Hepcidin Inhibits Erythroid Colony Formation In Vitro at Decreased Erythropoietin Concentrations.

The anemia of chronic disease (ACD) results from three major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. In this study, the effects of hepcidin on erythroid colony formation in vitro are examined. At the standard recombinant human erythropoietin (rhEPO) concentration used for erythroid colony-forming unit (CFU-E) assays in vitro (1 U/mL), hepcidin had no significant effects on colony formation. However, ACD is a syndrome associated with relative reductions in EPO concentration. The effects of hepcidin 100 ng/mL on CFU-E colony formation were evaluated at lower rhEPO concentrations (0.1 _ 0.5 U/mL). Colony formation was significantly decreased in the presence of hepcidin. The regulatory pathways controlling apoptosis and proliferation of HCD57 erythroleukemia cells replicate those observed in primary human erythroid progenitors. This cell line was studied in order to more fully define the effects of hepcidin on erythropoiesis. HCD57 cells were incubated in Iscove’s modified Dulbecco’s medium with 10% fetal calf serum with 0.3 U/mL rhEPO at 37°C for 24 hrs, with or without hepcidin 100 ng/mL. No difference in cell proliferation was seen. Apoptosis in HCD57 cells is regulated by the Bad/Bcl-x_L pathway. Total Bad and Bcl-x_L protein expression were unchanged by exposure to hepcidin; however, the proportion of the anti-apoptotic protein pBad was decreased approximately 50%. A recent report has demonstrated that hepcidin reduces macrophage iron efflux by binding to the iron transporter ferroportin, causing its internalization and subsequent degradation. It is unlikely that the inhibition of CFU-E colony formation reported here results from this effect: although macrophages are present in the marrow cell population, and iron is required for erythropoiesis in vitro, the amount of iron-saturated transferrin contributed to the culture medium by FCS is comparable to the amount required for maximal CFU-E colony formation in serum-free medium. CONCLUSION: At reduced rhEPO concentrations, CFU-E colony formation is inhibited by hepcidin. Short-term exposure to hepcidin induces a pro-apoptotic pattern in HCD57 erythroleukemia cells. The studies reported here do not disprove the possibility that hepcidin-induced impairment of iron flow to erythroid cells contributes to ACD: rather, they suggest an additional mechanism by which hepcidin can inhibit erythropoiesis.