Iron Uptake Mediated by Binding of H-Ferritin to the
TIM-2 Receptor in Mouse Cells
Jian Han1, William E. Seaman2,3, Xiumin Di4, Wei Wang4, Mark Willingham5,6, Frank M. Torti4,6, Suzy V.
1Department of Biology, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, United States of America, 2Department of Medicine,
University of California San Francisco, California, United States of America, 3Veterans Affairs Medical Center, San Francisco, California, United States of America,
4Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 5Department of Pathology, Wake Forest
School of Medicine, Winston-Salem, North Carolina, United States of America, 6Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, North
Carolina, United States of America, 7Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America
Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned.
TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin
domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the
immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve
as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately
expressed on the cell surface. HFt was labeled with55Fe and55Fe-HFt was incubated with TIM-2 positive cells or controls.
55Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express
endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of55Fe-HFt was specific and temperature-
dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal
compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron
delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased
levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.
Citation: Han J, Seaman WE, Di X, Wang W, Willingham M, et al. (2011) Iron Uptake Mediated by Binding of H-Ferritin to the TIM-2 Receptor in Mouse Cells. PLoS
ONE 6(8): e23800. doi:10.1371/journal.pone.0023800
Editor: Martin W. Brechbiel, National Institute of Health, United States of America
Received May 19, 2011; Accepted July 25, 2011; Published August 19, 2011
Copyright: ? 2011 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants from the National Institutes of Health (R01 AI061164 [WES], R01 DK071892 [SVT], and R37 DK42412 [FMT]).
The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Ferritin is a ubiquitously distributed protein principally known
for its role in iron storage and detoxification [1,2]. It is composed
of two subunit types, termed H and L; twenty-four of these
assemble to form the ferritin protein. The ratio of subunits within
the assembled protein is dictated by tissue type and is also
modulated by exogenous stimuli . The H subunit of ferritin
possesses ferroxidase activity , and enables the oxidation of iron,
whereas the L subunit facilitates iron nucleation within the central
core. Up to 4500 atoms of iron can be stored in a non-toxic but
bioavailable form within the central cavity of the ferritin protein.
Ferritin is regulated post-transcriptionally by iron through the
action of iron regulatory proteins (IRPs), which act as ferritin
translational repressors (see [4,5] for review). When levels of
intracellular iron rise, IRPs are inactivated, and ferritin mRNA is
translated. IRPs also control the levels of transferrin receptor 1
(TfR1), a protein that mediates uptake of iron bound to
transferrin, the principal source of iron in mammalian cells. In
the case of TfR1, regulation by IRPs destabilizes TfR1 mRNA
under conditions of iron repletion. Conversely, TfR1 mRNA and
protein increase under conditions of iron depletion, such as that
induced by an iron chelator.
Ferritin exists in multiple compartments and exhibits functions
in addition to its classic role in intracellular iron storage. Ferritin is
present in the cytosol of most cells, as well as in the nucleus or
mitochondria of some cell types. In addition, ferritin is present in
the bloodstream of mammals, and its levels can increase
dramatically in inflammation and cancer [6,7,8]. The role of
extracellular ferritin remains uncertain. Exogenous ferritin exerts
[9,10,11] and affecting chemokine receptor-mediated signal
transduction . Ferritin also acts as a pro-inflammatory
signaling molecule in hepatic stellate cells  and inhibits the
anti-angiogenic effects of kininogen . Exogenous ferritin also
can serve as an iron delivery vehicle. For example, ferritin released
by Kupffer cells is efficiently taken up by hepatocytes , and
ferritin secreted by macrophages serves as an iron source for
erythroid precursor cells .
Ferritin binds specifically and saturably to a variety of cell types
[10,17,18,19,20,21], and several ferritin receptors have recently
been cloned. Mouse TIM-2 was the first ferritin receptor to be
cloned . It is a specific receptor for HFt (i.e. ferritin comprised
of the H subunit), and is present in germinal center B cells, liver
bile duct epithelial cells, and kidney renal distal tubule cells, as well
as mouse oligodendrocytes . Since oligodendrocytes do not
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express transferrin receptor (which is responsible for iron uptake in
most mammalian cells), TIM-2 was postulated to be the primary
mechanism for iron uptake by these cells . Another cell surface
receptor for ferritin is Scara5, a mouse scavenger receptor . In
contrast to TIM-2, which is a HFt receptor, Scara5 preferentially
binds LFt (ferritin rich in the L subunit). Scara5 plays an important
role in kidney organogenesis, and serves as an iron delivery vehicle
to kidney capsular cells during development . The only ferritin
receptor identified in human cells to date is transferrin receptor 1,
which binds both HFt and transferrin .
The consequences of ferritin binding to its receptor TIM-2 have
not been assessed. TIM-2 is a member of the T cell immuno-
globulin and mucin domain containing (TIM) protein family 
and it plays an important role in immunity . TIM-2 deficient
mice have a heightened Th2 immune response, and they
demonstrate increased lung inflammation following allergic
challenge . Ectopic expression of TIM-2 also impairs
induction of NFAT and AP-1 in T cells in vitro . However
the expression of TIM-2 outside the immune system indicates that
this receptor may have broader roles. For example, TIM-2
regulates the differentiation of mouse fetal hepatocytes .
In this study, we tested whether ferritin binding to TIM-2 can
serve as an iron delivery mechanism in cells outside the brain. We
observed that iron-loaded HFt is taken up by TIM-2 positive cells
via an endocytic pathway, with delivery to lysosomes. Iron
delivered via this pathway becomes metabolically available,
resulting in increased levels of intracellular ferritin. We conclude
that ferritin can deliver iron to cells expressing TIM-2.
Materials and Methods
Chemicals and cell cultures
Ferrous ammonium sulfate, nitrilotriacetic acid (NTA), ferric
ammonium sulfate, phenylarsine oxide (PAO), and mouse apo-
transferrin were purchased from Sigma (St Louis, MO).55FeCl3
was purchased from Perkin Elmer (Fremont, CA). The TCMK-1
mouse kidney epithelial cell line and the A20 mouse B cell line
were obtained from the American Type Culture Collection
(ATCC, Rockville, MD). TCMK-1 cells were maintained in PC-
1 serum-free complete medium purchased from Lonza (Allendale,
NJ) supplemented with 100 units/ml penicillin, 100 mg/ml
streptomycin, and 2 mM glutamine. A20 cells were maintained
in RPMI 1640 medium from Invitrogen (Carlsbad, CA)
supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicar-
bonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium
pyruvate, 0.05 mM 2-mercaptoethanol, and 10% fetal bovine
serum (Invitrogen [Carlsbad, CA]). Cells were incubated in a
humidified atmosphere containing 5% CO2at 37uC.
Establishment of TIM-2 stable clones
TCMK-1 cells from the ATCC were transfected with TIM-2
containing and empty vector plasmids (BSR-a-FLAG ). After
24 hours, 1000 mg/ml G418 was added to the medium for clonal
selection. After selection, stable clones were maintained in medium
containing 350 mg/ml G418.
One million A20 cells were harvested and washed with PBS.
The cells were blocked with inactivated human AB serum
(Innovative Research [Novi, Michigan]) for 30 min and then
incubated with 1–2 mg biotin-linked anti-TIM-2 monoclonal
antibody  in 1% BSA (Sigma Aldrich [St. Louis, Missouri])/
PBS for 1 hour at 4uC in the absence of permeabilizing agents.
After incubation, the cells were washed with PBS and then
incubated with allophycocyanin (APC) linked streptavidin for
1 hour. The cells were washed and analyzed on a FACS Aria flow
cytometer (Becton Dickinson) using Diva and DakoCyotmation
Summit 4.3 analysis software.
Cells were lysed in lysis buffer (0.12% Triton X-100, 0.15 M
NaCl, 2.0 mM triethanolamine, pH 7.8, 2.5 mM CaCl2, 1 mM
MgSO4, 0.01% sodium azide) containing freshly added 1 mg/ml
aprotinin, 0.5 mg/ml leupeptin, 1 mM PMSF and 1 mM vann-
date. Lysates were incubated 2–3 h on ice and clarified by
centrifugation at 16,000 x g for 20 min at 4uC. Sixty mg protein
were used for immunoprecipiatation and 15 mg for western blot.
For immunoprecipitation, protein extracts were incubated with
anti-TIM-2 monoclonal antibody  and protein G beads
[Invitrogen, CA] at 4uC overnight. Beads were then washed three
times with FROP buffer (150 mM NaCl, 5 mM CHAPS, 50 mM
Tris-HCl, pH 8.0), suspended in 2xSDS gel loading buffer, heat
denatured and resolved on a 12% SDS-PAGE gel. The gel was
transferred onto a nitrocellulose membrane and blotted for TIM-2
protein using rabbit polyclonal anti-TIM-2 antibody  followed
by HRP conjugated anti-rabbit IgG and incubation with
chemiluminescent substrate (SuperSignal West Pico, Thermo
Scientific). Pre-stained standards (Full-range rainbow molecular
weight markers, Cat# RPN800E, GE Healthcare Life Sciences
[Piscataway, NJ]) were used to estimate molecular weights.
Recombinant H Ferritin purification and iron loading
Mouse HFt was produced in E. coli and purified as described
. Endotoxin was removed by adsorption to immobilized
polymyxin B (Detoxi-GelTM, pierce Chemical Co.), according to
the methods of the manufacturer. Endotoxin removal was
confirmed using the limulus amebocyte lysate assay (QCL-1000,
Cambrex Bio Science). To load iron into ferritin, 450 mM ferrous
ammonium sulfate was added to 50 mg/ml H ferritin diluted in 0.1
M HEPES pH 6.0 . Iron uptake was monitored spectropho-
Figure 1. Transfected TCMK-1 cells express TIM-2. A: Stable
transfectants were examined for TIM-2 protein expression by immuno-
precipitation followed by western blot. B: A TIM-2 positive clone (#3)
and a control vector transfectant (#3) were tested for TIM-2 mRNA
expression by realtime RT-PCR (means and standard deviation, n=4).
Iron Uptake Mediated by TIM-2
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tometrically at 310 nm. To label mouse recombinant HFt with
55Fe, 125 mCi
containing 20 mM citric acid, 2 mM ascorbate, and 0.1 M HEPES
(pH 6.0). This was followed by the addition of 450 mM unlabeled
ferrous ammonium sulfate and incubation for an additional hour.
The sample was then dialyzed in 0.1 M HEPES at 4uC overnight
. The final ratio of iron:ferritin was approximately 2000:1 as
measured by ferrozine assay . Specific activity of the
ferritin varied among preparations, and direct comparisons were
always made using the same batch of55Fe-ferritin. Ferritin was
filtered through a 0.45 mm syringe filter prior to cell culture use.
55FeCl3 was added to 50 mg/ml HFt in buffer
Real time RT-PCR
Real-time PCR was carried out on an ABI Prism 7000 sequence
detection system (Applied Biosystems, Foster City, CA) as
described . The standard curve method was used for
quantification. Total RNA was isolated using Trizol reagent
according to the manufacturer’s instructions. 30 mg RNA was
treated with 35U DNase I (Promega) for 30 min at 37uC. After
DNase I digestion, RNA was purified using Absolutely RNA RT-
PCR Miniprep kit (Stratagene) following the manufacturer’s
protocol. An oligodT primer was used in cDNA synthesis. b-actin
was measured as an internal control. Primers for PCR were
designed with IDT PrimerQuest software (Intergrated DNA
Technologies, Inc.). The forward primer for mouse TIM-2 was:
59-CCAACACCAGCACACACAGAGACCT-39, and the reverse
primer was: 59-TGGCTTCTGTGGAGGGATTACTTCA-39.
Figure 2. TIM-2 is present on the cell surface of transfected
cells. A: Flow cytometry was performed on non-permeabilized TCMK
TIM-2 transfectants and control vector transfectants. B: Flow cytometry
was performed on A20 cells (mouse lymphocyte that expresses
endogenous TIM-2) and A20 cells transfected with TIM-2 for over-
expression. Cells were incubated with anti-TIM-2 monoclonal antibody
(100 mg/ml), followed by FITC-linked anti-mouse secondary antibody
(100 mg/ml). Primary antibody was omitted in controls. C: TIM-2 was
immunoprecipitated from TIM-2 transfected TCMK cells and from A20
cells and analyzed by western blotting. Lanes were derived from the
same blot exposed for the same length of time; intervening irrelevant
lanes have been cropped out.
Figure 3. Specific uptake of HFt in TIM-2 expressing cells. A. HFt
was labeled with55Fe and incubated at a final concentration of 4 nM at
37uC with TIM-2 positive TCMK cells or TCMK vector controls. B. TIM-2
TCMK cells were incubated with55Fe-HFt in the presence or absence of
100-fold unlabeled iron-saturated HFt. C. A20 cells were incubated for
30 minutes with
unlabeled HFt. Shown are means and standard deviations of triplicate
determinations in a single experiment. The experiment was repeated 3
times with similar results.
55Fe-HFt in the presence or absence of 100-fold
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Electron microscopy observation
A total of 36106TIM-2 or vector transfectants were plated on
100 mm cell culture dishes and allowed to attach overnight before
150 mg HFt (fully saturated with iron) was added. The cells were
fixed in 2.5% gluteraldehyde and 2% osmium, dehydrated in
ethanol, scraped, and placed into a microcentrifuge tube. The
samples were infused with resin, cut into 80 nm sections, stained
with 33% lead citrate, and viewed with a Philips 400 transmission
electron microscope (Eindhoven, the Netherlands) at 60Kv.
Biotinylation of ferritin and capture of ferritin using
250 mg HFt was labeled with biotin using EZ-Link Sulfo-NHS-
LC-Biotin kit (Pierce Chemical Co., IL) following the protocol
provided by the manufacturer. Biotinylated HFt was dialyzed in
PBS overnight at 4uC. 125 mCi55Fe was loaded into biotinylated
HFt as described above, and biotinylated-55Fe-HFt was then
dialyzed in 0.1 M HEPES overnight. Three million TIM-2 or
vector transfectants were plated in 100 mm cell culture dishes and
allowed to attach overnight. Twomg/ml biotinylated-55Fe-HFt
(approximately 4 nM) was added to each plate and incubated at
37uC for 2 hours. Plates were then washed 3 times with PBS and
placed in growth media. Cells and media were collected at 0, 2, 4, 8,
24, and 48 h after incubation with biotinylated-55Fe-HFt. Cell
lysates were prepared by homogenization in whole cell lysis buffer
(25 mM Tris pH 7.4, 1% Triton X-100, 1%SDS , 1% sodium
deoxycholate, 150 mM NaCl , 2 mg/ml aprotinin, 1 mM PMSF,
complete protease inhibitor [Roche]) for 5 seconds. Samples were
Figure 4. Endogenous TIM-2 is not regulated by iron chelation. Triplicate cultures of A20 cells were incubated with control medium or
medium containing 1 mM ferric ammonium citrate (FAC), 10 mM deferoxiamine (DFO) or 50 mM deferoxiamine for 24 hrs. Cells were harvested and
expression of TIM-2 and TfR1 analyzed by western blotting. GAPDH was used as a loading control. B. Image intensities of the triplicate determinations
shown in (A) were quantified and normalized to GAPDH; means and standard deviations are shown. The experiment was repeated 3 times
independently with similar results.
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clarified by centrifugation at 12,000 x g at 4uC for 15 minutes.
Biotin-55Fe-HFt was immunoprecipitated from the supernatant by
incubation with streptavidin conjugated beads (Streptavidin –APC
[Jackson Immuno Research, PA]), followed by western blot
detection for HFt using polyclonal anti-HFt antibody . Extracts
depleted of biotinylated ferritin by incubation with streptavidin
beads were used for western blot detection of endogenous HFt.
TIM-2 mediates uptake of ferritin-bound iron
To test whether TIM-2 could function in iron uptake, we used a
mouse kidney epithelial cell line, TCMK-1. Since these cells do not
express detectable endogenous TIM-2, they provided a clean
to ferritin-dependent iron uptake. TCMK cells were transfected
with a TIM-2 expression vector or an empty vector control, and
stable transfectants were selected. TIM-2 expression in the
transfectants was examined at both protein and mRNA levels
(Figure 1). As expected, some TIM-2 transfectants expressed TIM-
2, but no TIM-2 was discernable in vector controls as measured by
western blot (Figure 1A) or real time RT-PCR (Figure 1B).
To confirm that TIM-2 was appropriately displayed on the cell
surface of transfected cells, we used flow cytometry. We compared
levels of cell surface TIM-2 in transfectants to A20 cells, a mouse
lymphocyte cell line that expresses endogenous TIM-2, as well as
to A20 cells that had been transfected with a TIM-2 expression
vector to increase expression of TIM-2 . As expected, A20
cells expressed endogenous TIM-2, and this increased in TIM-2
transfected A20 cells (Fig. 2). As shown in Figure 2, TIM-2
expression on the cell surface of TCMK-TIM-2 cells was
significantly higher than in vector controls, and of the same order
of magnitude as seen in A20 cells expressing endogenous TIM-2.
Immunoprecipitation confirmed that levels of TIM-2 in untreated
TCMK-TIM-2 transfectants were approximately equivalent to
levels of endogenous TIM-2 in untreated A20 cells (Fig. 2C).
To test whether cell surface TIM-2 could function as an uptake
mechanism for ferritin-bound iron, recombinant HFt was labeled
with55Fe. TCMK-TIM-2 and vector controls were incubated with
55Fe-HFt at 37uC. Cells were collected at intervals, washed, and
uptake increased in a time-dependent manner in TIM-2 transfec-
tants. No uptake was seen in vector controls. Uptake in TIM-2 cells
was specific, and could be effectively competed by a 100 fold molar
excess of unlabeled ferritin (Fig. 3B).55Fe-HFt was also taken up by
A20 cells, and uptake was inhibited by ferritin (Fig. 3C).
55Fe was measured. As shown in Figure 3A, iron
TIM-2 expression does not increase with iron chelation
Since iron transporters are frequently regulated by iron , we
queried whether levels of TIM-2 were responsive to changes in
levels of exogenous iron. We first examined the sequence of TIM-2
for the presence of a canonical iron responsive element (IRE). This
element is present in the untranslated regions of mRNAs encoding
proteins of iron uptake, storage, and export and serves as a target
site for binding of IRPs. No canonical IRE-like sequence was
evident in TIM-2 (data not shown).
We then directly measured the effect of altered iron levels on
expression of endogenous TIM-2 in A20 cells. Cells were either
left untreated, or treated with 1 mM ferric ammonium citrate,
10 mM deferoxamine (DFO, an iron chelator) or 50 mM DFO for
24 h, and TIM-2 expression was assessed by western blotting.
Expression of TfR1 was measured simultaneously as a control for
Figure 5. HFt bound to TIM-2 traffics to endosomes and lysosomes. A. TCMK-TIM-2 and vector controls were incubated with iron-loaded HFt
for 15 and 120 minutes at 37uC and observed by electron microscopy. A. TCMK-TIM-2 cells without added HFt. B. Vector transfectants treated with
iron-loaded HFt at 37uC for 120 minutes. C. HFt in early endosomes of TCMK-TIM-2 cells following 15 minutes of incubation with iron-loaded HFt at
37uC. D. HFt in endosomes or multivesicular bodies after 15 minutes incubation with TCMK-TIM-2 cells at 37uC. E. HFt in lysosomes after 120 minutes
incubation with TCMK-TIM-2 cells at 37uC. Arrows indicate iron-loaded HFt. 55,000X magnification. Scale bar equals 0.36 mm.
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effects of iron treatment. As shown in Fig. 4, treatment with iron
decreased TfR1, whereas treatment with the iron chelator DFO
increased TfR1, as expected. In contrast, there was no significant
effect of DFO on TIM-2, and iron appeared to increase rather
than decrease TIM-2. These results indicate that TIM-2 is not
regulated by iron status in the same way as TfR1.
Ferritin uptake by TIM-2 proceeds via an endocytic
To track the fate of iron-containing ferritin following uptake by
TCMK TIM-2 cells, we used electron microscopy, which enables
visualization of ferritin due to its electron-dense iron core. As
shown in Figure 5, when TIM-2 cells were incubated with iron-
saturated HFt for 15 minutes at 37uC, HFt was observed in coated
pits and endosomes (Fig. 5C and 5D). At 120 minutes, ferritin
appeared in lysosomes (Figure 5E). Uptake of ferritin was
(Fig. 6A–C). These observations suggested that ferritin uptake
was mediated by an endocytic pathway. To confirm this result, we
measured the uptake of
endocytosis inhibitor, phenylarsine oxide (PAO). As shown in
55Fe-HFt in the presence of the
Figure 6D, ferritin-mediated iron uptake was completely blocked
in cells treated with 10 mM PAO.
Intracellular iron released from HFt enters a bioavailable
To trace the fate of HFt-bound iron in cells, we prepared
biotinylated HFt, labeled it with
TCMK-TIM-2 cells for two hours. Cells were then washed, and
biotinylated HFt and its associated radioactivity were measured by
pulldown using streptavidin beads.55Fe in the cell lysate depleted
of biotinylated HFt was also measured. As shown in Figure 7A,
under these conditions iron was released from exogenous
(biotinylated) ferritin and transferred to a cellular fraction. Release
of iron occurred concomitantly with degradation of biotinylated
ferritin, consistent with processing through the lysosome (Fig. 5).
To test whether iron released from exogenous ferritin became
biologically available, we measured levels of endogenous ferritin,
since this protein is translationally upregulated by iron . As
shown in Figure 7B, release of iron from exogenously supplied
(biotinylated) ferritin was paralleled by an increase in endogenous
ferritin. The kinetics of the increase in endogenous ferritin were
55Fe, and incubated it with
Figure 6. Uptake of HFt by TIM-2 is temperature dependent. A. TCMK TIM-2 cells were incubated without HFt at 37uC. B. TCMK-TIM-2 cells
were prechilled and incubated on ice for 2 hours in the presence of HFt saturated with iron. C. TCMK-TIM-2 cells were incubated with iron-saturated
HFt at 37uC for 2 hours. Scale bar equals 0.36 mm. D. TIM-2 transfectants were incubated with55Fe-HFt in the presence or absence of of 10 mM
phenylarsine oxide (PAO), an endocytosis inhibitor. Shown are means and standard deviations of triplicate determinations. The experiment was
repeated 3 times independently with similar results.
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similar to those previously described following degradation of
ferritin in the lysosome . These results suggest that
released from HFt delivered through TIM-2 is able to enter a
biologically available pool, increase levels of intracellular ferritin,
and alter cellular iron status.
We previously demonstrated that TIM-2 serves as a specific
receptor for HFt in mouse cells . Results presented here
demonstrate that iron bound to HFt can serve as a cellular iron
source in mouse B cells or in mouse kidney cells that express TIM-
2. Following uptake in TIM-2-positive cells, HFt traffics to
endosomes and subsequently enters lysosomes (Fig. 5). This
pathway differs from the classical pathway of iron delivery
involving transferrin-mediated uptake of iron via TfR1, in which
transferrin recycles to the cell surface following iron release in the
endosome. Nevertheless, iron released from ferritin enters a
metabolically available pool, and is able to upregulate synthesis of
endogenous ferritin (Fig. 7).
Our work is consonant with other reports demonstrating both
the ability of ferritin to enter the lysosome and to serve as an iron
source. For example, recent work has shown that iron chelation
can induce entry of cytosolic ferritin into lysosomes, where it
undergoes degradation . Treatment of cells with cationic
ferritin similarly led to delivery of ferritin to lysosomes, iron
release, and induction of cytosolic ferritin synthesis . In human
cells, TfR1 serves as a receptor for HFt , and may functionally
substitute for TIM-2 . Receptor-mediated uptake of HFt
similarly directs ferritin to lysosomes in these cells .
Collectively, these results imply the existence of a mechanism for
the shuttling of iron from the lysosome to the cytosol. Candidate
transport mechanisms that may mediate iron efflux out of the
lysosome have recently been identified [39,40].
The relationship between TIM2 and Scara5, another recently
identified ferritin receptor that functions in iron trafficking during
development , is unclear. Unlike TIM-2, which selectively
binds HFt, Scara5 binds LFt. Similar to our results with TIM-2,
Scara5 mediates endocytosis of ferritin and ensuing delivery of
iron to cells. It was suggested that Scara5 may functionally
substitute for TfR1 in selected tissues of the kidney during
development . The relationship between expression of Scara5
and TIM-2 has not been examined. In the developing mouse,
Scara5 is expressed in stromal and capsular cells of the kidney as
Figure 7. Iron enters a metabolically available pool following TIM-2-mediated uptake of HFt. TCMK TIM-2 transfectants were incubated
with 4 nM biotinylated55Fe-HFt at 37uC for 2 hrs. The cells were washed 3 times with PBS and then changed to normal growth medium. Cell lysates
were prepared at 0, 2, 4, 8, 24, 48 hrs and biotin-55Fe-HFt was precipitated by incubation with streptavidin conjugated beads. Extracts depleted of
exogenous biotinylated ferritin following incubation with streptavidin beads were used to assess endogenous HFt. A.55Fe in biotinylated HFt and
cellular fraction depleted of biotinylated HFt. Means and standard deviations of triplicate determinations are shown. B. Biotinylated HFt and
endogenous ferritin were detected by western blotting. GAPDH was used as a loading control.
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well as in the airway, developing aorta, muscle bundles and
gonadal epithelia ; in the adult mouse, Scara5 is expressed in
epithelial cells of the testis, bladder, trachea, adrenal, skin, lung,
and ovary . This spatial distribution demonstrates little overlap
with TIM-2, which is expressed in adult B cells, bile duct epithelial
cells, renal tubules and oligodendrocytes, suggesting that in the
adult, expression patterns of these ferritin receptors are largely
independent. However, further work will be required to determine
whether Scara5 and TIM-2 exhibit any temporal or functional
Whether the TIM-2 pathway contributes in a substantial way to
iron import remains to be determined. Our results demonstrate
that TIM-2 can mediate uptake of ferritin and its associated iron;
however, the amount of iron delivered to cells through this
pathway will depend on the iron content of the ferritin, which can
vary over more than 2 orders of magnitude .
It is possible that the iron content of ferritin may influence the
ultimate cellular effect of TIM-2. For example, ferritin exhibits
immunosuppressive effects, inhibiting the proliferation of T and B
cells and the differentiation of myeloid cells [9,11,43,44]. Since
lymphocytes express TIM-2, it has been suggested that this anti-
proliferative effect of ferritin could be mediated through TIM-2
. We speculate that if ferritin contains little or no iron, it may
serve as a signaling molecule to induce such anti-proliferative or
apoptotic  effects. Alternatively, if receptor-bound ferritin
contains iron, it may serve as an iron source to support cell
proliferation. Delivery of iron through ferritin has previously been
proposed to occur in the developing kidney , and in
macrophage-mediated delivery of ferritin-bound iron to erythroid
precursors . Our results indicate that TIM-2 has the potential
to function as an iron delivery mechanism, but further work will be
required to elucidate the circumstances under which that
mechanism is activated.
Although specific binding of ferritin to cell surfaces has been
repeatedly documented [21,46,47,48,49,50], the source of ferritin
that binds to TIM-2 and other ferritin receptors in vivo remains
unknown. In our experiments, cells were exposed to 4 nM ferritin,
which is within the range found in the serum of patients with iron
overload and other inflammatory conditions such as Stills disease,
hemophagocytic syndrome, etc. . However, the majority of
subunits in serum ferritin resemble ferritin L more closely than
ferritin H [52,53], and since TIM-2 preferentially binds HFt, it is
not clear that serum ferritin represents a likely source of ferritin
ligand for TIM-2. Nevertheless, ferritin is a 24 subunit protein,
and most natural ferritins are heteropolymers of both H and L
subunits. Since the number of H subunits required for effective
binding of TIM-2 has not yet been determined, ferritin proteins
that contain a preponderance of L subunits may nonetheless bind
to TIM-2. In addition, small amounts of H subunit-rich ferritin are
present in the serum and can increase in certain pathological
conditions [53,54]. An alternative potential source of ferritin is
local secretion. For example, the macrophage, which can serve as
a source of circulating ferritin  may also secrete ferritin locally
[16,55], and this may serve as a paracrine source of ferritin for
uptake by TIM-2. Indeed HFt was identified as a soluble ligand
secreted by macrophage cell lines during the cloning of TIM-2
. HFt is also released by hepatocytes . Future studies will
be required to trace the physiological source of the HFt ligand.
Our results indicate that expression of TIM-2 is not increased
by iron chelation (Fig. 4). This distinguishes TIM-2 from other
iron uptake pathways. For example, TfR1, which mediates Tf-
dependent uptake of iron, and DMT-1, which mediates transport
of ferrous iron, are post-transcriptionally regulated by iron status
. For these transporters, regulation is mediated by IRE elements
on the 39 end of the mRNA. Our sequence inspection
demonstrated no obvious candidate IRE elements in the sequence
of TIM-2. However, since IRE elements may exhibit substantial
divergence in primary sequence , we cannot rule out the
presence of a non-canonical IRE element in TIM-2. The lack of
response of TIM-2 to iron chelation that we observed is different
from observations in oligodenodrocytes, in which expression of
TIM-2 was reported to be iron-regulated . Oligodendrocytes
have no TfR1 to mediate iron uptake , may depend heavily on
the TIM-2 pathway, and may have alternative mechanisms of
regulation not found in other cell types. In contrast, in A20 cells,
iron uptake is mediated by two pathways: the classical TfR1
pathway  and the TIM-2 pathway described here. It is possible
that uncoupled regulation of TfR1 and TIM-2 may permit
differential activation of these pathways. For example, in A20 cells
(and other cells that express both TfR1 and TIM-2), TIM-2 may
serve as a backup pathway that can respond to environmental
changes in HFt regardless of cellular iron status.
We thank Ken Grant and the Microscopy core facility of the
Comprehensive Cancer Center of Wake Forest University for assistance
with electron microscopy.
Conceived and designed the experiments: SVT WES FMT. Performed the
experiments: JH XD WW. Analyzed the data: MW. Contributed reagents/
materials/analysis tools: WES. Wrote the paper: JH SVT FMT WES.
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