Figure 1.

Effect of serum and hepcidin treatments on membrane-associated ferroportin and iron-related gene expression in isolated hepatocytes. Hepatocytes were isolated from 3–4 month old female C57Bl/6 mice (A, B), or 2 month old WT and hepcidin KO mice from the same littermate (C) as reported.¹⁸ After plating in presence of 10% fetal bovine serum for 4h, hepatocytes were cultured without serum for different time periods (from 24, 48 or 72h in A) or treated, as indicated, either with serum (B, C) or with synthetic hepcidin (B, C). For the Western blots, membrane proteins (50 μg/lane) were separated on SDS-PAGE, electro-transferred onto nitrocellulose membrane and analyzed with anti-ferroportin antibody as described.⁷ Molecular weight markers are indicated in kDa. Anti-α-tubulin was used as loading controls in blots B, C, and D. A typical experiment, representative of at least three independent experiments, is shown. For gene expression (A′, B′, and C′), relative changes in mRNA levels were quantified by real-time qRT-PCR performed using cDNA synthetized from 2 μg total RNA (GAPDH normalized, arbitrary units). Mean ± SD for n=3 independent samples in one experiment. The experiment was performed at least twice and a representative result is shown. Statistical analysis was performed using Student’s t test (unpaired, two-tailed). (A) Membrane-associated ferroportin levels were studied in hepatocytes cultured 24, 48 or 72h in serum-free medium. Anti-L-ferritin antibodies were used for assessing the hepatocytic iron content in cytosolic protein extracts (25 μg/lane). (A′) Relative changes in hepcidin, ferroportin and ferritin mRNA levels. Results are expressed relative to hepatocytes cultured without serum for 24h. (B) Membrane-associated ferroportin levels were studied in WT hepatocytes cultured 72h in serum-free medium (0%S), 48h in serum-free medium + 24h in the presence of 10% serum (10%S) or 48h in serum free medium + 24h in presence of 350 nM human synthetic hepcidin (0%S+hepc). (B′) Relative changes in hepcidin, ferroportin, and ceruloplasmin mRNA levels. Results are expressed relative to hepatocytes cultured without serum for 72h. ***P<0.0001 as compared to untreated hepatocytes. (C) Membrane-associated ferroportin levels were studied in hepcidin KO hepatocytes cultured 72h in serum-free medium (0%S), 48h in serum-free medium + 24h in the presence of 10% serum (10%S) or 48h in serum free medium + 24h in presence of 350 nM human synthetic hepcidin from Peptide International (0%S+hepc). Membrane proteins (50 μg) from iron-treated monocyte-macrophage cell line J774 were used as a positive control. (C′) Relative changes in ferroportin, and ceruloplasmin mRNA levels. Hepcidin mRNAs were undetectable (not shown). Results are expressed relative to hepatocytes cultured without serum for 72h. *P=0.02 as compared to untreated hepatocytes.