Interleukin-6 induces hepcidin expression through STAT3

Abstract

Iron homeostasis is maintained through meticulous regulation of circulating hepcidin levels. Hepcidin levels that are inappropriately low or high result in iron overload or iron deficiency, respectively. Although hypoxia, erythroid demand, iron, and inflammation are all known to influence hepcidin expression, the mechanisms responsible are not well defined. In this report we show that the inflammatory cytokine interleukin-6 (IL-6) directly regulates hepcidin through induction and subsequent promoter binding of signal transducer and activator of transcription 3 (STAT3). STAT3 is necessary and sufficient for the IL-6 responsiveness of the hepcidin promoter. Our findings provide a mechanism by which hepcidin can be regulated by inflammation or, in the absence of inflammatory stimuli, by alternative mechanisms leading to STAT3 activation.

Figure 1.

IL-6 induces hepcidin expression directly. Hep G2/2.2.1 cells were maintained without induction (□) or were induced with 20 ng/mL IL-6 () for 8 hours in the absence or presence of 10 μg/mL cycloheximide (Cyclx). Following treatment, total RNA was isolated and real-time quantification of hepcidin mRNA transcripts was performed in triplicate using a 2-step RT-PCR. The ratio of hepcidin (hepc) to β-actin (act, internal control) PCR products was used to determine the relative levels of hepcidin expression. Error bars represent standard deviation of the mean.

Figure 2.

The 5′ flanking region of the hepcidin gene contains conserved elements. The transcription start site of human hepcidin was determined by 5′ RACE (+1). A putative TATA box (light gray shading) and 10 conserved elements (CEs; dark gray shading) were identified by aligning 5′ flanking sequences of the hepcidin coding region from mouse, rat, human, and dog. Identical bases in all 4 species (*), conserved purines (#), and conserved pyrimidines (^) are indicated.

Figure 3.

Conserved element 9 mediates IL-6 responsiveness. (A) A diagram of hepcidin mini shows conserved elements (CEs) 1-10 and the luciferase reporter gene (dark gray box). (B) Hep G2/2.2.1 cells were transiently transfected with control plasmid-expressing Renilla luciferase along with one of the following firefly luciferase reporter genes: a 1.3-kb (long) or 0.6-kb (mini) fragment of the 5′ flanking region of the hepcidin gene or a promoterless negative control (basic). At 48 hours after transfection, cells were serum starved for 6 hours and induced with 20 ng/mL IL-6. After 16 hours, firefly luciferase activity (normalized to Renilla luciferase as a transfection control) was measured and compared with activity from uninduced cell lysates. (C) Mutations in hepcidin mini were introduced into CEs 1-4, 6-9, and 10 by site-directed mutagenesis. Each mutant promoter–firefly luciferase plasmid was transiently transfected into HepG2/2.2.1 cells and induced with IL-6 as described in “Materials and methods.” Fold luciferase activity above uninduced controls was compared with that of hepcidin mini. Error bars represent standard error. *P < .001.

Figure 4.

STAT3 binds the hepcidin promoter. HepG2/2.2.1 cells were treated with IL-6 (20 ng/mL) or left untreated. After cells were fixed, we performed chromatin immunoprecipitation with no antibody, anti-STAT3, or anti-STAT1 and used PCR to amplify a 150 bp fragment containing the consensus STAT binding site of the hepcidin promoter. We fractionated PCR products and determined relative product amounts normalized to input chromatin. Fold promoter binding was calculated as the ratio of IL-6–treated to untreated output/input volume ratios of PCR products.

Figure 5.

STAT3 is necessary and sufficient to activate the hepcidin promoter downstream of IL-6. HepG2/2.2.1 cells were transiently transfected with a firefly luciferase reporter gene under the control of hepcidin mini, the CE9 mutant or the IL-6 responsive m67 promoter in conjunction with a Renilla luciferase vector with or without constructs expressing mutant STAT3 proteins or siRNA. (A) Constitutively active STAT3 mutant STAT3-C (2.5 μg cDNA). (B) Dominant-negative STAT3 mutant STAT3-DN (2.5 μg cDNA). (C) 2.5 μg STAT3 short hairpin siRNA or vector control. Cells in panels B and C were treated for 12 to 16 hours with 20 ng/mL IL-6. Cell lysates were analyzed for luciferase activity 48 hours after transfection. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity and is expressed as a multiple of the activity of unstimulated cells transfected with reporter alone. Error bars represent standard deviation of the mean.