Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung

1997 ◽  
Vol 273 (1) ◽  
pp. L227-L233 ◽  
Author(s):  
V. C. Venkatesh ◽  
H. D. Katzberg
Keyword(s):  
Rna Polymerase Ii ◽  
Actinomycin D ◽  
Critical Role ◽  
Fetal Lung ◽  
Explant Culture ◽  
Late Gestation ◽  
Na Channel ◽  
Second Trimester ◽  
Cyclic Monophosphate ◽  
Human Fetal Lung

Pulmonary epithelial Na+ channels (ENaC), composed of three distinct subunits (alpha, beta, and gamma), play a critical role in the regulation of fluid reabsorption from airspaces of late-gestation fetal lung. We studied the expression of ENaC subunit genes in cultured human fetal lung. All three mRNAs were expressed at low levels in second trimester lung (13-32% of adult values at 24 wk gestation). There was a spontaneous increase of approximately threefold over preculture values of all three subunits within 24 h of explant culture in serum-free Waymouth's medium. Dexamethasone (Dex) induced all three mRNAs by two- to threefold. Maximal induction was noted by 8 h with 30-100 nM Dex and half-maximal stimulation with 3-10 nM Dex. Cycloheximide decreased basal expression of all three subunits by 8 h but did not alter the response to Dex. Actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), inhibitors of RNA polymerase II, decreased the basal and the Dex-induced expression of all three subunits with a more marked effect on human hENaC-gamma than on hENaC-alpha or hENaC-beta. Under conditions where transcription was blocked by actinomycin D or DRB, Dex did not alter the stability of the three mRNAs. Triiodothyronine (T3) at low (2 nM) or high (100 nM) concentrations had no effect on the expression of the three subunits in the presence or absence of low (10 nM) or high (100 nM) concentrations of Dex for 8 or 24 h. Similarly, 8-bromoadenosine 3',5'-cyclic monophosphate (2 microM) had no effect on basal or Dex-induced increase in the three subunits. We conclude that the three Na+ channel subunit genes are expressed in second trimester human fetal lung and are coordinately upregulated by glucocorticoid hormones but not by T3 or adenosine 3',5'-cyclic monophosphate. Glucocorticoid induction is receptor mediated, is primarily transcriptional, and does not require the induction of an intermediate protein for transcriptional enhancement. We speculate that induction of lung ENaC may contribute to the beneficial effects of antenatal glucocorticoids in premature babies.

Differentiation of Type II Cells during Explant Culture of Human Fetal Lung Is Accelerated by Endogenous Prostanoids and Adenosine 3′,5′-Monophosphate*

Endocrinology ◽  
1991 ◽  
Vol 128 (6) ◽  
pp. 2916-2924 ◽  
Author(s):  
PHILIP L. BALLARD ◽  
LINDA W. GONZALES ◽  
MARY C. WILLIAMS ◽  
JAMES M. ROBERTS ◽  
MARK M. JACOBS
Keyword(s):  
Fetal Lung ◽  
Explant Culture ◽  
Type Ii Cells ◽  
Type Ii ◽  
Human Fetal Lung

Surfactant protein B processing in human fetal lung

1998 ◽  
Vol 275 (3) ◽  
pp. L559-L566 ◽  
Author(s):  
Susan H. Guttentag ◽  
Michael F. Beers ◽  
Bert M. Bieler ◽  
Philip L. Ballard
Keyword(s):  
Polyclonal Antibodies ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Explant Culture ◽  
Normal Lung ◽  
Surfactant Protein B ◽  
Multistep Process ◽  
Human Fetal Lung ◽  
Protein B

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8(Phe201–Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145–Leu160, Gln186–Gln200, and Gly284–Ser304). Western blot analysis revealed a novel intermediate at ∼9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of ∼10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.

Expression of CFTR and presence of cAMP-mediated fluid secretion in human fetal lung

1992 ◽  
Vol 262 (4) ◽  
pp. L472-L481 ◽  
Author(s):  
P. B. McCray ◽  
W. W. Reenstra ◽  
E. Louie ◽  
J. Johnson ◽  
J. D. Bettencourt ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Tissue ◽  
Light And Electron Microscopy ◽  
Fetal Lung ◽  
Fluid Secretion ◽  
Developmental Expression ◽  
Normal Lung ◽  
Second Trimester ◽  
Significant Change ◽  
Human Fetal Lung

We studied the developmental expression of the cystic fibrosis (CF) gene in human lung tissue from normal and CF-affected fetuses. Two unrelated CF fetuses, both homozygous for the delta F508 deletion, were examined. Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was present in second-trimester CF lung and in first- and second-trimester normal lung as assessed by amplification of reverse transcribed total RNA with the use of the polymerase chain reaction. CFTR protein was identified by immunoprecipitation in normal second-trimester fetal lung explants. To evaluate possible functional consequences of CF in the fetus, lung tissue explants were grown in submersion organ culture. By light and electron microscopy, the CF fetal lung explants appeared normal. When explants from normal fetal lung were exposed to 8-(4-chlorophenylthio) adenosine 3',-5'cyclic monophosphate (CPT-cAMP), and 3-isobutyl-1-methylxanthine (IBMX) for 24 h, the intraluminal fluid content increased, as assessed by a 40 +/- 4% increase in cross-sectional diameter. In contrast, identically treated CF explants showed no significant change in explant diameter (3 +/- 1.6%). The transepithelial potential (psi t) across fetal lung explants was measured with microelectrodes. In normal second-trimester explants, CPT-cAMP and IBMX caused hyperpolarization of psi t (-0.93 +/- 14 mV to -4.3 +/- 1.2 mV); in contrast, CF fetal lung explants showed no significant change in psi t with CPT-cAMP and IBMX (-0.84 +/- 0.07 mV to -1.21 +/- 0.26 mV). This study confirms the presence of CFTR mRNA and protein in human fetal lung and suggests that although the CF fetal lung appears normal morphologically, there is a defect in cAMP-mediated fluid secretion in the lung of the CF fetus.

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts

2015 ◽  
Vol 308 (4) ◽  
pp. L391-L402 ◽  
Author(s):  
Hui Zhang ◽  
Neil B. Sweezey ◽  
Feige Kaplan
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Conditioned Medium ◽  
Fetal Lung ◽  
Late Gestation ◽  
Lung Fibroblasts ◽  
And Migration ◽  
The Impact ◽  
Human Fetal Lung ◽  
Lung Branching

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1+/−mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5LGL1KD). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5LGL1KDsuppressed cell growth and increased apoptosis of annexin V+staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5LGL1KDcells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

scholarly journals β-Adrenergic Receptors and cAMP Response Increase during Explant Culture of Human Fetal Lung: Partial Inhibition by Dexamethasone

1990 ◽  
Vol 28 (3) ◽  
pp. 190-195 ◽  
Author(s):  
Deborah J Davis ◽  
Mark M Jacobs ◽  
Philip L Ballard ◽  
Linda K Gonzales ◽  
James M Roberts
Keyword(s):  
Adrenergic Receptors ◽  
Fetal Lung ◽  
Explant Culture ◽  
Partial Inhibition ◽  
Response Increase ◽  
Β Adrenergic Receptors ◽  
Human Fetal Lung

LGL1, a novel branching morphogen in developing kidney, is induced by retinoic acid

2007 ◽  
Vol 293 (4) ◽  
pp. F987-F993 ◽  
Author(s):  
Jacklyn Quinlan ◽  
Feige Kaplan ◽  
Neil Sweezey ◽  
Paul Goodyer
Keyword(s):  
Retinoic Acid ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
Explant Culture ◽  
Late Gestation ◽  
Ureteric Bud ◽  
Fetal Kidney ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Late-gestation lung protein 1 (LGL1) is a glycoprotein secreted by fetal lung mesenchyme that stimulates branching morphogenesis of the developing lung bud. We show that Lgl1 mRNA and protein are also expressed in mesenchymally derived lineages of fetal kidney. Although Lgl1 expression is stimulated by glucocorticoids in kidney cells, cortisol (10−7 M) actually suppresses ureteric bud branching of fetal kidneys from HoxB7/GFP mice in explant culture. However, early branching morphogenesis in the lung and kidney is stimulated by retinoic acid, and we identified putative retinoic acid response elements in the Lgl1 promoter. All- trans-retinoic acid (10−6 M) stimulated Lgl1 promoter activity and endogenous Lgl1 mRNA expression in vitro. Branching of cultured fetal kidney explants was increased in the presence of all- trans retinoic acid (10−6 M). Heterozygous Lgl1 knockout mice were crossed to HoxB7/GFP mice to visualize the extent of ureteric bud branching at fetal stages. At embryonic (E) days E12.5–E13.0, mutant Lgl1+/− embryos showed a 20% reduction in ureteric bud branching compared with wild-type littermates. We propose a model in which retinoic acid stimulates branching morphogenesis by activating Lgl1 early in development. The prominent effects of glucocorticoids on Lgl1 expression in late lung development suggest a second role for LGL1 in alveolar maturation.

Collagen secretion mediated by NMDR activation in human fetal lung fibroblasts

2010 ◽  
Vol 34 (8) ◽  
pp. S70-S70
Author(s):  
MingJie WANG ◽  
ZiQiang LUO ◽  
Mei LU ◽  
LiHong SHANG ◽  
ShaoJie YUE
Keyword(s):  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Collagen Secretion ◽  
Human Fetal Lung

scholarly journals Molecular regulation of lung maturation in near-term fetal sheep by maternal daily vitamin C treatment in late gestation

2021 ◽  
Author(s):  
Erin V. McGillick ◽  
Sandra Orgeig ◽  
Beth J. Allison ◽  
Kirsty L. Brain ◽  
Youguo Niu ◽  
...  
Keyword(s):  
Vitamin C ◽  
Lung Development ◽  
Fetal Lung ◽  
Late Gestation ◽  
Lung Maturation ◽  
Maternal Vitamin ◽  
Healthy Pregnancy ◽  
Daily Vitamin ◽  
Expression Of Genes ◽  
Fetal Lung Maturation

Abstract Background In the fetus, the appropriate balance of prooxidants and antioxidants is essential to negate the detrimental effects of oxidative stress on lung maturation. Antioxidants improve respiratory function in postnatal life and adulthood. However, the outcomes and biological mechanisms of antioxidant action in the fetal lung are unknown. Methods We investigated the effect of maternal daily vitamin C treatment (200 mg/kg, intravenously) for a month in late gestation (105–138 days gestation, term ~145 days) on molecular regulation of fetal lung maturation in sheep. Expression of genes and proteins regulating lung development was quantified in fetal lung tissue. The number of surfactant-producing cells was determined by immunohistochemistry. Results Maternal vitamin C treatment increased fetal lung gene expression of the antioxidant enzyme SOD-1, hypoxia signaling genes (HIF-2α, HIF-3α, ADM, and EGLN-3), genes regulating sodium movement (SCNN1-A, SCNN1-B, ATP1-A1, and ATP1-B1), surfactant maturation (SFTP-B and ABCA3), and airway remodeling (ELN). There was no effect of maternal vitamin C treatment on the expression of protein markers evaluated or on the number of surfactant protein-producing cells in fetal lung tissue. Conclusions Maternal vitamin C treatment in the last third of pregnancy in sheep acts at the molecular level to increase the expression of genes that are important for fetal lung maturation in a healthy pregnancy. Impact Maternal daily vitamin C treatment for a month in late gestation in sheep increases the expression of gene-regulating pathways that are essential for normal fetal lung development. Following late gestation vitamin C exposure in a healthy pregnancy, an increase in lung gene but not protein expression may act as a mechanism to aid in the preparation for exposure to the air-breathing environment after birth. In the future, the availability/development of compounds with greater antioxidant properties than vitamin C or more specific targets at the site of oxidative stress in vivo may translate clinically to improve respiratory outcomes in complicated pregnancies at birth.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

Fabrication and Cytotoxicity Studies of the TiO2 Doped with Copper-Based Nanocomposite Particles

2011 ◽  
Vol 695 ◽  
pp. 393-396 ◽  
Author(s):  
Chang Mao Hung
Keyword(s):  
Fetal Lung ◽  
Copper Nitrate ◽  
Tissue Cell ◽  
Nanocomposite Particles ◽  
Toxicological Effect ◽  
Cytotoxicity Studies ◽  
Cultured Human Cells ◽  
Cytotoxicity Assessment ◽  
Human Fetal Lung ◽  
Dispersion Character

Since the growing interest in the manufacture and environmental applications of nanocomposites consisting of CuO and TiO2nanoparticles (NPs), related toxicological effect and interaction with cellular structures for these newly developed materials are still unknown. Recent literature reveals that nanosized CuO and TiO2particles have cytotoxicity risks and ability to cause oxidative stress on health. This work considers the CuO doped TiO2nanocomposite particles were synthesized via a coprecipitation method with aqueous solutions as precursors of copper nitrateand titanium dioxide. Moreover, the nanocomposite particles were characterized using TGA-DTA, UV-Vis and TEM measurements. The calcination temperature was selected at 873 K. The nanocomposite particles were characterized by TEM, as the primary particles, aggregates ranged from 30 to 100 nm and have a good dispersion character. Cell cytotoxicity assessment and the percentage cell survival was determined by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazoli um (MTS) assay on human fetal lung tissue cell (MRC-5). The experimental results show that the CuO doped TiO2nanocomposite particles cause potential cytotoxicity effect in cultured human cells.

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