Induction of Mdr1b expression by tumor necrosis factor-alpha in rat liver cells is independent of p53 but requires NF-kappa B signaling

The multidrug resistance protein Mdr1b in rats is upregulated during liver regeneration after partial hepatectomy or after endotoxin treatment. We hyp othesize that up-regulation of Mdr1b in these models is TNF-alpha -dependen t. The mechanism of Mdr1b activation by TNF-alpha is unknown as TNF-alpha c an signal through various pathways, including NF-kappaB and p53, transcript ion factors for which binding sites in the Mdr1b promoter have been identif ied. We aimed to elucidate the mechanism of up-regulation of Mdr1b by TNF-a lpha, We selectively used constructs expressing dominant negative Fas-assoc iated death domain protein (FADD), TNF receptor associated factor-2 (TRAF2) or I kappaB to inhibit pathways downstream of the TNF receptor. Further, t he proteasome inhibitor MG-132 was used, which prevents the breakdown of I kappaB, We show a critical role for NF-kappaB in activation of Mdr1b gene e xpression both in primary rat hepatocytes and in rat hepatoma H-4-II-E cell s, Because p53 is up-regulated by TNF-alpha in an NF-kappaB-dependent manne r and the Mdr1b promoter contains a p53 binding site, we used liver cells e xpressing a dominant negative p53 to show that TNF-alpha up-regulation of M dr1b is independent of functional p53, Using transient transfection assays, we show that Mdr1b up-regulation correlates with activation of the promote r. Mutation of the NF-kappaB site in the Mdr1b promoter prevents its induct ion by TNF-alpha. In conclusion our results show that activation of the rat Mdr1b gene by TNF-alpha is a result of NF-kappaB signaling and independent of p53.