Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is sugge sted by its ferroxidase activity and by the tissue iron overload in heredit ary Cp deficiency patients. In addition, plasma Cp increases markedly in se veral conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure , sickle cell disease, pregnancy, and inflammation. However, little is know n about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in hu man hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increa se in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism under lying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examinatio n of the sequence showed three pairs of consensus hypoxia-responsive elemen ts (HREs). Deletion and mutation analysis showed that a single HRE was nece ssary and sufficient for gene activation. The involvement of hypoxia-induci ble factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1 alpha and HIF-1 beta binding to a radiolabeled oligonucleotid e containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia ) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient i n HIF-1 beta, as shown functionally by the inactivity of a transfected Cp p romoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a mole cular mechanism that may help to understand these observations.