Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier.
The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, 14642, United States of America.
Peter V
PV
DiStefano
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, 14642, United States of America.
Alan V
AV
Smrcka
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, 14642, United States of America.
Angela J
AJ
Glading
D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000502:physiology; D055785:Gene Knockdown Techniques; D006801:Humans; D000075927:KRIT1 Protein; D008049:Lipase / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D020687:cdc25 Phosphatases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D008049:Lipase; D020687:cdc25 Phosphatases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020742:rhoA GTP-Binding Protein