Phospholipase D2 regulates endothelial permeability through cytoskeleton reorganization and occludin downregulation.

Endothelial permeability is controlled by adhesive strengths which connect cells to each other through interendothelial junctions and by contractile forces associated with cytoskeleton reorganization. Phospholipase D (PLD) activation resulting in the generation of phosphatidic acid (PA) is increasingly recognized as a key event in the initiation of various cell responses. In human umbilical vein endothelial cells (HUV-EC), enhancement of intracellular PA by a variety of approaches increased the permeability of endothelial cell monolayers and induced stress fibre formation. Using adenovirus-mediated overexpression and siRNA silencing, we showed that PLD2 but not PLD1 was involved in the enhancement of basal permeability through cytoskeleton reorganization. Furthermore, PLD2 overexpression induced ERK1/2 activation and downregulated the expression of occludin, a major component of tight junctions. A substantial part of PLD2 protein was associated with the low-density caveolin-rich fractions isolated on sucrose gradients. The Raf-1 specific inhibitor GW-5074 drastically reduced hyperpermeability induced by PLD2 overexpression, and inhibited PA-mediated increase of endothelial permeability and ERK1/2 activation. On the whole, the present results demonstrate the selective role of PLD2 isoform in the control of endothelial permeability through a mechanism involving both stress fibre formation and contraction, and occludin downregulation, possibly resulting from PA-mediated activation of Raf-1.