Phorbol Ester-Induced Endothelial Cytoskeletal Remodeling

Abstract

Proper functioning of intracellular membrane trafficking in endothelial cells is indispensable for maintaining endothelial barrier, as well as for regulated secretion of a variety of pro-and anticoagulants and inflammatory proteins. While the involvement of several heterotrimeric G proteins in regulation of membrane trafficking has been documented, there is only one work published on the possible role of G␣12 in this respect in PC12 cells, where G␣12 has been reported to inhibit exocytosis. In the present study, we addressed possible roles of G␣12 in membrane trafficking in endothelial cells, using human umbilical vein endothelial cells (HUVEC) as a model. Using confocal microscopy, ECIS measurements, and velocity sedimentation assays, we examined the effects of overexpression of wild-type or constitutively active G␣12, as well as siRNA-mediated depletion of endogenous G␣12, on several markers in resting or thrombin-stimulated HUVEC. Our data indicate that G␣12 has stimulatory effect on exocytosis in HUVEC. Moreover, we were able to detect macromolecular complexes of G␣12 with transported proteins, suggesting its direct involvement in membrane trafficking.