Fig. 4. The EGFR inhibitor gefitinib does not reverse hepcidin suppression in male mice, but hepatocyte ablation of Smad1 and Smad5 abolishes hepcidin suppression by EGF.

(A) Eight-week-old male and female Smad15;Alb-Cre+ and Smad158;Alb-Cre+ mice and their respective littermate Cre- controls were analyzed for hepatic Egfr mRNA (n=5–8 per group). (B-D) C57BL/6 wildtype male mice at 7 weeks were treated with 200 mg per kg gefitinib or DMSO vehicle via oral gavage for 5 days (n=5–6 per group). Mice were sacrificed 6 hours after the final dose and livers were analyzed for (B) EGFR phosphorylation and (D) SMAD5 phosphorylation levels by immunoblot and chemiluminescence quantitation and (C) Hamp mRNA by qRT-PCR. (E-F) Primary hepatocytes were isolated from 7-week-old Smad15;Alb-Cre+ and littermate Cre- control mice. Hepatocyte Hamp mRNA was analyzed by qRT-PCR in cells treated with (E) 100 nM LDN-193189 in 2% 2-hydroxypropyl-β-cyclodextrin or (F) 20 ng/ml mouse recombinant EGF or vehicle alone for 24 hours. Data from 4 (E) or 3 (F) independent experiments, each performed in triplicate, are shown. Transcripts were normalized to Rpl19 and the average of male Smad15;Alb-Cre- or vehicle-treated Cre- control mice were set to 1. Data are presented in scatter plots with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to female mice or vehicle-treated control mice of the same genotype or as otherwise noted by Student’s t test.