Abstract
Surfactant B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation. Expression of SP-B by respiratory epithelial cells is regulated by developmental and hormonal influences at the level of gene transcription. Previous studies supported the role of retinoic acids (RA) and their receptors (RARs) in SP-B gene transcription. In the present study, RARalpha was detected in mouse alveolar type II epithelial cells where SP-B is synthesized and processed. Deletion and site-specific mutagenesis analysis identified clustered retinoic acid-responsive element sites in the 5'-flanking enhancer region of the hSP-B gene that bound RARalpha proteins. RAR coactivators ACTR, SRC-1, and transcriptional intermediary factor 2 (TIF2) stimulated human (h) SP-B promoter activity in a dose-dependent fashion in pulmonary adenocarcinoma H441 cells. In addition, an RAR-associated protein, CREB-binding protein (CBP), potentiated the effects of RAR on hSP-B promoter activity in H441 cells. Importantly, RA stimulation of the hSP-B promoter depends on tissue-specific thyroid transcription factor (TTF-1) DNA-binding sites. TTF-1 protein synergistically stimulated the hSP-B promoter with RARalpha, CBP, and nuclear receptor coactivators in H441 cells. In addition, TTF-1 interacted directly with RARalpha and TIF2 in the mammalian two-hybrid system. These findings support a model in which RAR/retinoid X receptor, TTF-1, coactivators, and CBP form a transcription activation complex in the upstream enhancer region of the hSP-B gene.
Highlights
Pulmonary surfactant is synthesized and secreted primarily by type II epithelial cells in the alveoli of the lung preventing atelectasis during the respiratory cycle
RAR␣ was cotransfected with equal concentrations of RXR␥ and 0.25 g of the hSP-B 500 luciferase reporter construct into H441 cells
RAR␣/RXR␥ stimulated the hSP-B 500 luciferase reporter construct in H441 cells (Fig. 2)
Summary
Pulmonary surfactant is synthesized and secreted primarily by type II epithelial cells in the alveoli of the lung preventing atelectasis during the respiratory cycle. RAR consists of a DNA-binding domain that contains 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, including CBP/p300 and nuclear receptor coactivators. In addition to nuclear receptors, coactivators with HATs interact with each other to form a large transcription activation complex. In the absence of hormone ligands, nuclear receptors interact with corepressors, such as SMRT/TRAC and N-CoR/RIP13 to repress gene activation [46, 47]. These corepressors possess histone deacetylase activity [48]. Histone acetylation/deacetylation is critical to nuclear receptor signaling in epithelial cells
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