Hemin-mediated regulation of an antioxidant-responsive element of the human ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells.
An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. Here, we have found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5' regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE.
Iwasaki
Kenta
K
Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, North Carolina 27695, USA.
Mackenzie
Elizabeth L
EL
Hailemariam
Kiros
K
Sakamoto
Kensuke
K
Tsuji
Yoshiaki
Y
D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology*; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D041905:Erythroid Cells / Q000166:cytology*; D005293:Ferritins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D051549:Glutathione S-Transferase pi / Q000378:metabolism; D006427:Hemin / Q000494:pharmacology*; D006801:Humans; D020014:K562 Cells; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
D000975:Antioxidants; D050976:Basic-Leucine Zipper Transcription Factors; D012333:RNA, Messenger; D006427:Hemin; D005293:Ferritins; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D051549:Glutathione S-Transferase pi; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase