Abstract
Thyroid transcription factor 1 (TTF-1/Nkx-2.1) plays a critical role in lung morphogenesis and regulates the expression of lung-specific genes, including the surfactant proteins required for pulmonary function after birth. The activity of TTF-1 is influenced by its interactions with other transcription factors and coactivators, including CBP/p300 and SRC-1. In this study, we have identified poly(ADP-ribose) polymerases (PARP-2 and PARP-1) as TTF-1 interacting proteins that influence its transcriptional activity. Endogenous PARP-2 was coimmunoprecipitated from transformed mouse lung epithelial cell (MLE15) extracts with TTF-1 and was identified by mass spectrometry. PARP-1 and Ku70/Ku80 were also coimmunoprecipitated from the cell extracts with TTF-1. The E domain of PARP-2 interacted via the C-terminal domain of TTF-1. Both PARP-1 and PARP-2 enhanced the activity of the promoter of surfactant protein-B (Sftpb gene) but not other surfactant proteins in vitro. PARP-2 was selectively expressed in epithelial cells of the conducting and peripheral lung tubules of the fetal mouse lung from embryonic day 12.5 and was detected in bronchial epithelial cells in the adult lung at cellular sites consistent with that of surfactant protein B. PARP-2 and PARP-1 interact with TTF-1 and regulate the expression of surfactant protein B, a protein required for lung function.
Highlights
Thyroid transcription factor 1 (TTF-1) plays a critical role in lung morphogenesis, respiratory epithelial cell differentiation, and gene expression [8, 9]
TTF-1 Binds to Poly(ADP-ribose) polymerase (PARP)-2 in Immortalized Lung Epithelial Cells—To isolate TTF-1-interacting proteins, a FLAG-tagged TTF-1 (FLAGTTF-1) expression vector was transiently transfected into SV40 large T antigen immortalized lung epithelial cells (MLE15 cells)
PARP-2 and PARP-1 Bind to TTF-1—To further verify that TTF-1 interacted with PARP-2, Ku70 and Ku80, the complex obtained by immunoprecipitation with FLAG-TTF-1, was immunoblotted with antibodies of PARP-2, Ku70, and Ku80
Summary
Thyroid transcription factor 1 (TTF-1) plays a critical role in lung morphogenesis, respiratory epithelial cell differentiation, and gene expression [8, 9]. To further understand the mechanisms by which TTF-1 activates gene transcription in the lung, we isolated TTF-1-interacting proteins by immunoprecipitation using anti-TTF-1 antibody with nuclear extracts from SV40 large T antigen immortalized mouse lung epithelial cells (MLE15 cells). PARP-2 interacts with the transcription factor TTF-1 and influences target gene expression in pulmonary cells.
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