Background
Hereditary hemochromatosis (HH) accounts for most of the iron overload disorders that occur in individuals of European descent. It is an autosomal-recessive condition that is characterized by increased absorption of iron from the gastrointestinal tract and progressive accumulation of catalytically active iron in parenchymal organs. This iron excess can cause tissue damage and result in serious medical complications, including cirrhosis, primary liver cancer, diabetes, cardiomyopathy, endocrine dysfunction, and arthritis 1. In Northern Europe, most patients with HH are homozygous for a single mutation (C282Y) in the HFE gene (which encodes the hereditary hemochromatosis [HFE] protein) 2. Although the C282Y mutation disrupts a disulfide bond required for proper folding of the HFE molecule, the exact mechanisms by which HFE regulates iron homeostasis remain elusive. HFE expression can result in either the accumulation or the depletion of intracellular iron stores, depending on the cell type, suggesting that HFE interacts with other proteins that are involved in either the import or the export of iron 34. The challenge remains to identify these proteins.
Despite its high prevalence (approximately 5/1,000 individuals of Northern European descent), C282Y homozygosity is characterized by a low penetrance 5, and family studies have shown that genetic factors contribute to this reduced penetrance 6. Polymorphisms of modifier genes may have profound effects on the dominance of the HFE gene defect itself and explain individual variations in excess iron absorption and their pathologic consequences among carriers of the HH-predisposing genotype. However, the exact nature of these modifier genes in HH remains unknown, which currently precludes accurate prediction of who, among C282Y homozygotes, is likely to develop clinically significant iron-storage disease.
Murine models of iron loading, such as Hfe knockout mice (Hfe-/-), provide a useful alternative to humans in which to elucidate the physiologic pathways that are involved in the HH disease process and identifying modifier loci 78. We previously reported that, compared with the inbred mouse strain C57BL/6 (B6), the strain DBA/2 (D2) was particularly susceptible to iron loading in response to Hfe disruption 9, suggesting the existence of genes other than HFE that modify the severity of iron accumulation. We therefore took advantage of the marked phenotypic differences between these two strains to localize five chromosomal intervals that control hepatic iron loading 10. Analysis of recombinant inbred strains and exploration of strain-specific gene expression changes that result from Hfe disruption should facilitate the identification of the Hfe modifiers that account for variable disease expression in these intervals.
Thus far, investigations of regulatory circuits in response to Hfe disruption haves not addressed possible strain differences and have been limited to IronChip cDNA microarrays customized to analyze a selection of 300 genes encoding proteins that are directly involved in iron metabolism or in linked pathways 11. Of note, expression of genes that may still have unsuspected importance in iron metabolism cannot be explored using these customized microarrays. Our goal in the present study was to identify functional classes of genes and individual candidates that are involved in the perturbation of mechanisms of iron homeostasis that results from Hfe disruption, and to identify differences in gene expression profiles between the inbred mouse strains B6 and D2 that could explain their difference in iron accumulation. To achieve this goal, we used Affymetrix GeneChip® Mouse Genome 430 2.0 arrays containing 45,101 probe sets for over 39,000 transcripts, including 34,000 well characterized mouse genes, and bioinformatics tools to characterize expression networks in the duodenum and the liver of wild-type control and Hfe-/- B6 and D2 mice.
Results
Differential gene expression between Hfe-deficient and wild-type mice
Microarray studies of liver and duodenum from Hfe-/- mice identified 1,343 transcripts that were upregulated or downregulated in liver of either B6 or D2 Hfe-/- mice, as compared with wild-type mice of the same genetic backgrounds. Much fewer genes, namely 370, were upregulated or downregulated in the duodenum of these mice. A list of the transcripts differentially regulated between Hfe-deficient and wild-type mice is provided in Additional data files 1 (liver) and 2 (duodenum). As shown in Figure 1, more transcripts were regulated in Hfe-deficient D2 mice than in B6 mice, and this difference was particularly striking in duodenum.
<p>Figure 1</p>
Number of genes regulated by Hfe disruption by mouse strain and organ studied
Number of genes regulated by Hfe disruption by mouse strain and organ studied. Genes regulated by Hfe disruption identified by statistical analysis of microarrays (SAM) were filtered to summarize the number of upregulated or downregulated genes in liver and duodenum. Genes were included if the mean S-score across three independent comparisons was ≥2 or ≤-2.
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In liver, clustering analysis detected groups of transcripts that were similarly regulated in response to Hfe disruption in B6 and D2 mice (specifically, they were either downregulated [Figure 2, cluster 4] or upregulated [cluster 5] in both strains). However, most of the transcripts modulated after Hfe disruption had expression patterns that were strain specific (regulated only in D2 mice [clusters 1 and 6] or only in B6 mice [clusters 3 and 8]).
<p>Figure 2</p>
Genes regulated by Hfe deficiency in D2 and B6 liver
Genes regulated by Hfe deficiency in D2 and B6 liver. A tree view image of k-means clustering for 1,343 genes regulated by Hfe disruption in liver of D2 or B6 mice is shown. Genes were selected by statistical filtering of knockout (KO) versus wild-type (WT) S-scores, as described in Materials and methods. Corresponding values for wild-type D2 versus B6 S-scores are also shown. Red indicates upregulation by Hfe deficiency or more highly expressed in D2 mice; green indicates downregulation by Hfe deficiency or more highly expressed in B6 mice; and black indicates no difference.
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In duodenum of B6 mice, the expression of fewer than 20 genes was significantly modified by Hfe deficiency (Figure 1). Consequently, clustering analysis was essentially based on expression changes in D2 mice. Two main clusters were therefore identified in duodenum, one with genes upregulated (cluster 1, Additional data file 2) and the other with genes downregulated (cluster 3, Additional file 2) in response to Hfe disruption in D2 mice.
Enriched functional categories in the liver of Hfe-deficient mice
The Database for Annotation, Visualization, and Integrated Discovery (DAVID) annotation tool was used to search for over-representation of functional categories within the different gene clusters from Figure 2. Categories found to be enriched within the clusters of genes similarly regulated in the liver of Hfe-/- compared with wild-type mice are summarized in Table 1. As detailed below, they mainly concern detoxification mechanisms in response to oxidative stress, fatty acid β-oxidation, cholesterol catabolism, and circadian rhythm.
<p>Table 1</p>
Functional categories over-represented in clusters of genes similarly regulated by Hfe-disruption in the liver
Category
Term
n
EASE score
Cluster 1 (284 Affy IDs [248 genes])
GOTERM_BP
Steroid metabolism
11
1.4 × 10-5
GOTERM_MF
Mono-oxygenase activity
10
3.7 × 10-5
GOTERM_MF
UDP glucuronosyltransferase activity
5
2.9 × 10-2
Cluster 3 (218 Affy IDs [196 genes])
No functional category overrepresented
Cluster 4 (145 Affy IDs [139 genes])
GOTERM_BP
Rhythmic process
6
6.5 × 10-5
KEGG_PATHWAY
Fatty acid metabolism
7
3.2 × 10-6
GOTERM_BP
Defense response
14
4.7 × 10-3
GOTERM_BP
Nitrogen compound metabolism
9
5.6 × 10-3
Cluster 5 (94 Affy IDs [84 genes])
KEGG_PATHWAY
Glutathione metabolism
8
5.8 × 10-8
GOTERM_MF
Iron ion binding
8
2.7 × 10-4
Cluster 6 (364 Affy IDs [315 genes])
SP_PIR_KEYWORDS
Fatty acid metabolism
15
2.5 × 10-14
SP_PIR_KEYWORDS
Oxidoreductase
31
1.1 × 10-8
GOTERM_MF
Iron ion binding
19
1.6 × 10-6
KEGG_PATHWAY
Bile acid biosynthesis
6
1.1 × 10-3
GOTERM_BP
Cholesterol metabolism
6
3.2 × 10-3
Cluster 8 (219 Affy IDs [209 genes])
No functional category overrepresented
Affymetrix probesets in the different k-means clusters shown in Figure 2 were compared with Affymetrix MG-430 2.0 probe sets for over-representation of gene categories, using the DAVID (Database for Annotation, Visualization, and Integrated Discovery) functional annotation tool. The Category column shows the original database/resource from which the terms originate. The Term column indicates enriched terms associated with the gene list. The n column indicates the number of genes involved in the term. The expression analysis systematic explorer (EASE) score is a modified Fisher exact P value [51]. BP, biological processes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular functions; PIR, Protein Information Resource; UDP, Uridine 5'-diphospho.
Detoxification mechanisms in response to oxidative stress
The 84 genes from cluster 5 (Figure 2) and the 248 genes from cluster 1 that were induced by Hfe-deficiency in the liver were particularly enriched for functional categories associated with response to oxidative stress and iron ion binding (Table 2). Excess iron is known to generate reactive oxygen species that promote cell damage and fibrosis, and may be responsible for the induction of the aldehyde oxidase and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase genes observed in these mice. This appears to be counterbalanced by upregulation of genes involved in the glutathione metabolism pathway, in particular genes encoding enzymes that are responsible for glutathione synthesis (Gclc, Gclm, and Gss) and glutathione S-transferases, which catalyze the conjugation of reduced glutathione to electrophilic centers on a wide variety of substrates; the latter activity is useful in the detoxification of endogenous compounds such as peroxidized lipids. Excess iron also appears to be counterbalanced, particularly in Hfe-/- D2 mice, by upregulation of genes encoding uridine 5'-diphospho (UDP)-glucuronosyltransferases, which catalyze the glucuronidation reaction (the addition of sugars to lipids), which is an important step in the body's elimination of endogenous toxins. In addition, there was an enrichment, most notably in Hfe-/- D2 mice, of genes with mono-oxygenase activity, particularly genes encoding several cytochrome P450 isoforms and flavin-containing mono-oxygenase-5, which are considered to be xenobiotic detoxication catalysts and believed to protect mammals from lipophilic nucleophilic chemicals 12. The iron ion binding category, also enriched in the liver of both strains, includes the genes for ferroportin, ferritin light chain, and heme oxygenase, which catalyzes the degradation of heme into carbon monoxide and biliverdin. Of note, although expression of vanin1 was downregulated in mice lacking Hfe in both strains (cluster 4), this regulation is worth noting because mice deficient in vanin-1 exhibit a glutathione-mediated tissue resistance to oxidative stress 13.
<p>Table 2</p>
Main genes regulated by Hfe deficiency in liver and pertaining to enriched functional categories related to response to oxidative stress
Gene
Protein
S-score
D2 KO versus WT
B6 KO versus WT
D2 WT versus B6 WT
Glutathione metabolism pathway
Gclc
Glutamate-cysteine ligase, catalytic subunit
3.68
4.74
3.24
Gclm
Glutamate-cysteine ligase, modifier subunit
NS
4.11
NS
Gss
Glutathione synthetase
NS
2.15
NS
Gsta2
Glutathione S-transferase alpha2
8.83
9.10
NS
Gsta3
Glutathione S-transferase alpha3
2.44
2.24
NS
Gsta4
Glutathione S-transferase alpha4
4.64
5.99
4.35
Gstm1
Glutathione S-transferase mu1
NS
2.65
NS
Gstm3
Glutathione S-transferase mu3
3.04
4.04
2.56
Gstm6
Glutathione S-transferase mu6
3.36
2.26
NS
UDP glucuronosyltransferase activity
Ugt2b1
UDP glucuronosyltransferase 2B1
3.72
NS
NS
Ugt2b5
UDP glucuronosyltransferase 2B5
2.56
3.91
NS
Ugt2b34
UDP glucuronosyltransferase 2B34
NS
2.25
NS
Ugt2b35
UDP glucuronosyltransferase 2B35
2.56
3.91
NS
Ugt2b36
UDP glucuronosyltransferase 2B36
4.66
NS
-4.89
Mono-oxygenase activity
Cyp1a2
Cytochrome P450 1A2
3.14
2.60
3.49
Cyp2c29
Cytochrome P450 2C29
NS
2.79
4.84
Cup2c44
Cytochrome P450 2C44
3.07
NS
-5.89
Cyp2c55
Cytochrome P450 2C55
3.94
6.22
4.43
Cyp2c70
Cytochrome P450 2C70
5.55
4.70
4.17
Cyp2j6
Cytochrome P450 2J6
2.70
NS
NS
Cyp2j9
Cytochrome P450 2J9
NSD
2.91
2.14
Cyp2u1
Cytochrome P450 2U1
3.50
NS
NS
Fmo5
Flavin mono-oxygenase 5
2.12
NS
NS
Iron ion binding
Ftl1
Ferritin light chain 1
1.70
2.24
NS
Slc40a1
Ferroportin
3.18
4.89
3.94
Hmox1
Heme oxygenase 1
5.27
2.40
-3.66
Blvrb
Biliverdin reductase (for information)
2.50
3.06
NS
Vnn1
Vanin 1 (for information)
-4.27
-2.73
2.69
S-scores were obtained as described in Materials and methods and are proportional to fold changes. Positive S-scores indicate that the genes are more highly expressed in knockout (KO) than in wild-type (WT) mice, or in WT D2 than in WT B6 mice. NS, not significant; UDP, Uridine 5'-diphospho.
Fatty acid β-oxidation and cholesterol catabolism
The 139 genes from cluster 4 (Figure 2) and the 315 genes from cluster 6, which were repressed in liver by Hfe deficiency, were particularly enriched for functional categories associated with lipid metabolism (Table 3). In particular, genes encoding the rate-limiting enzyme for β-oxidation of long-chain fatty acids (Cpt) and the transcripts for enzymes involved in the three steps of β-oxidation were all significantly downregulated. The expression of the Cyp4a10 and Cyp4a14 genes was also repressed in Hfe-/- mice of both strains, which could be a physiologic response in the context of the reduced fatty acid β-oxidation. With a decrease in acetyl-coenzyme A generated by decreased β-oxidation, a decrease in citrate (the first intermediate generated in the tricarboxylic acid [TCA] cycle) would be expected in the mitochondria of Hfe-/- mice, with a subsequent slowing of the TCA cycle. Indeed, a downregulation of mitochondrial aconitase and isocitrate dehydrogenase suggests that the flux through the TCA cycle is maintained at a low level in order to adapt to the downregulated β-oxidation in these Hfe-deficient mice. Interestingly, the cholesterol metabolism category is also enriched among genes downregulated by Hfe deficiency in D2 mice, and this mainly affects genes that are involved in the catabolism of cholesterol into bile acids (Cyp7a1 and Cyp39a1).
<p>Table 3</p>
Main genes regulated by Hfe deficiency in liver and pertaining to the enriched functional categories fatty acid β-oxidation and cholesterol metabolism
Gene
Protein
S-score
D2 KO versus WT
B6 KO versus WT
D2 WT versus B6 WT
Fatty acid β-oxidation
Cpt1a
Carnitine palmitoyl transferase 1a
-2.95
-1.94
NS
Cpt2
Carnitine palmitoyl transferase 2
-2.59
NS
NS
Acadm
Acyl-CoA dehydrogenase, medium chain
-2.97
-1.80
NS
Acadl
Acyl-CoA dehydrogenase, long chain
-3.00
NS
NS
Acadvl
Acyl-CaA dehydrogenase, very long chain
-2.40
NS
NS
Ehhadh
Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase
-2.30
NS
NS
Hadha
Tripartite protein, alpha subunit
-2.08
-1.58
NS
Hadhb
Tripartite protein, beta subunit
-2.96
-1.90
NS
Hadh2
Hydroxyacyl-CoA dehydrogenase type II
NS
-4.05
-4.61
Acox1
Acyl-CoA oxydase 1, palmitoyl (peroxisomal)
-2.10
-1.63
-4.27
Cyp4a10
Cytochrome P450 4A10
-6.76
-2.46
NS
Cpy4a14
Cytochrome P450 4A14
-9.21
-3.09
NS
TCA cycle
Aco2
Aconitase 2, mitochondrial
-2.14
-1.82
NS
Idh2
Isocitrate dehydrogenase 2, mitochondrial
-2.09
NS
NS
Cholesterol catabolism
Cyp7a1
Cholesterol 7α-hydroxylase
-3.15
NS
NS
Cyp39a1
Oxysterol 7α-hydroxylase
-2.60
NS
NS
S-scores were obtained as described in Material and methods and are proportional to fold changes. Negative S-scores indicate that the genes are more highly expressed in wild-type (WT) than in knockout (KO) mice, or in WT B6 than in WT D2 mice. Variations in the expression of genes involved in the tricarboxylic acid (TCA) cycle are provided for information. CoA, coenzyme A; NS, not significant.
Circadian rhythm
Hfe-/- mice of both strains exhibit reduced expression of genes encoding Period (Per2 and Per3), D site albumin promoter binding protein (Dpb), and the nuclear receptor subfamily 1 (Nr1d1). Although surprising, this can be related to the recent observation that the circadian clock and heme biosynthesis are reciprocally regulated in mammals 14 and may be correlated with the upregulation of δ-aminolevulinate synthase (Alas2) in the liver of these mice.
Other variations of potential interest
Hfe-/- D2 mice exhibit increased expression of the gene encoding 3β-hydroxysteroid dehydrogenase (Hsd3b5), which is thought to be involved in the inactivation of steroid hormones, for example dihydrotestosterone 15. They also exhibit induction of the dopachrome tautomerase gene (Dpt), which affects pigmentation 16. It would be interesting to investigate whether these variations in gene expression are related to the deficit in testosterone and melanodermia observed in patients with severe hemochromatosis.
Enriched functional categories in the duodenum of Hfe-deficient mice
As shown in Table 4, there was no clearly enriched functional categories among the 177 genes (cluster 1) that were induced in #Hfe-/- D2 mice. Conversely, there was significant enrichment of genes involved in the immune defense among the 131 genes that were repressed in the same mice (cluster 3), particularly for genes involved in apoptosis (Casp4, Cdca7l, Ifit1 and Ifit2, Oasl2, and Scotin), innate antiviral or antimicrobial activity (Defcr4, Ddx58, and Lzp-s), and B and T cell mediated immune response (Mpa2l, Psme1, Trfrsf13b, and Tnfrsf17). This suggests a link between the control of iron metabolism and the immune system that should be explored.
<p>Table 4</p>
Functional categories over-represented in clusters of genes similarly regulated by Hfe disruption in duodenum
Category
Term
n
EASE score
Cluster 1 (209 Affy IDs [177 genes])
No functional category overrepresented
Cluster 3 (141 Affy IDs [131 genes])
GOTERM_BP
Defense response
21
1.6 × 10-7
GOTERM_BP
Induction of apoptosis
6
1.7 × 10-3
Affymetrix probesets in the different k-means clusters shown in Additional data file 2 were compared with Affymetrix MG-430 2.0 probe sets for over-representation of gene categories, using the DAVID (Database for Annotation, Visualization, and Integrated Discovery) functional annotation tool. The Category column shows the original database/resource from which the terms originate. The Term column indicates enriched terms associated with the gene list. The n column indicates the number of genes involved in the term. The expression analysis systematic explorer (EASE) score is a modified Fisher exact P value [51]. BP, biological process; GO, Gene Ontology.
Although mRNAs for duodenal iron transporters were not found to be significantly upregulated, expression levels of other metal ion transporters were increased in duodenum of Hfe-/- D2 mice, most notably the zinc transporters Slc39a4 and Slc39a14. The copper transporter Slc31a1 and, more anecdotally, the sodium-dependent vitamin C transporter Slc23a2 (previously observed to be increased in response to dietary iron deprivation 17) were also induced in D2 mice lacking Hfe. In addition, Hfe-/- D2 mice had increased expression of the mucin (Muc3) and spermin synthase (Sms) genes, which encode proteins that both may modulate iron uptake 1819.
Changes in expression of genes encoding proteins of iron metabolism
The Affymetrix GeneChip® Mouse Genome 430 2.0 arrays contain probe sets for the transcripts of all the genes directly or indirectly involved in iron metabolism 20. Significant alterations in their expression in liver or duodenum of Hfe-/- mice and gene expression differences between wild-type strains are summarized in Table 5. Specifically in the D2 strain, Hfe disruption induces expression of the Cybrd1 gene in duodenum; this gene encodes Dcytb, which converts dietary ferric iron into its ferrous form for transport. In the liver, Hfe-deficient mice of both strains exhibit upregulated expression of the gene encoding the ferritin light chain, which is responsible for cytosolic iron storage, and of the ferroportin gene, which is consistent with the notion that this protein plays a protective role by facilitating the release of excess iron 21. Somewhat unexpectedly, we observed significant downregulation of the sideroflexin gene (Sfxn2) and upregulation of the mitoferrin gene (Slc25a37) and the Bcrp gene (Abcg2), which encode three molecules that are involved in the mitochondrial import/traffic of iron and heme export. Also worthy of mention are several strain-specific modifications of the messengers of some regulators of iron metabolism in Hfe-deficient mice. First, we confirmed that wild-type B6 and D2 diverge in terms of the amounts of the two hepcidin messengers, namely Hamp1 and Hamp2 22, and we observed a downregulation of the two genes in Hfe-/- D2 mice. Conversely, we observed significant upregulation of the gene encoding the upstream transcription factor Usf2, which was recently found to be involved in the control of hepcidin expression 23, in the B6 strain. Finally, and worthy of note within the context of modifiers of iron loading severity, wild-type D2 mice have significantly lower expression of the Smad4 transcription factor, also involved in the control of hepcidin expression, than wild-type B6 mice.
<p>Table 5</p>
Changes in expression of genes involved in iron metabolism
Gene
Protein
Major biochemical activity
Role
Organ
S-score
D2 KO versus D2 WT
B6 KO versus B6 WT
D2 WT versus B6 WT
Iron storage
Ftl1
Ferritin L chain
Fe mineralization
Cytosolic storage
Liver
+1.70
+2.24
NS
Iron transport
Slc40a1
Ferroportin
Membrane transporter
Cellular export
Liver
+3.18
+4.89
+3.94
Abcg2
Bcrp
Membrane transporter
Possible mitochondrial heme export
Liver
+2.97
NS
NS
Sfxn2
Sideroflexin2
Membrane transporter
Mitochondrial traffic
Liver
-2.38
-2.16
NS
Slc25a37
Mitoferrin
Membrane transporter
Mitochondrial traffic
Liver
NS
+2.28
NS
Lcn2
Lipocalin2
Siderophore iron binding
Traffic of siderophore-bound iron
Liver
-2.91
NS
NS
Receptors
Tfrc
Transferrin receptor1
Transferrin binding
Transferrin iron uptake
Duodenum
-2.07
NS
NS
Lrp1
LRP/CD91
Hemoplexin receptor
Hemoplexin uptake
Liver
NS
-2.03
NS
Regulators
Ireb2
IRP2
RNA binding
Control of cellular iron
Duodenum
+2.26
NS
-2.64
Hamp1
Hepcidin 1
Ferroportin binding
Control of systemic iron
Liver
-6.27
NS
-3.57
Hamp2
Hepcidin 2
?
?
Liver
-3.40
NS
+3.36
Hfe
HFE
TfR1 binding
?
Liver
-7.96
-8.96
-3.16
Duodenum
-5.70
-7.32
NS
Hfe2
HJV
Neogenin binding
Control of hepcidin expression
Liver
NS
-2.01
NS
Fxn
Frataxin
Iron binding
Chaperon for Fe-S synthesis
Liver
NS
NS
-3.09
Smad4
Smad4
Transcription factor
Control of hepcidin expression
Liver
NS
NS
-3.59
Duodenum
NS
NS
-5.05
Usf2
Usf2
Transcription factor
Control of hepcidin expression
Liver
NS
+2.08
NS
Oxidoreductases
Cybrd1
Dcytb
Fe(III) reduction
Facilitates duodenal transport by DMT1
Duodenum
+2.97
NS
NS
Iron metabolism genes are cited in this table where significant expression variations in Hfe-/- mice (knockout [KO]) or expression differences between wild-type (WT) strains were detected. S-scores were obtained as described in Material and Methods and are proportional to fold changes. Positive S-scores indicate that the genes are more highly expressed in KO than in WT mice, or in WT D2 than in WT B6 mice. NS, not significant.
Confirmation of differential gene expression by quantitative PCR
Quantitative real-time PCR was performed on 21 genes expressed in the liver and four genes expressed in the duodenum. The selection of these genes was based on different criteria. The first group included genes of an enriched functional category identified using the DAVID annotation tool (Aox1, Ftl1, Fpn1, Hmox1, Vnn1, Por, Cpt1a, Aco2, Cyp7a1, and Hsd3b5). The second group of genes encode proteins of iron or heme metabolism, and their expression was either induced or repressed in Hfe-/- mice (Hfe2, Hamp1, Hamp2, Usf2, Lcn2, Sfxn2, Alas2, Slc25a37, and Abcg2). The third group encode proteins that might modulate iron absorption in the duodenum (Dcytb, Slc39a4, and Muc3). The fourth group includes genes that, although their involvement in iron metabolism regulation cannot be assumed, were highly regulated in liver (Lcn13 and Fmo3) or duodenum (Clca4) of Hfe-/- mice. A further 20 mice that were not analyzed using Affymetrix arrays (five per genotype/strain combination) were included in the analysis to test the validity of the results.
Concordant results were obtained for 24 out of 25 genes selected. Downregulation of the hemojuvelin gene (Hfe2) in Hfe-/- B6 mice was not confirmed. Downregulation of Lcn2, Hamp1, and Hamp2 in Hfe-/- D2 mice was confirmed in the samples used for Affymetrix array hybridizations but not in the additional samples used for validation, although a trend toward downregulation was observed in the validation set for Hamp1 and Hamp2. The upregulation of Usf2 and Slc25a37, originally found only in the liver of Hfe-/- B6 mice, was observed by quantitative PCR in both strains. Interestingly, Lcn13 and Fmo3 - which had highly significant S-scores of 11.06 and -6.66, respectively, in the liver of Hfe-/- D2 mice - were confirmed to be regulated by Hfe deficiency in both datasets. Because neither of these two genes is regulated by dietary iron content in wild-type mice (data not shown), these variations appear specific to Hfe disruption and warrant further investigation.
Correlation of expression profiling with studies on Hfe modifiers
Differences in liver or duodenal expression of specific genes between B6 and D2 wild-type mice could contribute to the divergent phenotypes induced by Hfe disruption in the two strains. We therefore established a list of the 1,538 transcripts with differential expression between wild-type D2 and B6 mice (Additional data file 3). In order to relate genomic results to severity of hemochromatosis, we first identified 210 genes exhibiting differences in basal expression between strains or with expression regulation in response to Hfe disruption, which reside within the five Hfe-modifier regions that we previously mapped on chromosomes 3, 7, 8, 11, and 12 10. To identify those that could be potential candidates for disease severity, we used the WebQTL interface to map the loci that regulate the expression of these genes. The information necessary to map these regulatory loci was available for a subset of 139 of these 210 genes.
We found that two genes on chromosome 3, four on chromosome 7, six on chromosome 8, 17 on chromosome 11, and one on chromosome 12 exhibited highly significant evidence for cis regulation (for regulation by a polymorphic variant between B6 and D2 mice located in the region of the gene itself; Table 6). None of them, except for Hamp, has yet been implicated in iron metabolism.
<p>Table 6</p>
Genes differentially expressed between wild-type strains or regulated by Hfe deficiency, located within the chromosomal regions containing Hfe-modifiers, and with evidence for cis regulation
Gene name
Chromosome
Position (Mb)
Type
Position of linkage peak (Mb) for cis regulator
Max LRS for cis regulator
Clca2
3
144.73
D
144.70 to 144.94
46.7
Lphn2
3
148.87
S
149.36 to 151.27
15.5
Uble1a
7
15.49
S
15.19 to 15.53
51.53
Ckap1
7
29.93
S
29.49 to 30.12
53.8
Hamp1/Hamp2
7
30.63
L, S
30.43 to 34.11
18.9
Fxyd5
7
30.74
D
34.41 to 34.62
15.2
Gpsn2
8
86.46
S
83.77 to 85.83
15.2
Ddx39
8
86.61
L
86.07 to 88.74
12.7
2410018C20Rik
8
87.14
S
86.07 to 88.74
20.6
Ier2
8
87.55
L
86.07 to 88.74
15.9
Gadd45gip1
8
87.72
S
86.07 to 88.74
116.4
Prdx2
8
87.86
S
83.77 to 85.83
36.1
Pttg1
11
43.26
S
42.87 to 44.25
68.9
5730409G07Rik
11
45.79
S
42.21 to 46.06
16.4
2900006B13Rik
11
51.43
L, S
50.95 to 53.90
38.9
Tnip1
11
54.75
S
50.95 to 53.90
60.6
Sparc
11
55.24
S
55.24 to 55.92
26.1
Guk1
11
59.00
S
58.93 to 59.04
50.8
Sat2
11
69.44
S
69.42 to 70.27
133.2
Mpdu1
11
69.47
S
72.49 to 72.98
43.3
Asgr2
11
69.91
S
69.42 to 70.27
69.9
Rabep1
11
70.66
L
73.93 to 75.08
18.3
Txnl5
11
72.02
S
67.96 to 68.74
11.1
Pafah1b1
11
74.49
L, S
75.29 to 76.41
14.5
Crk
11
75.50
L
76.76 to 76.83
10.5
Ccl9
11
83.39
L, S
88.48 to 89.36
36.9
Bcas3
11
85.17
S
89.57 to 89.92
12.2
Dhx40
11
86.59
S
83.52 to 88.25
36.8
Scpep1
11
88.74
L, S
83.52 to 88.25
39.2
9030617O03Rik
12
101.18
S
100.97 to 102.71
51.5
The Chromosome and Position columns indicate, respectively, the chromosome number and position (in megabases [Mb]) within one of the five Hfe-modifier intervals of the gene with expression variation. In the Type column, S indicates that expression differed between wild-type strains, D that expression was modulated by Hfe deficiency in duodenum, and L that expression was modulated by Hfe deficiency in liver. Max LRS indicates the maximum likelihood ratio statistic in favor of the cis regulator. Position of linkage peak for cis regulator and maximum LRS were retrieved from the WebQTL interface.
Discussion
Recent advances in the field of iron metabolism have elucidated basic processes of iron absorption and distribution in mammals 24. However, many aspects of iron metabolism remain obscure, in particular the mechanisms by which HFE regulates iron absorption. In this study we investigated the expression patterns of 34,000 well characterized mouse genes in liver and duodenum of wild-type and Hfe-/- mice of two inbred strains with different susceptibilities to iron accumulation.
Variations in duodenal gene expression in Hfe-deficient mice, as compared with wild-type mice, are consistent with our previously reported hypothesis 9 that hyperabsorption of iron in these mice reflects an inappropriate iron deficiency signal that is sensed by duodenal enterocytes. Indeed, expression of the Cybrd1 gene (encoding Dcytb, which converts dietary ferric iron to its ferrous form for transport by the divalent metal iron transporter Dmt1 to the duodenum) and the expression levels of several metal ion transporters, most notably the zinc transporters Zip4 (Slc39a4) and Zip14 (Slc39a14), were increased in the duodenum of Hfe-/- D2 mice. Although Hfe knockout was previously shown to increase Cybrd1 expression 11 and mucosal reductase activity near the villus tips 25, the increase in expression of the two zinc transporters has not yet been observed and is interesting within the context of recent reports indicating that Zip4 is a minor intestinal iron importer 26 and that Zip14 mediates non-transferrin-bound iron uptake into cells 27. Of note, Hfe-/- D2 mice also have increased duodenal expression of mucin and spermine synthase. Increased binding of Dmt1 to mucin in vesicles near the intestinal surface was observed in iron-deficient animals, which is believed to facilitate iron internalization 19, and recent studies have suggested that polyamines such as spermine modulate iron uptake 18.
Although it cannot be excluded that a slight upregulation of the Cybrd1, Slc39a4, and Muc3 messengers also exists in Hfe-/- B6 mice but does not reach a level detectable by microarray or RT-PCR analysis, the differential expression of these genes between Hfe-/- D2 and B6 mice does not appear to be related to the individual capacity of the two strains to respond to an iron-deficiency signal. Indeed, as shown in Figure 3, wild-type mice of both B6 and D2 genetic backgrounds fed an iron-deficient diet have induced duodenal expression of Cybrd1, Slc39a4, and Muc