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 re verse transcription-polymerase chain reaction analysis demonstrated that th e inducible nitric oxide synthase gene expression is increased by PWM treat ment. Since iNOS transcription has recently been shown to be under the cont rol of the nuclear factor (NF)-kappa B/Rel family of transcription factors, the effects of PWM on NF-kappa B/Rel activation were examined using a tran sient transfection assay and an electrophoretic mobility shift assay (EMSA) . Transient expression assays with NF-kappa B/Rel binding sites linked to t he chloramphenicol acetyltransferase gene suggest that the PWM-induced incr ease in transcription is mediated by the NF-kappa B/Rel transcription facto r complex. Using DNA fragments containing the NF-kappa B/Rel binding sequen ce, PWM was shown to activate the protein/DNA binding of NF-kappa B/Rel to its cognate site as measured by EMSA. Supershift EMSA showed the presence o f p50 and c-Rel protein in the complex at the kappa B site. Western blot an alysis of isolated nuclear fractions, using p65 and c-Rel-specific antibodi es, provided further evidence that c-Rel is increased by PWM treatment. N-T osyl-1-phenylalanine chloromethyl ketone, a potent inhibitor of NF-kappa B/ Rel activation, inhibited PWM-induced nitrite generation in a dose-dependen t manner. Collectively, the results of these experiments indicate that c-Re l is positively regulated by PWM to assist in the initiation of iNOS gene e xpression. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.