From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295
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ABSTRACT |
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Phospholipase D (PLD) has been implicated in
vesicle trafficking in the Golgi and hence secretion. In this study, we
show that the secretion of matrix metalloproteinase-9 (MMP-9) from HT
1080 human fibrosarcoma cells was stimulated by phorbol 12-myristate 13-acetate in a time- and dose-dependent manner that
involved protein kinase C. The phorbol ester also increased PLD
activity in the cells. Evidence that PLD was involved in the
stimulation of MMP-9 secretion was provided by the observations that
the secretion of MMP-9 was stimulated by the introduction of
short-chain phosphatidic acid (PA) into the growth medium and that
inhibition of PA production by 1-propanol inhibited secretion. Using a
short-chain diacylglycerol we excluded the possibility that MMP-9
secretion was induced by diacylglycerol formed from PA by phosphatidic
acid phosphatase. Furthermore, propranolol, an inhibitor of this
enzyme, had no effect on secretion induced by either phorbol
12-myristate 13-acetate or PA. The data presented here indicate that
activation of protein kinase C increases MMP-9 secretion in HT 1080 cells and implicate PLD and PA formation in the effect.
ADP-ribosylation factors
(ARFs)1 in their
myristoylated and GTP-bound form are essential for COPI (coatamer) and
clathrin coat assembly on the Golgi and for maintenance of this
organelle (1-5). ARFs also activate phospholipase D (PLD), and it has
been suggested that the Golgi-associated PLD mediates some of the
effects of ARF on the Golgi (6). Recently, it has been shown that
phosphatidic acid (PA), the product of phosphatidylcholine hydrolysis
by PLD, promotes vesicular transport from the endoplasmic reticulum to the Golgi (7) and also budding of secretory vesicles from the trans-Golgi network (8, 9). Furthermore, it has been suggested that PA
stimulates the assembly of COPI on the Golgi (10). PLD has also been
reported to regulate the recruitment of the adapter proteins required
for clathrin coat assembly on the Golgi (11). In summary, although the
exact role of PLD remains unclear, there is much evidence that it
participates in the control of vesicular trafficking to, through, and
from Golgi components.
Secretion of matrix metalloproteinases (MMPs) from cancer cells is an
important stage in the metastatic spread. MMPs hydrolyze collagen, a
major component of the extracellular matrix, and allow the invasion of
cancer cells from their primary site to the circulation and secondary
sites (12-14). Various stimuli, including phorbol 12-myristate
13-acetate (PMA), have been shown to induce the secretion of MMP-9 from
cancer cell lines (15) including the human fibrosarcoma line HT 1080 (16, 17). Focusing on the mechanisms regulating Golgi functions, we
investigated the regulation of MMP-9 secretion from HT 1080 cells. PMA
stimulated both MMP-9 secretion and PLD activity in a time- and
dose-dependent manner. A role for PLD was indicated by the
observation that inhibition of PA production blocked MMP-9 secretion.
Furthermore, the addition of dioctanoylphosphatidic acid (DOPA) induced
high secretion of MMP-9. The effect of DOPA seemed to be direct because
the product of its hydrolysis by phosphatidic acid phosphatase (PAP)
dioctanoylglycerol (DOG) had only a minor effect on secretion.
Moreover, inhibition of PAP with propranolol had no effect on secretion
induced by DOPA or PMA. These findings and those to be reported
elsewhere2 implicate ARF and
PLD in the stimulation of MMP-9 secretion by protein kinase C (PKC).
Materials--
Essentially fatty acid-free bovine serum albumin
(BSA) and PMA were products of Sigma. Dulbecco's modified Eagle's
medium (DMEM), fetal calf serum, and all other supplements for growth media were purchased from Life Technologies, Inc. Lipids were purchased
from Avanti Polar Lipids Inc. [9,10-3H]Myristic acid was
a product of NEN Life Science Products. Polyacrylamide 10% zymogram
gels, zymogram renaturing, and developing buffers were products of
Novex. All organic solvents were of fine grade and were obtained from Fisher.
Cell Culture--
Cells were maintained in DMEM, 10% fetal calf
serum, 10 units/ml penicillin, and 10 µg/ml streptomycin (growth
medium) at 37 °C and in 10% CO2 atmosphere. For MMP-9
assays 6 × 105 cells were seeded in 60-mm plates and
then allowed to grow for 24 h. Prior to the experiments, cells
were serum-deprived for 18 h in DMEM, 0.1% BSA. For PLD assays,
35-mm plates were seeded with 3.5 × 105 cells. After
24 h, the cells were serum-deprived and labeled in DMEM, 0.1% BSA
with 1 µCi/ml [3H]myristic acid for 18 h.
PLD Assay--
The serum-deprived and labeled cells were washed
with DMEM, 0.1% BSA, and following a 20-min preincubation in DMEM,
0.1% BSA, and 0.3% 1-butanol, they were stimulated with PMA at the
concentrations and times given in the figure legends. Cells were washed
with phosphate-buffered saline (1.68 mM KCl, 1.47 mM KH2PO4, 8.05 mM Na2PO4, 137 nM NaCl) and 0.1% BSA,
scraped with 1 ml of ice-cold CH3OH, and transferred into
glass tubes. CHCl3 and 0.1 M HCl were added to
a final ratio of 1:1:1. The lipid-containing lower phase was collected,
dried under a nitrogen stream, and dissolved in 30 µl of
CH3OH:CHCl3 (1:1). The samples were loaded on
thin layer chromatography plates that were developed in the lower phase
of H2O:ethyl acetate:acidic acid:iso-octane
(100:110:20:50). Tritiated phosphatidylbutanol (PtdBut) was measured
after the band corresponding to the PtdBut standard was scraped (Avanti
Polar Lipids Inc.).
MMP-9 Secretion and Activity Assay--
Before the assays, the
medium was replaced with fresh DMEM, 0.1% BSA containing, unless
otherwise described, either 100 nM PMA or 80 µg/ml DOPA.
Medium samples were collected, unless otherwise described, after
7.5 h and loaded with nonreducing sample buffer (2% SDS, 10%
glycerol in 62.5 mM Tris, pH 6.8) on 10% zymogram gels.
Before developing, the gels were rinsed for 30 min in renaturing buffer
and then for 30 min in developing buffer at room temperature. Gels were
incubated in fresh developing buffer for 18 h at 37 °C. MMP-9
activity was indicated by clear bands at 92 kDa that appeared after
staining with Coomassie Brilliant Blue and removal of excess dye by an
18-h rinse in water. Gels were dried and scanned, and then the image
was inverted (clear to black and black to clear) for presentation.
MMP-9 Secretion from HT 1080 Cells in Response to PMA
Treatment--
To investigate the role of PLD and PA in the induction
of MMP-9 secretion from HT 1080 cells, we established whether PMA
stimulated MMP-9 secretion from HT 1080 cells. The cells were treated
with PMA for various times, and then medium samples were assayed for collagenolytic activity. Fig.
1A illustrates that MMP-9
activity accumulated in the medium of cells treated with PMA in a
time-dependent manner, whereas no activity was observed in
untreated cells or cells treated with dimethyl sulfoxide, the solvent
for PMA. The effect of PMA was dose-dependent, with
secretion being detected with concentrations as low as 0.5-1
nM and reaching a plateau at 50-100 nM PMA
(Fig. 1B). Finally, the expected involvement of PKC in the
PMA effect was confirmed by the inhibition of secretion in cells
treated with the PKC blocker Ro 31-8220 (Fig. 1C).
PLD Activation Is Involved in the Induction of MMP-9 Secretion by
PMA--
PKC isozymes have a broad spectrum of effects and among these
is the activation of PLD (18). There is also evidence that PLD and its
product PA are important for vesicular trafficking involving the Golgi
(6-11). Therefore, we tested for the possible involvement of PLD in
the stimulation of MMP-9 secretion by PMA. We checked if PMA induced
PLD activation in HT 1080 cells. HT 1080 cells prelabeled with
[3H]myristate were stimulated with PMA for various times,
and PLD activity was measured by the formation of
[3H]PtdBut from 1-butanol. As shown in Fig.
2, PtdBut formation was rapidly induced
and reached a maximum at 90 min. The response was detectable with 1 nM PMA and maximal at 100 nM PMA (data not shown).
Although the preceding experiments showed that PLD was activated in
PMA-treated HT 1080 cells, they provided no evidence that PLD was
involved in the secretory pathway. To test this, the cells were treated
with various concentrations of a short-chain (dioctanoyl) PA (DOPA).
Fig. 3 shows that DOPA induced MMP-9
secretion in a dose-dependent manner with secretion
reaching a plateau at 80 µg/ml. A role for PLD (PA) in the pathway
leading to MMP-9 secretion received further support when 1-propanol was
used to block PA production by PLD by virtue of the formation of
phosphatidylpropanol through the transphosphatidylation reaction. Cells
were first treated with various concentrations of 1-propanol or, for
control, 2-propanol and then stimulated with PMA. Medium samples were
collected after 7.5 h and analyzed for MMP-9 secretion. Secretion
was inhibited by 100 mM 1-propanol but not by the same
concentration of 2-propanol (Fig.
4A). At 200 mM,
both alcohols were inhibitory, but the effect of 1-propanol was
complete. In an additional experiment we checked secretion from cells
treated with 133 mM 1- or 2-propanol prior to stimulation
with various PMA concentrations. Although MMP-9 secretion was increased
by increasing concentrations of PMA in the presence of 2-propanol,
1-propanol almost totally inhibited secretion (Fig. 4B).
These results demonstrate that PLD activity and the intracellular
accumulation of PA are importantly involved in
PKC-dependent MMP-9 secretion.
Prolonged PLD Activity Is Required for MMP-9 Secretion--
In the
preceding experiments, medium samples for the MMP-9 secretion assay
were taken after 7.5 h. This is because the MMP-9 activity in
shorter incubations was too low to give reliable quantitative measurements (Fig. 1A). Several reasons could account for
this, but one is that prolonged PLD activity is necessary for MMP-9 secretion. To address this possibility, cells were treated with PMA,
and 133 mM 1-propanol was added at hourly intervals after the addition of PMA. After 7.5 h, medium samples were collected and analyzed for MMP-9 secretion (Fig.
5). The figure illustrates that the
presence of 1-propanol during the first 2-3 h almost fully inhibited
MMP-9 secretion. At later times, there was partial suppression of
secretion. These data indicate that prolonged formation of PA is
important in the action of PMA on MMP-9 secretion.
MMP-9 Secretion Induced by PA Is Not Mediated by Phosphatidic Acid
Phosphatase--
Although the preceding results implicated PLD and PA
formation in the regulation of MMP-9 secretion, they did not prove that PA was the signaling molecule involved. This is because it was possible
that PA was converted to a diacylglycerol. Two approaches were used to
show that PA rather than diacylglycerol had a role in inducing
secretion. Cells were incubated with various concentrations of DOG, and
then medium samples were assayed for collagenolytic activity. As is
shown in Fig. 6A, DOG had only
a minor effect on secretion, even at a concentration higher than that
of DOPA. In additional experiments, the secretion of MMP-9 was assayed in cells pretreated with propranolol, an inhibitor of PAP activity (19). Fig. 6B shows that propranolol had no effect on the
secretion of MMP-9 from cells treated with either PMA or DOPA.
MMP-9 secretion from cancerous cells (15-17) leads to hydrolysis
of the extracellular matrix, thus enabling cells to break out of their
primary site into the circulation and from there to secondary sites
(12-14). MMP-9 secretion is induced by various agonists that differ
among various cell lines (15). We have confirmed that in human
fibrosarcoma HT 1080 cells, MMP-9 is secreted in response to PMA (17).
As expected, the secretory pathway involves the Golgi as revealed by
the inhibition of secretion by brefeldin A and other agents that
interfere with ARF activation or action.2
The most novel finding in the present study is the apparent involvement
of PLD in the effect of PMA (PKC) on MMP-9 secretion. Although PMA was
shown here to activate PLD in HT 1080 cells as in many other cell types
(18), the phorbol ester undoubtedly induced many other changes that
could be involved in the secretory response. However, the evidence
favors PLD as an important target of PKC in the stimulation of MMP-9
secretion. Importantly, inhibition of PA formation through the addition
of the primary alcohol 1-propranol blocked MMP-9 secretion, whereas
2-propanol, which does not participate in transphosphatidylation (20),
had much less effect. Furthermore, direct addition of a short-chain PA
to the cells was as effective as PMA in stimulating MMP-9 release,
supporting the postulated role of PLD.
The present study does not define the cellular site of action of PLD.
However, it is very probable that it is the Golgi, because there is
much evidence that PLD is involved in protein trafficking involving
this organelle (6-11), and exogenous PA can relieve the inhibition of
MMP-9 secretion induced by brefeldin A, an inhibitor of Golgi
function.2 In addition, prolonged activation of PLD was
required for the effect of PMA (Fig. 5) consistent with its involvement
in a relatively slow process. The PLD isoform (PLD1) that has been
localized to the Golgi (21) is known to be responsive to both PKC and
ARF (22), unlike the PLD2 isoform (21). These observations provide additional support for a site of action at this organelle.
Because PA can be metabolized to other lipids such as diacylglycerol
(by PAP) or lysophosphatidic acid (by phospholipase A2), the initial results provided no assurance that the observed effects of
added PA were because of this lipid per se. However, the
possibility that diacylglycerol was the active lipid was rendered
unlikely by the observation that the product of PAP action on DOPA
(DOG) was largely ineffective. Furthermore, propranolol, an inhibitor of PAP, did not diminish the effect of DOPA. With regard to the possibility that lysophosphatidic acid was the active agent, this seems
improbable because the addition of this lipid to HT 1080 cells did not
affect MMP-9 secretion.3
Finally, we describe here the characterization of a new model system
for the study of Golgi-dependent secretion. In comparison with other methods, the assay of MMP-9 secretion is quick and convenient. It offers an easier and faster way for studying membrane vesiculation and trafficking in response to extracellular signals. Unlike most other methods, it does not require cell permeabilization, virus infection, or any other stressful procedure. Therefore, the cells
are in a more physiological state providing information that is as
valid as possible in a tissue culture experiment.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
PMA induces MMP-9 secretion in HT 1080 cells. A, overnight serum-deprived cells were incubated
in freshly added DMEM, 0.1% BSA. 100 nM PMA was added at
various times to give the indicated durations of exposure to the
phorbol ester. Dimethyl sulfoxide (DMSO) solvent was used as
the control for PMA. After 7.5 h, medium samples, 5 µl each,
were separated on 10% zymogram gels, and MMP-9 activity was assayed as
described under "Experimental Procedures." B, cells were
incubated with the indicated concentrations of PMA. After 7.5 h,
medium samples were taken and assayed for MMP-9 activity. C,
cells were preincubated with or without 5 µM PKC
inhibitor Ro 31-8220 for 1 h and then stimulated with dimethyl
sulfoxide control or 100 nM PMA for 7.5 h. Medium
samples were taken and assayed for MMP-9 activity as described. The
data shown for all panels are representative of three independent
experiments.
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Fig. 2.
Activation of PLD in PMA-treated HT 1080 cells. The cells, at their log phase of growth, were
serum-deprived and labeled with 1 µCi/ml [3H]myristic
acid. Following washing and preincubation with DMEM, 0.1% BSA, 0.3%
1-butanol for 20 min, 100 nM PMA was added, and cells were
incubated for the indicated times at 37 °C. Lipids were extracted
and analyzed, and the production of [3H]PtdBut was as
described under "Experimental Procedures." [3H]PtdBut
values were normalized by dividing the measured counts/min by the
counts/min in the total lipid fraction. Data are expressed as the
mean ± S.E. of the fold activations in three independent
experiments performed in triplicate.
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Fig. 3.
MMP-9 secretion in response to DOPA
treatment. Overnight serum-deprived cells were incubated in
freshly added DMEM, 0.1% BSA with the indicated concentrations of
DOPA. After 7.5 h, 5-µl media samples were assayed for MMP-9
activity as described under "Experimental Procedures." The data
shown are representative of three independent experiments.
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Fig. 4.
MMP-9 secretion is inhibited by
1-propanol. Overnight serum-deprived cells were incubated with
freshly added DMEM, 0.1% BSA. A, the indicated
concentrations of either 2-propanol or 1-propanol were then added.
After 45 min, 100 nM PMA was added, and cells were
incubated for 7.5 h after which 5-µl medium samples were
analyzed for MMP-9 secretion. B, the cells were incubated
with 133 nM 2-propanol or 1-propanol for 45 min. PMA was
then introduced to give the indicated concentrations. The cells were
incubated for 7.5 h after which 5-µl medium samples were
analyzed for MMP-9 secretion. The data shown are representative of
three independent experiments.
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Fig. 5.
PLD activity is required during the early
stages of PMA induction of MMP-9 secretion. Serum-deprived cells
were incubated with fresh DMEM, 0.1% BSA. PMA (100 nM) was
added at 0 h, and 1-propanol (final concentration of 133 mM) was added at the indicated times thereafter. The medium
was sampled after 7.5 h and analyzed for MMP-9 secretion. The data
shown are representative of three independent experiments.
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Fig. 6.
Phosphatidic acid phosphatase is not involved
in the stimulation of MMP-9 secretion in DOPA-treated cells.
Overnight serum-deprived cells were incubated with fresh DMEM, 0.1%
BSA. A, the cells were then stimulated by the indicated
concentrations of DOG (dried and solubilized by a 30-s sonication in
DMEM, 0.1% BSA) or DOPA. B, the cells were incubated with
the indicated concentrations of propranolol for 30 min, and then 100 nM PMA or 80 µg/ml DOPA were added. After 7.5 h,
5-µl medium samples were assayed for MMP-9 activity. The data shown
are representative of three independent experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank Dr. Lynn Matrisian for helpful discussions and also Judy Childs for invaluable help in the preparation of this manuscript.
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FOOTNOTES |
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* 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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton{at}mcmail.vanderbilt.edu.
The abbreviations used are: ARF, ADP-ribosylation factor; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PA, phosphatidic acid; DOPA, dioctanoylphosphatidic acid; MMP-9, matrix metalloproteinase-9; PAP, phosphatidic acid phosphatase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PLD, phospholipase D; PtdBut, phosphatidylbutanol; DOG, dioctanoylglycerol.
2 B.-T. Williger, W.-T. Ho, S. Gharachoulou, and J. H. Exton, manuscript in preparation.
3 B.-T. Williger, and J. H. Exton, unpublished observations.
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REFERENCES |
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