Transcriptional Activation of the Human Inducible Nitric-oxide Synthase Promoter by Krüppel-like Factor 6

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (17), p.14812-14819
• Main Authors: Warke, Vishal G., Nambiar, Madhusoodana P., Krishnan, Sandeep, Tenbrock, Klaus, Geller, David A., Koritschoner, Nicolas P., Atkins, James L., Farber, Donna L., Tsokos, George C.
• Format: Article
• Language: English
• Subjects: Adult; Animals; Binding Sites; COS Cells; Humans; Hypoxia - chemically induced; Jurkat Cells; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Nitric Oxide - biosynthesis; Nitric Oxide Synthase - biosynthesis; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase - physiology; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Proto-Oncogene Proteins; T-Lymphocytes - metabolism; Trans-Activators; Transcription Factors - genetics; Transcription Factors - physiology; Transcriptional Activation; Transfection; Zinc Fingers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M300787200

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.