Abstract
Surfactant protein B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation and is developmentally regulated. Previous studies demonstrated that retinoic acid receptors (RARs) and thyroid transcription factor 1 (TTF-1) stimulated SP-B gene expression in respiratory epithelial cells. Clustered retinoic acid-responsive element and TTF-1 binding sites were identified in the enhancer region of the SP-B gene and were required for retinoic acid stimulation of the human SP-B (hSP-B) promoter. In addition, RAR and TTF-1 were colocalized in mouse bronchiolar and alveolar type II epithelial cells, the cellular site of SP-B synthesis. In the present studies, RAR and TTF-1 were colocalized in the nucleus of H441 cells. RAR and TTF-1 synergistically stimulated the hSP-B promoter in H441 cells. Direct protein-protein interactions between RAR and TTF-1 were demonstrated by the glutathione S-transferase pull-down assay and the mammalian cell two hybrid assay. Truncation/deletion studies showed that the RAR-TTF-1 interaction was mediated through the RAR DNA binding domain (DBD) and the TTF-1 homeodomain. RAR DBD greatly enhanced TTF-1 homeodomain DNA binding activity to a hSP-B enhancer oligonucleotide, in which retinoic acid-responsive element and TTF-1 DNA binding sites overlap. Chromatin immunoprecipitation assay demonstrated that retinoic acid treatment of H441 cells greatly stimulated both RAR and TTF-1 DNA binding to the hSP-B enhancer region in H441 cells. These findings support a model in which RAR/retinoid X receptor, TTF-1, and coactivators (p160 members and CBP) form an enhanceosome in the enhancer region of the hSP-B gene.
Highlights
Alveolarization of the fetal lung in vitro [3,4,5]
Co-localization of retinoic acid receptors (RARs)␣ and transcription factor 1 (TTF-1) in H441 Cells by Immunofluorescent Double Staining Assay—To study whether RAR␣ and TTF-1 interact with each other to regulate human SP-B (hSP-B) gene expression, RAR␣ and TTF-1 colocalization was assessed in H441 cells by double immunofluorescent staining
The retinoic acid (RA)/ RAR signaling pathway is well known to be critical to epithelial cell differentiation and proliferation in many tissues, little is known about how this pathway interacts with and is determined by tissue-specific factors in the respiratory system
Summary
Alveolarization of the fetal lung in vitro [3,4,5]. RA enhanced SP-B mRNA and surfactant protein B (SP-B) expression in lung epithelial cells and explant cultures of fetal lungs [3, 5,6,7,8]. Deletion of the enhancer sequence significantly reduced transcriptional activity of the hSP-B promoter [14] These sites were required for RA stimulation of hSP-B gene expression in respiratory epithelial cells. Both RAR and TTF-1 bound to the clustered RARE and TTF-1 binding sites in the enhancer region of the hSP-B gene [14, 15]. RAR consists of a DNA binding domain containing Zn2ϩ finger motifs, a ligand-binding/dimerization domain, a ligand-independent AF-1 transcription activation domain, and a ligand-dependent AF-2 transcription activation domain Through these various functional domains, RAR interacts with other transcription factors and coactivators to stimulate gene transcription. The DNA binding of RAR and TTF-1 to the hSP-B enhancer plays a critical role for enhanceosome formation.
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