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
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.
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,
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
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
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.
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.
Glucose-6-phosphate dehydrogenase: a historical perspective. Blood 111:16, 2008.
Iron storage disease: facts, fiction, and progress. Blood Cells Mol. Dis. 39:140,
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.
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,
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,
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.