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Originally published In Press as doi:10.1074/jbc.M300787200 on February 16, 2003

J. Biol. Chem., Vol. 278, Issue 17, 14812-14819, April 25, 2003
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Transcriptional Activation of the Human Inducible Nitric-oxide Synthase Promoter by Krüppel-like Factor 6*

Vishal G. WarkeDagger §, Madhusoodana P. NambiarDagger , Sandeep KrishnanDagger , Klaus TenbrockDagger , David A. Geller||, Nicolas P. Koritschoner**, James L. AtkinsDagger , Donna L. FarberDagger Dagger , and George C. TsokosDagger §§§

From the Dagger  Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, the § Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, the  Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, the Dagger Dagger  Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, the || Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and the ** Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina

Nitric oxide is a ubiquitous free radical that plays a key role in a broad spectrum of signaling pathways in physiological and pathophysiological processes. We have explored the transcriptional regulation of inducible nitric-oxide synthase (iNOS) by Krüppel-like factor 6 (KLF6), an Sp1-like zinc finger transcription factor. Study of serial deletion constructs of the iNOS promoter revealed that the proximal 0.63-kb region can support a 3-6-fold reporter activity similar to that of the full-length 16-kb promoter. Within the 0.63-kb region, we identified two CACCC sites (-164 to -168 and -261 to -265) that bound KLF6 in both electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutation of both these sites abrogated the KLF6-induced enhancement of the 0.63-kb iNOS promoter activity. The binding of KLF6 to the iNOS promoter was significantly increased in Jurkat cells, primary T lymphocytes, and COS-7 cells subjected to NaCN-induced hypoxia, heat shock, serum starvation, and phorbol 12-myristate 13-acetate/A23187 ionophore stimulation. Furthermore, in KLF6-transfected and NaCN-treated COS-7 cells, there was a 3-4-fold increase in the expression of the endogenous iNOS mRNA and protein that correlated with increased production of nitric oxide. These findings indicate that KLF6 is a potential transactivator of the human iNOS promoter in diverse pathophysiological conditions.

* This work was supported by Medical Research Materiel Command of the United States Army STEP C and by United States Public Health Service Grant RO1 49954.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.

§§ To whom correspondence should be addressed: Dept. of Cellular Injury, Walter Reed Army Inst. of Research, Bldg. 503, Rm. 1A32, Robert Grant Ave., Silver Spring, MD 20910. Tel.: 301-319-9911; Fax: 301-319-9133; E-mail: gtsokos@usa.net.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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