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 transdu ction and membrane traffic. We have synthesized a photolyzable derivat ive of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events, 1-(2-Nitrophenyl)diazoethan e, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl- PA to yield a 1-(2-nitrophenyl)ethyl ester of Pk Photolysis of the com pound by ultraviolet Light resulted in the formation of phosphatidic a cid, The structure of the compound and of its photolytic products was verified by NMR spectroscopy, The utility of cPA was examined in HT 10 80 metastatic fibrosarcoma cells, in which the formation of PA by phos pholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)), The uptake of cPA by BT 1080 cel ls reached a plateau after 120 min of incubation, Ultraviolet illumina tion 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 elevatio n in the release of MMP-2 to the medium, whereas nonphotolyzed cPA cau sed no change in MMP-2 release, Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly intr oduced Pk Thus, the effect of laminin on MMP-2 secretion can be mimick ed by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis, These res ults indicate that cPA could serve as a unique tool for studying the c ellular roles of PA.