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Phospholipase D mediates matrix metalloproteinase-9 secretion in phorbol ester-stimulated human fibrosarcoma cells
B T Williger1, W T Ho, J H Exton
1Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
Abstract:
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.
Insights
Phospholipase D (PLD) activation stimulates matrix metalloproteinase-9 (MMP-9) secretion in fibrosarcoma cells. This process involves protein kinase C and phosphatidic acid (PA) formation, crucial for MMP-9 release.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Phospholipase D (PLD) plays a role in vesicle trafficking and secretion.
- Matrix metalloproteinase-9 (MMP-9) is involved in extracellular matrix degradation and cell invasion.
Purpose of the Study:
- To investigate the role of Phospholipase D (PLD) in the secretion of matrix metalloproteinase-9 (MMP-9).
- To elucidate the signaling pathway involving protein kinase C and phosphatidic acid (PA) in MMP-9 secretion.
Main Methods:
- Stimulation of HT 1080 fibrosarcoma cells with phorbol 12-myristate 13-acetate.
- Measurement of MMP-9 secretion and PLD activity.
- Inhibition of PA production using 1-propanol.
- Exclusion of diacylglycerol involvement using short-chain diacylglycerol and propranolol.
Main Results:
- Phorbol 12-myristate 13-acetate dose-dependently stimulated MMP-9 secretion via protein kinase C.
- PLD activity and phosphatidic acid (PA) formation were increased by the phorbol ester.
- Exogenous PA stimulated MMP-9 secretion, while 1-propanol inhibited PA production and secretion.
- Diacylglycerol and phosphatidic acid phosphatase were excluded from the signaling pathway.
Conclusions:
- Protein kinase C activation enhances MMP-9 secretion in HT 1080 cells.
- PLD activation and subsequent phosphatidic acid (PA) formation are critical mediators of this enhanced secretion.