Characterization of rabbit SP-B promoter region responsive to downregulation by tumor necrosis factor-alpha

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. Tumor necro sis factor-alpha (TNF-alpha), an important mediator of lung inflammation, i nhibits surfactant phospholipid and surfactant protein synthesis in the lun g. In the present study, we investigated the TNF-alpha inhibition of rabbit SP-B promoter activity in a human lung adenocarcinoma cell line (NCI-H441) . Deletion experiments indicated that the TNF-alpha response elements are l ocated within -236 bp of SP-B 5'-flanking DNA. The TNF-alpha response regio n contained binding sites for nuclear factor-kappaB (NF-kappaB), Sp1/Sp3, t hyroid transcription factor (TTF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors. Inhibitors of NF-kappaB activation such as dexametha sone and N-tosyl-L-phenylalanine chloromethyl ketone and mutation of the NF -kappaB element did not reverse TNF-alpha inhibition of SP-B promoter, indi cating that TNF-alpha inhibition of SP-B promoter activity occurs independe ntly of NF-kappaB activation. TNF-alpha treatment decreased the binding act ivities of TTF-1 and HNF-3 elements without altering the nuclear levels of TTF-1 and HNF-3 alpha proteins. Pretreatment of cells with okadaic acid rev ersed TNF-alpha inhibition of SP-B promoter activity. Taken together these data indicated that in NCI-H441 cells 1) TNF-alpha inhibition of SP-B promo ter activity may be caused by decreased binding activities of TTF-1 and HNF -3 elements, 2) the decreased binding activities of TTF-1 and HNF-3 alpha a re not due to decreased nuclear levels of the proteins, and 3) okadaic acid -sensitive phosphatases may be involved in mediating TNF-alpha inhibition o f SP-B promoter activity.