Expression of CFTR and presence of cAMP-mediated fluid secretion in 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.

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Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization.

Journal of Clinical Investigation ◽

10.1172/jci115901 ◽

1992 ◽

Vol 90 (2) ◽

pp. 619-625 ◽

Cited By ~ 38

Author(s):

P B McCray ◽

C L Wohlford-Lenane ◽

J M Snyder

Keyword(s):

Cystic Fibrosis ◽

In Situ Hybridization ◽

Lung Tissue ◽

Fetal Lung ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Cystic Fibrosis Transmembrane ◽

Human Fetal Lung

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Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l227 ◽

1997 ◽

Vol 273 (1) ◽

pp. L227-L233 ◽

Cited By ~ 23

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.

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Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles

10.1101/2022.01.12.476078 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Lina Antounians ◽

Rebeca Lopes Figueira ◽

Martin Post ◽

Augusto Zani

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Amniotic Fluid Stem Cell ◽

Human Fetal Lung

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17~92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.

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Developing bronchopulmonary epithelium of the human fetus secretes fluid

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.262.3.l270 ◽

1992 ◽

Vol 262 (3) ◽

pp. L270-L279 ◽

Cited By ~ 9

Author(s):

P. B. McCray ◽

J. D. Bettencourt ◽

J. Bastacky

Keyword(s):

Human Fetus ◽

Chloride Transport ◽

Fetal Lung ◽

Fluid Secretion ◽

Chloride Secretion ◽

Beta Agonist ◽

Lung Fluid ◽

Luminal Area ◽

Fluid Production ◽

Human Fetal Lung

We studied human fetal lung tissue in submersion organ culture to determine whether the bronchopulmonary epithelium secretes fluid during development. In this system the acinar tubules continued to grow, secrete fluid, and become progressively dilated. Baseline transepithelial potential differences (psi t) of -0.5 to -11 mV (mean, -3.8 mV, lumen negative, n = 27) were measured with microelectrodes after 3-8 days in culture, suggesting active electrolyte transport. Bumetanide (500 microM), an inhibitor of chloride secretion in other systems, decreased the basal psi t from -5 +/- 1.5 to -3.2 +/- 1.6 (SE) mV (P less than 0.05, n = 6), suggesting that chloride transport contributed to the voltage. Isoproterenol (5 microM) increased the baseline psi t from -5.6 +/- 2.1 to -9.2 +/- 2.5 (SE) mV (P less than 0.05, n = 4). Subsequent addition of bumetanide inhibited the isoproterenol-induced stimulation of the psi t by 20% (P less than 0.05). 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. (CPT-cAMP, 50 microM) and 3-isobutyl 1-methylxanthine (IBMX, 100 microM) had similar effects, causing an increase in the psi t from -2.2 +/- 0.5 to -8 +/- 1.6 (SE) mV, an effect that was inhibited by the addition of bumetanide (P less than 0.005, n = 6). Both isoproterenol and CPT-cAMP/IBMX produced significant increases in the percentage luminal area of the explants at 12 and 24 h after exposure compared with control. We conclude that 1) the developing bronchopulmonary epithelium (acinar tubules) contributes to lung fluid production in the human fetus, 2) fetal lung fluid secretion is chloride dependent, and 3) chloride secretion and fluid secretion may be stimulated by a beta-agonist and cAMP.

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ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00207.2001 ◽

2002 ◽

Vol 282 (3) ◽

pp. L501-L507 ◽

Cited By ~ 10

Author(s):

Rebecca D. Edmonds ◽

Ian V. Silva ◽

William B. Guggino ◽

Robert B. Butler ◽

Pamela L. Zeitlin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Lung Disease ◽

Chloride Channel ◽

Chloride Channels ◽

Chloride Transport ◽

Premature Death ◽

Fetal Lung ◽

Chloride Secretion ◽

Normal Lung ◽

Ribonuclease Protection Assay

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.

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Surfactant protein B processing in human fetal lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.275.3.l559 ◽

1998 ◽

Vol 275 (3) ◽

pp. L559-L566 ◽

Cited By ~ 20

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.

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Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.4.l542 ◽

1998 ◽

Vol 274 (4) ◽

pp. L542-L551 ◽

Cited By ~ 1

Author(s):

Jonathan M. Klein ◽

Louis J. Dewild ◽

Troy A. McCarthy

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Lung Tissue ◽

Receptor Tyrosine Kinase ◽

Lung Development ◽

Egf Receptor ◽

Fetal Lung ◽

Surfactant Protein ◽

Mrna Levels ◽

Human Fetal Lung

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.

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A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.271.2.l287 ◽

1996 ◽

Vol 271 (2) ◽

pp. L287-L299 ◽

Cited By ~ 9

Author(s):

P. P. Young ◽

C. R. Mendelson

Keyword(s):

Gene Expression ◽

Lung Tissue ◽

Fusion Gene ◽

Fetal Lung ◽

Inducible Expression ◽

Nuclear Proteins ◽

Type Ii Cells ◽

Type Ii ◽

Nuclear Extracts ◽

Human Fetal Lung

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2. Binding activity of CRESP-A2 for nuclear proteins from human fetal lung tissue before culture was manifest as two complexes of different mobilities and equivalent intensity. By contrast, upon differentiation of the human fetal lung in culture in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), the higher mobility complex was decreased to undetectable levels. By UV cross-linking analysis, using nuclear extracts from midgestation human fetal lung before culture and radiolabeled CRESP-A2 as a probe, we observed binding of proteins of approximately 50, 36, and 30 kDa. When nuclear extracts from human fetal lung cultured in the presence of DBcAMP were analyzed, binding of only the 50- and 36-kDa proteins was apparent. On the other hand, when the canonical CRE (TGACGTCA) known to bind the transcription factor CREB (M(r) approximately 43,000) was used as a probe, binding of only a approximately 43-kDa protein was evident using nuclear extracts from human fetal lung before and after culture. In type II cells transfected with an SP-A2(-296):hGH fusion gene in which CRESP-A2 was mutated, there was a marked reduction of basal and cAMP-stimulated fusion gene expression. These findings indicate that CRESP-A2 serves an important role in mediating basal and cAMP-inducible expression of the human SP-A2 gene in type II cells, that the fetal lung nuclear proteins bound to CRESP-A2 differ from those bound to the canonical palindromic CRE, and that changes in the complex of nuclear proteins bound to CRESP-A2 accompany induction of SP-A gene expression.

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