Abstract
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.
Highlights
Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic
We show that caged PA (cPA) photolysis leads to a rise in intracellular phosphatidic acid (PA) and subsequently to secretion of MMP-2 in HT 1080 cells
The synthesis of cPA is based on alkylation of the weakly ionized phosphate group of PA by 1-(2-nitrophenyl)diazoethane (Fig. 1A)
Summary
Materials—All chemicals and solvents used were of reagent grade. Dipalmitoylphosphatidic acid (sodium salt), imidazole, and bovine serum albumin (BSA) were obtained from Sigma. Preparation of the Free Acid Form of PA—Phosphatidic acid (sodium salt) was dissolved in chloroform to yield a final concentration of 14 mg/ml. The lower phase containing the free acid form of PA was collected, and the pH was adjusted to 4 –5 with 10% triethylamine in chloroform. Synthesis of cPA—The preparation of 1-(2-nitrophenyl)hydrazonoethane was carried out by dissolving 0.826 g (5 mmol) of 2-nitroacetophenone, 0.562 g (11.2 mmol) of hydrazine hydrate, and 0.32 ml (5 mmol) of glacial acetic acid in 10 ml of ethanol in a 20-ml reflux receptacle. The final stage in the preparation of 1-(2-nitrophenyl)ethyl ester of PA (namely cPA) was accomplished by stirring PA-free acid solution (10.35 mol) together with the newly prepared 1-(2-nitrophenyl)diazoethane (155.25 mol) for 45 min. Results are expressed as the measured intensity relative to that of control
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