Protein kinase C-alpha signals Rho-guanine nucleotide dissociation inhibitor phosphorylation and Rho activation and regulates the endothelial cell barrier function

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and pho rbol la-myristate 13-acetate induced rapid phosphorylation of GDI and the a ctivation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosph orylation and Rho activation. Depletion of PKC prevented the thrombin-induc ed GDI phosphorylation and Rho activation, thereby indicating that these ev ents occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the t hrombin-induced decrease in transendothelial electrical resistance (a measu re of increased transendothelial permeability), thus indicating that PKC-in duced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venul ar endothelial cells with thrombin induced rapid association of PKC-alpha w ith Rho, Activated PKC-alpha but not PKC-epsilon induced marked phosphoryla tion of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in sign aling Rho-dependent endothelial barrier dysfunction.