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Scientific Report 2008


Molecular and Experimental Medicine




Studies in Iron Homeostasis


E. Beutler, K. Crain, T. Gelbart, P. Lee, H. Peng, J. Truksa, J. Waalen

Iron is essential to all forms of life, but an excess of iron can be injurious to organisms, probably by facilitating the generation of free radicals. Consequently, all organisms have developed mechanisms for regulating the amount of iron that they obtain from the environment and the exchange of iron between storage depots and functional compartments (Fig. 1).


Fig. 1. Iron absorption under various conditions. A, Normal iron absorption. B, Iron deficiency. C, Iron overload. D, Anemia of inflammatory disease. E, Hemochromatosis.

In higher organisms, a 25 amino acid antimicrobial peptide, hepcidin, has emerged as the central regulator of iron homeostasis. The cognate receptor for hepcidin is ferroportin, an iron-transport protein that is required for intestinal cells to release their iron to the blood and for macrophages to release their iron stores. When hepcidin levels are high, the serum iron level decreases, and intestinal iron absorption is diminished. Thus, overexpression of hepcidin results in iron deficiency. Conversely, when hepcidin levels are low, iron absorption from the gastrointestinal tract is facilitated, as is the release of iron from macrophages. This release leads to iron excess. Hepcidin is translated as a prohormone, prohepcidin, which undergoes cleavage to the active 25 amino acid peptide. Regulation of hepcidin appears to be largely or entirely transcriptional.

Fig. 1., continued.

We have focused on the regulation of hepcidin transcription. Transcription is upregulated by the inflammatory cytokines IL-1 and IL-6, by bone morphogenetic proteins (BMPs) 2, 4, and 9, by overexpression of another iron-regulating protein, hemojuvelin, and in vivo, by iron. The regulation of hepcidin by iron is particularly important physiologically, but it is difficult to study because it does not occur in vitro. In last year's report, we described our development of an in vivo method for measuring hepcidin transcription that is based on the luminescence of a reporter in intact mice.

We are seeking to dissect the various parts of the promoter involved in the regulation of hepcidin transcription and the complex of transcription factors that are involved in this process. The transcription factors SMAD4, C/EBPα, and HNF4α have been implicated in the regulation of hepcidin. Animals that have a liver-specific deletion of the genes for SMAD4 and C/EBPα have significantly reduced hepcidin expression and hepatic iron accumulation. Animals that have a liver-specific deletion of the gene for HNF4α have significantly increased hepcidin expression. Chromosome immunoprecipitation has indicated that the transcription factor STAT3 binds the hepcidin promoter in response to IL-6. Although the STAT3-binding site has been mapped to the proximal promoter region, it was not known where SMAD4 and C/EBPα bind on the hepcidin promoter.

We previously reported that the BMP- and iron-responsive region of the hepcidin promoter mapped to a region 1.6—1.8 kb upstream of the start of translation. If hemojuvelin works through the BMP—BMP receptor pathway by activating SMAD1 and SMAD4, then we would expect that the hemojuvelin- and SMAD1/4-responsive region of the hepcidin promoter to localize to the 1.6- to 1.8-kb region. Such localization was indeed the case. Using the same approach that we used to map the BMP- and iron-responsive region of the hepcidin promoter, we found that the hemojuvelin- and SMAD-responsive region localized to the 1.6- to 1.8-kb region of the distal hepcidin promoter. Nevertheless, this region had no identifiable SMAD1/4-responsive motif.

Deletion analyses narrowed the BMP-responsive element primarily to a HNF4α/COUP transcription factor binding site, although neighboring motifs seemed to also contribute to the responsiveness. Deletion of the HNF4α/COUP site or the STAT site resulted in a significantly lower basal level expression of the hepcidin promoter. Binding of recombinant transcription factors to the HNF4α/COUP site verified that HNF4α bound to a probe containing the HNF4α/COUP motif. C/EBPα bound to a probe encompassing the MEL transcription factor binding motifs. These data suggest that transcriptional activators and repressors regulating hepcidin expression might assemble into a complex with the liver-specific transcription factors HNF4α and C/EBPα at the core. This notion is consistent with the observation that hepcidin is expressed predominantly in hepatocytes.

Bruce Beutler and his group, Department of Genetics, discovered a mutant strain of mice that was designated Mask because of a phenotype in which body hair is missing but facial hair remains. This phenotype is due to a splicing mutation in the Tmprss6 gene, a gene that encodes a membrane serine protease. These mutant mice also have a severe microcytic anemia secondary to iron deficiency. Feeding the animals a diet very rich in iron or injecting them with iron corrected not only the anemia but also the loss of body hair. We were able to show that Mask mice maintained inappropriately high levels of hepatic hepcidin mRNA despite being severely iron deficient and anemic, both conditions that independently result normally in the suppression of hepcidin transcription. Moreover, in tissue culture cells subjected to stimuli that normally increase hepcidin, overexpression of Tmprss6 inhibited the increase. We have shown that Tmprss6 functions far downstream in the signaling sequence that stimulates hepcidin transcription and that it does so even when only a short fragment of the promoter sequence is present. Currently, studies are directed at better understanding the mechanism by which Tmprss6 downregulates hepcidin.

Because underexpression of hepcidin produces iron deficiency in mice, we posited that some patients with iron deficiency anemia resistant to treatment with iron might have mutations of the human ortholog. We have already detected 2 families with hereditary iron deficiency in which mutations of TMPRSS6 are present.

We have also continued to study clinical aspects of hereditary hemochromatosis. Although our study of the Kaiser Permanente Health Appraisal Clinic population clearly showed that the penetrance of hereditary hemochromatosis was extremely low, some people want to screen for this disorder. The approach that is generally used is to either perform DNA analysis to find homozygotes for the C282Y mutation of the HFE gene or to measure the serum transferrin saturation. But although the screening methods indicate that most or all of the patients are homozygous, only 1% to 2% of them will need treatment. We have therefore suggested and tested an alternative approach, viz, the measurement of serum ferritin levels. This approach seemed attractive because it is only patients with serum ferritin levels of more than 1000 ng/mL who have cirrhosis of the liver, the main clinical manifestation of hemochromatosis. We found that among 29,699 white patients participating in the study, only 59 had serum ferritin levels of more than 1000 ng/mL. Of these, 24 had homozygous mutant or compound heterozygous HFE genotypes. In all but 5 of the other patients, the causes of elevated ferritin were excessive alcohol intake, cancer, or liver disease. Thus, we were able to show that screening for serum ferritin levels not only detects all of the hemochromatosis patients at risk for cirrhosis but also detects patients with other medical problems that may require attention.

Publications

Aslan, D., Crain, K., Beutler, E. A new case of human atransferrinemia with a previously undescribed mutation in the transferrin gene. Acta Haematol. 118:244, 2007.

Barton, J.C., Acton, R.T., Lee, P.L., West, C. SLC40A1 Q248H allele frequencies and Q248H-associated risk of non-HFE iron overload in persons of sub-Saharan African descent. Blood Cells Mol. Dis. 39:206, 2007.

Beutler, E. Carrier screening for Gaucher disease: more harm than good [comment]? JAMA 298:1329, 2007.

Beutler, E. Consensus recommendations. Br. J. Haematol. 138:673, 2007.

Beutler, E., Waalen, J. Genetic screening for low-penetrance diseases. Annu. Rev. Genomics Hum. Genet., in press.

Beutler, E. Glucose-6-phosphate dehydrogenase: a historical perspective. Blood 111:16, 2008.

Beutler, E. Iron storage disease: facts, fiction, and progress. Blood Cells Mol. Dis. 39:140, 2007.

Beutler, E. Erythrocyte enzymopathies. In: Warrell, D.A., Cox, T.M., Firth, J.D. (Eds.). Oxford Textbook of Medicine. Oxford University Press, New York, in press.

Beutler, E. Hematopoietic cell transplantation in the future. In: Forman, S.J., Negrin, R.S., Blume, K. (Eds.). Thomas' Hematopoietic Cell Transplantation, 4th ed. Blackwell Science, Boston, in press.

Beutler, E., Duparc, S. Glucose-6-phosphate dehydrogenase deficiency and antimalarial drug development. Am. J. Trop. Med. Hyg. 77:779, 2007.

Flanagan, J.M., Truksa, J., Peng, H., Lee, P., Beutler, E. In vivo imaging of hepcidin promoter stimulation by iron and inflammation. Blood Cells Mol. Dis. 38:253, 2007.

Gallagher, P.G., Beutler, E. Membrane and enzyme abnormalities of the erythrocyte. In: Crowther, C., et al. (Eds.). Evidence-Based Hematology. Blackwell Science, Boston, 2008, p. 238.

Higgins, T., Beutler, E., Doumas, B.T. Hemoglobin, iron, and bilirubin. In: Burtis, C.A., Ashwood, E.R., Bruns, D.E. (Eds.). Tietz Fundamentals of Clinical Chemistry, 6th ed. Saunders, Philadelphia, 2008, p. 509.

Lee, P . Commentary to: "Post-translational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin,” by Erika Valore and Tomas Ganz. Blood Cells Mol. Dis. 40:139, 2008.

Lee, P., Beutler, E. Hepcidin and iron-overload disease. Annu. Rev. Pathol. Mech. Dis. in press.

Lee, P., Rice, L., McCarthy, J.J., Beutler, E. Severe iron overload with a novel aminolevulinate synthase mutation and hepatitis C infection: a case report. Blood Cells Mol. Dis., in press.

Lee, P., Waalen, J., Crain, K., Smargon, A., Beutler, E. Human chitotriosidase polymorphisms G354R and A442V associated with reduced enzyme activity. Blood Cells Mol. Dis. 39:353, 2007.

Lee, P.L., Gelbart, T., West, C., Barton, J.C. SLC40A1 c.1402Gg A results in aberrant splicing, ferroportin truncation after glycine 330, and an autosomal dominant hemochromatosis phenotype. Acta Haematol. 118:237, 2007.

Murugan, R.C., Lee, P.L., Kalavar, M., Barton, J.C. Early age-of-onset iron overload and homozygosity for the novel hemojuvelin mutation HJV R54X (exon 3; c.160Ag T) in an African American male of West Indies descent. Clin. Genet. 74:88, 2008.

Mañú Pereira, M., Gelbart, T., Ristoff, E., Crain, K.C., Bergua, J.M., López LaFuente, A., Kalko, S.G., Garcia-Mateos, E., Beutler, E., Vives-Corrons, J.-L. Chronic nonspherocytic haemolytic anemia associated with severe neurological disease due to γ -glutamylcysteine synthetase (GGCS) deficiency in a patient of Moroccan origin. Haematologica 92:e102, 2007.

Spear, G.S., Beutler, E., Hungs, M. Congenital Gaucher disease with nonimmune hydrops/erythroblastosis, infantile arterial calcification, and neonatal hepatitis/fibrosis: clinicopathologic report with enzymatic and genetic analysis. Fetal Pediatr. Pathol. 26:153, 2007.

Truksa, J., Lee, P., Beutler, E. The role of STAT, AP-1, E-box and TIEG motifs in the regulation of hepcidin by IL-6 and BMP-9: lessons from human HAMP and murine Hamp1 and Hamp2 gene promoters. Blood Cells Mol. Dis. 39:255, 2007.

Truksa, J., Lee, P., Peng, H., Flanagan, J., Beutler, E. The distal location of the iron responsive region of the hepcidin promoter. Blood 110:3436, 2007.

Truksa, J., Peng, H., Lee, P., Beutler, E. Different regulatory elements are required for response of hepcidin to IL-6 and bone morphogenetic proteins 4 and 9. Br. J. Haematol. 139:138, 2007.

Waalen, J., Felitti, V.J., Gelbart, T., Beutler, E. Screening for hemochromatosis by measuring serum ferritin levels: a more effective approach. Blood 111:3373, 2008.

Weinreb, N.J., Andersson, H.C., Banikazemi, M., Barranger, J., Beutler, E., Charrow, J., Grabowski, G.A., Hollak, C.E.M., Kaplan, P., Mankin, H., Mistry, P.K., Rosenbloom, B.E., vom Dahl, S., Zimran, A. Prevalence of type 1 Gaucher disease in the United States [commentary]. Arch. Intern. Med. 168:326, 2008.

 

Ernest Beutler, M.D.
Chairman and Professor
Head, Division of Hematology



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