Inflammation up-regulates hepcidin expression via phosphorylation of the Smad effectors 1, 5, and 8 independently of Bmp6. Groups of 6 CD1 mice (3 wild-type and 3 Bmp6^−/−) were killed at different time points after administration of LPS (1 μg/g of body weight). (A) Hepcidin (Hamp) mRNA levels were measured by quantitative RT-PCR. Values shown are means of −ΔCt (ie, −[Ct Hamp − Ct Hprt]) ± SD. The higher the −ΔCt, the greater the amount of Hamp amplicon. Four hours after LPS administration, hepcidin mRNA levels were increased on average 2.5-fold (−ΔΔCt = 8.31-6.97 = 1.34; 2^−ΔΔCt = 2.53) in wild-type mice and approximately 40-fold (−ΔΔCt = 5.62-0.25 = 5.37; 2^−ΔΔCt = 41.35) in Bmp6^−/− mice. Means of −ΔCt values between LPS-challenged and unchallenged mice of each genotype were compared by Student t tests. ***P < .001; **P < .01; *P < .05. (B) Protein extracts were prepared from the livers of 6 mice at each time point. Phospho–Stat-3, total Stat-3, phospho–Smad1/5/8, and total Smad5 were detected by immunoblot techniques in wild-type (left) and Bmp6^−/− (right) mice. The blots shown are representative of 3 independent experiments for each time point and each mouse genotype. Densitometry was performed using the ImageJ 1.45s gel-analysis method and results are shown in supplemental Figure 6.