Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression

Tomohiko Nakamura1, Mingyao Liu2,3, Eric Mourgeon2, Art Slutsky2,4, and Martin Post1,5,6

1 Lung Biology Program, Hospital for Sick Children Research Institute, 2 Thoracic Surgery Research Laboratory, The Toronto Hospital Research Institute, and Departments of 5 Paediatrics, 6 Physiology, 4 Medicine, and 3 Surgery, University of Toronto, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Physical forces derived from fetal breathing movements and hormones such as glucocorticoids are implicated in regulating fetal lung development. To elucidate whether the different signaling pathways activated by physical and hormonal factors are integrated and coordinated at the cellular and transcriptional levels, organotypic cultures of mixed fetal rat lung cells were subjected to static culture or mechanical strain in the presence and absence of dexamethasone. Tropoelastin and collagen type I were used as marker genes for fibroblasts, whereas surfactant protein (SP) A and SP-C were used as marker genes for distal epithelial cells. Mechanical strain, but not dexamethasone, significantly increased SP-C mRNA expression. Tropoelastin mRNA expression was upregulated by both mechanical strain and dexamethasone. No additive or synergistic effect was observed when cells were subjected to mechanical stretch in the presence of dexamethasone. Neither mechanical strain nor dexamethasone alone or in combination had any significant effect on the expression of SP-A mRNA. Dexamethasone decreased collagen type I mRNA expression, whereas mechanical strain had no effect. The increases in tropoelastin and SP-C mRNA levels induced by mechanical strain and/or dexamethasone were accompanied by increases in their heterogeneous nuclear RNA. In addition, the stretch- and glucocorticoid-induced alterations in tropoelastin and SP-C mRNA expression were abrogated with 10 µg/ml actinomycin D. These findings suggest that tropoelastin and SP-C genes are selectively stimulated by physical and/or hormonal factors at the transcriptional level in fetal lung fibroblasts and distal epithelial cells, respectively.

glucocorticoids; gene transcription; fetal lung cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FETAL LUNG DEVELOPMENT requires coordinated lung growth and maturation. This is a highly regulated process controlled by many factors including hormones and physical forces. The latter force derived from normal fetal breathing movements supports fetal lung growth, whereas an altered breathing pattern, e.g., reduced amplitude, may result in pulmonary hypoplasia (4). One of the most important markers of pulmonary maturation is the ability to produce surfactant, a complex of lipids and protein that lines the alveolar surface of the lung. Recent studies have shown that physical forces affect pulmonary surfactant metabolism. A static stretch of adult type II cells stimulated the release of surfactant phospholipids (31), whereas a cyclic stretch of fetal type II cells increased the synthesis of surfactant phospholipids (22). The effect of mechanical strain on surfactant protein (SP) production by fetal lung cells is as yet unknown. Antenatal glucocorticoid administration, however, has been shown to increase SP expression by fetal type II cells (16, 18). The importance of endogenous glucocorticoids for lung maturation is demonstrated by the delayed pulmonary development of mice with targeted mutation of the glucocorticoid receptor (2) or corticotropin-releasing hormone (CRH) (10, 11) genes. The lungs of CRH-deficient mice exhibit retarded lung maturation as indicated by delayed SP-A, SP-B, and fatty acid synthetase expression as well as by increased cellular proliferation (12). Additional features of morphological maturation of the fetal lung include increased air space formation, vascularization, and thinning of alveolar septa, leading to a mature blood-gas interface. Lung elastogenesis is intimately associated with these morphological processes. Moreover, tropoelastin expression in the fetal lung coincides with the initiation of fetal breathing and the rise of circulating glucocorticoids (15, 17). Both glucocorticoids (14, 36) and mechanical strain (24) have been shown to stimulate tropoelastin production in cultured mesenchyme-derived cells. Several other extracellular matrix molecule genes, including collagen type I, have been shown to be influenced by mechanical stress (33, 35) and glucocorticoids (1). Thus it is evident that physical forces and glucocorticoids influence the expression of a number of maturational genes in the fetal lung; however, the interaction between physical forces and hormonal factors in controlling fetal lung development is unknown. The objective of this study was to elucidate whether the signaling pathways induced by physical and hormonal factors during development are integrated and coordinated at the cellular and transcriptional levels.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of organotypic cultures. The organotypic culture of mixed fetal lung cells has been previously described (6). Briefly, the rats were killed by an excess of diethyl ether during the canalicular stage of fetal lung development at 19 days of gestation (term = 22 days). The fetuses were aseptically removed, and the fetal lungs were dissected out in cold Hanks' balanced salt solution without calcium or magnesium (HBSS-) and cleared of major airways and vessels. The lungs were washed twice in HBSS-, minced, and suspended in HBSS-. The lung tissue was digested for 20 min in an enzymatic solution of 0.125% (wt/vol) trypsin and 0.4 mg/ml DNase. After filtration through 100-µm mesh nylon blotting cloth, Eagle's minimum essential medium (MEM) with 10% (vol/vol) fetal bovine serum (FBS) was added to the single-cell suspension to neutralize trypsin activity, and the mixture was centrifugated at 300 g for 10 min. The pellet was resuspended in MEM plus 10% FBS and inoculated onto 2 × 2 × 0.25-cm Gelfoam sponges at a density of 1.6 × 106 cells/sponge. After inoculation, the cells were incubated for 1 h before the addition of 3 ml of MEM plus 10% (vol/vol) FBS to the culture dish. After a 24-h incubation, the sponges were washed three times with serum-free MEM and then incubated in serum-free MEM for 24 h before treatment.

Exposure of mixed fetal rat lung cells to stretch and dexamethasone. The mechanical stretch device used in these studies has been described in detail elsewhere (6). It consists of a programmable burst timer, a control unit, a dual-regulated DC power supply, and a set of solenoids. A culture dish with a Gelfoam sponge was placed in front of each solenoid. One end of each sponge was glued to the bottom of the dish. The other end was attached to a movable metal bar, which was wrapped and sealed in plastic tubing. The strain of cells cultured on sponges was driven by the magnetic force generated through the solenoids. The sponges were subjected to a 5% elongation from their original length at 60 cycles/min for 15 min/h, which optimally enhanced DNA synthesis and cell division without cell injury (6). The cells were incubated in MEM with and without dexamethasone (10-7 M) and subjected to static culture or intermittent strain for 24 h.

Northern analysis. Total cellular RNA was isolated from organotypic cultures by lysing the cells in 4 M guanidinium thiocyanate followed by centrifugation on a 5.7 M cesium chloride cushion to pellet RNA. Total RNA (15 µg) was size-fractionated on 1% (wt/vol) agarose gel containing 3% (wt/vol) formaldehyde, transferred to Hybond-N+ membranes, and immobilized by ultraviolet (UV) cross-linking. Tropoelastin and collagen type I cDNAs were labeled with [alpha -32P]dCTP with the random-primer method. Prehybridization and hybridization were performed in 50% (vol/vol) formamide, 5× sodium chloride-sodium phosphate-EDTA, 0.5% (wt/vol) SDS, 5× Denhardt's solution, and 100 µg/ml denatured herring sperm DNA at 42°C. After hybridization, the blots were washed with 2× saline-sodium citrate (SSC) containing 0.2% (wt/vol) SDS at 42°C for 10 min, followed by 1× SSC with 0.2% (wt/vol) SDS at 42°C for 10 min. The blots were exposed to Kodak XAR-5 film at -80°C. The blots were then stripped and rehybridized with radiolabeled rat beta -actin cDNA for normalization.

RT-PCR and Southern blotting. Steady-state mRNA levels of SP-A and SP-C and heterogeneous nuclear RNA (hnRNA) levels of tropoelastin and SP-C were amplified by RT-PCR. Specifically, total lung RNA was treated with RNase-free DNase to remove contaminating DNA. Total RNA (3 µg) in 5 µl of sterile water with 2.5 µM random hexanucleotide primer (pdN6) was heated to 70°C for 10 min, quick-chilled on ice, and then added to the reverse transcription reaction in microcentrifuge tubes. Each reaction contained 4 µl of 5× PCR buffer (100 mM Tris · HCl, pH 8.3, and 500 mM KCl), 0.2 µM 1,4-dithiothreitol, and 7.5 mM deoxynucleotide triphosphates (dNTPs). After incubation at 37°C for 2 min, 200 U of Superscript RT were added to a total volume of 20 µl. The samples were incubated at 37°C for 60 min and heated to 70°C for 15 min, followed by cooling at 5°C for 5 min. The cDNAs (5 µl) from the reverse transcription reaction were then incubated with 5 µl of 10× PCR buffer, 2.5 mM MgCl2, 10 mM dNTPs, 0.2 µM each primer, and 2.5 U of Taq polymerase in a total volume of 50 µl at 95°C for 3 min. The samples were amplified for 15 (mRNA) and 25 (hnRNA) cycles, each consisting of 53°C annealing for 30 s, 72°C extension for 30 s, and denaturation for 30 s at 95°C. Duration of the final elongation reaction was increased to 7 min at 72°C. For Southern blotting, amplified DNA (25 µl of total PCR) was transferred to Hybond-N+ membranes and immobilized by UV cross-linking. SP-A, SP-C, and beta -actin probes for detection of amplified mRNA were labeled by random priming with [gamma -32P]dCTP. Tropoelastin and SP-C oligomer probes for detection of amplified hnRNA were labeled with [alpha -32P]ATP with T4 polynucleotide kinase. After Southern hybridization, the blots were exposed to Kodak XAR-5 film for 6-12 h at -80°C. Autoradiographic signal was quantified by densitometry and normalized to the relative amount of beta -actin mRNA. No signals were detected after RNase treatment of samples as well as after PCR without the initial addition of RT. The oligonucleotide primer sequences are listed in Table 1, and PCR strategy is presented in Fig. 1.

                              
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Table 1.   Oligonucleotide primer sequences



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Fig. 1.   Schematic presentation of location of primers and probes used for RT-PCR and Southern blotting. SP, surfactant protein; TE, tropoelastin; hnRNA, heterogeneous nuclear RNA.

Statistical analysis. Statistical significance was determined by one-way ANOVA followed by assessment of differences with the Student-Newman-Keuls test for nonpaired groups. Significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical strain selectively stimulated SP-C but not SP-A mRNA expression. To examine the effects of physical and hormonal factors on fetal lung epithelial cell maturation, organotypic cultures of mixed fetal lung cells incubated with and without 10-7 M dexamethasone were subjected to either intermittent strain (60 cycles/min, 15 min/h, 5% elongation) or static culture for 24 h. Equal amounts of total RNA (3 µg) were analyzed by low-cycle RT-PCR followed by Southern hybridization with epithelium-specific SP-A and SP-C probes (5, 30, 32). Mechanical strain, but not dexamethasone treatment, significantly increased the steady-state mRNA level of SP-C (Fig. 2). When the cells were subjected to both mechanical strain and dexamethasone treatment, SP-C transcript levels were not increased further (Fig. 2). In contrast, neither mechanical strain nor dexamethasone affected steady-state mRNA levels of SP-A in organotypic cultures of fetal rat lung cells (Fig. 2).



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Fig. 2.   Mechanical strain increased mRNA expression of SP-C, but not SP-A, in organotypic cultures of fetal rat lung cells. Day 19 organotypic cultures of mixed lung cells incubated with and without 10-7 M dexamethasone (Dex) were subjected to a 24-h intermittent strain regimen (60 cycles/min, 15 min/h, 5% elongation) or static culture. Equal amounts of total RNA (3 µg) were analyzed by RT-PCR followed by Southern hybridization with 32P-labeled SP-A, SP-C, and beta -actin probes (A). Intensity of amplified mRNA bands was quantified by densitometry (B). Results are expressed as a ratio over beta -actin and normalized to control value. Values are means ± SE of 3 separate experiments. * P < 0.05 vs. control.

Mechanical strain and dexamethasone stimulated tropoelastin but not collagen type I mRNA expression. The effects of physical and hormonal factors on gene expression in fetal lung mesenchymal cells were determined with the use of tropoelastin and collagen type I as marker genes (19, 35, 36). Mechanical strain did not affect mRNA expression of collagen type I (Fig. 3), whereas dexamethasone treatment showed a tendency to decrease the number of collagen type I transcripts. In contrast, mechanical strain and dexamethasone treatment alone significantly increased mRNA levels of tropoelastin. However, when both mechanical strain and dexamethasone were applied to fetal lung cells, no additive or synergistic effect on either collagen type I or tropoelastin mRNA expression was observed (Fig. 3).



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Fig. 3.   Mechanical strain and Dex increased mRNA expression of tropoelastin, but not type I collagen, in organotypic cultures of fetal rat lung cells. Day 19 organotypic cultures of mixed lung cells incubated with and without 10-7 M Dex were subjected to a 24-h intermittent strain regimen (60 cycles/min, 15 min/h, 5% elongation) or static culture. Equal amounts of total RNA (10 µg) were analyzed by Northern hybridization with 32P-labeled tropoelastin, type I collagen, and beta -actin probes (A). Intensity of amplified mRNA bands was quantified by densitometry (B). Results are expressed as a ratio over beta -actin and normalized to control value. Values are means ± SE of 3 separate experiments. * P < 0.05 vs. control.

Mechanical strain and dexamethasone increased SP-C and tropoelastin gene expression at the transcriptional level. To determine whether the increased mRNA levels of SP-C and tropoelastin required new gene transcription, cells incubated with and without dexamethasone were subjected to either mechanical strain or static culture in the presence of 10 µg/ml actinomycin D, an inhibitor of gene transcription. Actinomycin D abrogated mechanical strain-induced SP-C and tropoelastin mRNA expression as well as the dexamethasone-induced increase in tropoelastin mRNA (Fig. 4). Thus it appears that the upregulation in SP-C and tropoelastin mRNA expression by mechanical strain and/or dexamethasone is controlled primarily at the level of transcription. To confirm that mechanical strain and/or dexamethasone treatment triggers transcriptional activation of the SP-C and tropoelastin genes, hnRNAs of SP-C and tropoelastin were analyzed by RT-PCR followed by Southern hybridization (25). In principle, hnRNA (or preprocessed mRNA) is a complete copy of a DNA template, which contains both introns and exons. After being spliced, intron sequences are removed and exons are linked to mRNA. Because splicing is a rapid process, measurement of hnRNA should estimate ongoing transcription. To measure hnRNA levels, PCR primers were designed to include intron sequences of the gene of interest, and the PCR products (SP-C, 643 bp; tropoelastin, 478 bp) were detected by Southern hybridization with oligoprobes containing SP-C- or tropoelastin-specific sequences (Fig. 1). Mechanical strain significantly increased the level of SP-C hnRNA, whereas dexamethasone alone had no effect (Fig. 5). The combination of mechanical strain and dexamethasone also increased the amount of SP-C hnRNA (Fig. 5). Stretch and dexamethasone alone as well as the combined treatment significantly increased hnRNA expression of tropoelastin (Fig. 6). These results corroborate that the upregulation of SP-C gene expression by mechanical strain and tropoelastin gene expression by mechanical strain and/or dexamethasone are mainly due to an increase in the rate of transcription.


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Fig. 4.   Increased SP-C and tropoelastin mRNA expression in organotypic cultures of fetal rat lung cells subjected to mechanical strain and/or Dex were blocked by actinomycin D. Day 19 organotypic cultures of mixed lung cells incubated with and without 10-7 M Dex were subjected to a 24-h intermittent strain regimen (60 cycles/min, 15 min/h, 5% elongation) or static culture in presence of 10 µg/ml actinomycin D. Equal amounts of total RNA were analyzed by either RT-PCR and Southern hybridization (SP-C) or by Northern hybridization (TE). Intensity of amplified mRNA bands was quantified by densitometry. Results are expressed as a ratio over beta -actin and normalized to control value. Values are means ± SE of 3 separate experiments. * P < 0.05 vs. control.



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Fig. 5.   Increased transcription of SP-C gene in organotypic cultures of fetal rat lung cells subjected to mechanical strain. Day 19 organotypic cultures of mixed lung cells incubated with and without 10-7 M Dex were subjected to a 24-h intermittent strain regimen (60 cycles/min, 15 min/h, 5% elongation) or static culture. Amounts of hnRNA were analyzed by RT-PCR followed by Southern hybridization (A; see Fig. 1). Intensity of amplified hnRNA bands was quantified by densitometry (B). Results were normalized to control value. Values are means ± SE of 3 separate experiments. * P < 0.05 vs. control.



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Fig. 6.   Increased transcription of TE gene in organotypic cultures of fetal rat lung cells subjected to mechanical strain and/or Dex. Day 19 organotypic cultures of mixed lung cells incubated with and without 10-7 M Dex were subjected to a 24-h intermittent strain regimen (60 cycles/min, 15 min/h, 5% elongation) or static culture. hnRNA expression was determined by RT-PCR followed by Southern hybridization (A; see Fig. 1). Intensity of amplified hnRNA bands was quantified by densitometry (B). Results were normalized to control value. Values are means ± SE of 3 separate experiments. * P < 0.05 from control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A variety of growth factors, hormones, and cytokines as well as physical factors act as positive or negative regulators of lung growth and development (27). Because fetal breathing movements and endogenous hormones are concurrently present during lung development, it is difficult to distinguish their relative contribution to the maturational process. To elucidate the interactions between physical and hormonal factors on fetal lung cell maturation at the cellular and molecular levels, we exposed mixed fetal rat lung cells in organotypic cultures to mechanical strain and dexamethasone alone or as a combination. Marker gene analysis of this complex cellular model allowed us to study the influence of mechanical strain and/or glucocorticoids on the maturation of lung epithelial cells and fibroblasts separately. We found that mechanical stress and dexamethasone selectively stimulated gene expression in both epithelial and mesenchymal cells. However, both stimulants appear not to form an integrative signaling network during lung development.

Selective stimulation of SP-C gene expression by mechanical strain. On the basis of previous in vivo (12, 21, 26) and in vitro (13, 28) studies in fetal rats, we anticipated that glucocorticoids would increase SP-A and SP-C mRNA expression. Our findings are not consistent with these earlier observations. A possible explanation may be the longer duration of exposure (>48 vs. 24 h) of the rat lung to glucocorticoids in the previous studies (13, 21, 26, 28). Although physical forces have been shown to affect pulmonary surfactant lipid metabolism of fetal lung cells (22), to our knowledge, this is the first demonstration that mechanical strain influences SP-C gene expression by fetal lung epithelial cells. The observation that SP-C and not SP-A mRNA expression is regulated by an intermittent mechanical strain in vitro suggests that fetal breathing movements may specifically contribute to SP-C gene expression during development. Indeed, the appearance of SP-C mRNA in the fetal rat lung coincides with the initiation of fetal breathing movements at 16-17 days of gestation, whereas SP-A transcripts are first detectable at 18-19 days of gestation (20). Whether mechanical strain directly affects SP-C expression in fetal epithelial cells remains to be elucidated. We used an organotypic cell culture system in which mesenchymal-epithelial cell interactions are preserved to mimic the cellular environment in vivo. Because the developmental response to mechanical strain is determined by the mesenchyme (34) and SP-C gene expression is controlled by mesenchymal-epithelial interactions (3, 23), it is possible that a mesenchyme-regulatory mechanism is involved in the mechanical strain-induced SP-C gene expression.

Selective stimulation of tropoelastin gene expression by mechanical strain and dexamethasone. The physiological importance of elastic fibers lies in the unique elastometric properties of elastin, which is the functional component of the mature fiber. During fetal development, the lung is characterized by the increase in collagenous and elastic matrices and by the dynamic changes in the number and spatial relationship of collagen- and elastin-producing cells. In this study, we demonstrated that mechanical force and glucocorticoids are important regulators of gene expression of tropoelastin, the soluble precursor of elastin. The induction of tropoelastin production coincides with the initiation of fetal breathing movements (8, 17). Yee et al. (36) have previously reported that glucocorticoid-induced tropoelastin gene expression in fetal lung fibroblasts is mediated via transforming growth factor (TGF)-beta 3. Because mechanical strain has been found to stimulate TGF-beta gene expression in various cells (7, 37), it is plausible that strain-induced tropoelastin gene expression in fetal lung cells is mediated via TGF-beta 3. No significant additive or synergistic effect in tropoelastin gene expression was noted when cells were subjected to mechanical strain in the presence of dexamethasone. This suggests that the signaling pathways activated by physiological levels of mechanical strain and glucocorticoids in fetal lung fibroblasts are probably not integrated or coordinated at the transcriptional level. Alternatively, it is possible that this level of mechanical strain and dexamethasone resulted in a maximal effect and that suboptimal levels would have shown additive or synergistic effects.

Transcriptional regulation of SP-C and tropoelastin by mechanical strain and dexamethasone. To assess the molecular mechanism by which physical force and glucocorticoids control tropoelastin and SP-C mRNA expression, we first inhibited transcription with actinomysin D. Because this treatment blocked mechanical strain-induced SP-C mRNA expression as well as mechanical strain- and/or dexamethasone-induced tropoelastin mRNA expression, we examined transcriptional regulation by determining the tropoelastin or SP-C hnRNA levels. Although this strategy does not directly measure the transcriptional activity, hnRNA levels reflect the rate of active, ongoing transcription (25). Compared with the commonly used nuclear runoff assays, the hnRNA approach is more sensitive and requires less material. A great advantage is that it can be combined with regular RT-PCR to measure both mRNA and hnRNA from the same RT products. In the present study, we found that hnRNA levels of both SP-C and tropoelastin were increased by mechanical strain, whereas dexamethasone also increased the amount of tropoelastin hnRNA. The magnitude of changes in mRNA and hnRNA levels induced by either mechanical strain or dexamethasone were in the same range, suggesting that the elevation in mRNAs was mainly due to an increase in transcription of the SP-C and tropoelastin genes. In contrast to tropoelastin expression, Xu et al. (35) have recently found that mechanical strain regulates the expression of various extracellular matrix molecules in fetal lung cells by a posttranscriptional mechanism. Mechanical strain increased soluble fibronectin content by increasing protein synthesis and secretion of fibronectin while decreasing fibronectin message levels (9). A similar effect of mechanical stress was observed for collagen type I expression (35). Mechanical strain of fetal lung cells has also been shown to increase the secretion of proteoglycans and glycosaminonglycans without affecting core protein expression (33, 35). Thus it appears that the effects of mechanical forces on extracellular matrix remodeling during fetal lung development are regulated at various levels depending on the extracellular matrix molecule.


    ACKNOWLEDGEMENTS

This study was supported by a Medical Research Council of Canada Group Grant (to M. Post) and Operating Grant MT-13270 (to M. Liu) and an operating grant from the James H. Cumming Foundation (to M. Liu).


    FOOTNOTES

E. Mourgeon is a recipient of Fellowships from the Société Française du Anesthesize et de Réanimation (SFAR) and the Dean's Office, Faculty of Medicine, University of Toronto (Toronto, Canada). M. Liu is a Scholar of the Medical Research Council of Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Post, Lung Biology Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: mppm{at}sickkids.on.ca).

Received 9 July 1999; accepted in final form 9 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Beck, JC, Mitzner W, Johnson JW, Hutchins GM, Fiodart JM, Lomdon WT, Palmer AE, and Scott R. Betamethasone and the rhesus fetus: effect on lung morphometry and connective tissue. Pediatr Res 15: 235-240, 1981[Abstract].

2.   Cole, TJ, Blendy JA, Monaghan P, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi Hummler E, Unsicker K, and Schütz G. Targeted disruption of the glucocorticoid receptor blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9: 1611-1621, 1995.

3.   Deimling, J, Thompson K, Tseu I, and Post M. Mesenchyme determines epithelial morphogenesis but not differentiation in lung recombinants at late fetal gestation (Abstract). Am J Respir Crit Care Med 157: A14, 1998.

4.   Harding, R, and Albuquerque C. Pulmonary hypoplasia: role of mechanical factors in prenatal lung growth. In: Lung Development, edited by Gaultier C, Bourbon J, and Post M.. Oxford, UK: Oxford University Press, 1999, p. 364-394.

5.   Kalina, M, Mason RJ, and Shannon JM. Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung. Am J Respir Cell Mol Biol 6: 594-600, 1992[ISI][Medline].

6.   Liu, M, Skinner SJM, Xu J, Han RNN, Tanswell AK, and Post M. Stimulation of fetal rat lung cell proliferation in vitro by mechanical strain. Am J Physiol Lung Cell Mol Physiol 263: L376-L383, 1992[Abstract/Free Full Text].

7.   Li, Q, Muragaki Y, Hatamura I, Ueno H, and Ooshima A. Stretch-induced collagen synthesis in cultured smooth muscle cells from rabbit aortic media and a possible involvement of angiotensin II and transforming growth factor-beta. J Vasc Res 35: 93-103, 1998[ISI][Medline].

8.   Mariani, TJ, and Pierce RA. Development of lung elastic matrix. In: Lung Development, edited by Gaultier C, Bourbon J, and Post M.. Oxford, UK: Oxford University Press, 1999, p. 28-45.

9.   Mourgeon, E, Xu J, Tanswell AK, Liu M, and Post M. Mechanical strain-induced posttranscriptional regulation of fibronectin production in fetal lung cells. Am J Physiol Lung Cell Mol Physiol 277: L142-L149, 1999[Abstract/Free Full Text].

10.   Muglia, LJ, Jenkins NA, Gilbert DJ, Copeland NG, and Majzoub JA. Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells. J Clin Invest 93: 2066-2072, 1994[ISI][Medline].

11.   Muglia, L, Jacobson Dikkes P, and Majzoub JA. Cortocotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 373: 427-432, 1995[ISI][Medline].

12.   Muglia, LJ, Bae DS, Brown TT, Vogt SK, Alvarez J, Sunday ME, and Majzoub JA. Proliferation and differentiation defects during lung development in corticotropin-releasing hormone-deficient mice. Am J Respir Cell Mol Biol 20: 181-188, 1999[Abstract/Free Full Text].

13.   Nichols, KV, Floros J, Dynia DW, Veletza SV, Wilson CM, and Gross I. Regulation of surfactant protein A mRNA by hormones and butyrate in cultured fetal rat lung. Am J Physiol Lung Cell Mol Physiol 259: L488-L495, 1990[Abstract/Free Full Text].

14.   Noguchi, A, Fisching K, Kursar JD, and Reddy R. Developmental changes in tropoelastin synthesis by rat pulmonary fibroblasts and effect of dexamethasone. Pediatr Res 28: 379-382, 1990[Abstract].

15.   Noguchi, A, and Samaha H. Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization. Am J Respir Cell Mol Biol 5: 571-578, 1991[ISI][Medline].

16.   Odom, MW, and Ballard PL. Developmental and hormonal regulation of the surfactant system. In: Lung Growth and Development, edited by McDonald J.. New York: Dekker, 1997, p. 495-575.

17.   Pierce, RA, Mariencheck WI, Sandefur S, Crouch EC, and Parks WC. Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development. Am J Physiol Lung Cell Mol Physiol 268: L491-L500, 1995[Abstract/Free Full Text].

18.   Post, M, and Smith BT. Hormonal control of surfactant metabolism. In: Pulmonary Surfactant: From Molecular Biology to Clinical Practice, edited by Robertson B, van Golde LMG, and Batenburg JJ.. Amsterdam: Elsevier, 1992, p. 379-424.

19.   Rolland, G, Xu J, Tanswell AK, and Post M. Ontogeny of extracellular matrix related gene expression by rat lung cells at late fetal gestation. Biol Neonate 73: 112-120, 1998[ISI][Medline].

20.   Schellhase, DE, Emrie PA, Fisher JH, and Shannon JM. Ontogeny of surfactant proteins in the rat. Pediatr Res 26: 167-174, 1989[Abstract].

21.   Schellhase, DE, and Shannon JM. Effects of maternal dexamethasone on expression of SP-A, SP-B and SP-C in the fetal rat. Am J Respir Cell Mol Biol 4: 304-312, 1991[ISI][Medline].

22.   Scott, JE, Yang S-Y, Stanik E, and Anderson JE. Influence of strain on [3H]thymidine incorporation, surfactant-related phospholopid synthesis, and cAMP levels in fetal type II alveolar cells. Am J Respir Cell Mol Biol 8: 258-265, 1993[ISI][Medline].

23.   Shannon, JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev Biol 166: 600-614, 1994[ISI][Medline].

24.   Sutcliffe, MC, and Davidson JM. Effect of static stretch on elastin production by porcine aortic smooth muscle cells. Matrix 10: 148-153, 1990[ISI][Medline].

25.   Swee, MH, Parks WC, and Pierce RA. Developmental regulation of elastin production. J Biol Chem 270: 14899-14906, 1995[Abstract/Free Full Text].

26.   Sweezey, N, Mawdsley C, Ghibu F, Song L, Buch S, Moore A, Antakly T, and Post M. Differential regulation of glucocorticoid receptor expression in fetal rat lung cells. Pediatr Res 38: 506-512, 1995[Abstract].

27.   Tanswell, AK, Liu M, and Post M. Bronchopulmonary dysplasia: strategies for therapeutic intervention. In: Intensive Care in Childhood, edited by Tibboel D, and van der Voort E.. Heidelberg, Germany: Springer-Verlag, 1996, vol. 25, p. 53-65.

28.   Vletza, SV, Nichols KV, Gross I, Lu H, Dynia DW, and Floros J. Surfactant protein C: hormonal control of SP-C mRNA levels in vitro. Am J Physiol Lung Cell Mol Physiol 262: L684-L687, 1992[Abstract/Free Full Text].

29.   Wang, J, Kuliszewski M, Yee W, Sedlackova L, Xu J, Tseu I, and Post M. Cloning and characterization of glucocorticoid-induced genes in fetal rat lung fibroblasts: transforming growth factor beta 3. J Biol Chem 270: 2722-2728, 1995[Abstract/Free Full Text].

30.   Wang, J, Souza P, Kuliszewski M, Tanswell AK, and Post M. Expression of surfactant proteins in embryonic rat lung. Am J Respir Cell Mol Biol 10: 222-229, 1994[Abstract].

31.   Wirtz, HRW, and Dobbs LG. Calcium mobilization and exocytosis after one mechanical strain of lung epithelial cells. Science 250: 1266-1269, 1990[ISI][Medline].

32.   Wohlford-Lenane, CL, and Snyder LM. Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization. Am J Respir Cell Mol Biol 7: 335-343, 1992[ISI][Medline].

33.   Xu, J, Liu M, Liu J, Caniggia I, and Post M. Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells. J Cell Sci 109: 1605-1613, 1996[Abstract/Free Full Text].

34.   Xu, J, Liu M, Tanswell K, and Post M. Mesenchymal determination of mechanical strain-induced fetal lung cell proliferation. Am J Physiol Lung Cell Mol Physiol 275: L545-L550, 1998[Abstract/Free Full Text].

35.   Xu, J, Liu M, and Post M. Differential regulation of expression of extracellular matrix molecules by mechanical strain of fetal lung cells. Am J Physiol Lung Cell Mol Physiol 276: L728-L735, 1999[Abstract/Free Full Text].

36.   Yee, W, Wang J, Liu J, Tseu I, Kuliszewski M, and Post M. Glucocorticoid induced tropoelastin expression is mediated via transforming growth factor-beta 3. Am J Physiol Lung Cell Mol Physiol 270: L992-L1001, 1996[Abstract/Free Full Text].

37.   Zhuang, H, Wang W, Tahernia AD, Levitz CL, Luchetti WT, and Brighton CT. Mechanical strain-induced proliferation of osteoblastic cell parallels increased TGF-beta 1 mRNA. Biochem Biophys Res Commun 13: 449-453, 1996.


Am J Physiol Lung Cell Mol Physiol 278(5):L974-L980
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