Role of protein kinase Czeta in thrombin-induced RhoA activation and inter-endothelial gap formation of human dermal microvessel endothelial cell monolayers.
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.
Minshall
Richard D
RD
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA. rminsh@uic.edu
Vandenbroucke
Emily E
EE
Holinstat
Michael
M
Place
Aaron T
AT
Tiruppathi
Chinnaswamy
C
Vogel
Stephen M
SM
van Nieuw Amerongen
Geerten P
GP
Mehta
Dolly
D
Malik
Asrar B
AB
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D053119:Benzophenanthridines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D020405:Dermis / Q000098:blood supply* / Q000201:enzymology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D020868:Gene Silencing; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism; D055806:Microvessels / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis*; D000099221:Protein Kinase C zeta
D053119:Benzophenanthridines; D002118:Calcium; D006490:Hemostatics; D018994:Myosin Light Chains; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D000099221:Protein Kinase C zeta; C066737:RHOA protein, human; C016299:chelerythrine