We report here that the transcriptional activity of NF-kappaB is negatively regulated by protein disulfide isomerase (PDI). Overexpression of PDI in RAW 264.7 cells strongly suppressed the LPS-induced production of inflammatory cytokines as well as NF-kappaBdependent luciferase activity. This negative regulation of NF-kappaB was reversed by bacitracin, a PDI inhibitor. Interestingly, NF-kappaB/ DNA complex formation and phosphorylation of NF-kappaB subunits was intact in PDI-expressing cells following stimulation with LPS. In addition, PDI and another redox regulator, thioredoxin (TRX), had opposite effects on NF-kappaB-dependent gene expression: activation of the NF-kappaB pathway by TRX was suppressed by expression of PDI in a dose-dependent manner. Finally, PDI expression was induced by the anti-inflammatory cytokine IL-10, and IL-10-mediated inhibition of LPS-induced IL-6 expression was reduced by bacitracin. These findings clearly demonstrate that PDI is a negative regulator of NF-kappaB, and may act downstream of IL-10 in this signaling pathway. (C) 2004 Elsevier Inc. All rights reserved.