(Received for publication, March 20, 1995; and in revised form, May 1, 1995)
From the
To understand the roles of intracellular calcium levels on
gelatinase/type IV collagenase expression, we analyzed the effects of
calcium ionophores on the expression of 92- and 72-kDa gelatinases
(MMP-9 and MMP-2) in human fibrosarcoma cells (HT-1080). Calcium
ionophores ionomycin and A23187 reduced the levels of pericellular
gelatinolytic activity in both untreated and phorbol 12-myristate
13-acetate (PMA) or tumor necrosis factor-
The family of matrix metalloproteinases (MMPs)
A major step in the regulation of
pericellular matrix metalloproteinase activity is the activation of
latent proenzyme by cleavage of a 10-kDa amino-terminal fragment.
Interstitial collagenase, stromelysin, and 92-kDa gelatinase are
activated by soluble enzymes like plasmin, stromelysin, and
chymase(3, 24, 25, 26, 27, 28) .
However, activation of 72-kDa gelatinase by these enzymes is
inefficient and seems to depend on the action of a cell membrane-bound
activator (see (3) and (29) -32). The 72-kDa
gelatinase processing activity is induced by phorbol esters and
concanavalin A and inhibited by retinoic
acid(33, 34) . The 72-kDa gelatinase activator is
found in purified plasma membrane fractions of various tumor cells, and
its action is inhibited by inhibitors of metalloproteinases like
1,10-phenanthroline, EDTA, and tissue inhibitor of metalloproteinases-2
(TIMP-2), suggesting that the activator would be a
metalloproteinase(29, 30) . Recently, Sato et
al.(31) cloned a cDNA with homology to other matrix
metalloproteinases and a transmembrane domain at the COOH terminus and
named it membrane-type matrix metalloproteinase (MT-MMP).
Overexpression of the MT-MMP in COS-1 and Sf-9 cells induces
proteolytic processing of exogenous 72-kDa gelatinase to the active 64-
and 62-kDa forms, resembling those seen in PMA- or concanavalin
A-treated HT-1080 cells. In fact, MT-MMP seems to be a functional cell
surface receptor for 72-kDa gelatinase, and TIMP-2 is needed for this
interaction(35) .
Numerous signal transduction pathways
utilize ionized calcium as a second messenger. Signal transduction
through tyrosine kinase and G-protein-linked receptors converges to the
formation of diacylglycerol and inositol trisphosphate by the
activation of phospholipase C isoforms bound to the plasma membrane.
Binding of inositol trisphosphate to its receptor in the endoplasmic
reticulum and other intracellular Ca
To analyze the approximate molecular
weights of the gelatinolytic proteins, conditioned medium from HT-1080
cells was assayed by gelatin zymography essentially as
described(16, 43) . Samples (15 µl) of conditioned
medium were dissolved in nonreducing Laemmli sample buffer and
separated by electrophoresis using discontinuous 3.5:7% polyacrylamide
gels containing 1 mg/ml gelatin(44) . After electrophoresis the
gels were washed twice with 50 mM Tris-HCl buffer, pH 7.6,
containing 5 mM CaCl
Transfection
was performed using lipofectamine according to manufacturer's
instructions (Life Technologies, Inc.). Subconfluent 60-mm cultures of
HT-1080 cells were washed twice with serum-free MEM. For each
transfection, 6 µg of DNA and 18 µl of lipofectamine were mixed
and incubated for 30 min at room temperature. DNA/liposome mixture was
then transferred onto cells and incubated for 6 h. Subsequently, the
cells were washed with MEM containing 0.2% bovine serum albumin for 30
min, re-washed with MEM, and incubated in MEM overnight. Next day PMA
(4 nM) and ionomycin (500 nM) were added, and the
incubation was continued for 30 h. At the end of this incubation the
cells were lysed in reporter lysis buffer (Promega) and analyzed for
luciferase and
For stable
transfections, a cDNA containing a 2.1-kb fragment of the 5`-flanking
region of 92-kDa gelatinase gene (-2112 to +20) linked to
luciferase gene was cloned to pcDNA3 expression vector (Invitrogen
Corp.) containing the neomycin resistance gene to substitute the
cytomegalovirus promoter of the vector. HT-1080 cells were transfected
as described above and selected for 2 weeks in MEM containing 0.7 mg/ml
neomycin analogue G418. Maintenance culture of cells was performed
using MEM containing 0.25 mg/ml G418. For experiments, confluent
cultures of transfected cells were washed with serum-free MEM without
G418 extensively for 6 h and treated with PMA or ionomycin for 12 h.
Subsequently, the cells were lysed and assayed for luciferase activity
as described above.
Figure 1:
Calcium ionophores and thapsigargin
decrease pericellular gelatinolytic activity and secretion of 92-kDa
gelatinase and inhibit the activation of 72-kDa gelatinase in
PMA-treated fibrosarcoma cells. A, degradation of substratum
bound
Figure 2:
Ionomycin (iono) inhibits the
induction of pericellular gelatinolytic activity and 92-kDa gelatinase
secretion and 72-kDa gelatinase activation in PMA, TNF-
Figure 3:
Ionomycin and thapsigargin prevent the
PMA- and TNF-
It has
recently been reported that overexpression of the MT-MMP causes
proteolytic processing of the 72-kDa gelatinase to the active 62-kDa
form in COS-1 and Sf-9 cells(31) . We analyzed therefore the
levels of MT-MMP expression in this system. The level of MT-MMP mRNA in
serum-starved HT-1080 cells was relatively high and was enhanced
2-3-fold by PMA. Ionomycin did not cause any notable effects in
the levels of MT-MMP mRNA (Fig. 3A).
Figure 4:
Analysis of temporal relationships between
PMA induction and the inhibitory effect of ionomycin. A,
ionomycin treatment does not change the temporal profile of the
PMA-mediated induction of 92-kDa gelatinase. Confluent cultures of
HT-1080 cells were treated with PMA and ionomycin under serum-free
conditions for 1-24 h as indicated on the figure. Northern
hybridization analysis of total RNA indicated that the induction
profiles of 92-kDa gelatinase were similar in PMA and PMA +
ionomycin-treated cells, but the amplitude of induction was lower in
ionomycin-treated cells. In contrast, induction of collagenase was
potentiated by ionomycin. B, induction of 92-kDa gelatinase by
PMA cannot be reversed by subsequent ionomycin treatment. Confluent
cultures of HT-1080 cells were first treated with PMA under serum-free
conditions for 14 h to induce the expression of 92-kDa gelatinase.
Subsequently, ionomycin (500 nM) was added, and the incubation
was continued for 3-48 h as indicated. Total RNA was then
isolated and analyzed by Northern hybridization using 92-kDa gelatinase
cDNA as a probe. C, induction of 92-kDa gelatinase by PMA is
inhibited by ionomycin: time dependence. Confluent cultures of HT-1080
cells were treated with PMA under serum free conditions for 12 h.
Ionomycin (500 nM) was added to the cell cultures at the time
points indicated on the figure. At the end of incubation, total RNA was
isolated and analyzed by Northern hybridization using 92-kDa gelatinase
and collagenase cDNAs as probes. After about 5 h the induction of
92-kDa gelatinase by PMA could not be prevented by ionomycin. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Figure 5:
Ionomycin inhibits PMA-induced
transcription of 92-kDa gelatinase. A, effect of actinomycin D
on 92-kDa gelatinase mRNA levels. Confluent cultures of HT-1080 cells
were treated with PMA and ionomycin under serum-free conditions for 12
h. Actinomycin D (5 µg/ml) was then added, and incubation was
continued for 2-8 h. At the end of incubation, total RNA was
isolated and analyzed for the expression of the 92-kDa gelatinase. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B,
analysis of 92-kDa gelatinase promoter activity in stably transfected
cells. HT-1080 cells were transfected with human -2.1-kb 92-kDa
gelatinase promoter-luciferase construct, and stably transfected cells
were selected by treatment of cells with G418 for 2 weeks (see
``Materials and Methods''). Confluent cultures were treated
with PMA (10 nM) and ionomycin (500 nM) under
serum-free conditions for 12 h. The cells were then lysed and analyzed
for luciferase activity. The values represent the mean of three
independent experiments. Standard deviation is indicated by error
bars. Activity of the untreated control sample is arbitrarily set
as 1.
The harsh treatment of cells used in transient transfections
could affect the ability of cells to react to PMA treatment, e.g. by increasing cell permeability to calcium. To avoid these
artifacts we studied the regulation of the reporter construct in stably
transfected cells and constructed a plasmid vector containing a 2.1-kb
fragment of the 5`-flanking region of 92-kDa gelatinase gene
(-2112 to +20) linked to luciferase gene and neomycin
resistance gene. HT-1080 cells were transfected with this construct
followed by selection with neomycin analogue G418 for 2 weeks. The
mixed population of transfected cells was then treated with PMA and
ionomycin for 12 h, and the level of luciferase in cell lysates was
measured. Untreated cells showed a quite high background level of
luciferase expression, which was decreased about 30% by treatment of
the cells with ionomycin (Fig. 5B). Treatment of cells
with PMA increased the level of luciferase expression 2-3-fold,
and this increase could be inhibited by simultaneous treatment with
ionomycin. Increase in the luciferase activity in cells treated with
PMA was maximal as early as 6 h after the onset of PMA treatment, and
at 30 h the level of luciferase expression was decreased almost to the
control levels, suggesting that the induction of the transcription of
the 92-kDa gelatinase gene is transient. Clones derived from single
transfected cells were produced by dilution cloning and assayed for the
regulation of luciferase activity by PMA and ionomycin. Basal levels of
luciferase expression varied about 5-fold between different clones, but
the regulation by PMA and ionomycin was similar to that seen in the
mixed pool of transfected cells (data not shown).
In the present work we have characterized the effects of
elevated intracellular calcium levels on the expression and proteolytic
processing of 92- and 72-kDa gelatinases. To modulate intracellular
calcium concentration, cultured cells were treated with two different
calcium ionophores, A23187 and ionomycin, which function as mobile ion
carriers and increase the permeability of plasma membrane and
intracellular calcium storage compartments to divalent cations. To
ensure that the effects observed were due to increased intracellular
calcium levels, we also used another chemical, thapsigargin, which
releases calcium from intracellular stores by inhibiting the function
of endoplasmic reticulum Ca
Despite extensive efforts the mechanisms of 72-kDa
gelatinase activation in vitro and in vivo are still
unraveled. The 72-kDa gelatinase processing activity of PMA- or
concanavalin A-treated cells has been localized to the plasma membrane
fraction, and it is sensitive to inhibition by metal ion chelators such
as EDTA and 1,10-phenanthroline as well as
TIMP-2(29, 30) . Overexpression of a novel
membrane-associated metalloproteinase, MT-MMP in COS-1 and Sf-9 cells,
leads to the appearance of 72-kDa gelatinase processing activity in the
plasma membrane fraction of the cells, and this activity could be
inhibited by TIMP-2(31) . Accordingly, MT-MMP is a good
candidate for the enzyme responsible for proteolytic processing and
activation of 72-kDa gelatinase in phorbol ester or concanavalin
A-treated HT-1080 cells.
In our study we found that MT-MMP mRNA is
expressed in high levels even in serum-starved HT-1080 cells, which
express only limited 72-kDa gelatinase processing activity.
Furthermore, we found that treatment of the cells with PMA caused only
2-3-fold induction of MT-MMP expression, and this induction was
not affected by calcium ionophores. The 72-kDa gelatinase processing
activity was, however, strongly induced by PMA, and this induction was
completely inhibited by ionomycin. In our studies, TNF
Like other matrix metalloproteinases, MT-MMP has an
NH
Recent evidence indicates that the
presence of some TIMP-2 is mandatory for the binding of 72-kDa
gelatinase to the MT-MMP at the cell surface and for the subsequent
membrane activation of 72-kDa gelatinase(35) . However, the
optimal concentration of TIMP-2 for 72-kDa gelatinase activation is
quite low and narrow, since an excess of TIMP-2 leads to the inhibition
of the activation reaction. In our experiments we could not see
remarkable differences in the mRNA or protein levels of TIMP-2 between
cells treated with PMA alone or PMA together with ionomycin (data not
shown). Accordingly, regulation of TIMP-2 levels seems not to be the
cause of ionomycin-mediated inhibition of 72-kDa gelatinase activation.
In the assays for pericellular gelatinolytic activity, PMA and
TNF
To analyze the mechanisms of the effects of calcium ionophores on
the expression of 92-kDa gelatinase, total RNAs were extracted and
analyzed by Northern hybridization. The basal level of 92-kDa
gelatinase expression in serum-starved HT-1080 cells was quite low and
was strongly induced by PMA and TNF
When analyzing the promoter activity of 92-kDa gelatinase
using transient transfection of cells with promoter/luciferase
constructs, we observed only very limited induction of promoter
activity in PMA-treated cells. However, promoter activity was
significantly decreased by ionomycin. The low levels of luciferase
expression could be caused by leakage of the plasma membranes during
the transfection process, which would allow influx of calcium. To
circumvent the harsh treatment of transient transfections, we produced
cells that were stably transfected with the 92-kDa
gelatinase-luciferase reporter construct. The basal level of luciferase
expression in these cells was relatively high, which could be explained
by the absence of a silencer that would exist in the vicinity of the
92-kDa gelatinase gene but which would be lacking in the transfected
cells. In stably transfected cells promoter activity was induced by PMA
and TNF-
Treatment of cells with calcium ionophores or thapsigargin
causes a transient increase in the intracellular calcium levels as
calcium flows into the cytoplasm from extracellular space and
intracellular calcium stores (see (37, 38, 39) ). After the initial surge of
calcium the intracellular calcium concentrations reaches a stable level
that is higher than before stimulation. However, in our study the
actual calcium levels were not measured. Absence of calcium waves and
oscillations typical of in vivo calcium signaling may also
modulate the effects of calcium ionophores. Another phenomenon that is
associated with prolonged treatment of cells with calcium ionophores or
thapsigargin is depletion of intracellular calcium stores, which might
have diverse and unappreciated effects on cellular signal transduction
and even on protein folding in the Golgi apparatus(38) . The in vivo counterpart to the effects of calcium ionophores
remains also unraveled. Possible candidates are growth factors and
peptides, which signal through tyrosine kinase or G-protein-linked
receptors to increase intracellular calcium levels. Cancer cells often
have abnormal calcium signaling mechanisms; they need much less
external calcium for proliferation, and they no longer obey calcium
signals to differentiate or die by apoptosis(36) . This
aberrance of the calcium signaling network might also include absence
of calcium-mediated inhibition of 72-kDa gelatinase activation and
92-kDa gelatinase expression. In any case, our data suggest the
presence of a calcium-dependent signaling mechanism leading to two
separate phenomena capable of decreasing the pericellular gelatinolytic
activity. Specific inhibition of 72-kDa processing activity by calcium
ionophores may also serve as a valuable tool in the further analysis of
the 72-kDa gelatinase activation cascade.
We thank Drs. Tapio Vartio, Ulpu Saarialho-Kere, and
Jussi Taipale for critical comments and Sami Starast for fine technical
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TNF
)-stimulated
cells as determined by degradation of radiolabeled gelatin. Gelatin
zymography and immunoblotting revealed a dose-dependent decrease in the
levels of secreted 92-kDa gelatinase, which was paralleled by a
decrease of its mRNA. Treatment of cells with thapsigargin caused
similar decreases of 92-kDa gelatinase mRNA and protein. The decrease
of 92-kDa gelatinase expression was due to lower transcription rate as
determined by transfection assays with 92-kDa gelatinase/luciferase
construct. The expression of 72-kDa gelatinase was only slightly
decreased by ionophores. Treatment of HT-1080 cells with PMA, TNF
,
or concanavalin A resulted in the conversion of 72-kDa gelatinase
proenzyme to its presumed 64- and 62-kDa active forms as determined by
gelatin zymography and immunoblotting. Simultaneous treatment with the
ionophores or thapsigargin resulted in inhibition of PMA-induced
gelatinase activation. The expression of membrane-type matrix
metalloproteinase, a potential activator of 72-kDa gelatinase, was not
affected by ionophores. The results indicate that calcium ionophores
decrease gelatinolysis by repressing both the expression of 92-kDa
gelatinase and the activation of the 72-kDa gelatinase.
(
)comprises at least nine enzymes that collectively
are capable of degrading most if not all components of the
extracellular matrix. Different MMPs have varying substrate specifities
and divergent regulation of gene expression and proenzyme
activation(1, 2, 3) . The expression of the
interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), matrilysin
(MMP-7), and 92-kDa gelatinase (MMP-9) genes is often coregulated and
is enhanced by epidermal growth factor, inflammatory cytokines like
TNF
and interleukin-1
, and the phorbol ester, phorbol
12-myristate 13-acetate
(PMA)(1, 4, 5, 6, 7, 8, 9) .
The common mediator of these inductions is supposed to be the
12-O-tetradecanoylphorbol-13-acetate-responsive element, which
is present in the promoter regions of all above-mentioned MMPs and
which binds the AP-1 transcription factor complex. In addition, the
NF-
B and Sp-1 binding sites in the 92-kDa gelatinase promoter are
essential for PMA- or TNF
-mediated induction(10) .
Glucocorticoids, retinoic acid, and transforming growth factor-
are known as repressive factors for these
MMPs(11, 12, 13, 14) . The 92-kDa
gelatinase is secreted by normal alveolar macrophages,
polymorphonuclear leukocytes, osteoclasts and keratinocytes, and
invading trophoblasts and by several transformed cell lines, but not by
fibroblastic
cells(8, 15, 16, 17, 18, 19, 20) .
The 72-kDa gelatinase is constitutively expressed in most fibroblastic
cells and by some transformed epithelial cells, and its expression is
enhanced by transforming growth factor-
but not by
PMA(14, 21, 22) . The promoter of the 72-kDa
gelatinase (MMP-2) gene contains an AP-2 binding site and two Sp-1
sites but no 12-O-tetradecanoylphorbol-13-acetate-responsive
element (23) .
storage
compartments causes then release of ionized calcium to cytoplasm and a
transient increase in the intracellular calcium level. Changes in
intracellular calcium concentration control numerous cellular
processes, including cell growth, differentiation, and
transformation(36, 37, 38, 39) .
Interstitial collagenase and stromelysin-1 genes are induced by an
increase in the intracellular calcium level, and treatment of rabbit
synovial fibroblasts with calcium ionophores results in endogenous
activation of interstitial collagenase(40, 41) .
Treatment of HT-1080 fibrosarcoma cells with carboxy amido-triazole, an
inhibitor of receptor-mediated calcium influx, reduces the levels of
both latent and activated forms of 72-kDa gelatinase, although an
initial increase in the level of 62-kDa activated form is
seen(42) . In the present study we have analyzed the effects of
calcium ionophores and thapsigargin, an inhibitor of endoplasmic
reticulum Ca
-ATPase, on the expression and
proteolytic processing of the 92-kDa (MMP-9) and 72-kDa gelatinases
(MMP-2) in human fibrosarcoma cells (HT-1080). We found that calcium
ionophores reduced the basal and PMA-inducible pericellular
gelatinolytic activities by inhibiting 92-kDa gelatinase expression and
72-kDa gelatinase activation. The present work describes a novel role
for intracellular calcium signaling as a regulator of pericellular
gelatinolytic activity.
Reagents
PMA, calcium ionophore A23187,
ionomycin, cycloheximide, actinomycin D, concanavalin A, and
thapsigargin were purchased from Sigma. Human recombinant TNF was
obtained from Boehringer Mannheim GmbH (Mannheim, Germany).
Cell Cultures
Human fibrosarcoma HT-1080 cells
(CCL-121, American Type Culture Collection, Rockville, MD) were
cultivated in Eagle's minimal essential medium (MEM) containing
10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100
IU/ml penicillin, and 50 µg/ml streptomycin. The cultures were
incubated at 37 °C in a humidified 5% CO atmosphere
until confluence. Before experiments, cultures were washed twice with
serum-free medium and then incubated under serum-free conditions for 6
h. All experiments were carried out under serum-free conditions. To
assay the toxicity of treatments, general protein synthesis was
measured by metabolic labeling followed by trichloroacetic acid
precipitation. Confluent cultures of HT-1080 cells were incubated under
serum-free conditions for 6 h followed by incubation with PMA (4
nM) and ionomycin (50-500 nM) for 12 h. L-[4,5-
H]Leucine (2 µCi/ml)
(Amersham, Buckinghamshire, United Kingdom) was then added and
incubation was continued for 12 h. Conditioned medium was then
collected and clarified by centrifugation, and secreted proteins were
precipitated by adding bovine serum albumin (3 mg/ml final) and
trichloroacetic acid (10% final concentration) followed by incubation
on ice for 30 min and centrifugation. Protein pellet was solubilized in
0.3 N NaOH, and radioactivity was measured by liquid
scintillation counter.
Assays for Gelatinolytic Activity
To assay the
pericellular gelatinolytic activity of cultured cells, tissue culture
wells were coated with radiolabeled gelatin. Briefly, 1 µl (60 nCi)
of N-[propionate-2,3-H]propionylated rat
type I collagen (0.19 mCi/mg; DuPont NEN) was diluted with 60 µl of
1 mg/ml unlabeled type I gelatin and heated at 80 °C for 30 min.
Gelatin was then diluted with MEM, applied onto culture dishes, and
allowed to attach for 2 h. Unbound radioactivity was removed by
extensive washing with MEM, and HT-1080 cells were seeded on the dish.
One day later the cultures were washed with serum-free MEM for 6 h,
after which fresh serum-free MEM was changed, supplemented with PMA (4
nM), ionomycin (500 nM), TNF
(10 ng/ml), and
concanavalin A (50 µg/ml), and incubation was continued for 30 h.
Subsequently, 500-µl aliquots of the conditioned medium were
analyzed by liquid scintillation counter to measure the release of
H-labeled peptides.
, 1 µM ZnCl
, 2.5% Triton X-100 (v/v) for 15 min to remove
SDS, followed by a brief rinsing in washing buffer without Triton
X-100. The gels were then incubated at 37 °C for 1-2 days in
50 mM Tris-HCl buffer containing 5 mM CaCl
, 1 µM ZnCl
, 1% Triton
X-100, 0.02% NaN
, pH 7.6. The digestion was terminated by
10% acetic acid followed by staining with Coomassie Brilliant Blue
R-250 and destaining with 10% acetic acid, 10% methanol. Zones of
enzymatic activity were seen as negatively stained bands.
Immunoblotting Assay for 72- and 92-kDa
Gelatinases
For the immunoblotting analysis, confluent cultures
of HT-1080 cells were treated with PMA (4 nM) and ionomycin
(500 nM) under serum free conditions. After 24-h incubation
the conditioned medium was harvested, clarified by centrifugation, and
an aliquot (15 µl) was dissolved in Laemmli sample buffer and
subjected to 4-15% gradient SDS-PAGE under nonreducing
conditions. Proteins were electrophoretically transferred to
nitrocellulose (Schleicher & Schuell, Dassel, Germany) using a
semi-dry blotting apparatus at 0.8 mA/cm for 1 h. Membranes
were saturated with 5% milk in TBST (10 mM Tris-HCl buffer, pH
8.0, containing 150 mM NaCl, and 0.05% Tween 20) and incubated
with monoclonal antibodies against human 72- and 92-kDa gelatinases
(clones 42-5D11 and 7-11C, respectively, Oncogene Science, Inc.,
Cambridge, MA) (1 and 2 µg/ml in TBST, respectively). After
several washes with the same buffer, the bound antibodies were detected
using biotinylated anti-mouse IgG antibodies and peroxidase-conjugated
streptavidin (Dakopatts, Copenhagen, Denmark) and enhanced
chemiluminescence Western blotting detection system (Amersham
International PIC, Amersham, UK) as described(45) .
Isolation of RNA and Northern Hybridization
Analysis
Confluent cultures were washed twice with serum-free
medium and then incubated under serum-free conditions for 6 h.
Subsequently, PMA (4 nM), TNF (10 ng/ml), concanavalin A
(50 µg/ml), calcium ionophores A23187 (10-2000 nM)
or ionomycin (10-2000 nM), or thapsigargin
(10-2000 nM) were added into the medium as indicated,
and incubation was continued for 24 h or as indicated. The cultures
were then washed with phosphate-buffered saline (170 mM NaCl,
10 mM sodium phosphate buffer, pH 7.4) and lysed for RNA
extraction. RNA was purified by pelleting a guanidinium
thiocyanate-N-laurylsarcosyl cell lysate through a CsCl
cushion(46) . Total RNA (15 µg) was electrophoresed on a
0.8% formaldehyde-agarose gel and transferred to a Zeta-Probe filter
(Bio-Rad) in 20
SSC (1
SSC is 150 mM NaCl, 15
mM sodium citrate buffer, pH 7.0) and immobilized by UV
cross-linking. The filters were hybridized to probes labeled with
[
-
P]dCTP (Amersham) by the random priming
method (Pharmacia LKB, Uppsala, Sweden). Hybridization was performed in
50% deionized formamide, 7% SDS, 250 mM NaCl, 250 mM
NaH
PO
, 1 mM EDTA at 42 °C for 24
h. Subsequent washing was carried out in 0.2
SSC containing
0.1% SDS at 63 °C. Stripping, when needed, was performed in 0.1
SSC, 0.5% SDS at 100 °C for 10 min. The following human
cDNA probes were used: collagenase(47) , 72-kDa
gelatinase(21) , and 92-kDa gelatinase(14) . Rat
glyceraldehyde-3-phosphate dehydrogenase (48) cDNA was used to
standardize the loading of RNAs. For detection of human MT-MMP mRNA, a
699-base pair cDNA probe corresponding to bases 884-1582 of the
published MT-MMP sequence (31) was generated by reverse
transcription-polymerase chain reaction from HT-1080 cDNA.
(
)
Transient and Stable Transfections of
HT-1080 Cells with 92-kDa Gelatinase Promoter-Luciferase
Construct
To study the transcription of the 92-kDa gelatinase
gene by transient transfection, we constructed a reporter plasmid
containing a 2.2-kb fragment of the 5`-flanking region of 92-kDa
gelatinase gene (-2161 to +20) linked to luciferase gene in
pGL2-Basic vector (Promega Corp., Madison, WI). Promoter activity was
then monitored by determining the luciferase activity of transiently
transfected HT-1080 cells using a Luciferase Assay System kit according
to manufacturer's instructions (Promega). Transfection efficiency
was controlled by cotransfecting the promoter/luciferase construct with
pCMV control plasmid containing cytomegalovirus promoter linked to
the
-galactosidase gene (Clontech Laboratories Inc., Palo Alto,
CA) and correcting the luciferase activities for
-galactosidase
activity, which was measured as described(49) .
-galactosidase activities.
Calcium Ionophores Inhibit the Gelatinolytic Activity
of HT-1080 Cells
Cultured human fibrosarcoma cells (HT-1080)
were used as a model to study the effects of elevated intracellular
calcium levels on the pericellular gelatinolytic activity. HT-1080
cells were seeded on culture plates coated with denatured H-labeled rat type I collagen (gelatin). After overnight
attachment and 6-h incubation in serum-free medium, the cultures were
treated with the calcium ionophore ionomycin together with PMA for 30
h, and the radioactivity released in the conditioned medium was
measured by a scintillation counter (Fig. 1A). Basal
level of released radioactivity (including both enzymatic and
non-enzymatic release) was increased about 3-fold by 4 nM PMA.
Simultaneous treatment of cells with 50 nM ionomycin led to a
notable decrease in the released radioactivity, and 500 nM ionomycin completely blocked the gelatinolysis-inducing activity
of PMA.
H-labeled gelatin. HT-1080 cells were seeded on
[
H]gelatin-coated tissue culture plates and
incubated for 18 h (see ``Materials and Methods'').
Subsequently, the cultures were washed, incubated with serum-free
medium for 6 h, washed again, and incubated with PMA and ionomycin as
shown on the figure for 30 h. Conditioned medium was then collected,
clarified by centrifugation, and the amount of released radioactivity
was measured. The values represent the mean of three independent
experiments. Standard deviation is indicated by error bars. B,
gelatin zymography. Confluent cultures of HT-1080 cells were treated
with A23187, ionomycin, or thapsigargin and PMA under serum-free
conditions for 24 h as shown on the figure (see ``Materials and
Methods''). Aliquots of conditioned medium (15 µl) were
analyzed by gelatin zymography using 6% SDS-PAGE. C,
immunoblotting of 72- and 92-kDa gelatinases. Confluent cultures of
HT-1080 cells were treated with PMA (4 nM) and ionomycin (500
nM) for 24 h. Aliquots (15 µl) of conditioned medium were
analyzed by 4-15% gradient SDS-PAGE under nonreducing conditions
followed by immunoblotting using monoclonal antibodies against 72- and
92-kDa gelatinases. Note the inhibition of PMA-induced 72-kDa
gelatinase processing and decrease of 92-kDa gelatinase expression by
ionomycin.
To study the molecular forms of gelatinolytic enzymes
responsible for the increased gelatinolytic activity of PMA-treated
cells, and the effect of calcium ionophores on their secretion,
conditioned medium of HT-1080 cells was analyzed by gelatin zymography.
Untreated cells secreted mainly 72-kDa (pro)gelatinase, whereas only
very low levels of 92-kDa gelatinase were observed (Fig. 1B). Treatment of the cells with PMA resulted in
enhancement of 92-kDa gelatinase secretion and partial
conversion/proteolytic processing of 72-kDa gelatinase proenzyme to the
presumably activated 64- and 62-kDa forms of 72-kDa
gelatinase(33) . Cotreatment with ionomycin or another calcium
ionophore A23187 resulted in the disappearance of 92-, 64-, and 62-kDa
gelatinolytic protein bands, suggesting that calcium ionophores inhibit
both the PMA-induced secretion of 92-kDa gelatinase and activation of
72-kDa gelatinase. The changes in the gelatinolytic activities were
paralleled by a decrease in the level of 92-kDa gelatinase and by
inhibition of the PMA-induced processing of 72-kDa gelatinase secreted
into the conditioned medium as determined by immunoblotting (Fig. 1C). Treatment of cells with ionomycin alone
caused also a moderate decrease in the level of secreted 72-kDa
gelatinase. Thapsigargin, an inhibitor of endomembrane
Ca-ATPase, is another chemical capable of increasing
intracellular calcium levels. Accordingly, treatment of HT-1080 cells
with thapsigargin and PMA caused a dose-dependent decrease of 92-kDa
gelatinase expression and inhibition of proteolytic
processing/activation of 72-kDa gelatinase (Fig. 1B).
Metabolic labeling of PMA- and ionomycin-treated cells with
[
H]leucine followed by trichloroacetic acid
precipitation of secreted macromolecules indicated a slight decrease
(20%) in general protein synthesis at maximal doses tested (data not
shown), but the decrease was much lower than the decrease of
gelatinolytic activity.
Processing of 72-kDa Gelatinase to Lower Molecular Weight
Forms by PMA, TNF-
We analyzed next whether ionomycin could also block
the effects of other known inducers of 72-kDa gelatinase activation and
92-kDa gelatinase expression. HT-1080 cells were cultivated on
[, or Concanavalin A Stimulation Is Prevented by
Ionomycin
H]gelatin-coated dishes as described above,
followed by treatment with PMA, TNF-
, or concanavalin A in the
presence of ionomycin for 30 h as indicated (Fig. 2A).
Scintillation counting of the supernatant fluids showed 2-3-fold
increases in the levels of released radioactivity in cultures treated
with PMA, TNF-
, or concanavalin A, indicating an increase in the
pericellular gelatinolytic activity (Fig. 2A). Addition
of ionomycin completely prevented this increase. To characterize the
regulation of different gelatinases and their activation by the
above-mentioned factors, aliquots of conditioned medium of a similar
experiment were assayed by gelatin zymography. Accordingly, treatment
of the cells with PMA or TNF-
resulted in an increase in 92-kDa
gelatinase secretion and partial conversion of 72-kDa gelatinase
proenzyme to 64- and 62-kDa forms. Treatment of cells with concanavalin
A caused almost complete conversion of 72-kDa gelatinase to 64- and
62-kDa forms, but did not affect the secretion of 92-kDa gelatinase.
Treatment with ionomycin resulted both in the inhibition of the
induction of 92-kDa gelatinase secretion and reduced activation of
72-kDa gelatinase (Fig. 2B).
, and
concanavalin A (ConA)-treated cells. A, degradation
of substratum bound
H-labeled gelatin. HT-1080 cells were
seeded on [
H]gelatin-coated tissue culture plates
and incubated for 18 h and washed (see legend to Fig. 1). The
cultures were then incubated with TNF-
(10 ng/ml), concanavalin A
(50 µg/ml), or PMA (4 nM) in the presence of ionomycin
(500 nM) for 30 h as shown on the figure. Conditioned medium
was then collected, clarified by centrifugation, and the amount of
released radioactivity was measured. The values represent the mean of
three independent experiments. Standard deviations are indicated by error bars. B, Gelatin zymography. Confluent cultures of
HT-1080 cells were treated with TNF-
(10 ng/ml), concanavalin A
(50 µg/ml), or PMA (4 nM) and ionomycin (500 nM)
under serum-free conditions for 24 h as shown on the figure. Aliquots
of conditioned medium were analyzed by gelatin zymography using 6%
SDS-PAGE.
Induction of 92-kDa Gelatinase Gene Expression Is
Inhibited by Calcium Ionophores and Thapsigargin
To study the
effect of elevated intracellular calcium on the expression of 92- and
72-kDa gelatinase mRNAs, HT-1080 cells were treated with PMA and
increasing concentrations of ionomycin. At the termination of
incubation the cells were lysed, and total RNA was extracted and
analyzed by Northern hybridization. In accordance with earlier results (4, 14) PMA increased markedly the mRNA levels of
collagenase and 92-kDa gelatinase, but had no effect on the expression
of 72-kDa gelatinase (Fig. 3A). Ionomycin (40
nM) reduced the levels of 92-kDa gelatinase mRNA in
PMA-treated cells, and at 500 nM it was barely detectable. As
expected, the expression of interstitial collagenase gene was induced
by ionomycin, and this induction was further potentiated by PMA. The
expression of 72-kDa gelatinase mRNA, when compared with
glyceraldehyde-3-phosphate dehydrogenase mRNA levels, was not affected
by ionomycin. Treatment of the cells with thapsigargin caused a
dose-dependent decrease of 92-kDa gelatinase expression with notable
effect at 10 nM and maximal effect at 500 nM (Fig. 3B). Collagenase mRNA levels were induced by
both PMA and thapsigargin, and the induction was potentiated by
simultaneous treatment of cells. Similar results on the regulation of
the mRNA levels for 92- and 72-kDa gelatinases and collagenase were
also obtained with ionophore A23187 (data not shown).
mediated induction of 92-kDa gelatinase mRNA.
Confluent cultures of HT-1080 cells were treated with ionomycin or
thapsigargin under serum-free conditions for 24 h as indicated. PMA (4
nM), concanavalin A (50 µg/ml), or TNF-
(10 ng/ml)
were added where indicated to induce the expression of 92-kDa
gelatinase. Total RNA was then isolated and analyzed by Northern
hybridization using random labeled cDNA probes for 92- and 72-kDa
gelatinases. Interstitial collagenase and MT-MMP were used as controls.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels
served as an internal standard. A, dose dependence analysis of
ionomycin treatment. B, dose dependence analysis of
thapsigargin treatment. C, effects of ionomycin treatment on
mRNA levels in TNF-
and concanavalin A-treated
cells.
To compare the
effects of ionomycin on the mRNA levels of 92- and 72-kDa gelatinases
in PMA-stimulated cells to those obtained by TNF- or concanavalin
A, the cells were treated as indicated for 12 h. Northern hybridization
analysis revealed that the expression of 92-kDa gelatinase was induced
by both PMA and TNF-
, and the induction was prevented by ionomycin (Fig. 3C). mRNA levels of 72-kDa gelatinase were only
slightly decreased by ionomycin. Concanavalin A did not have any effect
on the mRNA levels of either 92- or 72-kDa gelatinases.
Induction of 92-kDa Gelatinase by PMA Cannot Be Reversed
by Subsequent Ionomycin Treatment
We analyzed next the temporal
profiles of the induction and inhibition of 92-kDa gelatinase
expression by PMA and ionomycin. After 6-h serum starvation, HT-1080
cells were treated with PMA and/or ionomycin as indicated for
1-24 h followed by Northern hybridization analysis. Induction of
92-kDa gelatinase in PMA-stimulated cells was detectable at 5 h and
maximal at 12 h, after which the level of 92-kDa gelatinase mRNA
declined slightly to 24 h. Simultaneous treatment of cells with
ionomycin prevented almost completely the induction, but did not affect
the temporal profile (Fig. 4A).
To study further the
temporal relationships between PMA induction and the inhibition of
92-kDa gelatinase expression by ionomycin, HT-1080 cells were first
treated with PMA for 14 h to induce 92-kDa gelatinase expression to a
measurable level. Subsequently, ionomycin was added, and the incubation
was continued for an additional 3-48 h. Unexpectedly, the
PMA-induced mRNA level of 92-kDa gelatinase was not affected,
suggesting that the induction cannot be reversed by subsequent
ionomycin treatment (Fig. 4B). To determine the
critical time of the inhibition by ionomycin, HT-1080 cells were
incubated 12 h with PMA, and ionomycin was added 0-11 h after the
onset of the incubation. At 12 h total RNA was isolated and analyzed by
Northern hybridization. It was found that a time lag of 1-3 h
between addition of PMA and ionomycin notably attenuated the inhibitory
effect of ionomycin, and after 5-7 h ionomycin had no effect on
the 92-kDa gelatinase mRNA levels (Fig. 4C). Both PMA
and ionomycin induced collagenase expression, and maximal induction was
seen when ionomycin was added 3 h after PMA.
Ionomycin Inhibits PMA-induced Transcription of 92-kDa
Gelatinase
It is known that PMA induces the 92-kDa gelatinase
mRNA levels by increasing the transcription of the gene(10) .
To find out whether the inhibition of induction by ionomycin was caused
by inhibition of gene transcription or by destabilization of 92-kDa
gelatinase mRNA, the stability of mRNA was studied by actinomycin D
treatment. HT-1080 cells were treated with PMA and ionomycin for 12 h,
after which actinomycin D (5 µg/ml) was added to inhibit mRNA
synthesis, and the incubation was continued for 4-8 h. Total RNA
was then isolated and analyzed by Northern hybridization. The level of
92-kDa gelatinase mRNA was not affected by 8-h treatment with
actinomycin D in either PMA or PMA and ionomycin-treated cells,
suggesting that the half-life of 92-kDa gelatinase is quite long and is
not affected by ionomycin (Fig. 5A).
To analyze the
transcription of the 92-kDa gelatinase gene, we constructed a reporter
plasmid containing a 2.2-kb fragment of the 5`-flanking region of the
92-kDa gelatinase gene (-2160 to +20) linked to luciferase
gene. Transient transfection of HT-1080 cells was performed as
described under ``Materials and Methods.'' After
transfection, the cultures were treated with PMA and/or ionomycin for
30 h, after which cells were harvested, luciferase activity was
measured and correlated to -galactosidase activity. Treatment of
cells with PMA caused only a slight but reproducible increase in
luciferase activity. Ionomycin (500 nM) reduced the levels of
luciferase activity in both untreated and PMA-treated cells (data not
shown).
-ATPase. It was found that
the conditioned medium of HT-1080 cells contained only very low levels
of soluble gelatinase activity as measured by degradation of soluble
radiolabeled gelatin substrate. However, when radiolabeled gelatin
attached to cell culture plates was used as a substrate, we found a
steady level of basal pericellular gelatinolytic activity, which was
decreased by calcium ionophores and thapsigargin. Pericellular
gelatinolytic activity could be induced by treatment of the cells with
PMA, TNF
, and concanavalin A, and their effects were abrogated by
simultaneous treatment with calcium ionophores. Analysis of
gelatinolytic activity by gelatin zymography revealed two distinct
phenomena that could decrease the gelatinolytic potential of the cells
treated with ionophores. Both the basal and PMA- or TNF
-stimulated
expression of 92-kDa gelatinase was decreased, and the proteolytic
processing of 72-kDa gelatinase was inhibited. In contrast, treatment
of rabbit synovial fibroblasts with calcium ionophores has been
reported to result in endogenous activation of interstitial collagenase (40) , suggesting that the mechanisms regulating the activation
processes of these two MMPs are quite different. Our data are
suggestive but do not prove that the pericellular gelatinolytic
activity would be totally attributable to the two gelatinases. However,
these gelatinases are the only major gelatinolytic proteases present in
the conditioned medium of HT-1080 cells as assayed by gelatin
zymography.
and
concanavalin A had no effect on MT-MMP mRNA levels. This is in contrast
to the results of Sato et al.(31) reporting an
induction of MT-MMP mRNA levels in concanavalin A-treated HT-1080
cells. Thus it appears that the level of MT-MMP mRNA expression is not
directly connected with the level of 72-kDa gelatinase processing
activity.
-terminal latency-associated domain, which is presumably
removed upon activation. Activation of 72-kDa gelatinase by PMA and
concanavalin A could thus be mediated by induction of proteolytic
processing and activation of latent MT-MMP, which could then activate
the 72-kDa gelatinase. We have reported earlier that treatment of
isolated extracellular matrices of human embryonal lung fibroblasts
with urokinase-type plasminogen activator, thrombin, plasmin, and two
mast cell-derived enzymes, tryptase and chymase, causes processing of
matrix-bound 72-kDa gelatinase to 64- and 62-kDa
forms(43, 50) . On the other hand, in experimental
settings using purified 72-kDa gelatinase as a substrate,
urokinase-type plasminogen activator, plasmin, stromelysin, and even
trypsin have lacked the ability to activate 72-kDa
gelatinase(30, 51) . These contradictory results could
be explained by a model of a cell surface-associated proteolytic
cascade, which would involve activation of MT-MMP by some of the
above-mentioned serine proteases, perhaps receptor-bound urokinase-type
plasminogen activator, as also proposed by Strongin et
al.(35) . Active cell surface-bound MT-MMP would then bind
and activate 72-kDa gelatinase to achieve cell surface-targeted
gelatinolytic activity. Constitutive production of 72-kDa gelatinase by
stromal fibroblasts and its deposition in the pericellular matrix would
provide a potent, rapidly and targetedly activable store of latent
gelatinolytic enzyme. Still another possibility is that some other
membrane-associated metalloproteinase like a homologue of MT-MMP is
involved. In addition, there are also membrane metalloproteinases that
are not homologous to matrix metalloproteinases like
endothelin-converting enzyme-1 (ECE-1), but their possible effects in
the activation of matrix metalloproteinases are yet to be
characterized(52) .
were more potent inducers of gelatinolysis than concanavalin A (Fig. 2A). However, in gelatin zymography the
processing of 72-kDa gelatinase to lower molecular weight forms was
more complete in the conditioned medium of concanavalin A-treated
cells, whereas PMA- or TNF
-treated cells secreted high levels of
92-kDa gelatinase. Although 92-kDa gelatinase was found to be in the
92-kDa progelatinase form and not in the 84-kDa proteolytically
processed form, it may still be active in the pericellular environment.
. Maximal levels of 92-kDa
gelatinase expression were achieved at 12 h. Because the half-life of
the mRNA is rather long, the lacking of further accumulation suggests
that the induction of transcription is only transient. Addition of
ionomycin at the same time with PMA almost completely blocked the
induction, but if ionomycin was added only about 1 h after PMA, its
inhibitory effect on 92-kDa gelatinase expression was significantly
attenuated. If ionomycin was added 12 h after the onset of PMA
stimulation, no effect on the 92-kDa gelatinase expression could be
observed, suggesting that the effect of ionomycin is mediated by
inhibition of gene transcription rather than destabilization of 92-kDa
gelatinase mRNA. This was also confirmed by our observation that the
half-life of 92-kDa gelatinase is rather long and not affected by
ionomycin.
, and these inductions were prevented by ionophores, which
confirms that 92-kDa gelatinase is regulated at the transcriptional
level.
, tumor necrosis factor-
;
PMA, phorbol 12-myristate 13-acetate; TIMP-2, tissue inhibitor of
metalloproteinases, type 2; MT-MMP, membrane-type matrix
metalloproteinase; MEM, Eagle's minimal essential medium; PAGE,
polyacrylamide gel electrophoresis; kb, kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.