From the Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Received for publication, October 21, 2002
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ABSTRACT |
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Insulin-like growth factor-binding
protein-3 (IGFBP-3) is inhibitory to the growth of many breast
cancer cells in vitro; however, a high level of expression
of IGFBP-3 in breast tumors correlates with poor prognosis, suggesting
that IGFBP-3 may be associated with growth stimulation in some breast
cancers. We have shown previously in MCF-10A breast epithelial cells
that chronic activation of Ras-p44/42 mitogen-activated protein (MAP)
kinase confers resistance to the growth-inhibitory effects of IGFBP-3
(Martin, J. L., and Baxter, R. C. (1999) J. Biol.
Chem. 274, 16407-16411). Here we show that, in the same cell
line, IGFBP-3 potentiates DNA synthesis and cell proliferation
stimulated by epidermal growth factor (EGF), a potent activator of Ras.
A mutant of IGFBP-3, which fails to translocate to the nucleus and has
reduced ability to cell-associate, similarly enhanced EGF action in
these cells. By contrast, the structurally related IGFBP-5, which
shares many functional features with IGFBP-3, was slightly inhibitory
to DNA synthesis in the presence of EGF. IGFBP-3 primes MCF-10A cells
to respond to EGF because pre-incubation caused a similar degree of EGF
potentiation as co-incubation. In IGFBP-3-primed cells, EGF-stimulated
EGF receptor phosphorylation at Tyr-1068 was increased relative to unprimed cells, as was phosphorylation and activity of p44/42 and p38
MAP kinases, but not Akt/PKB. Partial blockade of the p44/42 and p38
MAP kinase pathways abolished the potentiation by IGFBP-3 of
EGF-stimulated DNA synthesis. Collectively, these findings indicate
that IGFBP-3 enhances EGF signaling and proliferative effects in breast
epithelial cells via increased EGF receptor phosphorylation and
activation of p44/42 and p38 MAP kinase signaling pathways.
Insulin-like growth factor-binding protein-3
(IGFBP-3),1 a 45-kDa
glycoprotein abundant in the circulation and extracellular environment,
is a key regulator of the peptide hormones IGF-I and IGF-II (1). By
virtue of its high affinity for these growth factors, IGFBP-3 competes
for ligand binding with the receptor primarily responsible for
mediating the actions of IGF-I and -II, the type I IGF receptor (IGFR1)
(2), and thereby blocks mitogenic and anti-apoptotic signaling
initiated by its activation. An important role for IGFBP-3 in
modulating the proliferative effects of IGFs in many cell types is well
recognized, and both exogenous and endogenous IGFBP-3 have been shown
to block IGF action in breast cancer cells in vitro (3-6).
An additional role for IGFBP-3 also exists as a growth modulator with
intrinsic bioactivity in breast cancer cells and other cells in
vitro. The antiproliferative effects of a number of antitumor agents including transforming growth factor- By contrast with these findings, however, IGFBP-3 may also be
growth-stimulatory in vitro. In MCF-7 breast cancer cells,
IGFBP-3 enhanced IGF-stimulated DNA synthesis (11) similar to its
effects in fibroblasts reported previously (12). More recent studies have shown that proliferation of airway smooth muscle cells is stimulated by IGFBP-3 in the presence of serum (13), and IGFBP-3 increases proliferation of LNCaP prostate cancer cells in the absence
of serum or IGFs (14). In another cell model, T47D breast cancer cells
transfected with IGFBP-3 cDNA were initially growth-inhibited by
the expressed protein, but with increasing passage number became resistant to its growth-inhibitory effects, and were instead
growth-stimulated by the endogenous IGFBP-3 (15).
Such observations of growth-stimulation by IGFBP-3 in breast cancer
cells, although difficult to reconcile with the many reports of its
antiproliferative actions, are consistent with clinical data, which
indicate that IGFBP-3 may be associated with indicators of poor
outcome. Thus, IGFBP-3 in breast tumors correlates inversely with
estrogen receptor expression, and is positively associated with
aneuploidy, S-phase fraction, and tumor size (16-19). Although the
significance of these associations is unclear, they imply that there
may be changes in sensitivity to the effects of IGFBP-3 on breast
epithelial cells in vivo, losing growth-inhibitory
bioactivity and perhaps even accelerating tumor growth.
While investigating mechanisms that may underlie IGFBP-3 insensitivity
in breast cancer cells, we found that, whereas IGFBP-3 inhibits DNA
synthesis in MCF-10A breast epithelial cells, this response is lost
when the cells have undergone malignant transformation via expression
of oncogenic Ras (20). Blockade of p44/42 MAP kinase-activation
downstream of Ras restored IGFBP-3 sensitivity, implicating this
pathway in the development of resistance to IGFBP-3. The present study
was initiated to determine whether activation of Ras and the p44/42 MAP
kinase pathway by epidermal growth factor (EGF), a potent mitogen for
normal breast epithelial cells and many breast cancer cell lines, could
similarly induce resistance to the inhibitory effects of IGFBP-3 in
MCF-10A cells. We now report that IGFBP-3 enhanced the growth
stimulatory effects of EGF in this cell line, and that the p44/42 and
p38 MAP kinase pathways appear to be involved in this potentiation.
Reagents--
Tissue culture reagents and plasticware were from
Trace Biosciences (North Ryde, New South Wales, Australia) and
Nunc (Roskilde, Denmark). Bovine serum albumin (BSA), bovine
insulin, hydrocortisone, and EGF were purchased from Sigma, and cholera
enterotoxin was from ICN Biomedicals Australasia (Seven Hills, New
South Wales, Australia). Transforming growth factor- Cell Culture--
The MCF-10A cell line was a kind gift from
Drs. Robert Pauley and Herbert Soule of the Karmanos Cancer Institute
(Detroit, MI) (24). Cells were maintained routinely in a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 15 mM Hepes, 5% horse serum, 10 µg/ml bovine insulin, 20 ng/ml EGF, 100 ng/ml cholera enterotoxin, and 0.5 µg/ml
hydrocortisone ("growth medium"). Cultures were passaged every 5-7
days by trypsin/EDTA detachment and used between passages 158 and 165.
DNA Synthesis and Cell Proliferation Assays--
DNA synthesis
was assessed by incorporation of
[methyl-3H]thymidine (Amersham, Bucks, United
Kingdom). MCF-10A cells were dispensed into 48-well plates at 1 × 105 cells/well in growth medium, and incubated for 48 h. Cells were changed to serum-free medium (Dulbecco's modified
Eagle's medium/F-12 containing 1 g/liter BSA) and incubated for
48 h prior to treatment. Test reagents (e.g. EGF,
IGFBP-3) were added in 0.2 ml/well serum-free medium, and incubation
was continued at 37 °C for another 24 h. For the final 4 h
of this incubation period, 1 µCi/well [3H]thymidine was
added in 25 µl of serum-free medium. Monolayers were rinsed with cold
saline (9 g/liter NaCl) and fixed in cold methanol:acetic acid (3:1)
for 2 h at 4 °C. Cells were solubilized in 0.4 ml of 1 M NaOH, and lysates were mixed with 3 ml of OptimaGold (Packard Instrument Co., Meriden, CT) before counting for 1 min in a
Hewlett-Packard
To measure cell proliferation, cells were dispensed into six-well
plates at 5 × 105 cells/well in growth medium,
allowed to attach for 24 h, then changed to serum-free medium as
above. Reagents were added, and incubation continued for 7 days. Cells
were trypsinized and counted using a hemacytometer.
Analysis of Cell-associated IGFBP-3--
Cell-associated IGFBP-3
was determined immunologically, as described previously (25). Briefly,
media were removed from cell monolayers after treatment, and cells were
washed twice with serum-free medium. Anti-IGFBP-3 antibody R30 was
added to each well (final concentration, 1/5000 in serum-free medium),
and incubation was continued overnight at 22 °C. Monolayers were
washed as before, and then 125I-protein A was added to each
well (20,000 cpm in 200 µl of serum-free medium) for 2 h at room
temperature. Cells were washed and solubilized with 5 g/liter SDS
before counting in a EGF Binding Assays--
Binding of 125I-EGF to
monolayers was carried out at 4 °C. Monolayers (with and without
IGFBP-3 pretreatment) were washed, and 50,000 cpm 125I-EGF
was added per well in 200 µl of serum-free medium, in the absence or
presence of 100 ng/ml unlabeled EGF (to determine nonspecific binding).
Incubation at 4 °C was continued for 2 h, after which cells
were washed twice with ice-cold saline, solubilized in 5 g/liter SDS,
and counted. Full displacement curves were generated by co-incubation
of cells with 125I-EGF in the presence of 0.1-100 ng/ml
unlabeled EGF under the same conditions.
Analysis of Signaling Intermediates--
Cells were plated and
grown in six-well plates as described above for cell proliferation
assays. Pre-incubation with IGFBP-3 with or without inhibitors was
performed when required for the second 24-h period of the 48-h
serum-free incubation. Media were then removed and replaced by
serum-free medium (1 ml/well) without or with EGF. Incubation was
continued at 37 °C for 8 min, after which media were removed, and
the monolayers were washed immediately in ice-cold saline, then
solubilized in 500 µl of 2× SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 20 g/liter SDS, 100 ml/liter glycerol, 1 g/liter bromphenol blue, and 50 mM
dithiothreitol) at 4 °C for 10 min. Lysates were scraped into cold
Eppendorf tubes and frozen immediately at SDS-PAGE and Western Analysis--
Prepared lysates were
subjected to SDS-PAGE (7.5% separating gel for EGFR, 12% separating
gel for p44/42, p38, and Akt) and electrophoretic transfer, as
described previously (20). After transfer, filters were blocked in
either 50 g/liter skim milk powder (for p44/42 and p38 MAPK) or 50 g/liter BSA (for EGFR and Akt) in Tris-buffered saline with Tween 20 (10 mM Tris, 150 mM NaCl, pH 7.4 containing 1 ml/liter Tween 20). Primary antibody incubation was carried out at
4 °C for 16 h, with antibodies diluted 1:1000 in blocking
buffer. Filters were washed three times for 10 min each in cold
Tris-buffered saline with Tween 20, and then incubated with the
appropriate horseradish peroxidase-linked secondary antibody for 1-2 h
at room temperature. Filters were washed as before and developed by
enhanced chemiluminescence using Pierce reagents.
In Vitro Kinase Assays--
Activity of p38 MAPK and p44/42 MAPK
in lysates from IGFBP-3- and EGF-treated MCF-10A cells was determined
by assessing phosphorylation of Elk-1 (for p44/42 MAPK) and Atf-2 (for
p38 MAPK) using reagents from Cell Signaling (Beverley, MA). Confluent
monolayers in six-well plates were preincubated with or without IGFBP-3
for 16 h and stimulated with EGF for 10 min. Cells were rinsed
with ice-cold PBS, and lysed in 200 µl of cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10 g/liter Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Statistical Analyses--
All experiments were performed a
minimum of two times and are shown as data pooled from the two
experiments, unless indicated otherwise. Significance on pooled data
was determined by analysis of variance using the Statview package for Macintosh.
We have shown previously that, in the absence of exogenous growth
factors, IGFBP-3 is inhibitory to DNA synthesis in MCF-10A breast
epithelial cells over 24 h, but that constitutive up-regulation of
Ras-MAPK signaling abolishes the growth-inhibitory effect of IGFBP-3
(20). Because EGF stimulates activation of the Ras-MAPK pathway in
these cells (data not shown), we therefore investigated whether it
similarly affected their growth response to IGFBP-3. As shown in Fig.
1A, EGF is a potent stimulator
of DNA synthesis in MCF-10A cells over 24 h, with a significant
effect apparent with 0.1 ng/ml EGF. In the absence of EGF, natural
human IGFBP-3 inhibited DNA synthesis in MCF-10A cells, as reported
previously (20) (data not shown). However, IGFBP-3 enhanced the
stimulatory effect of low concentrations (0.1 and 1 ng/ml) of EGF (Fig.
1A), with a significant and maximal effect at 10 ng/ml
IGFBP-3. The potentiating effect of IGFBP-3 was lost at high (10 ng/ml)
EGF concentrations (Fig. 1A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
), vitamin D,
and retinoic acid in breast cancer cells appear to be mediated, at
least in part, by IGFBP-3 acting independently of IGF sequestration (7,
8). The IGF independence of IGFBP-3 actions is inferred from an
inability of other IGFBPs to mimic the effect, persistence of the
growth-inhibitory effect of IGFBP-3 in the presence of insulin or
IGF analogs, which activate the IGFR1 but which do not bind IGFBP-3,
and no clear evidence of IGFs being present in the system under
investigation (7). In addition to its anti-mitogenic effects, IGFBP-3
exhibits pro-apoptotic activity in vitro. IGFBP-3 may
sensitize breast cancer cells to apoptotic inducers such as ionizing
radiation (9) and ceramide (10), and directly effect apoptosis via
induction of pro-apoptotic proteins such as Bax and Bad (9).
Collectively, these observations suggest that IGFBP-3 is an important
antiproliferative agent in breast cancer cells, acting both through
IGF-independent and IGF-modulatory pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) and
heregulin (heregulin-
, EGF domain) were from Upstate Biotechnology,
Inc. (Lake Placid, NY). Signaling pathway inhibitors were purchased from Calbiochem-Novabiochem (Alexandria, New South Wales, Australia): MEK inhibitor PD98059, PI 3-kinase inhibitor LY294002, and p38 MAP
kinase inhibitor SB203580. Inhibitors were made up as 50 mM stock solutions in dimethyl sulfoxide, and stored at
20 °C. The following antibodies for Western blotting were purchased from Cell
Signaling (Beverly, MA): phospho-Thr-202/Tyr-204 and total p44/42 MAP
kinase; phospho-Ser-473 and total Akt; phospho-Thr-180/Tyr-182 and
total p38 MAP kinase; phospho-Tyr-1068 EGFR and total EGFR. Natural
human IGFBP-3 was purified from Cohn fraction IV of human plasma, as
reported previously (21). Recombinant human IGFBP-3 and IGFBP-5 were
expressed in human 911 retinoblastoma cells using an adenoviral
expression system, and purified by IGF-I affinity chromatography and
reverse-phase high performance liquid chromatography (22, 23).
Electrophoresis and ECL reagents were purchased from Bio-Rad,
Amrad-Pharmacia (Ryde, New South Wales, Australia), and Pierce. EGF and
protein A were radiolabeled with 125I (ICN) using
chloramine T.
-counter.
-counter.
80 °C. Prior to SDS-PAGE
analysis, thawed lysates were sonicated for 15 s, heated at
90 °C for 10 min, cooled on ice, and then centrifuged at 15,000 rpm
for 2 min.
-glycerol
phosphate, 1 mM sodium vanadate, 1 µg/ml leupeptin) for
10 min at 4 °C. Lysates were scraped into Eppendorf tubes on ice and
sonicated four times for 10 s each, then clarified by
centrifugation at 15,000 rpm for 10 min at 4 °C. Supernatants (200 µl containing 200 µg of protein) were transferred to fresh
Eppendorf tubes, and phospho-p44/42 MAPK (Thr-202/Tyr-204) and
phospho-p38 MAPK (Thr-180/Tyr-182) immunoprecipitated overnight at
4 °C using phosphospecific antibodies immobilized onto agarose
hydrazide beads (Cell Signaling). Beads were washed twice with 500 µl
of cell lysis buffer, once with kinase buffer (25 mM Tris,
pH 7.5, 5 mM
-glycerol phosphate, 2 mM
dithiothreitol, 0.1 mM sodium vanadate, 100 mM
MgCl2), and resuspended in 50 µl of kinase buffer. Two
micrograms of recombinant Elk-1 or Atf-2 was added to tubes containing
the relevant kinase, with ATP to a final concentration of 200 µM. Phosphorylation reactions were carried out over 30 min at 30 °C and terminated by addition of 25 µl of 3× Laemmli
sample buffer (187.5 mM Tris-HCl, pH 6.8, 60 g/liter SDS,
30% glycerol, 150 mM dithiothreitol) and boiling for 5 min. Samples were vortexed, centrifuged for 2 min, and then loaded onto
10% polyacrylamide gels for Western analysis using antibodies against
phosphorylated Elk-1 (Ser-383) or phosphorylated Atf-2 (Thr-71).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IGFBP-3 potentiates EGF-stimulated DNA
synthesis and proliferation in MCF-10A cells. Panel
A, MCF-10A cells were treated in the absence of serum with
EGF at the indicated concentration, and IGFBP-3 at 1 (open
squares), 10 (open circles), or 100 (open triangles) ng/ml, or in the absence of
IGFBP-3 (filled circles). DNA synthesis was
determined 20-24 h later, as described under "Experimental
Procedures." Data are expressed as a percentage of counts
incorporated in the absence of additions, and derive from pooled data
of four to six experiments performed in quadruplicate. *,
p < 0.05; **, p < 0.001, compared
with the same concentration of EGF in the absence of IGFBP-3. For
panel B, cells were treated with EGF (1 ng/ml) in
the absence or presence of IGFBP-3 at 10 or 100 ng/ml as indicated, in
serum-free medium, and cell numbers were determined 7 days later. Data
are expressed as a percentage of the cell number in control (untreated)
cultures, and are pooled from two experiments each carried out in
triplicate wells. Significant differences shown are p < 0.05 (*) and p < 0.001 (**).
The effects of IGFBP-3 and EGF on cell proliferation were determined over 7 days in the absence of serum or other exogenous growth factors. EGF alone (1 ng/ml) increased cell number by ~30% compared with untreated cultures (p < 0.05, Fig. 1B), and this was enhanced in a dose-dependent manner by 10 and 100 ng/ml IGFBP-3. Final cell numbers in MCF-10A cultures treated with EGF and 100 ng/ml IGFBP-3 were increased by 40% relative to control (p < 0.001).
IGFBP-3 shares many structural and functional features with another
IGF-binding protein, IGFBP-5 (26). Therefore, we investigated whether
IGFBP-5 was also able to potentiate EGF action in MCF-10A cells, using
purified recombinant human IGFBP-5 expressed in an adenoviral
expression system. As shown in Fig.
2A, adenovirus-derived recombinant human IGFBP-3 (adIGFBP-3) enhanced EGF-stimulated DNA
synthesis similarly to plasma-derived IGFBP-3, with a significant effect at 1 ng/ml, and maximal enhancement at 10 ng/ml. In the absence
of EGF, adIGFBP-5 had no significant effect on DNA synthesis in MCF-10A
cells (data not shown). By contrast with adIGFBP-3, adIGFBP-5 did not
potentiate EGF-stimulated DNA synthesis, and in fact was slightly
inhibitory at low concentrations in the presence of EGF (Fig.
2B).
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We then investigated whether a recombinant human IGFBP-3 mutant
that exhibits decreased cell binding and nuclear import (27, 28)
retains EGF potentiating activity. This mutant, IGFBP-3(mut), has 5 residues in the basic domain in the C-terminal region of IGFBP-3
substituted with the analogous sequence of IGFBP-1, i.e. 228KGRKR MDGEA. Exogenous IGFBP-3(mut) showed
significantly decreased cell association compared with wild type
IGFBP-3 in MCF-10A cells (Fig. 2C), similar to that shown
previously for endogenous mutant protein in Chinese hamster ovary cells
(27). However, in MCF-10A cells some binding was evident at high
concentrations of IGFBP-3(mut), with a significant increase in
cell-associated IGFBP-3 detected with 1000 ng/ml IGFBP-3(mut),
comparable to the binding of 100 ng/ml wild type IGFBP-3 (Fig.
2C). In the presence of EGF, IGFBP-3(mut) enhanced DNA
synthesis to a degree similar to that for adIGFBP-3 (Fig.
2D), suggesting that the potentiating effect of IGFBP-3 occurs in the absence of its nuclear localization and under conditions where its cell association is markedly reduced.
The ability of IGFBP-3 to enhance the effects of other growth factors
in MCF-10A cells was then investigated. TGF-, which binds and
activates the EGF receptor (also known as ErbB1), stimulated DNA
synthesis in MCF-10A cells (Fig.
3A). The stimulation caused by
1 ng/ml TGF-
(~40%) was increased to ~120% in the presence 100 ng/ml IGFBP-3 (Fig. 3B). As observed for EGF, the level of potentiation by IGFBP-3 decreased with increasing concentrations of
TGF-
, so that with 10 ng/ml TGF-
, DNA synthesis was increased only an additional 20% by IGFBP-3 (Fig. 3C).
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To examine whether IGFBP-3 enhanced the effects of hormones that act through other members of the EGF receptor/ErbB family, we tested its effects on DNA synthesis stimulated by heregulin, which binds the EGF receptor family members ErbB3 and ErbB4. In the absence of IGFBP-3, heregulin stimulated DNA synthesis in MCF-10A cells (Fig. 3D), with the maximum dose tested, 25 ng/ml, inducing a 12-fold increase in DNA synthesis. The stimulatory effects of low (0.3 ng/ml, Fig. 3E) or high (10 ng/ml, Fig. 3F) heregulin were not enhanced significantly by IGFBP-3. IGFBP-3 had no effect on DNA synthesis stimulated by platelet-derived growth factor or long-[Arg3]IGF-I, an IGF analog with affinity for IGF receptors but not IGFBPs (data not shown).
To determine whether co-incubation of IGFBP-3 and EGF was necessary for
potentiation, MCF-10A cells were preincubated with IGFBP-3 for 24 h prior to its removal, then EGF was added for an additional 24 h.
As shown in Fig. 4, pre-exposure to
IGFBP-3 resulted in a similar degree of enhancement of EGF-stimulated DNA synthesis to that seen when EGF and IGFBP-3 were added together for
24 h, suggesting that preincubation with IGFBP-3 sensitizes MCF-10A cells to EGF.
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We then investigated the effect of IGFBP-3 preincubation on subsequent
binding of EGF to cell monolayers. Cells were preincubated with or
without IGFBP-3 for 16 h, and then binding of 125I-EGF
to cells over 2 h at 4 °C was determined. As shown in Fig. 5A, the amount of
125I-EGF bound to MCF-10A cells was not changed by
pre-exposing the cells to IGFBP-3, and displacement curves generated by
incubating cells with tracer in the presence of unlabeled EGF were
superimposable in IGFBP-3-pretreated and untreated cells. These data
suggest that exposure to IGFBP-3 does not alter EGF receptor affinity or number.
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The effects of IGFBP-3 on EGFR activation were then examined by assessing Tyr-1068-phosphorylated EGFR in cells pretreated with IGFBP-3 and then exposed to EGF for 5, 15, or 60 min. In the absence of IGFBP-3 pretreatment, EGF-stimulated phosphorylation of EGFR was apparent within 5 min (Fig. 5B); there was no effect of IGFBP-3 on the degree of receptor phosphorylation at this time point. However in cells pretreated for 24 h with either 10 or 100 ng/ml IGFBP-3, EGFR phosphorylation was increased relative to non-preincubated cells within 15 min of exposure to EGF (Fig. 5B). The enhancing effect of IGFBP-3 on receptor phosphorylation was transitory, being lost within 60 min after addition of EGF. Analysis of data from two similar experiments confirmed that pre-exposure to IGFBP-3 significantly enhanced EGF-stimulated phosphorylation of the EGFR at Tyr-1068 (Fig. 5C). IGFBP-3 tested over similar doses, but without subsequent exposure to EGF, did not stimulate phospho-EGFR (data not shown).
Next, we examined whether IGFBP-3 modulates EGF activation of
intracellular signaling, targeting particularly some pathways downstream of the EGFR. MCF-10A cells were preincubated with IGFBP-3 for 24 h and then exposed to EGF for 8 min. Cell lysates were analyzed by Western blot using phosphospecific antibodies directed against p44/42 MAPK, Akt, and p38 MAPK. In the absence of EGF, there
was no detectable phosphorylation of p44/42 or Akt, whereas a low level
of phosphorylation of p38 MAPK was apparent (Fig. 6A). Phosphorylation of each
of these signaling intermediates was stimulated within 8 min of
treatment with 1 ng/ml EGF. Pre-incubation with IGFBP-3 had no affect
on phosphorylation of Akt in response to EGF, but EGF-induced
phosphorylation of p44/42 MAPK and p38 MAPK was enhanced by
pre-incubation with IGFBP-3 with a maximal effect apparent between 8 and 15 min of EGF stimulation. Densitometric analysis of these data
(Fig. 6B) indicated that IGFBP-3 enhanced EGF-stimulated
phosphorylation of p44/42 MAPK and p38 MAPK by ~2-fold and 30%,
respectively. Analysis of lysates from cells stimulated for 1, 4, 8, 12, 18, and 24 h with EGF indicated that, although IGFBP-3
preincubation increased the magnitude of p44/42 and p38 MAP kinase
phosphorylation stimulated by EGF at early time points, this was not
sustained. Within 1 h of EGF treatment, phosphorylation of p44/42
and p38 MAP kinases had returned to basal levels, regardless of whether
there had been pre-incubation with IGFBP-3 (data not shown). A second
smaller peak of phosphorylation of p38 MAP kinase, but not p44/42 MAP
kinase, was apparent 18 h after addition of EGF, but this was not
enhanced in IGFBP-3-preincubated cells.
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The effect of IGFBP-3 preincubation on EGF-stimulated p44/42 and p38
MAP kinase activity was then assessed using in vitro phosphorylation of substrates for these enzymes, Elk-1 and Atf-2, as
markers of kinase activity. As shown in Fig.
7, lysates prepared from MCF-10A cells
treated for 10 min with EGF showed increased activity of both p44/42
and p38 MAP kinases. The activity of both kinases was enhanced further
in lysates from cells incubated with 10 or 100 ng/ml IGFBP-3 prior to
exposure to EGF, indicating that IGFBP-3 potentiates EGF-stimulated
p44/42 and p38 MAP kinase activity.
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To determine the involvement of these pathways in the growth potentiating effects of IGFBP-3 on EGF action, we examined whether attenuation of the p44/42 MAPK or p38 MAPK signaling pathways altered the ability of IGFBP-3 to enhance the effects of EGF. MCF-10A cells were co-incubated for 24 h with IGFBP-3 and EGF in the presence of inhibitors of MEK upstream of p44/42 MAP kinase (PD98059), Akt (LY294002), or p38 MAPK (SB203580). DNA synthesis was determined after 24 h. The dose of inhibitors used in these experiments was intentionally submaximal, because concentrations high enough to block signaling fully through these pathways were cytotoxic over 24 h. As shown in Table I, the doses used were sufficient to abolish the stimulatory effect of EGF on DNA synthesis, and Western blot analysis confirmed that these concentrations were sufficient to reduce the IGFBP-3-induced enhancement of EGF-stimulated phosphorylation of p44/42 and p38 MAPK (data not shown). As shown in Fig. 8, the potentiating effect of IGFBP-3 on EGF action was absent in the presence of the p44/42 MAPK inhibitor PD98059 (20 µM, Fig. 8A); in fact, 100 ng/ml IGFBP-3 caused significant inhibition of DNA synthesis in the presence of EGF when p44/42 MAPK activation was blocked (p < 0.05). Similarly, inhibition of the p38 MAPK pathway using SB203580 (10 µM) abolished the potentiating effect of IGFBP-3 on DNA synthesis in MCF-10A cells (Fig. 8B). Consistent with its lack of effect on Akt phosphorylation shown in Fig. 6, IGFBP-3 was still able to potentiate DNA synthesis stimulated by EGF when the PI 3-kinase pathway was blocked using LY294002 (10 µM, Fig. 8C). Collectively, these data suggest that IGFBP-3 potentiates EGF signaling in MCF-10A cells by enhancing its activation of p44/42 and p38 MAPK signaling pathways.
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DISCUSSION |
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Numerous studies have shown that IGFBP-3 exerts growth-inhibitory effects in a variety of cell types, either through blockade of IGF-stimulated mitogenesis and cell survival (1) or via antiproliferative activity unrelated to its ability to bind IGFs (7, 9, 10, 20, 29); however, growth-stimulatory effects of IGFBP-3 are less well documented. Early studies in fibroblasts (12, 30) and MCF-7 breast cancer cells (11) suggested that ligand interaction was involved in the stimulatory effect of IGFBP-3 on IGF activity, with its mechanism of action thought to involve either prevention of ligand-induced down-regulation of the IGFR1 (11) or processing of the IGFBP-3 to forms with altered affinity for IGFs (30). A study in airway smooth muscle cells also indicated a growth-stimulatory role for IGFBP-3 in the presence of fetal calf serum (13). In a bovine mammary epithelial cell line transfected to express IGFBP-3, DNA synthesis was increased in response to IGF-I, insulin, and long-[Arg3]IGF-I (31), implying that IGF-IGFBP-3 interaction was not required for a potentiating effect. However, growth-stimulatory interactions between IGFBP-3 and systems other than the IGF axis have not been reported previously.
In this study we have shown that, in MCF-10A breast epithelial cells,
IGFBP-3 enhances the potent growth-promoting effects of members of the
EGF system, which has been implicated in the development and
progression of malignant disease, by priming cells to respond to EGF
and TGF-. Although the underlying mechanism by which IGFBP-3
potentiates EGF action in these cells remains unclear, we made the
novel and significant observation that in cells pre-exposed to IGFBP-3,
EGF-stimulated phosphorylation of the EGFR at Tyr-1068 was increased.
Phosphorylation of this residue is crucial to linking EGFR activation
with the Ras-MAPK signaling pathway via Grb2 and Sos (32) and, although
we did not explicitly determine whether EGFR kinase activity was
increased, our observation of increased phosphorylation and activation
of p44/42 and p38 MAPK signaling intermediates downstream of Ras in
IGFBP-3-primed cells is consistent with increased Ras activation
occurring consequent to increased Tyr-1068 phosphorylation of the EGFR.
At present it is unclear how IGFBP-3 might be bringing about an increase in EGFR phosphorylation. Preincubation with IGFBP-3 did not markedly affect steady-state binding of EGF to cells, suggesting that an overall increase in EGF·EGFR interaction is not involved in the potentiating effect of IGFBP-3, although more dynamic effects on receptor availability arising from changes in internalization or heterodimerization (33) were not investigated. It is possible that IGFBP-3 is modulating the expression or activity of molecules involved in regulating receptor interaction with binding partners such as the Ras exchange factor Sos1 (34), or EGFR dephosphorylation, such as phosphotyrosine phosphatases. IGFBP-3 activation of a phosphotyrosine phosphatase that dephosphorylates the IGFR1 was recently proposed (35), although the IGF independence of IGFBP-3 action in this model has not been clearly demonstrated. Increased phosphorylation of the EGFR, as in the present study, would suggest decreased activity of phosphotyrosine phosphatases rather than increased, and the possibility that this is involved in IGFBP-3-enhancement of receptor phosphorylation is currently under investigation.
In MCF-10A cells, IGFBP-3 enhanced activation of the p44/42 and p38 MAP kinase signaling pathways, but not the PI 3-kinase pathway, in response to EGF stimulation. Involvement of the p44/42 and p38 MAP kinase pathways in progestin priming of breast cancer cells to respond to EGF has also been demonstrated in T47D breast cancer cells (36), associated with induction and activation of multiple proteins, including EGF receptor family members and Stat (signal transducers and activators of transcription) proteins (36, 37). In experiments not presented in this report, there was no clear change in expression of total Stat1, Stat5a or Stat5b protein in IGFBP-3-preincubated cells, suggesting that progestin and IGFBP-3 priming of cells to respond to EGF may involve distinct pathways and intermediates.
Although the structural elements of IGFBP-3 required for its
interaction with EGF signaling have not been elucidated, our experiments suggest that its interaction with major cell-surface moieties is not required. The 228KGRKR motif of IGFBP-3 has
been implicated in its cell association (27), and, indeed, we found
that the mutant with this region replaced with the corresponding region
of IGFBP-1, IGFBP-3(mut), exhibited greatly reduced binding to MCF-10A
cells compared with plasma-derived IGFBP-3. Despite this, mutant
IGFBP-3 enhanced EGF activity with similar potency to wild type
IGFBP-3, implying that IGFBP-3-priming of cells to respond to EGF
occurs in the absence of IGFBP-3 binding to a major cell-surface
component, although not ruling out the possibility of interaction with
a low abundance cell-surface protein. Although a clear relationship between cell association of IGFBP-3 and its biological activity has not
been demonstrated, the existence of, and requirement for, an IGFBP-3
receptor capable of mediating a growth-inhibitory signal has been
inferred from studies indicating a correlation between IGFBP-3
interaction with cell surfaces and effects on cellular growth (38, 39).
We have shown that IGFBP-3 can initiate inhibitory signaling in breast
cancer cells through the TGF- receptor/Smad pathway (40, 41);
however, the primary site of cell interaction of IGFBP-3 in this system
was not identified. Indeed, a plasma membrane receptor with IGFBP-3
signal transduction capability, either for growth stimulation or growth
inhibition, has not yet been identified in any cell system.
Nuclear localization of IGFBP-3 has been identified by a number of
research groups (28, 42, 43); however, the function of IGFBP-3 in the
nucleus remains unknown. It has been suggested that interaction between
IGFBP-3 and the retinoid X receptor- and regulation of its
transcriptional activity in the nucleus is essential for the apoptotic
effects of IGFBP-3 in prostate cancer cells (43). The findings of the
present study indicate that intranuclear interactions between IGFBP-3
and transcriptional regulators are not required for its
growth-stimulatory interaction with EGF signaling, because the
C-terminal basic motif of IGFBP-3 that is required for its nuclear
localization (28), 228KGRKR, is not necessary for
enhancement of EGF signaling by IGFBP-3. Consistent with this, IGFBP-5,
which has a similar basic motif and shares a common pathway of nuclear
transport with IGFBP-3 (28), did not enhance EGF action, and was in
fact slightly inhibitory in the presence of EGF.
Other studies from our laboratory have also shown that neither
significant cell binding nor nuclear localization of IGFBP-3 is
required for its bioactivity. Exogenous IGFBP-3(mut) induced phosphorylation of TGF- signaling intermediates similar to
plasma-derived IGFBP-3 in a T47D cell line (41), and IGFBP-3(mut)
overexpression inhibited growth and induced apoptosis in T47D breast
cancer cells (44). In the latter case, endogenous mutant IGFBP-3 may
have direct intracellular effects, thereby overcoming the need for secretion and re-uptake, which might require cell-surface binding. Bioactivity of exogenous mutant IGFBP-3 as in the present study and
that of Fanayan et al. (41) is somewhat more difficult to explain, although, as we demonstrated, cell binding of IGFBP-3 was not
completely abolished by substitution of the basic residues, and
residual binding of IGFBP-3(mut), perhaps to low abundance cell-surface
moieties, may be sufficient to elicit a response. Notably, N-terminal
fragments of IGFBP-3 that lack this domain are biologically active in a
number of systems (6, 45, 46), frequently with increased potency
compared with intact wild type IGFBP-3. Understanding the mode of
action of IGFBP-3, as either a growth inhibitor or stimulator, and the
structural determinants of such action remain important goals.
The concentrations of plasma-derived IGFBP-3 required for stimulation of EGF activity in MCF-10A cells (10-100 ng/ml) are similar to those required for its inhibitory activity in this cell line in the absence of EGF (20), up to an order of magnitude lower than those used in the demonstration of IGFBP-3 bioactivity in some other cell systems (7, 41), and similar to the levels expressed by many cell types (1). In view of the fact that MCF-10A cells secrete ~30 ng/ml IGFBP-3 under the conditions used in these experiments (20), it is somewhat surprising that they remain sensitive to exogenous IGFBP-3 at similar concentrations, and may reflect differences between the cell-derived IGFBP-3 and exogenous IGFBP-3 used in this study. It is noteworthy that, when compared within the same experiment, recombinant IGFBP-3 appeared to show increased potency compared with plasma-derived IGFBP-3, with significant enhancement of EGF activity with 1 ng/ml adenoviral IGFBP-3, compared with 10 ng/ml plasma-derived IGFBP-3. Adenoviral IGFBP-3 is essentially unphosphorylated, whereas plasma-derived IGFBP-3 has ~1 mol/mol serine phosphorylation (data not shown), raising the possibility that differences in the potency of IGFBP-3 from alternative sources may be caused by its differential phosphorylation.
Our observation of activation of the p44/42 and p38 MAPK signaling pathways, but not Akt/PKB, is in contrast with a study that showed, in fibroblasts, the PI 3-kinase pathway appears to be involved in IGFBP-3 potentiation of IGF action, with IGFBP-3 increasing the sensitivity of Akt/PKB to phosphorylation by IGF-I (47). This implies that IGFBP-3 may impact on numerous growth factor-regulated pathways, perhaps in a cell- or growth factor-specific manner, to enhance cell proliferation.
Although attenuation of either the p44/42 or p38 MAP kinase pathways
was sufficient to block the potentiating effect of IGFBP-3, inhibition of the p44/42 MAP kinase pathway alone had the additional effect of reinstating the growth-inhibitory activity of IGFBP-3 in
MCF-10A cells. We have shown previously a similar reversal of
refractoriness to growth inhibition by IGFBP-3 by blocking p44/42
MAP kinase activation in MCF-10Aras cells, in
which chronic activation of this pathway occurs as a result of
transfection with oncogenic ras (20). The results of the
present study confirm that activation of Ras-MAPK signaling ablates the
growth-inhibitory activity of IGFBP-3, and, importantly, extend these
findings to show that interactions between IGFBP-3 and
Ras-dependent signaling pathways may result in enhanced
growth-stimulatory signaling in breast cells. Inhibition of this
pathway has the potential both to block stimulatory activity arising
from the interaction of IGFBP-3 with other growth factor systems and to restore its inhibitory activity. Clearly, the identification of the
factors involved in the potentiation of IGFBP-3 of EGF action will be
the next important step in delineating its growth-stimulatory role in
breast cancer cells, and explaining its association with highly
malignant cancers with poor prognosis.
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ACKNOWLEDGEMENT |
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Recombinant IGFBP-3 and IGFBP-5 were kindly provided by Dr. Sue Firth (Kolling Institute of Medical Research, Sydney, Australia).
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FOOTNOTES |
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* This work was supported by National Health and Medical Research Council of Australia Grant 107244 (to J. L. M. and R. C. B.).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.
To whom correspondence should be addressed. Tel.: 61-2-9926-8486;
Fax: 61-2-9926-8484; E-mail: janetlm@med.usyd.edu.au.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M210739200
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ABBREVIATIONS |
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The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; EGF, epidermal growth factor; IGF, insulin-like growth factor; IGFR, insulin-like growth factor receptor; MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; BSA, bovine serum albumin; Stat, signal transducer and activator of transcription; PI 3-kinase, phosphatidylinositol 3-kinase.
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