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Role of protein kinase Czeta in thrombin-induced RhoA activation and inter-endothelial gap formation of human dermal microvessel endothelial cell monolayers.
Minshall, Richard D; Vandenbroucke, Emily E; Holinstat, Michael; Place, Aaron T; Tiruppathi, Chinnaswamy; Vogel, Stephen M; van Nieuw Amerongen, Geerten P; Mehta, Dolly; Malik, Asrar B.
Afiliação
  • Minshall RD; Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA. rminsh@uic.edu
Microvasc Res ; 80(2): 240-9, 2010 Sep.
Article em En | MEDLINE | ID: mdl-20417648
We studied the potential involvement of the Ca(2+)-independent atypical protein kinase C isoform PKCzeta in mediating the thrombin-induced increase in endothelial permeability. Studies were done using human dermal microvessel endothelial cells (HMEC), which we showed constitutively expressed PKCzeta. 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 approximately 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 PKCzeta activation using the PKCzeta pseudosubstrate peptide (PSI), depletion of PKCzeta protein with siRNA, and competitive inhibition of PKCzeta activity using dominant-negative (dn) PKCzeta mutant all prevented the thrombin-induced decrease in TER and MLC phosphorylation. Expression of dn-PKCzeta also inhibited thrombin-induced RhoA activation. These findings reveal a novel Ca(2+)-independent, PKCzeta-dependent mechanism of thrombin-induced increase in endothelial permeability. The results raise the possibility that inhibition of PKCzeta may be a novel drug target for thrombin-induced inflammatory hyperpermeability.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteína Quinase C / Hemostáticos / Trombina / Derme / Proteína rhoA de Ligação ao GTP / Células Endoteliais / Microvasos Limite: Humans Idioma: En Ano de publicação: 2010 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteína Quinase C / Hemostáticos / Trombina / Derme / Proteína rhoA de Ligação ao GTP / Células Endoteliais / Microvasos Limite: Humans Idioma: En Ano de publicação: 2010 Tipo de documento: Article