PARP-2 interacts with TTF-1 and regulates expression of surfactant protein-B.

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.

Conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli.

To identify relationships amongst tracheal and alveolar epithelial cells during lung development, we used conditional systems controlled by the rat CCSP and human SFTPC gene promoters to express Cre-recombinase in the developing mouse lung, thereby permanently labeling cells by expression of alkaline phosphatase or green fluorescent protein. When controlled by the rat CCSP promoter, continuous exposure of the fetus to doxycycline caused widespread recombination in conducting airway epithelial cells, including cells of the trachea, bronchi, and bronchioles before birth, and in both conducting and peripheral airways after birth. Neuroepithelial cells, identified by CGRP staining, were never labeled. Recombination and permanent labeling were observed in both ciliated and nonciliated respiratory epithelial cells, demonstrating their derivation from common progenitor cells during lung morphogenesis. Remarkable dorsal-ventral and cephalo-caudal labeling patterns, established before birth, were identified by recombination controlled by the rat CCSP gene promoter. In the trachea, subsets of epithelial cells labeled by the CCSP promoter were organized horizontally along the dorsal-ventral axis of the trachea, where selective labeling of cells juxtaposed to tracheal and bronchial cartilage was observed. In sharp contrast, recombination controlled by the human SFTPC gene promoter identified related cells that were organized in linear patterns along the cephalo-caudal axis of the conducting airways. Conditional expression of Cre-recombinase in the respiratory epithelium provides a useful model for the study of gene expression and function in the mouse respiratory tract and in the lung.

beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis.

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.