Transcriptional regulation of the murine surfactant protein-A gene by B- Myb

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.