Abstract
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.
Highlights
Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant
surfactant protein (SP)-B mRNA is induced during fetal lung development, and in adult lung its expression is restricted to alveolar epithelial cells and bronchiolar epithelial (Clara) cells [4, 5]
During lung development HNF-3␣ and transcription factor 1 (TTF-1) proteins are expressed at the onset of lung morphogenesis [16, 17] at which time expression of SP-B mRNA is not detected, and in fully developed lung the expression of HNF3␣, TTF-1, and SP-B co-localize to cells of distal epithelium
Summary
Surfactant protein B (SP-B) is essential for maintenance of biophysical properties and physiological function of pulmonary surfactant. We previously isolated and sequenced rabbit SP-B gene and determined that a minimal SP-B promoter region spanning Ϫ236 to ϩ39 nucleotides is sufficient for high level expression of CAT reporter gene in a cell-specific manner in NCI-H441 cells, a human pulmonary adenocarcinoma cell line with characteristics of Clara cells [13]. TTF-1 and HNF-3 are expressed in tissues other than lung, and during lung development TTF-1 and HNF-3 are expressed before differentiation of alveolar type II cells and expression of SP-B mRNA [16, 17] These observations suggest that TTF-1 and HNF-3 are not sufficient for cell type-specific activation of SP-B gene transcription and that additional factors might be required for activation of SP-B gene transcription. Electrophoretic mobility shift assays, and DNase I footprinting to identify DNA sequence elements and interacting protein factors important for the functional activity of SP-B promoter. Some of the findings reported in the present study have been presented in preliminary form [18]
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