Inhibition of TNF-α-induced NF-κB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132

The working hypothesis of the studies described herein was that inhibition of proteasome-mediated I kappa B degradation would inhibit TNF-alpha-induced nuclear factor-kappa B (NF-kappa B) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappa B Site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of I kappa B from the cytoplasm of cells, associated with a corresponding increase in NF-kappa B-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 mu M) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on I kappa B degradation as determined by Western blot analysis. I kappa B phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappa B binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated I kappa B degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappa B-mediated gene transcription.