1 Department of Pharmaceutics, Royal Danish School of Pharmacy; and 2 Department of Zoophysiology, August Krogh Institute, DK-2100 Copenhagen, Denmark
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human intestinal
cell line Caco-2 was used as a model system to study the effects of
epidermal growth factor (EGF) on peptide transport. EGF decreased
apical-to-basolateral fluxes of [14C]glycylsarcosine
([14C]Gly-Sar) up to 50.2 ± 3.6%
(n = 6) of control values. Kinetic analysis of the
fluxes showed that maximal flux (Vmax) of
transepithelial transport decreased from 3.00 ± 0.17 nmol · cm2 · min
1 in
control cells to 0.50 ± 0.07 nmol · cm
2 · min
1 in cells
treated with 5 ng/ml EGF (n = 6, P < 0.01). The apparent Michaelis-Menten constant
(Km) was 2.71 ± 0.31 mM (n = 6) in control cells and 1.89 ± 0.28 mM (n = 6, not significantly different from control) in EGF-treated cells.
Similarly, apical uptake of [14C]Gly-Sar decreased in
cells treated with EGF, with an ED50 value of 0.36 ± 0.06 ng/ml (n = 6) EGF and a maximal inhibition of
80 ± 0.02% (n = 6). Vmax
decreased from 2.61 ± 0.4 to 1.06 ± 0.1 nmol · cm
2 · min
1
(n = 3, P < 0.05), whereas
Km remained constant. Basolateral Gly-Sar uptake
showed no changes in Vmax or
Km after EGF treatment (n = 3).
RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We
conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells
by decreasing the number of peptide transporter molecules in the apical membrane.
intestinal oligopeptide transporter; growth factor; immunocytochemistry; laser scanning confocal microscopy
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN MAMMALIAN SMALL
INTESTINE, intact di- and tripeptides are transported from the
intestinal lumen into the cells via a H+-peptide
cotransporter, PepT1. The absorption of several important orally
pharmacologically active compounds such as -lactam antibiotics, angiotensin-converting enzyme, and renin inhibitors also depends to
some extent on human (h)PepT1-mediated transport (8, 17, 19,
21). The transporter has been cloned from human
(23), rat (29), rabbit (3, 10)
mouse (11), and Caco-2 (36) cells. hPepT1
consists of 708 amino acids and has 12 putative membrane-spanning
domains and a core molecular mass of ~79 kDa (23). PepT1 is exclusively located in the apical
membrane of mature enterocytes in the absorptive epithelium covering
the villi (26). Transport of peptides from the cytosol to
the blood is mediated via a not yet fully characterized di-/tripeptide
transport system located in the basolateral membrane (28,
34).
Little is known about regulation of PepT1 transport activity. Two
potential sites for protein kinase C phosphorylation have been
identified in hPepT1 (23), and it has been shown that
peptide transporter activity is downregulated in Caco-2 cells exposed to phorbol esters (4). Peptide transporter activity is
also influenced by cAMP (25), insulin (33),
the -receptor ligand (+)-pentazocine (13), and luminal
dipeptides (30, 32, 36).
Overall, the information on hormonal and growth factor-mediated regulation of peptide transport activity is sparse. In the present study we used the Caco-2 cell line to investigate the effects of treatment with epidermal growth factor (EGF) on peptide transport and hPepT1 expression. EGF is a peptide growth factor consisting of 53 amino acids that stimulates the proliferation of epidermal cells and a variety of other epithelial and nonepithelial cell types (12). The present results showed a decrease in transcellular transport and apical uptake of the nonhydrolyzable peptide glycylsarcosine (Gly-Sar), a decrease in total hPepT1 protein content, and a decrease in hPepT1 mRNA after long-term EGF treatment of Caco-2 cells. We conclude that EGF downregulates Gly-Sar transport in Caco-2 cells by decreasing hPepT1 mRNA and thereby hPepT1 expression.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). Cell culture media and human recombinant lyophilized EGF were purchased from Life Technologies (Høje Taastrup, Denmark). Hanks' balanced salt solution (HBSS) was obtained from Life Technologies. BSA, 2-(N-morpholino)ethanesulfonic acid (MES), and HEPES were from Sigma (St. Louis, MO). 14C-labeled Gly-Sar with a specific activity of 49.94 mCi/mmol and [3H]mannitol with a specific activity of 51.50 mCi/mmol were from NEN (Boston, MA). Restriction enzymes PstI, SacI, and EcoRI were from Amersham Pharmacia Biotech (Little Chalfont, UK). Rabbit-anti-hPepT1, raised against a peptide corresponding to the last 15 carboxy-terminal amino acid residues of the human peptide transporter hPepT1, was a generous gift from Dr. Wolfgang Sadée (San Francisco, CA) (15). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Bio-Rad (Hercules, CA). Alexa 488-conjugated goat anti-rabbit IgG, Alexa 488-conjugated phalloidin, and propidium iodide were from Molecular Probes (Eugene, OR).
Cell culture.
Caco-2 cells at passage 20 were seeded in culture flasks and
passaged in DMEM supplemented with 10% fetal bovine serum,
penicillin-streptomycin (100 U/ml and 100 µg/ml, respectively), 1%
L-glutamine, and 1% nonessential amino acids. When cells
reached passages 30-50, they were seeded onto tissue
culture-treated Transwells (4.7 cm2, 0.4-µm pore size) at
a density of 105 cells/cm2. Monolayer cultures
were grown in an atmosphere of 5% CO2-95%O2 at 37°C. Growth media were replaced every other day. EGF was
dissolved in sterile water to a concentration of 1 µg/ml and stored
in aliquots at 20°C. On the day of use, EGF was dissolved in the
culture medium and added to the monolayers (at both the apical and the basolateral sides), except when used examinations of the sidedness (i.e., apical vs. basolateral) of the inhibitory effect of EGF on
dipeptide transport. Transepithelial electrical resistance (TEER) was
measured during growth in tissue resistance measurement chambers
(Endohm) with a voltohmmeter (EVOM), both of which were from World
Precision Instruments (Sarasota, FL). Dipeptide transport activity
reached a steady maximal level at days 24-30. Transport experiments were subsequently performed on days 26-28
after seeding; isolation of protein and RNA was performed on day
24.
Transport experiments. Transport of [14C]Gly-Sar was measured in HBSS supplemented with 0.05% BSA. Apical media were buffered with 10 mM MES, and pH was adjusted to 6.0; basolateral media were buffered with 10 mM HEPES and adjusted to pH 7.4. The cell monolayers were rinsed once in prewarmed HBSS and placed on a shaking plate heated to 37°C. The cells were allowed to equilibrate for 1 h in the relevant experimental solutions without radioisotopes. The experiment was initiated by adding fresh apical buffer containing varying amounts of Gly-Sar (0-5 mM), 0.5 µCi/well [14C]Gly-Sar, and 0.5 µCi/well [3H]mannitol. Twenty-microliter samples were taken from the apical solution at t = 0, 60, and 120 min. One hundred-microliter samples were taken at 15-min intervals from the basolateral solution and replaced with fresh buffer (t = 0-120 min). Samples were transferred to counting vials, scintillation fluid was added (Ultima Gold; Packard, Canberra, Australia), and radioactivity was counted in a liquid scintillation analyzer. Fluxes of Gly-Sar and mannitol were constant after 60 min. The steady-state flux values of Gly-Sar and mannitol were therefore obtained as the means of the flux values at 90, 105, and 120 min.
Glycylsarcosine uptake experiments. Uptake of [14C]Gly-Sar was initiated as described for transport experiments. However, the cells were allowed to equilibrate for 15 min in apical and basolateral solutions without Gly-Sar, after which the experiment was started by adding fresh apical buffer containing the relevant Gly-Sar concentration (0-5 mM) and 0.5 µCi/well [14C]Gly-Sar or fresh basolateral buffer containing the relevant Gly-Sar concentration (0-10 mM) for basolateral uptake experiments. Apical uptake of Gly-Sar into the cells was terminated after 5 min and basolateral uptake after 15 min by gentle suction of the uptake medium followed by four washes of the monolayer with ice-cold HBSS. The polycarbonate filters were cut from the Transwell supports and placed into scintillation vials. Two milliliters of scintillation fluid was added, and the radioactivity was counted in a liquid scintillation analyzer.
Kinetic analysis.
Uptake of Gly-Sar as a function of apical or basolateral Gly-Sar
concentration was fitted to a Michaelis-Menten-type equation
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
Protein extraction, Western blotting, and immunoprecipitation. Caco-2 cells cultured for 24 days in either the absence or the presence of 5 ng/ml EGF in the culture media were lysed in lysis buffer containing 10 mM Tris · HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 2% NP-40. and a Complete Protease Inhibitor Cocktail Tablet (Boehringer Mannheim, Mannheim, Germany). Protein content in lysates free of cellular debris was measured using Bio-Rad protein assay dye reagent concentrate according to the manufacturer's instructions. Equal amounts of cell lysate were dissolved in 2× Laemmli buffer (22) and subjected to SDS-PAGE. Subsequently, the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Immunoreactive proteins were made visible using horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad) and enhanced chemiluminescence reagents according to the manufacturer's instructions (Biological Industries). Immunoprecipitates were collected by adding 100 µl of a 10% protein A-Sepharose 4B slurry (Pharmacia, Uppsala, Sweden) for 1 h followed by brief centrifugation. The precipitates were washed three times in 10 mM Tris · HCl, pH 7.4, 62.5 mM sucrose, 0.25 mM EDTA, 0.25 mM EGTA, 0.5% NP-40 containing a Complete Protease Inhibitor Cocktail Tablet and dissolved in 2× Laemmli buffer, and proteins were separated by SDS-PAGE. The proteins were transferred to nitrocellulose membranes, and proteins were made visible as described above. The band densities on immunoblots were measured with a densitometer (Imagestation 440 CF, Eastman Kodak). All densitometry was performed on exposures within the linear range of the film and the densitometer.
RNA isolation, cDNA synthesis, PCR, and enzyme digestion.
Total RNA was isolated from Caco-2 cells, either unstimulated or
stimulated with 5 ng/ml EGF for 24 days, using total RNA isolation
reagent (Advanced Biotechnologies, Epsom, UK) according to the
manufacturer's instructions. First-strand cDNA was synthesized with an
anchored oligo(dT) primer using Reverse-iT (Advanced Biotechnologies). PCR was carried out using 2 µl of reverse-transcribed RNA added to a
solution of 10 µM of both forward and reverse primers (DNA Technology, Aarhus, Denmark), 1.5 mM MgCl2, PCR buffer
[7.5 mM Tris · HCl, pH 8.8, 20 mM
(NH4)2SO4, and 0.01% (vol/vol)
Tween 20], 40 µM dATP, TTP, and GTPs, 20 µM dCTP, 1.25 units of
Red Hot DNA polymerase (all from Advanced Biotechnologies), and 2.5 µCi of 3,000 Ci/mmol [-32P]dCTP (NEN) and distilled
H2O in a total volume of 50 µl. Temperature cycling
proceeded as follows: 1 cycle at 94°C for 5 min and 20 cycles at
94°C for 30s, 55°C for 60s, and 72°C for 60s followed by 72°C
for 10 min. hPepT1 expression was measured using the sense strand
5'-GTGGCTTCAATTTCACCTCCT-3' (corresponding to bases 1107-1127) and
the ntisense strand 5'-CAGCTGTCATTCTTCCTTTGGACTA-3'
(corresponding to bases 1750-1772) resulting in a 643-bp PCR
product. The expression of glucose 6-phosphate dehydrogenase (G6PDH)
was measured (as an internal control) using the following primers:
5'-AGCTCTGACCGGCTGTCCAA-3' (sense strand, corresponding to bases
1005-1025) and 5'-ATCGGGGTTCCCCACGTACT-3' (antisense strand,
corresponding to bases 1409-1429), resulting in a 404-bp PCR
product. The PCR products were electrophoresed on 1% agarose gels. For
semiquantitative purposes, the reaction products were separated on a
6% polyacrylamide gel, which was dried and exposed to PhosphorImager
storage screens overnight. The screens were analyzed using the
Molecular Dynamics Storm 840 (Sunnyvale, CA), and band intensities were
calculated using rectangle mode/local background/volume integration.
All quantitations were normalized to the internal control G6PDH
(20). To confirm the identity of the PCR products, these
were subjected to restriction enzyme digestion as follows. The products
were digested with EcoRI, SacI, or
PstI. EcoRI should not lead to fragments, whereas
SacI should reveal two fragments of hPepT1 (441 and 204 bp)
and none of G6PDH. PstI should give two fragments from G6PDH
(264 and 142 bp) but none of hPepT1. All digestions were performed
directly on PCR products with appropriate buffers according to the
manufacturers' instructions.
Laser scanning confocal microscopy. Cells grown on filters as described in Cell culture were rinsed in HBSS (room temperature), fixed for 10 min in HBSS with 3% paraformaldehyde, and then permeabilized for 5 min in 0.1% Triton X-100 in PBS. The cells were blocked for 30 min with PBS plus 2% normal goat serum (NGS) followed by incubation with anti-hPepT1 (1:200) for 2 h. The filters were rinsed three times in PBS plus 2% NGS and then incubated with the secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG) for 2 h. Filters were rinsed three times in PBS plus 2% NGS. Some preparations were counterstained with 0.5 µM propidium iodide in PBS for 1 min. All preparation steps were preformed at room temperature (20°C). After washing in PBS, filters were mounted on coverslips and confocal imaging was performed on a Zeiss LSM 510 laser scanning confocal microscope, using a Zeiss plan apochromat ×63 oil-immersion objective with a numerical aperture of 1.4. Fluorophores were excited using an argon laser line at 488 nm and a HeNe laser line at 543 nm.
Statistical analysis. Values are given as means ± SE. The statistical significance of the results was determined using a two-tailed paired Student's t-test. When means with different variances were compared, the Welch t-test was used. Throughout this report, n is the number of cell passages used. P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EGF-treated Caco-2 cells display dose-dependent decrease in
transepithelial transport and apical uptake of
[14C]Gly-Sar.
Caco-2 cells were grown in the presence of varying amounts of EGF in
the culture medium for 26-28 days. The apical-to-basolateral unidirectional flux of [14C]Gly-Sar was measured with 2.5 mM Gly-Sar in the apical solution (Fig.
1A). The initial apical uptake
of [14C]Gly-Sar was determined over 5 min with a total
concentration of 1 mM Gly-Sar in the apical solution (Fig.
1B). EGF inhibited transepithelial Gly-Sar transport in a
dose-dependent manner. The flux values were significantly lower than
the control value at EGF concentrations of 2, 5, and 20 ng/ml
(P < 0.02). The maximal inhibition value was 50.2 ± 3.66% (n = 6) of the control flux. Apical-to-basolateral [3H]mannitol fluxes did not change
significantly after EGF treatment, although there was a slight
(nonsignificant, P = 0.23) tendency to an increase at
high EGF concentrations {[3H]mannitol flux was 2.48 ± 0.856 × 107 and 3.5 ± 0.885 × 10
7
nmol · cm
2 · min
1 at 0 and
20 ng/ml EGF, respectively (n = 6 for both)}. The
effects of EGF on TEER varied between experiments. TEER was 860 ± 120
· cm2 in control cells and 721 ± 215
· cm2 in cells treated with the highest dose of
EGF, 20 ng/ml (n = 6, P = 0.59). Thus
no significant changes in TEER were observed, although EGF treatment
increased the variance in TEER values. EGF caused a dose-dependent
decrease in apical Gly-Sar uptake. The uptake values were significantly
lower than the control value at EGF concentrations of 2, 5, and 20 ng/ml (P < 0.02). Inhibition constants were obtained
by fitting experimental data to Eq. 3. The maximal decrease
in initial uptake of Gly-Sar was 80 ± 0.02% (n = 6). The ED50 for EGF inhibition of initial apical uptake was estimated as 0.36 ± 0.06 ng/ml (n = 6).
|
EGF changes morphology of Caco-2 cells but does not induce
multilayer formation.
Earlier studies addressing the effect of EGF on Caco-2 cells indicated
that long-term treatment with EGF at high doses can induce the
formation of multiple cell layers and thereby change transport
parameters (6). Therefore, a series of vertical and horizontal scans of Caco-2 cell layers grown in the absence or presence
of 5 ng/ml EGF were performed using laser scanning confocal microscopy
(Fig. 2). The cell nuclei were labeled
with propidium iodide, and the actin was labeled with Alexa
488-conjugated phalloidin as described in MATERIALS AND
METHODS. The actin skeleton lies just beneath the cell membrane
and thereby visualizes the outline of the cells. In EGF-treated
monolayers, cells and cell nuclei appeared to be larger and more
disordered than in controls. The vertical scans showed that both
controls and EGF-treated cells formed a monolayer, with cell heights
ranging from 18 to 25 µm.
|
EGF treatment changes Vmax of transepithelial transport
and apical uptake of [14C]Gly-Sar but does not affect
basolateral uptake.
A series of experiments was performed in which transepithelial
transport and apical and basolateral uptake of Gly-Sar were determined
in control cells and in cells treated with 5 ng/ml EGF at varying
Gly-Sar concentrations. Apical-to-basolateral transport and apical
uptake of [14C]Gly-Sar were measured over a concentration
range of 0-5 mM Gly-Sar in the apical solution, and basolateral
uptake was measured over a concentration range of 0-10 mM Gly-Sar
in the basolateral solution. Transepithelial fluxes for this
concentration range (Fig. 3A) were used to generate transport kinetic constants. This yielded a
Vmax in control cells of 3.00 ± 0.17 nmol · cm2 · min
1 compared
with 0.50 ± 0.07 nmol · cm
2 · min
1 in the
EGF-treated cells (n = 6, different from control cell, P < 0.01). Apparent Km was
2.71 ± 0.31 mM in the control cells and 1.89 ± 0.28 mM in
the EGF-treated cells (n = 6, not significantly different from control). The k value (i.e., slope of the
linear component) was 5 × 10
5 ± 2 × 10
5 cm/min (n = 6) in the EGF-treated
cells but absent in control cells. Figure 3B shows the
apical uptake of Gly-Sar in controls and cells treated with 5.0 ng/ml
EGF. Data were fitted to the Michaelis-Menten equation (Eq. 1), and kinetic constants were obtained. The control curve showed
saturable kinetics, with an apparent Km of
0.66 ± 0.3 mM and a Vmax of 2.61 ± 0.4 nmol · cm
2 · min
1
(n = 3). The corresponding curve for cellular uptake of
Gly-Sar in cells treated with EGF showed an apparent
Km of 0.57 ± 0.2 mM and a
Vmax of 1.06 ± 0.1 nmol · cm
2 · min
1
(n = 3). The decrease in maximal uptake capacity was
~60% without any change in Km. As seen in
Fig. 3C, the basolateral uptake control curve showed
saturable kinetics, with an apparent Km of
13.09 ± 1.05 mM and a Vmax of 1.21 ± 0.10 nmol · cm
2 · min
1
(n = 3). The corresponding curve for cellular uptake of
Gly-Sar in cells treated with 5.0 ng/ml EGF showed an apparent
Km of 15.83 ± 1.10 mM and a
Vmax of 1.38 ± 0.10 nmol · cm
2 · min
1
(n = 3). There were thus no significant differences in
kinetic parameters for basolateral uptake between control and
EGF-treated cells. This indicates that EGF decreases transepithelial
peptide transport by decreasing the apparent
Vmax of apical peptide uptake, suggesting that
the EGF treatment reduced the population of active peptide transporters
in the apical membrane of the Caco-2 cell monolayers.
|
Effects of EGF on Gly-Sar transport are caused by long-term
stimulation.
In the experiments described above, EGF was present in the culture
medium throughout the growth period. We investigated the time course of
the EGF-mediated inhibition of peptide transport using Caco-2 cells
grown for 26 days. Cells were exposed to EGF at 5 ng/ml for various
time intervals before the experiment. A significant decrease in peptide
transport was observed after 5 days of treatment (n = 3; Fig. 4). The inhibition of Gly-Sar
flux was maximal when monolayers had been treated for >15 days,
indicating that the decrease in peptide transport was caused by
long-term treatment with EGF. Mannitol fluxes did not change
significantly as a function of time of EGF treatment (n = 3; Fig. 4, inset).
|
Effect of EGF on peptide transport is mediated via basolateral
receptors.
EGF receptors are present on both apical and basolateral membranes of
Caco-2 cells (2). A series of experiments were performed to investigate whether the observed effects were caused by apical stimulation, basolateral stimulation, or both. EGF (5 ng/ml) was added
to either the basolateral or the apical solution throughout the culture
period. Cells grown in the absence of EGF and cells cultured with EGF
on both sides were used as controls (Fig.
5). Cells treated with EGF in the apical
solution displayed a Gly-Sar flux identical to the control Gly-Sar flux
without treatment of EGF (n = 3). In cells treated with
EGF in the basolateral solution the Gly-Sar flux was significantly
lower than in untreated cells (n = 3, P < 0.01) and identical to the Gly-Sar flux in cells treated with EGF in
both the apical and basolateral solutions, which was significantly
lower than that in untreated cells (n = 3, P < 0.01). The inhibitory effect of EGF on
transcellular peptide transport thus appears to be mediated solely via
the basolateral receptors. No statistical significant changes in
mannitol fluxes were observed under the different experimental
conditions (Fig. 5, inset).
|
Expression of hPepT1 mRNA and hPepT1 protein in EGF-treated Caco-2
cells.
The localization of hPepT1 in 26-day-old Caco-2 cell monolayers is
shown in Fig. 6. The peptide transport
protein was primarily localized in the apical membrane and in vesicles
just below, with little or no staining in the basolateral membranes.
The degree of expression of hPepT1 varied between individual cells, as
judged by both the vertical x-y image (Fig. 6A)
and the horizontal x-z image (Fig. 6B). mRNA levels of
hPepT1 were analyzed by RT-PCR to investigate whether there was a
relationship between the decrease in transepithelial of Gly-Sar and the
expression of hPepT1. Total RNA was obtained from untreated monolayers
or monolayers treated with 5 ng/ml EGF throughout the culture period.
RNA isolation and RT-PCR were carried out as described in
MATERIALS AND METHODS. Agarose gel electrophoresis
demonstrated the presence of PCR products of the expected sizes (hPepT1
~643 bp, G6PDH ~404 bp; Fig.
7A). mRNA levels were
quantified using 32P-labeled cDNAs followed by
polyacrylamide gel electrophoresis and imaging as described in
MATERIALS AND METHODS. Figure 7B shows hPepT1
mRNA levels in controls and EGF-treated cells normalized to the
internal control G6PDH. The level of hPepT1 mRNA was significantly reduced (to ~65%; P < 0.05, n = 3)
in cells grown in the presence of EGF compared with controls. Protein
levels of hPepT1 were visualized using Western blotting and anti-hPepT1
antibody. Figure 8 shows a blot of total
cell lysate (lanes 1 and 2) and cell protein
immunoprecipitated with anti-hPepT1 (lanes 3 and
4). In all lanes, a prominent band at ~80 kDa was
apparent, corresponding to hPepT1. Quantification of the bands from
total cell lysate showed a 35 ± 3% (n = 3)
decrease in band intensity in EGF-treated cells compared with controls.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study demonstrates, for the first time, that a growth factor (EGF) decreases hPepT1-mediated transport of Gly-Sar in a dose-dependent manner. EGF caused a decrease in transepithelial transport of Gly-Sar in Caco-2 cells treated with EGF throughout the culture period. Apical and transepithelial kinetic data showed a decrease in Vmax without significant changes in Km. EGF influenced the apical peptide transport step but did not regulate the peptide transport activity in the basolateral membrane. Using semiquantitative RT-PCR we demonstrated a decrease in hPepT1 mRNA, and Western blotting indicated a decreased expression of hPepT1. Together, these data suggest that EGF decreased the number of hPepT1 transporters in the apical membrane of the Caco-2 cell monolayers. PCR data indicated that this was caused by a decrease in hPepT1 mRNA, thereby causing a decrease in the expression of the hPepT1 gene product.
Studies concerning the regulation of hPepT1 expression are sparse. Evidence indicates that protein kinase C (4), cAMP (25), and insulin (33) are involved in the regulation of PepT1. However, for these stimuli the regulation is on the level of transport function or protein recruitment to the plasma membrane. Both EGF and insulin act on tyrosine kinase receptors. The effect of insulin on hPepT1 was an insertion of preformed hPepT1 transporters after 1 h of insulin stimulation (33), whereas the downregulation of hPepT1 transport activity was caused by a stimulation of EGF for >5 days. This is probably because EGF acts at the level of gene expression whereas the effects of insulin are caused by short-term stimulation of protein kinases, which in turn phosphorylates cellular targets, eventually leading to the observed insertion of transporters. A study by Shiraga et al. (30) clearly shows that hPepT1 can be regulated at the levels of gene expression by specific amino acids and peptides. Thamotharan et al. (32) showed that increased hPepT1 expression and increased levels of hPepT1 mRNA could be caused by stimulation of luminal peptides, and Walker et al. (36) showed that a similar upregulation of hPepT1 was mediated in part via an increase in hPepT1 mRNA stability. The downregulation of sucrase-isomaltase (SI) in Caco-2/15 cells after long-term EGF treatment showed that EGF acts primarily at the pretranslational level by influencing SI gene transcription and/or SI mRNA stability (6).
The basolateral transport activity has been shown by some groups to be coupled to protons (34, 35), whereas others have shown the transport process to be independent of or only slightly dependent on protons (28, 31), suggesting the existence of distinct apical and basolateral transporters. In this study we showed a differential regulation of apical and basolateral peptide transport activity by EGF. Whereas apical peptide transport activity was markedly downregulated after EGF treatment, basolateral transport parameters remained unchanged. Furthermore, hPepT1 was found (using a hPepT1 antibody and laser scanning confocal microscopy) to be located in the apical membrane of the Caco-2 cells (Fig. 6). Similar results were found by Walker et al. (36), who showed that anti-hPepT1 immunostaining was present predominantly in the apical membrane in Caco-2 cells (using an anti-hPepT1 antibody distinct from the one used in our study). Our data thus add to the body of evidence indicating that two different transporters might be involved in transcellular transport of intact di-/tripeptides and certain peptidelike compounds (for references, see Refs. 28, 31, and 34-36).
Previous studies have indicated that EGF receptors are present on both apical and basolateral membranes of Caco-2 cells (2). Estimates of the basolateral-to-apical receptor ratio range from 3 to 15 (for references, see Ref. 2). However, only basolateral stimulation with EGF mediated the decrease in peptide transport, as demonstrated in the present study. Only a few studies have examined from which side (apical vs. basolateral) EGF exerts its effect in Caco-2 cells. Bishop and Wen (2) showed that Caco-2 cell proliferation by EGF was mediated exclusively by basolateral EGF receptors (EGFR). EGF and related substances mediate their effects on epithelial cells in vivo through binding to high-affinity EGFR at their basolateral surface (for review, see Ref. 5). Caco-2 cells thus functionally resemble human enterocytes, in which only basolateral EGFR have been found (27).
The decrease in Gly-Sar transport displayed an inhibition constant fairly close to binding constant (Kd) values for EGF binding to the EGFR observed in other tissues. The ED50 value found in this study was 0.36 ng/ml (0.7 nM) for the apical uptake. Kd values have been estimated to be 0.67 nM in Caco-2 cells (18), 0.86 nM in human urothelial cells (24), 0.83 nM in the jejunal crypt cell line IEC-6 (1), and 2.31 nM in rat enterocytes (14). The concentrations at which EGF decreased peptide transport activity are thus in the physiological relevant range for EGF-EGFR interaction.
Despite the large body of literature describing the effects of EGF in various biological systems, its exact role in tissue development and differentiation still remains unclear. The overall result of EGFR activation is a change in the steady-state RNA concentration of a number of cellular genes (for references, see Ref. 16). In Caco-2 cells it appears that treatment with EGF blocks differentiation and keeps the cells in a proliferative stage. Daniele and Quaroni (7) showed that Caco-2 cells grown in the presence of EGF throughout the culture period showed a decrease in expression of the differentiation marker dipeptidyl peptidase IV compared with controls, and Cross and Quaroni (6) showed the same to be the case with respect to differentiation-marker SI (6). In the present study we demonstrated that the differentiation marker hPepT1 displayed a decrease after EGF treatment. This was not caused only by a delayed differentiation of the cells, because cells treated without EGF for 21 days and then subsequently treated with EGF showed a significant decrease in Gly-Sar transport (Fig. 4), indicating an actual dedifferentiation.
It appears that the decrease in the expression of hPepT1 is part of a general pattern of effects after long-term stimulation of the basolateral EGF receptor in Caco-2 cells, namely, increased proliferation and a decrease in the number of differentiation-specific brush border marker proteins. However, a correlation of the present studies to the in vivo situation is required to fully understand the physiological role of EGF in downregulation of apical brush border proteins and enterocyte proliferation. In vivo, the luminal membrane will be more or less constantly exposed to EGF, because EGF is secreted from the gastrointestinal glands and is present in the intestinal lumen at fairly high concentrations (for references, see Ref. 12). Effective basolateral EGF concentrations are not easily estimated, because EGF acts in an autocrine/paracrine manner and serum concentrations (which are extremely low in adults) are not likely to reflect the effective concentrations encountered by the basolateral receptors on the enterocytes. Furthermore, EGF is released enzymatically from an extracellular domain of a large transmembrane precursor, which also is able to activate EGF receptors of neighboring cells while still anchored in the membrane. The exact role of EGF released in the lateral spaces by neighboring enterocytes or by other cell types still remains unclear, and there is a possibility that native enterocytes might experience a tonic stimulation by soluble EGF originating from the local environment and from EGF precursor molecules on neighboring cells.
In conclusion, the results of the present study provide evidence that long-term treatment of Caco-2 cell monolayers with EGF causes a decrease in transepithelial transport and apical uptake of Gly-Sar. We showed that this was due to a decrease in hPepT1 protein expression caused by the decrease in hPepT1 mRNA. Further studies are needed to investigate cellular events linking EGFR activation and hPepT1 expression in Caco-2 cells.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors acknowledge Dr. Wolfgang Sadeé for providing anti-hPepT1 antiserum, technicians Susanne Nørskov Sørensen and Bettina Dinitzen for their assistance, and Dr. Lars Hovgaard for the use of confocal microscopy facilities.
![]() |
FOOTNOTES |
---|
The Centre for Drug Design and Transport (a project grant from the Danish Medical Research Council) funded B. Brodin and C. U. Nielsen. J. Amstrup was supported by the Danish Natural Science Research Council.
Address for reprint requests and other correspondence: B. Brodin, Dept. of Pharmaceutics, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark (E-mail: bbr{at}dfh.dk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 November 2000; accepted in final form 6 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barnard, JA,
Polk WH,
Moses HL,
and
Coffey RJ.
Production of transforming growth factor- by normal rat small intestine.
Am J Physiol Cell Physiol
261:
C994-C1000,
1991
2.
Bishop, WP,
and
Wen JT.
Regulation of Caco-2 cell proliferation by basolateral membrane epidermal growth factor receptors.
Am J Physiol Gastrointest Liver Physiol
267:
G892-G900,
1994
3.
Boll, M,
Markovich D,
Weber WM,
Korte H,
Daniel H,
and
Murer H.
Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors.
Pflügers Arch
429:
146-149,
1994[ISI][Medline].
4.
Brandsch, M,
Miyamoto Y,
Ganapathy V,
and
Leibach FH.
Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line.
Biochem J
299:
253-260,
1994[ISI][Medline].
5.
Chailler, P,
and
Menard D.
Ontogeny of EGF receptors in the human gut.
Front Biosci
4:
D87-D101,
1999[Medline].
6.
Cross, HS,
and
Quaroni A.
Inhibition of sucrase-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2.
Am J Physiol Cell Physiol
261:
C1173-C1183,
1991
7.
Daniele, B,
and
Quaroni A.
Effects of epidermal growth factor on diamine oxidase expression and cell growth in Caco-2 cells.
Am J Physiol Gastrointest Liver Physiol
261:
G669-G676,
1991
8.
Dantzig, AH,
and
Bergin L.
Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, caco-2.
Biochim Biophys Acta
1027:
211-217,
1990[ISI][Medline].
9.
De Lean, A,
Munson PJ,
and
Rodbard D.
Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves.
Am J Physiol Endocrinol Metab Gastrointest Physiol
235:
E97-E102,
1978
10.
Fei, YJ,
Kanai Y,
Nussberger S,
Ganapathy V,
Leibach FH,
Romero MF,
Singh SK,
Boron WF,
and
Hediger MA.
Expression cloning of a mammalian proton-coupled oligopeptide transporter.
Nature
368:
563-566,
1994[ISI][Medline].
11.
Fei, YJ,
Sugawara M,
Liu JC,
Ganapathy V,
Ganapathy ME,
and
Leibach FH.
cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1.
Biochim Biophys Acta
1492:
145-154,
2000[ISI][Medline].
12.
Fisher, DA,
and
Lakshmanan J.
Metabolism and effects of epidermal growth factor and related growth factors in mammals.
Endocr Rev
11:
418-442,
1990[Abstract].
13.
Fujita, T,
Majikawa Y,
Umehisha S,
Okada N,
Yamamoto A,
Ganapathy V,
and
Leibach FH.
Receptor ligand-induced up-regulation of the H+/peptide transporter PepT1 in the human intestinal cell line Caco-2.
Biochem Biophys Res Commun
261:
242-246,
1999[ISI][Medline].
14.
Gallo, PN,
and
Hugon JS.
Epidermal growth factor receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture.
Endocrinology
116:
194-201,
1985[Abstract].
15.
Gonzalez, DE,
Covitz KM,
Sadee W,
and
Mrsny RJ.
An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2.
Cancer Res
58:
519-525,
1998[Abstract].
16.
Haley, JD.
Regulation of epidermal growth factor receptor expression and activation: a brief review.
Symp Soc Exp Biol
44:
21-37,
1990[Medline].
17.
Hashimoto, N,
Fujioka T,
Toyoda T,
Muranushi N,
and
Hirano K.
Renin inhibitor: transport mechanism in rat small intestinal brush-border membrane vesicles.
Pharm Res
11:
1448-1451,
1994[ISI][Medline].
18.
Hidalgo, IJ,
Kato A,
and
Borchardt RT.
Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers.
Biochem Biophys Res Commun
160:
317-324,
1989[ISI][Medline].
19.
Inui, K,
Yamamoto M,
and
Saito H.
Transepithelial transport of oral cephalosporins by monolayers of intestinal epithelial cell line caco-2: specific transport systems in apical and basolateral membranes.
J Pharmacol Exp Ther
261:
195-201,
1992[Abstract].
20.
Jensen, J,
Serup P,
Karlsen C,
Nielsen TF,
and
Madsen OD.
mRNA profiling of rat islet tumors reveals nkx 6.1 as a beta-cell-specific homeodomain transcription factor.
J Biol Chem
271:
18749-18758,
1996
21.
Kim, JS,
Oberle RL,
Krummel DA,
Dressman JB,
and
Fleisher D.
Absorption of ACE inhibitors from small intestine and colon.
J Pharm Sci
83:
1350-1356,
1994[ISI][Medline].
22.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
259:
680-685,
1970.
23.
Liang, R,
Fei YJ,
Prasad PD,
Ramamoorthy S,
Han H,
Yang FT,
Hediger MA,
Ganapathy V,
and
Leibach FH.
Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.
J Biol Chem
270:
6456-6463,
1995
24.
Messing Em, E,
and
Reznikoff CA.
Normal and malignant human urothelium: in vitro effects of epidermal growth factor.
Cancer Res
47:
2230-2235,
1987[Abstract].
25.
Muller, U,
Brandsch M,
Prasad PD,
Fei YJ,
Ganapathy V,
and
Leibach FH.
Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP.
Biochem Biophys Res Commun
218:
461-465,
1996[ISI][Medline].
26.
Ogihara, H,
Saito H,
Shin BC,
Terado T,
Takenoshita S,
Nagamachi Y,
Inui K,
and
Takata K.
Immuno-localization of H+/peptide cotransporter in rat digestive tract.
Biochem Biophys Res Commun
220:
848-852,
1996[ISI][Medline].
27.
Playford, RJ,
Hanby AM,
Gschmeissner S,
Peiffer LP,
Wright NA,
and
McGarrity T.
The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract.
Gut
39:
262-266,
1996[Abstract].
28.
Saito, H,
and
Inui K.
Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2.
Am J Physiol Gastrointest Liver Physiol
265:
G289-G294,
1993
29.
Saito, H,
Okuda M,
Terada T,
Sasaki S,
and
Inui K.
Cloning and characterization of a rat H+/peptide cotransporter mediating absorption of beta-lactam antibiotics in the intestine and kidney.
J Pharmacol Exp Ther
275:
1631-1637,
1995[Abstract].
30.
Shiraga, T,
Miyamoto KI,
Tanaka H,
Yamamoto H,
Taketani Y,
Morita K,
Tamai I,
Tsuji A,
and
Takeda E.
Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1.
Gastroenterology
116:
354-362,
1999[ISI][Medline].
31.
Terada, T,
Sawada K,
Saito H,
Hashimoto Y,
and
Inui K.
Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2.
Am J Physiol Gastrointest Liver Physiol
276:
G1435-G1441,
1999
32.
Thamotharan, M,
Bawani SZ,
Zhou X,
and
Adibi SA.
Mechanism of dipeptide stimulation of its own transport in a human intestinal cell line.
Proc Assoc Am Physicians
110:
361-368,
1998[ISI][Medline].
33.
Thamotharan, M,
Bawani SZ,
Zhou X,
and
Adibi SA.
Hormonal regulation of oligopeptide transporter pept-1 in a human intestinal cell line.
Am J Physiol Cell Physiol
276:
C821-C826,
1999
34.
Thwaites, DT,
Brown CDA,
Hirst BH,
and
Simmons NL.
Transepithelial glycylsarcosine transport in intestinal caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranes.
J Biol Chem
268:
7640-7642,
1993
35.
Thwaites, DT,
Brown CDA,
Hirst BH,
and
Simmons NL.
H+-coupled dipeptide (glycylsarcosine) transport across apical and basolateral borders of human intestinal Caco-2 cell monolayers display distinctive characteristics.
Biochim Biophys Acta
1151:
237-245,
1993[ISI][Medline].
36.
Walker, D,
Thwaites DT,
Simmons NL,
Gilbert HJ,
and
Hirst BH.
Substrate upregulation of the human small intestinal peptide transporter, hPepT1.
J Physiol (Lond)
507:
697-706,
1998