Thyroid transcription factor-1 (TTF-1) gene: identification of ZBP-89, Sp1, and TTF-1 sites in the promoter and regulation by TNF-α in lung epithelial cells

• Published in: American journal of physiology. Lung cellular and molecular physiology 2011-10, Vol.301 (4), p.L427-L440
• Main Authors: Das, Aparajita, Acharya, Sunil, Gottipati, Koteswara Rao, McKnight, James B, Chandru, Hemakumar, Alcorn, Joseph L, Boggaram, Vijay
• Format: Article
• Language: English
• Subjects: Alveoli; Base Sequence; Cell Line, Tumor; Cell Survival - drug effects; Chromatin; Cytokines; Differentiation; DNA; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Electrophoretic mobility; Epithelial cells; Epithelial Cells - metabolism; Epithelial Cells - pathology; Gene deletion; Gene expression; Hepatocytes; Humans; Immunoprecipitation; Inflammation; Lung; Lung - metabolism; Lung - pathology; Molecular Sequence Data; Morphogenesis; Mutation; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phosphorylation; Plasmids; Promoter Regions, Genetic; Promoters; Protein Binding; Respiratory distress syndrome; Respiratory Distress Syndrome, Adult - genetics; Respiratory Distress Syndrome, Adult - metabolism; Respiratory Distress Syndrome, Adult - pathology; Reverse Transcriptase Polymerase Chain Reaction; Serine; Sp1 protein; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Sp3 Transcription Factor - genetics; Sp3 Transcription Factor - metabolism; Surfactants; Threonine; Thyroid; Thyroid Nuclear Factor 1; Thyroid transcription factor 1; Transcription factors; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptional Activation; Transfection; Tumor necrosis factor- alpha; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00090.2011

Thyroid transcription factor-1 (TTF-1/Nkx2.1/TITF1) is a homeodomain-containing transcription factor essential for the morphogenesis and differentiation of the lung. In the lung, TTF-1 controls the expression of surfactant proteins that are essential for lung stability and lung host defense. In this study, we identified functionally important transcription factor binding sites in the TTF-1 proximal promoter and studied tumor necrosis factor-α (TNF-α) regulation of TTF-1 expression. TNF-α, a proinflammatory cytokine, has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) and inhibits surfactant protein levels. Deletion analysis of TTF-1 5'-flanking DNA indicated that the TTF-1 proximal promoter retained high-level activity. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and mutational analysis experiments identified functional ZBP-89, Sp1, Sp3, and TTF-1 sites in the TTF-1 proximal promoter. TNF-α inhibited TTF-1 protein levels in H441 and primary alveolar type II cells. TNF-α inhibited TTF-1 gene transcription and promoter activity, indicating that transcriptional mechanisms play important roles in the inhibition of TTF-1 levels. TNF-α inhibited TTF-1 but not Sp1 or hepatocyte nuclear factor-3 DNA binding to TTF-1 promoter. Transactivation experiments in A549 cells indicated that TNF-α inhibited TTF-1 promoter activation by exogenous Sp1 and TTF-1 without altering their levels, suggesting inhibition of transcriptional activities of these proteins. TNF-α inhibition of TTF-1 expression was associated with increased threonine, but not serine, phosphorylation of Sp1. Because TTF-1 serves as a positive regulator for surfactant protein gene expression, TNF-α inhibition of TTF-1 expression could have important implications for the reduction of surfactant protein levels in diseases such as ARDS.