From the Extracellular Matrix Pathology Section, Laboratory of Pathology, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-1500
Received for publication, September 6, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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The tissue inhibitors of metalloproteinases
(TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell
proliferation, migration, and invasion that are associated with
extracellular matrix (ECM) turnover. Here we demonstrate a
direct role for TIMP-2 in regulating tyrosine kinase-type growth factor
receptor activation. We show that TIMP-2 suppresses the mitogenic
response to tyrosine kinase-type receptor growth factors in a fashion
that is independent of MMP inhibition. The TIMP-2 suppression of
mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and
implicates cAMP as the second messenger in these effects. TIMP-2
neither altered the release of transforming growth factor In mature normal tissues, the structure and composition of the
extracellular matrix (ECM)1
functions to maintain tissue homeostasis and cellular quiescence. These
anti-proliferative and differentiation promoting effects of the ECM are
attributable both to its composition and three-dimensional spatial
organization, as well as the presence of soluble growth inhibitors,
such as TGF- In addition to disrupting the structural organization of the ECM, MMP
proteolysis of ECM can result in release and/or activation of
sequestered growth factors (1, 3). In addition, MMP activity may expose
cryptic sites in the ECM or directly modify cell surface receptors or
ligands involved in both cell-matrix, as well as cell-cell adhesion (1,
3, 9). The endogenous metalloproteinase inhibitors, tissue inhibitors
of MMPs (TIMPs), negatively regulate the proteolytic activity of MMPs
during ECM turnover. Reduction or ablation of TIMP gene expression
results in enhanced ECM proteolysis concomitant with up-regulation of
cell invasive activity of nontransformed differentiated cells (10, 11).
In comparison, TIMP overexpression results in decreased invasion of
endothelial and tumor cells both in vitro and in
vivo (12, 13). Recent transgenic animal studies have demonstrated
that alteration of the MMP/TIMP balance in vivo in favor of
TIMP-1 activity can block neoplastic proliferation in the SV40 T
antigen-induced model of murine hepatocellular carcinoma (14). The
mechanism of this TIMP-1 effect was mediated by direct inhibition of
MMP processing of insulin-like growth factor-binding protein-3
(IGFBP-3), thereby preventing the release of insulin-like growth
factor II and thus suppressing mitogenic activity. These and other
studies demonstrate that, through inhibition of MMP activity and
prevention of ECM turnover, TIMPs can suppress cell proliferation,
invasion and reduce metastasis formation, i.e. TIMPs act as
tumor suppressors. As a result of such studies, targeting MMP activity
with synthetic MMP inhibitors has become an attractive strategy for
therapeutic intervention in cancer progression (15). However, recent
studies suggest that TIMPs may also directly modulate cell growth in an
MMP-independent fashion, although many of these studies lack detailed
mechanistic insight (16-20). Thus, in addition to their action as
inhibitors of metalloproteinases, it is important to investigate
whether TIMPs function to directly modulate cell growth and the
potential mechanisms for these effects.
Epidermal growth factor receptor (EGFR) is highly expressed in human
cancers and is detectable at low levels in many normal tissues (21).
Overexpression of EGFR has been observed in a variety of human tumors,
and EGF-related growth factors play a role in human cancer growth
through autocrine and paracrine mechanisms (22). Overexpression of EGFR
(23) or structural alterations in the receptor protein, such as
truncation of the cytoplasmic domain, may elicit ligand-independent
signaling and autonomous cell growth (24). Ligand binding to EGFR
initiates receptor dimerization, autophosphorylation of tyrosyl
residues on the cytoplasmic domain of EGFR, and subsequently
Src-mediated activation of the extracellular signal-regulated kinase
mitogen-activated protein (MAP) kinase pathway (25). Mitogenic
signaling of the EGFR seems to critically depend on activation of the
extracellular signal-regulated kinase/mitogen-activated kinase cascade.
Here we study the role of TIMP-2 in the regulation of cell growth in
response to tyrosine kinase-type receptor (TKR) growth factor
stimulation. In addition to soluble ligand binding, membrane-anchored ligands can also stimulate TKR-mediated mitogenic responses at high
cell densities or following proteolytic processing from the cell
surface. Examples are the membrane-anchored EGFR ligands, which include
heparin-bound epidermal growth factor, amphiregulin, transforming
growth factor- We have examined the direct modulation of TKR growth factor-stimulated
proliferation of human, A549 lung carcinoma, MCF7 mammary carcinoma,
HT1080 fibrosarcoma, and Hs68 dermal fibroblast cells using both wild
type TIMP-2 (wt-TIMP-2) and a null-inhibitor form of TIMP-2,
Ala+TIMP-2. Both forms of TIMP-2 abrogate the TKR-mediated mitogenic
responses in these cells. We also investigated the mechanism of the
diminished mitogenic response following TIMP-2 pretreatment prior to
growth factor stimulation. The results demonstrate that these
suppressive effects are mediated by disruption of TKR activation proximal to the extracellular signal-regulated kinase pathway. To our
knowledge this is the first demonstration that TIMP-2 can directly
suppress activation of a mitogenic response through suppression of TKR
activation in an MMP-independent fashion.
Reagents--
Recombinant human EGF, PDGF, and bFGF were
obtained from R&D Systems, Minneapolis, MN. The following commercially
available antibodies were obtained: human anti-EGFR, clone 528, mouse,
monoclonal IgG2a (Santa Cruz Biotechnology, Santa Cruz,
CA); human anti-phosphotyrosine, clone PY-20, mouse IgG2b,
monoclonal, (Transduction Labs, Lexington, KY); human anti-MT-1-MMP,
clone 113-5B7 or 114-6G6 (catalytic), mouse, monoclonal (Chemicon
International, Temecula, CA), human anti-Grb2, clone C-23, rabbit,
polyclonal, (Santa Cruz, Santa Cruz, CA); human anti-SH-PTP1, clone
C-19, rabbit, polyclonal (Santa Cruz Biotechnology); human
anti-SH-PTP2, clone C-18, rabbit, polyclonal (Santa Cruz
Biotechnology); mouse and rabbit IgG-horseradish peroxidase conjugate,
(Santa Cruz); and goat anti-mouse IgG (H+L) (Kirkegaard & Perry,
Gaithersburg, MD). MMP synthetic hydroxamate inhibitor, BB-94, was a
gift from British Biotechnology, Ltd. (Oxford, United Kingdom).
Adenylate cyclase inhibitor, SQ22536, and PKA inhibitor, H89 were
purchased from Calbiochem (La Jolla, CA). The recombinant MT-1-MMP
catalytic domain (150 units/mg) was purchased from Chemicon
International and metalloproteinase activity was determined by the
thiopeptolide assay as described previously (28). RIPA buffer consists
of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS.
The rTIMP-2 protein was expressed using a vaccinia virus expression
system and purified as described (29). TIMP-2 was also expressed in
Escherichia coli with the authentic sequence (wt) or with an alanine residue appended to the amino-terminal cysteine (Ala+TIMP-2) as described previously (28). TIMP-2 and Ala+TIMP-2 were
purified by gel filtration in 4 M guanidine HCl, folded, and oxidized, and then purified by gel filtration under native conditions. Recombinant TIMP-1 was isolated from the conditioned medium
of EPA-transfected Chinese hamster ovary cells (8/8 2G EPA2) (Genetics
Institute, Cambridge, MA) as described (30), and then purified by high
performance liquid chromatography gel permeation chromatography using
50 mM Tris-HCl, 150 mM NaCl, pH 7.5. All TIMP
preparations were endotoxin tested using the Limulus amoebocyte lysis assay and found to contain less than 2 EU/mg of protein.
Cell Culture Conditions--
Human lung adenocarcinoma cells
(A549; ATCC CCL 185), human fibrosarcoma cells (HT1080; ATCC CCL 121),
human breast adenocarcinoma (MCF7; ATCC HTB 22), and human dermal
fibroblasts (Hs68; ATCC CRL 1635) were obtained from American Tissue
Culture Collection (Manassas, VA). Cells were grown to 80% confluence
in Dulbecco's modified Eagle's media (DMEM; Life Technologies Inc.)
containing 4500 mg/liter of D-glucose,
D-glutamine, sodium pyruvate, 100 units/ml penicillin-G,
100 µg/ml streptomycin sulfate, and 10% heat-inactivated fetal
bovine serum, unless otherwise indicated. Cells were trypsinized using
trypsin-EDTA (Life Technologies, Inc., Bethesda, MD).
Cell Growth Assays--
A549, Hs68, HT1080, and MCF7 cells were
plated at 5 × 105 cells/well on a 96-well Costar
plate for 18 h in DMEM with 10% fetal bovine serum. The cells
were then starved for 18 h in DMEM without serum to synchronize
cells in G1 (or G0) phase of the cell cycle. Fresh serum-free DMEM was added to the wells prior to treatment with
TIMP-2. Cells were routinely incubated with TIMP-2 at the indicated
concentrations for 30 min, followed by incubation in DMEM with or
without EGF (100 ng/ml, R & D Systems), bFGF (50 ng/ml, R & D Systems),
or PDGF (50 ng/ml, R & D Systems) and incubated for 24 h. TIMP-2
pretreatment could be reduced to 1 min prior to addition of growth
factor without loss of an effect on growth factor stimulation.
Following growth factor stimulation, the cells were incubated for
1 h with the CellTiter 96TM AQueous One
Solution reagent (Promega, Madison, WI) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium, inner salt, and phenazine ethosulfate. The quantity of formazan product
was determined by the 490 nm absorbance, and was directly proportional
to the number of living cells in culture. The mean and S.D. for
triplicate determinations were recorded for all incubation conditions.
Alternatively, the mitogenic response to growth factor stimulation with
and without TIMP-2 pretreatment was quantitated by [3H]thymidine incorporation assays. Cells were
synchronized in serum-free conditions and treated with TIMP-2 and
growth factors as described above. Following growth factor treatment
[3H]thymidine (0.1 µCi/ml; Amersham Pharmacia Biotech)
was added and incubated for 2 h at 37 °C. The percentage of
thymidine incorporated in a 2-h pulse correlated in a linear fashion
with the cell number. The culture medium was subsequently discarded,
the wells were washed twice with phosphate-buffered saline (PBS), and
the cells were fixed in methanol:glacial acetic acid (3:1). The
incorporated [3H]thymidine was extracted as described
previously and quantitated by liquid scintillation counting (31). The
mean and S.D. of triplicate assays were determined for all incubation
conditions. SQ22536 or 9-(tetrahydro-2-furyl)adenine
(Calbiochem/Novabiochem) was solubilized in sterile deionized
H2O and added to cells at a final concentration of 100 µM (32). H-89 was dissolved in sterile, deionized
H2O and added to give a final concentration of 0.1 µM (33).
The results of the growth assays are presented as the percentage of
maximal growth factor response for the mitogen being tested after
correcting for nonstimulated growth in basal medium. This allows
comparison of the effects of TIMP-2 or Ala+TIMP-2 on the mitogenic
response to various growth factors, as well as between cell lines.
Immunoprecipitation and Western Blotting--
HT1080, Hs68,
A549, or MCF7 cells were grown in a 6-well Costar plate, pretreated
with TIMP-2 or Ala+TIMP, followed by growth factors (described above).
Following incubation with growth factor for 5 min, 37 °C, cells were
washed with PBS and treated for 10 min at 4 °C with RIPA lysis
buffer containing freshly added protease inhibitors (10 µg/ml
aprotinin, 30 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 100 µM sodium orthovanadate). Cells were disrupted by
repeated aspiration through a 21-gauge needle. Cell lysates were
pre-cleared with normal mouse-IgG and Protein A/G (Pierce, Rockford,
IL), and the supernatants were then incubated with anti-EGFR monoclonal
antibodies (clone LA22, Upstate Biotech, Lake Placid, New York) at
4 °C, 1 h. Immune complexes were precipitated with Protein
A/G-agarose and washed extensively with RIPA buffer, 4 °C.
Immunoprecipitated EGFR was resolved by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane
(NOVEX, San Diego, CA) using a blotting apparatus (Bio-Rad). Tyrosine
phosphorylation of the EGFR was visualized by incubating the membrane
with anti-phosphotyrosine antibodies (primary), followed by anti-mouse
IgG horseradish peroxidase antibodies (secondary), and detection using
the ECL system (DuPont Renaissance, PerkinElmer Life Science, Boston,
MA). Total EGFR (loading control) or Grb2 was visualized by incubating
the membrane with stripping buffer (2% SDS, 62.5 mM
Tris-HCl, pH 7.4, 100 mM 125I-EGF Binding Assay--
A549 cell monolayers
were washed with PBS, trypsinized off the tissue culture, and
resuspended in DMEM to give a suspension of 1 × 106
cells/ml. Cells were equilibrated in DMEM containing 10% fetal calf
serum for 1 h, 23 °C, followed by washing with PBS (3 times). TIMP-2, Ala+TIMP-2 ± EGF (unlabeled) was added to cell
suspensions, in binding buffer (Amersham Pharmacia Biotech), followed
by addition of 125I-EGF (100 µCi/ml, >75 Ci/mmol,
Amersham Pharmacia Biotech) and incubated for 3 h, 4 °C. The
cells were diluted in ice-cold binding buffer, collected by gentle
centrifugation (<1000 × g), and washed three times
with cold PBS. EGF bound to the A549 cells was determined by Fluorescent Labeling and TIMP-2 Binding Assay--
TIMP-2 and
Ala+TIMP-2 were labeled with BODIPY-Fl by addition of three molar
equivalents (total) of BODIPY-Fl (Molecular Probes) in three batches
over 2 h, 23 °C. This labeling reaction was protected from
light by covering the reaction vessel with aluminum foil. The reaction
was quenched by addition of 1.5 M Tris-HCl, pH 7.5, to give
a final concentration of 50 mM Tris-HCl. The crude reaction mixture was then passed over a Superose 6 (Amersham Pharmacia Biotech)
gel filtration column using 50 mM Tris-HCl, pH 7.5, 100 mM NaCl as eluate. The BODIPY-labeled TIMP-2 or Ala+TIMP-2
containing peaks were collected and the degree of labeling was
calculated by determining the ratio of absorbance at 450 nm/280 nm. An
optimum value between 0.3 and 0.7 was obtained for these labeling reactions.
Binding assays of BODIPY-labeled TIMP-2 to A549 and MCF7 cells were
performed in triplicate as follows. Cells were grown to 80% confluence
in DMEM containing 10% fetal calf serum, in white-walled, clear-bottom
96-well Costar plates. Cells were washed with PBS and pretreated to
dissociate preformed ligand-receptor complexes with 3 M
glycine buffer in 0.9% saline, pH 3.0, for 3 min. The cells were again
washed in PBS prior to addition of TIMP-2-BODIPY in PBS containing
0.1% bovine serum albumin for 1 h, 37 °C. The supernatant was
removed from cells and placed into empty wells for determination of
unbound TIMP-2-BODIPY. Cell monolayers were washed three times with
ice-cold (4 °C) PBS containing 0.1% bovine serum albumin. Amounts
of bound TIMP-2-BODIPY were analyzed using a plate reader (PerkinElmer
Life Sciences HTS7000), and measuring fluorescence from each well ( Confocal Fluorescent Microscopy--
A549 and MCF7 cells were
plated at a range of 1-3 × 105 cells/ml in a Lab-Tek
chambered glass slide (Nalgene). The growth medium was discarded and
cells were washed with PBS. Nonspecific binding was blocked using 1 mg/ml bovine serum albumin (fatty acid-free), for 1 h. Cell were
washed with PBS 3 times and incubated with BODIPY-labeled TIMP-2 or
Ala+TIMP-2, for 30 min, 37 °C. Following incubation with
BODIPY-labeled TIMP-2, the cells were washed with PBS 5 times and fixed
in paraformaldehyde. For double staining experiments cells were first
incubated with BODIPY-labeled TIMP-2, washed, and fixed as described
above. These cells were then incubated with the appropriate primary
antibody for 1 h, followed by washing with PBS, prior to
incubation with secondary rhodamine-conjugated antibody for 45 min,
23 °C. Following this secondary antibody incubation the cells were
washed three times with PBS and fixed as before. Two drops of
Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA)
containing DAPI (for nuclear counter staining) were added prior to
coverslipping. Cell associated BODIPY-labeled TIMP-2 or Ala+TIMP-2, as
well as anti-MT-1-MMP antibody staining was localized using confocal
laser microscopy. All preparations were examined with a Leica confocal
microscope, model TCS4D/DMIRBE, equipped with argon and argon-krypton
lasers. Cells were originally photographed at × 80 magnification.
Protein-tyrosine Phosphatase Assays--
Protein-tyrosine
phosphatase activities were assayed following immunoprecipitation of
either SHP-1 (clone C-19, Santa Cruz Biotechnology) or SHP-2 (clone
N-16, Santa Cruz Biotechnology) utilizing selective antibodies.
Phosphotyrosine phosphatase activity of the immunoprecipitates was
measured as the amount of phosphotyrosine content remaining after
addition of known amounts of phosphotyrosine-containing peptide
substrates, (Roche Molecular Biochemicals). Hs68 or A549 cells were
plated on 75-cm2 Nunc flasks (2 × 106/flask). Cells were preincubated with TIMP-2, followed
by stimulation with growth factor (described above). Cells were
harvested in lysis buffer containing 10 mM MOPS, pH 6, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM
NaF, 25 mM glycerol phosphate, 10 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 30 µg/ml aprotinin at
4 °C for 10 min. The relative protein concentrations were determined using the BCA assay (Pierce). Equal amount of protein (200 µg) were
immunoprecipitated with anti-SHP-1 or SHP-2 antibodies (Transduction Labs) at 4 °C for 1 h, followed by incubation with anti-mouse agarose beads (Cappel) for 1 h. The beads were subsequently washed 3 times with lysis buffer (without orthovanadate), and 3 times with
phosphatase assay buffer. The immunoprecipitates were resuspended in
assay buffer and the reaction initiated by addition of phosphotyrosine peptides (Roche Molecular Biochemicals). The optical densities were
measured at 405 nm. Data was normalized to blank assay values and
plotted as relative OD units of phosphotyrosine. Enhanced phosphatase
activity is represented by a decrease in phosphotyrosine levels.
TIMP-2 Suppresses Tyrosine Kinase Growth Factor-stimulated Cell
Proliferation--
Previous reports have shown that TIMP-2 and
Ala+TIMP-2 can stimulate the growth of quiescent (serum starved) cells
in culture (28, 34). However, this system does not represent a
physiologic setting in which multiple stimuli, both positive and
negative, are integrated to determine the cellular response. The
effects of TIMP-2 on the mitogenic response to TKR growth factors in
several cells lines, including Hs68, HT1080, A549, and MCF7 cells were examined in vitro. Treatment of these quiescent cells with a
variety of TKR growth factors, including EGF, bFGF, and PDGF,
results in approximately a 2-fold stimulation of cell growth. A
representative example of this growth stimulation is presented in the
inset in Fig. 1A.
Preincubation of quiescent A549, HT1080, HS68, or MCF7 cells with
increasing TIMP-2 concentrations followed by addition of a TKR growth
factor, such as EGF, results in dose-dependent inhibition
of the mitogenic response (Fig. 1A). Preincubation of cells
with TIMP-2, prior to addition of growth factor, was routinely
performed for 30 min at 37 °C, but identical effects were obtained
with preincubation periods as short as 1 min, as previously reported
(28). This suppressive effect on growth factor stimulation was not
observed if TIMP-2 was added concurrent with EGF stimulation or after
treatment of the cells with EGF (data not shown).
The observed effects of TIMP-2 on mitogenic response are not specific
for A549 cells. The effects of TIMP-2 on the EGF-stimulated responses
of HT1080 human fibrosarcoma and MCF7 human mammary carcinoma cells are
essentially identical to those observed with the A549 cells (Fig.
1A). TIMP-2 maximally inhibited (student t test,
p < 0.01) the mitogenic response in all cell lines
tested to 50-60% of the level achieved following EGF stimulation
alone (Fig. 1A). Similarly, TIMP-2 reduced the mitogenic
response to bFGF and PDGF in these cell lines (data not shown). The
effects of TIMP-2 on the mitogenic responses were observed at low
nanomolar concentrations (<10 nM) with the maximal
suppression of growth factor-mediated proliferation obtained at 20-50
nM TIMP-2.
TIMP-2 Effect on Mitogenic Response Is Not Dependent on MMP
Inhibition--
To determine whether the effect of TIMP-2 on growth
factor mitogenic response in these cells was dependent on inhibition of MMP activity, we examined the effect of other MMP inhibitors on the
mitogenic response. We utilized the endogenous MMP-inhibitor, TIMP-1, a
synthetic MMP inhibitor (BB-94, Batimastat), and Ala+TIMP-2, a form of
TIMP-2 that lacks MMP inhibitor activity. Neither TIMP-1 nor the
synthetic hydroxamate inhibitor, BB-94, demonstrated any modulating
effects on mitogenic stimulation in any of the cell lines tested
(data not shown, see below). However, Ala+TIMP-2 was effective at
inhibiting the proliferative response stimulated by EGF, bFGF, and PDGF
treatment of A549 and Hs68 cells (Fig. 1). Pretreatment with 50 nM Ala+TIMP-2 prior to exposure to bFGF or PDGF suppressed
the growth factor-mediated mitogenic response of Hs68 and A549 cells
(Fig. 1B). Ala+TIMP-2 suppressed cell growth to the levels
observed with TIMP-2 stimulation alone, without addition of growth
factors (Fig. 1B). The suppressive effect of Ala+TIMP-2 also
demonstrated a dose dependence (Fig. 1C), however, Ala+TIMP-2 suppressed the mitogenic response to lower levels than those
achieved with TIMP-2.
TIMP-2 Inhibition of Growth Factor Response Requires Adenylate
Cyclase Activity--
We previously reported that the mitogenic
effects of TIMP-2 on quiescent Hs68 or HT1080 cell proliferation were
dependent on activation of a heterotrimeric G protein, and a subsequent increase in cytosolic cAMP (34). In the present study, we examined the
effects of an adenylate cyclase inhibitor (SQ22536) on TIMP-2 suppression of EGF-stimulated cell proliferation. Pretreatment of A549
cells with SQ22536 (100 µM), followed by TIMP-2 (50 nM) and then EGF, ablated the suppressive effect of TIMP-2,
and restored the EGF-stimulated mitogenesis to levels observed in the
absence of TIMP-2 (Fig. 2). In addition,
the suppressive effects of TIMP-2 on mitogenesis were mimicked in these
cells by use of nonhydrolyzable cAMP analogues, such as dibutryl-cAMP
or Sp-cAMP (100 µM). Treatment of
cells with cAMP analogues prior to stimulation with EGF suppressed the
proliferative response in these cells to similar levels as observed
with TIMP-2 or Ala+TIMP-2 (data not shown). These results are identical
to our previous study (28) on the effect of TIMP-2 on the growth
of serum-starved quiescent fibroblasts and suggest that G protein
activation and stimulation of adenylate cyclase are common mechanisms
for both effects.
TIMP-2 Does Not Compete for EGF Binding but Binds to the Cell
Membrane Independent of MT-1-MMP--
The mechanisms of the TIMP-2
mediated effects on stimulated mitogenesis were examined using A549
cells. Among possible mechanisms for the observed effects of TIMP-2 on
cell growth are the inhibition of protease-mediated release of cell
surface-bound EGF ligands or competition and displacement of exogenous
EGF from its cognate receptor. An alternative possibility is the direct
binding of TIMP-2 to the cell surface and activation of adenylate
cyclase activity required for inhibition of growth factor stimulation.
The effect of TIMP-2 on shedding of TGF-
Next, we examined the effects of TIMP-2 on cell surface binding of EGF.
Experiments with 125I-EGF showed that TIMP-2 does not
directly compete with EGF binding to the EGFR (Fig.
3). Incubation of A549 cells with TIMP-2
or Ala+TIMP-2 (100 nM), followed by addition of
125I-EGF (0.2 nM), did not interfere with the
binding of 125I-EGF to the EGFR protein.
25I-EGF binding was competed by addition of nonlabeled EGF,
as expected (Student's t test, p < 0.01).
Addition of TIMP-2 or Ala+TIMP-2 did not alter the competition of EGF
for 125I-EGF bound to EGFR (Fig. 3). These results
definitively demonstrate that TIMP-2 or Ala+TIMP-2 do not alter the
mitogenic response in the cells tested by interfering with the binding
of EGF to its cognate receptor.
The direct binding of TIMP-2 to the surface of A549 and MCF7 cells was
quantified by use of a direct, fluorescent binding assay. Previous
reports from several laboratories have shown that TIMP-2 can bind to
the membrane-type matrix metalloproteinase-1 (MT-1-MMP) (8, 35-38).
This interaction is mediated predominately through interaction of the
NH2-terminal inhibitory domain of TIMP-2 with the catalytic
active site of MT-1-MMP. In our study of TIMP-2 binding to the surface
of A549 cells we have utilized both TIMP-2 and the null inhibitor form
Ala+TIMP-2. Data shown in Fig.
4A demonstrate that Ala+TIMP-2
does not inhibit the ability of MT-1-MMP to degrade synthetic peptide
substrate, compared with the potent inhibitory activity of TIMP-2.
These results are similar to our previous report that TIMP-2 inhibits
MMP-2 activity, while Ala+TIMP-2 does not (28).
In the binding experiments, TIMP-2-BODIPY was added to a monolayer of
cells, grown to 80-90% confluence, and the amount of bound (B)
versus free (F) fluorescent TIMP-2-BODIPY was determined by
quantitation of fluorescence (Fig. 4B). The concentration
dependence of BODIPY-TIMP-2 binding was determined at each
concentration in six replicate measurements in the presence
(nonspecific binding) and absence (total binding) of unlabeled TIMP-2.
The data were plotted as the amount of bound TIMP-2-BODIPY
versus bound/free for Scatchard analysis (Fig.
4B). TIMP-2 bound to A549 cells in a specific and saturable
fashion with a subnanomolar dissociation constant,
Kd = 147 pM, and 115,00 receptors per
cell. For comparative purposes the binding parameters for BODIPY-TIMP-2 interaction with MCF7 cells was also determined. For these cells the
dissociation constant was low nanomolar Kd = 1.90 nM with 38,000 sites per cell. The data for TIMP-2 binding
to MCF7 cells was in excellent agreement with data previously published by others (36, 38, 39), as well as our own laboratory (35), using
125I-TIMP-2 for determination of cell binding parameters.
These findings suggest that our fluorescent-based method for
determination of TIMP-2 binding was comparable in sensitivity and
specificity to methods described previously.
The addition of a 10-fold excess of unlabeled Ala+TIMP-2 to A549 cells
treated with TIMP-2-BODIPY leads to a statistically significant
(Student's t test, p < 0.001) reduction in
binding of TIMP-2 to the cell surface. Ala+TIMP-2 competition results in a 65% decrease in BODIPY-TIMP-2 binding to A549 cells suggesting that it competes for binding to most, but not all, TIMP-2-binding sites
(Fig. 4B). In contrast, addition of a broad spectrum,
hydroxamate MMP inhibitor, BB-94 (0.5 µM), did not
significantly compete for binding of BODIPY-TIMP-2 to the cell surface
(Fig. 4B). This failure of BB-94 to compete with TIMP-2 cell
surface binding was in contrast to the effects of synthetic hydroxamate
MMP inhibitors which have been shown to inhibit the binding of TIMP-2
to MT-1-MMP (36, 38). Western blot analysis of A549 cell membranes
demonstrated that these cells have low but detectable levels of
MT-1-MMP compared with well characterized cell lines such as HT1080
(data not shown). However, addition of an MT1-MMP antibody, specific
for the catalytic domain, maximally reduced the binding of TIMP-2 to
cells by 35% (Fig. 4B). Together these data suggest that
TIMP-2 binds to the cell surface and that this interaction may consist
of at least two binding sites, an interpretation consistent with the
Scatchard analysis shown in Fig. 4A, as well as previous
reports (36).
Confocal, laser fluorescent microscopy was utilized to examine the
localization of TIMP-2 on the cell surface and colocalization with
MT-1-MMP. Confocal fluorescent microscopy demonstrated
fluorescent-labeled (BODIPY) TIMP-2 or Ala+TIMP-2 bound to the surface
of A549 cells (Fig. 5A).
Analysis of Nemarsky optic images of A549 cells with or without TIMP-2
or Ala+TIMP-2 treatment revealed no significant morphologic changes
following short term (30 min) exposure to TIMP-2 (Fig. 5, A,
top and middle left panels; B, top two
panels). Fluorescence localization of BODIPY-TIMP-2 or Ala+TIMP-2
demonstrates a linear, punctate pattern consistent with cell surface
localization (Fig. 5, A, lower panels, B, second panels from
top). xz-axis analysis of the confocal images
(Fig. 5A, bottom panel) confirms that TIMP-2 binding occurs
on the surface of A549 cells. By fluorescent antibody staining the
cells with rhodamine anti-MT-1-MMP antibody complexes, the majority of
TIMP-2 binds to the surface of A549 cells independent of MT1-MMP
localization (Fig. 5B, bottom panel right, and
arrowheads). A minor component of the TIMP-2 on the cell
surface colocalized with MT-1-MMP (Fig. 5B, bottom panel, and arrows). The MT-1-MMP colocalization was markedly less
apparent when the binding of Ala+TIMP-2 was examined (Fig. 5B,
bottom panel, left). Ala+TIMP-2 colocalization with MT1-MMP (Fig.
5, bottom panel, left, and arrows) was reduced
compared with that observed with wtTIMP-2 (Fig. 5, bottom panel,
right, and arrows). These findings are consistent with
our in vitro observations that the Ala+TIMP-2 mutant does
not inhibit MT-1-MMP activity (Fig. 4A) (i.e.
Ala+TIMP-2 does not bind to the MT-1-MMP active site).
TIMP-2 Disrupts EGFR Phosphorylation and Grb-2
Association--
From these studies so far, we have shown that TIMP-2
suppresses EGF mitogenisis without the requirement for MMP inhibition. TIMP-2 binds to the plasma membrane thereby activating an adenylate cyclase signaling pathway. This binding is independent of MT1-MMP, and
does not compete for EGF ligand binding to the EGFR. These findings
suggest that the effect of TIMP-2 on mitogenic stimulation should be
rapid and proximal in the EGF signaling pathway. To further study the
mechanism of TIMP-2 effects on the mitogenic response, we have examined
the activation status of the EGFR receptor (phosphorylation status,
Grb-2 association), as well as phosphatase activity that influences the
state of EGFR activation.
Ligand binding induced activation of the EGFR initiates
autophosphorylation of the receptor on the cytoplasmic, SH2 domain. The
amount of phosphorylated EGFR was measured by Western blot analysis of
EGFR immunoprecipitates prepared from equivalent numbers of A549 lung
adenocarcinoma or HT1080 fibrosarcoma cells. The total quantity of
immunoprecipitable EGFR did not change in response to treatment with
TIMP-2 or Ala+TIMP-2 and therefore served as a loading control for
these experiments (Fig. 6A, lower
gel panel). This finding also demonstrates that the effect of
TIMP-2 or Ala+TIMP-2 occurs in the absence and/or prior to any change
in the level of EGFR on the cell surface (i.e. EGFR
internalization and/or turnover). TIMP-2 pretreatment prior to EGF
stimulation of HT1080 cells results in a dose-dependent
decrease in EGFR-associated tyrosine phosphorylation (Fig.
6A). At the highest concentration of TIMP-2 tested (200 nM) in these experiments, the level of EGFR phosphorylation
approached that of basal levels under serum-free conditions. It should
be noted that under basal conditions the level of EGFR phosphorylation
in these HT1080 cells was low but significantly increased (greater than
10-fold) following EGF stimulation of cell growth. TIMP-2 and
Ala+TIMP-2 also showed a dose-dependent inhibition of EGFR
phosphorylation in MCF7 and A549 cells stimulated with EGF (data not
shown). Inhibition of EGFR tyrosine phosphorylation was not observed
following treatment with BB-94, or TIMP-1 prior to stimulation with EGF
(Fig. 6B). This finding supports the conclusion that TIMP-2
exhibits an early and immediate effect on EGFR activation by a
mechanism independent of MMP-inhibition.
We also examined the effects of an adenylate cyclase inhibitor,
SQ22536, on phosphorylation of EGFR tyrosyl residues. Preincubation of
A549 cells with SQ22536 (100 µM), followed by TIMP-2 (100 nM), and then stimulation with EGF, blocks the TIMP-2
suppressive effects on EGFR phosphorylation (data not shown). When
cells were preincubated with SQ22536, the amount of phosphorylated EGFR
remains similar to levels obtained with EGF stimulation alone. Thus,
the effect of TIMP-2 on EGFR phosphorylation, like the growth
suppressive effect reported in Fig. 2 depends on activation of
adenylate cyclase.
Following ligand-induced dimerization and autophosphorylation of EGFR,
there is specific recruitment of Grb-2 to phosphorylated EGFR (40).
Therefore, we determined the level of Grb-2 associated with EGFR to
confirm the effects of TIMP-2 on EGFR phosphorylation. Cells were
pretreated with TIMP-2 or Ala+TIMP-2 for 30 min, followed by incubation
with EGF for 5 min at 37 °C. Cells were lysed using RIPA buffer and
EGFR immunoprecipitates were prepared as before. The amount of 25-kDa
Grb-2 bound to EGFR was determined by Western blot for anti-Grb-2, of
EGFR immunoprecipitates. Addition of exogenous TIMP-2 prior to EGF
stimulation of MCF7 cells results in a decreased association of Grb-2
with EGFR (Fig. 6C). The decrease in amount of Grb-2
associated with EGFR correlated with the decreased EGFR phosphorylation
observed following TIMP-2 pretreatment. The results demonstrate that
TIMP-2 and Ala+TIMP-2 pretreatment results in a rapid (within 5 min of
EGF treatment) reduction in EGFR phosphorylation, that in turn results
in decreased Grb-2 association with EGFR.
Role of PKA in TIMP-2 Effects on TKR-stimulated Proliferation and
EGFR Phosphorylation--
Pretreatment of A549 cells with the PKA
inhibitor, H89 (0.1 µM), prior to EGF stimulation results
in a 60% reduction of the mitogenic response that is unchanged by the
addition of TIMP-2 prior to EGF stimulation (Fig.
7A). Although H89 did not
appear to reverse the suppressive effect of TIMP-2 on EGF-stimulated growth, the reduction in the EGF-induced mitogenic response by H89
alone prevents exclusion of a role for PKA in mediating the TIMP-2
suppression of EGF stimulated growth.
H89 was also used to investigate the possible role of PKA in the TIMP-2
reduction in EGFR phosphorylation observed following EGF stimulation.
The direct serine phosphorylation of the EGFR receptor by PKA catalytic
subunit reportedly results in down-regulation of EGFR mitogenic
signaling (41). We speculated that if PKA is required for the TIMP-2
down-regulation of EGFR tyrosine phosphorylation, that H89 would
reverse the effect of TIMP-2 in reducing EGFR phosphorylation. Fig.
7B presents the results of these experiments. The data
showed that the effects of H89 on reversal of TIMP-2 reduction in EGFR phosphorylation are at best only partial. The data from the two experiments on cell proliferation and EGFR phosphorylation using the
PKA inhibitor H89 do not definitively demonstrate a clear-cut requirement for PKA activity in the growth suppressive effects of
TIMP-2. However, the data suggest that an alternative pathway that is
not dependent on PKA activation may also function to mediate the
effects of TIMP-2 on cell growth and EGFR phosphorylation.
TIMP-2 Induces SH2 Protein Phosphatase-1 (SH-PTP1) Activity and
Association with EGFR--
The level of phosphorylation of activated
growth factor receptors with endogenous tyrosine kinase activity
depends upon the net result of both tyrosine specific
autophosphorylation and rapid dephosphorylation by phosphotyrosine
phosphatases (PTPs). Receptor dephosphorylation attenuates signaling
downstream from the activated receptor. The SH2-domain containing PTPs
(SH2-PTPs) have been shown to interact with multiple growth factor
receptors, including EGFR (42-44). Generally, SHP-1 (also known as
SH2-PTP-1 and PTP-1C) negatively regulates receptor signaling, while
SHP-2 (also known as SH2-PTP-1 and PTP-1D) reportedly enhances positive
signaling, although negative modulation of receptor signaling by SHP-2
has also been reported (42-44).
In this experiment, we determined if SHP-1 or SHP-2 was bound to EGFR
by immunoprecipitation of the EGFR complex by Western blot. The effects
of TIMP-2 on PTP association with this receptor complex were examined.
Cells (A549 or HT1080) were preincubated with and without TIMP-2 (100 nM), and stimulated with EGF as above. Analysis of
SH2-PTPs associated with EGFR immunoprecipitates demonstrates that, compared with EGF treatment alone, TIMP-2 pretreatment preserves the association of SHP-1 with EGFR immunoprecipitates to levels that are essentially identical with those observed in the basal state.
The levels of SHP-1 associated with EGFR complexes were inversely
correlated with the level of EGFR phosphorylation previously observed
(Figs. 6A, and 8, A and B). In
contrast, the association of SHP-2 with EGFR complex remains unchanged
following either TIMP-2 pretreatment or EGF stimulation of cells (Fig.
8, A and B). We
also assayed total cytoplasmic SHP-1 and SHP-2 activity utilizing an
in vitro tyrosine phosphatase assay following selective immunoprecipitation of total SHP-1 and SHP-2. The results of these experiments demonstrate a significant (Student's t test,
p < 0.01) increase in total SHP-1 activity in cells
pretreated with TIMP-2 prior to EGF stimulation (Fig. 8C,
decrease in optical density for phosphotyrosine staining reflects
enhanced phosphotyrosine activity). No increase in SHP-2 activity was
observed following TIMP-2 preincubation. In fact, EGF stimulation alone
results in a significant induction of SHP-2 activity and a slight
inhibition of SHP-2 activity was noted following TIMP-2 pretreatment
(Fig. 8C). These results demonstrate that TIMP-2 supression
of EGFR activation was mediated, at least in part, through persistent association of SHP-1 with EGFR and a selective increase total SHP-1
activity.
The tissue microenvironment is known to exert a profound influence
on cell proliferation and differentiation (45-47). Cell fate is the
net result of cellular integration of multiple signals derived from
soluble factors and adhesive interactions present in the
microenvironment (48-50). Evidence for the influence of this
integrative process on cell behavior is derived from studies using
reconstituted ECM, as well as alteration in the expression of cell
adhesion molecules, or the introduction of proteases to disrupt the
structure and/or composition of the microenvironment (3, 6, 46, 49).
ECM turnover is a critical event in development, morphogenesis, and
tissue remodeling (3, 6, 46, 49). Enhanced MMP activity results in
altered neonatal development and progression of pathologic conditions
(6, 49). Vis à vis their ability to inhibit MMP
activity, TIMPs can suppress ECM turnover associated with either
embryonic development or pathologic conditions (2, 9, 51), resulting
indirectly in suppression of cell growth and/or direct inhibition of
cellular invasion initiated by ECM remodeling (12-14, 52).
However, studies demonstrate that TIMPs also directly alter in
vitro cell growth and/or survival of a variety of cell types (16,
17, 18, 19, 31, 34, 53-57). These effects are independent of
TIMP-mediated MMP inhibitory activity. TIMP-3 overexpression induces
apoptosis (58, 59), an effect variably reproduced by synthetic MMP
inhibitors and possibly related to stabilization of TNF- In the present report we examine how TIMP-2 induction of intracellular
signaling is integrated with TKR growth factor induction of mitogenic
signals. TIMP-2 abrogates the mitogenic response of a variety of cell
types to several different TKR-type growth factors. The TIMP-2
inhibition of growth factor-stimulated mitogenesis occurs in a
concentration range identical to that observed for the maximal effect
on stimulation of growth in quiescent, dermal fibroblasts (28, 34).
These TIMP-2 concentrations are only slightly lower than reported for
the growth suppressive effect of TIMP-2 on bFGF-stimulated endothelial
cells (31). Mitogenic stimulation of various cell lines at subnanomolar
concentrations of TIMPs has also been reported (19, 53, 55). The
differences in effective TIMP-2 concentrations between these reports
are possibly due to variation in the innate sensitivity of the cell
lines tested, or, alternatively, different TIMP preparations may have
altered potency or contaminants (e.g. endotoxin). All of the
TIMP-2 preparations in this study are known to be free of significant
endotoxin contamination (see "Experimental Procedures"). It is also
possible that differences in the levels of active MMPs produced by the
cell lines may influence responsiveness to the growth modulating
activity of TIMP-2. This is because high concentrations of available
MMP active sites could sequester TIMP-2 and prevent it from binding to
the cell surface.
The suppressive effect of TIMP-2 on growth factor-stimulated
mitogenesis is specific and independent of MMP inhibition. No effects
on stimulated cell growth are observed with TIMP-1 or BB-94.
Ala+TIMP-2, lacking MMP inhibitor activity, remains active in
abrogating the TKR-stimulated response. No evidence of cell death or
apoptosis was observed in these experiments, and the cells remained
viable following exposure to TIMP-2 or Ala+TIMP-2 alone. In fact,
TIMP-2 or Ala+TIMP-2 treatment without growth factor stimulation
reproduces the modest mitogenic stimulation of quiescent cells as
previously reported (28, 34). Ala+TIMP-2 shows a somewhat more potent
effect on inhibiting EGF-stimulated growth compared with the wild type
protein. This is specific for EGF-stimulated growth and is not observed
with the other growth factors utilized in this study. Possible
explanations for this observation include intrinsic differences in the
growth factor-specific responses and/or greater availability of
Ala+TIMP-2, which did not bind effectively to the MT-1-MMP active sites
(see below).
TIMP-2 regulation of TKR-induced mitogenesis occurs immediately
downstream of receptor-ligand interaction during receptor activation.
Previous reports demonstrate that metalloproteinases contribute to
shedding of EGFR ligands, such as TGF- TIMP-2 binds directly to the cell surface (19, 34, 36, 55). However,
the identification of cell surface binding proteins for TIMP-2 is
complicated by the presence of MT-MMPs. These integral membrane
proteins contain a transmembrane domain and catalytic site that is
oriented toward the ECM (8, 37). Binding of TIMP-2 to MT-1-MMP involves
interaction of the NH2-terminal of TIMP-2 with the
catalytic site of MT-1-MMP (38), and is reportedly sensitive to
synthetic, hydroxamate MMP inhibitor (36). Our in vitro
analysis confirms that Ala+TIMP-2 does not inhibit MT-1-MMP activity,
as we have previously reported for MMP-2 (28). This finding suggests
that Ala+TIMP-2 will not bind to the MT-1-MMP catalytic site on the
cell surface.
We demonstrate specific and saturable binding of TIMP-2 and Ala+TIMP-2
to the surface of A549 and MCF7 cells. Cell surface binding of TIMP-2
to A549 cells was not competed by TIMP-1 or BB-94. Anti-MT-1-MMP
catalytic domain antibodies reduce TIMP-2 binding by only 35%,
compared with the 65% reduction following addition of excess unlabeled
Ala+TIMP-2. In fluorescence confocal microscopy experiments, minor
colocalization of TIMP-2 and MT-1-MMP in A549 cells is observed, but is
essentially absent when Ala+TIMP-2 and MT-1-MMP are visualized.
Collectively our studies on TIMP-2 binding and fluorescence
co-localization confirm the presence of a high affinity, TIMP-2-binding
site on A549 cells that is independent of MT-1-MMP. This is a principal
binding site for TIMP-2 in A549 cells that is specifically competed by
Ala+TIMP-2, but is not blocked by synthetic hydroxamate MMP inhibitors
nor anti-MT-1-MMP antibody. The presence of such sites has been
suggested in previous studies that demonstrated TIMP-2 binding in the
presence of synthetic MMP inhibitor (36), but such sites have remained poorly characterized. From these studies we conclude that TIMP-2 binds
to the cell surface and this interaction may consist of at least two
binding sites, one MT-1-MMP independent, as well as a MT-1-MMP site.
This interpretation is consistent with Scatchard analysis, as well as
previous reports of multiple TIMP-2-binding sites (36). TIMP-2 binding
to the MT-1-MMP-independent, high affinity site presumably results in
activation of adenylate cyclase that is required for TIMP-2 inhibition
of TKR-stimulated cell growth. TIMP-2 binding results in heterotrimeric
G protein activation and an increase in cytosolic cAMP level (34).
Activation of downstream signaling cascades by TKR(s) is dependent on
the net level of receptor phosphorylation. Net phosphorylation is
dependent on the level of autophosphorylation induced by ligand binding
and the level of associated protein-tyrosine phosphatase activity (62,
63). Cells treated with TIMP-2 or Ala+TIMP-2, prior to EGF,
down-regulate the level of EGFR autophosphorylation in a
dose-dependent fashion. This effect is again specific for TIMP-2 and is not observed with synthetic, hydroxamate MMP inhibitor, BB94, or TIMP-1. Furthermore, the decrease in EGFR phosphorylation is
confirmed by a concomitant decrease in Grb-2 association with EGFR.
This effect of TIMP-2 on EGFR autophosphorylation is dependent upon
adenylate cyclase activity, but is not completely reversed by the PKA
inhibitor, H89. The lack of PKA inhibitor to reverse the TIMP-2 effect
on cell growth or EGFR phosphorylation is in contrast to the direct
role of PKA in TIMP-2 mitogenic stimulation of quiescent cells
previously reported (34). Also, the positive effect of TIMP-2 on the
growth of quiescent cells is reversed at higher TIMP-2 concentrations,
this is not observed in the TIMP-2 suppression of stimulated growth or
EGFR down-regulation (34). These differences suggest that, although
similar in requirement for adenylate cyclase activity, these actions of
TIMP-2 are mechanistically distinct from one another. Direct
phosphorylation of EGFR by the serine kinase activity of PKA is
observed in vitro with purified, recombinant catalytic
subunit of PKA and in cells following administration of cAMP analogues
(41). The failure of H89 to completely reverse TIMP-2-mediated
reduction in EGFR phosphorylation is consistent with a TIMP-2 action
that is immediately downstream to receptor-ligand interaction and TKR
activation. To our knowledge this is the first demonstration that
TIMP-2 directly interferes with receptor signal transduction at the
level of TKR phosphorylation.
Activated, TKR(s) are rapidly dephosphorylated resulting in
down-regulation of their signaling activity (62, 63). The SH2-PTPs are
known to interact with multiple receptor systems, including the
erythropoietin receptor and interleukin-3 receptor in hematopoietic
cells, as well as the EGFR and vascular endothelial growth factor
(VEGF) receptor (Flt, KDR) in epithelial and endothelial cells,
respectively (42-44). The SH2-PTP, SHP-1, is involved in receptor
dephosphorylation and negative regulation of receptor signaling. SHP-2
has little effect on receptor phosphorylation and positively mediates
receptor signaling via mechanisms that are not well understood. The
direct interaction of SHP-1 with the EGFR receptor has been
demonstrated in vitro (42-44). The catalytic domain of
SHP-1 is important for EGFR dephosphorylation and cannot be substituted
by the catalytic domain in SHP-2 (43). It is not known if SHP-1
displays selectivity with respect to dephosphorylation of individual
phosphotyrosine residues on EGFR (43). Also, EGFR dephosphorylation
does not absolutely correlate with SHP-1 binding (43). This has been
interpreted to suggest that the SHP-1 active site may be sterically
hindered with respect to some phosphotyrosine sites on EGFR, and/or not
all SHP-1 activity is directly bound to EGFR in vivo. SHP-1
may also associate with the EGFR complex via an intermediary docking protein.
In our experiments TIMP-2 prevents dissociation and/or promotes
association of SHP-1 with the EGFR complex in a fashion that correlates
with the decrease in EGFR phosphorylation. These findings are confirmed
by direct measurement of phosphatase activity in SHP-1 and SHP-2
immunoprecipitates prepared from whole cell lysates. Comparison of the
results of SHP-1 levels associated with EGFR in the basal and
TIMP-2-treated cells with the direct assay of SHP-1 activity is
consistent with previous reports that suggest not all SHP-1 activity is
bound to EGFR (43). Alternatively there may be a selective increase in
the specific activity of the SHP-1 associated with EGFR following
TIMP-2 treatment. The observed increase in SHP-2 activity with EGF
treatment is consistent with the reported positive modulation of EGFR
signaling reported for this PTP (44). Our findings are consistent with
previous reports demonstrating that SHP-1 can negatively modulate EGFR activation and prevent downstream signal propagation from this receptor
(43, 44). Also, TIMP-2 may induce a selective increase in SHP-1
activity that is not directly bound to EGFR, but may require some
auxiliary docking protein that is as yet unidentified. Little is known
about regulation of SHP-1 levels or activity, but one report does
suggest that a cAMP-regulated pathway involving phosphorylation of
SHP-1 may function to regulate this activity (64).
In summary, TIMP-2 suppresses the mitogenic response to TKR growth
factor stimulation. TIMP-2 binds to the surface of A549 cells
independently of MT-1-MMP. TIMP-2 binding to the cell surface results
in activation of adenylate cyclase and increased cAMP levels in the
cytosol, presumably secondary to receptor-mediated activation of the
GTP-binding protein G from the
cell surface, nor epidermal growth factor (EGF) binding to the cognate
receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (Kd = 147 pM),
that is not competed by the synthetic MMP inhibitor BB-94 and is
independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation
of EGFR and a concomitant reduction in Grb-2 association.
TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation
from immunoprecipitable EGFR complex and a selective increase in total
SHP-1 activity. These studies represent a new functional paradigm for
TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by
short-circuiting EGFR activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(1-4). Compelling evidence for these effects also
comes from transgenic animal studies in which altered ECM expression or
organization, disruption of ECM attachments, or proteolytic
modification of ECM integrity results in altered developmental and
disease-related phenotypes (5-7). The matrix metalloproteinases (MMPs)
are a major determinant of ECM turnover in tissue morphogenesis.
Altered expression of MMP activity is associated with a variety of
pathologic conditions, including tumor progression and cancer invasion
(5-8).
(TGF-
), and betacellulin, which are shed from the
plasma membrane by proteolytic cleavage resulting in autocrine
activation of the receptor (26, 27). Synthetic metalloproteinase
inhibitors, such as BB-94 (Batimastat), reduce cell proliferation in
the human mammary epithelial cell line 184A1 by blocking TGF-
release (26). BB94 also inhibits EGFR trans-activation by
G-protein-coupled receptors that occurs via a metalloproteinase directed cleavage of pro-heparin-bound EGF (27). These findings suggest
that the metalloproteinase inhibitors prevent the release of
membrane-anchored EGFR ligands (e.g. TGF-
,
pro-heparin-bound EGF), thereby inhibiting autocrine activation of the
receptor protein (26). However, if soluble ligands that do not require metalloproteinase processing (e.g. EGF) are present, BB-94
did not inhibit the mitogenic response in these experiments (26). Thus
we have focused our experiments on TIMP inhibition of cellular responses to soluble mitogenic factors, in particular EGF.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-mercaptoethanol) for 2 h,
23 °C, followed by extensive washing (0.05% Tween 20 in PBS) and
blocking (0.25% nonfat milk, 0.12% Tween 20, SSC). The membrane was
incubated with anti-EGFR (primary), or anti-GRB-2 (primary) antibody,
followed by horseradish peroxidase-conjugated secondary antibody and
ECL development, as above.
counting (cpm) on a Packard, Cobra auto-
-counter (Canberra Co.,
Downes Grove, IL).
excitation = 494 nm and
emission = 520 nm). Specific
binding was calculated as the difference between bound BODIPY-TIMP-2 in
the presence or absence of excess (100-fold) unlabeled ligand.
Scatchard analysis of TIMP-2 binding was performed as previously
reported (35).
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Fig. 1.
TIMP-2 and Ala+TIMP-2 inhibit EGF, bFGF, and
PDGF-induced proliferation of A549, HT1080, Hs68, and MCF7 cells.
A, A549, MCF7, or HT1080 cells were seeded onto gelatin (10 µg/ml)-coated 96-well plates and were serum starved to quiescence,
followed by treatment with TIMP-2 (0-200 nM) for 30 min,
stimulated with EGF (200 ng/ml), and incubated for 24 h.
Proliferation of viable cells was determined by the change in
absorbance at 490 nm on reduction of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
salt or [3H]thymidine incorporation. Results shown are
the percentage of maximum proliferation obtained by stimulation of
cells with EGF alone (100%), after correction for basal rate of
proliferation in serum-free conditions. Each data point represents the
average ± S.D. of six determinations. B, A549 and Hs68
cells were treated with ±Ala+TIMP-2 (50 nM) for 30 min,
followed by stimulation with various growth factors (*GF) (bFGF (50 ng/ml with 1.13 units/mg of heparin), EGF (200 ng/ml), PDGF (50 ng/ml)), or with TIMP-2 (50 nM) alone (absence of growth
factors or serum). C, A549 and Hs68 cells were treated with
Ala+TIMP-2 (0-50 nM), followed by stimulation with EGF
(200 ng/ml).
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Fig. 2.
Adenylate cyclase inhibitor (SQ22536)
abrogates the suppressive effects of TIMP-2 on EGF-stimulated A549
growth. Cells were pretreated with SQ22536 (100 µM)
for 30 min, prior to treatment with TIMP-2 (100 nM) for 30 min, and stimulation with EGF (200 ng/ml), for 24 h. Preincubation
with SQ22536 prior to addition of TIMP-2 results in proliferation
levels comparable to EGF alone, i.e. reversing the
suppressive effect of TIMP-2. SQ22536 did not alter the proliferation
in response to EGF.
from the surface of A549
cells was examined by enzyme-linked immunosorbent assay measurement of
TGF-
released from A549 cells. Incubation of A549 cells with 10-100
nM TIMP-2 resulted in no detectable decrease in soluble
TGF-
concentration (<2.5 pg/ml). Whereas addition of 1-10
nM active gelatinase-A (MMP-2) resulted in an increase in
soluble TGF-
(>8 pg/ml) released from A549 cells. Thus, TIMP-2 did
not mediate growth suppressive effects by interfering with MMP-dependent proteolytic cleavage of membrane-anchored EGF ligands.
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Fig. 3.
TIMP-2 and Ala+TIMP-2 do not compete for
125I-EGF ligand binding to EGFR. A549 cells were
treated with 0.2 nM 125I-EGF and ± unlabeled
EGF (500 nM), ± TIMP-2 (100 nM), or ± Ala+
TIMP-2 (100 nM), allowed to bind to cells for 90 min. After
washing, the cell amount of bound 125I-EGF was measured in
a -counter. Each bar graph represents six individual
determinations ± S.D. Treatment with unlabeled EGF provided a
statistically significant (Student's t test,
p < 0.001) reduction in binding, compared with
control. Treatment with TIMP-2 and Ala+TIMP-2 alone, or in the presence
of excess unlabeled EGF did not have a statistically significant effect
on the binding of 125I-EGF to EGFR on the surface of A549
cells.
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Fig. 4.
TIMP-2 and Ala+TIMP-2 activity against
MT-1-MMP catalytic domain and cell binding experiments.
A, measurement of Ala+TIMP-2 suppressive activity against
MT-1-MMP. TIMP-2 and Ala+TIMP-2 inhibition of MT-1-MMP activity were
determined using the thiopeptolide assays as described previously (28).
These assays were performed in 50 mM MOPS, 150 mM NaCl, 1 mM CaCl2, and 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 7.0. Concentrations of the catalytic domain of MT-1-MMP and thiopeptolide
were 20 nM and 50 µM, respectively. TIMP-2
effectively inhibits MT-1-MMP activity, but Ala+TIMP-2 does not, as
indicated by no decrease in Vi over the range of
added Ala+TIMP-2 concentrations. B, measurement
of Kd and number of receptors per cell was
determined for TIMP-2-BODIPY binding to A549 cells by a 96-well plate
binding assay. A549 cells were seeded onto 96-well plates and treated
with TIMP-2-BODIPY (0-100 nM) for 30 min, 37 °C.
Supernatant was transferred to new wells and measured as the amount of
unbound TIMP-2-BODIPY. Cells were washed with PBS and the amount of TIMP-2-BODIPY was measured as the amount of
fluorescence remaining on the A549 cell surface. Concentration of bound
TIMP-2-BODIPY was determined with a standard curve (TIMP-2-BODIPY
concentration versus fluorescence units) and plotted against
Bound/Free, to give the resulting Scatchard plot. Scatchard analysis
was performed as described previously (35). Unlabeled TIMP-2 was added
to cells to determine specific binding from total and nonspecific
binding. C, a 10-fold excess of Ala+TIMP-2
(unlabeled) was added to cells, resulting in a reduction of bound
fluorescence (Student's t test, p < 0.01),
whereas addition of BB94 (0.5 µM) did not result in any
significant change. However, addition of 10 µg/ml anti-MT1-MMP (clone
114-6G6) resulted in a reduction in bound TIMP-2-BODIPY fluorescence
(Student's t test, p < 0.05).
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Fig. 5.
TIMP-2 and Ala+TIMP-2 binding to A549 cell
surface proteins and co-localization with MT-1-MMP.
A, A549 cells were treated with 3 M glycine,
0.9% NaCl, pH 3, to dissociate complexes, washed with PBS, blocked
with 1% bovine serum albumin, and treated with Ala+TIMP-2-BODIPY (100 nM) for 30 min, fixed with paraformaldehyde and stained for
4,6-diamidino-2-phenylindole. Ala+TIMP-2 cell surface binding is shown
in green, and nuclei are shown in blue. B, cells
were treated with TIMP-2 or Ala+TIMP-2 (100 nM) and
incubated with anti-MT1-MMP antibody (clone 113-5B7), followed by
IgG-tetramethylrhodamine B isothiocyanate. MT1-MMP is shown in
red and areas of colocalization with TIMP-2 appears
yellow (white arrows). Although some
colocalization with MT-1-MMP was observed, TIMP-2 binding without
MT-1-MMP was readily apparent (arrowheads). The TIMP-2 cell
surface localization-independent of MT-1-MMP was even more apparent
when BODIPY-Ala+TIMP-2 was utilized in these experiments
(arrowheads, lower left panel).
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Fig. 6.
TIMP-2 reduces tyrosyl phosphorylation of
EGFR and association of Grb-2 protein with EGFR. A,
HT1080 cells were serum-starved to quiescence, followed by treatment
with TIMP-2 (0-200 nM) for 30 min, and EGF (200 ng/ml) for
5 min. Cells were lysed with RIPA buffer, EGFR immunoprecipitates were
prepared, and PVDF membranes were probed with phosphotyrosine (PY-20)
antibody or EGFR antibody. Bands of interest were integrated for mean
pixel density using NIH Image. Data is presented as the percentage of
maximum phosphorylated EGFR, as from EGF treatment alone (100%).
Amount of EGFR did not change with increasing TIMP-2 concentration, and
was therefore used as an internal loading control. B, A549
cells were treated with BB94 (0.5 µM), TIMP-1 (100 nM), TIMP-2 (100 nM), or Ala+TIMP-2 (100 nM), and amounts of phosphorylated EGFR were determined by
Western blot. Each data point represents the average of three
determinations ± S.D. C, quiescent, serum-starved MCF7
cells were treated with TIMP-2 (100 nM) followed by
stimulation with EGF, and EGFR immunoprecipitates were prepared as
before. PVDF membranes of EGFR immunoprecipitates were probed for Grb-2
antibody. Treatment of cells with EGF enhanced the mean density of
Grb-2 bound to EGFR, whereas TIMP-2 treatment resulted in a
statistically significant reduction (Student's t test,
p < 0.01) in Grb-2 association.
View larger version (18K):
[in a new window]
Fig. 7.
Inhibition of PKA activity reduces
EGF-stimulated proliferation of A549 cells and only partially reverses
TIMP-2 reduction of EGFR phosphorylation. A, cells were
pretreated with H-89 (0.1 µM) for 30 min, prior to
treatment with TIMP-2 (100 nM) for 30 min, and stimulation
with EGF (200 ng/ml), for 24 h, 37 °C. Proliferation was
measured as described under "Experimental Procedures."
B, serum-starved A549 cells were treated ± H89 (0.1 µM) for 30 min, followed by ± TIMP-2 (50 nM), 30 min, and then EGF (200 ng/ml), 5 min EGFR
immunoprecipitates were prepared as described in the legend to Fig. 6.
Each data point represents the average of three replicate
measurements ± S.D.
View larger version (19K):
[in a new window]
Fig. 8.
TIMP-2 enhances binding of SH2
protein-tyrosine phosphatase-1 (SH-PTP1), but not phosphatase-2
(SH-PTP2) to EGFR. A, A549 cells were serum starved to
quiescence and treated with TIMP-2 (100 nM) followed by EGF
(200 ng/ml) for 5 min. EGFR immunoprecipitates were prepared, and
polyvinylidene difluoride membranes were probed for SH-PTP1 or SH-PTP2.
B, treatment of cells with EGF reduced SH-PTP1 association,
but TIMP-2 restored levels to that seen without EGF stimulation.
Amounts of SH-PTP2 were not significantly altered on treatment with EGF
or TIMP-2. C, cells were plated on 75-cm2 Nunc
flasks 2 × 106 cells per flask and following
attachment were incubated in serum-free DMEM for 2 h then in
serum-free DMEM for an additional 24 h. TIMP-2 (24 nM)
was preincubated for 5 min prior to a 10-min stimulation with growth
factor (EGF, 100 ng/ml). The cells were harvested in lysing buffer
containing vanadate. Equal amounts of protein from the lysates (200 µg) were immunoprecipitated with anti-SHP-1 or SHP-2 antibodies
(Transduction Labs) at 4 °C for 60 min followed by 60 min incubation
with anti-mouse agarose beads (Cappel). The beads were subsequently
washed three times in lysing buffer without orthovanadate and 3 times
more with phoshatase assay buffer. These immunoprecipitates were
resuspended in assay buffer, transferred to 96-well plates, and the
reaction initiated by adding a phosphotyrosine peptide. After 10 min at
30 °C, the reaction was stopped by addition of 100 µM
orthrovanadate. The relative levels of phosphatase activity in these
immunprecipitates was determined utilizing a phosphopeptide by
enzyme-linked immunosorbent assay as described by manufacturer's
instruction (Roche Molecular Biochemicals). Enzyme-linked immunosorbent
assay results were determined in a Molecular Devices microplate reader.
A decrease in optical density is indicative of enhanced phosphatase
activity against the phosphopeptide containing substrate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors
or inhibition of TNF-
converting enzyme (60).
, as well as EGFR (61). TIMP-2
treatment did not alter EGFR levels, compete for EGF binding to EGFR,
or alter TGF-
release. These findings are again consistent with
TIMP-2 growth modulation that is independent of inhibition of MMP
activity required for either ligand or receptor processing.
s. TIMP-2 modulates the phosphorylation of the EGFR following growth factor stimulation and
this effect is mediated by SHP-1. These effects of TIMP-2 on the
mitogenic response are observed with several different growth factors
(EGF, bFGF, and PDGF) and in multiple cell types (including neoplastic
cells as well as dermal fibroblasts). Based on these findings, we
propose TIMP-2 functions to suppress inappropriate growth factor
stimulation of cells in G1/G0 phase of the cell cycle, as well as to inhibit MMP-mediated ECM turnover. Consistent with
our observation that TIMP-2 needs to be available prior to growth
factor stimulation, and the proximal inhibition or down-regulation of
EGFR activation (phosphorylation), TIMP-2 must function during early
G1 prior to entry in the restriction point late in
G1. Both this G1 regulator-type and protease
inhibitor functions of TIMP promote tissue homeostasis. The
G1 regulator-type function may be a feedback mechanism that
informs cells that MMP-mediated remodeling of the ECM is complete. In
this proposal TIMP-2 would not function to suppress mitogenesis until
ECM remodeling is shutdown by inhibition of MMP activity. Saturation of
available MMP active sites in the remodeling matrix and/or on the cell
surface would block ECM turnover, only then would excess free TIMP-2
begin to suppress cell responsiveness to residual growth factor
stimulation. Our proposal is consistent with the recent finding that
timp-2-deficient mice display no abnormalities in fertility
or development (65, 66). As a G1 cell cycle phase
regulator, one would expect that TIMP-2 would be a nonessential gene
and that elimination from the germ line would not necessarily result in
disruption of fetal development (67). The results of the present study
demonstrate that TIMP-2 is multifunctional in suppressing growth factor
stimulation, modulating MT-1-MMP activation of pro-MMP-2, in addition
to the well established role of MMP inhibition, which prevents ECM
remodeling. The growth suppression and MMP inhibitor functions both act
to preserve tissue homeostasis. The control and integration of these
TIMP-2 functions is poorly understood and warrants further investigation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Larry Wahl for the Lumulus amoebocyte lysis (LAL) analysis, as well as Drs. Kazuyo Takeda and Zu-Xi Yu for technical support and assistance with the confocal mircoscopy. We also thank Drs. Liliana Gudez and David Roberts for helpful discussions.
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FOOTNOTES |
---|
* 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.
Current address: IGEN International Inc., 16020 Industrial Dr.,
Gaithersburg, MD 20877.
§ To whom correspondence should be addressed: Laboratory of Pathology, Div. of Clinical Sciences, NCI, National Institutes of Health, Bldg. 10, Room 2A33, MSC 1500, Bethesda, MD 20892-1500. Tel.: 301-496-2687; Fax: 301-402-2628; E-mail: sstevenw@mail.nih.gov.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008157200
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ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
TIMP, tissue inhibitor of matrix metalloproteinases;
MMP, matrix metalloproteinase;
Ala+TIMP-2, amino-terminal alanine appended
TIMP-2;
TKR, tyrosine kinase-type receptor;
bFGF, basic fibroblast
growth factor;
EGF, epidermal growth factor;
EGFR, epidermal growth
factor receptor;
TGF-, transforming growth factor
;
PDGF, platelet-derived growth factor;
ERK, extracellular regulated kinase;
PTP, protein-tyrosine phosphatase;
DMEM, Dulbecco's modified Eagle's
medium;
PBS, phosphate-buffered saline solution;
MT-1-MMP, membrane-type matrix metalloproteinase-1;
MOPS, 4-morpholinepropanesulfonic acid.
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