Thomas R. Korfhagen

GATA-6 activates transcription of surfactant protein A.

Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions −69 to −64 of the mouse SP-A gene. The transcriptional activity of wild-type SP-A reporter constructs in HeLa cells was increased 5–10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7–10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12–18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to acis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene.

Transgenic Mouse Models for the Study of Growth Factor Signaling During Lung Morphogenesis

Formation of the lung begins with an outpouching of endodermal tissue from the lateral esophageal laryngeal sulcus at approx 9 d postconception in the mouse. The endodermal-derived cells form the trachea and mainstem bronchi, and the esophagus separates from the trachea during the early embryonic period of lung morphogenesis. Endodermal tissue grows into the splanchnic mesenchyme and undergoes stereotypic dichotomous branching to form the bronchioles and acinar structures characteristic of mammalian lung. The columnar epithelial cells lining the endodermally derived tubules remain relatively undifferentiated until day 13–14 pc, when the conducting airways are virtually complete. Pulmonary vessels form along the airways to produce the highly vascularized capillary bed of the pulmonary vasculature. The splanchnic mesenchyme differentiates to form vascular, lymphatic, and stroma tissues that are distinct in proximal, as compared to peripheral, regions of the lung. Cell differentiation, expansion of the airspaces, and thinning of the splanchnic mesenchyme precedes birth. Successful adaptation to air breathing following birth requires production of pulmonary surfactant by type II epithelial cells, clearance of lung liquid, and continued growth of the gas exchange surface. Angiogenesis and vasculogenesis generate the extensive capillary network that forms during alveolarization. Thus, the orderly growth and differentiation of numerous cell types is required for the formation of the lung, and is thought to be mediated by tightly controlled paracrine signaling that occurs between the respiratory epithelium and the underlying mesenchyme.

Transgenic Models of Lung Development and Disease

Formatio of the lung requires the ordered control of cell proliferation and differentiation that are mediated by a myriad of autocrine and paracrine signals. Intracellular signaling controls cellular proliferation, migration, differentiation, and matrix deposition through complex networks of growth factors and their receptors. Growth factor signaling through interactions among specific cell surface receptors, in turn, controls gene expression. While in vitro cellular and biochemical studies are useful for identifying molecular detail of signaling pathways and control of gene expression, in vivo studies are useful in further defining precise molecular events controlling formation of the lung. The complex intracellular and intercellular events involved in normal lung development are thought to be recapitulated in recovery from lung injury, underscoring the importance of deciphering the complex pathways regulating pulmonary morphogenesis. This chapter focuses on studies of critical molecular pathways regulating the growth and differentiation of the respiratory epithelium using transgenic mouse models in vivo.