. CM isolated from day 6 cultures of untreated and PMA-stimulated primary rat hepatocytes failed to alter rat lung fibroblast tropoelastin mRNA expression (data not shown).

Repression of tropoelastin expression occurs in various lung fibroblasts and aortic smooth muscle cells. To determine if the repressive effect of +PMA CM was dependent on the developmental stage of the lung fibroblasts, we assessed its effect on primary cultures of NRLF as well as the fetal rat lung fibroblast RFL-6 cell line. Although the basal levels of tropoelastin expression vary between the fetal, neonatal, and adult cells (RFL-6 > NRLF > ALF), +PMA CM had a repressive effect of similar magnitude on all cell types (Fig. 5). Thus the repressive activity of the +PMA CM is not restricted to ALFs, suggesting that the ability of epithelial cells to regulate tropoelastin expression may not be confined to any particular developmental period. We also investigated the effects of this CM on elastogenic cells from other tissues. Similar to our results with lung fibroblasts, rat aortic smooth muscle cell tropoelastin mRNA expression was strongly repressed by +PMA CM but not by -PMA CM or by PMA alone (Fig. 5C). This suggests that the activity of the repressing factor(s) is not restricted to the lung.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Alveolar epithelial cell CM represses Tropo expression in fetal and neonatal rat lung fibroblasts (NRLFs) and neonatal aortic smooth muscle cells. Confluent cultures of the fetal rat lung fibroblast-derived RFL-6 cell line, primary NRLFs, or primary neonatal rat aortic smooth muscle cells were treated with 50% alveolar epithelial cell CM as described in EXPERIMENTAL PROCEDURES. A: densitometric quantitation of autoradiographic signal from duplicate experiments expressing the ratio of Tropo mRNA to GAPDH mRNA in treated cultures as a percentage of the level in the control cultures that received nonconditioned medium (%control). Values are means ± SD. * P < 0.01 by t-test. B: representative Northern blots of fibroblast RNA probed for Tropo and GAPDH mRNAs. C: representative Northern blot of RNA isolated from aortic smooth muscle cells, treated as described above, and probed for Tropo and GAPDH mRNAs.

Previous work in our laboratory has indicated that primary cultures of elastogenic cells, isolated from a variety of tissues, represent a heterogeneous population of cells with respect to tropoelastin gene expression. Figure 6 shows an in situ hybridization experiment for tropoelastin mRNA in confluent cultures of ALFs. These cultures are composed of cells expressing tropoelastin at various levels (large vs. small arrows) as well as nonelastogenic cells. A marked decrease in the total amount of signal for tropoelastin mRNA (bright grains in dark-field panels) is apparent in the +PMA-treated culture (Fig. 6, E and F) compared with control (Fig. 6, A and B).


View larger version (118K):
[in this window]
[in a new window]
 


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


View larger version (118K):
[in this window]
[in a new window]
 


View larger version (104K):
[in this window]
[in a new window]
 


View larger version (114K):
[in this window]
[in a new window]
 


View larger version (75K):
[in this window]
[in a new window]
 


View larger version (113K):
[in this window]
[in a new window]
 


View larger version (81K):
[in this window]
[in a new window]
 
Fig. 6.   In situ hybridization analysis confirms repression of Tropo expression in primary ALFs. Shown are paired bright- and dark-field views of cultured ALFs hybridized in situ for Tropo mRNA. A-D: lung fibroblasts treated with nonconditioned medium as control. E-H: lung fibroblasts treated with +PMA CM. Heterogeneity of cultured fibroblasts with respect to Tropo expression is readily apparent (large arrows vs. small arrows). Repression of Tropo mRNA levels is also seen as decreased signal (bright grains in dark-field views) in +PMA CM-treated cells (F and H) compared with control (B and D).

Alveolar epithelial cell-mediated tropoelastin repression acts at the level of transcription. Although inducers of tropoelastin expression usually act at the level of transcription, repression of tropoelastin expression has generally been found to occur at the posttranscriptional level (29). We used a recently reported, highly sensitive reverse transcription-PCR assay (49) to determine the level of tropoelastin pre-mRNA in control, -PMA CM-treated, and +PMA CM-treated ALFs. These data provide a quantitative assessment of the relative rate of transcription for the tropoelastin gene in these cells. The data in Fig. 7 show that steady-state levels of tropoelastin pre-mRNA in treated and untreated cultures reflect steady-state levels for tropoelastin mRNA in the same cultures. The magnitude of repression of tropoelastin pre-mRNA levels is similar to that found for mRNA levels. This indicates that the epithelial cell-mediated repression of tropoelastin expression occurs, at least in part, at the level of transcription.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Alveolar epithelial cell-mediated Tropo repression occurs at the level of transcription. RNA isolated from ALFs treated as previously described was digested with DNase and was subjected to quantitative reverse transcription (RT)-polymerase chain reaction (PCR) for the detection of Tropo pre-mRNA. A: densitometric quantitation of autoradiographs giving means ± SD of Tropo pre-mRNA from each sample relative to control. * P < 0.01 by t-test. B: representative Southern blot analysis for products of Tropo RT-PCR reaction.

Partial characterization of repressive factor. Several previously described candidate factors could potentially account for the repressive activity of the PMA-treated alveolar epithelial cell CM. We considered prostaglandins as potential mediators of tropoelastin regulation in this model. Prostaglandins are produced by cultured alveolar epithelial cells (24) and regulate production of matrix components (6). To address the possibility that prostaglandins were mediating the repression of tropoelastin expression, isolated alveolar epithelial cells were grown and were allowed to condition medium in the presence or absence of PMA as previously described while supplemented with 1-100 µM indomethacin. Indomethacin cotreatment (at all concentrations) of cultured alveolar epithelial cells had no affect on the tropoelastin-repressive activity of +PMA CM (data not shown).

All experiments to this point had been conducted in the presence of 10% calf serum, allowing for the possibility that serum factors were involved in the +PMA CM-related tropoelastin repression. To test for a dependence on serum, epithelial cells (at day 5 of culture) were washed with PBS and were allowed to condition serum-free medium for 24 h in the absence or presence of PMA. Adult rat lung fibroblast cultures were grown to confluence as described above, then washed with PBS and treated with the serum-free CM. The repressive effect of +PMA CM on tropoelastin mRNA expression was found to be independent of serum (Fig. 8A, untreated).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8.   Partial characterization of alveolar epithelial cell-expressed repressor of Tropo production. A: serum-free -PMA CM or +PMA CM was subjected to various manipulations and then was used to treat ALFs as described in EXPERIMENTAL PROCEDURES. Ratio of fibroblast Tropo mRNA to GAPDH mRNA was determined by densitometry of Northern blot autoradiographs. Level of Tropo expression for fibroblasts receiving pretreated +PMA CM is expressed relative to the level of Tropo expression for fibroblasts receiving -PMA CM pretreated under the same conditions. Values represent means from duplicate experiments. Repressive activity in +PMA CM is sensitive to boiling and pepsin digestion, yet is resistant to lipid extraction and acidification. Control, fibroblasts receiving nonconditioned medium; untreated, fibroblasts receiving unmanipulated serum-free +PMA CM. B: RNA was isolated from ALFs treated with -PMA CM (-) or +PMA CM (+) that were previously untreated (Untr) or acidified (Acid). Shown is a representative Northern blot probed for Tropo and GAPDH mRNAs. C: RNA was isolated from alveolar epithelial cells cultured for 6 days as previously described, the last 24 h being in the absence (-) or presence (+) of 50 ng/ml PMA. Shown is a representative Northern blot probed for transforming growth factor (TGF)-beta 1 and GAPDH mRNAs.

To determine the nature of the tropoelastin regulatory factor, the epithelial cell CM was subjected to various treatments before assaying for effect on fibroblasts. As shown in Fig. 8A, the repressive effect of +PMA CM was resistant to brief heating and to lipid extraction. The inability to ablate the CM-related repressive activity by lipid extraction further indicates that the effect is not dependent on the presence of PMA. The activity was, however, sensitive to boiling and pepsin digestion. Together, these data strongly suggest the factor is a protein.

Acidification of the CM (both -PMA CM and +PMA CM) led to stimulation of tropoelastin mRNA expression in fibroblasts; however, the repression specific to +PMA CM remained (Fig. 8B). This acidification-dependent stimulatory activity was pepsin and trypsin labile and was suspected of being due to latent transforming growth factor-beta produced by the epithelial cultures. Mature transforming growth factor-beta is an inducer of lung fibroblast tropoelastin expression (25) and is expressed by the cultured alveolar epithelial cells in the absence and presence of PMA (Fig. 8C).

Size fractionation of tropoelastin-repressing activity. To further characterize the tropoelastin-repressing activity produced by alveolar epithelial cells, we used gel-exclusion chromatography to size fractionate serum-free +PMA CM. Fractionation was performed on a 120-ml Sephacryl S-100HR column in PBS. After dialysis against PBS, +PMA CM was concentrated 10-fold by lyophilization, and 1 ml was separated into fractions at a flow rate of 0.5 ml/min. Collected fractions were dialyzed against serum-free culture medium and were assessed for activity in RFL-6 cells. Confluent RFL-6 cells were treated for 48 h in 50% dialyzed +PMA CM from varying fractions, and the steady-state level of tropoelastin mRNA relative to GAPDH mRNA was determined for each fraction. Northern blot analysis of treated RFL-6 cells from two different experiments is shown in Fig. 9. Tropoelastin-repressing activity was concentrated in fractions corresponding to relative molecular masses of ~25 and 50 kDa. Each of these fractions displayed only partial tropoelastin-repressing activity. Maximal repression for each peak was to 45-60% compared with 20% of control in unfractionated +PMA CM (Fig. 5).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 9.   Size fractionation of Tropo-repressing activity. Serum-free +PMA CM was dialyzed against phosphate-buffered saline (PBS), concentrated 10-fold, and then fractionated on a Sephacryl S-100HR column in PBS at 4°C. Collected fractions were tested for Tropo-repressing activity in RFL-6 cells. Densitometric quantitation of Northern blot autoradiographs was used to determine the ratio of Tropo mRNA to GAPDH mRNA in cultures treated with each fraction. Shown are the results from 2 separate experiments. Relative molecular masses were determined by analyzing elution profile of known molecular mass standards from the same column. Two "peaks" of Tropo-repressing activity were observed in each experiment at the same relative molecular masses of ~25 and 50 kDa.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Communication between epithelial and mesenchymal cells is thought to be of fundamental importance in the development of many tissues, including the lung. Numerous studies have indicated that these two cell types can modulate the phenotype of each other during development and in disease. As we report here, alveolar epithelial cells from normal adult rat lungs can regulate the expression of the gene encoding tropoelastin, an interstitial lung extracellular matrix component, in lung fibroblasts isolated from the same tissue.

The effect of alveolar epithelial cell CM on lung fibroblast tropoelastin mRNA expression is dependent on the state of the epithelial cells; untreated alveolar epithelial cell CM has a very modest stimulatory effect on fibroblast tropoelastin expression, whereas PMA-treated cell CM dramatically represses tropoelastin expression (Fig. 2). Importantly, in contrast to findings in other cell types (28), direct treatment of lung fibroblasts with PMA did not result in a significant repression of tropoelastin mRNA expression. Although PMA is a nonphysiological agent, it was chosen for its ability to be a very strong activator of protein kinase C and to mimic the action of cytokines such as interleukin-1, tumor necrosis factor, and gamma -interferon (34, 43). Preliminary observations indicate that certain peptide growth factors can mimic the action of PMA on alveolar epithelial cells, inducing the production of a tropoelastin repressor (Mariani, unpublished observations). We are currently evaluating the effects of these cytokines in this cell culture system.

The production of the tropoelastin-repressing activity by cultured alveolar epithelial cells coincided with a decrease in expression of the type I pneumocyte markers T1alpha and ICAM-1 (Fig. 1). Due to an incomplete repertoire of markers able to discriminate between type I and type II pneumocytes, it is impossible to determine the exact in vivo equivalent of our PMA-treated alveolar epithelial cell cultures. The phenotype of cultured alveolar epithelial cells can oscillate between type I-like and type II-like states, and these changes can be mediated by soluble factors (44). Our cultured alveolar epithelial cells continue to express T1alpha and ICAM-1 at reduced levels and do not express the type II cell marker surfactant protein C, suggesting that even after treatment with PMA, they display a type I pneumocyte-like phenotype.

We find that this tropoelastin-repressing activity of PMA-treated alveolar epithelial cells is effective on rat lung mesenchymal cells derived from fetal and neonatal as well as adult tissues (Fig. 5). This is of particular interest given the close association of differentiating epithelium and tropoelastin expression in the developing lung (33, 50). Recent work has shown that CM from cultured fetal lung epithelial cells, isolated from rat lungs during the phase of induction of tropoelastin expression, enhances tropoelastin mRNA expression in cultured fetal lung fibroblasts (M. C. Arikan and R. A. Pierce, unpublished observations). These findings suggest epithelial-mesenchymal interactions may be important for the induction of tropoelastin expression in the fetal rat lung.

Furthermore, we have found that the lung fibroblast response (of altered gene expression) to this alveolar epithelial cell CM is specific for tropoelastin in the absence of changes in fibroblast overall metabolic activity (Fig. 4, A and B) and nonelastic, extracellular matrix gene mRNA expression (Fig. 4C). The minor decrease in total protein synthesis noted in +PMA CM-treated fibroblasts may be accounted for by the loss of tropoelastin production alone. Although this specificity may be due to the conditions used in this study, it is interesting to note that elastin is a major product of interstitial fibroblasts from the lung alveolar wall. Conversely, CM from hepatocytes, which are derived from a nonelastic tissue, failed to alter lung fibroblast tropoelastin gene expression.

Two aspects of the +PMA CM activity observed in this model are of particular interest with respect to the cellular mechanisms of tropoelastin regulation. First, the activity appears to result in a sustained, phenotypic change in lung fibroblast tropoelastin mRNA expression (Fig. 3). Because tropoelastin expression is developmentally restricted, this phenotypic change may be indicative of a differentiation event induced by the activity. Second, we have determined that the repression occurs (at least in part) at the level of gene transcription, as evidenced by decreased steady-state tropoelastin pre-mRNA (Fig. 7) in +PMA CM-treated fibroblasts. This is particularly of interest with respect to the lung, where the developmental cessation of tropoelastin expression has been previously shown to occur at a posttranscriptional level (49).

We have partially characterized the factor(s) responsible for epithelial cell-mediated repression of tropoelastin expression. Analysis of cell proliferation of CM-treated fibroblasts indicates that regulation occurs in the absence of changes in fibroblast proliferative states (Fig. 4). The activity is sensitive to denaturation or protease digestion and therefore is likely to be a protein (Fig. 9). Furthermore, we have been able to fractionate +PMA CM by gel filtration into two separate peaks of partial activity, eluting at ~25 and 50 kDa. These may represent two biochemically distinct mediators that may function to repress tropoelastin expression in an additive fashion. Alternatively, they may be monomeric and dimeric forms of the same molecule, separated by the fractionation process, each showing partial activity. Identification and characterization of the factor(s) responsible for this effect are the focus of current investigation.

In other model systems, cultured lung epithelial cells have been shown to modulate mesenchymal interstitial matrix production. Griffin et al. (17) reported that alveolar type II cells stimulate dermal fibroblast secretion of type I collagen. This stimulation was inhibited by antibodies against insulin-like growth factor. Kawamoto et al. (22) reported that bronchial epithelial cells are capable of stimulating fibroblast type I collagen and fibronectin production. Our data, together with these previous findings, support the hypothesis that lung epithelial cells can influence interstitial extracellular matrix production. Retinal epithelial-stromal interactions have previously been shown to modulate fibroblast hyaluronic acid production (18), suggesting that the ability of the epithelium to influence fibroblast matrix production is not limited to the lung.

In this study, we demonstrate that PMA-stimulated alveolar epithelial cells are capable of potently repressing the expression of tropoelastin mRNA from lung fibroblasts. These data contribute to a limited body of information strongly supporting the hypothesis that epithelial cells can regulate interstitial extracellular matrix production by mesenchymal cells. The role of epithelial cells in controlling interstitial extracellular matrix production and turnover during development, homeostasis, and disease of the lung and other organs should be more thoroughly explored.

    ACKNOWLEDGEMENTS

We thank J. Roby, T. Tolley, K. Fliszar, and S. Sandefur for technical assistance; Drs. W. C. Parks, R. P. Mecham, G. A. Doyle, and A. Persson for helpful discussions; Dr. H. G. Welgus for support and sharing of reagents; and M. C. Williams (Boston University School of Medicine), F. Takei (University of British Columbia, Vancouver), and B. P. Hackett and J. D. Gitlin (Washington University School of Medicine, St. Louis) for cDNA clones.

    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-54049, HL-09179, 5T32-AR-07284, and HL-29594, an American Heart Association Grant-In-Aid, and the Washington University-Monsanto Biomedical Research Agreement. S. E. Dunsmore was supported as a Lucille P. Markey Pathway postdoctoral fellow.

Address for reprint requests: R. A. Pierce, Dept. of Internal Medicine, Washington Univ. School of Medicine at Barnes-Jewish Hospital, 216 South Kingshighway, St. Louis, MO 63110.

Received 24 June 1997; accepted in final form 22 September 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Adamson, I. Y., L. Young, and D. H. Bowden. Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am. J. Pathol. 130: 377-383, 1988[Abstract].

2.   Adamson, I. Y., L. Young, and K. M. King. Reciprocal epithelial-fibroblast interactions in the control of fetal and adult rat lung cells in culture. Exp. Lung Res. 17: 821-835, 1991[Medline].

3.   Barone, L. M., B. L. Wolfe, B. Faris, and C. Franzblau. Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures. Biochemistry 27: 3175-3182, 1988[Medline].

4.   Bernard, M. P., M.-L. Chu, J. C. Myers, F. Ramirez, E. F. Eikenberry, and D. J. Prockop. Nucleotide sequences of complementary deoxyribonucleic acids for the proalpha 1 chain of human type I procollagen. Statistical evaluation of structures that are conserved during evolution. Biochemistry 22: 5213-5223, 1983[Medline].

5.   Bernfield, M., S. D. Banerjee, J. E. Koda, and A. C. Rapraeger. Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction. In: The Role of Extracellular Matrix in Development, edited by R. L. Trestad. New York: Liss, 1984, p. 545-572.

6.   Bienkowski, R. S., and M. G. Gotkin. Control of collagen deposition in mammalian lung. Proc. Soc. Exp. Biol. Med. 209: 118-140, 1995[Abstract].

7.   Caniggia, I., I. Tseu, R. N. N. Han, B. T. Smith, K. Tanswell, and M. Post. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L424-L433, 1991[Abstract/Free Full Text].

8.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

9.   Christensen, P. J., S. Kim, R. H. Simon, G. B. Toews, and I. R. Paine. Differentiation-related expression of ICAM-1 by rat alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 8: 9-15, 1993[Medline].

10.   Crouch, E. C. Pathobiology of pulmonary fibrosis. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L159-L184, 1990[Abstract/Free Full Text].

11.   Cunha, G. R., R. M. Bigsby, P. S. Cooke, and Y. Sugimura. Stromal-epithelial interactions in adult organs. Cell Differ. 17: 137-148, 1985[Medline].

12.   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].

13.   Dobbs, L., M. 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].

14.   Dobbs, L. G., and R. J. Mason. Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to beta-adrenergic stimulation. J. Clin. Invest. 63: 378-387, 1979[Medline].

15.   Dunsmore, S. E., C. L. Osborne, R. A. Goodman, and D. E. Rannels. Composition of the extracellular matrix of type II pulmonary epithelial cells in primary culture. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L754-L765, 1995[Abstract/Free Full Text].

16.   Finch, P. W., J. S. Rubin, T. Miki, D. Ron, and S. A. Aaronson. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 215: 752-754, 1989.

17.   Griffin, M., R. Bhandari, G. Hamilton, Y.-C. Chan, and J. T. Powell. Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen. J. Cell Sci. 105: 423-432, 1993[Abstract/Free Full Text].

18.   Gross-Jendroska, M., G.-M. Lui, M.-K. Song, and R. Stern. Retinal pigment epithelium-stromal interactions modulate hyaluronic acid deposition. Invest. Ophthalmol. Vis. Sci. 33: 3394-3399, 1992[Abstract].

19.   Hackett, B. P., N. Shimizu, and J. D. Gitlin. Clara cell secretory protein gene expression in bronchiolar epithelium. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L399-L404, 1992[Abstract/Free Full Text].

20.   Horley, K. J., C. Carpenito, B. Baker, and F. Takei. Molecular cloning of murine intercellular adhesion molecule (ICAM-1). EMBO J. 8: 2889-2896, 1989[Abstract].

21.   Infeld, M. D., J. A. Brennan, and P. B. Davis. Human fetal lung fibroblasts promote invasion of extracellular matrix by normal human tracheobronchial epithelial cells in vitro: a model of early airway gland development. Am. J. Respir. Cell Mol. Biol. 8: 69-76, 1993[Medline].

22.   Kawamoto, M., D. J. Romberger, Y. Nakamura, Y. Adachi, L. Tate, R. F. Ertl, J. R. Spurzem, and S. I. Rennard. Modulation of fibroblast type I collagen and fibronectin production by bovine bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 12: 425-433, 1995[Abstract].

23.   Kornblihtt, A. R., K. Umezawa, K. Vibe-Pedersen, and F. E. Baralle. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 4: 1755-1759, 1985[Abstract].

24.   Maghni, K., C. Robidoux, J. Laporte, A. Hallee, and P. Sirois. Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes. Prostaglandins 40: 217-227, 1990[Medline].

25.   McGowan, S. E. Influences of endogenous and exogenous TGF-beta on elastin in rat lung fibroblasts and aortic smooth muscle cells. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L257-L263, 1992[Abstract/Free Full Text].

26.   Montesano, R., K. Matsumoto, T. Nakamura, and L. Orci. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67: 901-908, 1991[Medline].

27.   Niswander, L., C. Tickle, A. Vogel, I. Booth, and G. R. Martin. FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75: 579-587, 1993[Medline].

28.   Parks, W. C., M. E. Kolodziej, and R. A. Pierce. Phorbol ester-mediated downregulation of tropoelastin expression is controlled by a posttranscriptional mechanism. Biochemistry 31: 6639-6645, 1992[Medline].

29.   Parks, W. C., R. A. Pierce, K. A. Lee, and R. P. Mecham. Elastin. In: Advances in Molecular and Cell Biology, edited by H. K. Kleinman. Greenwich, CT: JAI, 1993, vol. 6, p. 133-182.

30.   Parks, W. C., and J. D. Roby. Consequences of prolonged inhalation of ozone on F344/N rats. IV. Effects on expression of extracellular matrix genes. Res. Rep. Health Eff. Inst. 65: 3-20, 1994[Medline].

31.   Pierce, R. A., S. B. Deak, C. A. Stolle, and C. D. Boyd. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning. Biochemistry 29: 9677-9683, 1990[Medline].

32.   Pierce, R. A., M. E. Kolodziej, and W. C. Parks. 1,25-Dihydroxyvitamin D3 represses tropoelastin expression by a posttranscriptional mechanism. J. Biol. Chem. 267: 11593-11599, 1992[Abstract/Free Full Text].

33.   Pierce, R. A., W. Mariencheck, S. Sandefur, E. C. Crouch, and W. C. Parks. Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L491-L500, 1995[Abstract/Free Full Text].

34.   Premecz, G., A. Markovits, and I. Foldes. Signal transduction mechanisms in interferon action. Acta Microbiol. Immunol. Hung. 40: 131-140, 1993.

35.   Prosser, I. W., K. R. Stenmark, M. Suthar, E. C. Crouch, R. P. Mecham, and W. C. Parks. Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am. J. Pathol. 135: 1073-1088, 1989[Abstract].

36.   Quinn, C. O., D. K. Scott, C. E. Brinckerhoff, L. M. Matrisian, J. J. Jeffrey, and N. C. Partridge. Rat collagenase: cloning, amino acid sequence comparison and parathyroid hormone regulation in osteoblastic cells. J. Biol. Chem. 265: 13521-13527, 1990[Abstract/Free Full Text].

37.   Rannels, S. R., and D. E. Rannels. Isolation and culture of type II pulmonary epithelial cells. In: Cell Biology: A Laboratory Handbook, edited by J. E. Celis. Orlando, FL: Academic, 1994, p. 116-123.

38.   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 lung alveolar type I cell gene in embryonic endodermal and neural derivatives. Dev. Biol. 167: 294-306, 1995[Medline].

39.   Ros, M. A., G. Lyons, R. A. Kosher, W. B. Upholt, C. N. D. Coelho, and J. F. Fallon. Apical ridge dependent and independent mesoderm domain of GHox-7 and GHox-8 expression in chick limb buds. Development 116: 811-818, 1993[Abstract/Free Full Text].

40.   Saarialho-Kere, U. K., E. S. Chang, H. G. Welgus, and W. C. Parks. Distinct localization of collagenase and tissue inhibitor of metalloproteinases expression in wound healing associated with ulcerative pyogenic granuloma. J. Clin. Invest. 90: 1952-1957, 1992[Medline].

41.   Saunders, J. The proximo-distal sequence of origin of the parts of the chick wing, and the role of the ectoderm. J. Exp. Zool. 108: 363-404, 1948.

42.   Sawyer, R. H., and J. F. Fallon. Epithelial-Mesenchymal Interactions In: Development. New York, New York: Praeger Publishers, 1983.

43.   Schutze, S., T. Machleidt, and M. Kronke. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J. Leukoc. Biol. 56: 533-541, 1994[Abstract].

44.   Shannon, J. M., S. D. Jennings, and L. D. Nielsen. 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].

45.   Shoji, S., K. A. Rickard, R. F. Ertl, J. Linder, and S. I. Rennard. Lung fibroblasts produce chemotactic factors for bronchial epithelial cells. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L71-L79, 1989[Abstract/Free Full Text].

46.   Smith, B. T., and M. Post. Fibroblast pneumocyte factor. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L174-L178, 1989[Abstract/Free Full Text].

47.   Smith, B. T., and M. Post. Tissue interactions. In: The Lung, edited by R. G. Crystal, and J. B. West. New York: Raven, 1991, p. 671-676.

48.   Solursh, M., and R. S. Reiter. Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis. J. Exp. Zool. 248: 147-154, 1988[Medline].

49.   Swee, M., W. C. Parks, and R. A. Pierce. Developmental regulation of elastin production. Expression of tropoelastin pre-mRNA persists after downregulation of steady-state mRNA levels. J. Biol. Chem. 270: 14899-14906, 1995[Abstract/Free Full Text].

50.   Vandersteen, P., A. Vanlommel, and J. M. Lauweryns. Elastogenesis is linked to epithelial-stromal interactions in postnatal hamster bronchioles. Cell Tissue Res. 284: 467-471, 1996[Medline].

51.   Wei, Y., E. V. Yang, K. P. Klatt, and R. A. Tassava. Monoclonal antibody MT2 identifies the urodele a1 chain of type XII collagen, a developmentally regulated extracellular matrix protein in regenerating newt limbs. Dev. Biol. 168: 503-513, 1995[Medline].

52.   Yang, E. V., D. T. Shima, and R. A. Tassava. Monoclonal antibody ST1 identifies an antigen that is abundant in the axolotl and newt limb stump but is absent from the undifferentiated regenerate. J. Exp. Zool. 264: 337-350, 1992[Medline].

53.   Yaoqi, W., and D. Sassoon. Ectoderm-mesenchyme and mesenchyme-mesenchyme interactions regulate Msx-1 expression and cellular differentiation in the murine limb bud. Dev. Biol. 168: 374-382, 1995[Medline].

54.   Zwilling, E. Limb morphogenesis. In: Advances in Morphogenesis, edited by M. Abercrombie, and J. Brachet. New York: Academic, 1961, p. 301-328.


AJP Lung Cell Mol Physiol 274(1):L47-L57
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society