Transcriptional Regulation of Human Inducible Nitric Oxide Synthase (NOS2) Gene by Cytokines: Initial Analysis of the Human NOS2 Promoter

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 3; pp. 1054 - 1059
• Main Authors: De Vera, Michael E., Shapiro, Richard A., Nussler, Andreas K., Mudgett, John S., Simmons, Richard L., Morris, Sidney M., Billiar, Timothy R., Geller, David A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 06.02.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Biochemistry; Blotting, Northern; Cell Line; Cell lines; Cytokines; Cytokines - pharmacology; Enzymes; Epithelial cells; Epithelium; Gene Expression Regulation, Enzymologic - drug effects; Genes; Hepatocytes; Humans; Interferon-gamma - pharmacology; Interleukin-1 - pharmacology; Kinetics; Liver; Luciferases - biosynthesis; Messenger RNA; Mice; Nitric Oxide Synthase - biosynthesis; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase - metabolism; Plasmids; Promoter regions; Promoter Regions, Genetic; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Regulatory Sequences, Nucleic Acid; Restriction Mapping; RNA, Messenger - biosynthesis; Thymidine Kinase - genetics; Transcription, Genetic; Transfection; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.3.1054

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor α , interleukin 1β , and interferon γ (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kb, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5′ flanking region is necessary to confer inducibility to lipopolysaccharide and interferon γ . These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5′ flanking region of the human NOS2 gene.