Michael D. Bruno

SPDEF regulates goblet cell hyperplasia in the airway epithelium

Goblet cell hyperplasia and mucous hypersecretion contribute to the pathogenesis of chronic pulmonary diseases including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In the present work, mouse SAM pointed domain-containing ETS transcription factor (SPDEF) mRNA and protein were detected in subsets of epithelial cells lining the trachea, bronchi, and tracheal glands. SPDEF interacted with the C-terminal domain of thyroid transcription factor 1, activating transcription of genes expressed selectively in airway epithelial cells, including Sftpa, Scgb1a1, Foxj1, and Sox17. Expression of Spdef in the respiratory epithelium of adult transgenic mice caused goblet cell hyperplasia, inducing both acidic and neutral mucins in vivo, and stainined for both acidic and neutral mucins in vivo. SPDEF expression was increased at sites of goblet cell hyperplasia caused by IL-13 and dust mite allergen in a process that was dependent upon STAT-6. SPDEF was induced following intratracheal allergen exposure and after Th2 cytokine stimulation and was sufficient to cause goblet cell differentiation of Clara cells in vivo.

GATA-6 Activates Transcription of Thyroid Transcription Factor-1

Thyroid transcription factor-1 (TTF-1) is expressed in respiratory epithelial cells, where it regulates the transcription of target genes expressed in a cell-selective manner. GATA-5 and -6, members of the zinc finger family of transcription factors, are also expressed in various cell types within in the developing lung. In the present work, GATA-6 mRNA was detected in adult mouse lung, purified mouse type II epithelial cells, and differentiated mouse pulmonary adenocarcinoma cells (MLE-15 cells), being co-expressed with TTF-1 mRNA. In order to test whether GATA factors regulated TTF-1 gene transcription, GATA-5 and -6 expression vectors were co-transfected with TTF-1 luciferase expression vector. GATA-6, but not GATA-5, markedly activated TTF-1 gene transcription in HeLa cells. EMSA and supershift analysis with GATA-6 antiserum demonstrated that GATA-6 in MLE-15 cell nuclear extracts bound to an element located 96–101 base pairs from major start of TTF-1 gene transcription. Site directed mutagenesis of the GATA element in the TTF-1 promoter region inhibited transactivation by GATA-6 in HeLa cells. GATA-6 is co-expressed with TTF-1 in the respiratory epitheliumin vivo and respiratory epithelial cells in vitro. GATA-6 strongly enhanced activity of the human TTF-1 gene promoter in vitro. These findings support the concept that GATA-6 may play an important role in lung cell differentiation and gene expression, at least in part by altering the expression of TTF-1 and its potential targets.

Characteristics of surfactant from SP-A-deficient mice

Mice that are surfactant protein (SP) A deficient [SP-A(-/-)] have no apparent abnormalities in lung function. To understand the contributions of SP-A to surfactant, the biophysical properties and functional characteristics of surfactant from normal [SP-A(+/+)] and SP-A(-/-) mice were evaluated. SP-A-deficient surfactant had a lower buoyant density, a lower percentage of large-aggregate forms, an increased rate of conversion from large-aggregate to small-aggregate forms with surface area cycling, increased sensitivity to inhibition of minimum surface tension by plasma protein, and no tubular myelin by electron microscopy. Nevertheless, large-aggregate surfactants from SP-A(-/-) and SP-A(+/+) mice had similar adsorption rates and improved the lung volume of surfactant-deficient preterm rabbits similarly. Pulmonary edema and death caused by N-nitroso- N-methylurethane-induced lung injury were not different in SP-A(-/-) and SP-A(+/+) mice. The clearance of125I-labeled SP-A from lungs of SP-A(-/-) mice was slightly slower than from SP-A(+/+) mice. Although the absence of SP-A changed the structure and in vitro properties of surfactant, the in vivo function of surfactant in SP-A(-/-) mice was not changed under the conditions of these experiments.

Sox2 activates cell proliferation and differentiation in the respiratory epithelium.

Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced epithelial cell proliferation within 3 days of expression. Epithelial cell proliferation was associated with increased Ki-67 and cyclin D1 staining. Expression of cell cycle genes, including FoxM1, Ccna2 (Cyclin A2), Ccnb2 (Cyclin B2), and Ccnd1 (Cyclin D1), was increased. Consistent with a role in cell proliferation, Sox2 activated the transcription of FoxM1 in vitro. In alveoli, Sox2 caused hyperplasia and ectopic differentiation of epithelial cells to those with morphologic and molecular characteristics of conducting airway epithelium. Sox2 induced the expression of conducting airway epithelial specific genes, including Scgb1a1, Foxj1, Tubb3, and Cyp2f2. Although prolonged expression of Sox2 caused cell proliferation and epithelial hyperplasia, Sox2 did not induce pulmonary tumors. Sox2 induces proliferation of respiratory epithelial cells and, subsequently, partially reprograms alveolar epithelial cells into cells with characteristics of the conducting airways.

MAPPING OF CELL-SPECIFIC TRANSCRIPTIONAL CONTROL ELEMENTS OF THE HUMAN SP-C GENE IN TRANSGENIC MICE. • 2125

MAPPING OF CELL-SPECIFIC TRANSCRIPTIONAL CONTROL ELEMENTS OF THE HUMAN SP-C GENE IN TRANSGENIC MICE. • 2125