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

1998 ◽  
Vol 274 (4) ◽  
pp. L542-L551 ◽  
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.

Regulation of human pulmonary surfactant protein gene expression by 1α,25-dihydroxyvitamin D3

2005 ◽  
Vol 289 (4) ◽  
pp. L617-L626 ◽  
Author(s):  
Sarabjit S. Phokela ◽  
Sara Peleg ◽  
Fernando R. Moya ◽  
Joseph L. Alcorn
Keyword(s):  
Lung Tissue ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Type Ii ◽  
Protein Levels ◽  
Human Fetal Lung ◽  
Dose Dependent

1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been reported to stimulate lung maturity, alveolar type II cell differentiation, and pulmonary surfactant synthesis in rat lung. We hypothesized that 1,25(OH)2D3 stimulates expression of surfactant protein-A (SP-A), SP-B, and SP-C in human fetal lung and type II cells. We found that immunoreactive vitamin D receptor was detectable in fetal lung tissue and type II cells only when incubated with 1,25(OH)2D3. 1,25(OH)2D3 significantly decreased SP-A mRNA in human fetal lung tissue but did not significantly decrease SP-A protein in the tissue. In type II cells, 1,25(OH)2D3 alone had no significant effect on SP-A mRNA or protein levels but reduced SP-A mRNA and protein in a dose-dependent manner when the cells were incubated with cAMP. SP-A mRNA levels in NCI-H441 cells, a nonciliated bronchiolar epithelial (Clara) cell line, were decreased in a dose-dependent manner in the absence or presence of cAMP. 1,25(OH)2D3 had no significant effect on SP-B mRNA levels in lung tissue but increased SP-B mRNA and protein levels in type II cells incubated in the absence or presence of cAMP. Expression of SP-C mRNA was unaffected by 1,25(OH)2D3 in lung tissue incubated ± cAMP. These results suggest that regulation of surfactant protein gene expression in human lung and type II cells by 1,25(OH)2D3 is not coordinated; 1,25(OH)2D3 decreases SP-A mRNA and protein levels in both fetal lung tissue and type II cells, increases SP-B mRNA and protein levels only in type II cells, and has no effect on SP-C mRNA levels.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

2002 ◽  
Vol 282 (3) ◽  
pp. L386-L393 ◽  
Author(s):  
Jonathan M. Klein ◽  
Troy A. McCarthy ◽  
John M. Dagle ◽  
Jeanne M. Snyder
Keyword(s):  
Gene Expression ◽  
Protein A ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Phosphorothioate Oligonucleotide ◽  
Myelin Formation ◽  
Surfactant Phospholipids ◽  
Human Fetal Lung

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3–5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

Oxygen modulates surfactant protein mRNA expression and phospholipid production in human fetal lung in vitro

1995 ◽  
Vol 268 (5) ◽  
pp. L818-L825 ◽  
Author(s):  
M. J. Acarregui ◽  
J. J. Brown ◽  
R. K. Mallampalli
Keyword(s):  
Mrna Expression ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Choline Incorporation ◽  
Associated Proteins ◽  
Dependent Protein ◽  
Surfactant Composition ◽  
Human Fetal Lung

We studied the effect of 20-95% O2 on mRNA levels for the surfactant-associated proteins (SP)-A, SP-B, and SP-C and [3H]choline incorporation into total phosphatidylcholine and type II cell-specific disaturated phosphatidylcholine (DPPC) in human fetal lung in culture. SP-A mRNA levels were increased by 25 and 39% in lung explants incubated in 70 and 95% O2, respectively, compared with levels in tissues incubated in 20% O2. SP-B mRNA levels were unaffected by O2, whereas SP-C mRNA levels were increased by 85, 102, and 115% in atmospheres of 35, 50, and 70% O2, respectively. [3H]choline incorporation into total phosphatidylcholine and DPPC were both increased in human fetal lung explants incubated in increased O2 concentrations compared with tissues incubated in 20% O2. Tissue levels of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) activity were not affected by O2 concentration, implying that the changes observed in SP mRNA levels and [3H]choline incorporation may not be mediated through alterations in PKA enzyme activity. These findings demonstrate that O2 regulates SP mRNA expression and phospholipid production in human fetal lung in vitro. We speculate that surfactant composition and possibly function may be regulated by O2 in human lung.

Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep

1999 ◽  
Vol 276 (2) ◽  
pp. L239-L245 ◽  
Author(s):  
A. Lines ◽  
L. Nardo ◽  
I. D. Phillips ◽  
F. Possmayer ◽  
S. B. Hooper
Keyword(s):  
Lung Tissue ◽  
Blot Analysis ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Lung Growth ◽  
Fetal Sheep ◽  
Protein Levels ◽  
Tracheal Obstruction ◽  
Lung Expansion

Obstruction of the fetal trachea is a potent stimulus for fetal lung growth, and it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. However, little is known about the effects of increased lung expansion on other aspects of lung development. Our aim was to determine the effect of increased and decreased lung expansion on the mRNA levels encoding surfactant protein (SP) A, SP-B, and SP-C in ovine fetal lungs. Lung tissue samples were collected from fetuses exposed to 2, 4, or 10 days of increased lung expansion caused by tracheal obstruction. The mRNA levels for SP-A, SP-B, and SP-C were determined by Northern blot analysis with specific ovine cDNA probes; SP-A protein levels were determined by Western blot analysis. Compared with age-matched (128-day gestational age) control fetuses, SP-A, SP-B, and SP-C mRNA levels in fetal lung tissue were significantly reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. However, SP-A protein levels were not reduced at 2 days of tracheal obstruction, tended to be reduced at 4 days, and were almost undetectable at 10 days. In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased SP-C, but not SP-A, mRNA levels in fetal lung tissue compared with age-matched control fetuses. Our results demonstrate that increases in fetal lung expansion, induced by obstruction of the fetal trachea, cause large simultaneous reductions in SP-A, SP-B, and SP-C mRNA levels in the fetal lung as well as a decrease in SP-A protein levels. These data suggest that expression of the genes encoding SPs in the fetal lung are specifically responsive to the degree of lung expansion.

Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin

1998 ◽  
Vol 274 (2) ◽  
pp. L177-L185 ◽  
Author(s):  
A. R. Kumar ◽  
J. M. Snyder
Keyword(s):  
Cell Line ◽  
Lung Surfactant ◽  
Protein A ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Model Systems ◽  
Mrna Levels ◽  
Mrna Transcripts ◽  
Lung Surfactant Protein ◽  
Human Fetal Lung

In the human fetal lung, surfactant protein A (SP-A) is encoded by two highly similar genes, SP-A1 and SP-A2, which are developmentally and hormonally regulated. Using primer extension analysis, we evaluated the levels of SP-A1 and SP-A2 mRNA transcripts in human fetal lung explants and in a human adult lung adenocarcinoma cell line (H441 cells) cultured in the absence or presence of either dibutyryladenosine 3′,5′-cyclic monophosphate (DBcAMP, 1 mM), dexamethasone (10−7 M), or insulin (2.5 μg/ml). In the human fetal lung explants, the content of SP-A1 mRNA was approximately four times that of SP-A2 mRNA. DBcAMP increased SP-A1 mRNA levels by 100% and SP-A2 mRNA levels by 500%, thus reducing the ratio of SP-A1 mRNA to SP-A2 mRNA to ∼1:1. Dexamethasone inhibited all of the SP-A1 and SP-A2 mRNA transcripts to the same extent, by ∼70%, whereas insulin inhibited all SP-A mRNA transcripts by ∼60%. The ratio of SP-A1 to SP-A2 mRNA in dexamethasone- or insulin-treated explants was the same as the ratio observed in controls. In the H441 cells, SP-A1 mRNA levels were ∼1.5 times that of SP-A2 mRNA levels. DBcAMP increased both SP-A1 and SP-A2 mRNA levels by 100%. Dexamethasone inhibited SP-A1 mRNA levels in the cell line by 60%, whereas SP-A2 mRNA levels were not significantly affected. Insulin inhibited SP-A1 mRNA levels in the cell line by 40% without affecting SP-A2 mRNA levels. These findings suggest that the two human SP-A genes are regulated differently in the two model systems.

Transcription and mRNA stability regulate developmental and hormonal expression of rabbit surfactant protein B gene

1995 ◽  
Vol 268 (3) ◽  
pp. L481-L490 ◽  
Author(s):  
R. K. Margana ◽  
V. Boggaram
Keyword(s):  
Gene Transcription ◽  
Mrna Stability ◽  
Lung Development ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Fetal Lung Development ◽  
Surfactant Protein B ◽  
Protein B

Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for the surface tension-reducing properties of surfactant. In the present study, we isolated and characterized cDNAs encoding rabbit SP-B, and used transcription run-on assays and Northern blot analysis to investigate the role of transcriptional and posttranscriptional mechanisms in the developmental and cAMP and dexamethasone induction of SP-B mRNA. We found two forms of SP-B cDNAs that differed by an insertion of 69 nucleotides in the 3' untranslated regions. We found that transcription across the SP-B gene is nonequimolar and the 3' end of the gene has high levels of antisense transcription. SP-B gene transcription and SP-B mRNA levels increased during fetal lung development. However, increased SP-B mRNA levels could not be accounted for primarily on the basis of increased transcription. These results suggested that enhanced SP-B gene transcription and enhanced SP-B mRNA stability mediate developmental induction of SP-B gene. In rabbit fetal lung in vitro, both dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and dexamethasone increased SP-B mRNA levels. DBcAMP-dependent increase in SP-B mRNA levels resulted from increased SP-B gene transcription, whereas dexamethasone-dependent increase resulted from combined effects on increased SP-B gene transcription and SP-B mRNA stability. In tissues treated with dexamethasone the half-life (t1/2) of SP-B mRNA increased > 2.5-fold (t1/2 control = 9 h; t1/2 dex-treated = 25 h). These data show that both transcription and mRNA stability regulate induction of SP-B gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

O2 regulates surfactant protein A mRNA transcription and stability in human fetal lung in vitro

1998 ◽  
Vol 274 (3) ◽  
pp. L343-L350 ◽  
Author(s):  
Michael J. Acarregui ◽  
Ashish R. Kumar ◽  
Scott T. Penisten ◽  
Jeanne M. Snyder
Keyword(s):  
Half Life ◽  
Protein A ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Mrna Transcription ◽  
Preferential Expression ◽  
Mrna Half Life ◽  
Human Fetal Lung

The effect of O2 on surfactant protein (SP) A mRNA transcription and half-life was determined in midtrimester human fetal lung tissue cultured in either 20 (control) or 70% O2. Incubation of tissues in 70% O2 resulted in a 133% increase in SP-A mRNA transcription rate compared with control tissues. The SP-A mRNA half-life was increased by 54% in lung tissues cultured in 70% O2 vs. control tissues. Western blot analysis indicated a threefold increase in SP-A in the 70% O2 condition, demonstrating that O2 regulation of SP-A mRNA levels results in corresponding changes in SP-A levels. Primer extension assays were performed to determine whether the observed increase in SP-A mRNA levels is secondary to the preferential expression of one of the human SP-A genes, SP-A1 or SP-A2. Transcripts of both the SP-A1 and SP-A2 genes were increased ∼100% in tissues maintained in 70% O2 compared with control tissues. These data demonstrate that O2regulates human SP-A mRNA levels by both transcriptional and posttranscriptional mechanisms. Furthermore, because there is no differential effect of O2 on the expression of SP-A1 vs. SP-A2 mRNA, the properties of these genes that mediate regulation by O2 must be conserved between the two genes.

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