C/EBP AND ALVEOLAR TYPE II CELL DIFFERENTIATION

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The type II cells of the alveolar epithelium are involved in the synthesis of pulmonary surfactant, a complex of lipids and proteins which lowers surface tension preventing alveolar collapse. Disorders of surfactant are postulated to cause respiratory distress syndrome in premature infants, congenital alveolar proteinosis in newborns and respiratory distress syndrome in adults. The hypothesis underlying this proposal is that the C/EBP family of transcription factors, known to play an important role in cells exhibiting specialized lipid metabolism, is involved in maintaining the differentiated state of the type II cell. C/EBPs can stimulate gene expression in a variety of epithelial cells. C/EBPs were first purified from liver; however, they are found in lungs as well. Preliminary data indicates that C/EBPs alpha, beta and delta are all present in adult rat type II cells. We have shown that when the type II cells are removed from the lung and purified, the level of C/EBPalpha rapidly declines. We have shown that levels of C/EBPalpha mRNA and protein increase late in fetal rat development while levels of C/EBPdelta increase in human fetal lung explants as lung maturation occurs. C/EBPalpha is detectable by immunoslot blotting in NC-H441, a lung-derived cell line which expresses SP-A and SP-B, while transfection of NC-H441 cells with antisense against C/EBPalpha reduces SP-A and SP-B gene expression. Introduction of C/EBPalpha expression vector into A549 cells, which are thought to be derived from type II cells but no longer express surfactant protein genes induces surfactant protein gene expression in these cells. Our hypothesis that C/EBPs play a role in the maintenance of the surfactant system will be tested by: 1) blocking c/EBP expression with antisense RNA or antisense adenoviral vectors in a cell line which produces surfactant proteins A and B as well as in rate type II cells; 2) using adenoviral vectors to induce synthesis of C/EBPs into type II-derived cell lines which no longer express surfactant proteins in order to restore their differentiated properties and also selecting stably transfected sublines which express C/EBPs and 3) examining the ability of C/EBPs to directly bid to sites in the rat SP-A gene and activate SP-A in transfection experiments.