ER stress induces hepcidin gene expression in vitro. (A to C) HepG2 cells were treated with different ER stressors, as indicated. RNA was extracted and subjected to semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) for unspliced (U, 442 bp) or spliced (S, 416 bp) XBP1 mRNA forms (shown in the upper panel by ethidium bromide stain) or to quantitative RT-PCR (qRT-PCR) for hepcidin mRNA (HAMP) (shown in the lower graph). Data are the mean ± SD of three independent triplicate experiments with mean control values set to 1.0 (*P < 0.01; **P < 0.001). (D) HepG2 cells were cultured in the absence or presence of actinomycin D (Act D, 1 μg/ml) and tunicamycin (Tm, 10 μg/ml). RNA was extracted and processed as above. Data are presented and analyzed as above (*P < 0.03; **P < 0.005).

ER stress induces hepcidin gene expression and perturbs iron homeostasis in vivo. 129S6/SvEvTac wild-type mice were treated with Tm or dextrose (Control) (7). Two independent experiments were performed with 10 mice per group. Reported data are from one representative experiment. (A) Hepatic XBP1 and HAMP mRNA were analyzed and the data are presented as in Fig. 1 (**P < 0.0002). (B) Serum iron and serum ferritin were measured in control and treated (Tm) animals (7) (**P < 0.0001). (C) Liver and spleen histopathologic pictures: Perls’ Prussian blue stain for iron showing iron accumulation in Kupffer cells (arrows) and splenic macrophages. Lower table: chemical iron assessment (**P < 0.0001).

CREBH is required for hepcidin promoter activity. (A) Upper graph: HepG2 cells were transfected with CREBH-specific siRNA or control siRNA and CREBH, hepcidin, and albumin mRNAs were analyzed by qRT-PCR. Data are the mean ± SD of five independent triplicate experiments with mean control values set to 1.0 (**P < 0.001). Lower graph: HepG2 cells were transfected as above and treated with Tm or dimethyl sulfoxide (DMSO, vehicle). Hepcidin mRNA was analyzed by qRT-PCR. Data are presented as above (*P < 0.03). (B) Upper graph: HepG2 cells were cotransfected with the pGL3-Basic plasmid (Vector) or the mHAMP-775/+14-luc plasmid and CREBH specific or control siRNA (**P < 0.005). Lower graph: the mHAMP-775/+14-luc plasmid was cotransfected with active (CREBH-N) or dominant negative (CREBH-DN) CREBH plasmids into HepG2 cells. Data are presented and analyzed are as in Fig. 1 (**P < 0.0005). (C) Different hepcidin promoter segments were cloned in the pGL3-Basic plasmid (Vector) and transfected in HepG2 cells. (D) HepG2 cells were transfected with wild-type mHAMP-775/+14-luc (WT) or mutant promoter constructs and luciferase activity assessed (**P<0.005). (E) HepG2 cells transfected with the pGL3-Basic (Vector) or WT or site A and B–mutated mHAMP-775/+14-luc plasmids were treated with either DMSO (Ctrl) or Tm; data are the mean ± SD of five independent experiments (*P < 0.04). (F) Immunoprecipitation of HepG2 chromatin from cells exposed to either DMSO (Ctrl) or Tm was performed with anti-CREBH antibody. Promoter regions (100 to 150 bp) spanning site A, site B, or an upstream control region were amplified, as depicted. Percentage of DNA immunoprecipitated with anti-CREBH antibody relative to input chromatin is shown; data are the mean ± SD of three independent experiments (*P < 0.05).

ER stress induction of hepcidin expression is defective in Crebh^−/− mice. (A) WT or Crebh^−/− mice were treated with either DMSO (Control) or Tm. Hepatic hepcidin mRNA expression was analyzed by qRT-PCR. Data are the mean ± SD from one representative experiment (four or five mice per group) of four experiments (**P < 0.0001). Mean control values were set to 1.0. (B) Serum iron, serum ferritin, and tissue iron were measured in control and treated (Tm) animals (four or five mice per group) (7)(*P < 0.02; **P < 0.004). (C) Effect of LPS administration on hepatic hepcidin expression in Crebh−/− mice. Wild-type or Crebh−/−mice (four or five mice per group) were treated with either saline or LPS (2 mg/kg) and killed at different time points, as indicated. Hepatic hepcidin mRNA expression was assessed by qRT-PCR and expressed for each time point as fold increase over the control set to 1. Data are the mean T SD (*P < 0.01; **P < 0.001).