Synergistic effect of ubiquitin on lipopolysaccharide-induced TNF-α production in murine macrophage cell line RAW 264.7 cells

AbstractUbiquitin synergistically augmented the production of tumor necrosis factor alpha (TNF-α) in the presence of lipopolysaccharide (LPS) in murine macrophage cell line RAW 264.7. To investigate the mechanism of this augmentation, we analyzed the effect of ubiquitin during TNF-α mRNA synthesis and degradation, and TNF-α degradation on RAW 264.7 cells stimulated by LPS. It is found that ubiquitin augmented TNF-α mRNA synthesis. Ubiquitin did not affect the degradation of TNF-α mRNA and TNF-α. In the presence of LPS, extracellular accumulation of TNF-α by ubiquitin was twice than those by LPS, but intracellular accumulation of TNF-α produced by ubiquitin with LPS or by LPS had no difference. These data indicate that ubiquitin might induce TNF-α accumulation mainly by up-regulation of the TNF-α gene transcription. Although extracellular functions of ubiquitin remain largely unknown, we postulate that ubiquitin might be involved in the modulatory mechanisms of immune response

Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-γ-treated RAW 264.7 cells

AbstractThe roles of PKC in iNOS induction by IFN-γ have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-γ-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-γ-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-γ-treated RAW 264.7 cells. PMA together with IFN-γ increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-γ-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-κB and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-γ in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-κB. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-γ-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-γ-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages