Abstract
Surfactant protein A (SP-A) is selectively synthesized in subsets of cells lining the respiratory epithelium, where its expression is regulated by various transcription factors including thyroid transcription factor-1 (TTF-1). Cell-specific transcription of the mouse SP-A promoter is mediated by binding of TTF-1 at four distinct cis-active sites located in the 5'-flanking region of the gene. Mutation of TTF-1-binding sites (TBE) 1, 3, and 4 in combination markedly decreased transcriptional activity of SP-A promoter-chloramphenicol acetyltransferase constructs containing SP-A gene sequences from -256 to +45. In contrast, the same mutations enhanced transcriptional activity in constructs containing additional 5' SP-A sequences from -399 to +45 suggesting that cis-acting elements within the region -399 to -256 influence effects of TTF-1 on SP-A promoter activity. A consensus Myb-binding site was identified within the region, located at positions -380 to -371 in the mouse gene. Mutation of the Myb-binding site decreased activity of SP-A promoter constructs in MLE-15 cells. MLE-15 cells, a cell line expressing SP-A mRNA, also expressed B-Myb. B-Myb bound to the MBS in the SP-A gene as assessed by electrophoretic mobility shift assay. While co-transfection of HeLa cells with a B-Myb expression plasmid activated the transfected SP-A promoter about 3-fold, co-transfection of B-myb with cyclin A and cdk-2, to enhance phosphorylation of B-Myb, increased transcriptional activity of SP-A constructs approximately 20-fold. Taken together, the data support activation of SP-A gene promoter activity by B-Myb which acts at a cis-acting element in the SP-A gene.
Highlights
Viruses, and fungi by macrophages and neutrophils, enhances production of free radicals (Refs. 1–3, for review, see Refs. 5 and 6), and enhances activity of the mannose receptor in macrophages [7, 8]
Transcription of the mouse Surfactant protein A (SP-A) gene is regulated by thyroid transcription factor-1 (TTF-1) which binds to four cis-active sites (TBE) [15]
These findings suggested that a stimulatory cis-acting element was located within sequences from Ϫ399 to Ϫ256 whose activity was influenced by TTF-1 binding at the previously identified TTF-1-binding sites
Summary
Viruses, and fungi by macrophages and neutrophils, enhances production of free radicals (Refs. 1–3, for review, see Refs. 5 and 6), and enhances activity of the mannose receptor in macrophages [7, 8]. The precise trans- and cis-active elements mediating SP-A gene expression have not been identified to date, SP-A mRNA in the lung increased with advancing gestational age and was stimulated by ␥-interferon, cAMP, and epidermal growth factor in fetal lung tissues. Transcription of the mouse SP-A gene is regulated by thyroid transcription factor-1 (TTF-1) which binds to four cis-active sites (TBE) [15]. Activation of SP-A and SP-B gene expression by TTF-1 is further enhanced by cAMP-dependent phosphorylation [19, 20]. TTF-1 functions in combination with other transcription factors including activator protein-1, nuclear factor-1, and hepatocyte nuclear factors to regulate expression of surfactant protein genes [17, 21]. In order to further study the mechanism and regulation of expression of SP-A, transfection analysis of additional 5Ј-flanking regions of the mouse SP-A gene was undertaken. The data support the hypothesis that B-Myb regulates transcription of the mouse SP-A gene
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