Induction of Matrix Metalloproteinase-9 Requires a Polymerized
Actin Cytoskeleton in Human Malignant Glioma Cells*
Shravan K.
Chintala
,
Raymond
Sawaya
,
Bharat B.
Aggarwal§,
Sadhan
Majumder¶,
Dipak K.
Giri§,
Athanassios P.
Kyritsis¶,
Ziya L.
Gokaslan
, and
Jasti S.
Rao
From the Departments of
Neurosurgery,
§ Molecular Oncology, Cytokine Research Laboratory, and
¶ Neuro-oncology, the University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030
 |
ABSTRACT |
Alterations in cytoskeleton and subsequent cell
shape changes exert specific effects on the expression of various
genes. Our previous results suggested that malignant human gliomas
express elevated levels of matrix metalloproteinases compared with
normal brain tissue and low grade gliomas. To understand the role of cell shape changes on matrix metalloproteinase expression in human glioma cells, we treated SNB19 cells with cytochalasin-D, an inhibitor of actin polymerization, and colchicine-B, a tubulin inhibitor, in the
presence of phorbol 12-myristate 13-acetate. Cytochalasin-D treatment
of SNB19 cells resulted in the loss of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 (also known as
gelatinase-B) expression and coincided with inhibition of actin
polymerization, resulting in cell rounding. Moreover, compared with
monolayers, cells grown as spheroids or cell aggregates failed to
express matrix metalloproteinase-9 in the presence of phorbol
12-myristate 13-acetate. Matrix metalloproteinase-9 expression was also
inhibited by calphostin-C, a protein kinase inhibitor, suggesting
the involvement of protein kinase C in matrix metalloproteinase-9
expression. Phorbol 12-myristate 13-acetate-induced invasion of SNB19
cells through Matrigel was inhibited by cytochalasin-D and
calphostin-C. These results suggest that the actin polymerization
transduces signals that modulate the expression of matrix
metalloproteinase-9 expression and the subsequent invasion of human
glioma cells.
 |
INTRODUCTION |
Basement membrane and extracellular matrix degradation by
proteolytic enzymes is the critical event that takes place during tumor
cell invasion and metastasis (1). Several proteases are secreted by
invading tumor cells, such as serine proteases, plasminogen activators,
and matrix metalloproteinases
(MMPs)1 (2, 3). Among the
proteases, elevated levels of MMPs have been shown in many tumors (3,
4) with strong association with the invasive phenotype (5). The role of
MMPs has been noted in a number of astrocytic tumors (6-8), and our
own results of glioblastoma studies suggest that both MMP-2 (also known
as gelatinase-A) and MMP-9 (also known as gelatinase-B) are elevated significantly at both the mRNA and protein levels in malignant glioblastomas (9) and contribute to the invasive ability of gliomas
both in vivo (9, 10) and in vitro (11, 12).
However, in vitro culturing of primary gliomas results in
the loss of MMP-9 expression
(13)2; MMP-9 is re-expressed
when these glioma cells are injected intracerebrally into nude mouse
brain.2
The production of MMPs is influenced by many factors. Physiologically
relevant cytokines such as interleukin-1 and tumor necrosis factor-
have been shown to induce the expression of MMPs (14, 15). Earlier
studies have shown that a number of pharmacological agents including
12-O-tetradecanoyl-13-phorbol acetate induce procollagenase,
MMP, and stromelysin expression (16-18) and the expression of various
biological markers associated with tumorigenesis (19). Alteration of
cell shape by growing cells on biological matrices also results in
dramatic changes in the phenotypes of a number of cell types (20), and
a strong correlation has been shown with a change in cell morphology;
it has been speculated that cell shape plays a major role in
chondrogenesis (21, 22) and adipogenesis (23, 24). Moreover, growth of
cells in three-dimensional collagen matrices alters the cytoskeleton
organization (25-27). Recent studies reported that changes in the
cytoskeleton activate MMP-2 (28) and inhibit MMP-9 (29).
Although the above studies have shown that cell shape changes result in
modulation of various phenotypic changes and MMP expression, studies on
extracellular matrix and cytoskeleton organization and the associated
changes in gliomas are of special importance because these tumors are
more highly infiltrative than all other tumor types. Despite a number
of studies on the role of MMPs and invasive phenotypes, the activation
of MMPs which contributes significantly to glioma invasiveness is not
clearly understood. In this study, we show that cell shape changes,
particularly those resulting from inhibition of actin polymerization,
suppress the activation of MMP-9 and the invasion properties of human
gliomas.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium/Ham's F-12
medium was obtained from Life Technologies, Inc. Cytochalasin-D,
colchicine-B, dexamethasone, phorbol 12-myristate 13-acetate (PMA), and
TRITC-phalloidin were obtained from Sigma. Calphostin-C, genistein,
HA1004, and 8-bromo-cAMP were purchased from Calbiochem. PolyHema
(poly[2-hydroxyethylmethylacrylate], also called Cellform Polymer)
was obtained from ICN biochemicals (Aurora, OH). Agar was obtained from
Difco Laboratories. Tissue culture plates were purchased from Becton
Dickinson (Franklin Lakes, NJ). Focal adhesion kinase (FAK) antibody
was obtained from Transduction Laboratories (Lexington, KY).
Cell Culture--
An established human glioma cell line SNB19
(30) was used in the current study. Cells were routinely grown in high
glucose Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum, 20 mM HEPES, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a humidified atmosphere containing 5% CO2
at 37 °C. Cells were passaged every 3-5 days. After being tested in
preliminary experiments, reagents were used at concentrations that have
maximal inhibitory or stimulatory effect on MMP-9 expression without
affecting cell viability (by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay): 50 ng/ml PMA, 1 µM
dexamethasone, 40 µM genistein, 1 µM 8-bromo-cAMP, 5 µM cytochalasin-D, 5 µM
colchicine-B, 10 µM HA1004, and 300 nM
calphostin-C.
Immunofluorescent Staining--
SNB19 cells grown in chamber
slides (Nunc Inc., Naperville, IL) for 24 h at 37 °C were
washed with PBS, fixed by adding 3% paraformaldehyde, and
permeabilized with 0.2% Triton X-100 for 5 min. Where indicated,
TRITC-phalloidin (200 nM/ml in PBS) was applied for 30 min
to Triton X-100-permeabilized cells to stain for F-actin.
Matrigel Invasion Assay--
Invasion of the glioma cells
in vitro was measured by the invasion of cells through
Matrigel-coated (Collaborative Research, Inc., Boston) Transwell
inserts (Costar, Cambridge, MA) according to a procedure described
previously (30). Briefly, Transwell inserts with an 8-µm pore size
were coated with a final concentration of 0.78 mg/ml Matrigel in cold
serum-free Dulbecco's modified Eagle's medium/Ham's F-12. Cells were
treated with trypsin, and 200 µl of cell suspension (1 × 106 cells/ml) from each treatment was added in triplicate
wells. After 48 h of incubation, the cells that passed through the
filter into the lower wells were stained with Hema-3 (CMS, Inc.,
Houston) and photographed under a microscope.
Gelatin Zymography--
Analysis of MMP-2/MMP-9 was performed on
SDS-polyacrylamide gels impregnated with 0.1% gelatin (w/v) and 10%
polyacrylamide (w/v) as described elsewhere (30). Cells were grown in
100-mm2 tissue culture plates in Dulbecco's modified
Eagle's medium/Ham's F-12 containing 10% fetal bovine serum until
they reached 80% confluence. Cells were washed and replaced with
serum-free medium, and the conditioned medium was collected after
48 h. Four parts of medium containing equal amounts of protein (20 µg) were mixed with one part of Laemmli sample buffer (minus
reductant) (31) before electrophoresis. Gels were run at a constant
current and then washed twice for 30 min in 50 mM Tris-HCl,
pH 7.5, plus 2.5% Triton X-100 and then incubated overnight at
37 °C in 50 mM Tris-HCl, pH 7.6, 10 mM
CaCl2, 150 mM NaCl, and 0.05%
NaN3. Gels were stained with Coomassie Brilliant Blue R-250
and then destained.
Expression of FAK--
Western blotting for p125FAK was
performed by lysing the cells with RIPA buffer (1% Nonidet P-40, 20 mM Tris, 150 mM NaCl, 1 mM
Na3VO4, 5 mM EDTA, 0.1 mg/liter
aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cells
grown in 100-mm tissue culture plates were washed with cold PBS before
the addition of RIPA buffer and incubated for 10 min. Cells were
scraped and pipetted into Eppendorf tubes and centrifuged for 20 min at
12,000 × g. Supernatants were transferred to fresh
Eppendorf tubes, and protein was determined by BCA protein assay kit
(Pierce Chemical Co.). 20 µg of this initial lysate was loaded onto
gels for electrophoresis and Western blotting. After reduction in the
sample buffer, samples were loaded onto a 10% resolving
SDS-polyacrylamide gel with a 4.5% stacking gel. Samples were
electrophoresed at a constant current (40 mA) for 2-4 h at 4 °C and
then electroblotted onto a nitrocellulose membrane overnight at 4 °C
at a constant current of 80 mA. Western blotting was performed with a
1:2500 dilution anti-FAK monoclonal antibody (Transduction
Laboratories). Immunoreactive bands for FAK were visualized using
horseradish peroxidase-conjugated anti-mouse IgG secondary antibody and
ECL reagents (Amersham Pharmacia Biotech). Film was analyzed with a
scanning densitometer, and the images were reproduced by UMAX scanner
with an Adobe Photoshop imaging system on a Macintosh computer.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed according to the procedures
described previously (32, 33). Briefly, 1 × 106 cells
were washed with cold Dulbecco's phosphate-buffered saline and
suspended in 0.4 ml of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml aprotinin, 0.5 mg/ml benzamidine). Cells were allowed to swell on ice
for 20 min followed by the addition of 12.5 µl of 10% Nonidet P-40.
The tubes were then vortexed vigorously for 10 s, and the homogenate was centrifuged for 30 s. The nuclear pellet was
resuspended in 25 µl of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin,
2.0 µg/ml aprotinin, 0.5 mg/ml benzamidine) and incubated on ice for
30 min with intermittent vortexing. Samples were centrifuged for 5 min
at 4 °C, and the supernatant (nuclear extract) was either used
immediately or stored at
70 °C. The protein content was measured
by the method of Bradford (34). Electrophoretic mobility shift assays
were performed by incubating 4 µg of nuclear extract with 16 fmol of
32P-labeled 45-mer double-stranded nuclear factor (NF)-
B
oligonucleotide from the HIV long term repeat,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (35), for 15 min at 37 °C. The incubation mixture included 2-3 µg of
poly(dI·dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet
P-40, 5% glycerol, 50 mM NaCl) (36). The DNA-protein
complex thus formed was separated from free oligonucleotide on 7.5%
native polyacrylamide gel using buffer containing 50 mM
Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA
(37), and the gel was then dried. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-
B to the DNA.
The specificity of binding was also examined by competition with the
unlabeled oligonucleotide. Visualization and quantitation of
radioactive bands were carried out by PhosphorImager (Molecular Dynamics) using IMAGEQUANT software (National Institutes of
Health, Bethesda, MD).
 |
RESULTS |
Phorbol Ester Induces MMP-9 Expression in SNB19 Cells--
SNB19
cells were treated with trypsin, and a single-cell suspension was
obtained. Cell suspensions containing 1 × 105
cells/ml were plated in six-well tissue culture plates and treated with
50 ng/ml PMA. PMA was added during the plating, after plating, or
during and after plating. Fig. 1 shows
that treatment of SNB19 cells with PMA resulted in the induction of
MMP-9, which is normally absent in this cell line, irrespective of the
time of the addition of PMA, although long term treatment resulted in
reduced expression of MMP-9 (results not shown). MMP-2 expression was
largely unaltered, although activation into the 66-kDa form resulted
occasionally.

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Fig. 1.
Induction of MMP-9 expression in SNB19
cells. SNB19 cells were plated (1 × 105
cells/well) in a six-well culture plate in the presence or absence of
PMA (50 ng/ml). Medium was replaced after overnight incubation at
37 °C, and fresh serum-free medium was added after two washes
including a 4-h incubation between washes. Finally, fresh serum-free
medium was added and allowed to condition for further 48 h.
Conditioned medium containing an equal amount of protein (20 µg) was
mixed with Laemmli loading buffer (without reducing agent) and run on
10% SDS-polyacrylamide gels containing gelatin (for details, see
"Experimental Procedures"). After the gels were washed with Triton
X-100 and incubated in buffer containing CaCl2, they were
stained with Coomassie Brilliant Blue and then destained. The
zymographic assay for MMPs was shown after the cells were treated with
PMA during plating and after washes with serum-free medium (+/+), at
the time of plating but not after washes with serum-free medium (+/ ),
only after washes with serum-free medium ( /+), and for untreated
cells ( / ). MMP-9 (92 kDa) expression was induced with PMA
treatment, irrespective of the time of PMA addition.
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MMP-9 Induction Is Inhibited by Cytochalasin-D--
To understand
the role of actin polymerization, we treated SNB19 cells with PMA
during the initial plating and then treated them with cytochalasin-D
(actin polymerization inhibitor) during plating, after plating, and
during and after plating. SNB19 cells treated with PMA expressed MMP-9,
and the expression of this enzyme was lost by the addition of
cytochalasin-D, provided it was added during or during and after
plating (Fig. 2). However, there was no
change in the expression of MMP-9 when cytochalasin-D was added after
spreading (Fig. 2). Similar results were obtained in another malignant
glioma cell line, UW5 (results not shown). Treatment of the cells with
colchicine-B (tubulin inhibitor) had no effect on the expression of
MMP-9, induced by PMA treatment (Fig. 3). To rule out a nonspecific effect of actin polymerization inhibition on
MMP-9 expression, similar experiments were conducted using the HT1080
fibrosarcoma cell line, which constitutively expresses both MMP-2 and
MMP-9. HT1080 cells treated with cytochalasin-D lost the ability to
express MMP-9 provided the agent was added during or during and after
spreading (Fig. 4); the ability to express proMMP-2 was unaltered by the addition of cytochalasin-D. On
the other hand, treatment of HT1080 cells with colchicine-B had no
effect on either MMP-2 or MMP-9 expression (Fig. 4). These results show
that the induced expression of MMP-9 in SNB19 cells and constitutive
expression of MMP-9 in HT1080 cells are lost by alterations in the
actin cytoskeleton.

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Fig. 2.
Addition of cytochalasin-D inhibits MMP-9
induction. SNB19 cells were plated (1 × 105
cells/well) in a six-well tissue culture plate in the presence of PMA
(50 ng/ml) and incubated overnight at 37 °C. Where indicated,
cytochalasin-D was added during the plating and after the washes (+/+),
added during the plating (+/ ), added after wash with serum-free
medium ( /+), or cells were not treated with cytochalasin-D ( / ).
Conditioned medium was collected after a 48-h incubation and run on
SDS-polyacrylamide gels containing gelatin. Zymographic analysis shows
that PMA-induced MMP-9 expression was inhibited when cytochalasin-D was
added during spreading but not after spreading.
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Fig. 3.
Colchicine-B treatment does not alter MMP-9
induction. SNB19 cells were plated as described in Fig. 2 and
treated with PMA (50 ng/ml). Cells were also treated with colchicine-B
during plating and after washes (+/+), added during plating (+/ ),
added after plating ( /+), or not treated ( / ). Conditioned medium
containing an equal amount of protein was run on SDS-polyacrylamide
gels containing gelatin. No alteration in PMA-induced MMP-9 expression
was observed, and cytochalasin-B had no effect on MMP-2
expression.
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Fig. 4.
Cytochalasin-D inhibits constitutive
expression of MMP-9 in HT1080 cells. HT1080 cells were plated
(1 × 105 cells/well) in six-well tissue culture
plates and treated with cytochalasin-D or colchicine-B during and after
plating (+/+), added during plating (+/ ), added after plating ( /+),
or not treated ( / ), in the absence of PMA. Additionally, in a
separate set of experiments HT1080 cells were also treated with PMA
alone. Conditioned medium was collected and run on SDS-polyacrylamide
gels containing gelatin. HT1080 cells constitutively expressed both
MMP-2 and MMP-9; however, expression of MMP-9 but not of MMP-2 is
inhibited significantly in the presence of cytochalasin-D. The addition
of PMA has no significant effect on the expression of both MMP-2 and
MMP-9.
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Inhibition of MMP-9 Is Caused by Alteration in Actin
Polymerization--
Because inhibitors of actin polymerization
resulted in MMP-9 inhibition, we performed cell spreading assays where
changes in cell shape and actin polymerization were observed during
spreading by staining the cells with TRITC-phalloidin. Fig.
5 shows that treatment of cells with
cytochalasin-D resulted in changes in the cell shapes as observed both
by light microscopy and fluorescence microscopy. Treatment with
cytochalasin-D resulted in the loss of actin cytoskeleton, provided the
agent was added during spreading or during and after spreading.
Although treatment with cytochalasin-D after spreading resulted in some
changes in cell shape, most of the cells were spread and retained
stress-fiber formation. However, colchicine-B had no effect on actin
polymerization in SNB19 cells. We treated SNB19 cells further with
dexamethasone, which induces a rapid change of nonpolymerized actin to
polymerized actin. Cells treated with PMA followed by
dexamethasone showed induction of MMP-9 as expected (Fig. 9).
Interestingly, cells treated with PMA and dexamethasone followed by
cytochalasin-D retained the ability to express MMP-9, whereas cells
treated with cytochalasin-D in the presence of PMA invariably lost the
ability to express MMP-9, as observed in earlier experiments (see Fig.
2). Treatment with dexamethasone alone did not result in the induction
of MMP-9 and had no effect on the constitutive expression of MMP-2 in
SNB19 cells. These results suggest that polymerization of the actin cytoskeleton induced by dexamethasone is resistant to disruption by
cytochalasin-D treatment.

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Fig. 5.
Cell morphology and cytoskeleton organization
are altered by cytochalasin-D. SNB19 cells were plated (1 × 102 cells/well) in chamber slides, treated with various
agents, and photographed under phase-contrast microscopy; or cells were
fixed with 4% paraformaldehyde, treated with Triton X-100, and stained
with TRITC-phalloidin to display actin filaments. Panel A
shows the actin cytoskeleton in cells treated with PMA (50 ng/ml) as
described in Fig. 1. Panel B shows the cell morphology under
a phase-contrast microscopy, and panel C shows the actin
cytoskeleton in cells treated with cytochalasin-D as described in Fig.
2. Panel D shows the cell morphology under phase-contrast
microscopy, and panel E shows the actin cytoskeleton in
cells treated with colchicine-B as described in Fig. 3. Cell rounding
and loss of actin polymerization were observed only in cells treated
with cytochalasin-D during spreading and during and after
spreading.
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Cell Shape Alteration by Cytochalasin-D Alters the Expression of
FAK--
As the cells treated with cytochalasin-D lost their
cytoskeletal organization, we were interested to see the changes in the expression of FAK where stress fibers terminate. Cells were plated as
described above and treated with cytochalasin-D or colchicine-B; after
the treatments, cells were extracted in RIPA buffer, and FAK protein
was examined by Western blotting. The Western blot for FAK in Fig.
6 shows that SNB19 cells without any
treatment or with PMA treatment expressed FAK protein. However,
treatment of cells with cytochalasin-D during or during and after
treatment resulted in the loss of FAK expression. On the other hand,
similar levels of FAK were expressed in colchicine-B-treated cells
compared with levels expressed by control and PMA-treated cells. These observations show that both the actin cytoskeleton and the expression of FAK are lost when cells are treated with cytochalasin-D. This in
turn results in altered cell shape and consequent loss of ability to
express MMP-9.

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Fig. 6.
Loss of FAK expression in SNB19 cells.
Cells were plated (1 × 105 cells/well) in six-well
tissue culture plates that were precoated with fibronectin (5 µg/ml)
in the presence of PMA (50 ng/ml). Where indicated, cells were treated
with cytochalasin-D as described in Fig. 2 and colchicine-B as
described in Fig. 3. Medium was aspirated, and the cells were washed
with cold PBS followed by scraping the cells in RIPA buffer (for
details, see "Experimental Procedures"). Samples containing equal
amounts of protein (20 µg), including a positive control, were run on
10% SDS-polyacrylamide gels under reducing conditions. Proteins were
transferred onto nitrocellulose membranes and incubated with blocking
buffer. Nitrocellulose membranes were incubated with anti-FAK antibody,
followed by a secondary antibody, and the bands were visualized on
x-ray film using ECL reagent according to the manufacturer's
instructions. The figure shows that 125-kDa FAK protein expression was
lost by treating the cells with cytochalasin-D during and during and
after spreading.
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Cells Cultured as Three-dimensional Spheroids Do Not Express
MMP-9--
Because cell "rounding" in the presence of
cytochalasin-D resulted in the loss of MMP-9 expression, we grew cells
on PolyHema or on agar-coated plates; this prevents the cells from
attaching to the tissue culture plastic and results in cell aggregation or spheroid formation. Cells are then treated with 50 ng/ml PMA. Gelatin zymography of the conditioned medium from cells grown as
spheroids (Fig. 7, left panel,
lane B) and cell aggregates (Fig. 7, lane C)
failed to express MMP-9 in the presence of PMA, whereas cells grown as
monolayers expressed MMP-9 in response to PMA (Fig. 7, lane
A), indicating that cell spreading and subsequent actin
polymerization are necessary for MMP-9 expression. MMP-2 expression was
unaltered by growing the cells on PolyHema or on agar-coated
plates.

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Fig. 7.
Expression of MMPs in cells grown in
three-dimensional spheroids. SNB19 cells were grown as aggregates
on PolyHema or as spheroids on agar-coated tissue culture plates. Cell
aggregates or spheroids were treated with PMA and incubated for 48 h at 37 °C. Conditioned medium was collected and run on 10%
SDS-polyacrylamide gels containing gelatin. The left panel
shows cells grown as monolayers and treated with PMA (lane
A); cells grown as spheroids and treated with PMA (lane
B); and cells grown as cell aggregates and treated with PMA
(lane C). The right panel shows the morphology of
cells grown as monolayers, spheroids, and aggregates. SNB19 cells grown
in three-dimensional configuration failed to express MMP-9 in response
to PMA.
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Expression of NF-
B Is Lost by Cytochalasin-D Treatment--
It
is speculated that cell shape changes exert specific effects on gene
expression by modulating the activity of transcriptional factors that
reside in the cytoplasm of the unstimulated cells in an inactive form
and migrate to the nucleus in response to various stimuli. One such
factor is NF-
B, a dimeric complex that activates transcription of a
variety of genes, including MMP-9 expression, activation of cell
surface receptors, and activation of cell adhesion molecules. SNB19
cells were treated with PMA in the presence and absence of cell shape
modulators, cytochalasin-D and colchicine-B as described above. As
shown in Fig. 8, untreated cells have no
DNA binding activity of NF-
B. Stimulation of NF-
B was observed
when the cells were treated with PMA. Subsequently, PMA-induced NF-
B
binding was decreased in the presence of cytochalasin-D, whereas
significant binding of NF-
B was observed in colchicine-B-treated cells. These observations suggest that PMA-induced NF-
B binding was
inhibited by disruption of the actin cytoskeleton.

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Fig. 8.
Induction of NF- B binding is altered by
cytochalasin-D. Cells were plated (1 × 105
cells/well) in six-well tissue culture plates and treated with PMA (50 ng/ml) during spreading. Cells were also treated with cytochalasin-D as
described in Fig. 2 and colchicine-B as described in Fig. 3. Nuclear
extracts were prepared as described under "Experimental
Procedures," and mobility shift assays were performed. The figure
shows that PMA induced NF- B binding, which was almost absent in
untreated control cells. PMA-induced NF- B binding was inhibited by
cytochalasin-D but not by colchicine-B.
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Inhibition of MMP-9 Expression by Protein Kinase C
Inhibitors--
It has been shown that MMP induction in glioma cells
is dependent on protein kinase C (PKC) expression. To understand the role of actin cytoskeleton in PKC-dependent MMP expression,
cells were treated with PMA and incubated with various PKC and tyrosine kinase inhibitors. MMP-9 expression was induced by PMA as expected (Fig. 9), and the PMA-induced MMP-9
expression (and to some extent MMP-2 expression) was inhibited by
calphostin-C, a specific inhibitor of PKC, but not by the tyrosine
kinase inhibitors genistein and HA1004 (Fig. 9). This showed that
induction of both MMP-2 and MMP-9 is dependent on PKC expression in
SNB19 human glioma cells.

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Fig. 9.
MMP-9 induction is inhibited by the PKC
inhibitor calphostin-C. Cells were plated (1 × 105 cells/well) in six-well tissue culture plates and
treated with PMA (50 ng/ml) during spreading. Cells were also treated
with calphostin-C, cAMP, genistein, and HA1004 for 1 h before the
addition of PMA. In a separate set of experiments, cells were also
treated with dexamethasone after PMA treatment, or cells were treated
with PMA and dexamethasone followed by cytochalasin-D. Conditioned
medium was collected, and samples containing an equal amount of protein
(20 µg) from each treatment were run on SDS-polyacrylamide gels
containing gelatin. PMA-induced expression of MMP-9 and to some extent
MMP-2 was inhibited by calphostin-C.
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Invasion of SNB19 Cells Is Inhibited by Cytochalasin-D--
MMP-9
plays an important role in tumor cell invasion, and thus, experiments
were performed to examine the effect of actin polymerization inhibitors
on in vitro SNB19 cell invasion. Invasion of SNB19 cells
through Matrigel was induced 2-3-fold by PMA, and this induction was
inhibited subsequently by the addition of cytochalasin-D (Fig.
10). However, treatment of cells with
colchicine-B had no significant effect on invasion. Treatment of cells
with calphostin-C resulted in significant loss of invasion capacity of
SNB19 cells (Fig. 10), whereas other tyrosine kinase inhibitors had no
effect (results not shown). Calphostin-C-inhibited invasion of SNB19 cells was only minimally reversed by PMA treatment (Fig. 10).

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Fig. 10.
In vitro invasion of SNB19 cells is
inhibited by cytochalasin-D and calphostin-C. 200 µl of a
single-cell suspension (1 × 106 cells/ml) of SNB19
cells was placed in the upper wells of individual Transwell inserts
containing 8-µm pore size polycarbonate membranes precoated with
Matrigel (0.78 mg/ml). Before placing the cells in Transwell inserts,
cells were also treated with PMA (50 ng/ml) alone or in combination
with cytochalasin-D, colchicine-B, or calphostin-C. Cells were allowed
to invade for 48 h at 37 °C followed by the fixation and
staining of cells with Hema-3. Cells on the upper surface were removed
with a cotton swab, and the cells that passed through the polycarbonate
membrane were mounted onto microscope slides and photographed under a
light microscope at × 20 magnification. The figure shows that
invasion of SNB19 cells was inhibited significantly by cytochalasin-D
and calphostin-C.
|
|
 |
DISCUSSION |
Changes in cytoskeletal architecture reflected in cell shape
changes are well known to accompany changes in gene expression in a
number of cell types such as mammary epithelium (38), chondrocytes (22), adipocyte precursors (24), and cells from synovial tissues (39).
Unemori and Werb (40) reported that disruption of the actin
cytoskeleton stimulated procollagenase and stromelysin secretion in
rabbit synovial fibroblasts, which led to the speculation that perturbation of the actin microfilaments might be linked to the expression of genes involved in the initiation of extracellular matrix degradation. In addition, two recent reports demonstrating that
MMP-9 was suppressed by an alteration in cell shape in melanoma cells
(29) and that MMP-2 activation was regulated by organization of the
polymerized actin in human palmar fascial fibroblasts (28) show that an
alteration in cell shape influences MMP-9 and as well as MMP-2 in
different cell types.
In the current study, we performed experiments to understand whether
changes in cytoskeleton polymerization, a dynamic process that occurs
during tumor cell invasion, modulate the expression of MMPs using
agents that change the cell shape in vitro. Gelatin zymographic analysis of medium from the SNB19 glioma cell line revealed
that the expression of MMP-9 is induced by PMA, which is normally
absent in this cell line (Fig. 1). Treatment of the cells with
cytochalasin-D, which causes disruption of actin stress fibers,
resulted in decreased or loss of MMP-9 expression (Fig. 2). In
contrast, treatment of cells with colchicine-B, an inhibitor of tubulin
polymerization, had no effect on the expression of MMP-9 or MMP-2 (Fig.
3). To understand whether the constitutive expression of MMP-9 in other
cell types is altered by cell shape changes, we used HT1080
fibrosarcoma cells, which constitutively express both MMP-2 and MMP-9,
because glioma cells do not express constitutive MMP-9 in in
vitro culturing conditions. Cytochalasin-D inhibited constitutive
expression of MMP-9 in HT1080 cells (Fig. 4), similar to the results
observed in SNB19 glioma cells (Fig. 2), whereas colchicine-B treatment
had no effect on the expression of either MMP-9 or MMP-2. These results
confirm that induced production of MMP-9 in SNB19 cells and
constitutive expression of MMP-9 in HT1080 cells were lost when the
cytoskeletal organization was altered, suggesting that the loss of
MMP-9 could be caused by the alteration in actin polymerization and
subsequent shape modulation of SNB19 cells. Total actin content by
fluorescent estimation of rhodamine phalloidin showed that the total
quantity of the actin is not changed during the treatment conditions
(results not shown), ruling out the idea that the observed effects are caused by changes in actin content. Cells grown as three-dimensional spheroids were treated with PMA to find out whether these cells (which
do not spread and do not form polymerized actin) respond to PMA and
express MMP-9. Interestingly, these spheroids failed to express MMP-9
(Fig. 7), unlike monolayer cultures.
Because cytochalasin-D is known to alter the polymerization of actin,
experiments were performed to examine the changes in cell spreading and
actin polymerization by TRITC-phalloidin staining. Cytochalasin-D-treated cells showed inhibition of actin polymerization when the cells were treated during spreading, whereas cells treated after spreading retained efficient cytoskeleton. In contrast, colchicine-B-treated cells, although showing some morphological changes, expressed organized cytoskeleton efficiently (Fig. 5). Actin
cytoskeletons terminate at focal adhesion contacts in fully spread
cells, and it has been shown that expression of FAK, phosphorylation of
FAK, or both modulate the expression of a variety of events including
migration and invasion (41). Results in Fig. 6 show that control cells
as well as PMA-treated cells expressed FAK protein as observed on
Western blots. Interestingly, FAK expression was lost completely in
cytochalasin-D cells only when the cells were treated during spreading,
whereas cells allowed to spread and then treated with cytochalasin-D
retained FAK expression. On the other hand, colchicine-B-treated cells
always expressed FAK irrespective of when colchicine-B was added. These
results suggest that cytoskeletal organization and FAK expression are essential for the induction of MMP-9 but not of MMP-2 as MMP-2 is
always expressed in these cells, including HT1080 cells.
To understand whether NF-
B (an upstream regulator that induces the
expression of MMP-9) was altered by changes in actin polymerization, nuclear extracts prepared from various treatment conditions were examined for the NF-
B expression by gel mobility shift assays. Interestingly, PMA-induced NF-
B expression was reduced to some extent in cytochalasin-D-treated cells but not in colchicine-B-treated cells, showing that NF-
B expression also is regulated by actin polymerization. Although the exact biochemical process by which depolymerization of actin leads to activation of NF-
B remains to be
elucidated, the present findings establish a role for NF-
B in
sensing the changes in the state of the cytoskeleton and converting them to changes in gene activity. Because cytoskeletal changes are
likely to be induced by cell-substrate and cell-cell interaction, this
process would provide signal transduction pathways by which these
physical interactions can modulate gene expression and thereby affect
glioma tumor cell invasion.
PKC, an enzyme essential to the cellular response of phorbol ester
(19), plays an important role in MMP signal transduction. In previous
studies by other investigators, abnormally high levels of PKC activity
in glioblastomas were reported compared with those of nontransformed
glia (42, 43). Moreover, PKC also plays an important role in cell
migration, invasion (44, 45) and metastatic spread of tumors (46), and
glioma cell invasion (47). In our study, treating cells with
calphostin-C (a highly specific inhibitor of PKC) led to a decrease in
MMP-9 and to some extent in MMP-2 in SNB19 cells (Fig. 9) with a
concomitant decrease in invasion through Matrigel (Fig. 10). Other
highly selective inhibitors of tyrosine kinase and
cAMP-dependent kinase inhibitors had no effect on MMP
expression (Fig. 9). These results are similar to the earlier reports
wherein phorbol ester induced the expression of MMP with a concomitant
increase in glioma cell invasion (47). Moreover, earlier studies showed
that PMA-induced PKC also controlled actin polymerization by
maintaining the cell shape and F-actin levels (48). Our results show
that cell shape alteration, particularly inhibition of actin
polymerization, results in inhibition of MMP-9 but not MMP-2 in
glioblastomas; this indicates that these two enzymes are activated by
distinct signaling mechanisms.
 |
FOOTNOTES |
*
This work was supported in part by Grant CA56792 from the
NCI, National Institutes of Health, by American Cancer Society Grant EDT-91 (to J. S. R.), and by a grant from the Anthony D. Bullock III
Foundation (to R. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Neurosurgery, Box 064, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
1
The abbreviations used are: MMP(s), matrix
metalloproteinase(s); PMA, phorbol 12-myristate 13-acetate; TRITC,
tetramethylrhodamine B isothiocyanate; FAK, focal adhesion kinase; PBS,
phosphate-buffered saline; NF-
B, nuclear factor-
B; PKC, protein
kinase C.
2
S. K. Chintala, R. Sawaya, and J. S. Rao,
unpublished observation.
 |
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