Characterization of rabbit SP-B promoter region responsive to downregulation by tumor necrosis factor-alpha.

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. Tumor necrosis factor-alpha (TNF-alpha), an important mediator of lung inflammation, inhibits surfactant phospholipid and surfactant protein synthesis in the lung. In the present study, we investigated the TNF-alpha inhibition of rabbit SP-B promoter activity in a human lung adenocarcinoma cell line (NCI-H441). Deletion experiments indicated that the TNF-alpha response elements are located within -236 bp of SP-B 5'-flanking DNA. The TNF-alpha response region contained binding sites for nuclear factor-kappa B (NF-kappa B), Sp1/Sp3, thyroid transcription factor (TTF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors. Inhibitors of NF-kappa B activation such as dexamethasone and N-tosyl-L-phenylalanine chloromethyl ketone and mutation of the NF-kappa B element did not reverse TNF-alpha inhibition of SP-B promoter, indicating that TNF-alpha inhibition of SP-B promoter activity occurs independently of NF-kappa B activation. TNF-alpha treatment decreased the binding activities of TTF-1 and HNF-3 elements without altering the nuclear levels of TTF-1 and HNF-3 alpha proteins. Pretreatment of cells with okadaic acid reversed TNF-alpha inhibition of SP-B promoter activity. Taken together these data indicated that in NCI-H441 cells 1) TNF-alpha inhibition of SP-B promoter activity may be caused by decreased binding activities of TTF-1 and HNF-3 elements, 2) the decreased binding activities of TTF-1 and HNF-3 alpha are not due to decreased nuclear levels of the proteins, and 3) okadaic acid-sensitive phosphatases may be involved in mediating TNF-alpha inhibition of SP-B promoter activity.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene.

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising -236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, -236 to -140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, -236 to -170 and -170 to -140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1, and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription.

Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA expression is restricted to alveolar type II epithelial cells and bronchiolar epithelial cells (Clara cells) of adult lung. We previously (Margana, R. K., and Boggaram, V. (1996) Am. J. Physiol. 270, L601-L612) found that a minimal promoter region (-236 to +39) of rabbit SP-B gene is sufficient for high level expression of chloramphenicol acetyltransferase reporter gene in NCI-H441 cells, a cell line with characteristics of Clara cells. In the present study we used mutational analysis, electrophoretic mobility shift assays, and DNase I footprinting to identify cis-DNA regulatory elements and trans-acting protein factors required for lung cell-specific expression of SP-B gene. We found that in addition to thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3alpha (HNF-3alpha) binding sites, two spatially separate DNA sequences that bind Sp1 and Sp3 factors are necessary for the maintenance of SP-B promoter activity. Mutation of any one of the transcription factor binding sites caused a significant reduction in SP-B promoter activity suggesting that Sp1, Sp3, and TTF-1 and HNF-3alpha interact cooperatively with SP-B promoter to activate gene transcription.

Rabbit surfactant protein B gene: structure and functional characterization of the promoter.

Surfactant protein B (SP-B) is essential for physiological function of pulmonary surfactant. In the present investigation, we isolated rabbit SP-B gene and determined its nucleotide sequence, transcription start site, and exon/intron organization. The coding region of rabbit SP-B gene is comprised of 6.8 kb of sequence and is organized into 10 introns and 11 exons. By deletion analysis we determined that a region of SP-B gene extending from -236 to +39 nucleotides is sufficient for high-level expression of CAT reporter gene in a cell type-specific manner in the pulmonary adenocarcinoma cell line NCI-H441. Deletion of 5'-flanking sequence to -140 and -71 nucleotides significantly reduced SP-B promoter activity, suggesting that the -236 to +39 region contains cis-DNA elements required for cell type-specific expression. The proximal promoter region of SP-B gene contained DNA sequence motifs for binding thyroid transcription factor 1 (TTF-1) (-113 to -97) and hepatocyte nuclear factor 3(HNF-3) (-91 to -81). Although SP-B gene sequence -140 to +39 did not support high level expression of CAT gene in NCI-H441 cells, it was capable of activation by TTF-1 in HeLa cells, suggesting that the -236 to -140 sequence plays an important role in cell type-specific activation of SP-B promoter.

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

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.

Developmental and hormonal regulation of surfactant protein C (SP-C) gene expression in fetal lung. Role of transcription and mRNA stability.

Pulmonary surfactant protein C (SP-C) gene expression is developmentally and hormonally regulated in fetal lung. In the present study, we investigated the role of transcriptional and posttranscriptional mechanisms in the developmental, cAMP, and dexamethasone induction of SP-C mRNA. We found that developmental induction of SP-C mRNA was not coincident with induction of SP-C gene transcription. SP-C mRNA levels reached approximately 90% of levels in adult lung on day 24 of gestation, whereas SP-C gene transcription was only approximately 4% of level in adult lung and did not increase until day 28 of gestation (term in rabbit = 31 days). Treatment of fetal lung tissues in vitro with dibutyryl cyclic AMP (Bt2cAMP) and dexamethasone increased SP-C mRNA accumulation by different mechanisms. Increase in SP-C mRNA accumulation by Bt2cAMP was the result of increased SP-C gene transcription, whereas increased SP-C mRNA accumulation by dexamethasone was due to stabilization of RNA. In control tissues the SP-C mRNA half-life (t1/2) was 11.2 h, and after dexamethasone treatment it increased to 30 h. These data show that both transcriptional and mRNA stabilization mechanisms regulate induction of SP-C gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.

Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80-90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10-20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene.