Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-kappa B-repressing factor (NRF) in basal repression of the hiNOS gene.
Although nuclear factor (NF)-kappaB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-kappaB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-kappaB responsive genes. By sequence comparison with the IFN-beta and IL-8 promoters, we identified an NRE in the hiNOS promoter located at -6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the -6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.
Feng
Xuesheng
X
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Guo
Zhong
Z
Nourbakhsh
Mahtab
M
Hauser
Hansjorg
H
Ganster
Ray
R
Shao
Lifang
L
Geller
David A
DA
D002460:Cell Line; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C111027:NKRF protein, human; D012097:Repressor Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II