Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)

Abstract

Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Hepcidin, a small peptide synthesized in the liver, is a key regulator of iron absorption and homeostasis in mammals. Hepcidin production is increased by iron overload and decreased by anemia and hypoxia; but the molecular mechanisms that govern the hepcidin response to these stimuli are not known. Here we establish that the von Hippel-Lindau/hypoxia-inducible transcription factor (VHL/HIF) pathway is an essential link between iron homeostasis and hepcidin regulation in vivo. Through coordinate downregulation of hepcidin and upregulation of erythropoietin and ferroportin, the VHL-HIF pathway mobilizes iron to support erythrocyte production.

Figure 1. Iron deficiency downregulates hepcidin in an HIF-1–dependent fashion.

(A) Hepcidin mRNA level in livers of WT mice under regular or low-iron diet (3 weeks), determined by real-time RT-PCR. Results, normalized to 18S ribosomal RNA expression, are expressed as mean ± SD (n = 5 in each group). (B) HIF-1 expression in liver extracts of iron-starved WT mice by Western blotting. (C) Hepcidin mRNA expression in livers of WT and Albumin-Cre/HIF-1αflox/flox (HIF-1^–/–) iron-starved mice by real-time RT-PCR (n = 8).

Figure 2. Albumin-Cre/VHL^flox/flox mice develop erythrocytosis and iron deficiency.

(A) WT and Albumin-Cre/VHL^flox/flox (VHL^–/–) mice (4 weeks old). Right: Spleen and liver weights of 3- to 4-week-old WT and Albumin-Cre/VHL^flox/flox mice (n = 8 in each group). (B) H&E stainings of liver sections from WT and Albumin-Cre/VHL^flox/flox mice. Solid arrow indicates steatosis, dashed arrow inflammatory cell infiltrate. (C) EPO mRNA expression in kidney and liver of WT (black bars) and Albumin-Cre/VHL^flox/flox (gray bars) mice by real-time RT-PCR (n = 8). EPO, rbc, hematocrit, and hemoglobin levels in blood or serum from 5-week-old mice. n = 8 in each group. (D) Peripheral blood smears from WT and Albumin-Cre/VHL^flox/flox mice. Solid arrow indicates hypochromasia, dashed arrow anisocytosis. Right: mean corpuscular hemoglobin (MCH) of WT and Albumin-Cre/VHL^flox/flox mice. Original magnification, ×200. (E) Quantification of liver iron level in WT and Albumin-Cre/VHL^flox/flox mice using the method of Torrance et al. (33) (n = 5 in each group). (F) Western blot analysis of ferritin in liver extracts from WT and Albumin-Cre/VHL^flox/flox mice. (G) Iron staining of splenic sections by Perls Prussian blue. Original magnification, ×200.

Figure 3. Binding of HIF-1 to the promoter of hepcidin and downregulation of hepcidin in Albumin-Cre/VHL^flox/flox.

(A) Sequence of murine (C57BL/6) hepcidin promoter; HREs are in bold; arrows indicate primers selected for ChIP. ChIP assay in vivo on liver extracts of WT and Albumin-Cre/VHL^flox/flox mice. (B) DFO (150 μM) induces binding of HIF-1 as shown by ChIP assay. (C) Luciferase-reporter constructs under the control of the regulatory region of the human hepcidin gene. HEK293 cells transiently transfected with pGL3 basic or pGL3-Hepc/HRE vector. (D) The “native” (CCACGTG) and mutated (CAA-TG) HREs (indicated by an X) are shown. HEK293 cells were transiently transfected with pGL3 basic, pGL3-Hepc/HRE, or pGL3-Hepc/mutHRE. (E) Hepcidin mRNA expression in livers of WT and Albumin-Cre/VHL^flox/flox by real-time RT-PCR (n = 8). HIF-1 and hepcidin expression in liver extracts of WT and Albumin-Cre/VHL^flox/flox mice. (F) Hepcidin mRNA expression in livers of WT, Albumin-Cre/VHL^flox/flox, and Albumin-Cre/VHL^flox/flox/ARNT^flox/flox (VHL^–/–ANRT^–/–) mice (n = 4). (G) IL-6 and IL-1β mRNA levels in livers of WT and Albumin-Cre/VHL^flox/flox mice.

Figure 4. Upregulation of ferroportin in Albumin-Cre/VHL^flox/flox mice.

(A) Immunostaining for ferroportin in duodenum and liver sections from WT and Albumin-Cre/VHL^flox/flox mice. Solid arrow indicates a hepatocyte, dashed arrow a Kupffer cell. (B) Ferroportin expression in liver extracts of WT and Albumin-Cre/VHL^flox/flox mice. (C) Ferroportin mRNA levels in livers of WT and Albumin-Cre/VHL^flox/flox mice. Results are expressed as mean ± SD (n ≥ 4 in each group); statistical analysis was done using Student’s t test (unpaired, 2 tailed).