Role for NF-κB in the Regulation of Ferritin H by Tumor Necrosis Factor-α

Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor-α (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5′-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located ∼4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides −4776 and −4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-κB family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-κB consensus sequence and may bind a different member of the NF-κB/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress. Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor-α (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5′-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located ∼4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides −4776 and −4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-κB family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-κB consensus sequence and may bind a different member of the NF-κB/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress.