Nrf2-dependent and -independent induction of ABC transporters ABCC1, ABCC2, and ABCG2 in HepG2 cells under oxidative stress.
Nrf2, an NF-E2-related transcription factor, plays a critical role in transcriptional upregulation of many target genes, including those for metabolizing enzymes and transporters essential for cellular defense in response to oxidative and/or electrophilic stress. In the present study, we have studied the potential involvement of Nrf2 in induction of human ABC transporter genes under oxidative stress. We created a real-time PCR primer set to quantitatively investigate the induction of human ABC transporters by a redox-active compound tert-butylhydroquinone (tBHQ) in HepG2 cells. We found that mRNA levels of ABCC1, ABCC2, ABCC3, and ABCG2 were significantly elevated in dose- and time-dependent manners. Translocation of Nrf2 into the nuclei occurred concomitantly with the induction of ABCC1 and ABCC2 as well as both heavy and light chains of gamma-glutamylcysteine synthetase (gamma-GCSh and gamma-GCSI) during tBHQ treatments. To examine the potential involvement of Nrf2 in upregulation of the ABC transporters, we treated cells with siRNA to knockdown the expression of Nrf2. Under such Nrf2-knockdown conditions, tBHQ-induced mRNA levels of ABCC2 and ABCG2 were significantly suppressed as were mRNA levels of gamma-GCSh and gamma-GCSI. Interestingly, however, the elevated mRNA level of ABCC1 was little affected by Nrf2 siRNA treatment. We also addressed the involvement of Keap1, which is a negative regulator of Nrf2 by retrieving it in the cytoplasm. When HepG2 cells were treated with Keap1-specifc siRNA, a significant increase was observed in mRNA levels of ABCC1, ABCC2, and ABCG2 as well as gamma-GCSI, suggesting that induction of ABCC2 and ABCG2 by tBHQ is mediated by the Nrf2/Keap1 system, whereas the induction of ABCC1 may involve a Keap1-dependent but Nrf2-independent mechanism.
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-60 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Tatsuhiko
T
Adachi
Hiroshi
H
Nakagawa
Ivy
I
Chung
Yuichiro
Y
Hagiya
Kazuyuki
K
Hoshijima
Noriko
N
Noguchi
Macus Tien
MT
Kuo
Toshihisa
T
Ishikawa
D045744:Cell Line, Tumor; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D000072019:Kelch-Like ECH-Associated Protein 1; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D026901:Membrane Transport Proteins / Q000235:genetics*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D051267:NF-E2-Related Factor 2 / Q000502:physiology*; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000032:analysis*
D006873:Hydroquinones; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; C000715087:ABCC2 protein, human; C431364:KEAP1 protein, human; C495635:NFE2L2 protein, human; C114462:multidrug resistance-associated protein 3; C018855:2-tert-butylhydroquinone; C421109:multidrug resistance-associated protein 1