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【摘要】
Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease.
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Iron is essential for life, but its excess is toxic because of its potential participation in the generation of reactive oxygen species. Heavy iron overload is observed in primary (hereditary) and secondary hemochromatosis. Excessive intestinal absorption, as it occurs in hereditary hemochromatosis (HH), leads to iron deposition in the parenchymal cells of various organs, such as the liver, heart, and pancreas. In the liver, common complications include progressive fibrosis resulting in cirrhosis and hepatocellular carcinoma.1 In secondary hemochromatosis, as seen in patients with thalassemia receiving regular transfusion therapy, excess iron initially accumulates in reticuloendothelial macrophages and, later, when reticuloendothelial iron stores saturate, iron deposition starts to increase in parenchymal tissues such as the endocrine glands, liver, and myocardium. Cardiac iron deposition produces arrhythmias, systolic and diastolic dysfunction, and congestive heart failure.2
Recently, several genes have been identified that, when mutated, can elicit HH. HH type 1, the most common form, is a late-onset autosomal recessive disease caused by mutations in HFE, which codes for a ubiquitously expressed3,4 major histocompatibility complex class I-like molecule.5 HFE requires ß2-microglobulin (ß2m) for appropriate cell surface expression,6 and in fact both HfeC/C and ß2mC/C mice recapitulate human HH and develop iron overload in the liver because of iron hyperabsorption in the duodenum.7-9 HFE influences iron homeostasis through its binding to transferrin receptor 1 (TfR1), which is part of the major cellular iron uptake pathway, and by reducing the affinity of TfR1 for transferrin (Tf), thus competing with Tf binding.10,11
Juvenile hemochromatosis (JH) or type 2 hemochromatosis, a rare autosomal disease, shares several features with HFE hemochromatosis. However, because the rate of iron accumulation is much faster than in the classical form, all of the clinical manifestations develop earlier, typically in the first or second decade of life. In JH, the most prominent clinical features are heart failure and endocrine manifestations.12 JH has been shown to have two causative factors: loss of function mutations in hepcidin (HAMP)13 or hemojuvelin (HJV)14 genes.
Hepcidin is a hormone discovered independently by three groups as a mouse peptide expressed in response to iron levels and lipopolysaccharide (LPS)15 and as a human antimicrobial peptide of the ß-defensin family found in urine16 and blood.17 In all these reports, hepcidin was found to be highly expressed in the liver. Furthermore, mice deficient in hepcidin are iron-overloaded18 whereas, conversely, transgenic mice overexpressing hepcidin are severely anemic,19 suggesting that hepcidin is a negative regulator of iron absorption. Hepcidin expression in the liver is regulated by iron levels and inflammation,20 and its mechanism of action includes posttranslational regulation of the cellular iron exporter ferroportin 1 (Fp1) in intestinal epithelial cells and macrophages.21
Comparatively, much less is known about HJV. HJV encodes for a glycosylphosphatidylinositol-anchored protein that has a partial von Willebrand factor type D domain and an Arg-Gly-Asp motif, usual in the structure of integrins. HJV is uniformly expressed in skeletal muscle and is present selectively in periportal hepatocytes.22 HJV shares high similarity with repulsive guidance molecules (RGMs), which are involved in axonal guidance.23 In the mouse, the Rgm family comprises three members, Rgma, Rgmb, and Rgmc, the mouse ortholog of HJV. In fact, Rgmc-deficient mice, like hepcidin-deficient mice, develop severe hemochromatosis, with deposition of excess iron, mostly in parenchymal cells of the liver, heart, and pancreas.22,24
A common feature in both HFE-linked HH and JH caused by HJV mutations is lower than expected hepcidin basal levels.14,25 Although it is now clear that Rgmc participates in the maintenance of iron homeostasis, it is not yet fully understood how the gene is regulated, through which signaling pathways, and whether there is any relationship between Rgmc and HFE for the control of hepcidin basal levels.
In this study, to gain new insights into the regulation of Rgmc, we used mouse models of HH (HfeC/C and ß2mC/C) and toll-like receptor 4 (Tlr4)-deficient mice to investigate adaptive changes of Rgmc levels. In addition, the role of individual cytokines, namely, interleukin (IL)-6 and tumor necrosis factor (TNF)-, in the regulation of Rgmc was examined both in vivo and in vitro.
【关键词】 repression repulsive guidance molecule inflammation independent involves necrosis
Materials and Methods
Animals
All procedures were performed in accordance with Canadian Council on Animal Care guidelines after approval by the institutional Animal Care Committee of the Centre Hospitalier de l??Universit? de Montr?al. HfeC/C mice were kindly provided by Dr. Nancy C. Andrews, Howard Hughes Medical Institute and Harvard Medical School, Children??s Hospital, Boston, MA, in the 129/SvEvTac background9 and were backcrossed onto the C57BL/6 (B6) background for 10 generations (N10). C57BL/6 controls, ß2mC/C (C57BL/6 background, N11), B6.129S6-Il6tm1Kopf (IL-6-deficient; C57BL/6 background, N11), and the C3H substrains, C3H/HeOuJ (wild type or WT) and C3H/HeJ (Tlr4-deficient), were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). All animals were housed in a light- and temperature-controlled environment with free access to tap water and food.
Animal Treatments
Control mice were fed a commercial diet (Teklad Global 18% protein rodent diet; Harlan Teklad, Madison, WI). Dietary iron overload was produced by giving 8-week-old mice the same commercial diet supplemented with 2.5% (w/w) carbonyl iron (Sigma-Aldrich, St. Louis, MO) for 2 weeks. Iron deprivation was induced by feeding the mice the same commercial diet deficient in iron for 2 weeks.
Ten-week-old mice were injected with saline (LPS; Escherichia coli serotype 055:B5, 5 mg/kg26,27 or 25 mg/kg28 i.p.; Sigma-Aldrich), recombinant mouse IL-6 (1 µg i.p.29,30 ; Cederlane Laboratories Ltd., Hornby, ON, Canada) or recombinant mouse TNF- (1 µg i.p.;30 Cederlane). Control mice were similarly injected with an equivalent volume of sterile saline solution. Livers were excised 6, 17, 24, and 48 hours after LPS administration and 3 and 6 hours after IL-6 or TNF- injections.
Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)
Tissue samples were stored in RNALater (Qiagen, Mississauga, ON, Canada), and total RNA was extracted with Trizol reagent (Invitrogen, Burlington, ON, Canada). RT was performed with the Omniscript RT kit (Qiagen), using random hexamers and RNase inhibitor (Invitrogen). mRNA levels of Rgmc, ß-actin, and hepcidin were measured by real-time PCR in a Rotor Gene 3000 real-time DNA detection system (Montreal Biotech Inc., Kirkland, QC, Canada) with the QuantiTect SYBR+Green I PCR kit (Qiagen).31 The following primers were used: ß-actin 5'-TGTTACCAACTGGGACGACA-3' and 5'-GGTGTTGAAGGTCTCAAA-3'; Rgmc 5'-AATTTCACACATGCCGTGTC-3' and TCAAAGGCTGCAGGAAGATT-3'; hepcidin 5'-AGAGCTGCAGCCTTTGCAC-3' and 5'-GAAGATGCAGATGGGGAAGT-3'. Expression levels were normalized to the housekeeping gene ß-actin.
Hepatocyte Isolation and Treatments
Hepatocytes were isolated from adult mouse livers according to a procedure already described.32 Isolated cells were resuspended in Williams?? E media with 5% fetal bovine serum and seeded on collagen-coated plates at a density of 2.4 x 105 cells per well. After 3 hours, unattached cells were removed, and the medium replaced by fresh medium. Twenty-two hours after isolation, the medium was changed and replaced by fresh medium alone or medium containing IL-6 (20 ng/ml), TNF- (20 ng/ml), or LPS (100 ng/ml) for 24 hours.
Serum Iron (SI) and Transferrin Saturation (TS) Measurements
SI, total iron binding capacity, and TS were assessed by colorimetric assay with the Kodak Ektachem DT60 system (Johnson & Johnson, Ortho Clinical Diagnostics, Mississauga, ON, Canada).31
Measurements of Tissue Iron Concentration
Liver iron concentrations were assessed by acid digestion of tissue samples, followed by iron quantification with atomic absorption spectroscopy.33
Statistical Analysis
Student??s t-test (unpaired, two-tailed) was applied for comparison between two groups. Comparisons between more than two groups were performed by one-way analysis of variance, followed by the Bonferroni multiple comparison test.
Results
Effects of Iron Levels on Rgmc Expression
Feeding mice on diets with different iron content resulted in statistically significant differences in body iron levels, as evidenced by measuring liver iron concentrations (Figure 1A) . Mice fed the low-iron diet had a 2.5-fold reduction in liver iron concentrations compared to mice fed the standard diet, whereas animals kept on the high-iron diet had an approximately twofold increase in hepatic iron stores. Hepatic Rgmc mRNA levels, however, did not differ among mice subjected to the different diets (Figure 1B) . Thus, changes in body iron levels through diet do not affect Rgmc quantitatively at the mRNA level. In contrast, and as expected,15,34 hepcidin levels were clearly regulated by iron stores, increasing with iron loading and decreasing when the dietary iron level was low (Figure 1C) .
Figure 1. Liver iron concentration, hepatic Rgmc, and hepcidin expression in control, iron-deficient, iron-loaded, and HH mouse models. Liver iron concentration was measured by atomic absorption spectroscopy (A and D). Rgmc and hepcidin mRNA expression were quantified by real-time RT-PCR (B, C, E, and F). In ACC, mice were fed for 2 weeks with an iron-deficient diet (CFe), standard diet (Std) or iron-enriched diet (+Fe). In DCF, WT, HfeC/C, and ß2mC/C mice were fed Std. The Rgmc/ß-actin x 102 and hepcidin/ß-actin ratios are shown. The animals were 10 weeks old. The results are presented as means ?? SEM, n = 6 mice per group. Statistical analysis was performed by one-way analysis of variance. n.s., not significant.
Hfe-Deficient Mice Show Normal Basal Rgmc Expression
Next, we investigated whether alterations in basal Rgmc levels in Hfe-deficient mice, namely ß2m and Hfe knockout mice, contribute to inappropriate hepcidin levels found in these mice.25,35 As expected,7,9 ß2mC/C and HfeC/C mice had approximately threefold more iron in the liver than WT mice (Figure 1D) . Basal Rgmc and hepcidin mRNA levels in the livers of Hfe-deficient mice were similar to those in WT mice (Figure 1, E and F) . Because, unlike hepcidin, Rgmc mRNA levels were not regulated by iron stores (Figure 1B) , this indicates that Rgmc was expressed at appropriate basal levels in the liver of Hfe-deficient mice.
LPS Represses Rgmc Expression in Both the Liver and Heart
To investigate changes in iron parameters and Rgmc expression in response to inflammation, we initially determined the effect of a single LPS administration at various times up to 48 hours. SI and TS were significantly lower for up to 24 hours after LPS challenge, returning to normal or even to higher than normal levels by 48 hours (Figure 2, A and B) , indicating that mice become hypoferremic, at least during the first 24 hours. Hepatic Rgmc mRNA levels decreased significantly by 6 hours to 9.7% that of the control value and, in contrast to hypoferremia, the reduction persisted for 48 hours, albeit a modest recovery was observed after the lowest point at 6 hours (Figure 2C) .
Figure 2. SI, TS, and Rgmc mRNA levels in the liver and heart at various times after LPS administration. SI (A) and TS (B) were measured at 0, 6, 17, 24, and 48 hours after LPS administration (n = 4 mice per group per time). Rgmc mRNA expression was quantified by real-time RT-PCR in the liver (C) and heart (D). The animals were 10 weeks old. The Rgmc/ß-actin x 102 ratio is shown. The results are presented as means ?? SEM. Statistical analysis was performed by one-way analysis of variance: n.s., not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with 0 hours.
Because hepatic Rgmc expression was down-regulated in the mouse liver after LPS administration, we investigated whether Rgmc expression was altered in the heart, which also expresses Rgmc and is an organ severely affected in JH.12 As in the liver, LPS treatment caused an 50% decrease in Rgmc mRNA level in the heart that persisted for 48 hours (Figure 2D) . Thus, the LPS-induced acute phase response reduces Rgmc expression in both the mouse liver and heart.
Rgmc Repression by LPS Is Independent of Hfe
Next, to assess whether changes in Rgmc mRNA expression are appropriately elicited in Hfe-deficient mice, we measured Rgmc mRNA expression in HfeC/C and ß2mC/C mice after LPS administration. As shown in Figure 3 , Hfe-deficient mice responded, like WT mice, by dramatically down-regulating their hepatic Rgmc expression (Figure 3A) and up-regulating hepcidin (Figure 3B) . This demonstrates that the ability to down-regulate Rgmc during the acute phase response remains intact in the absence of functional Hfe.
Figure 3. Hepatic Rgmc and hepcidin mRNA levels in HH mouse models after LPS administration. Hepatic Rgmc (A) and hepcidin (B) mRNA expression was quantified by real-time RT-PCR in WT, HfeC/C, and ß2mC/C, saline (control, Ctrl)-, and LPS-treated (LPS) mice, 6 hours after treatments. The animals were 10 weeks old. The Rgmc/ß-actin x 102 and hepcidin/ß-actin ratios are shown. The results are presented as means ?? SEM, n = 5 to 6 mice per group. Statistical analysis was performed by Student??s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the controls.
Suppression of Rgmc Expression by LPS Involves Signaling through Tlr4
Pathogen-associated molecular patterns (PAMP), including LPS, are recognized by TLRs, which, upon activation, lead to the production of proinflammatory cytokines. LPS is a PAMP that is recognized by Tlr4 and the CD14 co-receptor.36 To establish whether hypoferremia and Rgmc down-regulation observed during LPS-induced acute inflammation are dependent on the Tlr4 pathway, we tested two C3H mouse substrains: mice deficient in Tlr4 (C3H/HeJ) and WT (C3H/HeOuJ) mice. We observed higher SI and TS values in saline-treated mice from this mouse strain (C3H) compared to C57BL/6 mice (Figure 2, A and B) . Strain to strain differences in SI and TS, with C57BL/6 mice having the lowest values, have been observed by others37 ; thus, these differences are attributable most likely to strain variations.
Both WT and Tlr4-deficient mice developed hypoferremia 6 hours after LPS treatment, as evidenced by decreases in SI, by 30% in WT and 32% in Tlr4-deficient mice at 5 mg/kg and by 72% in WT and 51% in Tlr4-deficient mice at 25 mg/kg doses of LPS (Figure 4A) . TS decreased by 40% in both mouse strains at 5 mg/kg and by 71% in WT and 60% in Tlr4-deficient mice at 25 mg/kg doses of LPS (Figure 4B) . We found that Rgmc repression and hepcidin up-regulation in the liver were suppressed in Tlr4-deficient animals at both 5 mg/kg and 25 mg/kg doses of LPS (Figure 4, C and D) . In addition, Rgmc down-regulation in the heart was repressed (Figure 4E) . Thus, the pathways involved in Rgmc and hepcidin regulation during inflammation, but not the hypoferremic response, is Tlr4-dependent.
Figure 4. SI, TS, Rgmc, and hepcidin modulation by LPS in Trl4-deficient mice. SI (A) and TS (B) were measured 6 hours after 5 mg/kg or 25 mg/kg of LPS administration. Rgmc (C) and hepcidin (D) mRNA expressions were quantified by real-time RT-PCR in the liver (n = 4 to 5 mice per group) and Rgmc in the heart (E; n = 3 mice per group) of WT and Tlr4-deficient (Tlr4d) mice. The animals were 10 weeks old. The Rgmc/ß-actin x 102 and hepcidin/ß-actin ratios are shown. The results are presented as means ?? SEM. Statistical analysis was performed by two-way analysis of variance: n.s., not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the saline-treated controls (Ctrl).
TNF- Is Involved in the Suppression of Rgmc Expression by LPS
The Tlr4 signaling pathway leads to activation of nuclear factor (NF)-B, which initiates the transcription of proinflammatory cytokine genes such as Tnf- and Il-6. Consequently, in Tlr4-deficient C3H/HeJ mice, LPS induces markedly less production of TNF- and IL-6 in serum, as well as hepatic mRNA levels (data not shown), than in Tlr4-normal C3H/HeOuJ mice,27,36,38,39 suggesting that cytokines might be mediators of the LPS-induced fall in Rgmc mRNA levels. To determine whether these cytokines are able to decrease Rgmc mRNA and cause hypoferremia in vivo, C57BL/6 mice were treated with 1 µg of recombinant mouse IL-6 or TNF- for 3 or 6 hours. As shown in Figure 5, A and B , TNF- led to hypoferremia, because SI and TS were reduced to similar levels, as observed after LPS administration. Likewise, as reported in humans40 and rats,41 IL-6 caused hypoferremia at earlier time points (90 minutes), with a 29% decrease in SI (P < 0.0001) and a 22% decrease in TS (P < 0.001). TNF- and IL-6 were both able to suppress Rgmc mRNA, albeit less than LPS treatment. In fact, TNF- induced a 65% decrease at 3 hours and 48% at 6 hours, whereas IL-6 caused only a 38% decline at 3 hours (Figure 5C) . In contrast, hepcidin expression was up-regulated twofold by IL-6 at 3 hours but remained unaffected by TNF- treatment (Figure 5D) .
Figure 5. SI, TS, Rgmc, and hepcidin modulation by proinflammatory cytokines and LPS. SI (A) and TS (B) in saline (control, Ctrl)-, TNF--, IL-6-, and LPS-treated mice. Hepatic Rgmc (C) and hepcidin (D) mRNA expression was quantified by real-time RT-PCR. The animals were 10 weeks old. The Rgmc/ß-actin x 102 and hepcidin/ß-actin ratios are shown. The results are presented as means ?? SEM, n = 4 mice per group. E and F: Primary hepatocytes treated with TNF-, ll-6, and LPS. The fold changes in Rgmc (E) and hepcidin (F) mRNA levels, normalized to ß-actin, are shown. The results are presented as means ?? SEM. Statistical analysis was performed by one-way analysis of variance: *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the controls.
To verify whether IL-6 and TNF- were able to target Rgmc expression directly on hepatocytes, primary cultured mouse hepatocytes were prepared from perfused livers and treated with the two different cytokines. As illustrated in Figure 5E , exogenous TNF- decreased Rgmc mRNA to 38% that of control levels, whereas no significant response was observed in IL-6-treated hepatocytes. Differently, and as expected,42 hepcidin levels were up-regulated only by IL-6 (Figure 5F) . These results show that TNF-, but not IL-6, is sufficient for the repression of Rgmc mRNA levels in hepatocytes.
IL-6 Is Not Required for Rgmc Down-Regulation in Response to LPS
To exclude further the possible requirement for IL-6 in Rgmc down-regulation during inflammation, we tested IL-6 knockout mice. As seen in Figure 6, A and B , IL-6-deficient mice developed hypoferremia, as SI and TS, respectively, decreased to 42 and 45% of control levels. Furthermore, IL-6-deficient mice retained their capacity to mount a robust response to LPS by down-regulating Rgmc expression by 87% (Figure 6C) . In contrast, and as reported previously,43 IL-6-deficient mice presented an impaired hepcidin response, as observed in Figure 6D . Thus, although IL-6 is necessary for hepcidin regulation, it is not needed for the modulation of Rgmc expression during inflammation induced by LPS.
Figure 6. C SI, TS, Rgmc, and hepcidin modulation in IL-6-deficient mice. SI (A) and TS (B) in saline (control, Ctrl)-, LPS-treated WT and IL-6-deficient (Il-6C/C) mice. Hepatic Rgmc (C) and hepcidin (D) mRNA expression were quantified by real-time RT-PCR. The animals were 10 weeks old and were analyzed 6 hours after treatments. The Rgmc/ß-actin x 102 and hepcidin/ß-actin ratios are shown. The results are presented as means ?? SEM, n = 7 mice per group. Statistical analysis was performed by Student??s t-test: n.s., not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the controls.
Discussion
Rgmc has recently emerged as a key molecule involved in iron metabolism along with hepcidin, whose expression in the liver is modulated by iron levels. Two recent studies, probing whether Rgmc is similarly regulated by iron levels, used parenteral iron loading as a model and found different outcomes depending on the treatment given. In fact, after a single injection of iron polyisomaltosate (600 mg/kg), no significant changes were observed in Rgmc expression.44 However, a significant increase in Rgmc expression was reported in a mouse model with daily iron-dextran injections for 5 days (1 mg of iron dextran/day).45 Importantly, parenteral iron loading results in excess iron being accumulated in reticuloendothelial macrophages and Kupffer cells, whereas dietary iron loading, the model in our study, leads to iron loading predominantly in parenchymal cells of the liver,7 mimicking the pattern in HH and JH.46 Our results indicate that Rgmc mRNA levels are not regulated by dietary iron deprivation or iron supplementation, ie, by varying amounts of iron concentration in hepatocytes. Thus, iron sensing by Rgmc does not seem to involve modulation of Rgmc mRNA expression levels.
Despite the fact that Rgmc mRNA levels are not regulated by iron, recent evidence emerging from Rgmc knockout mouse studies indicate that Rgmc plays an essential role in the iron-sensing pathway at the systemic level, through the control of basal hepcidin levels. In fact, Rgmc knockout mice have low basal hepcidin levels and develop severe iron overload in the liver, heart, and pancreas, as do JH patients.22,24 Eventually, the lack of Rgmc regulation by iron at the mRNA level does not exclude the possibility of posttranscriptional regulation, either at the cell membrane, intracellular compartments, or even as a secreted factor.47
Besides iron levels, iron homeostasis is also altered during inflammation, which is characterized by the occurrence of hypoferremia and the regulation of expression levels of iron metabolism-related genes, including Rgmc.44 The response to LPS seems to be tissue-specific because Rgmc down-regulation is not seen in the skeletal muscle of mice treated with LPS but is evident in the liver.22 We further show that down-regulation of Rgmc expression also occurs in the heart, where hepcidin is as well expressed,31 and persists for up to 48 hours in both the liver and heart. This indicates that Rgmc is important not only in the control of iron metabolism at the systemic level, as it impacts intestinal iron absorption through the regulation of hepcidin levels,22 but also locally, in the heart. This could be pertinent because cardiomyopathy is considerably more frequent in JH than in HH.46
We also explored the possible connection between Hfe and Rgmc because HH patients and Hfe-deficient mice,25,35,48,49 like Rgmc knockout mice,22,24 have lower than expected basal hepcidin levels. We investigated whether Hfe-deficient mice express appropriate basal Rgmc levels and are able to respond to LPS challenge. Our finding that both Hfe-deficient strains tested show basal Rgmc levels similar to WT mice, coupled with the observation that they retain the ability to down-regulate Rgmc after LPS administration, excludes any involvement of Hfe in Rgmc regulation and participation in the pathogenesis of classical HH. Thus, Hfe and Rgmc control of basal hepcidin expression occurs through two independent pathways. Further support for the existence of these pathways in the regulation of basal hepcidin mRNA levels comes from the findings that Rgmc knockout mice are unable to respond to an increase in iron stores by up-regulating their hepcidin expression22 ; in contrast, HfeC/C mice seem to retain this capacity.35,50 Thus, it appears unlikely that Hfe lies downstream of the Rgmc-dependent pathway for hepcidin regulation by iron, but Hfe may instead regulate basal hepcidin levels through another pathway, as summarized by the model in Figure 7 .
Figure 7. Model showing the iron-sensing and inflammatory pathways for Rgmc and hepcidin regulation. Iron-sensing pathway: Hfe/ß2m and Rgmc regulate basal hepcidin expression independently. Iron-sensing by Rgmc does not involve modulation of Rgmc mRNA expression levels, but Rgmc is necessary for the regulation of hepcidin levels through the iron sensing-pathway.22 Inflammatory pathway: mRNA Rgmc down-regulation by LPS involves signaling through Tlr4 and consequent production of TNF-. Down-regulation of Rgmc suppresses hepcidin control through the iron-sensing pathway and allows hepcidin induction by IL-6.
The molecular signaling mechanism for Rgmc by LPS seems to involve Tlr4 because Rgmc down-modulation was abrogated in both the liver and heart of Tlr4-deficient mice treated with LPS. However, we have shown that Tlr4-deficient mice and mice lacking downstream elements, such as IL-6-deficient mice, are still able to respond with hypoferremia, indicating that iron metabolism changes elicited by LPS may occur through both Tlr4- and non-Tlr4-dependent mechanisms.
Activation of the Tlr4-dependent signaling pathway ultimately results in the production of proinflammatory cytokines, including IL-6 and TNF-, which are known to directly influence iron metabolism and provoke hypoferremia. The mechanism by which IL-6 is believed to induce hypoferremia is related to its ability to stimulate hepcidin expression,40 a finding confirmed in this study (Figure 5D) , which in turn causes iron sequestration in macrophages and reduces intestinal iron absorption51 by binding to Fp1 and inducing its internalization.21 However, there is ample evidence that TNF- administration is also sufficient to cause hypoferremia52 by increasing iron sequestration within macrophages.53 A potential mechanism for TNF--induced iron sequestration in both the liver and spleen54 can be linked to the ability of TNF- to directly down-regulate Fp1 in a variety of cells, including macrophages,55 hepatocytes,35 and endothelial cells.56 Thus, TNF- can cause hypoferremia by a mechanism independent of hepcidin induction.54
We found that in vivo both TNF- and IL-6 treatments led to down-regulation of Rgmc expression. However, when tested in primary hepatocyte cultures, only TNF- was able to reduce Rgmc expression. Proinflammatory cytokines are known to induce each other??s expression,57 which may explain why, in vivo, IL-6 treatment had a modest repressive effect on Rgmc expression. In addition, we further excluded IL-6 requirement for Rgmc regulation by demonstrating that IL-6-deficient mice retain the ability to repress Rgmc expression after LPS administration. Taken together, these results indicate that TNF-, but not IL-6, is sufficient to induce Rgmc down-regulation in hepatocytes. The data contrast with those reported for hepcidin, in which IL-6, but not TNF-, was shown to up-regulate hepcidin expression in primary hepatocytes,40,43 a finding that we reproduced in our study.
Regulation of the expression of genes involved in iron metabolism by different cytokines may allow for finer and possibly time-dependent control of iron metabolism changes during the response to inflammation. In fact, the signaling cascade triggered by LPS leads to time-dependent and concerted release of proinflammatory cytokines, which is initiated by transduction of the LPS signal across the cell membrane by Tlr4. This is followed by TNF- production, which, as we demonstrate here, can directly elicit Rgmc down-regulation. The initial surge in TNF- production is a prerequisite to the subsequent production of other mediators, including IL-1ß58 and IL-6.57,59 These two cytokines, in turn, directly modulate the expression of hepcidin in hepatocytes.40,43,60 Consistent with our previous findings that the inflammatory pathway overrides the iron-sensing pathway for hepcidin regulation,35 Rgmc suppression during inflammation seems to be a prerequisite for the transcriptional regulation of hepcidin by cytokines (Figure 7) . These results provide further support for the mechanistic view proposed by others that the iron-sensing pathway is switched off during inflammation, which has emerged from their observation that, in Rgmc-deficient mice, the cytokine-dependent pathway for hepcidin regulation remains functional while the iron-sensing pathway is inoperative.22
In conclusion, we report that in vivo Rgmc mRNA levels are not regulated by hepatocyte iron stores and that their control during the inflammatory response is Hfe-independent. Furthermore, LPS-induced signaling involves the Tlr4 pathway and consequent production of TNF- and IL-6, which are sufficient for Rgmc and hepcidin regulation, respectively. These findings provide important insights into the regulatory pathways impacting iron metabolism changes during inflammation that may also be relevant to other disease settings affecting iron metabolism.
Acknowledgements
We thank Ovid Da Silva, Research Support Office, Research Centre, Centre Hospitalier de l??Universit? de Montr?al, for editing the manuscript; and Hortence Makui, Massar Jouga, and Wenlei Jiang for their technical help and support with animal care.
【参考文献】
Pietrangelo A: Iron, oxidative stress and liver fibrogenesis. J Hepatol 1998, 28:8-13
Wood JC, Enriquez C, Ghugre N, Otto-Duessel M, Aguilar M, Nelson MD, Moats REX, Coates TD: Physiology and pathophysiology of iron cardiomyopathy in thalassemia. Ann NY Acad Sci 2005, 1054:386-395
Parkkila S, Parkkila AK, Waheed A, Britton RS, Zhou XY, Fleming RE, Tomatsu S, Bacon BR, Sly WS: Cell surface expression of HFE protein in epithelial cells, macrophages, and monocytes. Haematologica 2000, 85:340-345
Parkkila S, Waheed A, Britton RS, Feder JN, Tsuchihashi Z, Schatzman RC, Bacon BR, Sly WS: Immunohistochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract. Proc Natl Acad Sci USA 1997, 94:2534-2539
Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon BR, Wolff RK: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996, 13:399-408
Feder JN, Tsuchihashi Z, Irrinki A, Lee VK, Mapa FA, Morikang E, Prass CE, Starnes SM, Wolff RK, Parkkila S, Sly WS, Schatzman RC: The hemochromatosis founder mutation in HLA-H disrupts beta2-microglobulin interaction and cell surface expression. J Biol Chem 1997, 272:14025-14028
Santos M, Schilham MW, Rademakers LH, Marx JJ, de Sousa M, Clevers H: Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man. J Exp Med 1996, 184:1975-1985
Ajioka RS, Levy JE, Andrews NC, Kushner JP: Regulation of iron absorption in Hfe mutant mice. Blood 2002, 100:1465-1469
Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC: The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999, 94:9-11
Feder JN, Penny DM, Irrinki A, Lee VK, Lebron JA, Watson N, Tsuchihashi Z, Sigal E, Bjorkman PJ, Schatzman RC: The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 1998, 95:1472-1477
Salter-Cid L, Brunmark A, Li Y, Leturcq D, Peterson PA, Jackson MR, Yang Y: Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci USA 1999, 96:5434-5439
Camaschella C, Roetto A, De Gobbi M: Juvenile hemochromatosis. Semin Hematol 2002, 39:242-248
Roetto A, Papanikolaou G, Politou M, Alberti F, Girelli D, Christakis J, Loukopoulos D, Camaschella C: Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet 2003, 33:21-22
Papanikolaou G, Samuels ME, Ludwig EH, MacDonald ML, Franchini PL, Dube MP, Andres L, MacFarlane J, Sakellaropoulos N, Politou M, Nemeth E, Thompson J, Risler JK, Zaborowska C, Babakaiff R, Radomski CC, Pape TD, Davidas O, Christakis J, Brissot P, Lockitch G, Ganz T, Hayden MR, Goldberg YP: Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis. Nat Genet 2004, 36:77-82
Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O: A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001, 276:7811-7819
Park CH, Valore EV, Waring AJ, Ganz T: Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001, 276:7806-7810
Krause A, Neitz S, Magert H-J, Schulz A, Forssmann W-G, Schulz-Knappe P, Adermann K: LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000, 480:147-150
Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, Vaulont S: Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 2001, 98:8780-8785
Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, Sirito M, Sawadogo M, Kahn A, Vaulont S: Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci USA 2002, 99:4596-4601
Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S: The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002, 110:1037-1044
Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J: Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004, 306:2090-2093
Niederkofler V, Salie R, Arber S: Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload. J Clin Invest 2005, 115:2180-2186
Monnier PP, Sierra A, Macchi P, Deitinghoff L, Andersen JS, Mann M, Flad M, Hornberger MR, Stahl B, Bonhoeffer F, Mueller BK: RGM is a repulsive guidance molecule for retinal axons. Nature 2002, 419:392-395
Huang FW, Pinkus JL, Pinkus GS, Fleming MD, Andrews NC: A mouse model of juvenile hemochromatosis. J Clin Invest 2005, 115:2187-2191
Bridle KR, Frazer DM, Wilkins SJ, Dixon JL, Purdie DM, Crawford DH, Subramaniam VN, Powell LW, Anderson GJ, Ramm GA: Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet 2003, 361:669-673
Liuzzi JP, Lichten LA, Rivera S, Blanchard RK, Aydemir TB, Knutson MD, Ganz T, Cousins RJ: Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response. Proc Natl Acad Sci USA 2005, 102:6843-6848
Li N, Choudhuri S, Cherrington NJ, Klaassen CD: Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4). Drug Metab Dispos 2004, 32:1265-1271
Camenisch TD, Koller BH, Earp HS, Matsushima GK: A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock. J Immunol 1999, 162:3498-3503
Kirchgessner TG, Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS: Tumor necrosis factor-alpha contributes to obesity-related hyperleptinemia by regulating leptin release from adipocytes. J Clin Invest 1997, 100:2777-2782
de Coupade C, Ajuebor MN, Russo-Marie F, Perretti M, Solito E: Cytokine modulation of liver annexin 1 expression during experimental endotoxemia. Am J Pathol 2001, 159:1435-1443
Makui H, Soares RJ, Jiang W, Constante M, Santos MM: Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading. Blood 2005, 106:2189-2195
Musallam L, Ethier C, Haddad PS, Bilodeau M: Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes. Am J Physiol 2001, 280:G1360-G1369
Miranda CJ, Makui H, Andrews NC, Santos MM: Contributions of beta2-microglobulin-dependent molecules and lymphocytes to iron regulation: insights from HfeRag1(C/C) and beta2mRag1(C/C) double knock-out mice. Blood 2004, 103:2847-2849
Mazur A, Feillet-Coudray C, Romier B, Bayle D, Gueux E, Ruivard M, Coudray C, Rayssiguier Y: Dietary iron regulates hepatic hepcidin 1 and 2 mRNAs in mice. Metabolism 2003, 52:1229-1231
Constante M, Jiang W, Wang D, Raymond V-A, Bilodeau M, Santos MM: Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation. Am J Physiol 2006, 291:G229-G237
Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S: Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999, 162:3749-3752
Dupic F, Fruchon S, Bensaid M, Loreal O, Brissot P, Borot N, Roth MP, Coppin H: Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice. Gut 2002, 51:648-653
Beutler B, Krochin N, Milsark IW, Luedke C, Cerami A: Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 1986, 232:977-980
Mengozzi M, Sironi M, Gadina M, Ghezzi P: Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate. J Immunol 1991, 147:899-902
Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T: IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004, 113:1271-1276
Kobune M, Kohgo Y, Kato J, Miyazaki E, Niitsu Y: Interleukin-6 enhances hepatic transferrin uptake and ferritin expression in rats. Hepatology 1994, 19:1468-1475
Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T: Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003, 101:2461-2463
Lee P, Peng H, Gelbart T, Beutler E: The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and beta 2-microglobulin-deficient hepatocytes. Proc Natl Acad Sci USA 2004, 101:9263-9265
Krijt J, Vokurka M, Chang KT, Necas E: Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin. Blood 2004, 104:4308-4310
Theurl I, Ludwiczek S, Eller P, Seifert M, Artner E, Brunner P, Weiss G: Pathways for the regulation of body iron homeostasis in response to experimental iron overload. J Hepatol 2005, 43:711-719
Pietrangelo A: Hereditary hemochromatosis??a new look at an old disease. N Engl J Med 2004, 350:2383-2397
Lin L, Goldberg YP, Ganz T: Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin. Blood 2005, 106:2884-2889
Ahmad KA, Ahmann JR, Migas MC, Waheed A, Britton RS, Bacon BR, Sly WS, Fleming RE: Decreased liver hepcidin expression in the Hfe knockout mouse. Blood Cells Mol Dis 2002, 29:361-366
Muckenthaler M, Roy CN, Custodio AO, Minana B, deGraaf J, Montross LK, Andrews NC, Hentze MW: Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet 2003, 34:102-107
Gehrke SG, Herrmann T, Kulaksiz H, Merle U, Bents K, Kaiser I, Riedel HD, Stremmel W: Iron stores modulate hepatic hepcidin expression by an HFE-independent pathway. Digestion 2005, 72:25-32
Roy CN, Andrews NC: Anemia of inflammation: the hepcidin link. Curr Opin Hematol 2005, 12:107-111
Tanaka T, Araki E, Nitta K, Tateno M: Recombinant human tumor necrosis factor depresses serum iron in mice. J Biol Response Mod 1987, 6:484-488
Alvarez-Hernandez X, Liceaga J, McKay IC, Brock JH: Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor. Lab Invest 1989, 61:319-322
Laftah AH, Sharma N, Brookes MJ, McKie AT, Simpson RJ, Iqbal TH, Tselepis C: Tumour necrosis factor alpha causes hypoferraemia and reduced intestinal iron absorption in mice. Biochem J 2006, 397:61-67
Ludwiczek S, Aigner E, Theurl I, Weiss G: Cytokine-mediated regulation of iron transport in human monocytic cells. Blood 2003, 101:4148-4154
Nanami M, Ookawara T, Otaki Y, Ito K, Moriguchi R, Miyagawa K, Hasuike Y, Izumi M, Eguchi H, Suzuki K, Nakanishi T: Tumor necrosis factor-{alpha}-induced iron sequestration and oxidative stress in human endothelial cells. Arterioscler Thromb Vasc Biol 2005, 25:2495-2501
Zetterstrom M, Sundgren-Andersson AK, Ostlund P, Bartfai T: Delineation of the proinflammatory cytokine cascade in fever induction. Ann NY Acad Sci 1998, 856:48-52
Nelson ME, Wald TC, Bailey K, Wesselius LJ: Intrapulmonary cytokine accumulation following BAL and the role of endotoxin contamination. Chest 1999, 115:151-157
Fattori E, Cappelletti M, Costa P, Sellitto C, Cantoni L, Carelli M, Faggioni R, Fantuzzi G, Ghezzi P, Poli V: Defective inflammatory response in interleukin 6-deficient mice. J Exp Med 1994, 180:1243-1250
Lee P, Peng H, Gelbart T, Wang L, Beutler E: Regulation of hepcidin transcription by interleukin-1 and interleukin-6. Proc Natl Acad Sci USA 2005, 102:1906-1910
作者单位：From the Research Centre, Centre Hospitalier de l??Universit? de Montr?al, Universit? de Montr?al, Hôpital Notre-Dame,* Montreal; and Hôpital Saint-Luc, Montreal, Canada