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.
Université de Lyon, F-69622, Lyon, France.
Caroline
C
Zeiller
Saïda
S
Mebarek
Rami
R
Jaafar
Luciano
L
Pirola
Michel
M
Lagarde
Annie-France
AF
Prigent
Georges
G
Némoz
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D021962:Membrane Microdomains; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D062793:Occludin; D010712:Phosphatidic Acids; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D000199:Actins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010712:Phosphatidic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1