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Phospholipase D Mediates Matrix Metalloproteinase-9 Secretion in Phorbol Ester-stimulated Human Fibrosarcoma Cells*

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.

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 12myristate 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)(2)(3)(4)(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)(13)(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 elsewhere 2 implicate ARF and PLD in the stimulation of MMP-9 secretion by protein kinase C (PKC).

EXPERIMENTAL PROCEDURES
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. [9,10-3 H]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 g/ml streptomycin (growth medium) at 37°C and in 10% CO 2 atmosphere. For MMP-9 assays 6 ϫ 10 5 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 ϫ 10 5 cells. After 24 h, the cells were serum-deprived and labeled in DMEM, 0.1% BSA with 1 Ci/ml [ 3 H]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 KH 2 PO 4 , 8.05 mM Na 2 PO 4 , 137 nM NaCl) and 0.1% BSA, scraped with 1 ml of ice-cold CH 3 OH, and transferred into glass tubes. CHCl 3 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 l of CH 3 OH:CHCl 3 (1:1). The samples were loaded on thin layer chromatography plates that were developed in the lower phase of H 2 O: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 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
was replaced with fresh DMEM, 0.1% BSA containing, unless otherwise described, either 100 nM PMA or 80 g/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 [ 3 H]myristate were stimulated with PMA for various times, and PLD activity was measured by the formation of [ 3 H]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 g/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. DISCUSSION MMP-9 secretion from cancerous cells (15)(16)(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)(13)(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 A 2 ), 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.