|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3974-3979
The Effect of Apotransferrin on Iron Release From Caco-2 Cells,
an Intestinal Epithelial Cell Line
By
Xavier Alvarez-Hernandez,
Margaret Smith, and
Jonathan Glass
From the Section of Hematology/Oncology, Department of Medicine and
The Center for Excellence in Cancer Research, Louisiana State
University Medical Center, Shreveport, LA.
 |
ABSTRACT |
The Caco-2 cell line grown in bicameral chambers was used to study
the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as 59Fe on the apical surface of the Caco-2 cells, transport
of 59Fe into the basal chamber was stimulated by 50 µmol/L apotransferrin. Here, we examined the effect on
59Fe transport of lower concentrations of apotransferrin,
as well as the effects on transport of ovo-, cobalt-, and
ferri-transferrin and of iron chelators with an affinity for iron
greater than that of transferrin. The stimulation of 59Fe
transport was more sensitive to the presence of apotransferrin with a
Km of 0.078 ± 0.008 µmol/L compared with
ferri-transferrin with a Km of 1.24 ± 0.39 µmol/L
(P < .006). 59Fe transport was less sensitive to
diethylenetriaminopenta-acetic acid (DTPA) than
apotransferrin with Kms of 1.52 ± 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of
59Fe transport. Analysis of laser scanning confocal
micrographs showed that apotransferrin labeled with Texas Red is
internalized by Caco-2 cells from the basal side and localizes in
distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of
apotransferrin with the basal surface of the intestinal epithelium.
| |
INTRODUCTION |
THE TRANSPORT OF IRON across the
intestinal epithelium occurs in three phases: an uptake phase during
which iron is transported from the lumen of the intestine into an
apical compartment of the epithelial cells; a transport phase during
which iron is transported across the cell to the serosal or basolateral
surface of the cell; and a transfer phase during which the iron is
transported across the basolateral membrane to the plasma. This report
advances our understanding of the mechanisms operating in the transfer
phase.
The mechanism(s) controlling the transfer phase are enigmatic. The role
of transferrin (Tf), the plasma iron transport protein, in the
transfer phase has been evaluated in a variety of
studies.1-3 Transferrin receptors are present on the
basolateral surface of the intestinal epithelium and undergo recycling
as in other cells.4,5 These receptors may be involved in
iron uptake into intestinal epithelium for normal cellular metabolism.
The question addressed by a variety of studies is whether transferrin,
presumably interacting through these receptors, is directly involved in
iron release. There is evidence that iron transported across the
intestinal mucosa is released into the portal circulation in a low
molecular weight form to be taken up by the liver and then released
from the liver onto transferrin.6 Recent evidence from
studies with the hypotransferrinemic mouse also suggests that
transferrin is not involved in release and that three distinct,
although as yet unidentified, species of nontransferrin iron can be
detected.2,3 Other studies suggest a constitutive release
of iron from the epithelial cells with the released iron rapidly
binding to transferrin in a process dependent on a dialyzable moiety,
presumably bicarbonate.7 The Caco-2 cell line, when grown
as a polarized cell layer in bicameral chambers, has been used to
delineate various aspects of the uptake and transfer phases of
intestinal iron transport by other groups8-14 and by
us.15-18 We have previously shown that the transfer phase
is facilitated by the presence of apotransferrin (apo-Tf) in the
basal chamber16 and recently we have reported that
apo-Tf undergoes a different endocytic cycle than
ferri-transferrin (FeTf).18
The present studies were initiated to determine the specificity and
sensitivity of the effect of apotransferrin and if apotransferrin could
be visualized by laser scanning confocal microscopy within the Caco-2
cells. As part of these studies, we determined if the stimulation of Fe
transport by apo-Tf merely reflected exchange of iron from the small
portion of ferri-transferrin in the apo-Tf preparations with iron
within the cells. In addition, we determined the effect of
ovo-transferrin (ovo-Tf), which is purported not to
interact with the transferrin receptor, and cobalt-Tf, as Co bound to
Tf, is kinetically inert.19 Finally, to determine if apo-Tf
were serving merely as a chelator of iron, we examined the effect on
the transfer phase of chelators with a greater affinity for iron than
transferrin. We found that apo-Tf has unique characteristics that allow
apo-Tf to enhance the transfer phase of the process of iron absorption.
| |
MATERIALS AND METHODS |
Cell culture.
Caco-2 cells, from American Type Culture Collection #HTB37 (Rockville,
MD), were maintained in Dulbecco's modified Eagle medium supplemented
with 10% fetal bovine serum (FBS; GIBCO, Gaithersburg, MD), 1%
nonessential amino acids, and antibiotics/antimycotic (100 U/mL penicillin-G, 100 U/mL streptomycin, and 250 ng/mL
fungizone; GIBCO). Cells were grown in Transwell bicameral chambers
with 3 µmol/L pore size membrane (Costar, Cambridge, MA) coated with collagen. The collagen film was applied to the filter as 50 µL of
collagen solution (0.1 mg/mL, 60% ethanol; rat tail, type I; Boehringer, Mannheim, Germany) and then the Transwells were inverted and dried under a sterile laminar air flow. Formation of a monolayer was monitored by measuring the transepithelial electrical resistance (TEER) with a Millicell electrical resistance system (Millipore, Bedford, MA). Cell layers were used only after TEER had increased to
greater than 250 Ohms.cm2, a level indicating
the formation of an intact
monolayer.15-18,20,21
Transport of 59Fe into the basal chamber.
The transport of 59Fe from the apical chamber across the
Caco-2 cells into the basal chamber was studied as previously
described.15,16 First, the cell monolayers were depleted of
serum proteins by washing the monolayers twice with Dulbecco's
modified Eagle medium and then incubating the monolayers overnight with
Dulbecco's modified Eagle medium without FBS. Next, 1 µmol/L
59Fe (59FeCl3, 110-740 Bq/mg;
NEN-Dupont, Boston, MA) was offered to the apical surface in 1 mmol/L
ascorbate in 50 mmol/L HEPES buffered saline solution (HBS), pH 7.4, prepared from 193 mmol/L HEPES diluted with an isotonic salt solution
consisting of 130 mmol/L NaCl, 10 mmol/L KCl, 1 mmol/L
CaCl2, and 1 mmol/L MgSO4. The basal chamber
buffer was HBS to which were added apotransferrin (apo-Tf), ovo-transferrin (ovo-Tf), cobalt-transferrin (Co-Tf), bovine
serum albumin (BSA), diethylenetriaminopentacetate (DTPA),
DTPA-dextran, or nitrilotriacetate (NTA) at the indicated
concentrations. The TEER was monitored during the course of the
incubations and was constant in the presence of these buffers for at
least 2 hours. Apo-Tf was prepared from human Tf (Boehringer, Mannheim,
Germany), which was brought to pH 5.0 with HCl, dialyzed extensively
against Chelex beads (Bio-Rad, Hercules, CA) and then titrated to pH
7.0 with NaOH. The Fe saturation, measured by monitoring the absorption at 280 nm and 470 nm, was always less than 2%. Ovo-Tf was obtained from Canadian Lysozyme (Abbottford, British Columbia, Canada) and
apo-ovo-Tf prepared as for apo-Tf. Co2-Tf was prepared by the addition of CoCl2 to apo-Tf in the presence of 0.04 mol/L NaHCO3 and the preparation allowed to stand at
4°C for 7 days.22 The saturation of Tf with Co,
determined by measuring the absorption at 405 nm and using an
extinction coefficient of 8,900 mol/L-1cm-1,
was always 94% to 96%. BSA, DTPA, and NTA were obtained from Sigma
Chemicals (St Louis, MO) and DTPA-dextran from Molecular Probes (Eugene, OR). The moles of DTPA coupled to dextran were determined by addition of 59Fe citrate at different
concentrations to a solution of dextran-DTPA. After incubation at
25°C for 60 minutes, the dextran-DTPA-59Fe was
separated from unbound 59Fe citrate by ultracentrifugation
through a Amicon UM-10 filter (Amicon, Beverly, MA), the radioactivity
of the bound 59Fe determined, and the moles of DTPA per
mole of dextran calculated assuming one 59Fe bound per
DTPA. The lot of dextran-DTPA used was determined to have 17.1 ± 0.12 mol of DTPA per mole of dextran.
Uptake of 59Fe from the basal chamber.
In some experiments, 59Fe uptake into the cells was
measured from 59Fe,125I-transferrin in the
basal chamber. In these experiments
59Fe,125I-transferrin was prepared as
previously described,23 added to the basal chamber at
various concentrations up to 10 µmol/L for 60 minutes at 37°C,
the cells harvested as previously described,15 and the
59Fe radioactivity in the cells determined. In preliminary
experiments uptake into the cells was linear for at least 2 hours and
was not affected by the presence of Fe(II) ascorbate in the apical chamber. Nonspecific binding of 59Fe and
125I-transferrin was determined from the linear portion of
the binding curves.24
Laser scanning confocal microscopy.
Texas Red-labeled Tf (Tf-TxR) was obtained from Molecular Probes. The
Tf-TxR was rendered Fe-free as previously described for fetal calf
serum (FCS).15 Briefly, the pH of 2 mL of a
2-mmol/L solution of Tf-TxR was lowered to 4.5 with HCl in the presence of 0.5 g of Chelex resin (Bio-Rad), the pH of the solution was slowly
increased to 7.4 by the addition of small aliquots of NaOH with
constant stirring. The resin was removed by sedimentation, the
apotransferrin filter sterilized, and kept at  20°C until used.
Apo-Tf-TxR was offered on the basolateral surface of Caco-2 cells for
20 minutes. Transferrin was maintained in its apo form by the addition
of 1 mmol/L desferroxiamine (DFO; Ciba-Geigy, Summit, NJ) an iron
chelator. After this incubation period, the cell layer was washed with
HBS and fixed with paraformaldehyde following Bacallao's protocol for
confocal microscopy.25 The cell monolayer was also stained
with ToPro-1 (Molecular Probes) to show the nucleus. Caco-2 cell
monolayers were observed under a Laser Scanning Confocal Microscope
(LSCM). The LSCM used consists of a Bio-Rad MRC 1024 scan head
connected to a Nikon Diaphot microscope (Hercules, CA). Illumination of
specimens is by Ar/Kr and Argon lasers. A 60×, 1.4 NA Nikon
objective was used to observe the Caco-2 cell monolayer. The images at
different wavelengths were recorded in sequential mode. Image analysis
was accomplished with NIH-Image v1.60 software (NIH Image
is a public domain program, developed at the US National Institutes of
Health, available by anonymous FTP from zippy.nimh. nih.gov).
Statistical analysis and curve fitting.
Statistical analyses were performed using Instat v2.04 (Graph Pad
Software, San Diego, CA) using an unpaired t-test. Curve fitting was performed with GraFit Software (Erithacus Software, Ltd) using nonlinear least-squares regression analysis.
| |
RESULTS |
The sensitivity of the transport of 59Fe from Caco-2 cells
to apotransferrin.
Caco-2 cells, grown to confluency as determined by measurement of TEER
>250 ohms · cm2, were offered 59Fe
ascorbate on the apical surface with different concentrations of apo-Tf
in the basal chamber. The transport of 59Fe into the basal
chamber was measured after 1 hour. Preliminary experiments for these
studies, as also noted previously,16 showed linear
59Fe transport into the basal chamber for at least 2 hours
after an initial lag period of about 15 minutes. As shown in
Fig 1, the transport was sensitive to the
concentration of apo-Tf. A constitutive release of about 0.211 ± 0.049 pmoles of 59Fe/insert/hour was noted in the absence
of apo-Tf (mean ± standard deviation [SD] of 12 experiments). The
transport of 59Fe did not significantly increase until
apo-Tf concentrations of greater than 0.01 µmol/L apo-Tf were added
to the basal chamber. Maximal stimulation occurred by about 0.4 µmol/L apo-Tf. As noted in Table 1, the
data for apo-Tf showed a Km of about 0.08 µmol/L and a
Vmax of about 0.8 pmoles of 59Fe/insert/hour.
Addition of NaHC03 to the basal chamber did not affect the
transport of Fe at any concentration of apo-Tf (data not shown). The
basal chamber contained only apo-Tf, serum proteins were removed before
the 59Fe transport experiment (see Materials and Methods).
At all concentrations of apo-Tf added, the 59Fe released
into the basal chamber was precipitated with 60% ammonium sulfate.
Hence, the released 59Fe was all bound to Tf. Also, we have
shown using high-performance liquid chromatography (HPLC)
that when apo-Tf was present in the basal chamber, only a
59Fe-Tf peak is observed in the chromatogram.16
When apo-Tf was presented covalently linked to Sepharose beads, no
stimulation was noted even at apo-Tf concentrations of 10 µmol/L
(data not shown).
View larger version (28K):
[in this window]
[in a new window]
| Fig 1.
Caco-2 cells grown in bicameral chambers and depleted of
serum proteins were offered 1 µmol/L 59Fe ascorbate in
the apical chamber with the basal chamber containing either 0 to 50 µmol/L apotransferrin ( ) or 0 to 10 µmol/L ferri-transferrin ( ). The cells were incubated at 37°C for 1 hour and the moles of
59Fe transported into the basal chamber were determined.
Presented are the mean ± SD of triplicates for three experiments.
When not apparent, the error bars are smaller than the symbols. The
curves were fit to the data with Michaelis-Menton kinetics using
nonlinear regression analysis.
|
|
Fe2-Tf also stimulated release of 59Fe as shown
in Fig 1. The Vmax was not significantly greater than that
for apo-Tf. However, stimulation of 59Fe transport required
about a 16-fold higher concentration of Fe2-Tf than with
apo-Tf. With Fe2-Tf in the basal chamber, 59Fe
transport continued at about the constitutive level until about 0.6 µmol/L Fe2Tf with a Km of 1.2 µmol/L and a
Vmax of 1.1 pmoles of 59Fe/insert/hour. In
contrast to apo-Tf stimulated 59Fe transport, only about
50% of the released 59Fe could be precipitated by 60%
ammonium sulfate at the Vmax for Fe2-Tf.
Does stimulation of 59Fe transport reflect exchange of Fe
by the Caco-2 cells with Fe on Fe-Tf in the basal chamber?
Despite the effort to maintain apo-Tf as the Fe depleted moiety, it is
difficult to maintain apo-Tf fully depleted of bound Fe, as the
affinity of Tf for Fe is high and Fe is ubiquitous in reagents. Hence,
it is possible that Fe release from the Caco-2 cells represented merely
exchange of Fe from the cell for Fe being delivered by Tf in the basal
chamber. Spectrophotometric determination of Fe on apo-Tf just before
the addition of apo-Tf to the basal chamber suggested no more than 1%
to 2% Fe-Tf in the apo-Tf preparation. Nonetheless, the following
experiments were undertaken to exclude the possibility that the
stimulation of Fe release from the cells merely represented exchange of
intracellular Fe with Fe carried on Tf added to the basal chamber. In
these studies, the rate of 59Fe uptake into cells was
measured from 59Fe-Tf in the basal chamber. If transport
merely represented exchange of Fe from Fe being taken up from Fe-Tf in
the basal chamber, then the rate of transport out of the cell should be
similar to the rate of 59Fe transport into the cell. The
uptake of 59Fe from 59Fe2-Tf in the
basal chamber showed saturation after 1 µmol/L
59Fe2-Tf. The data were also fitted with
Michaelis-Menton kinetics and exhibited a Km = 0.733 ± 0.197 µmol/L and a Vmax = 19.8 ± 1.6 pmoles of
59Fe/insert/hour (mean ± SD of three experiments). At
the Km for apo-Tf and assuming that the apo-Tf contained
1% to 2% Fe-Tf based on spectrophotometric determination, the
concentration of Fe-Tf would be about 0.0008 to 0.0016 µmol/L and the
predicted Fe uptake of about 0.02 to 0.04 pmoles of
Fe/insert/hour would be far less than the observed constitutive
release. Likewise, at the concentration of apo-Tf required to reach the
Vmax for apo-Tf, the predicted uptake of Fe from the 1% to
2% Fe-Tf present would be approximately 0.1 to 0.2 pmoles of
Fe/insert/hour; this uptake rate is about eightfold to 16-fold less
than the observed transport rate into the basal chamber. Hence, it
appears unlikely that exchange of Fe between the small fraction of Fe
in the apo-Tf preparation is accounting for the transport into the
basal chamber.
The effect of Co-Tf, ovo-Tf, and BSA on Fe transport.
Transferrin carrying other metals, homologues of transferrin,
and other metal binding proteins can participate in Fe uptake and
release from various cells and do so with characteristics different
from Tf. It was of interest then to see if any of these other proteins
added to the basal chamber could effect Fe transport. For example, Co
bound to Tf is kinetically inert.19 Hence, if the Fe
binding sites were occupied with Co and if these sites on Tf were
important in the stimulation of Fe transport, then 59Fe
transport into the basal chamber should be inhibited by Co-Tf. As seen
in Table 1, Co-Tf did not inhibit, but stimulated the rate of
transport. Unfortunately, the preparations of Co-Tf always contained
4% to 6% unoccupied metal binding sites and this amount of apo-Tf
could account for the observed increase in Vmax. This conclusion is supported by the finding that all of the 59Fe
released in the presence of Co-Tf was precipitated by 60% ammonium sulfate.
The effect of ovo-Tf in the basal chamber was examined because ovo-Tf
has an affinity for Fe similar to that of Tf, but is reported to
interact poorly with the transferrin receptor.26,27 Hence,
the addition of ovo-Tf would supply a protein, which binds Fe
analogously to Tf but which should not interact with the transferrin receptor. To our surprise, when we investigated 59Fe uptake
from 1 µmol/L 59Fe-ovo-Tf in the basal chamber, the
Caco-2 cells exhibited an uptake of about 10 pmoles
59Fe/insert/hour. This uptake was similar to that observed
from 59Fe2-Tf in the basal chamber. Despite the
Caco-2 cells being able to acquire 59Fe from ovo-Tf,
apo-ovo-Tf had a slight, nonsignificant effect of 59Fe
transport into the basal chamber. The slight effect had a sensitivity similar to that of apo-Tf and was not inhibited by the presence of
antibodies, which block the binding of ovo-Tf to the ovo-Tf receptor
(kindly provided by Dr Anne B. Mason, Department of
Biochemistry, University of Vermont College of Medicine, Burlington,
VT). Again, all of the 59Fe released from the cells in the
presence of ovo-Tf was precipitated with 60% ammonium sulfate,
suggesting that the released 59Fe was bound to the protein.
Finally, the effect of BSA in the basal chamber was examined because of
the known metal binding sites on BSA and the inability of BSA to
interact with the transferrin receptor. No increase in constitutive
release was observed until high concentrations of BSA were added and no
dose response curve was observed. At 2 and 10 µmol/L BSA, the
59Fe transport rate was 0.360 ± 0.059 and 0.31 ± 0.021 pmoles/insert/hour, respectively (mean ± SD of three
experiments with triplicates for each concentration).
The effect of metal chelators on 59Fe transport.
The effect of two chelators, NTA and DTPA, on Fe transport was
examined. The Ka for Fe(III) for these chelators is
1024 for NTA and 1028 for DTPA.28
These values compare with the Ka of 1021 of Tf
for Fe. NTA had no effect on transport until 40 µmol/L at which
concentration the observed transport of 59Fe was 0.393 ± 0.049 pmoles/insert/hour (mean ± SD of three experiments with
triplicates for each concentration). As noted in Table 1, DTPA did
stimulate transport with a Km of about 1.5 µmol/L and a
Vmax of nearly 3 pmoles/insert/hour, the highest observed
rate of transport. To be certain that DTPA was not merely shifting equilibrium by acting as a sink for intracellular 59Fe,
rates of transport were measured in the presence of 10 µmol/L DTPA
and were found to be linear for at least 2 hours. This observation supports DTPA as having an effect on the rate of release of iron from
the cells. To determine if the parameters measured for DTPA reflected
merely the relatively low molecular weight of DTPA and hence its
ability to find access to 59Fe, DTPA was also presented in
the basal chamber bound to dextran of molecular weight 70,000 daltons.
For the experiments using DTPA-dextran, the amount of DTPA per molecule
of dextran was determined by titrating the DTPA-dextran with increasing
amounts of 59Fe-citrate, separating the unbound
59Fe from the DTPA-dextran bound 59Fe by
ultrafiltration and then expressing the concentration of DTPA-dextran
as molarity of DTPA. The Km for DTPA-dextran was not
significantly different from DTPA alone and although the
Vmax was less than for DTPA, the Vmax was
significantly more than that observed for the various transferrins
(Table 1).
To determine if apo-Tf and DTPA were stimulating transport from the
same or different compartments, 59Fe transport was measured
with both apo-Tf and DTPA in the basal chamber and compared with the
transport with both entities alone. We found no additive effects of
apo-Tf and DTPA, suggesting that apo-Tf and DTPA were stimulating
59Fe transport from the same compartment.
The localization of apotransferrin by confocal microscopy.
Apotransferrin binds to the transferrin receptor with an affinity far
less that Fe-transferrin.29-32 As a consequence, in many cell types, it is not possible to show an interaction with apo-Tf. Hence, given the exquisite sensitivity of apo-Tf in stimulating Fe
transport from Caco-2 cells, it was important to show that apo-Tf could
gain entry into the Caco-2 cells. The internalization from basolateral
surface of apotransferrin into vesicles within the Caco-2 cells was
assessed by laser scanning confocal microscopy using apo-Tf labeled
with Texas Red.
In these studies, as shown in Fig 2A, the
Caco-2 cell monolayer was tilted in such a way that one could observe
the apical or supranuclear side, at the right, and the basal or
infranuclear side, at the left, of the cells in the same confocal
micrograph. The scheme in Fig 2A depicts the viewpoint for observation
and the angle at which the monolayer was tilted. Figure 2B is a typical fluorescent micrograph showing the Caco-2 cell nuclei, stained with
ToPro-1, in green, and vesicles, containing apo-Tf-TxRed, in red.
Because of the optical slicing power of laser confocal microscopy, it
is possible to determine clearly if vesicles are above or below the
nucleus. In similar studies (data not shown), Fe2-Tf-TxRed
could be located in vesicles mainly beneath the nucleus.
View larger version (70K):
[in this window]
[in a new window]
| Fig 2.
Confocal microscopy of apotransferrin in a Caco-2 cell
monolayer. Caco-2 cell monolayers were offered apo-Tf-TxRed from the basal side for 20 minutes. The cell layers were counterstained with
ToPro-1 to show the nucleus in green and the monolayers observed by
laser scanning confocal microscopy as described in Materials and
Methods. Shown is a photomicroghraph of a section through a typical
monolayer. As shown in the schematic (A), the cell layer was tilted so
that the optical section (B) passed through the basal and apical
portions of the cells constituting the monolayer. Note the vesicles
above the nucleus at the right side of the figure. Because of the
optical slicing power of laser confocal microscopy, it is possible to
determine clearly in this figure if a vesicle is above or below the
nucleus.
|
|
| |
DISCUSSION |
During intestinal Fe absorption, the mechanism of the transfer phase,
that is, how Fe is transported out of the basal surface of the
intestinal epithelium, remains elusive. The solubility and reactivity
of Fe dictate that Fe be bound to a cellular constituent during
transport through cellular compartments.33 Recently, a
divalent cation transporter (DCT1) has been implicated as a metal
transporter involved in the uptake step.34 Other studies have suggested that a newly described protein, Mobilferrin, and a
 3-integrin are also involved in the uptake
process.35,36 Studies from our laboratory suggest that a
moiety with a basic Iso-electric point (pI) and
transferrin, but not ferritin, are involved in intracellular
transport.16 Recently, we have reported that apo-Tf and
FeTf undergo a different endocytic cycle.18 How then is Fe
in various intracellular compartments transported across the basal
surface and how do the observations presented here, as well as others
studies, showing greater or lesser involvement of transferrin in iron
transport comport with the intracellular handling of Fe?
The Caco-2 cells exhibit a constitutive rate of transport of Fe that
was stimulated about fourfold by the presence of apo-Tf. While iron
depletion of the cells did not significantly alter the constitutive
rate, the stimulation of transport by apo-Tf was markedly enhanced by
iron depletion.16 The effect of the apotransferrin added to
the basal chamber was to stimulate the rate of release of Fe and not
merely to shift the equilibrium. This observation was supported by the
lack of stimulatory effect of (1) apo-Tf bound to Sepharose beads; (2)
ovo-Tf, which has a Ka for Fe similar to apo-Tf; and (3)
NTA, a chelator with a Ka for Fe greater than that of
apo-Tf. The stimulation of Fe transport observed with Fe-Tf and Co-Tf
represented the unsaturated metal binding sites present in the Fe-Tf
and Co-Tf preparations.
Our studies are also compatible with the Fe transport, which occurs in
the hypotransferrinemic mouse.2,3 As with the mice, the
Caco-2 cells have a constitutive rate of iron transport in the absence
of added apo-Tf. In the hypotransferrinemic mice, the intestinal mucosa
senses the anemia and increases intestinal Fe absorption. In the model
system of Caco-2 cells, the cells recognize the iron deficient state
and increase transport.15 As noted
previously,16 the Tf concentration in the
hypotransferinemic mice is about 0.1 µmol/L, a level that will
stimulate Fe transport from Caco-2 cells. In human plasma, Tf
concentration is about 50 µmol/L. As only about one third of the iron
binding sites on plasma Tf are occupied, there will be ample apo-Tf to
stimulate Fe transfer. These observations suggest a model in which
apo-Tf, or unoccupied Fe sites on Tf, reaches a compartment of newly
absorbed iron within the epithelial cell and accelerates transfer of
that iron out of the cell.
The sensitivity of transport to the presence of apo-Tf suggests that
apo-Tf has a unique ability to interact with the basal surface of the
Caco-2 cells. This hypothesis is supported in several ways. First, as
detailed in the Results, the release of 59Fe from the cells
could not be merely exchange of 59Fe in the cells for Fe on
Tf in the basal chamber. The interaction of apo-Tf with the cells to
stimulate transfer of 59Fe out of the cells need not be
through the transferrin receptor. Recently transferrin has been noted
to interact with heparin sulfate proteoglycan and this interaction can
affect the rate of release of Fe from transferrin.37
Second, the stimulation of 59Fe transfer is not an effect
of the affinity of a chelator for Fe. DTPA with an affinity for Fe
greater than that of Tf, had a Km for stimulating transport
of 59Fe that was more than 15-fold greater than for apo-Tf.
Neither ovo-Tf, with a Ka for Fe similar to apo-Tf, nor
NTA, with a Ka greater than apo-Tf, had an effect on Fe
release. These results suggest that apo-Tf has a greater ability to
interact with the Fe release site than does a homologue of Tf or the
chelators DTPA or NTA, which have a greater affinity for Fe. Third,
using confocal microscopy, it was possible to show that apo-Tf
proceeded into vesicular compartments within the Caco-2 cells and that
these apo-Tf vesicles reached the apical portions above the nucleus, while in contrast, Fe2Tf was located primarily beneath the
nuclei. As newly absorbed iron is found in intestinal epithelium at the terminal web, the ability of the cells to sort apo-Tf to this region
may explain why apo-Tf stimulates iron transport. In toto these
experiments suggest that apo-Tf has a unique mechanism independent of
the traditional Tf receptor for interacting with the basal surface of
Caco-2 cells.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted December 21, 1997.
Supported in part by Grants No. DK-41279 and DK-37866 from the National
Institutes of Health, Bethesda, MD, and grants from the
Center for Excellence in Cancer Research, Treatment, and Education, Louisiana State University Medical Center, Shreveport, LA.
Address reprint requests to Xavier Alvarez-Hernandez, PhD, 1501 Kings
Highway, Shreveport, LA 71130.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
REFERENCES |
1.
Savin MA,
Cook JD:
Mucosal iron transport by rat intestine.
Blood
56:1029,
1980[Abstract/Free Full Text]
2.
Buys SS,
Martin CB,
Eldridge M,
Kushner JP,
Kaplan J:
Iron absorption in hypotransferrinemic mice.
Blood
78:3288,
1991[Abstract/Free Full Text]
3.
Simpson RJ,
Lombard M,
Raja KB,
Thatcher R,
Peters TJ:
Iron absorption by hypotransferrinaemic mice.
Br J Haematol
78:565,
1991[Medline]
[Order article via Infotrieve]
4.
Anderson GJ,
Powell LW Halliday JW:
Transferrin receptor distribution and regulation in the rat small intestine: Effect of iron stores and erythropoiesis.
Gastroenterology
98:576,
1990[Medline]
[Order article via Infotrieve]
5.
Hughson EJ,
Hopkins CR:
Endocytic pathways in polarized Caco-2 cells: Identification of an endosomal compartment accessible from both apical and basolateral surfaces.
J Cell Biol
110:337,
1990[Abstract/Free Full Text]
6.
Morgan EH:
The role of plasma transferrin in iron absorption in the rat.
Q J Exp Physiol
65:239,
1980
7.
Snape S,
Simpson RJ:
Iron binding to, and release from, the basolateral membrane of mouse duodenal enterocytes.
Biochim Biophys Acta
1074:159,
1991[Medline]
[Order article via Infotrieve]
8. Halleux C, Schneider YJ: Iron absorption by intestinal epithelial
cells: 1. Caco-2 cells cultivated in serum-free medium, on
polyethyleneterephthalate microporous membranes, as an in vitro model.
In Vitro Cell Dev Biol 27A:293, 1991
9.
Halleux C,
Schneider YJ:
Iron absorption by Caco-2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier.
J Cell Physiol
158:17,
1994[Medline]
[Order article via Infotrieve]
10.
Han O,
Failla ML,
Hill AD,
Morris ER,
Smith JC:
Reduction of Fe(III) is required for uptake of nonheme iron by Caco-2 cells.
J Nutr
125:1291,
1995
11.
Sanchez L,
Ismail M,
Liew FY,
Brock JH:
Iron transport across Caco-2 cell monolayers. Effect of transferrin, lactoferrin and nitric oxide.
Biochim Biophys Acta
1289:291,
1996[Medline]
[Order article via Infotrieve]
12.
Brock JH,
Ismail M,
Sanchez L:
Interaction of lactoferrin with mononuclear and colon carcinoma cells.
Adv Exp Med Biol
357:157,
1994[Medline]
[Order article via Infotrieve]
13.
Garcia MN,
Flowers C,
Cook JD:
The Caco-2 cell culture system can be used as a model to study food iron availability.
J Nutr
126:251,
1996
14.
Glahn RP,
Wein EM,
Van Campen DR,
Miller DD:
Caco-2 cell uptake from meat and casein digests parallels in vivo studies: Use of a novel method for rapid estimation of iron bioavailability.
J Nutr
126:332,
1996
15.
Alvarez-Hernadez X,
Nichols GM,
Glass J:
Caco-2 cell line: A system for studying intestinal iron transport across epithelial cell monolayers.
Biochim Biophys Acta
1070:205,
1991[Medline]
[Order article via Infotrieve]
16.
Alvarez-Hernadez X,
Smith M,
Glass J:
Regulation of intestinal iron uptake by transferrin in Caco-2 cells, an intestinal cell line.
Biochim Biophys Acta
1192:215,
1994[Medline]
[Order article via Infotrieve]
17.
Nunez MT,
Alvarez-Hernandez X,
Smith M,
Tapia V,
Glass J:
Role of redox systems on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells.
Am J Physiol
267:C1582,
1994[Abstract/Free Full Text]
18.
Nunez MT,
Nunez-Millacura C,
Beltran M,
Tapia V,
Alvarez-Hernandez X:
Apotransferrin and holotransferrin undergo different endocytic cycles in intestinal epithelia (Caco-2) cells.
J Biol Chem
272:19425,
1997[Abstract/Free Full Text]
19.
Bali PK,
Harris WR,
Nesset-Tollefson D:
Kinetics of iron removal from monoferric and cobalt-labeled monoferric human serum transferrin by nitriotris(methylenephosphonic acid) and nitriloacetic acid.
Inorg Chem
30:502,
1991
20.
Hidalgo IJ,
Kato A,
Borchardt RT:
Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers.
Biochem Biophys Res Commun
160:317,
1989[Medline]
[Order article via Infotrieve]
21.
Audus KL,
Bartel RL,
Hidalgo IJ,
Borchardt RT:
The use of cultured epithelial and endothelial cells for drug transport and metabolism studies.
Pharm Res
7:435,
1990[Medline]
[Order article via Infotrieve]
22.
Aisen P,
Aasa R,
Redfield AG:
The chromium, manganese, and cobalt complexes of transferrin.
J Biol Chem
244:4628,
1969[Abstract/Free Full Text]
23.
Glass J,
Nuñez MT,
Fischer S,
Robinson SH:
Transferrin receptors, iron transport and ferritin metabolism in Friend erythroleukemia cells.
Biochim Biophys Acta
542:441,
1978
24.
Hamazaki S,
Glass J:
Non-transferrin dependent 59Fe uptake in phytohemagglotinin-stimulated human peripheral lymphocytes.
Exp Hematol
20:436,
1991
25. Bacallao R, Kiai K, Jesaitis L: Guiding principles of specimen
preservation for confocal fluorescence microscopy, in Pawley JB (ed):
Handbook of Biological Confocal Microscopy. New York, NY, Plenum, 1995, p 311
26.
Zapolski EJ,
Princiotto JV:
Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.
Biochim Biophys Acta
421:80,
1976[Medline]
[Order article via Infotrieve]
27.
Shimo-Oka T,
Hagiwara Y,
Ozawa E:
Class specificity of transferrin as a muscle trophic factor.
J Cell Physiol
126:341,
1986[Medline]
[Order article via Infotrieve]
28. Martell AE, Smith RM: Critical Stability Constants (vol 5). New
York, NY, Plenum, 1989
29.
Young SP,
Bomford A,
Williams R:
The effect of the iron saturation of transferin on its binding and uptake by rabbit reticulocytes.
Biochem J
219:505,
1984[Medline]
[Order article via Infotrieve]
30.
Dautry-Varsat A,
Ciechanover A,
Lodish HF:
pH and the recycling of transfemn during receptor-mediated endocytosis.
Proc Natl Acad Sci USA
80:2258,
1983[Abstract/Free Full Text]
31.
Klausner RD,
Ashwell G,
van Renswoude J,
Harford JB,
Bridges KR:
Binding of apotransferrin to K562 cells explanation of the transferrin cycle.
Proc Natl Acad Sci USA
80:2263,
1983[Abstract/Free Full Text]
32.
Harding C,
Stahl P:
Transferin recycling in reticulocytes: pH and iron are important detemminants of ligand binding and processing.
Biochem Biophys Res Commun
113:650,
1983[Medline]
[Order article via Infotrieve]
33.
Crichton RR,
Ward RJ:
Iron metabolism  new perspectives in view.
Biochemistry
31:11255,
1992[Medline]
[Order article via Infotrieve]
34.
Gunshin H,
Mackenzie B,
Berger U,
Gunshin Y,
Romero M,
Boron W,
Nussberger S,
Gollan J,
Hediger M:
Cloning and characterization of a mammalian proton-coupled metal-ion transporter.
Nature
388:482,
1997[Medline]
[Order article via Infotrieve]
35.
Conrad ME,
Umbreit JN,
Peterson RDA,
Moore EG,
Harper KP:
Function of integrin in duodenal mucosal uptake of iron.
Blood
81:517,
1993[Abstract/Free Full Text]
36.
Conrad ME,
Umbreit JN:
Iron absorption The mucin-mobilferrin-integrin pathway. A competitive pathway for metal absorption.
Am J Hematol
42:67,
1993[Medline]
[Order article via Infotrieve]
37.
Regoeczi E,
Chindemi PA,
Hu WL:
Interaction of transferrin and its iron-binding fragments with heparin.
Biochem J
299:819,
1994
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y. Ma, M. Yeh, K.-y. Yeh, and J. Glass
Iron Imports. V. Transport of iron through the intestinal epithelium
Am J Physiol Gastrointest Liver Physiol,
March 1, 2006;
290(3):
G417 - G422.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Moriya and M. C. Linder
Vesicular transport and apotransferrin in intestinal iron absorption, as shown in the Caco-2 cell model
Am J Physiol Gastrointest Liver Physiol,
February 1, 2006;
290(2):
G301 - G309.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. E. Serfass and M. B. Reddy
Breast Milk Fractions Solubilize Fe(III) and Enhance Iron Flux across Caco-2 Cells
J. Nutr.,
February 1, 2003;
133(2):
449 - 455.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. R. Follett, Y. A. Suzuki, and B. Lonnerdal
High specific activity heme-Fe and its application for studying heme-Fe metabolism in Caco-2 cell monolayers
Am J Physiol Gastrointest Liver Physiol,
November 1, 2002;
283(5):
G1125 - G1131.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Ma, R. D. Specian, K.-Y. Yeh, M. Yeh, J. Rodriguez-Paris, and J. Glass
The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium
Am J Physiol Gastrointest Liver Physiol,
October 1, 2002;
283(4):
G965 - G974.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. N. Roy and C. A. Enns
Iron homeostasis: new tales from the crypt
Blood,
December 15, 2000;
96(13):
4020 - 4027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Alvarez-Hernandez, M. Smith, and J. Glass
Apo-transferrin is internalized and routed differently from Fe-transferrin by Caco-2 cells: a confocal microscopy study of vesicular transport in intestinal cells
Blood,
January 15, 2000;
95(2):
721 - 723.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ferruzza, M.-L. Scarino, G. Rotilio, M. R. Ciriolo, P | |
Abstract
Introduction
Abstract