Induction of Mdr1b expression by tumor necrosis factor-alpha in rat liver cells is independent of p53 but requires NF-kappaB signaling.
The multidrug resistance protein Mdr1b in rats is up-regulated during liver regeneration after partial hepatectomy or after endotoxin treatment. We hypothesize that up-regulation of Mdr1b in these models is TNF-alpha-dependent. The mechanism of Mdr1b activation by TNF-alpha is unknown as TNF-alpha can signal through various pathways, including NF-kappaB and p53, transcription factors for which binding sites in the Mdr1b promoter have been identified. We aimed to elucidate the mechanism of up-regulation of Mdr1b by TNF-alpha. We selectively used constructs expressing dominant negative Fas-associated death domain protein (FADD), TNF receptor associated factor-2 (TRAF2) or IkappaB to inhibit pathways downstream of the TNF receptor. Further, the proteasome inhibitor MG-132 was used, which prevents the breakdown of IkappaB. We show a critical role for NF-kappaB in activation of Mdr1b gene expression both in primary rat hepatocytes and in rat hepatoma H-4-II-E cells. Because p53 is up-regulated by TNF-alpha in an NF-kappaB-dependent manner and the Mdr1b promoter contains a p53 binding site, we used liver cells expressing a dominant negative p53 to show that TNF-alpha up-regulation of Mdr1b is independent of functional p53. Using transient transfection assays, we show that Mdr1b up-regulation correlates with activation of the promoter. Mutation of the NF-kappaB site in the Mdr1b promoter prevents its induction 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.
Ros
J E
JE
Groningen University Institute for Drug Exploration (GUIDE), Division of Gastroenterology and Hepatology, Department of Internal Medicine, Groningen, the Netherlands. j.e.ros@med.rug.nl
Schuetz
J D
JD
Geuken
M
M
Streetz
K
K
Moshage
H
H
Kuipers
F
F
Manns
M P
MP
Jansen
P L
PL
Trautwein
C
C
Müller
M
M
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D005786:Gene Expression Regulation / Q000502:physiology*; D022781:Hepatocytes / Q000502:physiology; D020672:I-kappa B Proteins / Q000494:pharmacology; D008099:Liver / Q000502:physiology*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D020672:I-kappa B Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D000097202:ATP-Binding Cassette Sub-Family B Member 4