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J Biol Chem, Vol. 274, Issue 36, 25906-25912, September 3, 1999
From the Heparin-binding epidermal growth factor-like
growth factor (HB-EGF) transduces mitogenic signals through the EGF
receptor (EGFR). There are two forms of HB-EGF, the membrane-anchored
form (pro-HB-EGF) and the soluble form (sHB-EGF). We studied the
biological activity of pro-HB-EGF by using a model in which
pro-HB-EGF-expressing effector cells was co-cultured with
EGFR-expressing target cells. The DER cell, an EGFR-expressing
derivative of the interleukin-3-dependent hematopoietic 32D
cell line, grows well in the presence of EGF or sHB-EGF without IL-3.
When DER cells were co-cultured on a monolayer of Vero-H cells
overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis
were induced in the DER cells even in the presence of excess amounts of
EGF or sHB-EGF. Such growth inhibition of DER cells was abrogated when
specific antagonists for pro-HB-EGF were added in the culture medium or
when direct contact of DER cells with Vero-H cells was prevented,
indicating that pro-HB-EGF is involved in this inhibitory effect.
Pro-HB-EGF-induced apoptosis of DER cells was also observed even in the
presence of IL-3. This rules out the possibility of simple competition between soluble EGFR ligands and pro-HB-EGF. Moreover, 32D cells expressing EGFR mutant composed of the extracellular and the
transmembrane domain of EGFR and the cytoplasmic domain of
erythropoietin receptor did not undergo apoptosis by co-culture with
Vero-H cells, indicating that the inhibitory signal induced by
pro-HB-EGF-expressing Vero-H cells is mediated to DER cells via EGFR
and that the cytoplasmic domain of EGFR is essential for
pro-HB-EGF-induced apoptosis. From these results, we concluded that
pro-HB-EGF has unique biological activity through cell-cell contact
that is distinct from the activity of sHB-EGF.
In multicellular organisms, cells interact with each other to form
and maintain the cellular society. A number of molecules of different
functional categories play a role in this interaction. Growth factors,
cytokines, and their receptors are important constituents of
intercellular signaling. Until recently, growth factors or cytokines
had been recognized to be soluble proteins usually found in blood or
other body fluid, and many studies have been done to understand the
function of these soluble factors. However, recent studies revealed
that several growth factors and cytokines are synthesized as
membrane-anchored proteins and that such membrane-anchored forms may
have functions distinct from the soluble forms. For example, the
epidermal growth factor
(EGF)1 family of growth
factors, tumor necrosis factor- Heparin-binding EGF-like growth factor (HB-EGF) was first identified as
a 20-22-kDa glycoprotein in a conditioned medium of macrophage-like
cells (10). It is structurally a member of the EGF family, which
encompasses a number of structurally homologous mitogens including EGF,
transforming growth factor- Not only is pro-HB-EGF the precursor protein of sHB-EGF, but pro-HB-EGF
itself is a biologically active protein. Pro-HB-EGF is known to be the
specific receptor for diphtheria toxin (DT) and mediates endocytosis of
the receptor-bound DT, resulting in entry of its A fragment into the
cytoplasm (15, 16). As a growth factor, pro-HB-EGF has mitogenic
activity to neighboring cells in a juxtacrine mode when
pro-HB-EGF-expressing cells are fixed with formalin (17). A feature of
pro-HB-EGF that is distinct from other membrane-anchored growth factors
is that it forms a complex with other membrane proteins. CD9, a
tetramembrane-spanning protein family, forms complexes with pro-HB-EGF
(15, 18), thereby up-regulating the biological activity of pro-HB-EGF
by protein-protein interaction (15, 17). In addition to CD9, pro-HB-EGF
forms a complex with integrin We previously showed that pro-HB-EGF-expressing cells pretreated with
formalin, stimulate cell growth of neighboring cells in a juxtacrine
manner (17). Although these results clearly indicate that pro-HB-EGF
itself is a biologically active protein and affects the cell growth of
neighboring cells, formalin treatment may modulate the biological
functions of pro-HB-EGF. In order to examine the biological activities
of pro-HB-EGF under more physiological conditions, we used a co-culture
system in which intact cells expressing pro-HB-EGF were incubated with
EGFR-expressing cells. The juxtacrine activities of pro-HB-EGF were
evaluated by measuring the cell growth of the EGFR-expressing recipient cells. Using this system, we provide evidence of different biological activity of pro-HB-EGF from that of the soluble form.
Materials
Mouse anti-human EGFR mAb (LA-1) was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Recombinant human EGF was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Recombinant human HB-EGF, murine IL-3, and anti-human HB-EGF
neutralizing antibody were purchased from R & D Systems (Minneapolis,
MN). CRM197 was prepared as described previously (21). DNA ladder marker was purchased from New England Biolabs, Inc. (Beverly, MA).
Fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody
was purchased from Cappel (Durham, NC).
Binding of 125I-Labeled DT
Purified DT was labeled with Na125I (Amersham
Pharmacia Biotech) as reported previously (22), using Sepharose beads
conjugated with lactoperoxidase and glucose oxidase (Sigma) (23).
Binding of 125I-labeled DT to Vero cells and Vero-H cells
was carried out as described previously (24).
Cell Culture and Transfection
Vero cells, a cell line derived from the African green monkey
kidney, and Vero-H cells, Vero cells stably expressing human pro-HB-EGF
(13), were grown in Eagle's minimum essential medium supplemented with
nonessential amino acids MEM-NEAA supplemented with 10% FCS,
penicillin G (100 units/ml), and streptomycin (100 µg/ml). Murine 32D
cells and their derived cell lines were maintained in RPMI 1640 medium
supplemented with 10% FCS, penicillin G (100 units/ml), streptomycin
(100 µg/ml), and 5% WEHI-3 cell-conditioned medium as a source of
IL-3 (25). All cell lines used in this study were determined to be
mycoplasma-free. Contamination of mycoplasma would greatly affect the
experimental results in the present study.
To obtain stable transformants expressing EGFR and EGFR/EpoR chimera,
32D cells were transfected with plasmids containing human EGFR cDNA
(pTJNeo-EGFR) (26) and cDNA of EGFR-EpoR chimera (designated Flow Cytometry
Cells were allowed to react with anti-human EGFR mAb (LA-1)
followed by incubation with FITC-conjugated goat anti-mouse IgG. The
cells were then analyzed using an EPICS XL Flow Cytometer (Coulter Co.,
Miami, FL). The scatter window was set to eliminate dead cells and cell
debris. For each measurement, 1 × 104 cells were analyzed.
Co-culture Assay
Effector cells (Vero cells or Vero-H cells) were placed in
tissue culture dishes at a density of 1-2.5 × 104
cells/cm2 and cultured in MEM-NEAA containing 10% FCS for
18 h. The cells were washed twice with RPMI 1640 containing 10%
FCS. Then 1-10 × 104 cells/ml of target cells (DER
cells or 32D-derived cell lines) were added to the monolayer, followed
by incubation in RPMI 1640 containing 10% FCS. Recombinant EGF (0.3 nM), recombinant HB-EGF (1 nM), or recombinant
IL-3 (1.3 pM) was also added to the culture medium. In some
cases, Transwell (0.4-mm pore size, Millipore Corp., Bedford, MA) was
introduced into the well to separate the recipient cells from the
effector cells (Fig. 1). After co-culture for 24-48 h, the rate of DNA
synthesis or degree of apoptosis of the target cells was determined.
Measurement of DNA Synthesis
The degree of DNA synthesis was determined by measuring the
incorporation of [3H]thymidine into the DNA of the target
cells by incubation with [3H]thymidine (74 kBq/ml) for
4 h. Then the target cells were harvested by gentle pipetting, and
the radioactivity of the trichloroacetic acid-insoluble fraction was
measured with a scintillation counter.
Determination of Apoptosis
The degree of apoptosis was measured by three methods.
Detection of DNA Ladder--
Target cells were harvested by
gentle pipetting, washed once with phosphate-buffered saline, and lysed
in 300 µl of TE-T buffer (10 mM Tris, 10 mM
EDTA, 0.5% Triton X-100, pH 8.0). After centrifugation at 15,000 rpm
for 20 min, the supernatant was subject to digestion with ribonuclease
A (0.4 mg/ml) for 1 h at 37 °C, followed by incubation with
proteinase K (0.4 mg/ml) for 1 h at 37 °C. The sample was then
extracted with isopropyl alcohol overnight at Detection of Cytoplasmic Fragmented DNA-Histone Complex
(28)--
Target cells were harvested and analyzed by Cell Death
Detection ELISAPLUS (Roche Molecular Biochemicals), according to the manufacturer's instructions.
Detection of Phosphatidylserine Expression on Cytoplasmic
Membranes by Flow Cytometry (29)--
Target cells were harvested and
analyzed using the Annexin V-FITC Apoptosis Detection Kit (Genzyme,
Cambridge, MA), according to the manufacturer's instructions. The
percentage of apoptosis-positive cells was determined by the ratio of
the cell number in the second peak of the stronger relative
fluorescence intensity to the total cell number (1 × 104), which was set to 100%.
Pro-HB-EGF Antagonizes the Mitogenic Activity of EGF and sHB-EGF to
DER Cells--
In order to study the biological activity of
pro-HB-EGF, we employed a co-culture assay in which target cell lines
expressing EGFR were incubated with a monolayer of an effector cell
line expressing pro-HB-EGF (Fig. 1).
Vero-H cells (13), stable transformants of Vero cells that express
pro-HB-EGF at a level 20 times higher than the parental Vero cells,
were used as the effector cell line (Fig.
2a). As target cells, we used
the DER cell line, which is a stable transformant of
IL-3-dependent 32D cells expressing human EGFR (Fig.
2b). Similar to other 32D-derived cell lines expressing EGFR
(30), DER cells can grow in a medium containing EGFR ligands without
IL-3. Fig. 2c shows that DNA synthesis in DER cells was maximally promoted by EGF at concentrations over 30 pM or
by sHB-EGF at concentrations over 300 pM.
DER cells were co-cultured on a monolayer of Vero-H cells or Vero cells
in a medium containing 10% FCS. After incubation for 48 h,
[3H]thymidine was added to the medium, followed by
incubation for 4 h. The DER cells were then separated from the
Vero-H cells or Vero cells by gentle pipetting, and the amount of DNA
synthesis was determined. Vero-H cells and Vero cells do not detach by
this pipetting procedure. Furthermore, under these culture conditions, incorporation of [3H]thymidine into Vero-H cells or Vero
cells was negligible; thus, co-culture does not affect the
incorporation of [3H]thymidine into DER cells. As shown
in Fig. 3a, only a little DNA
synthesis was observed in DER cells under these co-culture conditions
with the monolayer of Vero cells or Vero-H cells, compared with that of
DER cells cultured alone in the presence of 0.3 nM EGF.
However, growth of DER cells, monitored by DNA synthesis, was enhanced
to a great degree when DER cells were separated from the Vero cells or
Vero-H cells by Transwell. Although Vero cells and Vero-H cells express
pro-HB-EGF molecules on their cell surface, they also constitutively
secrete significant amounts of sHB-EGF into the medium (13). Therefore,
DER cells would be affected by both pro-HB-EGF and sHB-EGF under these
co-culture conditions but affected by only sHB-EGF when separated by
Transwell. The present results, therefore, imply the possibility that
the secreted sHB-EGF in the medium is mitogenic, but the
membrane-anchored form (pro-HB-EGF) does not efficiently support DER
cell growth in a juxtacrine manner or rather antagonizes the mitogenic
activity of sHB-EGF.
In order to examine whether pro-HB-EGF exhibits the antagonizing
property to the mitogenic activity of soluble EGF receptor ligand, a
co-culture assay was carried out in the presence of 0.3 nM
of EGF in the culture medium. This concentration of EGF is enough to
support DER cell growth (Fig. 2c). As shown in Fig. 3b, DER cell growth was markedly reduced in the presence of
EGF when they were co-cultured with Vero-H cells, as compared with DER
cells cultured alone in the same amount of EGF. Co-culture with Vero
cells resulted in a slight reduction of DER cell growth. Moreover, the
inhibitory effect of Vero-H cells and Vero cells on the growth of DER
cells was abrogated when the DER cells were separated from the effector
cells by Transwell, indicating that direct contact of DER cells with
Vero-H cells is required for inhibitory activity.
The higher inhibitory activity seen by co-culture with Vero-H cells
than by co-culture with Vero cells, suggests that pro-HB-EGF is
involved in this inhibitory activity. To further show that pro-HB-EGF
is involved in the inhibitory activity of Vero-H cells, pro-HB-EGF
activity was neutralized by CRM197. CRM197, a nontoxic mutant protein
of DT (21), specifically binds to the EGF-like domain of HB-EGF; thus,
CRM197 neutralizes the activity of human HB-EGF but not other EGF
ligands (31). When CRM197 was added to the medium, it reduced the
growth-inhibitory activity of Vero-H cells (Fig. 3c). CRM197
per se did not affect the rate of DER cell growth (data not
shown). These results indicate that pro-HB-EGF is implicated in the
inhibitory activity of Vero-H cells.
The amount of pro-HB-EGF expressed on the cell surface of Vero-H cells
was about 1 × 106 molecules/cell, determined by DT
binding. Therefore, the concentration of pro-HB-EGF in the present
co-culture conditions is estimated at about 0.03 nM (0.03 pmol/2 × 104 cells/ml). Since the concentration of
EGF added to this co-culture assay was 0.3 nM, this
suggests that the inhibitory activity of pro-HB-EGF is unlikely to be
due to simple competition with EGF.
Pro-HB-EGF Induces Apoptosis of DER Cells--
The growth of DER
cells was inhibited by co-culture on the monolayer of Vero-H cells.
Since cell death was observed under a microscope in the co-culture of
DER cells with Vero-H cells (data not shown), we examined whether
apoptosis of DER cells is induced when their growth is inhibited by
co-culture on Vero-H cells. Apoptosis was examined by detection of DNA
ladder formation (Fig. 4a).
Consistent with cell growth, a DNA ladder was observed in DER cells
cultured in the absence of EGF, while a DNA ladder was not observed in
DER cells cultured in the presence of EGF. When DER cells were
co-cultured for 24 h on Vero-H cells, a DNA ladder was observed in
DER cells even in the presence of EGF. CRM197 abrogated such DNA ladder
formation of DER cells. A DNA ladder was also observed when DER cells
were co-cultured on Vero-H cells in the presence of recombinant HB-EGF
instead of EGF. A DNA ladder was not observed when DER cells and Vero-H
cells were separated by Transwell.
The level of apoptosis was examined more quantitatively by measuring
the amount of fragmented nucleosomal complex by enzyme-linked immunosorbent assay (28). As shown in Fig. 4b, the level of apoptosis of DER cells co-cultured on Vero-H cells was reduced by about
60% when DER cells and Vero-H cells were separated with Transwell and
by 90% by the addition of CRM197.
To examine the percentage of the population of DER cells that undergo
apoptosis when co-cultured with Vero-H cells, we also measured the
level of apoptosis of DER cells by flow cytometry using the
FITC-labeled Annexin V method (29). Under this assay condition,
apoptosis was detected in 50% of DER cells cultured alone for 36 h without any EGFR ligands (data not shown). When DER cells were
cultured with 0.3 nM EGF, only 2.5% of the cells were
counted as apoptosis-positive (Fig. 4c). When DER cells were co-cultured with Vero-H cells, more than 15% of DER cells were apoptosis-positive even in the presence of EGF. When the contact of DER
cells with Vero-H cells was abrogated by Transwell, the percentage of
apoptotic DER cells was reduced to about 6%. When the activity of
pro-HB-EGF was inhibited by CRM197 or anti-HB-EGF neutralizing
antibody, the percentage of apoptotic DER cells was reduced to less
than 6%. Neither CRM197 nor anti-HB-EGF neutralizing antibody
per se affected the rate of apoptosis of DER cells (data not
shown). These results indicate that pro-HB-EGF, or
pro-HB-EGF-expressing Vero cells, have the capability of inducing not
only growth inhibition but also apoptosis of DER cells, even in the
presence of the soluble form of EGF or sHB-EGF. Pro-HB-EGF-mediated
apoptosis was also observed when EGFR-expressing Ba/F3 cells were used
as the target cells (data not shown), indicating that the apoptosis is
not a specific phenomenon of the 32D cell lines.
Pro-HB-EGF Induces Apoptosis of DER Cells in the Presence of
IL-3--
We have shown that pro-HB-EGF induces apoptosis of DER cells
even in the presence of an abundant amount of EGF or sHB-EGF. This
inhibitory effect could be explained by the following possibility. Pro-HB-EGF might not have any biological activity, but it might inhibit
the mitogenic activity of EGF and sHB-EGF by competitive binding to
EGFR. To examine this possibility, we analyzed whether pro-HB-EGF
induces apoptosis of DER cells that are cultured in the presence of
IL-3. DER cells, derived from IL-3-dependent 32D cells,
grow well in the presence of IL-3, and DER cell growth would not be
affected by co-culture on Vero-H cells if the inhibitory effect of
pro-HB-EGF is due to competitive binding activity. However, this was
not the case. Apoptosis of DER cells was induced even in the presence
of IL-3 by co-culture with Vero-H cells, although the degree of
apoptosis was lower than that in the presence of EGF (Fig.
5). CRM197 and anti-EGFR mAb each
inhibited apoptosis of DER cells co-cultured on Vero-H cells in the
presence of IL-3, indicating that the apoptosis-inducing activity of
pro-HB-EGF is not due to simple competition with soluble EGFR ligands
to bind to EGFR. Anti-EGFR mAb did not affect the rate of apoptosis of
DER cells when used alone (data not shown).
The Cytoplasmic Domain of EGFR Is Essential for Pro-HB-EGF-induced
Apoptosis of 32D Cell Lines--
Vero-H cells that overexpress
pro-HB-EGF inhibit the growth of EGFR-expressing DER cells in a cell
contact-dependent manner, and they consequently induce
apoptosis of DER cells even in the presence of soluble mitogens. This
inhibitory effect was neutralized by anti-HB-EGF antibody and CRM197,
indicating that pro-HB-EGF is involved in this inhibitory activity. To
examine whether this inhibitory activity of pro-HB-EGF is mediated to
DER cells through EGFR, 32D cells expressing EGFR-EpoR chimeric
receptor (32D/EGFR-EpoR) were used as the effector cells instead of DER
cells. This chimeric receptor is composed of the extracellular and
transmembrane domains of EGFR and the cytoplasmic domain of the
erythropoietin receptor. 32D/EGFR-EpoR cells, similar to DER cells,
grow well in the presence of EGF or sHB-EGF without IL-3 (data not
shown). When 32D/EGFR-EpoR cells were co-cultured with Vero-H cells,
their cell growth was not inhibited; thus, apoptosis was not induced
(Fig. 6a). The level of
expression of the chimeric receptor on the cell surface was low
compared with that of EGFR on DER cells. However, the insusceptibility
of 32D/EGFR-EpoR cells is not due to lower expression of the chimeric
receptor, since apoptosis was also observed in another 32D cell line
expressing EGFR (DER2), which had a much lower number of EGFR than the
number of chimeric receptors on 32D/EGFR-EpoR cells (Fig.
6b). Furthermore, when 32D/EGFR-EpoR cells were cultured
alone in the absence of EGF or IL-3, apoptosis was observed similar to
DER cells and DER2 cells (data not shown). Thus, the difference of the
responsiveness of EGFR-expressing 32D cells and the chimeric
receptor-expressing 32D cells to apoptotic induction by Vero-H cells
would not be due to the difference in level of expression of receptor
molecules. A similar result was also observed when EGFR-EpoR-expressing
Ba/F3 cells were used as the target cells (data not shown). These
results indicate that the inhibitory activity toward cell growth and
induction of apoptosis by pro-HB-EGF are transmitted to target cells
through their EGFR and that the cytoplasmic domain of EGFR is required
for transducing the apoptotic signal.
Pro-HB-EGF-induced Growth Inhibition and Apoptosis--
In this
study, we have shown that co-culture of EGFR-expressing target cells
with pro-HB-EGF-expressing effector cells resulted in growth inhibition
and consequent apoptosis in the target cells. Neutralization
experiments on the inhibitory activity of Vero-H cells with CRM197 or
anti-HB-EGF antibody indicated that pro-HB-EGF is involved in this
inhibitory activity. This is also supported by the weak inhibitory
activity of Vero cells expressing a lower amount of pro-HB-EGF than
Vero-H cells. Moreover, experiments using Transwell indicated that
direct contact of effector cells with target cells is required for
growth inhibition and apoptosis. From these results, we concluded that
pro-HB-EGF is involved in inhibiting the growth of target cells and
induces apoptosis in a contact-dependent manner. The
results also show that pro-HB-EGF has biological activity that is
distinct from that of sHB-EGF.
Pro-HB-EGF did not induce apoptosis in 32D cells expressing EGFR-EpoR
chimeric receptors. This indicates that the apoptotic activity of
pro-HB-EGF-expressing cells is transmitted to the target cells via EGFR
and that the cytoplasmic domain of EGFR is required for induction of
apoptosis. Although recent reports have shown that HB-EGF binds to HER4
as well as EGFR (HER1) (12), at least HER1 is implicated in this
inhibitory response. Both direct contact of the target cells with
pro-HB-EGF-expressing cells and expression of EGFR with intact
cytoplasmic domain on the surface of the target cells were required for
induction of apoptosis of the target cells. This suggests that direct
interaction between pro-HB-EGF and EGFR in a juxtacrine manner is
necessary for the inhibitory activity of pro-HB-EGF.
Pro-HB-EGF-expressing effector cells inhibit the cell growth of target
cells. However, it has not been clear whether this growth inhibition
and apoptosis are due to innate activity of pro-HB-EGF itself or a
secondary effect induced by pro-HB-EGF. In either case, however, it
should be emphasized that interaction of pro-HB-EGF with EGFR is the
initial trigger for growth inhibition and apoptosis. In addition, the
present study does not discriminate between growth inhibition and
apoptosis. The cell lines used in our study are of 32D or Ba/F3 origin,
which are both IL-3-dependent. Apoptosis was concomitant
with growth inhibition in these cells when cultured without IL-3 or
EGFR ligands. Thus, it is difficult to determine whether pro-HB-EGF
induces growth inhibition, which consequently causes apoptosis, or if
pro-HB-EGF generates a direct apoptotic signal.
Pro-HB-EGF Generates a Different Signal from That of
sHB-EGF--
As shown in this study, EGF and sHB-EGF stimulate the
growth of DER cells, while pro-HB-EGF inhibits their growth even in the
presence of EGF or sHB-EGF. Why do two forms of HB-EGF, the soluble
form and membrane-anchored form, exhibit opposite biological effects?
One possible explanation might be that pro-HB-EGF is a mitogenically
inactive molecule but that it inhibits the mitogenic activity of EGF
and sHB-EGF by competitive binding to EGFR. However, this possibility
is denied by the following results: 1) growth inhibition and apoptosis
of target cells were observed in the presence of a sufficient amount of
EGF (300 pM), while the concentration of pro-HB-EGF
molecule in the co-culture conditions with Vero-H cells is estimated to
be about 30 pM; 2) apoptosis of target cells was also
observed in the presence of IL-3, which transduces mitogenic signal to
the target cells in an EGFR-independent manner; and 3) apoptosis
induced by the co-culture with Vero-H cells was strongly reduced in
target cells expressing EGFR mutant (EGFR-EpoR), although the EGFR
mutant binds to HB-EGF.
The addition of EGF to the culture medium of A431 cells, a cell line
that naturally overexpresses EGFR, causes growth inhibition (32). It is
plausible that a too strong signal generated by EGFR causes growth
inhibition of cells. In fact, constitutive and strong induction of Ras,
Raf or mitogen-activated protein kinase can lead to apoptosis (33-35).
The target cells used in this study also express a large amount of
EGFR. Therefore, we can assume that the binding of pro-HB-EGF to EGFR
causes constitutive activation and/or hyper-activation of EGFR and its
downstream signaling molecules. However, neither constitutive
phosphorylation nor hyperphosphorylation of EGFR was observed in DER
cells co-cultured with Vero-H cells (data not shown). In addition to
EGFR phosphorylation, activation of mitogen-activated protein kinase
was also examined by using the specific antibody to phosphorylated
mitogen-activated protein kinase. However, significant activation of
mitogen-activated protein kinase was not observed (data not shown).
Thus, pro-HB-EGF-induced growth inhibition and apoptosis cannot be
explained by hyperactivation of EGFR.
A more plausible explanation for pro-HB-EGF-induced growth inhibition
and apoptosis is that interaction of EGFR with pro-HB-EGF may generate
a downstream signal linked to growth inhibition and apoptosis. As shown
here, pro-HB-EGF-induced growth inhibition and apoptosis were not seen
in 32D cells expressing EGFR/EpoR chimeric receptor, despite the fact
that these cells can grow well in the presence of EGF. These suggest a
requirement of the cytoplasmic domain of EGFR for the inhibitory
activity of pro-HB-EGF and support the notion that a qualitatively
different signal is generated from EGFR by interaction with
pro-HB-EGF.
What differences in the molecular nature of pro-HB-EGF and sHB-EGF are
responsible for inducing an opposite biological response in
EGFR-expressing cells? The importance of the clustering of membrane-anchored ligands has generally been suggested. In the case of
the B type of ephrin, the secreted form has no function, but clustering
of the soluble form by antibody makes it active (8). It has also been
suggested that dimerization of the membrane-anchored c-Kit ligand is
important for its activity (36). Analogous to these previous findings,
we hypothetically propose the formation of a homodimer or homooligomer
of pro-HB-EGF molecules. Pro-HB-EGF-associating CD9 (15), integrin
Relevance to Physiological Role of Pro-HB-EGF--
The
growth-inhibitory activity of pro-HB-EGF may be helpful for maintaining
cultured cells in saturation-dependent growth arrest. Vero
cells and Vero-H cells, which we used as effector cells, express EGFR
as well as pro-HB-EGF, and both pro-HB-EGF and EGFR localize at
cell-cell contact sites (19). When these cell cultures reach confluent
states, the proliferation of these cells stops or decreases, as seen in
cultures of many other types of cells. Even at the confluent stage,
pro-HB-EGF and EGFR are expressed in these cells and localize at
cell-cell contact sites. Thus, it would be possible to transmit a
growth-inhibitory signal to a neighboring cell by virtue of pro-HB-EGF.
Vero-H cells express pro-HB-EGF at a level about 20 times higher than
Vero cells, and Vero-H cells show higher growth-inhibitory activity to
EGFR-expressing target cells, as shown in this paper. We have found
that the saturation density of Vero-H cells is lower than that of the
parental Vero cells,2
consistent with the notion that pro-HB-EGF is involved in arresting the
growth of confluent cells. It has also been reported that hepatoma
cells transfected with the construct expressing pro-HB-EGF, proliferate
slower than the same cells transfected with the construct expressing
only sHB-EGF (37). Thus, the growth-inhibitory activity of pro-HB-EGF
may partly fill the role of the growth arrest mechanism seen in
confluent cells or in so-called "contact inhibition" state.
We gratefully acknowledge Drs. Akihiko
Yoshimura (Kurume University, Kurume) and Masabumi Shibuya (University
of Tokyo, Tokyo) for kind gifts of EGFR-EpoR chimeric receptor cDNA
and EGFR cDNA, respectively.
*
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.
¶
Supported by grants from the Japan Science and Technology Corporation.
**
Supported in part by a grant from The Research for the Future
Program, the Japan Society for the Promotion of Science (Project No.
97L00303), and Grants-in Aid for Scientific Research 09254261 and
09480198 from the Ministry of Education, Science, Sports and Culture.
To whom correspondence should be addressed: Inst. of Life
Science, Kurume University, 2432-3 Aikawa, Kurume, Fukuoka 839-0861, Japan. Tel.: 81-942-37-6317; Fax: 81-942-31-3320; E-mail: emekada@lsi.kurume-u.ac.jp.
2
R. Iwamoto, K. Handa, and E. Mekada, unpublished observation.
The abbreviations used are:
EGF, epidermal
growth factor;
HB-EGF, heparin-binding EGF-like growth factor;
pro-HB-EGF, membrane-anchored form of HB-EGF;
sHB-EGF, secreted form of
HB-EGF;
IL-3, interleukin-3;
DT, diphtheria toxin;
EGFR, EGF receptor;
EpoR, erythropoietin receptor;
mAb, monoclonal antibody;
FITC, fluorescein isothiocyanate;
FCS, fetal calf serum.
Contact-dependent Growth Inhibition and Apoptosis
of Epidermal Growth Factor (EGF) Receptor-expressing Cells by
the Membrane-anchored Form of Heparin-binding EGF-like Growth
Factor*
§¶,§, and
**
Institute of Life Science, the Center
for Innovative Cancer Research,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, colony-stimulating factor-1, c-Kit
ligands 1 and 2, ligands for Eph family (B type of ephrins), and Fas
ligand are synthesized as membrane-anchored forms, and these
transmembrane forms are biologically active. In the case of
transforming growth factor-, the membrane-anchored form is
mitogenically active and transduces the mitogenic signal to neighboring
cells by cell-cell contact, termed "juxtacrine stimulation" (1-4).
In the case of c-Kit ligand (5-7), ephrins (8), and Fas ligand (9),
the membrane-anchored form is fully biologically active, but the
soluble form shows limited or no biological function. Thus, it is now
clear that the membrane-anchored forms of growth factors and cytokines
are not only precursor proteins of the soluble factors but also
biologically active proteins having unique roles in cell-to-cell
interaction. Despite the importance of studying membrane-anchored
growth factors, not many studies on the biological functions of
membrane-anchored growth factors and the molecular mechanisms through
which they act have been done due to the lack of suitable methods.
, vaccinia virus growth factor,
amphiregulin, 
-cellulin, epiregulin, and neuregulin-1 and -2 (for a
review, see Ref. 11). Similar to other EGF-family growth factors,
HB-EGF binds to the EGFR, thereby inducing phosphorylation. More
recently, HB-EGF has been shown to bind and stimulate HER4 as well as
EGFR (12). HB-EGF can bind to heparin and cell surface heparan sulfate
proteoglycans (11). Like other members of the EGF family, HB-EGF is
synthesized as a transmembrane protein (pro-HB-EGF) and can be cleaved
on the plasma membrane to yield soluble HB-EGF (sHB-EGF) (13, 14). sHB-EGF is a potent mitogen for a number of cells including NIH3T3 cells, smooth muscle cells, epithelial cells, keratinocytes, and kidney
tubule cells (for a review, see Ref. 11).
31 (19).
Heparan sulfate proteoglycan(s) also binds to the heparin-binding
domain of pro-HB-EGF (20). The fact that the complex composed of
pro-HB-EGF, CD9, and integrin 31
co-localizes at cell-cell contact sites supports the notion that
pro-HB-EGF plays a role in intercellular communication in a juxtacrine
manner (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
108,
obtained from Dr. A. Yoshimura, Kurume University) (27), respectively,
by electroporation using the Cell-Porator Electroporation System I
(Life Technologies, Inc.). The transfected cells were cultured in the
presence of 600 µg/ml of G418, and clones of G418-resistant cells
were isolated. The level of expression of EGFR in the stable
transformants was determined by flow cytometry analysis as described below.
20 °C. The
precipitated DNA was resuspended in 20 µl of TE buffer (10 mM Tris, 10 mM EDTA, pH 8.0) and analyzed by
electrophoresis on a 0.7 or 1.3% agarose gel in the presence of 0.5 µg/ml ethidium bromide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Diagram of the co-culture assay.
EGFR-expressing target cells were co-cultured on a monolayer of
pro-HB-EGF-expressing effector cells in the presence of an excess
amount of EGF (0.3 nM). a, target cells were
allowed direct contact with effector cells; b, target cells
were separated from the effector cells by Transwell.
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Fig. 2.
Characterization of Vero-H cells and DER
cells. a, the amount of pro-HB-EGF on the surface of
Vero cells (open circle) and Vero-H cells
(closed circle) was determined by binding assay
of 125I-labeled DT. Vero cells or Vero-H cells were
incubated with increasing amounts of 125I-labeled DT
(1.7 × 107 cpm/µg) for 10 h at 4 °C in
24-well plates at a cell density of 1 × 105
cells/well. The cells were then washed to remove unbound
125I-DT, and the cell-associated radioactivity was
determined. b, flow cytometric analysis of EGFR expressed on
32D cells and DER cells. Cells were incubated with (heavy traces) or without (dashed traces)
anti-EGFR mAb, followed by incubation with FITC-conjugated goat-anti
mouse IgG. The fluorescence intensity of the cells was measured by flow
cytometry. c, the mitogenic activity of EGF and sHB-EGF to
DER cells was determined by measuring the amount of DNA synthesis. DER
cells (1 × 104) were incubated with the soluble
recombinant form of EGF (open circle) or HB-EGF
(closed circle) for 48 h, followed by
incubation with [3H]thymidine for 4 h. The amount of
[3H]thymidine incorporated into DNA was measured. Data
show the mean ± S.D. obtained from three independent
experiments.
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Fig. 3.
Growth inhibition of DER cells by co-culture
with Vero-H cells. a, Vero cells and Vero-H cells do
not support DER cell growth. DER cells (1 × 104) were
cultured alone in the presence (bar 1) or absence
(bar 2) of 0.3 nM EGF or co-cultured
with 2 × 104 Vero cells (bar 3)
or 2 × 104 Vero-H cells (bar 4). DER cells were allowed direct contact with the effector
cells ( ), or direct contact was hampered by Transwell
(TW). After culture for 48 h, DER cells were collected
by gentle pipetting, and the incorporation of
[3H]thymidine into the DNA of DER cells was measured. The
rate of DNA synthesis is indicated as a percentage of the rate of DNA
synthesis in DER cells cultured alone in the presence of EGF. Data
represent the mean ± S.D. obtained in three independent
experiments. b, Vero-H cells antagonize growth of DER cells
even in the presence of EGF. DER cells (1 × 104) were
cultured alone in the absence (bar 1) or presence
(bar 2) of 0.3 nM EGF or co-cultured
with 2 × 104 Vero cells (bar 3)
or 2 × 104 Vero-H cells (bar 4)
in the presence of 0.3 nM EGF. DER cells were allowed
direct contact with the effector cells (), or direct contact was
hampered by Transwell (TW). After culture for 48 h, the
rate of DNA synthesis was determined. Data represent the mean ± S.D. obtained in three independent experiments. c,
neutralization of growth-inhibitory activity of Vero-H cells by CRM197.
DER cells (1 × 104 cells) were cultured alone
(bar 1), or co-cultured with Vero-H cells (2 × 104) (bars 2 and 3) in
the presence of 0.3 nM EGF (bars 1-3). CRM197, a nontoxic mutant protein of DT that
specifically inhibits mitogenic activity of HB-EGF, was added at a
concentration of 1 µg/ml (bar 3). After culture
for 48 h, the rate of DNA synthesis was determined. Data represent
the mean ± S.D. of the results obtained in three independent
experiments.
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Fig. 4.
Apoptosis of DER cells co-cultured on Vero-H
cells. a, detection of apoptosis by DNA ladder. DER
cells (1 × 106) were cultured alone (lanes 1 and 2) or co-cultured with 2 × 106 Vero-H cells (lanes 3-7) with
the following: 0.3 nM EGF (lanes 2, 4, and 5), 1 nM sHB-EGF
(lanes 6 and 7), 1 µg/ml CRM197
(lane 5), or no addition (lanes 1 and 3). In the case of lane 7, Vero-H cells and DER cells were separated by Transwell.
After culture for 24 h, fragmented DNA was prepared from DER cell
lysate and analyzed by electrophoresis on a 1.3% (left panel) or 0.7% (right panel) agarose
gel. The results shown are representative data obtained in five
independent experiments. M, DNA ladder marker. b,
apoptosis determined by means of fragmented DNA-histone complex. DER
cells (1 × 104) were co-cultured with 2 × 104 Vero-H cells in the presence of 0.3 nM EGF
under the following conditions. DER cells were separated from Vero-H
cells by Transwell (TW) or with the addition of 1 µg/ml
CRM197 (CRM). After culture for 48 h, the fragmented
chromatin fraction was prepared from DER cell lysate and analyzed using
the Cell Death Detection Kit. The rate of apoptosis was determined by
absorbance at 405 nm. Data are shown as specific absorbance values,
which were obtained by subtracting the background value determined by
DER cells growing randomly in the presence of EGF. Similar results were
obtained in two independent experiments. c, detection of
apoptosis by the Annexin-V-FITC method. DER cells (1 × 105) were cultured alone (bar 1) or
co-cultured with 2.5 × 105 Vero-H cells
(bars 2-5) with 0.3 nM EGF and under
the following conditions. Bar 4, in the presence
of 1 µg/ml CRM197; bar 5, in the presence of 1 µg/ml anti-HB-EGF neutralizing Ab; bar 3, DER
cells were separated from Vero-H cells by Transwell. After culture for
36 h, DER cells were harvested and analyzed using the Annexin
V-FITC apoptosis detection kit. The data were scored from histograms
obtained by flow cytometric analysis as described under "Experimental
Procedures." Similar results were obtained in two independent
experiments.
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Fig. 5.
Apoptosis of DER cells co-cultured with
Vero-H cells in the presence of IL-3. DER cells (1 × 106) were cultured alone (lanes 1 and
2) or co-cultured with 2 × 106 Vero-H
cells (lanes 3-5) with the following additions:
1.3 pM IL-3 (lanes 2 and
3), 1.3 pM IL-3 plus 1 µg/ml CRM197
(lane 4), 1.3 pM IL-3 plus 1 µg/ml
of anti-EGFR mAb (lane 5), or no addition
(lane 1). After a 24-h culture, the degree of
apoptosis was measured by ladder formation of fragmented DNA in DER
cells by electrophoresis on a 1.3% agarose gel. Representative results
from four independent experiments are shown. M, DNA ladder
marker.
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Fig. 6.
Insensitivity of 32D cells expressing
EGFR-EpoR chimeric receptors to pro-HB-EGF-induced apoptosis.
a, 32D-derived target cells (1 × 106) were
cultured alone ( ) or co-cultured with 2 × 106
Vero-H cells in the presence of 0.3 nM EGF. DER
and DER2, 32D cells expressing EGFR;
32D/EGFR-EpoR, 32D cells expressing chimeric receptor
composed of the extracellular domain of EGFR and the cytoplasmic domain
of EpoR. After culture for 24 h, fragmented DNA was prepared from
each target cell lysate, and the DNA ladder was examined. Similar
results were obtained in two independent experiments and also in
experiments using independently isolated 32D/EGFR-EpoR clones.
M, DNA ladder marker. b, level of expression of
EGFR and the chimeric receptor on the cell surface. The amounts of EGFR
and the chimeric receptor were determined by flow cytometry using
anti-EGFR antibody (LA-1), which recognizes the extracellular domain of
EGFR, and a fluorescent second antibody. Relative intensity of
fluorescence was scored as the mean ± S.D. obtained from 1 × 104 cells of each cell line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31 (19), or heparan sulfate proteoglycans (20) may induce this oligomer formation. Oligomerized pro-HB-EGF molecules on the cell surface may result in oligomerization of EGFR,
which may lead to recruitment of signaling molecules involved in growth
inhibition and apoptosis to EGFR in EGFR-expressing cells. We
previously reported that pro-HB-EGF on formalin-fixed cells has
mitogenic activity similar to sHB-EGF (17). One possible explanation
for this is perturbation of oligomer formation of pro-HB-EGF molecules
by fixation with formalin.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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