Transcriptional activation of the human inducible nitric-oxide synthase promoter by Kruppel-like factor 6.
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/ 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.
Warke
Vishal G
VG
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA.
Nambiar
Madhusoodana P
MP
Krishnan
Sandeep
S
Tenbrock
Klaus
K
Geller
David A
DA
Koritschoner
Nicolas P
NP
Atkins
James L
JL
Farber
Donna L
DL
Tsokos
George C
GC
D000328:Adult; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D006801:Humans; D000860:Hypoxia / Q000139:chemically induced; D019169:Jurkat Cells; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D013601:T-Lymphocytes / Q000378:metabolism; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection; D016335:Zinc Fingers
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II