Role of Hypoxia-inducible Factor-1 in Transcriptional Activation of Ceruloplasmin by Iron Deficiency

Abstract

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying 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. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1␣ and HIF-1␤ binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1␤, as shown functionally by the inactivity of a transfected Cp promoter-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 molecular mechanism that may help to understand these observations.

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance

“…As shown by Western blot analysis in Figure 2a, Hif-1a was stabilized as early as 6 h after exposure to hypoxia and Hif-1a protein levels steadily increased during a 72 h exposure in A204 RMS and A673 ES cells. To investigate whether stabilized Hif-1a is capable of recruiting ARNT (Hif-1b) and forming a functional DNA-binding complex, electrophoretic mobility shift assay (EMSA) was performed using a 3 0 HRE-derived oligonucleotide probe (Mukopadhyay et al, 2000). Hif-1a showed increased DNAbinding under hypoxic conditions in both cell lines (Figure 2b).…”

“…Oligonucleotide DNA probes containing the Hif-1a binding sequence 5 0 -TCTGTACGTGACCACACTCACCTC (Mukopadhyay et al, 2000) and a mutant probe 5 0 -TCTGTAAAA GACCACACTCACCTC, labeled with with g-32 P-ATP (Amersham, Freiburg, Germany) and annealed with complementary oligonucleotides, were used for EMSA. DNA-protein binding reactions were carried out at 41C for 30 min in a total volume of 20 ml, with 4-5 mg of nuclear protein in the presence of 0.5 mg of Salmon sperm DNA (Sigma) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 5 mM DTT and 5% glycerol.…”

Anemia upregulates lipocalin 2 in the liver and serum

“…Anemia results in tissue hypoxia, which is a known activator of the expression of many genes involved in iron metabolism, such as transferrin [17], TfR [18,19], ferritin [20], and ceruloplasmin [21]. To evaluate the effect of hypoxia on Lcn2 gene expression, mice were treated with CoCl 2 , a chemical inducer of hypoxia-like responses.…”

Differential protein profiling of renal cell carcinoma urinary exosomes

“…Finally, the levels of CAIX and CP are found significantly increased in RCC urinary exosomes: it has to be underlined that their promoters were reported to be activated by the transcriptional factor HIF-1a, known to be involved in RCC genesis. [48][49][50] In particular, gene expression profiling on renal tissue showed a marked CP mRNA overexpression in RCC patients compared to controls, 34,51 while CAIX is proven to be a powerful tissue marker for ccRCC and was recently shown to correlate with tumor size. 52 Both CP and CAIX have been detected in RCC patient serum.…”