IL-22, like IL-6, synergizes with BMP6 in inducing hepcidin and causes STAT3 phosphorylation in hepatoma cells. (A-B) BMP6 synergises with IL-6 in inducing hepcidin in Hep3B and HepG2 cells. qRT-PCR measurement of hepcidin mRNA expression relative to GAPDH in (A) Hep3B and (B) HepG2 hepatoma cells cultured for 18 hours in serum-free media with a titration of recombinant human BMP6 in the presence (dark gray bars) or absence (light gray bars) of recombinant human IL-6 (50 ng/mL, ∼ 2.4nM). The mean and range of 3 independent experiments are plotted; a significant synergistic effect of IL-6 was observed for both cell types (P < .001, Wilcoxon matched-pairs signed rank test, accounting for each condition from each of the 3 experiments), despite the variation in baseline hepcidin levels between the 3 experiments. (C-E) IL-22 stimulates hepcidin up-regulation in Hep3B cells independently of IL-6. Hepcidin expression relative to GAPDH in Hep3B cells cultured with combinations of recombinant human BMP6 (low and high concentrations, 1.8nM or 16.2nM, respectively), IL-6 (50 ng/mL), IL-22 (50 ng/mL, ∼ 3nM), and a mixture of neutralizing anti–IL-6 and anti–IL-6R antibodies (termed anti-IL6/R, 10 μg/mL each). The effects of IL-6, IL-22, and anti-IL6/R on hepcidin induction in the absence of BMP6, or presence of 1.8nM or 16.2nM BMP6 are shown in panels C through E, respectively. Note that for clarity the ranges of y-axis scales in panels C through E are not identical. (F) Immunoblots of Hep3B cells treated with BMP6 and/or IL-6/IL-22. Immunoblots for phospho-SMAD1/5/8 (pSMAD1/5/8) and phospho-STAT3 (pSTAT3) in Hep3B cell lysates after 40 minutes stimulation with BMP6 (1.8nM) and/or IL-6 (50 ng/mL) or IL-22 (50 ng/mL); equal loading is demonstrated by immunoblotting for SMAD1, STAT3, and β-actin. Data are representative of 3 independent experiments.