U.S. patent application number 12/775032 was filed with the patent office on 2010-11-18 for competitive regulation of hepcidin mrna by soluble and cell-associated hemojuvelin.
Invention is credited to TOMAS GANZ, YIGAL P. GOLDBERG, LAN LIN.
Application Number | 20100292171 12/775032 |
Document ID | / |
Family ID | 37561680 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100292171 |
Kind Code |
A1 |
GANZ; TOMAS ; et
al. |
November 18, 2010 |
Competitive Regulation of Hepcidin mRNA by Soluble and
Cell-Associated Hemojuvelin
Abstract
Disclosed herein are hemojuvelin-specific siRNAs that vary
hemojuvelin mRNA concentration. Also disclosed herein,
GPI-hemojuvelin positively regulated hepcidin mRNA expression,
independently of the IL-6 pathway, whereas soluble hemojuvelin
(s-hemojuvelin) suppressed hepcidin mRNA expression in primary
human hepatocytes in a log-linear dosedependent manner. Disclosed
are compositions and methods for modulating diseases of iron
metabolism and hepcidin expression or hepcidin levels.
Inventors: |
GANZ; TOMAS; (LOS ANGELES,
CA) ; LIN; LAN; (BEIJING, CN) ; GOLDBERG;
YIGAL P.; (VANCOUVER, CA) |
Correspondence
Address: |
Suzannah K. Sundby (UC);SMITH, GAMBRELL & RUSSELL, LLP
1130 Connecticut Avenue, NW, Suite 1130
WASHINGTON
DC
20036
US
|
Family ID: |
37561680 |
Appl. No.: |
12/775032 |
Filed: |
May 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12397589 |
Mar 4, 2009 |
7745407 |
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12775032 |
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11427095 |
Jun 28, 2006 |
7534764 |
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12397589 |
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60694676 |
Jun 29, 2005 |
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Current U.S.
Class: |
514/21.2 ;
435/320.1; 435/325; 436/86; 530/350 |
Current CPC
Class: |
A61P 3/02 20180101; A61P
3/00 20180101; A61K 38/1709 20130101 |
Class at
Publication: |
514/21.2 ;
530/350; 436/86; 435/320.1; 435/325 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/435 20060101 C07K014/435; G01N 33/68 20060101
G01N033/68; C12N 15/63 20060101 C12N015/63; C12N 5/07 20100101
C12N005/07; A61P 3/00 20060101 A61P003/00; A61P 3/02 20060101
A61P003/02 |
Claims
1-14. (canceled)
15. The purified polypeptide of claim 21, consisting of SEQ ID
NO:1.
16. A pharmaceutical composition comprising the purified
polypeptide of claim 21 and a pharmaceutically acceptable
carrier.
17. A method for monitoring or diagnosing a disease of iron
metabolism in a subject comprising assaying the amount of a
membrane-associated GPI-linked hemojuvelin, the amount of a soluble
hemojuvelin protein, or both in the subject and determining whether
the amount is normal or abnormal.
18. The method of claim 17, wherein determining whether the amount
is normal or abnormal comprises determining a difference in the
amount obtained from the subject relative to the amount in an
individual not so afflicted or at such risk; wherein said
difference indicates the amount is abnormal.
19. An expression vector capable of expressing the soluble
mammalian hemojuvelin protein of claim 21.
20. A recombinant host comprising the expression vector of claim
19.
21. A purified soluble mammalian hemojuvelin protein.
22. The purified soluble mammalian hemojuvelin protein of claim 21,
which lacks its glycophosphatidylinositol anchor.
23. The purified soluble mammalian hemojuvelin protein of claim 21,
having a sequence which comprises at least 335 consecutive amino
acid residues of SEQ ID NO:1 and suppresses hepcidin mRNA
production.
24. The purified soluble mammalian hemojuvelin protein of claim 21,
which comprises SEQ ID NO:1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/427,095, filed 26 Jun. 2006, pending, and claims the
benefit of U.S. Provisional Patent Application Ser. No. 60/694,676,
filed 29 Jun. 2005, both of which are herein incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to diseases of iron
metabolism, hepcidin and hemojuvelin.
[0004] 2. Description of the Related Art
[0005] Various diseases of iron metabolism are known in the art and
include hemochromatosis, ferroportin mutation hemochromatosis,
transferrin receptor 2 mutation hemochromatosis, juvenile
hemochromatosis, neonatal hemochromatosis, hepcidin deficiency,
transfusional iron overload, thalassemia, thalassemia intermedia,
alpha thalassemia, sideroblastic anemia, porphyria, porphyria
cutanea tarda, African iron overload, hyperferritinemia,
ceruloplasmin deficiency, atransferrinemia, congenital
dyserythropoietic anemia, anemia of chronic disease, anemia,
hypochromic microcytic anemia, iron-deficiency anemia, conditions
with hepcidin excess, Friedreich ataxia, gracile syndrome,
Hallervorden-Spatz disease, Wilson's disease, pulmonary
hemosiderosis, hepatocellular carcinoma, cancer, hepatitis,
cirrhosis of liver, pica, chronic renal failure, insulin
resistance, diabetes, atherosclerosis, neurodegenerative disorders,
multiple sclerosis, Parkinson's Disease, Huntington's Disease,
Alzheimer's Disease.
[0006] Juvenile hemochromatosis (JH) is an early-onset inherited
disorder of iron overload. Two phenotypically very similar forms
have been recently characterized, one due to the homozygous
disruption of the HJV gene encoding a protein named hemojuvelin,
and the other due to the homozygous disruption of the HAMP gene
encoding hepcidin. See Papanikolaou, G, et al. (2004) Nat. Genet.
36:77-82. Hepcidin is a key iron-regulatory peptide hormone which
controls extracellular iron concentration by regulating the major
iron flows into plasma, and normally constrains intestinal iron
absorption. See Ganz, T. (2005) Best Pract. Res. Clin. Haematol.
18:171-182. Although a few mutated forms of juvenile
hemochromatosis gene (HFE2A) have been identified and may be
suitable for detecting the mutations, no suitable therapeutic has
been identified and shown to have a therapeutic effect. See
Samuels, et al. WO 2004092405.
[0007] Anemia of chronic disease (alternatively known as anemia of
inflammation) is another disease of iron metabolism due to the
excessive production of the iron-regulatory hormone hepcidin. See
Rivera, S., et al. (2005) Blood 105:1797-1802; Nemeth, E., et al.
(2004) J. Clin. Invest 113:1271-1276; Roy & Andrews (2005)
Curr. Opin. Hematol. 12:107-111; Fleming & Sly (2001) PNAS USA
98:8160-8162; and Weiss & Goodnough (2005) N. Engl. J. Med.
352:1011-1023. Anemia of chronic disease is a condition associated
with inflammatory diseases including rheumatological disorders,
inflammatory bowel diseases, chronic infections, chronic renal
diseases, as well as with malignant disorders including various
forms of cancer, lymphomas and multiple myeloma, and the like.
[0008] In anemia of chronic disease (anemia of inflammation) the
production of hepcidin is stimulated by various cytokines including
interleukin-6 Hepcidin acts by binding to ferroportin, the sole
known cellular iron exporter, and inducing its degradation. Excess
hepcidin causes the loss of ferroportin from the surfaces of
macrophages engaged in the recycling of iron from senescent red
cells. See Nemeth, E., et al. (2004) Science 306:2090-2093. As a
result, iron is trapped in macrophages and blood iron
concentrations decrease, restricting the flow of iron to the bone
marrow, and thus slowing the production of hemoglobin and
consequently decreasing the production of red blood cells. See
Rivera, S., et al. (2005). Synthetic hepcidin causes rapid
dose-dependent hypoferremia and is concentrated in
ferroportin-containing organs, Blood (2005). Unfortunately,
suitable and effective therapies for anemia of chronic disease are
limited. Specifically, the three main therapies are based on (1)
treating the underlying disease which is usually not possible,
otherwise this diagnosis would not exist, (2) erythropoietin
administration which is effective in only about 50% of all the
patients and is associated with undesirable side effects, and (3)
transfusions which are undesirable due to contamination, infection
and iron overload.
[0009] Thus, a need still exists for compositions and methods for
treating diseases of iron metabolism, such as juvenile
hemochromatosis and anemia of chronic disease.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods for
modulating hepcidin and disease of iron metabolism.
[0011] In some embodiments, the present invention provides a method
of treating, preventing, modulating, or attenuating a disease of
iron metabolism in a subject which comprises administering to the
subject a therapeutically effective amount of a least one soluble
hemojuvelin protein. In some embodiments, the soluble hemojuvelin
protein lacks a glycophosphatidylinositol anchor. In some
embodiments, the soluble hemojuvelin protein is a polypeptide
consisting of at least 6 consecutive amino acid residues of SEQ ID
NO:1. In some embodiments, the polypeptide consists of at least 20
consecutive amino acid residues of SEQ ID NO:1. In some
embodiments, the polypeptide consists of at least 50 consecutive
amino acid residues of SEQ ID NO:1. In some embodiments, the
polypeptide consists of SEQ ID NO:1. In some embodiments, the
disease of iron metabolism is anemia of chronic disease also
sometimes referred to as anemia of inflammation.
[0012] In some embodiments, the present invention provides a method
of modulating hepcidin production or hepcidin levels in a subject
which comprises administering to the subject a membrane-associated
GPI-linked hemojuvelin or a soluble hemojuvelin protein. In some
embodiments, the soluble hemojuvelin protein lacks a
glycophosphatidylinositol anchor. In some embodiments, the soluble
hemojuvelin protein is a polypeptide consisting of at least 6
consecutive amino acid residues of SEQ ID NO:1. In some
embodiments, the polypeptide consists of at least 20 consecutive
amino acid residues of SEQ ID NO:1. In some embodiments, the
polypeptide consists of at least 50 consecutive amino acid residues
of SEQ ID NO:1. In some embodiments, administration of the
membrane-associated GPI-linked hemojuvelin increases hepcidin
production. In some embodiments, administration of the soluble
hemojuvelin protein decreases hepcidin production.
[0013] In some embodiments, the present invention provides a method
of treating, preventing, modulating, or attenuating a disease of
iron deficiency in a subject which comprises modulating hepcidin
production or hepcidin levels in the subject which comprises
administering to the subject a membrane-associated GPI-linked
hemojuvelin or a soluble hemojuvelin protein. In some embodiments,
the soluble hemojuvelin protein lacks a glycophosphatidylinositol
anchor. In some embodiments, the soluble hemojuvelin protein is a
polypeptide consisting of at least 6 consecutive amino acid
residues of SEQ ID NO:1. In some embodiments, the polypeptide
consists of at least 20 consecutive amino acid residues of SEQ ID
NO:1. In some embodiments, the polypeptide consists of at least 50
consecutive amino acid residues of SEQ ID NO:1. In some
embodiments, administration of the membrane-associated GPI-linked
hemojuvelin increases hepcidin production. In some embodiments,
administration of the soluble hemojuvelin protein decreases
hepcidin production.
[0014] In some embodiments, the present invention provides a
purified polypeptide consisting of at least 6 consecutive amino
acid residues of SEQ ID NO:1. In some embodiments, the polypeptide
consists of at least 20 consecutive amino acid residues of SEQ ID
NO:1. In some embodiments, the polypeptide consists of at least 50
consecutive amino acid residues of SEQ ID NO:1.
[0015] In some embodiments, the present invention provides a
pharmaceutical composition comprising at least one purified
polypeptide of the present invention and a pharmaceutically
acceptable carrier. In some embodiments, the purified polypeptide
consists of at least 6 consecutive amino acid residues of SEQ ID
NO:1. In some embodiments, the polypeptide consists of at least 20
consecutive amino acid residues of SEQ ID NO:1. In some
embodiments, the polypeptide consists of at least 50 consecutive
amino acid residues of SEQ ID NO:1.
[0016] In some embodiments, the present invention provides a method
for monitoring or diagnosing a disease of iron metabolism in a
subject comprising assaying the amount of membrane-associated
GPI-linked hemojuvelin in biopsy material or by non-invasive means
in human subjects, the concentration of soluble hemojuvelin protein
in blood, serum or plasma, or both in the subject and determining
whether the amount is normal or abnormal.
DESCRIPTION OF THE DRAWINGS
[0017] This invention is further understood by reference to the
drawings wherein:
[0018] FIGS. 1A and 1B show that suppression of hemojuvelin mRNA
results in the suppression of hepcidin mRNA. Each individual point
represents an experiment in which Hep3B cells were treated with one
of the siRNA preparations overnight, and then incubated for
additional 24 hours before mRNA extraction. In each experiment,
hemojuvelin and hepcidin mRNA were quantified by real time qRT-PCR
and normalized to the housekeeping gene G3PD. Control cells were
treated only with transfection reagents and their hemojuvelin/G3PD
and hepcidin/G3PD ratios were set as baseline=1.
[0019] FIG. 1A is a plot showing a regression line (all HJV siRNAs
experiments, R=0.64, with 95% confidence limit) indicates that as
HJV/G3PD ratio decreases, there is a corresponding decrease in the
hepcidin mRNA/G3PD ratio. Closed symbols represent hemojuvelin
siRNAs experiments (.box-solid.: HJVsi1, : HJVsi2, : HJVsi3,
.tangle-solidup.: HJVsi4).
[0020] FIG. 1B is a plot showing no consistent effect on hepcidin
is seen with control siRNAs. Note the larger horizontal scale
compared to panel A. Open symbols represent siRNA control
experiments (.DELTA.: NCsi1, .smallcircle.: NCsi2, .quadrature.:
NCsi3).
[0021] FIG. 2 is a graph showing hemojuvelin suppression decreased
hepcidin expression but did not affect its inducibility by IL-6.
Hep3B cells were first treated with hemojuvelin siRNA HJVsi3 (+) or
diluent (-), followed by 20 ng/ml IL-6 (- -) to induce hepcidin for
24 hours (n=6 separate experiments). Hepcidin mRNAs was assayed by
qRT-PCR and normalized to G3PD. In each experiment, expression of
each target/G3PD ratio in control cells (not treated with IL-6 or
siRNA) was set as the baseline=1. Treatment with hemojuvelin siRNA
significantly decreased both hemojuvelin and hepcidin mRNA levels
in the presence and absence of IL-6, but did not affect mRNA
expression of CEBP.delta.. Significant differences as judged by the
paired Student t-test are indicated by their p values. Regardless
of hemojuvelin siRNA treatment, IL-6 produced a similar fold
induction of hepcidin and CEBP.delta. mRNA expression, indicating
that the IL-6 effect is not modulated by hemojuvelin
expression.
[0022] FIG. 3 shows gels evidencing that hemojuvelin protein exists
in both cell-associated and soluble forms. HEK293 and Hep3B cells
were transfected with pcDNA3.1(+) or pcDNA-HJV in 6-well tissue
culture plates and incubated overnight, followed by a 24-hour
incubation in serum free medium (2 ml/well). Whole cell lysates
were collected in 150 .mu.l NETT buffer per well and 30 .mu.l of
cleared total protein solution was analyzed. Conditioned cell
culture media (2 ml/sample) were filter concentrated (5 kD cutoff)
and concentrates equivalent to 800 .mu.l starting material were
analyzed. Western blots after reducing SDS-PAGE were probed with
anti-G3pep2-3 antibody. Arrows indicate cell-associated hemojuvelin
(apparent MW=46 kD) in both human liver protein extracts (#1 and
#2) and whole cell lysate of HEK293 cells transfected with
pcDNA-HJV (pHJV), but not in HEK293 cells treated with control
vector (pcDNA). Soluble hemojuvelin (apparent MW=44 kD) is
indicated by "*", and seen in conditioned cell culture media from
HEK293 and Hep3B cells transfected with pcDNA-HJV (pHJV), as well
as in conditioned media from Hep3B cells transfected with control
vector (pcDNA) but not in media from HEK293 cells treated with
control vector (pcDNA).
[0023] FIG. 4 shows gels evidencing that soluble hemojuvelin is
present in human serum and plasma. Serum and plasma samples were
separated on reducing SDS-PAGE. In Western blot analysis of all
serum samples, Ab112 detected a protein band (*) of 30 kD (three
different donors #1, #2, and #3, left panel), and anti-G3pep2-2
antibody detected a protein band (**) of 16 kD (middle panel).
Pretreatment of Ab112 with excess s-hemojuvelin abolished the 30 kD
Western blot signal. See FIG. 9. Blood plasma (1 .mu.l, P, right
panel) probed with Ab112 contained bands identical to those of
serum from the same donor (1 .mu.l, S, right panel) indicating that
the hemojuvelin cleavage was not caused by the clotting
reaction.
[0024] FIGS. 5A and 5B show that iron loading reduces soluble
hemojuvelin release into cell culture medium. Each panel is
representative of at least three independent experiments.
[0025] FIG. 5A shows gels of HEK293 and Hep3B cells transfected
with hemojuvelin vector (pcDNA-HJV) in 6-well tissue culture plates
and incubated overnight, followed by a 24-hour incubation in serum
free medium (2 ml/well) with FAC concentrations ranging from about
0 to about 100 .mu.M. Conditioned cell culture medium (2 ml/sample)
was filter-concentrated (5 kD cutoff) and analyzed on a reducing
SDS-PAGE/western blot probed with anti-G3pep2-3. In both Hep3B
(upper panel) and HEK293 cells (lower panel), the amount of soluble
hemojuvelin decreased progressively with increasing FAC
concentrations.
[0026] FIG. 5B shows gels of Holo- and Apo-transferrin added to
pcDNA-HJV transfected HEK293 cells at various ratios to reach a
constant total transferrin concentration of 30 .mu.M. Conditioned
cell culture medium (2 ml/sample) was extracted by cation exchange
and filter-concentrated (5 kD cutoff) before being analyzed on a
non-reducing SDS-PAGE/Western blot probed with Ab112. Lane 1 shows
conditioned cell culture medium from pcDNA 3.1(+) vector
transfected HEK293 cells as a negative control. The amount of
soluble hemojuvelin decreased progressively with increasing iron
saturation of transferrin.
[0027] FIG. 6 is a graph showing dose-dependent suppression of
hepcidin mRNA by s-hemojuvelin in primary human hepatocyte culture.
Primary human hepatocyte cultures (n=5) from 4 different donors
were treated for 24 hours with purified s-hemojuvelin from two
different preparations. Hepcidin mRNA was quantified by real time
qRT-PCR and normalized to the housekeeping gene .beta.-actin. For
each experiment, the hepcidin/.beta.-actin ratio of untreated cells
was considered as baseline=1. Individual experiments (open symbols,
dotted lines) and the regression line with 95% confidence intervals
are shown. Hepcidin mRNA expression showed a significant log-linear
anti-correlation (R=-0.88, P<0.001) with added s-hemojuvelin
concentration.
[0028] FIG. 7 is a gel evidencing that recombinant soluble
hemojuvelin from baculovirus/insect cells is similar to that from
mammalian cells, both in size and reactivity with antibodies.
Soluble hemojuvelin was generated by transfecting HEK293 cells with
pcDNA-HJV in 75 cm.sup.2 flasks, 25 ml of conditioned cell culture
medium (with 10% FBS) were harvested after a 40 hour incubation,
then partially purified using cation exchange chromatography,
desalted and concentrated by filtration (5 kD cutoff) to 250 .mu.l
soluble hemojuvelin standard (s-). Protein samples were loaded with
or without reducing agent DTT, Western blot was then probed with
Ab112. Purified s-hemojuvelin preparation (80 ng, rs-, Lane 1 and
3) showed similar reactive bands as the soluble hemojuvelin
standard (5 .mu.l, s-, Lane 2 and 4).
[0029] FIG. 8 shows gels indicating the purity of recombinant
hemojuvelin. 500 ng of purified s-hemojuvelin was analysed on
non-reducing and reducing SDS-PAGE, followed by silver staining.
Purified s-hemojuvelin on non-reducing SDS-PAGE showed greater than
about 95% purity (left panel). Reducing SDS-PAGE showed that
purified s-hemojuvelin was partially cleaved into two major
fragments of 16 kD and 29 kD. The 40 kD band in reducing SDS-PAGE
(right panel) was identified by amino acid sequencing as the
non-reduced form of s-hemojuvelin (identical migration as in
non-reducing SDS-PAGE, left panel).
[0030] FIG. 9 shows gels indicating the specificity of soluble
hemojuvelin detection in human serum. Ab112 antibody were diluted
in antibody dilution buffer to final concentration (1:5000), and
rotated at 4.degree. C. overnight with or without about a 50-fold
excess s-hemojuvelin (antigen/specific IgG ratio, 2.8 .mu.g
s-hemojuvelin/1 .mu.l anti-serum). Two .mu.l of human serum sample
was loaded along with s-hemojuvelin (rs-, 50 ng) and soluble
hemojuvelin standard (s-, 3 .mu.l). One single blot was cut and
probed in parallel with two antibody solutions. Arrows indicate
that excess s-hemojuvelin completely abolished the 30 kD protein
band in human serum. The hemojuvelin bands generated by engineered
HEK293 and insect cells were also nearly abolished by antigen
competition.
[0031] FIGS. 10A and 10B are graphs showing the combined effects of
IL-6 and s-hemojuvelin on hepcidin mRNA. Primary human hepatocyte
cultures from two different donors were treated for 24 hours with
purified s-hemojuvelin and 20 ng/ml IL-6. Hepcidin mRNA was
quantified by real time qRT-PCR and normalized to the housekeeping
gene .beta.-actin.
[0032] FIG. 10A indicates that regardless of IL-6 treatment (IL-6
untreated: open symbol, dot line; 20 ng/ml IL-6: closed symbol,
dashed line), addition of s-hemojuvelin to primary human hepatocyte
showed a similar suppression of hepcidin mRNA expression.
Hepcidin/.beta.-actin ratio of s-hemojuvelin untreated cells was
used as baseline=1 within each experiment (with or without
IL-6).
[0033] FIG. 10B shows that IL-6 (20 ng/ml) induced hepcidin
expression 6 and 16-fold in the hepatocyte cultures from 2
different donors (closed symbols, 0 ng/ml s-hemojuvelin). The
addition of s-hemojuvelin significantly lowered hepcidin
expression; high dose (about 1000 to about 3000 ng/ml) treatment
restored hepcidin expression to a normal or nearly normal level.
Cells not treated with s-hemojuvelin or IL-6 in each pair of
experiments were used as controls and their hepcidin/.beta.-actin
ratio of control cells was set as baseline=1.
[0034] FIGS. 11A and 11B show two graphs indicating the effect of
s-hemojuvelin on the global gene expression pattern in primary
human hepatocytes. Each graph compares the gene expression in
mock-treated cells with gene expression in s-hemojuvelin treated
cells (3 .mu.g/ml). Each dot represents a single spot on the array,
corresponding to a single transcript. The dots on the diagonal
represent genes whose expression is unchanged. The black arrow
points to the dot representing the hepcidin transcript. The lines
show 2-, 3-, 10- and 30-fold change.
[0035] In FIG. 11A, light grey color is used to identify
transcripts that are absent or marginally detectable in both
treated and untreated cells, dark grey designates transcripts that
are absent or marginal in either treated or mock-treated cells, and
black color indicates transcripts that are present in both treated
and mock-treated cells.
[0036] In FIG. 11B, the colors are changed to indicate transcripts
that are significantly increased (black), unchanged (grey) or
decreased (black) with s-hemojuvelin treatment.
[0037] FIG. 12 shows human hemojuvelin protein sequence. Human
hemojuvelin protein includes an N-terminal signal peptide (1-35),
an RGD motif (98-100), a partial von Willebrand factor type D
domain (167-253) and a glycosylphosphatidylinositol (GPI) anchoring
site at the position of 400 followed by a c-terminal transmembrane
motif required for GPI anchor formation.
[0038] FIG. 13 shows human hemojuvelin-Alkaline phosphatase fusion
protein. The c-terminus of human GPI-hemojuvelin (1-400) is fused
with human placenta alkaline-phosphatase. Predicted molecular
weight was indicated for each corresponding fragment. Molecular
weights determined by mass-spectrometry were shown in brackets.
[0039] FIG. 14 shows mouse soluble hemojuvelin lentiviral
expression transfer vector pRRL-Hjv-FUR
[0040] While the above-identified drawings set forth preferred
embodiments of the present invention, other embodiments of the
present invention are also contemplated, as noted in the
discussion. This disclosure presents illustrative embodiments of
the present invention by way of representation and not limitation.
Numerous other modifications and embodiments can be devised by
those skilled in the art which fall within the scope and sprit of
the principles of the present invention.
DETAILED DESCRIPTION
[0041] As provided herein, the expression and regulatory roles of
GPI-hemojuvelin and soluble forms of hemojuvelin (s-hemojuvelin)
were studied and it was found that, in extracellular iron
homeostasis, GPI-hemojuvelin and s-hemojuvelin act as opposing
regulators of hepcidin. Therefore, the present invention provides
compositions and methods for regulating or modulating hepcidin.
[0042] The hemojuvelin (HJV) gene produces multiple alternatively
spliced mRNA isoforms. The longest isoform of hemojuvelin mRNA
encodes a 426 amino acid protein, which contains a C-terminal
putative transmembrane domain characteristic of a
glycosylphosphatidylinositol-linked membrane anchor (GPI-anchor).
See Niederkofler, V. et al. (2004) J. Neurosci. 24:808-818; and
Monnier, P. P., et al. (2002) Nature 419:392-395.
[0043] The genetic linkage between juvenile hemochromatosis due to
HJV mutations and nearly absent hepcidin excretion in the affected
individuals left open the possibility that hemojuvelin, like its
congener RgmA, is a developmental factor. See Rajagopalan, S., et
al. (2004) Nat. Cell Biol. 6:756-762, which is herein incorporated
by reference. RgmA and hemojuvelin are associated with cell
membranes but both lack cytoplasmic tails and contain consensus
sequences indicating that they are GPI-linked proteins. See
Niederkofler, V., et al. (2004) J. Neurosci. 24:808-818, which is
herein incorporated by reference. RgmA is involved in neural
development through binding to a protein ligand neogenin, a
transmembrane receptor. See Rajagopalan, S., et al. (2004) Nat.
Cell Biol. 6:756-762, which is herein incorporated by reference.
Thus, GPI-linked cell-associated hemojuvelin (GPI-hemojuvelin) may
also interact with a similar transmembrane receptor, to stimulate
the production of hepcidin.
[0044] In principle, the deficiency of hepcidin in subjects having
HJV mutations could be due to a developmental defect in hepatocyte
function or due to the involvement of hemojuvelin in hepcidin
regulation. To establish whether hemojuvelin controlled hepcidin
synthesis, a human hepatoma cell line Hep3B was used as a model for
in vitro studies. Hep3B cells spontaneously produce hemojuvelin
mRNA at a similar concentration as in primary human hepatocytes
(data not shown).
[0045] As provided herein, Hep3B human hepatocarcinoma cells and
HEK293T/17 cells (HEK293) were maintained in Dulbecco's Modified
Eagle Medium (DMEM; Invitrogen, Carlsbad, Calif.) supplemented with
10% fetal bovine serum (FBS). Human hepatocytes (Liver Tissue
Procurement and Distribution System, Minneapolis, Minn.) were
cultured in human hepatocyte maintenance medium (Clonetics, San
Diego, Calif.) at 37.degree. C. in 5% humidified CO.sub.2.
Hepatocytes were treated with purified recombinant
s-hemojuvelin
TABLE-US-00001 (SEQ ID NO: 1)
QCKILRCNAEYVSSTLSLRGGGSSGALRGGGGGGRGGGVGSGGLCRALRS
YALCTRRTARTCRGDLAFHSAVHGIEDLMIQHNCSRQGPTAPPPPRGPAL
PGAGSGLPAPDPCDYEGRFSRLHGRPPGFLHCASFGDPHVRSFHHHFHTC
RVQGAWPLLDNDFLFVQATSSPMALGANATATRKLTIIFKNMQECIDQKV
YQAEVDNLPVAFEDGSINGGDRPGGSSLSIQTANPGNHVEIQAAYIGTTI
IIRQTAGQLSFSIKVAEDVAMAFSAEQDLQLCVGGCPPSQRLSRSERNRR
GAITIDTARRLCKEGLPVEDAYFHSCVFDVLISGDPNFTVAAQAALEDAR
AFLPDLEKLHLFPSDAGV
for 24 hours before harvesting. Human recombinant IL-6 (R&D
Systems, Minneapolis, Minn.) was used at 20 ng/ml
concentration.
[0046] Human serum and plasma were obtained from volunteer donors
under an IRB-approved protocol. Frozen normal human liver tissue
was obtained from the UCLA Human Tissue Resource Center (Los
Angeles, Calif.) under an IRB-approved protocol.
Construction of siRNA
[0047] Four siRNA duplexes targeting human hemojuvelin mRNA and one
siRNA negative control were constructed using Silencer.RTM. siRNA
Construction Kit (Ambion, Austin, Tex.) according to the
manufacturer's instructions. HJV siRNA targets, commercially
available from Dharmacon, Inc., Lafayette, Colo., included:
TABLE-US-00002 HJVsi1: 5'-AACTCTAAGCACTCTCACTCT-3' (SEQ ID NO: 2)
HJVsi2: 5'-AACCATTGATACTGCCAGACG-3' (SEQ ID NO: 3) HJVsi3:
5'-AAGTTTAGAGGTCATGAAGGT-3' (SEQ ID NO: 4) HJVsi4:
5'-AAAGCTACAAATTCTTCACAC-3' (SEQ ID NO: 5)
[0048] A negative control, NCsi1 target: 5'-GCGCGCTTTGTAGGATTCG-3'
(SEQ ID NO: 6) was used.
[0049] The following siRNA negative control duplex were also
used:
NCsi2: 5'-AATTCTCCGAACGTGTCACGT-3' (SEQ ID NO:7) (Qiagen, Valencia,
Calif.)
[0050] NCsi3: Silencer.RTM. Negative Control #2 siRNA (Ambion,
Austin, Tex.).
Transfections
[0051] In all siRNA treatment experiments, Hep3B cells were seeded
at 10% confluence 24 hours before siRNA transfection. Hep3B cells
were transfected with 20 nM siRNA duplexes using Oligofectamine
Transfection Reagent (Invitrogen, Carlsbad, Calif.) according to
manufacturer's protocol for 24 hours, followed by 24-hour treatment
with 20 ng/ml human recombinant IL-6 (R&D Systems, Minneapolis,
Minn.) or its solvent. In hemojuvelin expression experiments, 24
hours before transfection, Hep3B cells were seeded at 50%
confluence and HEK293 cells were seeded at 10% confluence.
pcDNA-HJV was generated by cloning full length human HJV cDNA into
vector pcDNA3.1(+) plasmid (Invitrogen, Carlsbad, Calif.). The
pcDNA-HJV or the control plasmid vector pcDNA3.1(+) were
transfected using Lipofectamine.TM. 2000 Transfection Reagent
(Invitrogen, Carlsbad, Calif.) according to manufacturer's protocol
for 24 hours prior to further treatment.
Recombinant Soluble Hemojuvelin Production and Purification
[0052] To express recombinant soluble human hemojuvelin
(s-hemojuvelin), a cDNA of human hemojuvelin truncated by 72
nucleotides at the 3' end to remove the transmembrane segment and
with an added stop codon, was cloned into BaculoDirect baculovirus
expression system (Invitrogen) according to manufacturer's
instructions. Culture medium from infected Hi5 insect cell culture
was purified by cation exchange chromatography (CM Prep, Biorad,
Richmond, Calif.), followed by high performance liquid
chromatography on a C4 reverse phase column (Vydac, 214TP54) eluted
with an acetonitrile gradient.
RNA Isolation, mRNA Assay and Microarray Analysis
[0053] RNA from Hep3B cells and primary human hepatocytes was
prepared using TRIzol (Invitrogen) according to manufacturer's
instructions. Single-pass cDNA was synthesized using the iScript
cDNA synthesis kit (Bio-Rad, Hercules, Calif.). The quantitative
real-time polymerase chain reaction (qRT-PCR) was performed using
iQ SYBR Green Supermix (Bio-Rad). Human hepcidin and hemojuvelin
mRNA concentrations were normalized to human glyceraldehyde
3-phosphate dehydrogenase (G3PD) or human .beta.-actin. Human
CEBP.delta. was used for IL-6 response positive control.
[0054] The following primers were used in qRT-PCR:
TABLE-US-00003 hepcidin: (SEQ ID NO: 8) forward:
5'-CACAACAGACGGGACAACTT-3'; (SEQ ID NO: 9) reverse:
5'-CGCAGCAGAAAATGCAGATG-3'; hemojuvelin: (SEQ ID NO: 10) forward:
5'-CTCTTAGCTCCACTCCTTTCTG-3'; (SEQ ID NO: 11) reverse:
5'-GCCCTGCTTCCTTTAATGATTC-3'; G3PD: (SEQ ID NO: 12) forward
5'-TGGTATCGTGGAAGGACTC-3'; (SEQ ID NO: 13) reverse:
5'-AGTAGAGGCAGGGATGATG-3'; .beta.-actin: (SEQ ID NO: 14) forward
5'-ATCGTGCGTGACATTAAG-3'; (SEQ ID NO: 15) reverse:
5'-ATTGCCAATGGTGATGAC-3'; CEBP.delta.: (SEQ ID NO: 16) forward
5'-CAACGACCCATACCTCAG-3'; (SEQ ID NO: 17) reverse:
5'-GGTAAGTCCAGGCTGTAG-3'.
[0055] Affymetrix HG-U133 Plus2 (Affymetrix, Santa Clara, Calif.)
were used for microarray analysis according to manufacturer's
protocol.
Western Blot Analysis and Antibody
[0056] Cellular protein was extracted with 150 mM NaCl, 10 mM EDTA,
10 mM Tris (pH 7.4) (NETT), 1% Triton X-100 and a protease
inhibitor cocktail (Sigma-Aldrich, Saint Louis, Mo.) using methods
known in the art. Frozen normal human liver fragments were
pulverized in liquid nitrogen with a mortar and pestle. About 50 mg
of tissue was homogenized in 700 .mu.l NETT buffer, and about 150
.mu.g of total protein extract was analyzed. Human sera and plasma
samples were loaded directly at 1 or 2 .mu.l/lane. Cell culture
media were further processed before Western analysis. Serum-free
conditioned cell culture media were concentrated by 5 kD molecular
weight cut-off ultrafiltration with Amicon.RTM. Ultra-4 Centrifugal
Filter Units (Millipore, Bedford, Mass.) using methods known in the
art. Conditioned cell culture media that contained 30 .mu.M Apo-
and Holo-transferrin were extracted with the weak cation exchange
matrix CM Macroprep (Bio-Rad, Richmond, Calif.), the matrix was
eluted with 500 mM sodium chloride in 25 mM ammonium acetate buffer
(pH 6.5), and the eluate was concentrated by ultrafiltration using
methods known in the art. Conditioned cell culture media that
contained 10% FBS were partially purified by cation exchange
chromatography before concentration using methods known in the art.
Protein samples were separated on 4-20% iGels (SDS-Tris-Glycine)
(Gradipore, Hawthorne, N.Y.) with dithiothreitol (DTT) if not
mentioned specifically otherwise, and silver-stained or transferred
on Immobilon-P membrane (Millipore Corp., Bedford, Mass.) using
methods known in the art.
[0057] Three different anti-hemojuvelin polyclonal antibodies were
prepared by immunizing rabbits with peptide antigens: anti-G3pep2-2
and anti-G3pep2-3: Target sequence N-CRGDLAFHSAVHGIED-C, (SEQ ID
NO:18) (1:1000); Ab112: Target sequence N-CDYEGRFSRLHGRPPG-C (SEQ
ID NO:19) (1:5000). Western blots were visualized by
chemiluminescence using methods known in the art.
Results
[0058] Suppression of Hemojuvelin mRNA Results in the Suppression
of Hepcidin mRNA
[0059] Four different siRNA sequences, HJVsi1, HJVsi2, HJVsi3, and
HJVsi4, were used to target the coding and non-coding 3'
untranslated (3'-UTR) regions of hemojuvelin mRNA. As shown in
FIGS. 1A and 1B, each individual point represents an experiment in
which Hep3B cells were treated with one of the siRNA preparations
overnight, and then incubated for additional 24 hours before mRNA
extraction. In each experiment, hemojuvelin and hepcidin mRNA were
quantified by real time qRT-PCR and normalized to the housekeeping
gene G3PD using methods known in the art. Control cells, NCsi1,
NCsi2, and NCsi3, were treated only with transfection reagents and
their hemojuvelin/G3PD and hepcidin/G3PD ratios were set as
baseline=1. See FIG. 1B.
[0060] These siRNAs showed a wide range of efficiency (about 30% to
about 90%) in suppressing hemojuvelin mRNA level about 48 hours
after transfection. Decrease in hepcidin mRNA correlated with
decreased hemojuvelin mRNA levels (R=0.64). See FIG. 1A. No
significant suppression of hepcidin mRNA was observed when
hemojuvelin mRNA concentration was above about 50% of untreated
control. This is consistent with the observation that individuals
with only one copy of disrupted HJV do not develop iron overload.
See Papanikolaou, G., et al. (2004) Nat. Genet. 36:77-82, which is
herein incorporated by reference. The 3 different siRNA negative
controls showed slight suppression or induction of either
hemojuvelin or hepcidin mRNA, but no significant correlation or
specificity was observed as shown in FIG. 1B.
Hemojuvelin and IL-6 Independently Regulate Hepcidin mRNA
[0061] Next, whether hemojuvelin is necessary for the inflammatory
induction of hepcidin was examined. IL-6 is a well-defined inducer
of hepcidin during anemia of inflammation. See Nemeth, E., et al.
(2004) J. Clin. Invest. 113:1271-1276, which is herein incorporated
by reference. Hep3B cells were pretreated with hemojuvelin siRNA or
diluent for 24 hours, followed by 24 hours of treatment with 20
ng/ml human IL-6 to induce hepcidin. See FIG. 2. Suppression of
hemojuvelin to as low as about 10% to about 20% of the control
(cells not treated with siRNA or IL-6) caused a maximum of about
2-fold reduction of hepcidin baseline expression, but did not
interfere with its inducibility by IL-6 (a similar 4-fold induction
of hepcidin mRNA level in both hemojuvelin siRNA treated and
control cells). An IL-6 specific acute phase protein CEBP.delta.
was used as a positive control for IL-6 induction as well as a
negative control for hemojuvelin siRNA specificity. See Ramji, D.
P., et al. (1993) Nucl. Acids Res. 21:289-294; and Alam, T., et al.
(1992) J. Biol. Chem. 267:5021-5024, which are herein incorporated
by reference. The mRNA levels of CEBP.delta. were unaffected by
hemojuvelin siRNA treatment but were induced by approximately
4-fold with 20 ng/ml IL-6 in both hemojuvelin siRNA treated and
control cells. These data showed that IL-6 and hemojuvelin act
independently to regulate hepcidin mRNA levels.
Hemojuvelin Protein is Detected as Both Cell-Associated and Soluble
Forms
[0062] Total protein extract from human liver was analyzed on
reducing SDS-PAGE and the corresponding blot was probed with the
polyclonal anti-hemojuvelin antibody anti-G3pep2-3 targeted to the
N-terminus of hemojuvelin. One predominant protein band of about 46
kD was detected in human liver from 2 different donors. See FIG. 3,
Lane 1 and 2. Lysate of Hep3B cells (with endogenous hemojuvelin
mRNA expression), was also analyzed by western blot, but no signal
was detected using any of the available antibodies (data not
shown).
[0063] In order to confirm the specificity of antibody detection of
the 46 kD protein band in human liver, the full length hemojuvelin
cDNA was cloned into pcDNA 3.1(+) vector to generate the pcDNA-HJV
construct, and used it to transfect the Hep3B and HEK293T/17
(HEK293) cell lines (the latter with undetectable endogenous
hemojuvelin mRNA) as positive controls for cellular expression of
hemojuvelin. Hemojuvelin expression was compared in vector
(pcDNA3.1 (+)) alone or in construct (pcDNA-HJV)-treated cells.
Cell lysate and conditioned medium were analyzed by Western blot
with anti-G3pep2-3. In cell lysate of HEK293 cells, a unique
protein band of approximately 46 kD, identical in size to the band
seen in human liver protein extract, was identified in
pcDNA-HJV-treated cells but not in cells treated with control
vector. See FIG. 3, Lane 3 and 4. No hemojuvelin-specific band was
detected in the cell lysate of Hep3B cells transfected with
pcDNA-HJV or pcDNA3.1(+) (data not shown). This could be due to a
low transfection efficiency in Hep3B cells (generally about 10%,
compared to over 90% in HEL293T/17 cells, estimated by green
fluorescence) and low detection sensitivity of anti-G3pep2-3
antibody.
[0064] Next, whether hemojuvelin was present in the media derived
from cells expressing hemojuvelin was examined. In the conditioned
culture medium of HEK293 cells transfected with pcDNA-HJV, but not
with vector pcDNA 3.1(+), one unique prominent protein band of
approximately 44 kD was detected in Western blot using
anti-G3pep2-3. See FIG. 3. A similar result with the conditioned
culture medium of Hep3B cell transfected with both vectors was
obtained. See FIG. 3. The detection of s-hemojuvelin in
vector-treated Hep3B but not HEK293 cells is consistent with the
endogenous hemojuvelin mRNA expression in Hep3B cells.
[0065] An alternative antibody Ab112, targeting a region 35 amino
acids downstream from the region used to generate anti-G3pep2-3,
detected both GPI-hemojuvelin and s-hemojuvelin in transfected
Hep3B and HEK293 cells, but not in human liver. Using Ab112, under
reducing conditions, an additional 16 kD reactive protein band was
detected in both cell types but only one reactive protein band
appeared under non-reducing conditions, 46 kD for GPI-hemojuvelin,
and 44 kD for s-hemojuvelin (data not shown). There was about a 2
kD difference between the size of the GPI-hemojuvelin and
s-hemojuvelin (46 kD vs 44 kD), indicating that a cleavage near the
C-terminus of the cell-associated form caused the release of the
soluble form.
[0066] Thus, GPI-hemojuvelin may be detected in human liver and in
cultured cell lines engineered to express hemojuvelin. Moreover,
s-hemojuvelin can also be detected in the media conditioned by cell
lines expressing hemojuvelin.
Production of Recombinant Soluble Human Hemojuvelin
(S-Hemojuvelin)
[0067] Recombinant soluble human hemojuvelin (s-hemojuvelin) was
expressed in a baculovirus/insect cell expression system. Purified
s-hemojuvelin migrated as a single band in western blots of
non-reducing SDS-PAGE, but formed two bands in blots of reducing
SDS-PAGE, reactive with anti-hemojuvelin antibody Ab112 (FIG. 7,
Lane 1 and 3) but not with pre-immune serum (data not shown). The
purified s-hemojuvelin was similar in size to s-hemojuvelin
partially purified from HEK293 cell culture engineered to express
hemojuvelin (FIG. 7, Lane 2, 4). The non-reducing SDS-PAGE gel
staining indicated over 95% purity for s-hemojuvelin (FIG. 8, Lane
2). In addition to the full-length s-hemojuvelin (apparent MW of 44
kD), two additional bands of 29 kD and 16 kD (apparent MW) on
reducing SDS PAGE (FIG. 8, Lane 4) were also observed. These two
bands were not observed on a non-reducing SDS-PAGE (FIG. 8, Lane
2), suggesting that they were the proteolytic cleavage products of
s-hemojuvelin linked together by a disulfide bond(s). Edman
degradation was used to sequence the N-terminus of the two reduced
fragments and non-reduced s-hemojuvelin. Undetectable signal
indicated a characteristically blocked N-terminal glutamine at the
start of the N-terminal fragment (amino acid 36Q). The C-terminal
fragment generated the sequence PHVR . . . indicating that it was
generated from an Asp-Pro cleavage site after amino acid 172D
(FGD.dwnarw.PHVR). Non-reduced s-hemojuvelin was also N-terminally
blocked but generated a sequence suggestive of the exposure of a
second N-terminus (PHVR) by cleavage. These results agree with
previous report of three mouse RGMs (a, b, and c) and chicken RGM
which all showed identical cleavage sites (FGD.dwnarw.PH V/L R).
See Niederkofler, V., et al. (2004) J. Neurosci. 24:808-818; and
Monnier, P. P., et al. (2002) Nature 419:392-395, which are herein
incorporated by reference. The conserved Asp-Pro bond is known to
be unusually labile, and can undergo hydrolysis in acidic cellular
compartments or after treatment with mild acids. See Lidell, M. E.,
et al. (2003) J. Biol. Chem. 278:13944-13951, which is herein
incorporated by reference. The observation that s-hemojuvelin forms
a disulfide-linked two chain structure with one blocked N-terminus
explains the inconsistency between the apparent molecular weight
and sequencing results previously interpreted as glycosylation and
removal of the N-terminal fragment in native RGMs.
[0068] The unmodified hemojuvelin precursor protein (45.1 kD) could
be subject to a series of post-translational modifications, due to
the presence of an N-terminal signal peptide (3.57 kD), a
C-terminal transmembrane motif characteristic for GPI anchor (2.46
kD), and multiple putative glycosylation and protease cleavage
sites. After the removal of the signal peptide and C-terminal
transmembrane domain, the s-hemojuvelin has a predicted MW of 39.1
kD. Mass spectrometry (MALDI-TOF) of s-hemojuvelin (apparent MW of
44 kD on SDS-PAGE) yielded a mass of about 41.5 kD with multiple
peaks at about 160 D intervals, indicating a typical glycosylation
pattern.
Soluble Hemojuvelin can be Detected in Human Plasma and Serum
[0069] The release of s-hemojuvelin into cell culture indicated the
possibility that s-hemojuvelin exists in vivo and has a
physiological function. Two .mu.l human serum was separated on a
reducing SDS-PAGE and detected a single prominent protein band of
30 kD reactive with Ab112 (FIG. 4, left panel). Anti-G3pep2-2
antibody detected another specific protein band of 16 kD in the
same samples (FIG. 4, middle panel, bottom bands). To confirm the
30 kD protein band is specific for hemojuvelin, Ab112 was
neutralized with 50-fold excess of s-hemojuvelin (antigen/specific
IgG ratio) and performed a western blot of human serum. The
competition from excessive s-hemojuvelin completely abolished the
30 kD protein band in human serum (FIG. 9, Lane 2 and 5), as well
as the bands corresponding to s-hemojuvelin (FIG. 9, Lane 1 and 4,
rs-) and s-hemojuvelin from engineered HEK293 cells (FIG. 9, Lane 3
and 6, s-). In multiple serum samples, the relative signal
intensity of the 30 kD band correlated well with the signal
intensity of the 16 kD band, suggesting that they were both
components of s-hemojuvelin in human serum.
[0070] To rule out the possibility that the cleavage of soluble
human hemojuvelin in serum might be an artifact of the clotting
process, 1 .mu.l of human serum and plasma from the same donor on
reducing SDS-PAGE probed with Ab112 was analyzed. The identical 30
kD protein band was detected in both human serum and plasma (FIG.
4, right panel), indicating the cleaved product is present in human
blood.
[0071] The patterns of antibody reactivity of plasma hemojuvelin as
compared to s-hemojuvelin (FIG. 9) indicated that the plasma
hemojuvelin is cleaved between the two antigenic epitopes used for
antibody generation rather than at the 172D.dwnarw.P cleavage site
of s-hemojuvelin downstream of the epitope region for Ab112.
[0072] The strong signal detected in human serum by western blot
analysis indicates a substantial amount of s-hemojuvelin in human
blood, estimated to be in the .mu.g/ml range. Both the liver and
the large mass of skeletal muscle may be the source of
s-hemojuvelin, since both contain hemojuvelin mRNA at very high
concentrations.
Iron Treatment Reduces the Amount of Soluble Hemojuvelin Released
into Cell Culture Medium
[0073] To determine whether hemojuvelin protein expression or the
release of soluble form is regulated by iron, ferric ammonium
citrate (FAC) or apo/holo transferrin was added into cell cultures
of both HEK293 and Hep3B cell line transfected with either
pcDNA-HJV or vector alone. Western blot probed with anti-G3pep2-3
or Ab112 was used to analyze both whole cell lysate and conditioned
cell culture medium. No significant change in cell-associated
hemojuvelin could be detected (data not shown). However,
s-hemojuvelin in cell culture media from both cell lines
progressively decreased with increasing FAC concentration from 3 to
100 .mu.M. See FIG. 5A. Similar results were also observed when
treating hemojuvelin-transfected HEK293 cell with increasingly
iron-saturated transferrin at a constant total transferrin
concentration of 30 .mu.M. See FIG. 5B.
Recombinant Soluble Hemojuvelin Suppresses Hepcidin mRNA in a Dose
Dependent Manner in Cultured Primary Human Hepatocytes
[0074] According to previous reports, the mRNA concentrations of
hepatic RgmC (the HJV homolog in mouse) were not affected by iron
feeding. See Krijt, J., et al. (2004) Blood 104:4308-4310, which is
herein incorporated by reference. The inverse correlation of iron
loading and s-hemojuvelin concentration in vitro leads to the
hypothesis that s-hemojuvelin is a negative regulator of hepcidin
mRNA concentration.
[0075] Considering the amount of s-hemojuvelin detectable on
Western blot, the s-hemojuvelin protein level was estimated to be
less than about 5 ng/ml in hepatocyte culture medium after a 24
hour incubation. Primary human hepatocytes were treated for 24
hours with higher concentrations of s-hemojuvelin (about 20 to 3000
ng/ml), similar to the concentrations detected in human sera, and
observed that hepcidin mRNA concentrations decreased in a
dose-dependent manner. No cytotoxicity was observed as judged by
.beta.-actin mRNA expression and cell morphology. The decrease in
hepcidin mRNA level showed a striking log-linear anti-correlation
with s-hemojuvelin concentration (R.sup.2>0.9 in each individual
experiment, data not shown), and this log-linear anti-correlation
was consistent in hepatocyte cultures from 4 different donors and 2
independent preparations of s-hemojuvelin (FIG. 6), indicating a
possible competition for a hemojuvelin ligand.
[0076] A similar dose-dependent fractional suppression of hepcidin
mRNA by s-hemojuvelin in the presence of 20 ng/ml human IL-6 (FIG.
10A) was observed. This result indicated that the suppression of
hepcidin mRNA expression was IL-6 independent, consistent with the
observation from the hemojuvelin siRNA treatment that
cell-associated hemojuvelin regulated hepcidin mRNA expression in
an IL-6 independent manner. Nevertheless, treatment with high doses
of s-hemojuvelin (about 1 to about 3 .mu.g/ml) effectively reversed
the 6 to 16-fold induction of hepcidin mRNA by 20 ng/ml of IL-6.
See FIG. 10B. Therefore, the present invention provides methods of
inhibiting, decreasing, or suppressing hepcidin in a subject which
comprises administering to the subject s-hemojuvelin.
[0077] The suppression of hepcidin mRNA by s-hemojuvelin was highly
selective. Using the Affymetrix HG-U133 Plus2 microarray, the
global gene expression pattern in primary human hepatocytes treated
with s-hemojuvelin (3 .mu.g/ml) versus those treated with diluent
(FIG. 11) was compared. Hepcidin mRNA decreased about 5-fold after
treatment with s-hemojuvelin, the largest change of any transcript
that was present in both treated and mock-treated hepatocytes. This
decrease was significant at p<0.0001 using the statistics (at
default settings) of the Affymetrix GeneChip Operating Software
version 1.2.
[0078] Therefore, the present invention provides methods for
regulating or modulating hepcidin expression or levels in subjects
which comprises administering soluble hemojuvelin (s-hemojuvelin)
to the subjects. As used herein, "soluble hemojuvelin" refers to
natural and synthetic hemojuvelin proteins which lack the
glycophosphatidylinositol (GPI) anchor that binds hemojuvelin to
cell membranes. One of ordinary skill in the art may readily obtain
s-hemojuvelin by removing the GPI anchor using methods known in the
art, including protein cleavage and recombinant techniques. As used
herein, the terms "protein", "polypeptide", and "peptide" are used
interchangeably to refer to two or more amino acid residues linked
together. Preferred s-hemojuvelin proteins of the present invention
include polypeptides consisting of at least about 6, preferably at
least about 20, and more preferably at least about 50 consecutive
amino acid residues of SEQ ID NO:1. In some preferred embodiments,
the s-hemojuvelin protein consists of SEQ ID NO:1. However, it is
noted that other hemojuvelin proteins and fragments thereof known
in the art, including those recited in U.S. Publication No.
20060073497, which is herein incorporated by reference, may be used
in accordance with the present invention. Specifically, the
hemojuvelin proteins and fragments known in the art may be
engineered to lack the GPI anchor and used in accordance with the
present invention. Therefore, as used herein, "soluble hemojuvelin"
refers to hemojuvelin proteins and fragments thereof known in the
art which lack a GPI anchor.
[0079] As used herein, a "disease of iron metabolism" includes
diseases where aberrant iron metabolism directly causes the
disease, or where iron blood levels are disregulated causing
disease, or where iron disregulation is a consequence of another
disease, or where diseases can be treated by modulating iron
levels, and the like. More specifically, a disease of iron
metabolism according to this disclosure includes iron overload
disorders, iron deficiency disorders, disorders of iron
biodistribution, other disorders of iron metabolism and other
disorders potentially related to iron metabolism, etc. Even more
specifically diseases of iron metabolism includes hemochromatosis,
ferroportin mutation hemochromatosis, transferrin receptor 2
mutation hemochromatosis, juvenile hemochromatosis, neonatal
hemochromatosis, hepcidin deficiency, transfusional iron overload,
thalassemia, thalassemia intermedia, alpha thalassemia,
sideroblastic anemia, porphyria, porphyria cutanea tarda, African
iron overload, hyperferritinemia, ceruloplasmin deficiency,
atransferrinemia, congenital dyserythropoietic anemia, anemia of
chronic disease, anemia, hypochromic microcytic anemia,
iron-deficiency anemia, conditions with hepcidin excess, Friedreich
ataxia, gracile syndrome, Hallervorden-Spatz disease, Wilson's
disease, pulmonary hemosiderosis, hepatocellular carcinoma, cancer,
hepatitis, cirrhosis of liver, pica, chronic renal failure, insulin
resistance, diabetes, atherosclerosis, neurodegenerative disorders,
multiple sclerosis, Parkinson's disease, Huntington's disease,
Alzheimer's disease.
[0080] In some cases the diseases and disorders included in the
definition of "disease of iron metabolism" are not typically
identified as being iron related. It is recognized by the instant
invention that based on the tissue distribution of HFE2A
(hemojuvelin) and its related protein, hepcidin, that iron
metabolism may play a significant role in these disease processes.
For example, hepcidin is very highly expressed in the murine
pancreas suggesting that diabetes (Type I or Type II), insulin
resistance, glucose intolerance and other disorders may be
ameliorated by treating underlying iron metabolism disorders. See
Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which is herein
incorporated by reference. As such, these diseases are encompassed
under the broad definition. Those skilled in the art are readily
able to determine whether a given disease is a "disease or iron
metabolism" according to the present invention using methods known
in the art, including the assays of WO 2004092405, which is herein
incorporated by reference, and assays which monitor hepcidin,
hemojuvelin, or iron levels and expression.
[0081] It is important to note that the various diseases of iron
metabolism are caused by abnormal hepcidin production, either too
much or too little. As provided herein, hepcidin production is
regulated by hemojuvelin in such a way that GPI-hemojuvelin
induces, increases, or stimulates hepcidin production and
s-hemojuvelin suppresses, decreases, or inhibits hepcidin
production.
[0082] Thus, the present invention provides methods for treating,
preventing, or modulating diseases of iron metabolism in subjects
which comprise administering to the subject GPI-hemojuvelin to
increase hepcidin production or administering s-hemojuvelin to
decrease hepcidin production. For example, to treat juvenile
hemochromatosis in a subject, GPI-hemojuvelin is administered to
the subject in order to increase hepcidin production. To treat
anemia of chronic disease in a subject, s-hemojuvelin is
administered to the subject in order to decrease hepcidin
production.
[0083] The present invention also provides methods of monitoring or
diagnosing diseases of iron metabolism in subjects which comprise
assaying the levels of GPI-hemojuvelin, s-hemojuvelin, or both in
the subject and determining whether the levels are normal or
abnormal.
[0084] The present invention further provides compositions
comprising the s-hemojuvelin proteins described herein. The
compositions include pharmaceutical compositions which may be
readily formulated for desired routes of administration using
methods known in the art. Suitable formulations and
pharmaceutically acceptable carriers are known in the art.
Expression of Soluble Hemojuvelin in Mice
[0085] As provided herein an increased iron concentration (both
ferric ammonium citrate and increasingly saturated iron
transferrin) can suppress the release of soluble hemojuvelin into
cell culture media in an in vitro system (HEK293 cells transfected
with human hemojuvelin expressing vector). The release of soluble
hemojuvelin is dependent on the enzymatic activity of furin
convertase (encoded by FUR), which cleaved a conserved RXRR.dwnarw.
site in hemojuvelin. See FIG. 12. This cleavage activity was
sensitive to an inhibitor of furin convertase (Chloromethylketone).
The cleavage site of soluble hemojuvelin was confirmed by
amino-terminal sequencing of a human hemojuvelin-alkaline
phosphatase fusion construct, which was processed into an
N-terminal fragment with an identical migration pattern as soluble
hemojuvelin. See FIG. 13.
[0086] On the other hand, purified human recombinant soluble
hemojuvelin can suppress hepcidin mRNA expression in human primary
hepatocyte cultures. This suppression had a strong dose-dependent
log-linear anti-correlation with the added soluble hemojuvelin.
This pattern is characteristic for a ligand-receptor competition
model, where membrane associated hemojuvelin positively regulates
hepcidin mRNA concentration in human liver, and soluble hemojuvelin
acts as its natural antagonist. However, mouse primary hepatocyte
cultures treated with purified human recombinant soluble
hemojuvelin did not show downregulation of hepcidin mRNA
expression. This could be due to the difference between mouse and
human hemojuvelin (88.1% sequence identity in amino acid sequence).
Tail vein injection of purified human recombinant soluble
hemojuvelin in mice had no significant effect on hepatic hepcidin
expression either.
[0087] To evaluate the physiological effect of soluble hemojuvelin
in vivo, a lentiviral expression system for stable expression of
soluble hemojuvelin in mouse liver and skeletal muscles was used.
The expressed soluble hemojuvelin is expected to be released into
circulation (based on observations in in vitro cell culture system
using HEK293 cells).
[0088] The lentiviral expression construct (Transfer vector
pRRL-Hjv-FUR) express soluble hemojuvelin (s-Hjv) shown in FIG. 14
was constructed using methods known in the art.
[0089] The soluble mouse hemojuvelin protein sequence encoded by
pRRL-Hjv-FUR is as follows:
TABLE-US-00004 (SEQ ID NO: 20)
MGQSPSPRSPHGSPPTLSTLTLLLLLCGQAHSQCKILRCNAEYVSSTLSL
RGGGSPDTPRGGGRGGLASGGLCRALRSYALCTRRTARTCRGDLAFHSAV
HGIEDLMIQHNCSRQGPTAPPPARGPALPGAGPAPLTPDPCDYEARFSRL
HGRAPGFLHCASFGDPHVRSFHNQFHTCRVQGAWPLLDNDFLFVQATSSP
VSSGANATTIRKITIIFKNMQECIDQKVYQAEVDNLPAAFEDGSINGGDR
PGGSSLSIQTANLGSHVEIRAAYIGTTIIIRQTAGQLSFSIRVAEDVARA
FSAEQDLQLCVGGCPPSQRLSRSERNRR.
[0090] A control vector expressing mouse albumin (Transfer vector
pRRL-Abl1) was also constructed using the same transfer vector. The
lentiviral vectors may be packaged in a HEK293T cell line to
generate replication incompetent viral particles and concentrated
to prepare high titer viral supernatant. Subjects, such as C57BL/6
mice, are injected intravenously with 10.sup.8 viral
particles/subject. All subjects are allowed sufficient time, e.g. 1
week, for transgene integration and to recover from virus induced
inflammation before any further treatment. Then the effects of
soluble hemojuvelin on hepatic hepcidin expression and body iron
status are studied under the following physiological conditions:
[0091] 1. Acute inflammation: induced by injecting turpentine into
the interscapular fat pad. [0092] 2. Chronic inflammation: induced
by injecting Cytodex beads co-cultured with Staphylococcus
epidermidis (S. Epi) into the peritoneal cavity. [0093] 3. Dietary
iron loading: by putting mice on moderate iron diet (50 ppm)
through out experiment (before and after viral injection). [0094]
4. Acute iron ingestion: by switching experimental mice from low
iron diet (<4 ppm, before and after viral injection) to high
iron diet (10000 ppm) for over night.
[0095] All mice are then euthanized. Then the levels of serum iron
and transferrin saturation, hepatic hepcidin, hemojuvelin, soluble
hemojuvelin, CEBP/delta, IL-6 mRNA, skeletal muscle hemojuvelin,
beta-actin mRNA, transgene genome insertion are measured using
qRT-PCR. The level of plasma soluble hemojuvelin protein may also
be measured using ELISA.
[0096] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
[0097] Variations, modification, and other implementations of what
is described herein will occur to those of skill in the art without
departing from the spirit and scope of the invention and the
following claims.
Sequence CWU 1
1
201368PRTArtificialHuman recombinant soluble hemojuvelin 1Gln Cys
Lys Ile Leu Arg Cys Asn Ala Glu Tyr Val Ser Ser Thr Leu1 5 10 15Ser
Leu Arg Gly Gly Gly Ser Ser Gly Ala Leu Arg Gly Gly Gly Gly 20 25
30Gly Gly Arg Gly Gly Gly Val Gly Ser Gly Gly Leu Cys Arg Ala Leu
35 40 45Arg Ser Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg
Gly 50 55 60Asp Leu Ala Phe His Ser Ala Val His Gly Ile Glu Asp Leu
Met Ile65 70 75 80Gln His Asn Cys Ser Arg Gln Gly Pro Thr Ala Pro
Pro Pro Pro Arg 85 90 95Gly Pro Ala Leu Pro Gly Ala Gly Ser Gly Leu
Pro Ala Pro Asp Pro 100 105 110Cys Asp Tyr Glu Gly Arg Phe Ser Arg
Leu His Gly Arg Pro Pro Gly 115 120 125Phe Leu His Cys Ala Ser Phe
Gly Asp Pro His Val Arg Ser Phe His 130 135 140His His Phe His Thr
Cys Arg Val Gln Gly Ala Trp Pro Leu Leu Asp145 150 155 160Asn Asp
Phe Leu Phe Val Gln Ala Thr Ser Ser Pro Met Ala Leu Gly 165 170
175Ala Asn Ala Thr Ala Thr Arg Lys Leu Thr Ile Ile Phe Lys Asn Met
180 185 190Gln Glu Cys Ile Asp Gln Lys Val Tyr Gln Ala Glu Val Asp
Asn Leu 195 200 205Pro Val Ala Phe Glu Asp Gly Ser Ile Asn Gly Gly
Asp Arg Pro Gly 210 215 220Gly Ser Ser Leu Ser Ile Gln Thr Ala Asn
Pro Gly Asn His Val Glu225 230 235 240Ile Gln Ala Ala Tyr Ile Gly
Thr Thr Ile Ile Ile Arg Gln Thr Ala 245 250 255Gly Gln Leu Ser Phe
Ser Ile Lys Val Ala Glu Asp Val Ala Met Ala 260 265 270Phe Ser Ala
Glu Gln Asp Leu Gln Leu Cys Val Gly Gly Cys Pro Pro 275 280 285Ser
Gln Arg Leu Ser Arg Ser Glu Arg Asn Arg Arg Gly Ala Ile Thr 290 295
300Ile Asp Thr Ala Arg Arg Leu Cys Lys Glu Gly Leu Pro Val Glu
Asp305 310 315 320Ala Tyr Phe His Ser Cys Val Phe Asp Val Leu Ile
Ser Gly Asp Pro 325 330 335Asn Phe Thr Val Ala Ala Gln Ala Ala Leu
Glu Asp Ala Arg Ala Phe 340 345 350Leu Pro Asp Leu Glu Lys Leu His
Leu Phe Pro Ser Asp Ala Gly Val 355 360 365221DNAArtificialsiRNA
duplex targeting human hemojuvelin mRNA 2aactctaagc actctcactc t
21321DNAArtificialsiRNA duplex targeting human hemojuvelin mRNA
3aaccattgat actgccagac g 21421DNAArtificialsiRNA duplex targeting
human hemojuvelin mRNA 4aagtttagag gtcatgaagg t
21521DNAArtificialsiRNA duplex targeting human hemojuvelin mRNA
5aaagctacaa attcttcaca c 21619DNAArtificialsiRNA negative control
for targeting human hemojuvelin mRNA 6gcgcgctttg taggattcg
19721DNAArtificialsiRNA negative control for targeting human
hemojuvelin mRNA. 7aattctccga acgtgtcacg t
21820DNAArtificialforward primer qRT-PCR for hepcidin 8cacaacagac
gggacaactt 20920DNAArtificialreverse primer qRT-PCR for hepcidin
9cgcagcagaa aatgcagatg 201022DNAArtificialforward primer for
qRT-PCR human hemojuvelin 10ctcttagctc cactcctttc tg
221122DNAArtificialreverse primer for qRT-PCR human hemojuvelin
11gccctgcttc ctttaatgat tc 221219DNAArtificialforward primer for
qRT-PCR human G3PD 12tggtatcgtg gaaggactc
191319DNAArtificialreverse primer for qRT-PCR human G3PD
13agtagaggca gggatgatg 191418DNAArtificialforward primer for
qRT-PCR human beta-actin 14atcgtgcgtg acattaag
181518DNAArtificialreverse primer for qRT-PCR human beta-actin
15attgccaatg gtgatgac 181618DNAArtificialforward primer for qRT-PCR
human CEBPdelta 16caacgaccca tacctcag 181718DNAArtificialreverse
primer for qRT-PCR human CEBPdelta 17ggtaagtcca ggctgtag
181816PRTArtificialhuman antigen anti-G3pep2-2 18Cys Arg Gly Asp
Leu Ala Phe His Ser Ala Val His Gly Ile Glu Asp1 5 10
151916PRTArtificialhuman antigen anti-G3pep2-3 19Cys Asp Tyr Glu
Gly Arg Phe Ser Arg Leu His Gly Arg Pro Pro Gly1 5 10
1520328PRTArtificialsoluble mouse hemojuvelin protein sequence
encoded by pRRL-Hjv-FUR 20Met Gly Gln Ser Pro Ser Pro Arg Ser Pro
His Gly Ser Pro Pro Thr1 5 10 15Leu Ser Thr Leu Thr Leu Leu Leu Leu
Leu Cys Gly Gln Ala His Ser 20 25 30Gln Cys Lys Ile Leu Arg Cys Asn
Ala Glu Tyr Val Ser Ser Thr Leu 35 40 45Ser Leu Arg Gly Gly Gly Ser
Pro Asp Thr Pro Arg Gly Gly Gly Arg 50 55 60Gly Gly Leu Ala Ser Gly
Gly Leu Cys Arg Ala Leu Arg Ser Tyr Ala65 70 75 80Leu Cys Thr Arg
Arg Thr Ala Arg Thr Cys Arg Gly Asp Leu Ala Phe 85 90 95His Ser Ala
Val His Gly Ile Glu Asp Leu Met Ile Gln His Asn Cys 100 105 110Ser
Arg Gln Gly Pro Thr Ala Pro Pro Pro Ala Arg Gly Pro Ala Leu 115 120
125Pro Gly Ala Gly Pro Ala Pro Leu Thr Pro Asp Pro Cys Asp Tyr Glu
130 135 140Ala Arg Phe Ser Arg Leu His Gly Arg Ala Pro Gly Phe Leu
His Cys145 150 155 160Ala Ser Phe Gly Asp Pro His Val Arg Ser Phe
His Asn Gln Phe His 165 170 175Thr Cys Arg Val Gln Gly Ala Trp Pro
Leu Leu Asp Asn Asp Phe Leu 180 185 190Phe Val Gln Ala Thr Ser Ser
Pro Val Ser Ser Gly Ala Asn Ala Thr 195 200 205Thr Ile Arg Lys Ile
Thr Ile Ile Phe Lys Asn Met Gln Glu Cys Ile 210 215 220Asp Gln Lys
Val Tyr Gln Ala Glu Val Asp Asn Leu Pro Ala Ala Phe225 230 235
240Glu Asp Gly Ser Ile Asn Gly Gly Asp Arg Pro Gly Gly Ser Ser Leu
245 250 255Ser Ile Gln Thr Ala Asn Leu Gly Ser His Val Glu Ile Arg
Ala Ala 260 265 270Tyr Ile Gly Thr Thr Ile Ile Ile Arg Gln Thr Ala
Gly Gln Leu Ser 275 280 285Phe Ser Ile Arg Val Ala Glu Asp Val Ala
Arg Ala Phe Ser Ala Glu 290 295 300Gln Asp Leu Gln Leu Cys Val Gly
Gly Cys Pro Pro Ser Gln Arg Leu305 310 315 320Ser Arg Ser Glu Arg
Asn Arg Arg 325
* * * * *