Protein-protein interaction of retinoic acid receptor alpha and thyroid transcription factor-1 in respiratory epithelial cells.

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.