Hepcidin negatively regulates systemic
iron homeostasis in response to
inflammation and elevated serum
iron. Conversely,
hepcidin expression is diminished in response to
hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic
hepcidin regulation in HuH7
hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7
hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR).
Hepcidin promoter activity was measured using
luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human
hepcidin promoter, and constructs containing mutations in
bone morphogenetic protein (BMP)/SMAD4,
signal transducer and activator of transcription 3 (STAT3),
CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of
bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the
protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to
hypoxia or H(2)O(2),
hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to
hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin
mRNA, or
protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with
hypoxia caused a marked decrease in nuclear and cytosolic
SMAD4 protein and SMAD4
mRNA expression in cocultured HuH7 cells. Together these data indicate that
hypoxia represses
hepcidin expression through inhibition of BMP/SMAD signaling.