Role of Protein Kinase Cζ in Thrombin-induced RhoA Activation and Interendothelial Gap Formation of Human Dermal Microvessel Endothelial Cell Monolayers

• Published in: Microvascular research Vol. 80; no. 2; pp. 240 - 249
• Main Authors: Minshall, Richard D., Vandenbroucke, Emily E., Holinstat, Michael, Place, Aaron T., Tiruppathi, Chinnaswamy, Vogel, Stephen M., van NieuwAmerongen, Geerten P., Mehta, Dolly, Malik, Asrar B.
• Format: Journal Article
• Language: English
• Published: 24.04.2010
• ISSN: 0026-2862; 1095-9319
• DOI: 10.1016/j.mvr.2010.04.007

We studied the potential involvement of the Ca 2+ -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 Ca 2+ concentration [Ca 2+ ] 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 Ca 2+ -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 [Ca 2+ ] 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 Ca 2+ -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.