Identification of Betamethasone-Regulated Target Genes and Cell Pathways in Fetal Rat Lung Mesenchymal Fibroblasts

Endocrinology ◽  
2019 ◽  
Vol 160 (8) ◽  
pp. 1868-1884 ◽  
Author(s):  
Bennet K L Seow ◽  
Annie R A McDougall ◽  
Kelly L Short ◽  
Megan J Wallace ◽  
Stuart B Hooper ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Preterm Birth ◽  
Transcriptome Sequencing ◽  
Target Genes ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Matrix Remodeling ◽  
Rat Lung ◽  
Fetal Rat ◽  
Fetal Rat Lung

Abstract Preterm birth is characterized by severe lung immaturity that is frequently treated antenatally or postnatally with the synthetic steroid betamethasone. The underlying cellular targets and pathways stimulated by betamethasone in the fetal lung are poorly defined. In this study, betamethasone was compared with corticosterone in steroid-treated primary cultures of fetal rat lung fibroblasts stimulated for 6 hours and analyzed by whole-cell transcriptome sequencing and glucocorticoid (GC) receptor (GR) chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared with corticosterone for both induced and repressed genes. A total of 483 genes were significantly stimulated by betamethasone or corticosterone, with 476 stimulated by both steroids, indicating a strong overlap in regulation. Changes in mRNA levels were confirmed by quantitative PCR for eight induced and repressed target genes. Pathway analysis identified cell proliferation and cytoskeletal/cell matrix remodeling pathways as key processes regulated by both steroids. One target, transglutaminase 2 (Tgm2), was localized to fetal lung mesenchymal cells. Tgm2 mRNA and protein levels were strongly increased in fibroblasts by both steroids. Whole-genome GR ChIP-Seq analysis with betamethasone identified GC response element–binding sites close to the previously characterized GR target genes Per1, Dusp1, Fkbp5, and Sgk1 and near the genes identified by transcriptome sequencing encoding Crispld2, Tgm2, Hif3α, and Kdr, defining direct genomic induction of expression in fetal lung fibroblasts via the GR. These results demonstrate that betamethasone stimulates specific genes and cellular pathways controlling cell proliferation and extracellular matrix remodeling in lung mesenchymal fibroblasts, providing a basis for betamethasone’s treatment efficacy in preterm birth.

The adenylate cyclase response to parathyroid hormone in fetal lung fibroblasts is enhanced by cortisol

1987 ◽  
Vol 7 (6) ◽  
pp. 503-508 ◽  
Author(s):  
Carolyn Lowe ◽  
Peter M. Barling ◽  
Stephen J. M. Skinner
Keyword(s):  
Parathyroid Hormone ◽  
Epithelial Cells ◽  
Adenylate Cyclase ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Rat Lung ◽  
Osteosarcoma Cells ◽  
Fetal Rat ◽  
Fetal Rat Lung ◽  
Umr 106

Parathyroid hormone (PTH, <10−8 M) stimulated adenylate cyclase in fibroblasts, but not epithelial cells, isolated from fetal rat lung. In contrast to osteosarcoma cells (UMR 106), the response of fibroblasts to PTH was increased by pretreatment with cortisol (< 10−8-10−7 M).

Hyperoxia inhibits fetal rat lung fibroblast proliferation and expression of procollagens

1997 ◽  
Vol 273 (4) ◽  
pp. L726-L732 ◽  
Author(s):  
Naveed Hussain ◽  
Fengying Wu ◽  
Constance Christian ◽  
Mitchell J. Kresch
Keyword(s):  
Steady State ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type I ◽  
Fibroblast Proliferation ◽  
Rat Lung ◽  
Collagen Production ◽  
Fetal Rat ◽  
Fetal Rat Lung ◽  
Steady State Levels

The direct effects of hyperoxia on collagen production by fetal lung fibroblasts are unknown and would be important to the understanding of the molecular mechanisms involved in bronchopulmonary dysplasia in premature infants. We studied the effect of hyperoxia on 1) proliferation, 2) mRNA levels for type I and III procollagens, and 3) net collagen production in primary cultures of fetal rat lung fibroblasts. Fibroblasts from 19-day-old rat fetuses (term is 22 days) were obtained. Test plates were incubated in hyperoxia and controls in room air for varying time periods. Cell viability in both conditions was >97% as determined by trypan blue exclusion. Fibroblast proliferation in nonconfluent cultures was found to be significantly reduced with exposure to hyperoxia ( P< 0.001). Steady-state levels of type I and III procollagen mRNAs, analyzed on Northern blots hybridized to [32P]cDNA probes, were significantly decreased in hyperoxia ( P < 0.01). This effect was noted as early as 4 h of exposure to hyperoxia and persisted for 5 days. There was a significant inverse correlation between duration of exposure to O2 and steady-state levels of mRNA for α1(I)-procollagen ( r = −0.904) and α1(III)-procollagen ( r = −0.971). There were no significant changes in steady-state levels of β-actin mRNA. We also found a significant decrease in collagen synthesis in hyperoxia-exposed fibroblasts ( P < 0.05). We conclude that hyperoxia directly effects a reduction in fetal lung fibroblast proliferation and net collagen production at a pretranslational level.

scholarly journals Mechanical strain-induced posttranscriptional regulation of fibronectin production in fetal lung cells

1999 ◽  
Vol 277 (1) ◽  
pp. L142-L149 ◽  
Author(s):  
Eric Mourgeon ◽  
Jing Xu ◽  
A. Keith Tanswell ◽  
Mingyao Liu ◽  
Martin Post
Keyword(s):  
Cell Proliferation ◽  
Mechanical Strain ◽  
Fetal Lung ◽  
Northern Analysis ◽  
Normal Lung ◽  
Lung Cells ◽  
Mrna Levels ◽  
Rat Lung ◽  
Fetal Rat ◽  
Fetal Rat Lung

We have shown that intermittent mechanical strain, simulating fetal breathing movements, stimulated fetal rat lung cell proliferation. Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix remodeling, we investigated the effect of strain on fibronectin metabolism. Organotypic cultures of fetal rat lung cells, subjected to intermittent strain, showed increased fibronectin content in the culture media. Fibronectin-degrading activity in media from strained cells was similar to that of static cultures. Northern analysis revealed that strain inhibited fibronectin mRNA accumulation seen during static culture. Synthesis of fibronectin, determined by metabolic labeling, was increased by strain despite lower mRNA levels or presence of actinomycin D. This increase was not mediated via a rapamycin-sensitive mechanism. Strain stimulated prelabeled fibronectin secretion even in the presence of cycloheximide. These results suggest that strain differentially regulates fibronectin production of fetal lung cells at the transcriptional and posttranscriptional levels. Mechanical strain increases soluble fibronectin content by stimulating its synthesis and secretion without increasing fibronectin message levels.

Differential regulation of extracellular matrix molecules by mechanical strain of fetal lung cells

1999 ◽  
Vol 276 (5) ◽  
pp. L728-L735 ◽  
Author(s):  
Jing Xu ◽  
Mingyao Liu ◽  
Martin Post
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Mechanical Strain ◽  
Fetal Lung ◽  
Lung Cells ◽  
Gelatinase A ◽  
Rat Lung ◽  
Fetal Rat ◽  
Gene And Protein Expression ◽  
Fetal Rat Lung

We have previously shown that an intermittent mechanical strain regimen (5% elongation, 60 cycles/min, 15 min/h) that simulates fetal breathing movements stimulated fetal rat lung cell proliferation. Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix (ECM) remodeling, we subjected organotypic cultures of fetal rat lung cells ( day 19 of gestation, term = 22 days) to this strain regimen and examined alterations in ECM gene and protein expression. Northern analysis revealed that mechanical strain reduced messages for procollagen-α1(I) and biglycan and increased the levels of mRNA for collagen-α1(IV) and -α2(IV), whereas laminin β-chain mRNA levels remained constant. Regardless of mRNA changes, mechanical strain increased the protein content of type I and type IV collagen as well as of biglycan in the medium. Mechanical strain did not affect gene expression of several matrix metalloproteinases (MMPs), such as MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and MMP-3 (stromelysin-1). Neither collagenase nor gelatinase (A and B) activities in conditioned medium were affected by mechanical strain. Tissue inhibitor of metalloproteinase activities in conditioned medium remained unchanged during the 48-h intermittent mechanical stretching. These data suggest that an intermittent mechanical strain differentially regulates gene and protein expression of ECM molecules in fetal lung cells. The observed increase in matrix accumulation appears to be mainly a result of an increased synthesis of ECM molecules and not of decreasing activity of degradative enzymes.

Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts

2002 ◽  
Vol 282 (3) ◽  
pp. L448-L456 ◽  
Author(s):  
Juan Sanchez-Esteban ◽  
Yulian Wang ◽  
Lawrence A. Cicchiello ◽  
Lewis P. Rubin
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Progression ◽  
Fetal Lung ◽  
Mechanical Stretch ◽  
Lung Fibroblasts ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Flow Cytometry ◽  
Fetal Rat ◽  
Induced Apoptosis

Development of the pulmonary air sacs is crucial for extrauterine survival. Late fetal lung development is characterized by a thinning of the mesenchyme, which brings pneumocytes and endothelial cells into apposition. We hypothesized that mechanical stretch, simulating fetal breathing movements, plays an important role in this remodeling process. Using a Flexercell Strain Unit, we analyzed the effects of intermittent stretch on cell proliferation and apoptosis activation in fibroblasts isolated from fetal rat lungs during late development. On day 19, intermittent stretch increased cells in G0/G1 by 22% ( P = 0.001) and decreased in S phase by 50% ( P = 0.003) compared with unstretched controls. Cell proliferation analyzed by 5-bromo-2′-deoxyuridine incorporation showed a similar magnitude of cell cycle arrest ( P = 0.04). At this same gestational age, stretch induced apoptosis by two- to threefold over controls, assayed by DNA flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling, and caspase-3 activation. These results indicate that mechanical stretch of fibroblasts isolated during the canalicular stage inhibits cell cycle progression and activates apoptosis. These findings are cotemporal with the mesenchymal thinning that normally occurs in situ.

168 THY-1 EXPRESSION IS ASSOCIATED WITH UP-REGULATION OF WNT SIGNALING AND MYOFIBROBLAST PHENOTYPE IN FETAL RAT LUNG FIBROBLASTS.

2006 ◽  
Vol 54 (1) ◽  
pp. S109.2-S109
Author(s):  
C. Dasgupta ◽  
Y. Wang ◽  
R. Sakurai ◽  
J. Santos ◽  
J. S. Torday ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Lung Fibroblasts ◽  
Rat Lung ◽  
Myofibroblast Phenotype ◽  
Fetal Rat ◽  
Fetal Rat Lung

The oncogeneTrop2regulates fetal lung cell proliferation

2011 ◽  
Vol 301 (4) ◽  
pp. L478-L489 ◽  
Author(s):  
Annie R. A. McDougall ◽  
Stuart B. Hooper ◽  
Valerie A. Zahra ◽  
Foula Sozo ◽  
Camden Y. Lo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Fetal Lung ◽  
Alveolar Epithelial Cells ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Lung Growth ◽  
Ki 67 ◽  
Alveolar Epithelial ◽  
Fetal Rat ◽  
Lung Expansion

The factors regulating growth of the developing lung are poorly understood, although the degree of fetal lung expansion is critical. The oncogene Trop2 (trophoblast antigen 2) is upregulated during accelerated fetal lung growth, and we hypothesized that it may regulate normal fetal lung growth. We investigated Trop2 expression in the fetal and neonatal sheep lung during accelerated and delayed lung growth induced by alterations in fetal lung expansion, as well as in response to glucocorticoids. Trop2 expression was measured using real-time PCR and localized spatially using in situ hybridization and immunofluorescence. During normal lung development, Trop2 expression was higher at 90 days gestational age (GA; 4.0 ± 0.8) than at 128 days GA (1.0 ± 0.1), decreased to 0.5 ± 0.1 at 142 days GA (full term ∼147 days GA), and was positively correlated to lung cell proliferation rates ( r = 0.953, P < 0.005). Trop2 expression was regulated by fetal lung expansion, but not by glucocorticoids. It was increased nearly threefold by 36 h of increased fetal lung expansion ( P < 0.05) and was reduced to ∼55% of control levels by reduced fetal lung expansion ( P < 0.05). Trop2 expression was associated with lung cell proliferation during normal and altered lung growth, and the TROP2 protein colocalized with Ki-67-positive cells in the fetal lung. TROP2 was predominantly localized to fibroblasts and type II alveolar epithelial cells. Trop2 small interfering RNA decreased Trop2 expression by ∼75% in cultured fetal rat lung fibroblasts and decreased their proliferation by ∼50%. Cell viability was not affected. This study demonstrates that TROP2 regulates lung cell proliferation during development.

scholarly journals Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation

2002 ◽  
Vol 282 (3) ◽  
pp. L405-L410 ◽  
Author(s):  
J. S. Torday ◽  
H. Sun ◽  
L. Wang ◽  
E. Torres
Keyword(s):  
De Novo ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type Ii ◽  
Rat Lung ◽  
Parathyroid Hormone Related Protein ◽  
Fetal Rat ◽  
Type Ii Cell ◽  
Leptin Expression ◽  
Stimulation Of

Developing rat lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed mutually exclusively by fetal lung fibroblasts and type II cells, suggesting a paracrine signaling “loop.” This hypothesized mechanism is supported by the following experimental data: 1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400% · 100 ng−1 · ml−1 · 24 h−1) and adult human airway epithelial cells (85% · 100 ng−1 · 24 h−1); 2) leptin is secreted by lipofibroblasts in amounts that stimulate type II cell surfactant phospholipid synthesis in vitro; 3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP), PGE2, and dexamethasone stimulate leptin expression by fetal rat lung fibroblasts; 4) PTHrP or leptin stimulate the de novo synthesis of surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants, an effect that is blocked by a leptin antibody; and 5) a PTHrP receptor antagonist inhibits the expression of leptin mRNA by explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop that functionally cooperates to induce alveolar acinar lung development.

scholarly journals Fetal rat lung epithelium has a functional growth hormone receptor coupled to tyrosine kinase activity and insulin-like growth factor binding protein-2 production

1998 ◽  
Vol 21 (1) ◽  
pp. 73-84 ◽  
Author(s):  
DC Batchelor ◽  
RM Lewis ◽  
BH Breier ◽  
PD Gluckman ◽  
SJ Skinner
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Binding Protein ◽  
Fetal Lung ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Rat Lung ◽  
Lung Epithelium ◽  
Fetal Rat ◽  
Fetal Rat Lung

Although growth hormone (GH) receptor (GHR) mRNA and protein are present in fetal tissues such as the lung, there is little evidence that GH mediates growth in the fetus. We have identified functional responses to GH in fetal rat lung epithelia and suggest a possible role for GHR in the developing lung. GHR mRNA in lung extracts was high before birth at day 16 of gestation (16f), decreased to low levels at day 22f but increased again after birth. At day 20f GHR mRNA levels were higher in lung than in liver, whereas growth hormone binding protein mRNA levels were approximately equal in lung and liver. Stimulation of primary cell cultures of day 19f lung epithelia with GH caused increased tyrosine phosphorylation in specific proteins, demonstrating functional GHR. Lung fibroblasts isolated at the same time did not respond to GH. Ligand and Northern blot analysis of the epithelial cultures revealed that GH stimulation increased insulin-like growth factor binding protein-2 (IGFBP-2) activity and mRNA. These experiments demonstrate the functional activity of GHR, specifically in fetal lung epithelium. We suggest that one role for GH in vivo may be indirectly to modify insulin-like growth factor activity in the developing fetal lung by increasing IGFBP-2.

An Ultrastructural Comparison of Rat Lung Cells Grown in an Organotypic Culture System with Lung Tissue Found in the Whole Animal

1976 ◽  
Vol 34 ◽  
pp. 172-173
Author(s):  
Peter J. Del Vecchio ◽  
Edward P. Dougherty ◽  
William H. J. Douglas
Keyword(s):  
Lung Tissue ◽  
Organotypic Culture ◽  
Fetal Lung ◽  
Lung Cells ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Tissue Organization ◽  
Fetal Rat ◽  
Fetal Rat Lung

This study describes the ultrastructure of an organotypic system prepared from rat fetal lung and compares it to lung in rat fetuses. The preparation of the organotypic system has been described elsewhere (1).The organotypic systems in this study are prepared from 18-19 day fetal rat lung and spend two days in culture making the cells a total of 20-21 days old. The fine structure of the type II cells present in this system will be compared to the type II cells in fetal lung at 20-21 days gestation. Because the tissue organization of the organotypic system is more like the organization of fetal lung at an earlier stage (18-19 days gestation) , the cell to cell relationships of the organotypic system are compared to fetal lung tissue at this stage.The histology of the organotypic system is similar to that of the fetal lung in its glandular stage (Fig. 1). The epithelial cells are all columnar and are surrounded by relatively undifferentiated mesenchyme.

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