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.
Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
C
C
Yan
A
A
Naltner
J
J
Conkright
M
M
Ghaffari
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011510:Proteolipids / Q000096:biosynthesis / Q000235:genetics; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism; D011663:Pulmonary Surfactants / Q000096:biosynthesis / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D002843:Chromatin; D018398:Homeodomain Proteins; C000614412:NKX2-1 protein, human; C000614414:Nkx2-1 protein, mouse; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011510:Proteolipids; D011663:Pulmonary Surfactants; C000607071:RARA protein, human; C000607072:Rara protein, mouse; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D008156:Luciferases