No abstract

This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.

Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A 2 activity by both wortmannin and DMV with an IC 50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (ϳ10 nM). While it has not been possible to fully block in vitro phospholipase A 2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.The fungal metabolite wortmannin and its structural analogue demethoxyviridin (DMV) 1 have been demonstrated to have an inhibitory effect upon phosphatidylinositol 3-kinase (PI 3-kinase) activity at nanomolar concentrations (1). Inhibition has been demonstrated upon both PI 3-kinase activity in anti-p85 immunoprecipitates and the stimulation of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) generation in N-formylmethionylleucylphenylalanine (fMLP)-stimulated neutrophils (1). The binding of 17-[ 3 H]hydroxywortmannin to a number of neutrophil proteins has been demonstrated, and one of these has been identified as the 110-kDa subunit of PI 3-kinase (2).On the basis of these findings, it has been proposed that wortmannin and DMV are specific inhibitors of PI 3-kinase, and, thus, addition of these compounds to cells will result in the specific inhibition of the PI 3-kinase pathway. Consequently, an increasing number of papers have described the use of wortmannin and, on the sole basis of such experiments, assigned a role for PI 3-kinase in a number of physiological responses (e.g. Refs. 3-5).Wortmannin has also been reported to inhibit phospholipase D (PLD) (6), myosin light chain kinase, and plekstrin phosphorylation (see Ref. 1). Although these results could cast doubt upon the specificity of wortmannin, these effects have been shown to occur at concentrations greater than those reported to inhibit PI 3-kinase. However, the specificity of wortmannin for other lipid-metabolizing enzymes has not been examined. In this paper, we have examined the specificity of wortmannin and DMV and show that they inhibit stimulated PIP 2 -phospholipase C (PI-PLC), PLD, phospholipase A 2 (PLA 2 ) as well as PI 3-kinase in Swiss 3T3 cells and, in addition, in vitro PI 3-kinase and PLA 2 activities. We also demonstrate that both compounds are more potent inhibitors of PLA 2 than PI 3-kinase. EXPERIMENTAL PROCEDURESMaterials-Radiochemicals were from Amersham International plc, except for the inositol phosphate standards which were from DuPont NEN, all tissue culture media and sera were from Life Technologies, Inc., t...

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A 2 and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A 2 and 5-lipoxygenase activities are sites for potential therapeutic intervention.