Role of protein kinase Cζ in thrombin-induced RhoA activation and inter-endothelial gap formation of human dermal microvessel endothelial cell monolayers

We studied the potential involvement of the Ca2+-independent atypical protein kinase C isoform PKCζ in mediating the thrombin-induced increase in endothelial permeability. Studies were done using human dermal microvessel endothelial cells (HMEC), which we showed constitutively expressed PKCζ. We quantified the patency of inter-endothelial junctions (IEJs) and endothelial barrier function by measuring transendothelial electrical resistance (TER) in confluent HMEC monolayers. In control monolayers, thrombin decreased TER by ∼ 50%, indicating thrombin-dependent opening of IEJs. Thrombin also elicited increases in cytosolic Ca2+ concentration [Ca2+]i, actin stress fiber formation, and myosin light chain (MLC) phosphorylation. Pan-PKC inhibitors, calphostin C and chelerythrine, abrogated these responses. Thrombin also decreased TER after depletion of conventional and novel Ca2+-dependent PKC isoforms using phorbol 12-myristate 13-acetate (PMA). In these PMA-treated cells, thrombin induced inter-endothelial gap formation, MLC phosphorylation, and actin stress fiber formation, but failed to increase [Ca2+]i. Inhibition of PKCζ activation using the PKCζ pseudosubstrate peptide (PSI), depletion of PKCζ protein with siRNA, and competitive inhibition of PKCζ activity using dominant-negative (dn) PKCζ mutant all prevented the thrombin-induced decrease in TER and MLC phosphorylation. Expression of dn-PKCζ also inhibited thrombin-induced RhoA activation. These findings reveal a novel Ca2+-independent, PKCζ-dependent mechanism of thrombin-induced increase in endothelial permeability. The results raise the possibility that inhibition of PKCζ may be a novel drug target for thrombin-induced inflammatory hyperpermeability.