Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload

Abstract

Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.

Figure 1

Hjv expression in periportal hepatocytes. (A) Targeting strategy used for homologous recombination in ES cells to eliminate Hjv gene function. The Hjv locus contains 3 coding exons (yellow). A targeting construct containing eGFP (dark gray) followed by IRES-NLS-lacZ-pA (blue) and thymidine kinase neomycin (TK-neo) (light gray) cassettes was integrated in frame into the second coding exon of Hjv. The probe used for genomic Southern blot analysis is indicated in blue. Integrated cassette is not drawn to scale. STOP, carboxyterminal stop codon. (B) Genomic Southern blot of Hjv^+/+, Hjv^+/–, and Hjv^–/– genomic DNA using the probe indicated in A. (C) Northern blot analysis of total RNA isolated from P21 hindlimb muscles of Hjv^+/+ and Hjv^–/– mice probed for the expression of Hjv (top) and GAPDH (bottom). (D) Schematic drawing depicting the territories of liver lobules. Portal tracts (PT) are indicated in blue; central veins (CVs) are shown in red. Note that solid lines in D–G outline the hexagonally shaped hepatic lobule with PTs at the corners. (E–G) Detection of enzymatic lacZ activity in liver from 3-month-old Hjv^+/– mice analyzed on vibratome (E) or cryostat (F and G) sections. Red circles indicate CV, blue circles indicate PT. Inset in (G) depicts high magnification of individual binuclear hepatocytes that express lacZ. (H–K) Immunohistochemical detection of HNF4α (H, J, and K: red), lacZ (I, J, and K: green), and SYTOX green (nuclei; K: blue) in liver from 3-month-old Hjv^+/– mice. Arrows point to binuclear Hjv-expressing hepatocytes. Scale bar: 530 μm (E); 260 μm (F); 70 μm (G); 30 μm (inset in G); 40 μm (H–K)

Figure 2

Iron accumulation in Hjv-mutant mice. (A–H) Histological detection of iron content on cryostat sections of liver (A–D) and spleen (E–H) of wild-type (A, C, E, and G) and Hjv^–/– (B, D, F, and H) mice. Note uniform iron accumulation in the liver of 2.5-month-old Hjv-mutant mice and absence thereof in the red pulp of the spleen. (I) Quantitative determination of iron content (μmol/g dry weight) in various organs of 2.5-month-old wild-type (white), Hjv^+/– (gray), and Hjv^–/– (black) mice (n = 5 for each group). Asterisks indicate significant changes (P < 0.05) in Hjv^–/– mice as compared with wild-type littermates. (J) Time course (P12–P300) of iron content (μmol/g dry weight) determined in Hjv^–/– mice (squares) compared with pooled wild-type and Hjv^+/– mice (triangles). Liver (green) and spleen (blue) are depicted in the graph. At least 3 animals per time point and genotype were included in the analysis. Asterisks indicate significant changes (P < 0.05) in Hjv^–/– mice as compared with pooled wild-type and Hjv^+/– littermates. Scale bar: 270 μm (A and B); 45 μm (C and D); 1.2 mm (E and F); 100 μm (G and H).

Figure 3

Lack of hepcidin expression in Hjv-mutant mice. (A) Developmental time course (E13.5–P90) of Hjv, hepcidin, and GAPDH expression levels as determined by Northern blot analysis on total RNA isolated from liver. (B) Northern blot analysis of total RNA isolated from adult (P90) or P1.5 liver of wild-type and Hjv^–/– mice probed for the expression of Hjv, hepcidin, and GAPDH. (C) Northern blot analysis of total RNA isolated from adult (P90) liver of wild-type and Hjv^–/– mice sacrificed 7 days after sham injection (S) or injection with iron-dextran (ID) and probed for the expression of hepcidin and GAPDH. Scale bar: 100 μm. (D) In situ hybridization on cryostat sections of liver isolated from adult (P90) wild-type and Hjv^–/– mice probed for the expression of hepcidin.

Figure 4

Selective suppression of Hjv during inflammatory response. (A–C) Northern blot analysis of hepcidin (A), ferroportin (B), and Hjv (C) expression on total RNA isolated from liver of wild-type or Hjv-mutant mice. Before isolation of total RNA, mice were injected intraperitoneally with PBS (sham), LPS, IL-6, or TNF-α. At least 3 animals per experimental condition were analyzed, and 1 representative example is shown. Quantification of expression levels was performed by normalization of each sample to GAPDH expression probed sequentially on the same blots (data not shown). Histograms depict wild-type mice in black and Hjv-mutant mice in grey. Asterisks indicate significant changes (P < 0.05) in animals treated with LPS, IL-6, or TNF-α as compared with sham-injected animals of the same genotype. (D) Northern blot analysis of Hjv expression on total RNA isolated from skeletal muscle of wild-type mice after sham or LPS injection. Quantification was performed as described in (A–C). Histogram depicts sham-injected mice in black and LPS-injected mice in white.

Figure 5

An essential role for Hjv in the iron-sensing pathway. (A) Model depicting the dietary iron-sensing pathway in wild-type and Hjv-mutant mice. In wild-type mice, green arrows indicate responses in the presence of high iron; red arrows show responses in the presence of low iron. Balanced regulation of this pathway adjusts iron levels to the needs of the healthy organism (follow green or red arrows from left to right). In Hjv-mutant mice, iron-sensing is defective due to the absence of Hjv (indicated by gray double line). Despite high iron, this leads to an essentially complete absence of hepcidin expression, iron overload, and hemochromatosis (green arrows in Hjv mutant). Fpn, ferroportin. (B) Model depicting the impact of acute inflammation on the iron-sensing pathway (blue arrows). Acute-phase cytokines IL-6 and TNF-α act to coordinately downregulate Hjv expression in the liver while simultaneously inducing hepcidin expression. The reduction of Hjv results in a blockade of the dietary iron-sensing pathway (indicated by blue double line). This mechanism efficiently suppresses the iron-sensing pathway during the inflammatory response, which results in a low iron serum concentration inhibiting pathogenic growth.