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)-κB/Rel family of transcription factors, the effects of PWM on NF-κB/Rel activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-κB/Rel binding sites linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the NF-κB/Rel transcription factor complex. Using DNA fragments containing the NF-κB/Rel binding sequence, PWM was shown to activate the protein/DNA binding of NF-κB/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 κB 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-l-phenylalanine chloromethyl ketone, a potent inhibitor of NF-κB/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. Copyright (C) 1999 Elsevier Science Ireland Ltd.
- Pokeweed mitogen