Fig. 2.

Hepcidin promoter activity is upregulated under hypoxia via the STAT3 signaling cascade. (A) The induction of hepcidin under hypoxia (5% O₂) was efficiently blocked by the treatment with 0.1 µg/mL Actinomycin (**, P < 0.001). Results are presented as mean of hepcidin mRNA levels normalized to β2-microglobulin ± SD. (B) The deletion of the STAT3-binding site completely blocked the hypoxia-dependent induction of hepcidin promoter activity (*, P < 0.05). Huh7 cells were transfected with hepcidin promoter constructs including the wild type promoter (WT3kb), promoter regions with decreased length of the 5‘-flanking region (WT 1 kb, WT 855 bp and WT 165 bp) or the WT with specific deletions of transcription factor binding sites (STAT3-binding site (STAT3bs del) and the BMP-responsive elements 1–3 (bmp-RE1 to − 3 del)). The cells were transfected for 48 h and then conditioned to 5% O₂ in a hypoxia chamber for 24 h. Cells transfected with the WT3kb construct showed a 2-fold increased hepcidin promoter activity relatively the control (21% O₂). Renilla plasmid was used as control for expression and transfection. The results are expressed as fold induction ± SD of firefly/Renilla luciferase activity relatively to the 21% O₂ control of each construct. (C) Although the silencing of IL6-R and JAK2 decreased the promoter responsiveness to hypoxia, only the silencing of JAK1 and STAT3 completely blocked the hypoxia effect significantly. The cells were co-transfected with hepcidin wild type promoter containing firefly luciferase construct, Renilla control plasmid and siRNA directed against individual molecules of the JAK-STAT3 signaling or universal negative siRNA as a control. 48 h after transfection, the cells were conditioned to 5% O₂ in a hypoxia chamber for further 24 h. The results are presented as mean of the firefly/Renilla luciferase activity ratio ± SD. Significant differences are marked by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). n.s.: not significant.