Hemojuvelin is essential for dietary iron sensing, and its mutation leads
to severe iron overload (original) (raw)
Generation of Hjv-mutant mice and expression of Hjv in periportal hepatocytes. The recent genetic linkage of HJV to the iron overload disease juvenile hemochromatosis in humans (13) has opened the way to elucidation of the function of Hjv in iron homeostasis under normal physiological conditions and in disease by use of genetic studies in mice. We therefore generated _Hjv-_mutant mice, which coordinately express lacZ targeted to the nucleus from the Hjv locus (Figure 1, A and B). Previous work has shown that the strongest expression of Hjv in mice is found in skeletal muscles, but a lower level of expression has also been detected in the liver (14). Mice homozygous for the mutated Hjv allele showed a complete absence of Hjv mRNA in all tissues analyzed, including skeletal muscles, providing evidence for a complete null mutation (Figure 1C and data not shown).
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)
To determine the exact site of expression of Hjv in the liver, we processed vibratome sections of adult liver from Hjv+/– mice in order to determine the presence of lacZ activity (Figure 1E). Interestingly, we found a patterned distribution of Hjv expression in the liver whereas skeletal muscles were stained uniformly (Figure 1E and data not shown). To determine the identity of these cells, we analyzed lacZ expression on thin sections and found labeled cells surrounding portal tracts but not central veins (Figure 1, F and G). At high magnification, lacZ+ cells were often shown to contain 2 nuclei; this has previously been described as occurring frequently in hepatocytes (17, 18) (Figure 1G). In addition, double-labeling immunohistochemistry with an antibody against hepatocyte nuclear factor 4α (HNF4α), a transcription factor expressed in hepatocytes (19), confirmed the hepatocytic identity of these lacZ+ cells (Figure 1, H–K). In contrast, lacZ expression was not detected in any other of the several liver cell types examined, including sinusoidal endothelial cells expressing CD31 (20) (data not shown). Together, these findings indicate that Hjv expression in the liver is restricted to hepatocytes surrounding the portal tracts.
Hjv mutation in mice causes severe iron overload. To assess the consequences of Hjv mutation for iron homeostasis in various organs, we used both histological staining procedures and quantitative determination of iron content (Figure 2 and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI25683DS1). At 2.5 months of age, _Hjv-_mutant mice showed a severe increase in iron content in the liver (˜20 fold; iron accumulation in the parenchymal cells of the liver), pancreas (˜25 fold; iron accumulation in acinar tissue), and heart (˜4.5 fold) (Figure 2, A–D, I, and J, and Supplemental Figure 1). In contrast, we found a reduction in iron accumulation in the spleen (˜4.5 fold; Figure 2E–H, I, and J), probably due to the inability of reticuloendothelial macrophages residing in the red pulp to sequester iron. These findings are consistent with the previously observed distribution of iron content under conditions of hemochromatosis in both human patients and other mouse models of this disease (10, 11, 21–24).
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).
A time-course experiment for the determination of iron content in various tissues at several postnatal developmental stages of _Hjv-_mutant mice showed a rapid and permanent increase in iron accumulation, which reached plateau levels by 4 months of age (Figure 2J and Supplemental Figure 1E). Importantly, the first signs of hepatic iron overload were already detected by P30 (Figure 2J). These findings reveal that mutation of Hjv in mice leads to iron accumulation in multiple organs with a time course and tissue distribution comparable to that observed in patients suffering from juvenile hemochromatosis (10).
Although we found similarities detected in iron accumulation between human juvenile hemochromatosis patients and _Hjv-_mutant mice, we did not observe obvious features of cardiomyopathy in these mice as assessed by histology, analysis of heart weight, and mRNA expression analysis of a number of marker genes known to be altered in cardiomyopathy (Supplemental Figure 1, F and G, and data not shown). Moreover, _Hjv-_mutant mice did not experience an increase in mortality (up to 15 months of age) or show signs of diabetes (Supplemental Figure 1H and data not shown). While _Hjv-_mutant males were sterile, they did not show signs of hypogonadism as assessed by determination of testicular size, a phenotype frequently observed in human sufferers of juvenile hemochromatosis (10). Together, these findings suggest that _Hjv-_mutant mice show an iron homeostasis phenotype highly similar to that of human patients but, surprisingly, do not develop all of the associated pathological conditions.
Lack of hepcidin expression in Hjv-mutant mice. We next began to assess the molecular mechanism by which absence of Hjv leads to iron accumulation in mice. Hepcidin expression is a well-established indicator of iron levels and is upregulated by high body iron (25). In the liver of wild-type rats, hepcidin expression occurs in 2 waves: an early postnatal spike (P0–P3) that declines rapidly followed by a second increase that begins during the fourth postnatal week and continues into adulthood (26). A very similar time course can be detected in mice in which adult levels of hepcidin expression in the liver are reached at P24 (6) (Figure 3A). In contrast to the dynamic expression of hepcidin, Hjv expression in the liver was already detected at E13.5 and reached a steady level by late embryonic stages (6) (Figure 3A).
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.
We first determined the level of hepatic hepcidin expression in adult _Hjv-_mutant mice by Northern blot analysis and in situ hybridization experiments. We found that hepcidin mRNA was virtually undetectable in adult _Hjv-_mutant mice compared with wild-type littermates, in which expression was detected broadly throughout the liver (≤ 0.3% of wild type; Figure 3, B and D, and Figure 4A). Moreover, hepatic hepcidin expression in _Hjv-_mutant mice was also absent at early postnatal stages, when wild-type mice exhibit a naturally occurring spike of hepcidin expression (Figure 3B). To determine whether _Hjv-_mutant mice exhibit a general block of hepcidin regulation in response to iron, we assessed whether artificial elevation of iron levels in _Hjv-_mutant mice was capable of inducing hepcidin expression. We found that subcutaneous injection of iron-dextran (8) in _Hjv-_mutant mice did not increase hepcidin expression significantly whereas the same treatment consistently increased hepcidin in wild-type mice (Figure 3C).
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.
Together, these findings point to an essential role for Hjv in iron sensing, that of an upstream regulator of hepcidin expression. Moreover, the massive reduction in hepcidin provides a molecular explanation for the continued iron accumulation and lack of effective regulatory mechanisms to decrease iron uptake in _Hjv-_mutant mice.
Acute inflammation can induce hepcidin expression in Hjv-mutant mice. Does the lack of hepcidin expression in _Hjv-_mutant mice represent an absolute inability to induce hepatic hepcidin expression, or is it possible to bypass this deficiency by stimulation of the inflammatory pathway (4, 8)? We found that induction of acute inflammation by lipopolysaccharide (LPS) injection led to rapid and robust upregulation of hepcidin in _Hjv-_mutant mice compared with sham-injected mutant animals (˜300 fold; Figure 4A). To determine whether downstream products of LPS were also sufficient to mimic the effect of LPS on hepcidin expression in _Hjv-_mutant mice, we used injections of either proinflammatory cytokine IL-6 or TNF-α (27). We found that either IL-6 or TNF-α was sufficient to mimic the effect of LPS, albeit to a lesser extent (IL-6: ˜130 fold; TNF-α: ˜160 fold) (Figure 4A).
We also assessed whether, in _Hjv-_mutant mice, inflammation-mediated upregulation of hepcidin expression was capable of effectively eliciting appropriate downstream responses. The iron exporter ferroportin has been shown both to regulate cellular iron uptake by binding to hepcidin (3) and to be transcriptionally downregulated by high hepcidin levels (28). Consistent with the observed lack of hepcidin expression in _Hjv_-mutant mice, we found high expression of ferroportin in both untreated and sham-injected _Hjv-_mutant mice (Figure 4B and data not shown). In contrast, upon LPS injection (associated with hepcidin induction), ferroportin mRNA is significantly reduced in _Hjv-_mutant mice as well as in wild-type mice (Figure 4B), indicating the presence of intact downstream responses to hepcidin in _Hjv-_mutant mice. These findings show that the inflammatory pathway can efficiently bypass a requirement for Hjv in the induction of hepatic hepcidin expression and assign a specific role to Hjv in the iron-sensing pathway upstream of hepcidin regulation.
Inflammation induces selective downregulation of Hjv in liver but not muscle. Normal iron balance is subverted during inflammation when hepcidin levels are elevated to create a transient hypoferremic environment inhibitory to pathogenic growth (9). This low serum iron concentration should be perceived as hypoferremia by the dietary iron-sensing pathway and rapidly counteracted; however, this does not occur. Interestingly, previous experiments have shown that Hjv expression in the liver of wild-type mice is strongly downregulated upon induction of acute inflammation by LPS (6). These findings raise the question of whether the observed effect is selective to the liver and whether other genes involved in iron metabolism (1, 10–12) are regulated in a similar manner.
Interestingly, in contrast to the dramatic downregulation of Hjv expression observed in the liver of LPS-injected animals (6) (Figure 4C), no decrease in the expression of Hjv in skeletal muscles was detected under these conditions (Figure 4C). Moreover, we also found that the expression levels of several hemochromatosis- or iron metabolism–related genes, such as Hfe, transferrin receptor 2 (Tfr2), β_2-microglobulin,_ and ceruloplasmin, analyzed in the liver were unchanged following LPS injection (Supplemental Figure 2). Finally, a decrease in Hjv expression in the liver was also observed in response to IL-6 or TNF-α injection (Figure 4D). Together, these findings show that the inflammatory response induces a transcriptional downregulation of Hjv specifically in the liver and that such a response is not observed for other genes implicated in iron regulatory pathways.