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J Biol Chem, Vol. 275, Issue 11, 8183-8189, March 17, 2000

Molecular Basis of Cell-specific Endothelial Nitric-oxide Synthase Expression in Airway Epithelium^*

Zohre German, Ken L. Chambliss, Margaret C. Pace, Urs A. Arnet^§, Charles J. Lowenstein^§, and Philip W. Shaul^¶

From the  Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the ^§ Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

Nitric oxide (NO) plays an important role in airway function, and endothelial NO synthase (eNOS) is expressed in airway epithelium. To determine the basis of cell-specific eNOS expression in airway epithelium, studies were performed 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 increase in promoter activity versus vector alone. No activity was found in lung 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 for promoter activity and that the positive regulatory element between 248 and 79 is the Sp1 binding motif at 125. Electrophoretic mobility shift assays yielded two complexes with the GATA site and three with the Sp1 site. Immunodepletion with antiserum to GATA-2 prevented formation of the slowest migrating GATA complex, and antiserum to Sp1 supershifted the slowest migrating Sp1 complex. An electrophoretic mobility shift assay with H441 versus fibroblast nuclei revealed that the slowest migrating GATA complex is unique to airway epithelium. Thus, cell-specific eNOS expression in airway epithelium is dependent on the interaction of GATA-2 with the core eNOS promoter, and the proximal Sp1 binding site is also an important positive regulatory element.

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^* This work was supported by National Institutes of Health Grants HD30276 and HL63399.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063. Tel.: 214-648-2015; Fax: 214-648-2481; E-mail: pshaul@mednet.swmed.edu.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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