Induction of inducible nitric oxide synthase gene expression by Pokeweed mitogen.
The present study has characterized the expression of iNOS gene in Pokeweed mitogen (PWM)-driven murine macrophage RAW 264.7 cells. PWM significantly induced nitric oxide production in a dose-dependent manner. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that the inducible nitric oxide synthase gene expression is increased by PWM treatment. Since iNOS transcription has recently been shown to be under the control of the nuclear factor (NF)-kappaB/Rel family of transcription factors, the effects of PWM on NF-kappaB/Rel activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-kappaB/Rel binding sites linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the NF-kappaB/Rel transcription factor complex. Using DNA fragments containing the NF-kappaB/Rel binding sequence, PWM was shown to activate the protein/DNA binding of NF-KB/Rel to its cognate site as measured by EMSA. Supershift EMSA showed the presence of p50 and c-Rel protein in the complex at the kappaB site. Western blot analysis of isolated nuclear fractions, using p65 and c-Rel-specific antibodies, provided further evidence that c-Rel is increased by PWM treatment. N-Tosyl-1-phenylalanine chloromethyl ketone, a potent inhibitor of NF-kappaB/Rel activation, inhibited PWM-induced nitrite generation in a dose-dependent manner. Collectively, the results of these experiments indicate that c-Rel is positively regulated by PWM to assist in the initiation of iNOS gene expression.
Jeon
Y J
YJ
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Lee
J S
JS
Jeong
H G
HG
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000032:analysis; D011043:Pokeweed Mitogens / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D020569:Proto-Oncogene Proteins c-rel; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D009687:Nuclear Proteins; D011043:Pokeweed Mitogens; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse