From the Department of Surgery, McGill
University Health Center, Royal Victoria Hospital, Montreal, Quebec H3A
1A4, Canada, ¶ Department of Anatomy and Cell Biology, McGill
University, Montreal, Quebec H3A 2B2, Canada, and
Faculté
de Pharmacie Paris XI, INSERM U510, 92296 Chatenay-Malabry, France
Received for publication, January 2, 2001
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ABSTRACT |
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The receptor for the type 1 insulin-like
growth factor (IGF-I) has been implicated in cellular
transformation and the acquisition of an invasive/metastatic phenotype
in various tumors. Following ligand binding, the IGF-I receptor is
internalized, and the receptor·ligand complex dissociates as the
ligand is degraded by endosomal proteinases. In the present study we
show that the inhibition of endosomal IGF-I-degrading enzymes in human
breast and murine lung carcinoma cells by the cysteine proteinase
inhibitors, E-64 and CA074-methyl ester, profoundly altered receptor
trafficking and signaling. In treated cells, intracellular ligand
degradation was blocked, and although the receptor and two substrates,
Shc and Insulin receptor substrate, were hyperphosphorylated on
tyrosine, IGF-I-induced DNA synthesis, anchorage-independent growth,
and matrix metalloproteinase synthesis were inhibited. The results
suggest that ligand processing by endosomal proteinases is a key step
in receptor signaling and function and a potential target for therapy.
The receptor for the type 1 insulin like growth factor
(IGF-IR)1 and its ligands
IGF-I and IGF-II regulate normal somatic growth during embryogenesis
and post-natal development (1). However, IGF-I and its receptor have
also been implicated in the development of several human malignancies
such as glio- and neuroblastomas, breast, lung, colon, and prostate
carcinomas (2). Furthermore, IGF-I has been shown to regulate the
induction and maintenance of the transformed phenotype and the
acquisition of the invasive/metastatic phenotype (3, 4).
IGF-I binding to its cell surface receptor activates intrinsic tyrosine
kinase activity generating docking sites for src homology or
phosphotyrosine binding domain-containing proteins and leading to
activation of downstream signaling cascades (5). This is coupled to
ligand-mediated internalization of the receptor leading initially to
attenuation of the IGF-I response through down-regulation of plasma
membrane receptor levels and subsequently to receptor recycling to the
cell surface (6). These processes of receptor internalization and
recycling have long been regarded as key events in the regulation of
receptor activity (7). In the acidic milieu of the endosomes, ligand
dissociation from its receptor is thought to be required for
receptor recycling to the cell surface, and abrogation of ligand
dissociation leads to the intraendosomal accumulation of
ligand·receptor complexes (8). In the case of insulin,
ligand-receptor dissociation at acidic pH values is coupled to
intraendosomal proteolysis by an as yet undefined proteinase (9). For
other internalized ligands such as glucagon or epidermal growth factor
(10, 11), the cysteine proteinase cathepsin B has been implicated in
endosomal ligand degradation. These studies define a tight coupling
between receptor-ligand internalization, ligand dissociation and
proteolysis, and receptor trafficking in the endosomal/lysosomal compartments.
Previously one of our laboratories reported that treatment of murine
Lewis lung carcinoma subline H-59 cells with the cysteine proteinase
inhibitor E-64 blocked their invasiveness in vitro and
inhibited liver metastases formation in vivo (12). In the present work, we investigated further the molecular mechanism underlying the E-64 effects using H-59 and MCF-7 cells. We show here that E-64 and a second cathepsin B inhibitor, CA074-methyl ester
(ME), inhibited endosomal degradation of IGF-I, and this led to an
alteration of IGF-I receptor signaling. Under these conditions, the
IGF-I receptor, as well as the signaling molecules Shc and IRS-1, were
highly tyrosine-phosphorylated. On the other hand, IGF-I-induced DNA
synthesis, anchorage-independent growth, and MMP-2 synthesis were all
inhibited. Selective intervention at the internalization/degradation
level may be of therapeutic relevance in abrogating IGF-I-mediated
tumor formation and metastasis.
Cell Lines and Tissues--
H-59 is a highly metastatic subline
of the Lewis lung carcinoma with metastatic predilection for the liver,
developed by one of our laboratories (13). Human breast carcinoma cell
line MCF-7 (14) was a gift from Dr. Mader (Dept of Biochemistry,
University of Montreal, Quebec, Canada). Endosomal fractions
were prepared from livers of male Harlan Sprague-Dawley rats after an
18-h period of fasting. The livers were homogenized, and the endosomal
fractions were isolated by discontinuous sucrose gradient
centrifugation and collected at the 0.25 to 1.0 M sucrose
interface (9, 10, 15). The soluble extract (ENs) from the
endosomal fractions was isolated by freeze/thawing in 5 mM
sodium phosphate, pH 7.4, and disrupted in the same hypotonic medium
using a small Dounce homogenizer (15 strokes with the tight Type A
pestle) followed by centrifugation at 300,000 × g for
30 min as described previously (9, 10, 15).
Reagents and Antibodies--
E-64
(trans-epoxysuccinyl-L-leucylamido
(4-guanidino)-butane), protein A-Sepharose beads, and MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
thiazolyl blue) were purchased from Sigma. CA074-ME
(N-(L-3trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline), a pro-inhibitor of intracellular cathepsin B (16), was from Peptides
International (Louisville, KY). [3H]thymidine (2.0 Ci/mmol) was from DuPont. Recombinant Human IGF-I used for all the
biological assays was purchased from Intergen (Purchase, NY).
125I-labeled IGF-I (2000 Ci/mmol) used for the ligand
binding assay was obtained from Amersham Pharmacia Biotech.
Human recombinant IGF-I used for the IGF-I proteolysis assay was
radioiodinated by the lactoperoxidase method as described previously
for insulin (15) to specific activities of 350-500 Ci/mmol and
purified by gel filtration on Sephadex G-50. A 1.1-kb type IV
collagenase cDNA fragment was kindly provided by
Dr. W. Stetler-Stevenson (NIH, Bethesda, MD). A 700-bp IGF-IR
cDNA fragment was a kind gift from Dr. M. Pollak (Lady Davis
Research Institute, Montreal, PQ, Canada). The following antibodies
were used: rabbit antiserum to MMP-2 (Ab-45), a kind gift from
Dr. William Stetler-Stevenson (NIH); rabbit antisera against
IRS-1 were either obtained as a kind gift from Dr. Barry I. Posner
(McGill University) or purchased from Calbiochem; anti-phosphotyrosine
mAb PT-66 was from Sigma; horseradish peroxidase
(HRP)-conjugated mAb RC20-H was from Transduction Laboratories
(Mississauga, Ontario, Canada); mAb C-20 to the murine IGF-IR Functional Assays for IGF-IR--
Thymidine incorporation
and soft agar cloning assays were performed as follows: semiconfluent
cultures of H-59 or MCF-7 were cultured in serum free-medium for
24 h with or without E-64, dispersed, seeded onto 96-well
polystyrene plates (Falcon), and incubated with different
concentrations of IGF-I with or without E-64 for 54 h prior to
pulsing with 0.1 mCi/ml of [3H]thymidine for 18 h.
For soft agar cloning (17) the tumor cells were mixed with a solution
of 0.8% agar added to an equal volume of a 2× concentrated RPMI-fetal
calf serum medium with or without 10 µg/ml of E-64, plated on
solidified 2% agar at a concentration of 104 cells/plate,
and supplemented with 1 ml of RPMI-fetal calf serum containing or not
10 µg/ml of E-64. This medium was replenished on alternate days for
12 days. MMP-2 synthesis was analyzed by Northern and Western blotting
and by gelatin zymography, which were performed as previously described
(18). For Northern blotting, 32P-labeled 1.1-kb human MMP-2
and 800-bp rat cyclophilin (19) cDNA fragments were used as
hybridization probes. Western blotting and gelatin zymography were
performed on concentrated (60×) serum-free media conditioned by
H-59 cells for 48 h in the presence or absence of IGF-I with or
without 10 µg/ml E-64. Blots were probed with a 1:500 dilution of mAb
Ab-45 to MMP-2 and an alkaline phosphatase-conjugated affinity purified
goat anti-rabbit IgG, diluted 1:2000.
Measurement of Cell Surface IGF-I Receptors--
The ligand
binding assay and fluorocytometry were used to measure cell surface
IGF-I receptors on the murine H-59 and human MCF-7 cells, respectively.
2-day-old H-59 cultures were replenished with fresh medium containing
or not 10 µg/ml E-64, and the binding assay was performed 24 h
later using 8-1500 pM 125I-labeled IGF-I with
or without graded concentrations of unlabeled IGF-I. Incubation was for
1 h at 37 °C following which the cells were rinsed and lysed in
0.01 N NaOH containing 0.1% Triton X-100 and 0.1% SDS,
and the radioactivity was measured. The number of cells/plate at the
time of the assay was determined from triplicate control wells, which
were manipulated in a similar manner. The Ligand program (20, 21) was
used to calculate the number of IGF-I binding sites per cell. IGF-I
receptors on MCF-7 cells were immunofluorescence-labeled using 5 µg/ml mAb Ligand Proteolysis Assays--
Proteolysis of IGF-I was measured
using the soluble endosomal extract prepared from rat liver parenchyma
(1 ng), and cell lysates (3-15 mg) were derived from H-59 and MCF-7
cells cultured for 24 h with or without 10 µg/ml E-64, lysed by
incubation in 50 mM phosphate buffer, pH 7.4, containing
0.5% Triton X-100, 0.5% deoxycholate, and 0.2 M NaCl for
30 min at 4 °C and then clarified by centrifugation at 30,000 × g for 30 min. These preparations were incubated for
various lengths of time at 37 °C with 10 Immunoprecipitation and Western Blot Analysis--
MCF-7 and
H-59 cells were treated with 10 ng of IGF-I for 5 min following or not
pretreatment with E-64 or CA074-ME as described above. Cells were then
washed with phosphate-buffered saline, solubilized in 30 mM
Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100 and spun at
maximal speed in a microfuge for 15 min at 4 °C. Cell lysates (1 to
3 mg) were then immunoprecipitated, respectively, with anti-Shc,
anti-IRS-1 (from B. I. Posner), or anti-IGF-IR overnight at 4 °C.
Immunoprecipitates were collected by addition of protein A-Sepharose
beads, washed three times with lysis buffer, and resuspended in Laemmli
sample buffer (22). Immunoprecipitates were resolved by SDS
polyacrylamide gel electrophoresis and transferred onto nitrocellulose
membranes followed by either immunoblotting with anti-phosphotyrosine
antibodies (Sigma) or RC-20 (Transduction Laboratories) conjugated to
HRP or with anti-IRS-1 (Calbiochem), anti-Shc, or anti-IGF-IR
antibodies. The blots were revealed by enhanced chemiluminescence
followed by radioautography on Kodak X-Omat AR films.
Abrogation of IGF-IR Functions by the Cysteine Proteinase
Inhibitor E-64--
DNA synthesis, anchorage-independent growth, and
production of the matrix metalloproteinase MMP-2 are three
IGF-I-regulated cellular functions that are critical for the expression
of the malignant phenotype (4, 18). Treatment of MCF-7 and H-59 cells
with the cysteine proteinase inhibitor E-64 at the non-toxic concentration of 10 µg/ml (12) reduced by factors of 7 and 10, respectively, the cloning efficiency of these cells in semi-solid agar
(Fig. 1A) and abrogated
[3H]thymidine incorporation in response to IGF-I in both
cell lines (Fig. 1B). Furthermore, MMP-2 mRNA synthesis
in serum-containing medium, which was previously shown to depend on
IGF-IR expression in these cells (23), was reduced by a factor of 2, and this was reflected in reduced levels of MMP-2 protein levels in
H-59 conditioned medium analyzed by immunoblotting and gelatin
zymography (Fig. 1C). IGF-I-induced MMP-2 production and
activity measured following addition of 10 ng/ml IGF-I to serum-starved
cells were likewise reduced (Fig. 1C).
E-64 Inhibits Endosomal Proteolysis of IGF-I--
Endosomal
endopeptidases such as cathepsin B and L are inhibited by E-64 and have
been implicated in the processing of receptor·ligand complexes (9,
10). We postulated that IGF-I receptor-mediated cellular functions in
E-64-treated cells were altered as a consequence of perturbed endosomal
processing of the internalized receptor-bound IGF-I. Changes in IGF-I
proteolysis were therefore investigated in lysates of E-64-treated
tumor cells, as well as in isolated liver parenchymal endosomal
fractions, which were incubated with exogenous IGF-I at acidic pH
values. Reverse-phase HPLC analysis revealed that IGF-I degradation
products, which were detectable in the untreated preparations, were
absent following E-64 treatment (Fig. 2,
A and C). This was subsequently confirmed when
cell lysates and endosomal fractions were incubated with radioiodinated
IGF-I for 1 h, and trichloroacetic acid precipitation was
used to monitor ligand integrity. An increase in trichloroacetic
acid-soluble radioactivity over time was evident in the untreated
preparation, but this was completely abolished by E-64 pretreatment
(Fig. 2, B and D) indicating that IGF-I
proteolysis was dramatically reduced.
Reduced Cell Surface Levels of IGF-IR in E-64-treated
Cells--
One possible consequence of ligand proteolysis blockade is
the endosomal trapping of receptor·ligand complexes leading to a
decreased availability of free receptor for recycling at the cell
surface. We measured the effect of E-64 treatment on cell surface IGF-I
receptor levels on H-59 and MCF-7 cells. Ligand binding analysis
revealed that the number of IGF-I binding sites as measured after the
addition of 125I-IGF-I to H-59 cells was reduced by more
than 2-fold, from 3.9 × 104 sites/cell on untreated
to 1.8 × 104 sites/cell on E-64-treated cells (Fig.
3A). Flow cytometric analysis with a monoclonal antibody (mAb Increased Levels of Tyrosine-phosphorylated IGF-IR and
Substrates in Cells Treated with Cysteine Proteinase
Inhibitors--
One of the earliest molecular events in IGF-IR
ligand-induced signaling is the autophosphorylation of tyrosine
residues on the receptor Our results show that inhibition of cysteine proteinase
activity by E-64 resulted in reduced cell surface IGF-IR expression levels and abrogation of cellular responses to IGF-I. In an apparent paradox, however, treatment with this or a second cathepsin B inhibitor, CAO74-ME, also caused an increase in tyrosine
phosphorylation of the IGF-IR When taken together with our findings that IGF-I proteolysis was
blocked in E-64-treated liver parenchymal endosomes and in tumor cell
lysates, our results indicate that inhibition of processing of the
IGF-IR·IGF-I complex has the following two major consequences: (i)
receptor recycling to the plasma membrane is decreased, and (ii) the
IGF-IR
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit was from Santa Cruz Biotechnology (Santa Cruz, CA); mAb
IR3
to human IGF-IR was from Calbiochem; HRP-conjugated goat anti-mouse and
goat anti-rabbit IgG antibodies were from Bio-Rad (Mississauga,
Ontario, Canada); and alkaline phosphatase-conjugated affinity purified
goat anti-rabbit IgG was from Bio/Can Scientific, (Mississauga,
Ontario, Canada).
IR3 and a fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (diluted 1:50). Prior to labeling, the cells were
cultured for 24 h in serum free-RPMI with or without 10 µg/ml E-64 then dispersed, reseeded at a density of 105
cells/well into 96-well plates, stimulated with 10 ng/ml of IGF-I for
10 min, and incubated for an additional 30 min at 37 °C. Labeled cells were fixed in phosphate-buffered saline containing 1% formalin and analyzed using a FACS Calibur system (Becton-Dickinson, San Jose, CA).
6
M unlabeled or 50,000 cpm [125I]-labeled
IGF-I in 200 or 400 µl of 50 mM citrate-phosphate, pH 5, respectively. The integrity of the radiolabeled ligand was assessed by
precipitation with 10% trichloroacetic acid (9, 10, 15). To measure
proteolysis of the unlabeled IGF-I, the samples were acidified with
acetic acid (15%) and immediately loaded onto a reverse-phase HPLC
column. Reverse-phase HPLC was performed on a Waters model 600 liquid
chromatograph equipped with a model U6K sample injector fitted with a
500-ml loop and a mBondapak C18 column (0.39 × 30 cm, 10 mm
particle size; Waters). Samples were chromatographed using as eluent a
mixture of 0.1% trifluoroacetic acid in water (Solvent A) and 0.1%
trifluoroacetic acid in acetonitrile (Solvent B) with a flow rate of 1 ml/min. Elution was carried out using two sequential linear gradients followed by the following isocratic elution: an initial gradient of
0-20% Solvent B (30 min); a second gradient of 20-39% Solvent B (15 min); and a third isocratic elution of 39% Solvent B (15 min). Eluates
were monitored on-line for absorbance at 214 nm with a liquid
chromatography spectrophotometer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IGF-IR functions are blocked in tumor cells
treated with the cysteine proteinase inhibitor E-64. A,
reduced anchorage-independent growth. H-59 and MCF-7 cells were
cultured in semi-solid agar for 12 days in the absence (a
and c) or presence (b and d) of 10 µg/ml E-64. Colonies exceeding 250 µm in diameter were scored.
Shown are the results of one of two experiments performed in
triplicate. A light microscopic view of the agar colonies (× 250) is
shown on the right (a and b, H-59;
c and d, MCF-7). The number of colonies/plate
expressed as means and S.D. of three plates is shown on the
left. B, inhibition of IGF-I-induced
[3H]thymidine incorporation. Serum-starved, E-64-treated,
or untreated H-59 (left) and MCF-7 (right) cells
were seeded in 96-well microtiter plates and incubated for 72 h in
serum-free medium containing IGF-I with or without E-64. Shown are the
results of a representative experiment of three performed. They
are expressed as the increase in [3H]thymidine
incorporation relative to cells incubated without IGF-I and represent
means ± S.D. of triplicates. C, loss of MMP-2
synthesis. Top, Northern blot analysis was performed on
total RNA extracted from H-59 cells cultured for 48 h in the
presence of serum, with or without 10 µg/ml E-64. The blots were
probed consecutively with 32P-labeled MMP-2 and cyclophilin
cDNA probes, and the bands were analyzed by laser densitometry.
Results of the densitometry are shown in the bar graph and
are expressed as MMP-2:cyclophiline ratios. Bottom, proteins
in the concentrated media (× 60) collected from H-59 cells cultivated
for 48 h with or without 10 µg/ml E-64 and in the presence or
absence of 10 ng/ml IGF-I were analyzed by immunoblotting using mAb
Ab-45 to MMP-2 (I) and by gelatin zymography
(II). a, control (untreated); b,
E-64-treated; c, untreated, IGF-I stimulated; and
d, E-64 treated, IGF-I stimulated H-59 cells.
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Fig. 2.
The cysteine proteinase inhibitor E-64 blocks
endosomal IGF-I degradation. The effect of E-64 on IGF-I
degradation was tested using lysates of E-64-treated cells
(A and B) and endosomal fractions isolated from
whole rat livers (C and D). H-59 and MCF-7 cells
were cultured for 24 h with or without 10 µg/ml E-64. The cells
were lysed, and cell lysates were immediately incubated with
10 7 M IGF-I and analyzed by reverse-phase
HPLC. Shown are profiles of eluates monitored on-line for absorbance at
214 nm. Typically, two peaks corresponding to intact
(arrowhead) and degraded (arrow) IGF-I were
observed in untreated cells. E-64-treated cells produced essentially
one peak corresponding to the intact IGF-I. Loss of IGF-I degradation
was confirmed by assessing the integrity of the ligand in 10%
trichloroacetic acid precipitates following incubation of the cell
lysates for up to 1 h at 37 °C with
[125I]-labeled IGF-I (B). Similar patterns
were observed when soluble endosomal extracts prepared from rat
liver parenchyma were incubated with unlabeled (C) or
125I-labeled (D) IGF-I in the presence or
absence of 10
7 M E-64. EN,
endosome.
IR3) to the
subunit of the human IGF-I receptor revealed that 40 min after the addition of ligand
to serum-starved MCF-7 cells, there was a reduction of 45% in the
number of immunolabeled cells with the mean intensity of fluorescence
declining from 255 to 82 (Fig. 3B). In neither of these cell
types did E-64 treatment cause a reduction in IGF-IR mRNA levels
(Fig. 3C) or in the total level of immunoprecipitable receptor (Fig. 3D). These experiments suggested that the
reduction of IGF-IR expression at the cell surface was not because of a change in receptor transcription or translation.
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Fig. 3.
E-64 treatment causes a reduction in
post-ligand binding cell surface receptor expression without affecting
IGF-IR synthesis. A, analysis of cell surface IGF-IR by
125I-IGF-I binding. H-59 cultures were incubated with fresh
medium containing or not 10 µg/ml E-64. 24 h later, cultures
were incubated with 8-1500 pM 125I-labeled
IGF-I (triplicate wells were used for each concentration), with or
without graded concentrations of unlabeled IGF-I as described under
"Experimental Procedures." Counts were analyzed using the Ligand
program and are presented as a Scatchard plot. B/F,
bound/free. B, analysis of cell surface IGF-IR by flow
cytofluorometry. Serum-starved, MCF-7 cells incubated for 24 h
with or without 10 µg/ml E-64 were incubated for 10 min with or
without 10 ng/ml IGF-I and then allowed an additional 30 min of
incubation at 37 °C prior to labeling with mAb IR3 and a
fluorescein isothiocyanate-conjugated goat anti-mouse IgG serum for
flow cytofluorometry. Shown are results of a representative of two
experiments performed. Numbers on the right
indicate the proportion of positively labeled cells. MIF,
mean intensity of fluorescence. Analysis of IGF-IR mRNA
(C) and protein levels (D) is shown. Total RNAs
extracted from H-59 and MCF-7 cells that were cultured for 24 h in
the presence or absence of 10 µg/ml E-64 were analyzed by Northern
blotting (C) using 32P-labeled, 700-bp human
IGF-IR and 800-bp rat cyclophilin cDNA fragments as hybridization
probes. Protein levels (D) were analyzed by
immunoprecipitation and immunoblotting with mAb C-20 to the 95-kDa
IGF-IR
subunit.
subunit and the subsequent phosphorylation
of downstream substrates such as IRS-1 and Shc. We first measured
ligand-induced tyrosine phosphorylation of the receptor in E-64- and
ME-treated cells by immunoprecipitation with anti-IGF-IR antibodies
followed by immunoblotting with either anti-phosphotyrosine antibodies or anti-IGF-IR antibodies. Three experiments were performed for each
condition. In all the experiments, we found an increase in tyrosine-phosphorylated receptor
subunit in inhibitor-treated cells. For ME-treated cells the means of the increases were 3.75 (p < 0.0007, n = 3)- and 2.08 (p < 0.001, n = 3)-fold, and for E-64-treated cells they were 2.1 (p < 0.002, n = 3)- and 1.33 (p < 0.005, n = 3)-fold compared with IGF-I-treated H-59 and MCF-7 cells, respectively (Fig. 4, A
and B). The different responses to the two inhibitors may
reflect a higher efficiency of intracellular uptake of ME because of
its increased membrane permeability. Two major substrates of the IGF-IR
are Shc and IRS-1. Their interactions with the activated IGF-IR have
been mapped to the Tyr950 residue suggesting that
they could potentially compete for binding (24). In MCF-7 cells, a
1.7-fold increase in tyrosine-phosphorylated IRS-1 was noted in
ME-treated cells stimulated with IGF-I as compared with cells
stimulated with IGF-I only (Fig. 4D, n = 3, p < 0.0008), but no significant change in
p52Shc tyrosine phosphorylation was noted (data not shown).
In H-59 cells, on the other hand, p52Shc tyrosine
phosphorylation increased by 1.9-fold (n = 2) in
response to ligand binding in E-64- or ME-pretreated cells (Fig.
4C, upper and middle panels), whereas
Shc expression per se did not change (Fig. 4C,
bottom panel, n = 3). In these cells, no
detectable levels of IRS-1 were observed by Western blotting or
immunoprecipitation using experimental conditions that identified this
protein in MCF-7 cells. It appears therefore that in these cells,
differential expression levels of the adaptor proteins Shc and IRS-1
may determine their preferential IGF-IR binding and phosphorylation in
response to IGF-I.
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Fig. 4.
Increased levels of
tyrosine-phosphorylated IGF-I receptor in E-64-treated tumor
cells. Serum-starved H-59 (A) or MCF-7 (B)
cells were treated overnight with 10 µg/ml E-64 or CA-074 ME and then
stimulated for 10 min with 10 ng/ml IGF-I. The cells were lysed, and
the proteins were immunoprecipitated using 10 µg of mAb IR3 (for
MLF-7) or mAb c-20 (for H-59) and then immunoblotted with
anti-phosphotyrosine (PY) mAb PT-66 or anti-IGF-IR mAb C-20
using an HRP-conjugated goat anti-mouse IgG antiserum as a
secondary antibody. Each of these experiments were done three times.
Cell lysates were also immunoprecipitated with anti-Shc or anti-IRS-1
antibodies, and the immunoprecipitates were resolved by SDS
polyacrylamide gel electrophoresis and immunoblotted with either
anti-phosphotyrosine antibodies or antibodies to Shc or IRS-1. In H-59
cells (n = 2), Shc was found to be
tyrosine-phosphorylated (C) whereas in MCF-7 cells
(n = 3), IRS-1 was tyrosine-phosphorylated
(D).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit, IRS-1, and Shc.
subunit and the IRS-1/Shc substrates remain hyperphosphorylated, and this attenuates rather than activates IGF-IR-mediated biological functions such as the induction of DNA
synthesis and MMP-2 production. A direct effect of cysteine proteinase
inhibitors on the synthesis or activity of tyrosine phosphatases have
not, to our knowledge, been documented. Our data may therefore be best
reconciled in a model whereby inhibition of IGF-I proteolysis
"traps" the receptor·ligand complex in a subcellular compartment
that retains the receptor in a hyperphosphorylated state (as
represented in Fig. 5). This, in turn,
may result in retention of phosphorylated receptor substrates in the
same compartment, preventing them from accessing normal signaling
pathways in the cytoplasm. Alternatively, receptor retention in the
endosomes may trigger an alternate signal transduction cascade that
leads to inhibition rather than activation of DNA synthesis and MMP-2 production. Receptor phosphorylation and signaling within the endosomal
compartment have been documented for the insulin receptor kinase (15,
25), and epidermal growth factor receptor activation and Shc
recruitment within endosomes have also been observed (26, 27). Reports
have linked receptor internalization to the activation of the
Shc/mitogen-activated protein kinase pathway (28, 29). On the other
hand, inappropriate localization of signaling molecules after
ligand-induced activation of tyrosine kinase receptors may also lead to
the generation of abnormal signaling pathways (30, 31).
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Fig. 5.
Schematic representation of the
postulated effects of the proteinase inhibitors E-64/ME on
IGF-I-induced DNA synthesis. En, endosomes; E-64
Comp, E-64/ME-dependent compartment.
It has been clearly shown that ligand degradation is a key event in
receptor recycling (11, 15) and signaling (27). Recently it was
reported that anti-p185/HER2 antibody-mediated targeting of a cysteine
proteinase inhibitor to a cathepsin B-containing intracellular
compartment resulted in growth inhibition of two breast carcinoma cell
lines including MCF-7 (32). Our model offers mechanistic insight into
these observations, suggesting that growth impairment in these cells
was related to defective ligand processing in the endosomes.
Collectively, the results identify the endocytic apparatus as a
critical component of growth factor receptor signaling that can be
accessible and sensitive to specific proteinase inhibitors.
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ACKNOWLEDGEMENTS |
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We are indebted to Tarek Boutros and Lucia Fallavollita for excellent technical assistance and to Dr. JohnMort (Shriners Hospital, Montreal, Quebec) for reagents and helpful discussions.
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FOOTNOTES |
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* This study was supported mainly by a grant from the Medical Research Council of Canada (to P. B.) and also by a grant from the National Cancer Institute of Canada (to J. J. M. 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.
§ Present address: Lung Biology Research Laboratory, Hospital for Sick Children, Toronto, Ontario, Canada M4Y 2R8.
** Present address: Rm. 1075, Samuel Lunenfeld Research Inst., Mount Sinai Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada.
To whom correspondence should be addressed: Dept. of Surgery,
McGill University Health Center, Royal Victoria Hospital, Rm. H6.25,
Montreal, Quebec H3A 1A4, Canada. Tel.: 514-842-1231 (ext. 6692); Fax:
514-843-1411; E-mail: pnina.brodt@muhc.mcgill.ca.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M100019200
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ABBREVIATIONS |
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The abbreviations used are: IGF-IR, receptor for the type 1 insulin like growth factor; IRS-1, insulin receptor substrate-1; ME, methyl ester; kb, kilobase; bp, base pair; mAb, monoclonal antibody; MMP-2, matrix metalloproteinase-2; HRP, horeseradish peroxidase; HPLC, high pressure liquid chromatography.
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REFERENCES |
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