Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium

Nitric oxide (NO) plays an important role in airway function, and endotheli al NO synthase (eNOS) is expressed in airway epithelium. To determine the b asis of cell-specific eNOS expression in airway epithelium, studies were pe rformed in NCI-H441 human bronchiolar epithelial cells transfected with the human eNOS promoter fused to luciferase. Transfection with 1624 base pairs of sequence 5' to the initiation ATG (position -1624) yielded a 19-fold in crease in promoter activity versus vector alone. No activity was found in l ung fibroblasts, which do not express eNOS. 5' deletions from -1624 to -279 had modest effects on promoter activity in H441 cells. Further deletion to -248 reduced activity by 65%, and activity was lost with deletion to -79, Point mutations revealed that the GATA binding motif at -254 is mandatory f or promoter activity and that the positive regulatory element between -248 and -79 is the Spl binding motif at -125. Electrophoretic mobility shift as says yielded two complexes with the GATA site and three with the Spl site. Immunodepletion with antiserum to GATA-2 prevented formation of the slowest migrating GATA complex, and antiserum to Spl supershifted the slowest migr ating Spl complex, An electrophoretic mobility shift assay with H441 versus fibroblast nuclei revealed that the slowest migrating GATA complex is uniq ue to airway epithelium. Thus, cell-specific eNOS expression in airway epit helium is dependent on the interaction of GATA-2 with the core eNOS promote r, and the proximal Spl binding site is also an important positive regulato ry element.