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

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...

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Published in:	Proceedings of the National Academy of Sciences - PNAS 1996-02, Vol.93 (3), p.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:	Article
Language:	eng
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
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Summary:	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.
ISSN:	0027-8424 1091-6490