Deficiency of heme-regulated eIF2{alpha} kinase decreases hepcidin expression and splenic iron in HFE-/- mice

Heme-regulated eIF2 ${alpha}$ kinase (HRI) is essential for regulatingglobin translation in iron deficiency and in β-thalassemia.We investigated the role of heme-regulated eIF2 ${alpha}$ kinase in hemoglobinand red blood cell production as well as in iron homeostasisin a mouse model of iron overload. We show that HRI deficiencydoes not significantly affect red cell parameters of hemochromatosis(HFE^–^/^–) mice. Importantly, heme-regulated eIF2 ${alpha}$ kinase deficiency exacerbates decreases in hepcidin expressionand splenic macrophage iron in HFE^–^/^– mice. Furthermore,the serum level of bone morphogenic protein 2, which positivelyregulates hepcidin, is reduced in heme-regulated eIF2 ${alpha}$ kinasedeficiency, but not in HFE deficiency.

Key words: heme-regulated eIF2 ${alpha}$ kinase, hepcidin, iron deficiency.

Introduction

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Introduction

Hepcidin, a key regulator of iron homeostasis, controls plasmairon levels by inhibiting the absorption of dietary iron fromthe intestine and the release of iron from macrophages.^1,2 Hepcidinexerts its function by binding to the iron exporter ferroportinand targeting ferroportin for degradation.³ Hepcidin expressionis homeostatically regulated by body iron status, inflammationand erythropoietic needs. It is enhanced by iron overload⁴ andinflammation,^5–8 and is inhibited by anemia and hypoxia.⁵In addition, inappropriately low hepcidin production seems tobe the common mechanism for hereditary hemochromatosis, an ironoverload disease caused by mutations in HFE, hemojuvelin (HFE2),transferrin receptor 2 (TFR2) and hepcidin (HAMP).⁹ Recently,bone morphogenic protein (BMP) signaling was found to positivelyregulate hepcidin expression through hemojuvelin, a co-receptorfor BMP.^10–13

Heme-regulated eIF2 ${alpha}$ kinase (HRI) balances heme and globin productionby controlling globin protein synthesis via phosphorylationof the ${alpha}$ -subunit of the eukaryotic translational initiation factor(eIF2 ${alpha}$ ).¹⁴ Since HRI plays a critical role during iron/heme deficiencyin the production of hemoglobin and formation of red blood cellsthat contain nearly 70% of the total body iron, it may alsobe important in systemic and cellular iron homeostasis. Furthermore,HRI is activated by non-heme stresses, especially oxidativestress,¹⁵ which occurs in iron overload. We have already shownthat HRI is activated in β-thalassemia to reduce the severityof β-thalassemia including splenic and hepatic iron overload.¹⁶Recently, we demonstrated that HRI protein is also expressedin macrophages and is important for iron recycling and hepcidinexpression.¹⁷ In this study, we investigated the role of HRIin hemoglobin synthesis and iron homeostasis under iron overloadconditions in HFE^–^/^– mice¹⁸ by generating compoundHRI^–^/^– HFE^–^/^– mice.

Design and Methods

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Design and Methods

Mouse breeding and genotyping
Mouse production and experimentation were approved by the committeeon Animal Care at Massachusetts Institute of Technology. HR^–^/^–(mixed genetic backgrounds of C57BL and S129) and HFE^–^/^–(S129) mice were generated as previously described,^18,19 andwere used to generate double mutant mice. Genotyping of theHRI gene was as previously described.¹⁹ Genotyping of HFE genewas carried out by PCR using primers 5’GTCACGAAGTTG GGAGTGGTGTCC-GAGTC3’and 5’GCACAGTGAGGGTTTCCTACAGAG-GTCAC3’ for the knockoutallele, and primers 5’AAGAGGCAGTGAG-AGGCTGG3’ and5’TGGT-GA-AAGTGAC-TCGCCAC3’ for the wild type allele.

Hematologic and pathological analyses and non-heme iron assays
Blood, liver and spleen samples were collected from 4 month-oldmice. The complete blood count analyses and reticulocyte countswere performed as previously described.¹⁹ Liver and spleen non-hemeiron were assayed and stained by Prussian blue as previouslydescribed.¹⁸

Quantitative RT-PCR and serum BMP2
RNA samples were isolated from livers using a total RNA isolationkit (Promega). Quantitative measurements of mRNA were carriedout as previously described.¹⁷ eIF2 ${alpha}$ was used as an internalcontrol. Serum BMP2 and TGF-β1 were measured by ELISA accordingto instructions from R&D systems.

Data analysis
Statistical analyses were performed by the two-tailed Student’st test. p<0.05 was considered statistically significant.

Results and Discussion

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Results and Discussion

Enhanced changes in splenic and serum iron concentrations in HRI^–/– HFE^–/– mice
We analyzed 207 mice from 23 litters produced by mating HRI^+/–HFE^+/^–mice. Mice of each of the nine predicted genotypes were bornaccording to expected Mendelian ratios. Deletion of the HRIgene did not have an obvious effect on complete blood countsor erythrocyte parameters of HFE^–/– mice. HRI^–/–HFE^–/– mice did not develop anemia or polycythemia(data not shown). Therefore, in contrast to the essential roleof HRI in iron deficiency,¹⁹ HRI does not play a significantrole in regulating hemoglobin synthesis and red blood cell productionunder the condition of iron overload induced by HFE deficiency.HFE^–^/^– mice had a significant increase in the liveriron (p<0.005, Figure 1A). However, HRI deficiency did notaffect the liver iron content of HFE^–/– mice (Figure 1A).In contrast, splenic iron was further reduced in HFE^–^/^–mice (p=0.01, Figure 1B) compared with HFE^+/+ HFE^–^/^–mice. Decreased iron in HRI^–^/^–, HFE^–^/^–and HRI^–^/^– HFE^–^/^– mice was also evidentwhen spleen tissue sections were stained for iron (Figure 1C).Notably, the iron was seen mainly in macrophages. Serum ironin HFE^–^/^– mice was further increased when the micewere also deficient for HRI (p<0.05, Figure 1D). HRI^–^/^–HFE^+/+ mice also displayed decreased splenic iron and increasedserum iron compared to wild type (Wt) mice (p<0.05, Figure 1B),similar to the original HRI^–^/^– mice reported recently.¹⁷Altogether, these results demonstrate that splenic iron wasreduced in both HFE deficiency and HRI deficiency. The moredrastic decrease of splenic iron in combined HRI and HFE deficienciesis likely to be the result of the additive effect of the singlegene deficiencies.

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Figure 1. Effects of HRI on the tissue and serum iron contents in HFE^–^/^– mice. (A) Hepatic iron contents, (B) Splenic iron contents, (C) Iron stain of spleen tissue sections (D) Serum iron contents. Results are presented as mean±SEM (n=7–9).

Decreased hepcidin expression in HRI^–/– HFE^–/– mice
As expected, hepatic hepcidin expression in HRI^+/+ HFE^–^/^–mice was decreased by 64.0% compared with Wt (p<0.005, Figure 2A).HRI^–^/^– HFE^+/+ mice also had a 41.0% decrease inhepcidin expression compared with Wt (p<0.005, Figure 2A).Importantly, hepcidin expression was further decreased by 89.0%in HRI^–^/^– HFE^–^/^– mice (p<0.005, Figure 2A).The severe reduction of hepcidin expression in HRI^–^/^–HFE^–^/^– mice appeared to be of a magnitude expectedfrom the effects of the single knockouts combined. The moremoderate reduction of hepcidin in HRI^–^/^– mice mayhelp explain the lack of iron overload in HRI^–^/^–liver in contrast to HFE^–^/^– liver. In human patients,the decrease in urinary hepcidin levels correlated with theseverity of hemochromatosis.^20,21 It is unclear why HRI^–^/^–HFE^–^/^–mice did not develop more severe iron overloadin the liver at four months. There was no significant differencein the distribution of iron in hepatocytes and macrophages betweenHFE^+/+ HFE^–^/^– and HRI^+/+ HFE^–^/^–mice.Most of the iron stain was in hepatocytes.

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Figure 2. Effects of HRI, HFE and iron on hepatic hepcidin expression and serum BMP2 levels. (A) Hepatic hepcidin mRNA expression. (B) Serum BMP2 levels. Hepcidin mRNA expression was analyzed by qRT-PCR and normalized with eIF2 ${alpha}$ . Hepcidin expression in Wt is defined as 1. Results are presented as mean±SEM (n=7–9).

Decreased serum BMP2 in HRI deficiency
Since BMP signaling is important for hepcidin expression, wemeasured serum BMP2 levels of HRI^–^/^– and HFE^–^/^–mice. As shown in Figure 2, the serum BMP2 level was significantlydecreased in HRI^–^/^– mice (p<0.05) and in HRI^–^/^–HFE^–^/^– mice (p<0.005), but not in HFE^–^/^–mice. Furthermore, there was no significant difference in theserum BMP2 levels between HRI^–^/^– HFE^+/+ and HRI^–^/^–HFE^–^/^– mice. Therefore, the decrease in hepcidinexpression in HFE^–^/^– mice was independent of serumBMP2 level and is mediated by yet unknown factors. Since noELISA kit is available for other BMP family members, we couldnot test whether other BMP proteins are affected in HRI deficiency.However, there was no significant difference in serum TGF-β1levels between Wt and HRI^–^/^– mice (data not shown),consistent with the previous observation that members of theTGF-β subfamily do not have as important a role as theBMPs in regulating hepcidin expression.^10,11 It is importantto note that the serum BMP2 levels reported here are withinthe linear range of the dose response curve for hepcidin inductionby BMP2.^12,13 The reduced serum BMP2 level in HRI deficiencyis consistent with the reduced hepcidin expression in HRI^–^/^–mice. The decreases in serum BMP2 and hepatic hepcidin wereobserved in the original HRI^–^/^– mice (C57BL andS129 mixed genetic backgrounds, data not shown),¹⁷ as well asin HRI^–^/^– HFE^+/+ mice (Figure 2) derived from thecross with HFE^–^/^– mice (S129 genetic background).These results strongly suggest that genetic background doesn’thave a significant role in the reduction of BMP2 and hepcidinin HRI^–^/^– mice.

The mechanism and physiological significance of this decreasedserum BMP2 in HRI^–^/^– mice must be further investigated.It was suggested that BMP signaling for hepatic hepcidin expressionmight be an autocrine event.¹¹ However, intravenous administrationof BMP2 to mice resulted in increased hepcidin production anddecreased serum iron levels.¹¹ We found that hepatic BMP2 andBMP4 mRNA expression was not significantly different betweenWt and HRI^–^/^– mice (data not shown).

We showed that HRI is also expressed in macrophages, althoughat a lower level than in erythroid precursors.¹⁷ HRI deficiencyimpaired the maturation of macrophages and HRI^–^/^–mice showed a weaker anti-inflammatory response with reducedcytokine production upon lipopolysaccharide challenge.¹⁷ Therefore,defective maturation of HRI^–^/^– macrophages mightalso contribute to the lower hepcidin expression in HRI^–^/^–mice through reduced expression of yet unknown macrophage-derivedfactors. It is possible that HRI might be expressed in BMP producingcell types such as osteoblasts and bone marrow stromal cells,and might modulate BMP production.

Interestingly, the serum level of growth differentiation factor(GDF),¹⁵ which is a member of TGF-β superfamily, was foundto be elevated in β-thalassemic patients.²² Expressionof GDF15 was increased during erythroid maturation and was secretedinto medium of erythroblasts culture. Furthermore, hepcidinexpression in primary hepatocyes was inhibited by high concentrationsof GDF15.²² Therefore, GDF15 may be one of the factors generatedby increased erythropoietic activity, which is known to reducehepcidin expression.^23,24 While HRI^–^/^– mice arenot anemic, they do exhibit mild erythrohyperplasia.¹⁹ It ispossible that some factor similar to GDF15 may be produced byHRI^–^/^– erythroblasts to inhibit hepcidin expression.We found that GDF15 expression was not significantly alteredin HRI^–^/^– E14.5 fetal liver cells by genechip expressionanalysis (Liu SJ and Chen J-J, unpublished observation).

To summarize, this study demonstrates that HRI deficiency canaffect the phenotype of HFE^–^/^– mice by further decreasinghepcidin expression.

Footnotes

Funding: this work was supported in part by grants from NIHDK016272 (JJC), DK066373 (NCA) and from Cooley’s AnemiaFoundation (SJL, RNVSS and APH).

Authorship and Disclosures

SJL and JJC designed the research, analyzed the data and wrotethe paper; SJL, RNS, APH and WTZ performed experiments. NCAprovided HFE^–/– mice and helped write the paper.The authors reported no potential conflicts of interest.

Received for publication August 30, 2007. Revision received November 13, 2007. Accepted for publication December 10, 2007.

References

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