Release of gelatinase A (matrix metalloproteinase 2) induced by photolysis of caged phosphatidic acid in HT 1080 metastatic fibrosarcoma cells.
Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic. We have synthesized a photolyzable derivative of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events. 1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of PA. Photolysis of the compound by ultraviolet light resulted in the formation of phosphatidic acid. The structure of the compound and of its photolytic products was verified by NMR spectroscopy. The utility of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which the formation of PA by phospholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after 120 min of incubation. Ultraviolet illumination of cPA-loaded cells for 5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to the medium, whereas nonphotolyzed cPA caused no change in MMP-2 release. Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly introduced PA. Thus, the effect of laminin on MMP-2 secretion can be mimicked by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis. These results indicate that cPA could serve as a unique tool for studying the cellular roles of PA.
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
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BT
Williger
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R
Reich
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M
Neeman
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Bercovici
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Liscovitch
D002460:Cell Line; D005354:Fibrosarcoma; D018093:Gelatinases / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000378:metabolism; D009362:Neoplasm Metastasis; D010712:Phosphatidic Acids / Q000138:chemical synthesis / Q000494:pharmacology* / Q000528:radiation effects; D010782:Photolysis; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
C097201:1-(2-nitrophenyl)ethyl-phosphatidic acid; D010712:Phosphatidic Acids; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2